clinical contributions Abdominal Cutaneous Nerve Entrapment Syndrome (ACNES): A Commonly Overlooked Cause of Abdominal

By William V Applegate, MD, FABFP

Introduction two patients with this diagnosis for every 150 patients Abdominal cutaneous nerve entrapment syndrome overall but have seen as many as three such patients (ACNES) may sound like an esoteric condition rarely per consultation session in a busy evening clinic where seen by clinicians but is a common condition. When a 15 or more clinicians were on duty. patient is seen for abdominal pain without other clini- Acute cases of ACNES are usually seen in the evening, cally significant symptoms, ACNES should be high on especially in spring and summer, when people are more the list of likely diagnoses. active. Chronic and recurrent cases are more likely to Beginning in 1792 with J P Frank’s description of the be seen in the daytime throughout the year. condition he named “peritonitis muscularis,”1 a sam- To avoid causing the patient unnecessary anxiety and pling of pertinent medical literature on this subject2-9 tension, loss of work time, and both the expense and shows how often the subject has been written about possible hazard of multiple diagnostic procedures, the over the years. These articles state that abdominal wall first physician examining the patient must establish the pain is often wrongly attributed to intra-abdominal dis- diagnosis of ACNES if this condition is present. Com- orders and that this misdirected diagnosis can lead to piled from my own experience and that of other inves- unnecessary consultation, testing, and even abdominal tigators who have written about ACNES, the informa- surgery, all of which can be avoided if the initial exam- tion presented here should give readers the tools iner makes the right diagnosis. In a study of 117 pa- necessary for diagnosing and treating this condition. tients in 1999, Greenbaum10 estimated that the amount of money expended on unnecessary workup was $914 Pathophysiology of ACNES per patient. In 2001, Thompson et al11 noted that an Kopell and Thompson13 stated that peripheral nerve average of $6727 per patient was required for previous entrapment occurs at anatomic sites where the nerve diagnostic testing and hospital charges. Hershfield6 listed changes direction to enter a fibrous or osseofibrous preliminary diagnoses of patients re- tunnel or where the nerve passes over ferred to him as irritable bowel, spas- a fibrous or muscular band and that When a patient is tic colon, gastritis, psychoneurosis, de- entrapment can be at these sites be- seen for abdominal pression, anxiety, hysteria, and cause mechanically induced irritation pain without other malingering. Many of these patients is most likely to occur at these loca- clinically significant were given a psychiatric diagnosis tions. Muscle contraction at these sites symptoms, ACNES when the actual diagnosis could not may add additional insult by direct should be high on be determined. In fact, the most com- compression, although I believe that the list of likely mon cause of abdominal wall pain is traction on the nerve from muscle ac- diagnoses. nerve entrapment at the lateral bor- tivity also is likely. Mechanical irrita- der of the rectus abdominis tion causes localized swelling that may muscle;3,5,8,9,12 Carnett,3 in the early 20th century, called injure the nerve directly or compromise the nerve’s this syndrome “intercostal neuralgia” and claimed to circulation. Tenderness of the main nerve trunk may have seen three patients per week with this diagnosis be found proximal or distal to the affected portion and as many as three per day in consultation sessions. (Valleix phenomenon). Proximal tenderness may re- In my own primary care practice, I have seen one or sult from vascular spasm or from unnatural traction

William V Applegate, MD, FABFP, is a retired SCPMG Family Practice physician from San Diego. He is an Associate Clinical Professor at UCSD Medical School in San Diego, CA.

20 The Permanente Journal/ Summer 2002/ Volume 6 No. 3 clinical contributions Abdominal Cutaneous Nerve Entrapment Syndrome ACNES: A Commonly Overlooked Cause of Abdominal Pain

on the nerve trunk against the point of entrapment. In ceptible to mechanical irritation than are the posterior ACNES, all these mechanisms can be at work. and lateral branches, which enter the muscle at a more oblique angle. Lateral branches are affected by lateral Anatomy Pertinent to ACNES bending and twisting of the trunk; posterior branches The thoracoabdominal nerves, which terminate as are affected by bending, lifting, and twisting. the cutaneous nerves, are anchored at six points (Fig- Accessory branches perforate the muscle wall Symptoms of ure 1):14 1) the spinal cord; 2) the point at which the above and below the main branches but also ACNES can be posterior branch originates; 3) the point at which the exit from adjacent muscle mass. These acute or chronic … lateral branch originates; 4) the point at which the an- branches are difficult to palpate unless symp- The pain may terior branch makes a nearly 90° turn to enter the rec- tomatic and tender to touch. have started tus channel; 5) the point from which accessory branches during the night are given off in the rectus channel, shown (although Diagnosing ACNES but did not cause not labeled) in previously published microphoto- Clinical Presentation these patients to graphs;15 and 6) skin. Symptoms of ACNES can be acute or chronic. miss work the The most common cause of abdominal wall pain is The acute pain is described as localized, dull, next morning. nerve entrapment at the lateral border of the rectus or burning, with a sharp component (usually muscle. In the rectus channel, the nerve and its vessels on one side) radiating horizontally in the up- are surrounded by fat and connective tissue that bind per half of the abdomen and obliquely downward in the nerve, artery, and vein into a discrete bundle ca- the lower abdomen. The pain may radiate when the pable of functioning as a unit independently from sur- patient twists, bends, or sits up. Lying down may help rounding tissue. At a point located about three quar- but sometimes worsens the pain. Younger people, who ters of the way through the rectus muscle (from back are usually more physically active than older persons, to front), there is a fibrous ring that provides a smooth are more often seen for the first episode of acute pain. surface through which the bundle can slide. Positioned The pain may have started during the night but did not anterior to the ring, the rectus aponeurosis provides a cause these patients to hiatus for the exiting bundle. miss work the next The hypothesis that nerve ischemia is caused by local- morning. Nonetheless, ized compression of the nerve at the level of the ring is they come to the deduced from juxtaposition of the soft bundle to the evening clinic because hard ring. Herniation of the bundle through the ring the pain persists, wors- due to too much pressure from behind or from pulling ens, and causes them from in front will compress the bundle’s vessels and to be afraid that they the nerve itself. Too much traction on the bundle from won’t be able to work behind or from pulling in front will cause the bundle the next day. Young to be “strummed” against the ring, which then causes women often express irritation and swelling even before herniation occurs. concern about their Anything that increases pressure behind the abdomi- “ovaries,” “kidneys” nal wall can cause the bundle to herniate through the (the bladder is meant), fibrous ring and aponeurotic opening. Use of the ab- or both. dominal muscles can add additional insult. Enlarge- Brief discussion of the ment of the abdomen from any cause will put the nerves ovarian complaint here under greater traction. Scar or suture around the nerve is important because it in front of the rectus16-18 can directly compress the nerve occurs frequently and is or place it under further traction. Disparate motion the predominant initial between skin and muscle will aggravate this situation. reason for women with Although any main branch of the nerve may become ACNES to be seen in symptomatic, the anterior branches are most likely to the clinic.16,17,19,20 be affected, because stretching of the nerve is greatest Concern about their Figure 1. Anatomy of thoracoabdominal nerves. at the point most distant from its origin (ie, the spinal gonads is uppermost in (Adapted and reproduced by permission of the publisher, cord). Because the anterior branches enter the back of the minds of young of the author, and of the illustrator, Nelva M Bonucchi, from: Applegate WV. Abdominal cutaneous nerve entrapment the muscle at nearly a right angle, they are more sus- people who have re- syndrome. Am Fam Physician 1973 Sep;8(3):132-3.14)

The Permanente Journal/ Summer 2002/ Volume 6 No. 3 21 clinical contributions Abdominal Cutaneous Nerve Entrapment Syndrome ACNES: A Commonly Overlooked Cause of Abdominal Pain

cently matured sexually. Because the testicles are lo- was happy to hear my explanation of its cause. If a cated in the externally positioned scrotal sac, men have patient says, “I have this pain in my stomach, and no- the advantage of being able to examine their testicles body seems able to find the cause,” the examiner should easily, whereas women’s ovaries, being located inside immediately think of ACNES. the abdomen, are inaccessible to examination except ACNES-related pain is well localized and usually af- by medical personnel. Consequently, women may at- fects only one side. However, the pain can occur on tribute any abdominal complaint to an ovarian disor- both sides at the same level (usually in the lower ab- der until a different cause of pain is shown. Given a domen), or more than one nerve can be affected on chief complaint of “pain in the ovary,” the examiner opposite sides and at different levels. Pain radiating should certainly examine the ovaries but should re- into the scrotum or vulva suggests involvement at the member that this is often the way ACNES clinically T12/L1 level, but inguinal or femoral hernia and pain manifests itself. arising from the adductor muscles of the thigh must Unfortunately, women are not the only be ruled out. Pain and tenderness ones to attribute abdominal pain to gy- posterolaterally just below the iliac crest Chronic necologic pathology when the source of can occur with involvement at the T12/ ACNES pain is actually in the abdominal wall. L1 level. This finding is useful because it patients suffer Noting that between 30% and 76% of di- is present with abdominal wall pain but considerable agnostic laparoscopic procedures done is absent if the pain arises from inside anxiety and for pelvic pain show normal tissues, the abdomen.3 Pain radiating from T11 worry that Slocomb20 expressed concern about sur- and T12 runs at an oblique angle and they may have gical exploration with removal of pelvic follows the course of these nerves. Such some horrible structures for normal variants in women pain can suggest urolithiasis; however, condition … with chronic pelvic pain when the prob- patients with urolithiasis are usually seen lem was actually traceable to the abdomi- writhing in pain, whereas patients with nal wall. One of my patients was a woman who had ACNES tend to lie quietly on the table with their hand surgery first for “ovarian cyst” and then for “adhesions” placed over the area of discomfort. T11 involvement but still complained of the same pain, which, I discov- on the right side may suggest appendicitis, and involve- ered, was caused by ACNES.5 A study of 120 emer- ment on either side may suggest ovarian involvement gency admissions to the hospital for abdominal pain21 or spigelian hernia; all these conditions should be iden- showed that 23 of 24 patients who had abdominal sur- tified by proper physical examination. Pain on the right gery with a positive Carnett’s sign (see below) had no side at the T8 or T9 level may suggest cholecystitis or intra-abdominal disorder; instead, the pain was traced peptic ulcer; however, as Carnett3 has suggested, deep to the abdominal wall. tenderness is not detected without peritoneal involve- Young men with ACNES are often seen in the daytime ment. Pain at the T6, T7, or T8 levels can suggest pleu- for a chief complaint of “hernia” or “ulcer,” complaints risy, costochondritis, or slipping rib syndrome (which considered more common in men. Older men and is probably a form of ACNES caused by traction). Pain women may express concern about cancer (not an un- and numbness laterally in the thigh and hip may be reasonable concern among seniors). These patients may caused by meralgia paresthetica, mentioned here as a need further examination, even if ACNES caused the matter of interest because it is also caused by nerve pain that brought them to the doctor. A history of mul- entrapment; in this case, the lateral femoral cutaneous tiple abdominal operations should raise suspicion about nerve is entrapped between the iliac ligament and the ACNES. Finding several surgical scars on the abdomen anterosuperior aspect of the iliac spine.13 For a com- should alert the examiner to this possibility. plete list of conditions other than ACNES that can cause Chronic complaints due to ACNES are usually seen abdominal wall pain, the reader is referred to Carnett,3 during the day in older patients. Medical history in Hershfield,6 Suleiman and Johnston,9 Gallegos and these patients shows that acute exacerbation of pain Robsley,17 and Greenbaum.22 may occur over several days or weeks and then disap- Chronic ACNES patients suffer considerable anxiety pear for varying lengths of time, sometimes for years. and worry that they may have some horrible condition One of my male patients with ACNES reported that he as yet undiscovered. As a result, they may be given a had pain intermittently for 47 years.5 He had long ago psychiatric diagnosis (eg, anxiety, somatization, or de- decided that the pain was of no great consequence but pression) and therefore often take antidepressant drugs,

22 The Permanente Journal/ Summer 2002/ Volume 6 No. 3 clinical contributions Abdominal Cutaneous Nerve Entrapment Syndrome ACNES: A Commonly Overlooked Cause of Abdominal Pain

tranquilizers, muscle relaxants, or pain relievers. Such it is associated with symptoms and localized tenderness. a medical history should raise the question of ACNES. A description of how the anterior foramina actually feels will help examiners to find them. Approaching Physical Examination the opening with the hand resting lightly on the abdo- A suggestive medical history should direct the exam- men from the lateral side, the middle fingertip is moved iner to precisely locate the tender spot by asking the over the rounded edge of the rectus, where the exam- patient, “Where exactly is the pain?” The patient usu- iner may feel an oval-shaped depression oriented hori- ally responds by placing several fingers over the area, zontally but sloped posteriorly on the edge of the rec- whereupon the examiner says, “Show me with one tus at levels T8 through T12/L1. As pressure from the finger.” As patients place a fingertip on the exact spot, straight finger tuft is gradually increased, the examiner pushing a little harder to find it, they usually say, “Right feels, in order: 1) firm skin; 2) spongy-textured subcu- here!” and flinch as the tender spot is pressed. taneous fat; 3) the oval, firm ring of the aponeurosis To proceed beyond this point in the examination, the examiner must be familiar with the exact location of each neuromuscular foramen. To do this, the exam- iner should practice finding these depressions on his or her own abdomen and on someone else. In addition, each time a patient’s abdomen is examined for any rea- son, the examiner should feel for these aponeurotic open- ings; their size differs widely among persons. Larger open- ings, usually found in obese patients, are easier to palpate and provide familiarity with the feel of a foramen so that the examiner will know what to look for when presented with smaller dimensions in another patient. The anterior exits are easiest to feel and are often best felt with the patient standing and pushing the ab- domen out: T10 is at the lateral edge of the rectus margin at the level of the umbilicus; T12/L1 is at the level of the internal inguinal ring; and T11 is halfway between T10 and T12/L1 at the rectus margin, which is closer to the midline for these last two points. T8 is at the junction of the rib margin (eighth rib) and the lat- eral rectus; and T9 is halfway between T8 and T10. T6 and T7 are located where their respective ribs meet Figure 2. “That’s it!” When localizing own pain, patient points left the edge of the rectus muscle. index finger, for example, to T-10 opening. Clinician reaches in from The lateral muscular foramen are more difficult to lateral side to confirm location is a neuromuscular foramen. palpate and are most easily felt with the patient lean- ing away from the side being palpated. Firmer pres- sure with the finger is required. These openings are in containing a morbilliform mass of fat (the fatty plug); the vertical groove found at the junction between the and 4) deep to these structures, the firm, round ring back and abdominal muscles. Lateral T10 is located at the which prevents further invasion of the channel. The point where the 10th rib meets the groove. Lateral L1 can aponeurotic openings for these nerves may vary in size be felt in the groove just above the iliac crest, and the from that which barely admits the tip of the finger tuft other two lateral branches are in the groove between T10 to a size that allows placement of the entire finger tuft and L1. The examiner should not be discouraged if find- into the depression. The ring felt deep in the channel ing such a foramen seems difficult; they are easier to find may feel too tough to push beyond. The fatty plug when they are symptomatic. varies in size from 2 mm to 2 cm, depending on how Posterior foramina are found in the groove between dilated the aponeurotic openings have become. In prac- the paravertebral muscles and the more lateral back tical terms, it is the aponeurotic openings and enclosed muscles. These, too, are more difficult to palpate, but fatty plug that are most easily distinguished from sur- the muscular depression at that site is easier to find when rounding tissue. These fatty plugs can often be pal-

The Permanente Journal/ Summer 2002/ Volume 6 No. 3 23 clinical contributions Abdominal Cutaneous Nerve Entrapment Syndrome ACNES: A Commonly Overlooked Cause of Abdominal Pain

pated in asymptomatic persons and may normally feel Practically, if the area of tenderness can be localized to uncomfortable to firm palpation, attesting to the fact one of the palpably identifiable nerve exits, these other that their location predisposes them to trauma. The tests (with the possible exception of Carnett’s sign) are anterior openings of T6, T7, and T8 feel more triangu- probably only of academic interest. lar and are oriented in the anteroposterior plane across Having come this far in the examination, if either the the posteroinferior part of the rib tip. Firmer pressure patient or the examiner needs further convincing of against the rib tip is necessary to feel these openings. the ACNES diagnosis, local injection of an anesthetic The same technique can be advised for the lateral and agent is appropriate (described later under “Treatment”). posterior openings, which usually will admit only 2 Complete relief of pain by the anesthetic agent estab- mm to 3 mm of finger tuft. Accessory nerve exits are lishes the diagnosis. located 2 mm to 3 mm above or below the main branch exits or over adjacent muscle and usually cannot be Recommended Treatment for ACNES palpated with certainty unless symptomatic. A properly administered local injection of an anes- The examiner now must confirm that the point lo- thetic agent completely relieves the pain of ACNES. cated by the patient is actually a nerve exit. With the Technique is critical for both diagnosis and treatment, hand resting gently on the patient’s abdomen lateral to and the tendency is to inject too deeply. the tender spot indicated by the patient’s The patient is given an injection of .5 mL to 1 mL of finger, the examiner’s straightened middle a 2% solution (or its equivalent) using a #21 Complete relief finger can be used to displace the patient’s or #22 needle of length appropriate for the thickness of pain by the finger medially by advancing in a tiny, of the subcutaneous tissue present. A needle of this anesthetic agent circular motion. As finger pressure is size best allows the clinician to feel the anatomic land- establishes the gradually increased, the patient with marks while administering the injection, but a #25 or diagnosis. ACNES will recoil or grab the examiner’s #26 needle can be used if the clinician is sufficiently hand, exclaiming, “That’s it!” (Figure 2).9 familiar with the landmarks. For patients with a thick (Hershfield6 calls this the Hover Sign.) From the loca- layer of adipose tissue, a spinal needle may be needed tion of the symptomatic spot and from the feel of the to reach the front of the muscle. muscular foramen, the examiner should be satisfied The injection serves two purposes: to relieve pain that this is a genuine example of ACNES. and to reduce herniation of the neurovascular bundle To further differentiate the source of the pain, Carnett’s through the fibrous ring. Sequentially as the needle is sign should be elicited.3 While in supine position with introduced, the clinician feels resistance to the needle the arms crossed over the chest, the patient should be from the patient’s skin, the nonresistant texture of the asked to raise his or her head or feet off the table subcutaneous fat, and then mild resistance to the needle while the examiner pushes on the tender spot. If splint- from the aponeurosis and fatty plug. (The needle should ing the muscles in this manner reduces the amount of not be introduced deeper than this level; deeper injec- pain, the source is probably intra-abdominal. If the pain tion can cause ecchymosis and may increase pressure is in the abdominal wall, splinting the muscles will not on the neurovascular bundle in an already tight fibro- reduce the pain and may actually increase it. muscular channel.) At this point, the needle should The “pinch test”3 can also be used if the examiner is already be in the center of the channel and fatty plug initially unable to identify the side on which the pain and just beneath the aponeurosis. If the examiner is originates. This test consists of picking up the patient’s unsure that the needle is positioned correctly, it may skin and subcutaneous fat with the thumb and index be pulled back into the subcutaneous fat to prepare finger, first on one side of the midline of the abdomen for another attempt at placing the tip of the needle and then on the other side. The patient will state whether beneath the structures in front of the fibrous ring. one side hurts more than the other. Cotton and pinprick As mentioned above, landmarks of the pertinent struc- technique can be used to check for hypoesthesia or tures can best be felt with the patient standing and hyperesthesia around the pain site, and Knockhaert23 bearing down, and the injection can be given in this notes that electromyelographic studies of the affected position. However, if the patient is more comfortable nerve show abnormalities in 60% of patients studied lying down, the injection may alternatively be given in (although this author23 admitted that the procedure has this position. low sensitivity). Carnett3 noted sensitivity along the proxi- To be sure the needle is positioned precisely (Figure 3)24 mal portion of the affected nerve (Valleix phenomenon). at the correct place for injection, the examiner should

24 The Permanente Journal/ Summer 2002/ Volume 6 No. 3 clinical contributions Abdominal Cutaneous Nerve Entrapment Syndrome ACNES: A Commonly Overlooked Cause of Abdominal Pain

first place the middle finger of one hand in the aponeurotic opening and then, without lifting the finger off the skin, move the fingertip infe- riorly, cleanse the skin with alcohol using the other hand, and with that hand introduce the needle above the tip of the examining finger. When the needle is properly situated beneath the aponeurosis, the clinician stabilizes the sy- ringe for injection by gripping it using the same hand used to locate the opening. The patient should be asked not to breathe during aspira- tion and injection. These instructions may seem rudimentary; however, if followed exactly, they will guarantee success in diagnosing and treat- ing ACNES. A patient who feels faint after receiving the injection should be allowed to lie down until s/he feels better; otherwise, the patient should be encouraged to move about the room. When the syringe has been disposed of and the pa- Figure 3. Clinician locating the precise injection site. tient has taken a few steps, the clinician should (Adapted and reproduced by permission of the publisher, of the authors, and of the illustrator, ask if the patient still feels pain. If the injection Marjorie Domenowske, from: Bonica JJ, Johansen K, Loeser JD. Abdominal pain caused by has been effective, patients often volunteer, other diseases. In: Bonica JJ, editor. The management of pain. 2nd ed Vol 2. Philadelphia: Lea & Febiger; 1990. p 1254-82.24) with a look of amazement on their faces, “It’s gone!” Clinical response sometimes takes more time than this if the injection has been made slightly pointment, even to evaluate new associated symptoms. off the locus. If the response is mediocre and the clini- An alternative is to schedule three return appointments cian suspects the reason may be because the injection a few days apart, a tactic that gives patients the option was not placed accurately, a second injection may be to cancel the appointment if they do not think they attempted after about ten minutes or on another day. need it. Some patients need multiple injections to elimi- Occasionally, a patient reports relief from pain upon nate the pain completely, but these patients seldom arriving home. In such circumstances, a patient should need more than four or five injections. Each injection be encouraged to return to the clinic if the pain recurs should provide relief for a longer and longer time until or if new symptoms arise. no more are needed. For patients who toler- Mehta4 and McGrady17 used a Teflon-coated needle ate local anesthesia well but must return ev- In many patients, with exposed tip to locate the nerve by electrical stimu- ery few weeks for another injection, alter- one injection gives lation. I tried this technique with several patients and native regimens are available. prolonged relief or found the procedure cumbersome and time-consum- The clinician must first decide whether fur- may sufficiently ing. After learning to locate the nerve as described ther evaluation is justified. Does the patient reassure younger here, clinicians can place the injection accurately in have musculoskeletal conditions (eg, scolio- patients that the minutes without using a nerve locator. sis or one short leg) that might subject a par- condition is In many patients, one injection gives prolonged re- ticular nerve to undue traction? Especially in benign … lief or may sufficiently reassure younger patients that older patients, are underlying medical prob- the condition is benign and will not require another lems causing abdominal enlargement? If for visit unless another injection is needed for pain relief. any reason the pain is recurrent or persistent, it can be Older persons should be advised to return whenever treated by destroying the symptomatic portion of the the pain recurs or when other symptoms develop so nerve. Some patients with ACNES have nerve entrap- that underlying causes can be addressed if necessary. ment in an abdominal scar.6,16-18 Excising this part of Because repeat injection requires only a few minutes the scar or removing the suture from around the nerve17 in patients who have already been evaluated, these may solve the problem by two mechanisms: 1) relief patients may often be scheduled for a same-day ap- of direct compression of the nerve and 2) relief of dis-

The Permanente Journal/ Summer 2002/ Volume 6 No. 3 25 clinical contributions Abdominal Cutaneous Nerve Entrapment Syndrome ACNES: A Commonly Overlooked Cause of Abdominal Pain

tal traction on the nerve, especially with disparate tion. Use of corticosteroid drugs is theoretically valuable motion between the abdominal wall and the skin. The because some inflammation is seen in ACNES; however, diagnosis of nerve entrapment in a scar is suggested injection of corticoid drugs into muscles can sometimes by exacerbation of the pain from pinch- cause considerable pain, and tissue atro- ing the scar or by moving the scar across phy can occur with repeated injection. In- Almost everyone the underlying muscle. For nerve entrap- jection with lidocaine and alcohol has who has written ment under the aponeurosis, injection of worked well in my practice, and I see no about abdominal phenol or absolute alcohol is an option. compelling reason to add corticoid agents wall pain Phenol, 5%-7% 1 mL, has been used by to the regimen. overlooks the some investigators;4,12,17 however, either Other treatment modalities may tempo- diagnosis of because the injection was given too rarily relieve pain of ACNES. Precise ap- ACNES … deeply or because they were side effects plication of an ice cube wrapped in a thin of the phenol, pain and systemic effects washrag can help by acting as a local an- occurred frequently. In my experience, using 1 mL of esthetic and by reducing swelling around the nerve. Ap- absolute alcohol mixed with 0.5 mL of a 2% lidocaine plication of an elastic bandage for counterpressure may solution achieves a good result and causes minimal be helpful. Heat applications may relieve associated local pain. Because lidocaine gives immediate relief muscle spasm. and prevents any burning sensation from the alcohol Most of these recommendations for treatment can be when it is first introduced, use of lidocaine helps the applied to the lateral, posterior, and accessory nerves. clinician to decide whether the injection was properly In fact, these recommendations could, theoretically, be placed. A phone call from the patient after a few days applied to any other anatomic area where nerves pass is all that is needed to confirm treatment success. Only through muscles or other tight structures. I strongly once have I reinjected alcohol into a patient who had suspect that the anatomic areas that Janet Travell called only partial relief from the previous injection. Surgery “trigger points”27 are actually areas where sensory nerves also is an option, although I would consider it only for are trapped in muscles that are in spasm. Acupuncture patients who had scar involvement or who for some points may also be located at nerve exits.13 My limited reason could not tolerate alcohol injection. The surgi- experience with acupuncture suggests that these points cal procedure should be done with the patient under can be located by identifying a sensitive depression in local anesthesia so that the patient can state whether the underlying muscle. traction on the nerve duplicates the symptoms. If this is the case, the nerve should be severed at the front of Summary and Conclusions the muscle to release distal traction. Over many years, investigators have warned their Some investigators7,12,25,26 have recommended use of readers that pain in the abdominal wall is too often corticosteroid drugs as part of the injec- misdiagnosed as intra-abdominal pain and that the time and effort spent looking for the cause (which is liter- ally right under their fingertips) are totally unjustified Practice Tips and may cause the patient considerable anxiety and The most common cause of abdominal wall pain is nerve even unnecessary surgery. The most common cause of entrapment at the lateral border of the rectus muscle. abdominal wall pain is nerve entrapment at the lateral Ask the patient, “Where exactly is the pain?” “Show me with one finger.” border of the rectus muscle. 3 Diagnosed and treated by local anesthetic injection into the In 1926, Carnett called this condition “intercostal muscular channel through which the affected nerve passes. neuralgia.” However, recent studies of the anatomy and The injection serves two purposes: to relieve pain and to histopathology of this condition indicated that it is not reduce herniation of the neurovascular bundle through the so much an inflammatory condition as a matter of nerve fibrous ring. entrapment. Accordingly, I prefer the name abdominal Precise application of an ice cube in a thin washrag can help cutaneous nerve entrapment syndrome (ACNES). This by acting as a local anesthetic and by reducing swelling condition is diagnosed and treated by local anesthetic around the nerve. injection into the muscular channel through which the Application of an elastic bandage for counterpressure may be helpful. affected nerve passes. This article discusses in detail Heat applications may relieve associated muscle spasm. how to identify the muscular neuroforamina by palpa- tion as well as the specific technique for injecting them.

26 The Permanente Journal/ Summer 2002/ Volume 6 No. 3 clinical contributions Abdominal Cutaneous Nerve Entrapment Syndrome ACNES: A Commonly Overlooked Cause of Abdominal Pain

Almost everyone who has written about abdominal wall 10. Abdominal wall pain easily differentiated from visceral pain overlooks the diagnosis of ACNES while admon- origin. Gastroenterology Observer 1990 Nov-Dec;9(6):1,8. ishing the medical profession against subjecting patients 11. Thompson C, Goodman R, Rowe WA. Abdominal wall syndrome: a costly diagnosis of exclusion. Gastroenterol- to unnecessary tests, but each writer also says that if a ogy 2001 Apr;120(5 Suppl 1):A637. patient does not respond to the usual treatments, the 12. Doouss TW, Boas RA. The abdominal cutaneous nerve patient should be further evaluated for underlying con- entrapment syndrome. N Z Med J 1975 May;81(540):473-5. tributing causes. This instruction is particularly impor- 13. Kopell HP, Thompson WA. Peripheral entrapment tant for older patients. Diagnostic procedures for these neuropathies. Malabar (FL): Robert E. Kreiger Publishing; patients are ultimately a matter of clinical judgment, but 1976. p 1-7, 85-8. certainly clinicians and patients can be spared much 14. Applegate WV. Abdominal cutaneous nerve entrapment syndrome. Am Fam Physician 1973 Sep;8(3):132-3. trouble if the diagnosis of ACNES is established at the 15. Applegate WV, Buckwalter NR. Microanatomy of the first visit. The information given in this article should structures contributing to abdominal cutaneous nerve make that early diagnosis of ACNES possible. Srinivasan entrapment syndrome. J Am Board Fam Pract 1997 Sep- and Greenbaum22 feel that an ACNES patient monitored Oct;10(5):329-32. very closely for three months without convincing evi- 16. Sippo WC, Burghardt A, Gomez dence that local anesthetic injection or other treat- AC. Nerve entrapment after … if new symptoms Pfannenstiel incision. Am J Obstet arise suggesting ment has really helped should receive further study Gynecol 1987 Aug;157(2);420-1. visceral disease, for visceral disease. Obviously, if new symptoms arise 17. McGrady EM, Marks RL. Treatment suggesting visceral disease, further diagnostic evalua- of abdominal nerve entrapment further diagnostic tion is justified at any time even though the treatment syndrome using a nerve stimulator. evaluation for ACNES seems to be effective. ❖ Ann R Coll Surg Engl 1988 is justified … May;70(3):120-2. 18. Gallegos NC, Hobsley M. Acknowledgments Abdominal wall pain: an alternative diagnosis. The clinical research on which this review article was based Br J Surg 1990 Oct;77(10):1167-70. was approved by the Institutional Review Board of the Southern 19. Thomson WH, Dawes RF, Carter SS. Abdominal wall California Permanente Medical Group. tenderness: a useful sign in chronic abdominal pain. Br J Juan Domingo provided original adaptations of the illustrations. Surg 1991 Feb;78(2):223-5. 20. Slocumb JC. Neurological factors in chronic pelvic pain: trigger points and the abdominal pelvic pain syndrome. References Am J Obstet Gynecol 1984 Jul 1;149(5):536-43. 1. Cited in: Murray GR. An address on myofibrositis as a 21. Thomson H, Francis DM. Abdominal-wall tenderness: simulator of other maladies. Lancet 1929 Jan 19;1:113-5. a useful sign in the acute abdomen. Lancet 1977 Nov 2. Cyriax EF. On various conditions that may simulate the 19;2(8047):1053-4. referred of visceral disease, and a consideration of 22. Srinivasan R, Greenbaum DS. Chronic abdominal wall these from the point of view of cause and effect. pain: a frequently overlooked problem. Practical approach Practitioner 1919;102:314-22. to diagnosis and management. Am J Gastroenterol 2002 3. Carnett JB. Intercostal neuralgia as a cause of abdominal Apr;97(4):824-30. pain and tenderness. Surg Gynecol Obstet 1926;42:625-32. 23. Knockaert DC, Boonen AL, Bruyninckx FL, Bobbaers HJ. 4. Mehta M, Ranger I. Persistent abdominal pain. Treatment Electromyographic findings in ilioinguinal-iliohypogastric by nerve block. Anaesthesia 1971 Jul;26(3):330-33. nerve entrapment syndrome. Acta Clin Belg 5. Applegate WV. Abdominal cutaneous nerve entrapment 1996;51(3):156-60. syndrome. Surgery 1972 Jan;71(1):118-24. 24. Bonica JJ, Johansen K, Loeser JD. Abdominal pain caused 6. Hershfield NB. The abdominal wall. A frequently by other diseases. In: Bonica JJ, editor. The management of overlooked source of abdominal pain. J Clin Gastroenterol pain. 2nd ed. Philadelphia: Lea & Febiger; 1990. p 1254-82. 1992 Apr;14(3):199-202. 25. Sharf M, Shvartzman P, Farkash E, Horvitz J. Thoracic 7. Peleg R. Abdominal wall pain caused by cutaneous nerve lateral cutaneous nerve entrapment syndrome without entrapment in an adolescent girl taking oral contraceptive previous lower abdominal surgery. J Fam Pract 1990 pills. J Adolesc Health 1999 Jan;24(1):45-7. Feb;30(2):211-2, 214. 8. Oesch A, Kupfer K, Bodoky A. [Intercostal nerve syn- 26. Tung AS, Tenicela R, Giovanitti J. Rectus abdominis nerve drome]. [Article in German]. Swiss Surg 2001;7(2):82-5. entrapment syndrome. JAMA 1978 Aug 25;240(8):738-9. 9. Suleiman S, Johnston DE. The abdominal wall: an 27. Travell JG, Simons DG. Myofascial pain and dysfunction: overlooked source of pain. Am Fam Physician 2001 Aug the trigger point manual. Baltimore: Williams & Wilkins; 1;64(3):431-8. 1983-92.

The Permanente Journal/ Summer 2002/ Volume 6 No. 3 27 Open Access Review Article DOI: 10.7759/cureus.3036

Symptomatology Correlations Between the Diaphragm and Irritable Bowel Syndrome

Bruno Bordoni 1 , Bruno Morabito 2

1. Cardiology, Foundation Don Carlo Gnocchi Irccs, Department of Cardiology, Institute of Hospitalization and Care with Scientific Address, S Maria Nascente, Via Capecelatro 66, Milan 20100, Italy., Milano, ITA 2. Osteopathy, School of Osteopathic Centre for Research and Studies, Rome, ITA

 Corresponding author: Bruno Bordoni, [email protected] Disclosures can be found in Additional Information at the end of the article

Abstract Irritable bowel syndrome (IBS) is one of the most debilitating and common gastrointestinal disorders; nevertheless, its pathophysiology is still unclear. It affects 11% of the world's population, and is characterized by alternating periods of pain and/or motility disorders with periods of remission and without any evidence of any structural and functional organic variation. It has been recently proposed that an altered contractile ability of the diaphragm muscle might adversely influence intestinal motility. The text reviews the diaphragm's functions, anatomy, and neurological links in correlation with the presence of chronic symptoms associated to IBS, like chronic low back pain, chronic pelvic pain, chronic headache, and temporomandibular joint dysfunction, vagus nerve inflammation, and depression and anxiety. The interplay between an individual's breath dynamic and intestinal behaviour is still an unaddressed point in the physiopathology of IBS, and the paucity of scientific studies should recommend further research to better understand the importance of breathing in this syndrome.

Categories: Pathology, Physical Medicine & Rehabilitation, Pulmonology Keywords: irritable bowel syndrome, pain, diaphragm, low back pain, chronic headache

Introduction And Background Irritable bowel syndrome (IBS) is the most commonly diagnosed gastrointestinal disorder. The patient complains of abdominal pain or discomfort and altered bowel habits in the absence of concomitant organic diseases that could explain symptoms [1]. IBS can be divided into subgroups, depending on the prevailing symptoms (based on the scale of Rome III) and stool consistency (based on the Bristol Stool Form Scale): diarrhea or IBS-D; with constipation or IBS-C; alternating constipation and diarrhea, or IBS-M; untyped IBS or IBS-U (insufficient stool Received 07/09/2018 abnormalities to be IBS-C, D or M) [1-2]. Patients may have variations in their symptoms, with Review began 07/13/2018 Review ended 07/15/2018 periods of remission; it is not easy to correctly classify patients with IBS [1-2]. Anyway, to Published 07/23/2018 diagnose IBS, symptoms have to persist for at least three months, with abdominal discomfort presenting 3-4 times per month, with constant feedback for 1-2 years; after 10 years, 50-70% of © Copyright 2018 Bordoni et al. This is an open access patients still present symptoms [3]. IBS currently refers to a disease of unknown aetiology. article distributed under the terms of the Creative Commons Attribution License CC-BY 3.0., which permits The most recent hypotheses unrestricted use, distribution, and The most recent hypotheses concern the presence of intestinal dysbiosis, which may both reproduction in any medium, provided the original author and the large and small bowel tract. Many factors participate in the alteration of intestinal source are credited. microbiota, such as a recent history of acute intestinal infections or reduction of intestinal motility [4]. Another hypothesis is that the presence of small intestinal bacterial overgrowth

How to cite this article Bordoni B, Morabito B (July 23, 2018) Symptomatology Correlations Between the Diaphragm and Irritable Bowel Syndrome. Cureus 10(7): e3036. DOI 10.7759/cureus.3036 (SIBO), which causes a non-physiological post-prandial fermentation with elevated gas production, results in rapid gastric (painful) distension. SIBO is probably responsible for chronic and sub-clinical inflammation of the intestinal mucosa and could also explain an elevation in a mucosal specific inflammatory , calprotectin, which may be found in these patients [5]. Other theories deal with the role of serotonin, a neurotransmitter involved in multiple intestinal functions, and able to influence motility, immune response, and visceral sensitivity [6]. Intestinal receptors and serotonin uptake may be possibly involved in its pathogenesis, especially in IBS-D and IBS-C, but additional data are certainly needed to clarify serotonin’s role in IBS aetiology [5]. The central may be also involved in the pathogenetic mechanism leading to IBS. The -gut axis is considered as the bi-directional relationship between the enteric nervous system and the central nervous system, including the hypothalamus-pituitary-adrenal gland axis [5]. When this mechanism is altered, some visceral could appear, due to the increased release of cortisol and pro-inflammatory cytokines, increasing symptoms [4]. Stress generates an endocrine response (corticotropin-releasing factor (CRF), activating in turn the hypothalamic-pituitary-adrenal axis (HPA). CRF is secreted by the paraventricular nucleus of the hypothalamus, the release of which is regulated by the amygdala (the limbic system). The CRF receptors (CRF1 and CRF2) are found on enteric neurons and at the mucosal layer. The constant presence of stress could cause a pathophysiological change in the intestine, altering its motility (mobility, relaxation, transit) through the activation of these receptors [4]. This fibromyalgia-like phenotype is often diagnosed in patients affected by IBS and it is characterized by chronic fatigue, chronic back pain, chronic pelvic pain, chronic headache, and temporomandibular joint dysfunction, at a rate almost twice the general population [3]. Some authors call such symptoms "functional somatic syndromes" and have therefore proposed to treat these patients with selective serotonin receptive inhibitors (SSRI) or tricyclic antidepressants. In addition, more than half of the patients reported symptoms of anxiety and depression; the risk of developing dementia in subjects (after 50 years) is increased [3-7]. The interest in the role of the diaphragm muscle in the context of functional intestinal disorders is justified by its autonomic dependent function and because it is the only striated muscle whose movement affects the small and large bowel. The purpose of this article is to review the importance of the diaphragm muscle in the context of IBS symptoms, since there are many studies that show that an alteration of its contractile ability might have a negative impact on the pathology.

Review Diaphragm The diaphragm muscle is innervated by the phrenic nerve (C3-C5) and the vagus nerve (cranial nerve X); the first receives pulses from groups of medullary neurons of the pre-Bötzinger complex and from neurons of the parafacial retrotrapezoid complex, connected in turn with the retroambiguus nucleus of medulla, although the underlying mechanisms of these connections are not completely clear. The vagus nerve is part of the parasympathetic autonomic system originating from the ambiguus nucleus of the medulla [8-9].

Symptomatology correlations The abdominal muscles, the rectus, and the oblique and transverse muscles not only play a biomechanical role in relation with the spine, but also in relation to intra-abdominal content, changing their electrical activity according to intestinal pressure [10]. In patients affected by IBS, this accommodation mechanism of the abdominal muscles does not work properly, and an electromyographic alteration can be observed in postprandial periods. The patients experience a paradoxical effect: a contraction of the diaphragm muscle and a relaxation of the upper portion of the abdominal wall, while in healthy subjects, concomitant relaxation of the diaphragm muscle and activation of the rectus abdominis (the upper part) and external oblique usually happen. This phenomenon causes abdominal swelling, and probably dyspepsia [10].

2018 Bordoni et al. Cureus 10(7): e3036. DOI 10.7759/cureus.3036 2 of 9 This lack of motor coordination between the diaphragm and the abdominal wall is called "abdomino-phrenic dyssynergia" [11]. During respiration, the muscles of the abdominal wall and the diaphragm muscle are controlled by the same centers of the retroambiguus phrenic nucleus in the medulla, in an electric combination that allows a perfect synergic contraction during inspiration and expiration [8]. A dysfunction of the diaphragm muscle may alter this functional synergy and cause an alteration in the motor scheme. This event could lead to swelling and dyspepsia in these patients.

Abnormalities in the pelvic area and in the upper airways The phrenic centers in the medulla control the pelvic floor muscles and the tongue during respiration [8-9]. During inspiration, the tongue protrudes (to open the upper airway), while the pelvic floor undergoes a downward movement; during expiration, the opposite happens. If this mechanism is altered for a dysfunction of the diaphragm, abnormalities in the pelvic area (chronic pelvic pain) and in upper airways can be observed [8-9]. Patients with IBS are affected by chronic pelvic pain in 35%-80% of cases worldwide; in addition, collapsibility of the upper airways during sleeping is observed in 15%-44% of cases, as reported in a pilot study [12-13].

Low back pain Low back pain can be also related to IBS, although its aetiology is not completely clear [14]. The diaphragm plays a fundamental role in defining posture, its maintenance, and body position changes. The dysfunction of the diaphragm is one of the recognized causes of low back and sacroiliac joint pain. People with low back pain often experience early fatigue of the diaphragm muscle, altered and reduced excursion during respiration, and inadequate proprioceptive activation [15]. The diaphragm dynamically stabilizes the lumbar spine. Lowering during inhalation together with the latest coasts, it stabilizes abdominal pressure, with a deeper movement in its ventral portion than in the dorsal area. In people with chronic lower back pain, the diaphragm remains higher and more flattened, with inadequate movement of the ventral portion. When the legs move, the diaphragm stabilizes the spine allowing the required movement; this does not happen correctly in people from chronic pain. The coasts do not drop and do not allow the diaphragm to have fixity; there is a minor drop in the diaphragmatic cupola with a reduced ability to manage the intra-abdominal pressure, consequently causing lumbar spine instability [15-17]. Since a dysfunction of the electrical activation of the diaphragm has been demonstrated in patients with IBS, we can assume that one of the causes leading to low back pain in this population may be altered activation of the main respiratory muscle.

Temporomandibular joint dysfunction Another somatic disorder affecting IBS patients is temporomandibular joint dysfunction with pain [3]. The functional mechanisms connecting the diaphragm and tongue during normal respiration are well known, as well as their neurological connections, either central (medulla) and peripheral (anastomosis of the phrenic nerve and hypoglossal nerve at the level of the ansa cervicalis) [8]. Upper airways abnormalities, together with difficult coordination of tongue activity, can alter kinematics of the temporomandibular joint [18]. This lets us presume even a close relationship between these symptoms, diaphragm dysfunction, and IBS.

Chronic headaches To better understand the presence of chronic headaches in IBS and its relations with diaphragmatic functions, the activity of the thoracolumbar fascia should be considered. The latter is a multi-layered fascial and muscular structure that covers the entire abdominal muscles and the myofascial area between the sacrum and the cervical area, from the surface to depth; the diaphragm is directly connected with the thoracolumbar fascia [8]. All fascial layers

2018 Bordoni et al. Cureus 10(7): e3036. DOI 10.7759/cureus.3036 3 of 9 are inseparable; they move simultaneously, relate to one another, and influence each other's activity [19]. The thoracolumbar fascia properly conveys the tensions generated by movements and breathing along the spine, creating a synergy between the diaphragm and daily activities [15]. The sub-occipital muscles (a couple of recti and a couple of oblique muscles) are part of the thoracolumbar fascia and are innervated by the first cervical branches [8]. The recti muscles and the superior oblique sub-occipital muscle have a myodural bridge to the dura mater [20]. In the connective tissue of the dura, near the arterial and venous vessels, there are some peculiar receptors with functions similar to Ruffini receptors and which have highly mechano-sensitive and chemical-sensitive responses, and [21]. Abnormal pressures recorded by the dura mater, caused by an abnormal tension derived from the myodural bridges of the sub- occipital muscles, may be the cause of the chronic headaches [22]. Diaphragmatic dysfunctions affect the whole system, including sub-occipital muscles [8-9]. The incorrect functioning of the diaphragm could cause an increase in work for the accessory respiratory muscles. We can assume that the overloading of the sternocleidomastoideus and scalene muscles could be another cause of headaches that result from the primary dysfunction of the diaphragm. A correlation between chronic headaches and the altered function of the diaphragm muscle can be assumed.

Gastroesophageal reflux Gastroesophageal reflux (GERD) is another symptom frequently observed in patients with IBS (around 40%) [23]. The diaphragmatic crura play a role as extrinsic sphincter in the region of the gastroesophageal junction to protect the esophagus from gastric reflux; a crural dysfunction can cause GERD [24]. Therefore, a relationship between IBS and GERD can be reliably established in case of diaphragmatic dysfunction.

Anxiety, depression, and perceived pain Psychiatric disorders such as depression and anxiety are quite common in patients affected by IBS; they have been observed in 46% and 34% of cases respectively [5]. These behavioural changes may be due to disease-related stress, since the severity of the emotional disorder is often correlated with the severity of the symptoms of IBS [5]. Chronic altered emotional states can cause changes in neural and structural patterns of emotional perception and thus increase pain perception [25]. Another hypothesis deals with the persistent solicitation of visceral nociceptors in response to mechanical stress (inappropriate or exaggerated colic distension, inflammation, and ischemia), potentially causing visceral or allodynia. This peripheral perception may induce changes in the central regulation, establishing a vicious circle between visceral and emotional information, and pain perception [25]. The visceral afferents, interoceptive as well as nociceptive (small caliber A delta and C fibers) reach lamina I and II of the spinal cord, and are then projected to supraspinal centers, such as the posterior ventromedial nucleus of the thalamus and the limbic area [26]. Another reason to study the presence of psychiatric disorders in this patient population is to research its correlation with dysfunction of the diaphragm muscle and the relationship of this dysfunction with emotions and pain perception. The perception of pain is reduced if patients hold their breath after a deep breath, when the diaphragm is lowered [27]. This observation lets us presume the potential role of baroreceptors.

During inspiratory apnea, systolic pressure increases with a decrease in cardiac frequency. When the baroreceptors located in the carotid body and in the aortic arch area, in the adventitia of the vessels, are stimulated by the cardiac cycle, in particular during the systolic phase, the nociceptive stimulus is attenuated by the activation of baroreceptors [27]. The baroreceptors’ activity also affects muscle tone, as it decreases the activity of the sympathetic nervous system, reducing the contractile state. The baroreceptors are activated if the vessels are stretched by blood passage; afferent fibers conduct pulses to the nucleus of the solitary tract (NTS), which

2018 Bordoni et al. Cureus 10(7): e3036. DOI 10.7759/cureus.3036 4 of 9 regulates the activation of the vagal system and sympathetic inhibition at the spinal level in the region of the nucleus ambiguus, dorsal motor nucleus, and rostral ventrolateral area of the medulla. The baroreceptorial afferents influence different areas of the central nervous system, with a generalized inhibitory effect. The NTS is interconnected with the reticular formation; the information will be then transmitted to the anterior (limbic area), latero-medial and prefrontal part of the insula and to the anterior cingulate cortex; even the thalamus, hypothalamus, and periaqueductal grey area receive baroreceptorial pulses from NTS [27]. The diaphragm with its movements changes the body pressure, as it facilitates venous and lymphatic return [8]. This pressure modulation influences blood re-distribution. This action may affect the response of baroreceptors and thus pain perception, but there are no scientific studies to support this statement yet. We can presume that the baroreceptors will not be adequately stimulated, in case of an alteration of the diaphragm motility; this would lead to an increased sensitivity to pain. A lower sensitivity of the baroreceptors and a higher pain perception have been demonstrated in IBS patients [28]. Chronic pain can alter the subject's emotional state [27]. We can speculate that the dysfunction of the diaphragm could be one of the causes of this psychiatric condition and high pain perception.

The diaphragm also influences emotions directly. The interaction between breathing and emotions reflects a complex interaction between the brainstem and some brain areas such as the limbic area and the cortex. The amygdala, which is part of the limbic system, and some respiratory areas located in the medulla, are connected to each other; thus, the amygdala is usually considered as the most important area in the interaction between breathing and emotional state. The amygdala is divided into three areas (basolateral, cortical and central); the basolateral amygdala conducts pulses to the central area, which is directly and indirectly connected to the hypothalamus and to the brainstem [27]. The amygdala is stimulated by dopamine production from the tegmental area of the midbrain; a recent study on animal models demonstrated the role of dopamine in the amygdala in the management of the emotional breath [29]. The efferent fibers of the amygdala are connected to some respiratory areas, such as the NTS and related areas. The breath stimulates the mechanoreceptors of the diaphragm and the visceral receptors of the organs moved during the respiratory acts, constituting a mechanism called interoception [27]. Interoception is the awareness of the body's condition based on information directly obtained from the body itself; it is also related to visceral movements when breathing in and out. The interoceptive afferent fibers are connected to autonomic and homeostatic centers in the spinal cord and in the brainstem, and then to the cingulate cortex and to the anterior dorsal posterior insula, through the thalamus- cortical tract. The interoception system can modulate the exteroceptive representation of the body, as well as the subject's tolerance of pain; the dysregulation of pathways managing or stimulating the interoception could cause a distortion of body image, affecting the subject's emotional state [27]. We can speculate that an altered function of the diaphragm may adversely affect the patient's emotional state, adversely affecting interoceptive afferents.

Pain and inflammation The peripheral nerve structure is subjected to daily mechanical stress, as when the joint moves, it undergoes alternating phases of contraction and stretching. The correct sliding of the fascial structures of the nerve, and the nerve position between the various innervated tissues, become essential for nerve adaptation and regeneration [6]. Altered sliding may lead to dysfunction, increasing the rigidity of fascial structures during joint or respiratory movements; the nerve may develop a diameter reduction, defined as transverse contraction, and an increase in the pressure in the endoneural compartment may be observed [30]. Repetitive elongations of a nerve with reduced elasticity will further hamper nerve sliding, decreasing the blood flow, with potential ischemic changes. The fascial structures become more sensitive to mechanical stress and, after a few days of local inflammation, an action potential (similar to the mechanical stimulus that has initially caused the dysfunction) can be generated, causing inflammation at

2018 Bordoni et al. Cureus 10(7): e3036. DOI 10.7759/cureus.3036 5 of 9 the ends of the neural pathways, such as in the spinal cord and innervated tissues, through anterograde and retrograde mechanisms. This mechanism is called ectopic electrogenesis [30]. The vagus nerve and the phrenic nerve, which innervate the diaphragm muscle, may be affected if muscular movement is limited for such a dysfunction, leading to altered sensitivity schemes and allodynia.

The vagus nerve is a mixed nerve, 20% consisting efferent cholinergic descendent fibers, and 80% afferent ascending fibers [31]. The vagus nerve, below the diaphragm, forms anastomoses with the sympathetic ganglia [24]. The vagal nerve contains large myelinated type A and B fibers, conducting efferent and visceral afferent information, and efferent preganglionic sympathetic and parasympathetic information respectively; it also includes small size, non- myelinated, visceral afferent fibers (type C), representing the largest part of the afferent compartment C [32]. The efferent fibers mainly derived from the retroambiguus nucleus (important for phrenic orders) and from the dorsal motor nucleus; the afferent fibers mainly connect to the area postrema, the spinal trigeminal nucleus, and the NTS (located in the medulla and receiving information on pain and emotions) [32]. The vagus nerve also plays an important role in the immune response and . The vagus nerve perceives the inflammation and has the ability to regulate the response, influencing the hypothalamic- pituitary-adrenal gland axis, modulating the autonomic system in controlling key immune organs (spleen, adrenal glands and probably the bone marrow), and even directly through colinergic myenteric neurons [24]. This mechanism is called “cholinergic anti-inflammatory via”, acting through the inhibition of the production of pro-inflammatory cytokines, with a strategy involving the alpha7nAChR (alpha7 nicotinic acetylcholine receptor subunit).

A reduction in vagal control could stimulate inflammation, increasing the production of inflammatory cytokines [33]. It is worthy to mention that, in the population of patients with IBS, there is a dysregulation in the vagal and sympathetic system and/or a reduction in vagal tone [34]. This reduced vagal tone may be induced by mechanical stress caused by a dysfunction of the diaphragm, resulting in a compression of the nerve (with an increase in endoneural compartment pressure), which induces abnormal vagal function. In this scenario of reduced vagal control, the baroreceptors are also involved, since they are influenced by the vagus nerve and breathing. The low response of baroreceptors can increase the inflammatory response, creating a vicious circle [35]. There is a close relationship between the vagus nerve and the perception of pain. The afferents of the vagus nerve are usually able to inhibit the activity of the second order nociceptive neurons in the spinal cord, in spinothalamic and spinoreticular tracts and in the trigeminal nucleus [36].

Recent studies, however, highlight the vagus nerve’s ability to carry painful afferents to the supraspinal centers, especially with regards visceral pain [18]. This also happens for a retrograde transport of biochemical molecules through the nerve [8]. The vagus nerve also collaborates in the formation and maintenance of central pain memory, also modulating descendant inhibitory pathways connected to the nociceptive areas in the spinal cord [37]. No precise information is currently available on these ascendant (probably involving NTS, parabrachial nuclei, the periaqueductal grey area, hypothalamus, limbic area, magnum raphe, locus coeruleus) and descendant mechanisms, but we can derive that vagal tone probably has an important influence on pain perception [38]. We know that a compression of the vagus nerve can alter its function and its transport ability, just like a dysfunction of a peripheral nerve, mimicking an entrapment syndrome [39]. We can assume that abnormal tension of the diaphragm in the region of the oesophagal hiatus could cause a compression of the vagus nerve, reducing its antinociceptive and anti-inflammatory activity. The phrenic nerve originates from the spinal cord (C3-C5) and descends along the deep fascial system of the neck, reaching the mediastinum and lying on the pericardial fascia; then it crosses the diaphragm [8]. In the sub- diaphragmatic region, the phrenic nerve forms anastomoses with the celiac and the superior mesenteric ganglion and constitutes small phrenic ganglia, to finally reach the adrenal

2018 Bordoni et al. Cureus 10(7): e3036. DOI 10.7759/cureus.3036 6 of 9 gland [24]. These connections with the sympathetic system influence the phrenic nerve with retrograde impulses from the sympathetic plexus and viscerosomatic communications. These connections are very complex and still partly unknown. The celiac and superior mesenteric ganglion are involved in the cholinergic anti-inflammatory action of the vagus nerve (to the spleen), emphasizing a parasympathetic and sympathetic action [40]. When the diaphragm muscle does not contract properly, it becomes stiff. We can assume that this could create a compression syndrome also on the phrenic nerve, altering the connections between the sympathetic and the parasympathetic system and the adrenal gland, and adversely affecting the perception of pain, the inflammatory status, and the emotional state. Based on what was previously described, this could contribute to an altered bowel function. The splanchnic nerves cross the diaphragm through small muscular spaces [8]. The sympathetic system, when activated, is able to amplify the pain and modulate the pain memory [41]. Previous studies demonstrated that the presence of vertebral osteophytes may alter the morphology of the sympathetic nerves and their function [42-43]. We can assume that if the sympathetic nerves are compressed at the level of their passage through the diaphragm, their function and morphology can change, negatively influencing the innervated tissues, including the colon.

Conclusions Irritable bowel syndrome (IBS) is the most commonly diagnosed gastrointestinal disorder. The patient complains of abdominal pain or discomfort and altered bowel habits in the absence of other conditions that may cause the syndrome. Patients undergo various comorbidities and a concomitant dysfunction of the diaphragm muscle. The comorbidities addressed in the article are chronic low back pain, chronic pelvic pain, chronic headache, and temporomandibular joint dysfunction, vagus nerve inflammation, and depression and anxiety; we can still find other co- morbidities, like abnormalities in upper airways and gastroesophageal reflux. Considering the neurological, fascial, and anatomical diaphragmatic connections with the lumbar area, the cervical and mandibular area, as well as its influence on the emotional sphere and on the perception of pain, we can conclude that there is a strong relationship between these co- morbidities and patients with IBS. Currently, there is no data on the pre-IBS functional status of the diaphragm muscle of these patients, as well as on their vagal and sympathetic functions. At the same time, there are no data on the potential impact of therapeutic approaches for IBS including diaphragmatic training; in the current context, we cannot quantify the influence of breathing on IBS and related pathologies. Further studies focused on the relation between breathing and IBS are needed in order to evaluate if this correlation could change therapeutic approaches to IBS.

Additional Information Disclosures Conflicts of interest: In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work. Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work.

References 1. Chey WD, Kurlander J, Eswaran S: Irritable bowel syndrome: a clinical review . JAMA. 2015, 313:949-58. 10.1001/jama.2015.0954 2. Talley NJ, Holtmann G: Irritable bowel syndrome and functional dyspepsia: what can epidemiology tell us about etiology?. Expert Rev Gastroenterol Hepatol. 2018, 12:633-635.

2018 Bordoni et al. Cureus 10(7): e3036. DOI 10.7759/cureus.3036 7 of 9 10.1080/17474124.2018.1476136 3. Canavan C, West J, Card T: The epidemiology of irritable bowel syndrome. Clin Epidemiol. 2014, 6:71-80. 10.2147/CLEP.S40245 4. Thompson JR: Is irritable bowel syndrome an infectious disease? . World J Gastroenterol. 2016, 22:1331-4. 10.3748/wjg.v22.i4.1331 5. Padhy SK, Sahoo S, Mahajan S, Sinha SK: Irritable bowel syndrome: Is it "irritable brain" or "irritable bowel"?. J Neurosci Rural Pract. 2015, 6:568-77. 10.4103/0976-3147.169802 6. Kim DY, Camilleri M: Serotonin: a mediator of the brain-gut connection . Am J Gastroenterol. 2000, 95:2698-709. 10.1111/j.1572-0241.2000.03177.x 7. Chen CH, Lin CL, Kao CH: Irritable bowel syndrome is associated with an increased risk of dementia: a nationwide population-based study. PLoS One. 2016, 11:0144589. 10.1371/journal.pone.0144589 8. Bordoni B, Zanier E: Anatomic connections of the diaphragm: influence of respiration on the body system. J Multidiscip Healthc. 2013, 6:281-91. 10.2147/JMDH.S45443 9. Bordoni B, Zanier E: The continuity of the body: hypothesis of treatment of the five diaphragms. J Altern Complement Med. 2015, 21:237-42. 10.1089/act.2015.29031.hme 10. Burri E, Barba E, Huaman JW, Cisternas D, Accarino A, Soldevilla A, et al.: Mechanisms of postprandial abdominal bloating and distension in functional dyspepsia. Gut. 2014, 63:395- 400. 10.1136/gutjnl-2013-304574 11. Sullivan SN: Functional abdominal bloating with distention . ISRN Gastroenterol. 2012, 2012:721820. 10.5402/2012/721820 12. Lessa LM, Chein MB, da Silva DS, et al.: Irritable bowel syndrome in women with chronic pelvic pain in a Northeast Brazilian city. Rev Bras Ginecol Obstet. 2013, 35:84-9. 13. Gold AR, Broderick JE, Amin MM, Gold MS: Inspiratory airflow dynamics during sleep in irritable bowel syndrome: a pilot study. Sleep Breath. 2009, 13:397-407. 10.1007/s11325-009- 0262-6 14. Vora AJ, Doerr KD, Wolfer LR: Functional anatomy and pathophysiology of axial low back pain: disc, posterior elements, sacroiliac joint, and associated pain generators. Phys Med Rehabil Clin N Am. 2010, 21:679-709. 10.1016/j.pmr.2010.07.005 15. Bordoni B, Marelli F: Failed back surgery syndrome: review and new hypotheses. J Pain Res. 2016, 9:17-22. 10.2147/JPR.S96754 16. Kolar P, Sulc J, Kyncl M, et al.: Postural function of the diaphragm in persons with and without chronic low back pain. J Orthop Sports Phys Ther. 2012;42(4, 352-362. 10.2519/jospt.2012.3830 17. Vostatek P, Novák D, Rychnovský T, Rychnovská S: Diaphragm postural function analysis using magnetic resonance imaging. PLoS ONE. 2013;8(3, 56724. 10.1371/journal.pone.0056724 18. Weber P, Corrêa EC, Bolzan Gde P, Ferreira Fdos S, Soares JC, Silva AM: Chewing and swallowing in young women with temporomandibular disorder. Codas. 2013, 25:375-80. 19. Stecco C, Schleip R: A fascia and the fascial system . J Bodyw Mov Ther. 2016, 20:139-40. 10.1016/j.jbmt.2015.11.012 20. Enix DE, Scali F, Pontell ME: The cervical myodural bridge, a review of literature and clinical implications. J Can Chiropr Assoc. 2014, 58:184-92. 21. Adeeb N, Mortazavi MM, Tubbs RS, Cohen-Gadol AA: The cranial dura mater: a review of its history, embryology, and anatomy. Childs Nerv Syst. 2012, 28:827-37. 10.1007/s00381-012- 1744-6 22. Kahkeshani K, Ward PJ: Connection between the spinal dura mater and suboccipital musculature: evidence for the myodural bridge and a route for its dissection--a review. Clin Anat. 2012, 25:415-22. 10.1002/ca.21261 23. Nagasako CK, Montes CG, Lorena SL, Mesquita MA: Irritable bowel syndrome subtypes: clinical and psychological features, body mass index and comorbidities. Rev Esp Enferm Dig. 2016, 108:59-64. 10.17235/reed.2015.3979/2015 24. Bordoni B, Marelli F, Morabito B, Sacconi B, Caiazzo P, Castagna R: Low back pain and gastroesophageal reflux in patients with COPD: the disease in the breath. Int J Chron Obstruct Pulmon Dis. 2018, 13:325-334. 10.2147/COPD.S150401 25. Greenwood-Van Meerveld B, Moloney RD, Johnson AC, Vicario M: Mechanisms of stress- induced visceral pain: implications in irritable bowel syndrome. J Neuroendocrinol. 2016, 28- 8. 10.1111/jne.12361

2018 Bordoni et al. Cureus 10(7): e3036. DOI 10.7759/cureus.3036 8 of 9 26. Jänig W: Mechanical allodynia generated by stimulation of unmyelinated afferent nerve fibres. J Physiol. 2011; 589, 18:4407-8. 10.1113/jphysiol.2011.217083 27. Bordoni B, Marelli F, Bordoni G: A review of and emotive breathing: a multidisciplinary approach. J Multidiscip Healthc. 2016, 9:97-102. 10.2147/JMDH.S101208 28. Davydov DM, Naliboff B, Shahabi L, Shapiro D: Baroreflex mechanisms in irritable bowel syndrome: part I (traditional indices). Physiol Behav. 2016, 157:102-8. 10.1016/j.physbeh.2016.01.042 29. Sugita T, Kanamaru M, Iizuka M, Sato K, Tsukada S, Kawamura M, et al: Breathing is affected by dopamine D2-like receptors in the basolateral amygdala. Respir Physiol Neurobiol. 2015, 209:23-7. 10.1016/j.resp.2014.09.020 30. Bordoni B, Bordoni G: Reflections on osteopathic fascia treatment in the peripheral nervous system. J Pain Res. 2015, 8:735-40. 10.2147/JPR.S89393 31. Spaziani R, Bayati A, Redmond K, Bajaj H, Mazzadi S, Bienenstock J, et al: Vagal dysfunction in irritable bowel syndrome assessed by rectal distension and baroreceptor sensitivity. Neurogastroenterol Motil. 2008, 20:336-42. 10.1111/j.1365-2982.2007.01042.x 32. Yuan H, Silberstein SD: Vagus Nerve and Vagus Nerve Stimulation, a Comprehensive Review: Part I. Headache. 2016, 56:71-8. 10.1111/head.12647 33. Das UN: Can vagus nerve stimulation halt or ameliorate rheumatoid arthritis and lupus? . Lipids Health Dis. 2011, 10:19. 10.1186/1476-511X-10-19 34. Mazurak N, Seredyuk N, Sauer H, Teufel M, Enck P: Heart rate variability in the irritable bowel syndrome: a review of the literature. Neurogastroenterol Motil. 2012, 24:206-16. 10.1111/j.1365-2982.2011.01866.x 35. Bassi GS, Brognara F, Castania JA, et al.: Baroreflex activation in conscious rats modulates the joint inflammatory response via sympathetic function. Brain Behav Immun. 2015, 49:140-7. 10.1016/j.bbi.2015.05.002 36. Busch V, Zeman F, Heckel A, Menne F, Ellrich J, Eichhammer P: The effect of transcutaneous vagus nerve stimulation on pain perception--an experimental study. Brain Stimul. 2013, 6:202-9. 10.1016/j.brs.2012.04.006 37. Zhang X, Cao B, Yan N, et al.: Vagus nerve stimulation modulates visceral pain-related affective memory. Behav Brain Res. 2013, 236:8-15. 10.1016/j.bbr.2012.08.027 38. Frøkjaer JB, Bergmann S, Brock C, et al.: Modulation of vagal tone enhances gastroduodenal motility and reduces somatic pain sensitivity. Neurogastroenterol Motil. 2016, 28:592-8. 10.1111/nmo.12760 39. Dahlin LB, Archer DR, McLean WG: Axonal transport and morphological changes following nerve compression: an experimental study in the rabbit vagus nerve. J Hand Surg Br. 1993, 18:106-10. 40. Pereira MR, Leite PE: The Involvement of Parasympathetic and Sympathetic Nerve in the Inflammatory Reflex. J Cell Physiol. 2016, 231:1862-9. 10.1002/jcp.25307 41. Schlereth T, Birklein F: The sympathetic nervous system and pain . Neuromolecular Med. 2008, 10:141-7. 10.1007/s12017-007-8018-6 42. Lipschitz M, Bernstein-Lipschitz L, Nathan H: Thoracic sympathetic trunk compression by osteophytes associated with arthritis of the costovertebral joint: anatomical and clinical considerations. Acta Anat (Basel). 1988, 132:48-54. 43. Nathan H: Osteophytes of the spine compressing the sympathetic trunk and splanchnic nerves in the thorax. Spine (Phila Pa 1976). 1987, 12:527-32.

2018 Bordoni et al. Cureus 10(7): e3036. DOI 10.7759/cureus.3036 9 of 9 Autonomic Neuroscience: Basic and Clinical 153 (2010) 106–115

Contents lists available at ScienceDirect

Autonomic Neuroscience: Basic and Clinical

journal homepage: www.elsevier.com/locate/autneu

Visceral organ cross-sensitization — An integrated perspective

P.R. Brumovsky a,b,⁎, G.F. Gebhart a,⁎ a Pittsburgh Center for Pain Research, Department of Anesthesiology, University of Pittsburgh, Pittsburgh, Pennsylvania, United States b School of Biomedical Sciences, Austral University, Buenos Aires, Argentina article info abstract

Article history: Viscero-somatic referral and sensitization has been well documented clinically and widely investigated, Received 14 April 2009 whereas viscero-visceral referral and sensitization (termed cross-organ sensitization) has only recently Received in revised form 9 July 2009 received attention as important to visceral disease states. Because second order neurons in the CNS have Accepted 10 July 2009 been extensively shown to receive convergent input from different visceral organs, it has been assumed that cross-organ sensitization arises by the same convergence-projection mechanism as advanced for viscero- Keywords: somatic referral and sensitization. However, increasing evidence also suggests participation of peripheral Thoracic organs fl Pelvic organs mechanisms to explain referral and sensitization. We brie y summarize behavioral, morphological and Dichotomizing afferents physiological support of and focus on potential mechanisms underlying cross-organ sensitization. Peripheral sensitization © 2009 Elsevier B.V. All rights reserved. Central sensitization

1. Cross-organ sensitization somatic referral and sensitization. However, there is also a long history associated with consideration of peripheral mechanisms to explain Around 600 BC, Sushruta, an Indian surgeon, described the Hritshoola, referral and sensitization. We briefly summarize behavioral, morpho- which literally means heart pain (Dwivedi and Dwivedi, 2007; Dwivedi logical and physiological support of and discuss potential mechanisms and Chaturvedi, 2000). However, it was not until the late 19th century underlying cross-organ sensitization. that potential mechanisms underlying this well known symptom — referred pain — were advanced as reflecting a “commotion” or “irritable 2. Clinical and experimental evidence of cross-organ sensitization focus” in spinal segments receiving input from an organ (Ross, 1888; Sturge, 1888). These concepts were later expanded and formulated as the Based on clinical (Table 1) and experimental (Table 2) studies of cross- “convergence–projection” theory of referred visceral sensation (Ruch, organ sensitization, the organs affected can be grouped into: 1) thoraco- 1965), convergence denoting input from both somatic and visceral upper abdominal (esophagus, heart, lower airways, stomach, duodenum structures onto the same second order spinal neuron (see Ness and and gallbladder) and 2) pelvic-lower abdominal (colon, rectum, ureter, Gebhart, 1990; Gebhart and Ness, 1991 for more extensive discussion). urinary bladder, pelvic urethra, uterus and prostate). The grouping is not Whereas viscero-somatic referral and sensitization has been well arbitrary. Cross-sensitization typically occurs between organs within documented clinically and widely investigated, viscero-visceral referral either thoraco-upper abdominal or pelvic-lower abdominal areas. and sensitization (termed cross-organ sensitization) has only recently However, organ cross-talk/modulation between thoraco-upper abdom- received attention as important to visceral disease states (e.g., see inal and pelvic-lower abdominal areas has also been reported (see Berkley, 2005; Foreman, 2007; Malykhina, 2007). Dmitrieva et al., 2001; Morrison et al., 2006; Qin et al., 2003, 2007e). Because second order neurons in the CNS have been widely documented to receive convergent input from different visceral organs, it has been assumed that cross-organ sensitization arises by 2.1. Thoraco-upper abdominal cross-organ sensitization a similar convergence–projection mechanism as advanced for viscero- 2.1.1. Heart/esophagus/lower airways/stomach/gallbladder Pain referred to the skin and muscle of the thorax and left arm

⁎ Corresponding authors. Gebhart is to be contacted at Pittsburgh Center for Pain during myocardial ischemia (angina) represents a landmark symptom Research, W1444BST, 200 Lothrop Street, Pittsburgh, PA 15213, United States. Tel.: +1 recognized not only clinically, but also by the general public. However, 412 383 5911; fax: +1 412 383 5466. Brumovsky, Pittsburgh Center for Pain Research, the same symptoms and pattern of referral thought to be derived from W1402BST, 200 Lothrop Street, Pittsburgh, PA 15213, United States, or School of the heart are replicated from the pathologic esophagus (Heatley et al., Biomedical Sciences, Austral University, Buenos Aires, Argentina, Av. Pte. Perón 1500, 2005). Growing evidence suggests that the heart is neuroanatomically B1629AHJ Pilar, Buenos Aires, Argentina. E-mail addresses: [email protected] (P.R. Brumovsky), and functionally related with the esophagus. Thus, it was shown both [email protected] (G.F. Gebhart). in cat (Garrison et al., 1992) and rat (Euchner-Wamser et al., 1993; Qin

1566-0702/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.autneu.2009.07.006 P.R. Brumovsky, G.F. Gebhart / Autonomic Neuroscience: Basic and Clinical 153 (2010) 106–115 107

Table 1 tion of the heart. Likewise, airway irritants (e.g., ammonia, cigarette Clinical observations of cross-organ sensitization. smoke) have been shown to excite rat thoracic spinal neurons that Organs involved References also respond to esophageal distension (Hummel et al., 1997)or Thoraco-upper Lower esophagus and upper Sarkar et al. (2000, 2001), intrapericardial administration of bradykinin (Euchner-Wamser et al., abdominal esophagus Frokjaer et al. (2005), 1994; Qin et al., 2007d). cross-organ Sarkar et al. (2006) sensitization Lower esophagus and duodenum Frokjaer et al. (2005) 2.1.2. Esophagus/stomach/duodenum Lower esophagus and rectum Frokjaer et al. (2005) Duodenum and esophagus Hobson et al. (2004) Several studies in have recently shown that noxious Hypoalgesia of esophageal and Dimcevski et al. (2006) stimulation of the lower esophagus (LE) can sensitize the upper duodenal distension in painful esophagus (UE). Thus, infusion in the LE induces hyperalgesia in chronic pancreatitis patients the UE (Frokjaer et al., 2005; Sarkar et al., 2000, 2001, 2006). Pelvic-lower Colon and other viscera Whorwell et al. (1986) Acidification of the duodenum also induces esophageal hypersensitivity abdominal Bladder and colon Alagiri et al. (1997) cross-organ Sigmoid colon and rectum (Naliboff et al., 1997; in humans (Hobson et al., 2004), a process that may involve central sensitization Lembo et al., 1997; sensitization of spinal cord neurons receiving convergent input from Munakata et al., 1997) both organs. Supporting this idea, it has been shown in rat that T9–T10 Reproductive organs and upper Giamberardino et al. (2001) spinal neurons receive both duodenal and gastric afferent input, and urinary system respond to gastric distension in the noxious range (Qin et al., 2007a,b).

2.2. Pelvic-lower abdominal cross-organ sensitization et al., 2004) that thoracic spinal neurons receiving somatic input also receive convergent esophageal and cardiac input. Moreover, these Cross-organ sensitization between the lower gut and pelvic neurons appear to be sensitized by noxious stimulation of the two urinary or gynecologic organs has been shown to be a common and related organs (Garrison et al., 1992; Qin et al., 2004). troublesome clinical circumstance, leading to significant problems in Convergence has also been described between the heart and diagnosing and treating the diseased organ (see Baranowski et al., stomach or gallbladder. Studies in cat (Ammons and Foreman, 1984), 2008; Berkley, 2005; Berkley et al., 2005, 2007; Malykhina, 2007; monkey (Ammons et al., 1984) and rat (Qin et al., 2007c) have shown Saini et al., 2008; Stanford et al., 2007; Theoharides et al., 2008; van de that distension of the gallbladder (Ammons et al., 1984; Ammons and Merwe et al., 2008; Warren et al., 2008). The organs that appear most Foreman, 1984) or stomach (Qin et al., 2007c) activates thoracic often involved in pelvic-lower abdominal cross-sensitization, both in spinothalamic tract neurons responsive to noxious chemical stimula- humans and animals, seem to be the colon/rectum, the urinary bladder/pelvic urethra, the uterus and the prostate (see Tables 1 and 2). Interestingly, the urinary bladder may be more vulnerable to Table 2 cross-modulation than other pelvic organs and, conversely, inflam- Experimental behavioral and physiological evidence of cross-organ sensitization. matory processes in the bladder appear less effective in inducing Organs involved Species References cross-sensitization in colon or uterus (see below). Thoraco-upper Esophagus and Male rat Garrison et al. (1992), abdominal heart Euchner-Wamser et al. (1993), 2.2.1. Colon/urinary bladder cross-organ Qin et al. (2004) In normal conditions, colon and bladder are functionally related. sensitization Stomach and heart Qin et al. (2007c) Studies by Denny–Brown and Robertson (1933), showing that Gallbladder and Cat Male rat Ammons and Foreman (1984), micturition and defecation are normally alternated (Vilensky et al., heart Ammons et al. (1984) Stomach and Male rat Qin et al. (2007a,b) 2004), and by Kock and Pompeius (1963), who noted also that duodenum motility of the bladder was inhibited by distension of the anal canal or Heart and lower Qin et al. (2007d) rectum and by stimulation of the perineal skin, suggested an respiratory airways anatomical and functional relationship between the bowel and the Pelvic-lower Colon and bladder Cat Floyd et al. (1978, 1979), abdominal Floyd et al. (1982) urinary bladder. Floyd et al. (1982) later showed in cats that colonic cross-organ Male rat Malykhina et al. (2004, 2006), distension results in graded inhibition of spontaneous bladder sensitization Qin et al. (2005), Noronha contractility. et al. (2007) Importantly, studies in humans reveal that colon-bladder cross- Female rat Pezzone et al. (2005), sensitization could also result in painful symptoms. For example, Ustinova et al. (2006, 2007) Male mouse Lamb et al. (2006) patients with irritable bowel syndrome (IBS) often exhibit signs of Colon and pelvic Male rat Peng et al. (2009) urinary bladder hypersensitivity: nocturia, frequency and urgency of urethra micturition, incomplete bladder emptying, back pain and, in women, Colon and uterus Female rat Winnard et al. (2006) dyspareunia (Whorwell et al., 1986). More recently, studies where Bladder and colon Cat Bouvier et al. (1990) Female rat Pezzone et al. (2005) acute (Pezzone et al., 2005) or chronic (Bielefeldt et al., 2006; Lamb Male mouse Bielefeldt et al. (2006) et al., 2006) colon irritation were induced in mouse (Pezzone et al., Female mouse Rudick et al. (2007) 2005) and rat (Lamb et al., 2006; Peng et al., 2009) confirmed ‘colon- Lower respiratory Male rat Qin et al. (2007e) to-bladder’ sensitization, showing increased frequency of bladder airways and colon contractions, reduced inter-contraction intervals (Pezzone et al., Bladder and uterus Female rat (Dmitrieva et al., (2001), fl Dmitrieva and Berkley (2002), 2005), and altered micturition re exes (Lamb et al., 2006). It is Winnard et al. (2006) noteworthy to indicate that these signs are usually indicators of Bladder and heart Male rat Qin et al. (2003) bladder/pelvic distress and pain, as their presence in humans often Uterus and colon Female rat Winnard et al. (2006) correlates with pain (Theoharides et al., 2008). Uterus and bladder Winnard et al. (2006), Morrison et al. (2006) Uterus and pelvic Peng et al. (2008a,b) 2.2.2. Urinary bladder/colon urethra It has been suggested that ‘bladder-to-colon’ cross-modulation Uterus and vagina Cason et al. (2003), may be less frequently observed than its counterpart (Winnard et al., Berkley et al. (2007)) 2006). However, an epidemiological study reported that patients 108 P.R. Brumovsky, G.F. Gebhart / Autonomic Neuroscience: Basic and Clinical 153 (2010) 106–115 diagnosed with interstitial cystitis (IC) presented concomitant chronic afferent input onto spinal neurons converged with afferent input from diseases, including IBS (Alagiri et al., 1997). Conversely, individuals somatic structures (subsequently projected to supraspinal sites), with IBS showed an increased association with IC when compared providing a central, spinal explanation for referred viscero-somatic with the general population. While these observations suggest the and viscero-visceral hypersensitivity. That is, organ disease or occurrence of cross-organ sensitization between bladder and colon, experimental inflammation increases the excitability of spinal they do not address which organ/system is first affected. Recent neurons which is reflected as increased responses to stimuli applied studies in rat (Pezzone et al., 2005) and mouse (Bielefeldt et al., 2006) in tissues providing convergent input onto the same spinal neuron. It confirm ‘bladder-to-colon’ cross-sensitization, by showing that ani- has since been widely documented that second order spinal neurons mals treated with cyclophosphamide, an antineoplastic drug that receive visceral afferent as well as somatic afferent input, if not also induces bladder inflammation (see Korkmaz et al., 2007; Laird et al., convergent input from another organ(s) in the same body area (e.g., 2002), develop hypersensitivity to colon distension. see Table 2 for references), suggesting a central basis for cross-organ sensitization (Fig. 1). 2.2.3. Uterus/prostate/ureter/urinary bladder and pelvic urethra/colon In addition to a spinal segmental contribution, convergence of Cross-organ modulation is a common observation in the fields of viscero-somatic and viscero-visceral input has been documented in gynecology and urology (see Alagiri et al.,1997; Berkley, 2005; Berkley brainstem and thalamus. Regarding viscero-visceral convergence, et al., 2005; Saini et al., 2008; Theoharides et al., 2008). Accordingly, morphological studies in Barrington's nucleus (the pontine micturi- pain originating from different organs of the lower abdomen and tion centre) demonstrated the existence of neurons responding both pelvis are often considered components of chronic pelvic pain to bladder and colon distension (Hubscher et al., 2004; Rouzade- syndrome (CPPs). Dominguez et al., 2003a,b). In the rat thalamus, neurons responding Several studies in animals have confirmed cross-organ modulation to electrical stimulation of the dorsal nerve of the penis and to colon among the lower urinary tract, colon and gynecologic structures distension were identified in a number of nuclei (Hubscher and (Table 2). Thus, bladder inflammation in female rats results in the Johnson, 2003). It is noteworthy that the general principle of reduction of uterine contractions (Dmitrieva et al., 2001). Moreover, convergence from adjacent or nearby organs may not necessarily uterine inflammation induces plasma extravasation in the urinary apply in the thalamus, which receive visceral inputs from widely bladder and to a lesser extent also in the colon, in an estrous cycle- separated organs (e.g., Apkarian et al., 1995). dependent manner, suggesting cross-organ inflammation (Winnard Transection of visceral nerves also provides support for central et al., 2006). Furthermore, experimental endometriosis in female rats contributions to cross-organ sensitization. For example, transection of not only induced bladder plasma extravasation, but also urinary the hypogastric nerve (a major source of afferent and efferent bladder hypersensitivity, reflected as a decrease in micturition innervation of the uterus (Berkley et al., 1993; Sato et al., 1996)in thresholds (Morrison et al., 2006). female rats was found to reduce the plasma extravasation in the Cross-organ sensitization has also been documented between the urinary bladder induced by inflammation of the colon or uterus uterus and the pelvic urethra, the ureter or the vagina (Table 2). The (Winnard et al., 2006), as well as the inhibitory effects of urinary pelvic urethra reflex, understood as the reflexive closure of the urethra bladder inflammation on uterine contractions (Dmitrieva et al., 2001). caused by bladder distension (see de Groat et al., 2001), and which has Convergent afferent inputs from the duodenum and colon onto vagal been related to neurogenic urethra hyperactivity (Lin, 2003, 2004), is pre-ganglionic efferents innervating the stomach have also been sensitized by the instillation of into the uterus of demonstrated in ferrets, and shown to inhibit gastric motility (Grundy anesthetized rats (Peng et al., 2008a,b). Moreover, studies in women et al., 1981). Finally, it has been hypothesized that antidromically (Giamberardino et al., 2001) and rat (Giamberardino et al., 2002) produced dorsal root reflexes (Willis, 1999), generated in micro- suggested that both existing (dysmenorrhea) and latent (pelvic circuits between dorsal horn interneurons receiving inputs from an congestion at ovulation/menstruation; asymptomatic endometriosis, inflamed organ and making synaptic contact with afferent inputs from ovarian cysts) conditions of female reproductive organs enhance an uninflamed organ, could promote cross-sensitization (Berkley, referred pain due to ureteral calculosis. And, in a rat model of 2005). endometriosis, Berkley et al. (2007) and Cason et al. (2003) showed Glutamate and glutamate receptors, as well as capsaicin and its that abnormal endometrial tissue growths directly influence devel- receptor, TRPV1, have been associated with central mechanisms of opment of vaginal hypersensitivity and that the resulting pain is cross-organ sensitization. Instillation of capsaicin into the uterus of exacerbated by estrogens (Berkley et al., 2007). female rats sensitizes evoked pelvic urethra reflex activity, with a In men there is potential for cross-organ sensitization between the parallel increase in the content of phosphorylated NMDA subunit prostate and other pelvic organs (Pontari, 2008; Saini et al., 2008). NR2B receptor in spinal neurons (Peng et al., 2008b). Interestingly, the Lower urinary tract symptoms such as urgency and nocturia often urethral as well as spinal effects were attenuated by intrathecal overlap with chronic pain in the perineum, testes or tip of the penis, application of NMDA (Peng et al., 2008b) or TRPV1 (Peng et al., 2008b) typical symptoms of chronic prostatitis (Pontari, 2008; Saini et al., receptor antagonists. Similar and supporting results were obtained 2008). when assessing the effects of mustard oil-induced colon inflammation on the rat pelvic urethra reflex (Peng et al., 2009). However, since local 3. Potential mechanisms of cross-organ sensitization blockade of the TRPV1 receptor in the inflamed colon (Peng et al., 2009) or uterus (Peng et al., 2008a) attenuated effects on the urethral 3.1. The ‘central’ theory reflex, it could be hypothesized that peripheral mechanisms may also have a role in cross-organ sensitization (see below). As briefly introduced above, the earliest theories on referred pain Finally, recto- (Neuhuber et al., 1993) and colo-spinal (Suckow and date to the late 19th century and work by MacKenzie, Sturge and Ross Caudle, 2008) afferent neurons have been reported in the rat, and (see Gebhart and Ness, 1991; Ness and Gebhart, 1990). In various partly because they express neuropeptides and receptors associated forms, these authors inferred from their clinical observations that with nociception, it has been proposed that they participate in visceral increased tenderness to palpation of overlying structures, or hyper- pain processing (see Suckow and Caudle, 2008). While the physiolo- sensitivity of skin in the area of referred sensation (e.g., left shoulder), gical significance of these neurons remains to be confirmed, one may arose from changes in the excitability of spinal neurons (the speculate that these neurons could transmit nociceptive information conceptual antecedent to ‘central sensitization’). These concepts from the distal gut to spinal neurons receiving convergent input from were later assimilated by Ruch (1965) into a proposal that visceral other organs, thus contributing to central sensitization. P.R. Brumovsky, G.F. Gebhart / Autonomic Neuroscience: Basic and Clinical 153 (2010) 106–115 109

Fig. 1. Hypothetical mechanisms of centrally mediated cross-organ sensitization. (A) The general model illustrates convergence of inputs from colon and bladder onto the same second order spinal neuron. An insult to colon (represented by a lightning bolt) increases the excitability of the spinal neuron (central sensitization) such that ‘normal’ input from the bladder is also amplified by the second order spinal neuron. (B) Alternatively, activation by input from the colon of an inhibitory spinal interneuron (filled circle) could lead to an increase in primary afferent depolarization, generation of dorsal root reflexes (DRR; Willis, 1999) and neurogenic inflammation in the bladder.

3.2. The ‘peripheral’ theory Dichotomizing sensory neurons would naturally cross-sensitize. In cultured lumbosacral bladder sensory neurons from rats with colitis, The principal peripheral mechanism advanced to explain referral significant increases in the net inward current induced by capsaicin of visceral sensation and cross-tissue sensitization is based on so- and in the peak amplitude of tetrodotoxin-resistant (TTX-R) Na+ called dichotomizing fibers (i.e., sensory endings of a single neuron currents were shown (Malykhina et al., 2004). In a subsequent study, innervating two different tissues), a concept most enthusiastically acute colitis in male rats was shown to decrease the voltage and supported by Sinclair et al. (1948). They and subsequent investigators reported electrophysiological (Pierau et al., 1982; Sinclair et al., 1948) Table 3 and morphological evidence suggesting the presence of dichotomiz- Anatomical demonstration of dichotomizing primary afferent neurons. ing fibers in the sacroiliac plexus nerves of pigeons (Taylor and Pierau, 1982) and rats (Taylor et al., 1983) (see Table 3). Subsequently, Organs involved Species References dichotomizing neurons projecting into the thoracic (intercostal) and Non-visceral Dichotomizing neurons Pigeon Taylor and visceral (splanchnic) nerves of rat were also described (Dawson et al., dichotomizing in the leg Pierau (1982) nerves Male rat Taylor et al. 1992). Although the number of dichotomizing sensory neurons in (1983) dorsal root ganglia (DRG) varies between animals and studies (from Thoraco-upper Dichotomizing neurons Male rat Dawson et al. 0.1% to 21% of all traced neurons) (Dawson et al., 1992; Taylor et al., abdominal nerves projecting to the intercostal (1992) 1983), their identification suggested an anatomical and physiological (somatic) and splanchnic (visceral) nerves basis for the occurrence of referred pain. Pelvic-lower Physiological evidence of dichotomizing primary afferents abdominal between the colon, the anus and the lower urinary tract was also dichotomizing reported (Bahns et al., 1986)(seeTable 3 and Fig. 2). Later, nerves morphological studies in rat (Chaban et al., 2007; Chen et al., 2005; Colon/bladder Convergent colon and Male rat Keast and de bladder DRG neurons Groat (1992), Christianson et al., 2007; Keast and de Groat, 1992; Malykhina et al., Malykhina et al. 2006), mouse (Christianson et al., 2007) and cat (de Groat et al., 1987) (2006) using two different retrogradely transported dyes injected in different Male rat Christianson et al. organs revealed the presence of dichotomizing afferents between and (2007) mouse colon and the urogenital and sexual organs (Table 3). In contrast, it Up-regulation of CGRP and Male rat Qiao and Grider seems that autonomic neurons present in the rat major pelvic TrkB in rat bladder afferent (2007) ganglion do not dichotomize, as shown by the absence of doubly neurons after TNBS labeled, colon/bladder neurons (Rouzade-Dominguez et al., 2003a). Uterus/Colon Convergent colon and uterus Female rat Chaban et al. Further characterization of dichotomizing primary afferent neurons DRG neurons (2007) TRPV1 and P2X3 expression Chaban et al. innervating the colon and uterus (Chaban et al., 2007) showed of dually projecting colon (2007) expression of TRPV1 and the purinoceptor P2X3, both involved in and uterus DRG neurons nociceptive mechanisms (see Brederson and Jarvis, 2008; Broad et al., Up-regulation of molecules Li et al. (2008) 2009). Also, up-regulation of CGRP was observed in rat bladder DRG in convergent colon and uterus neurons after colon inflammation (Qiao and Grider, 2007). DRG neurons 110 P.R. Brumovsky, G.F. Gebhart / Autonomic Neuroscience: Basic and Clinical 153 (2010) 106–115

Fig. 2. Hypothetical mechanisms of peripherally mediated cross-organ sensitization — the dichotomizing primary afferent neuron. The general model illustrates innervation of the colon and bladder by a single . The inset shows DRG neurons that innervate the colon (green; arrowheads), the bladder (red; double arrowheads) and both the colon and bladder, as evidenced by the colocalization of both green and red signals (yellow; arrows) in the merged channel. See Christianson et al. (2007) for details. Images (unpublished) were provided courtesy of Drs. Julie C. Christianson and Brian M. Davis, University of Pittsburgh. Scale bar: 20 µm. current thresholds for action potential firing in dichotomizing ~600 project to the bladder and/or colon (Christianson et al., 2007), capsaicin-sensitive lumbosacral DRG neurons, from 3 to 30 days only ~4% of the total number of L6/S1 DRG neurons project to these after the onset of colitis (Malykhina et al., 2006). organs. Accordingly, the proportion of dichotomizing visceral These changes in bladder or colon sensory neurons could also result neurons is small and their role in cross-organ sensitization remains in alterations in the sensitivity of their nerve terminals in the target to be confirmed as functionally significant. organ. For example, in vitro single fiber recordings of pelvic nerve There exist other peripheral mechanisms, discussed below, that bladder afferents from rats with acute (Ustinova et al., 2006) or chronic may potentially contribute to cross-tissue/organ sensitization. (Ustinova et al., 2007) colon irritation revealed sensitization of bladder afferents to both mechanical (innocuous and noxious bladder disten- 3.2.1. Alterations in afferent processing of DRG neurons and their sion) and chemical (capsaicin, bradykinin and SP) stimuli (Ustinova et projections al., 2006). These effects were abolished by afferent dennervation of the Sensory neurons innervating different organs could be cross-sensitized bladder (Ustinova et al., 2006) and by systemic capsaicin pretreatment in a number of additional ways that do not require dichotomizing fibers (Ustinova et al., 2007), suggesting a role for TRPV1-expressing bladder (Fig. 3). Cross excitation could occur at the level of the sensory neuron in sensory neurons in the generation of cross-organ sensitization. the DRG. Studies in cultured DRG neurons innervating the hindpaw Early reports on the existence of dichotomizing visceral sensory showed that A- and C-neurons (i.e., neurons with myelinated and neurons were generally discounted on technical grounds, but recent unmyelinated , respectively; see Lawson, 2002)areelectrically studies using improved tracers and experimental strategies have coupled (Amir et al., 2002, 2005; Amir and Devor, 2000). Interestingly, reinforced the presence of dichotomizing sensory neurons innervat- electrical excitability of neuron somata seems not to be required for ing different organs and stimulated additional studies. One caution through conduction of afferent input from the periphery to the spinal cord when considering dichotomizing sensory neurons as principal (Amir and Devor, 2003). If not electrical, then chemical coupling could players in the generation and maintenance of cross-organ sensitiza- play a role (Amir and Devor, 1996; Mantyh et al., 1994). Intraganglionic tion is their low percentage relative to the proportion of all visceral release of substance P (SP) (Harding et al., 1999; Huang and Neher, 1996; sensory neurons. Of all labeled colon and bladder sensory neurons, Neubert et al., 2000) and of calcitonin -related peptide (CGRP) only 5–27% dichotomizing neurons were found in rat (Christianson (Eberhardt et al., 2008; Ulrich-Lai et al., 2001)hasbeenshownin et al., 2007; Malykhina, 2007; Qiao and Grider, 2007) and ~21% in trigeminal (Eberhardt et al., 2008; Neubert et al., 2000; Ulrich-Lai et al., mouse (Christianson et al., 2007). Likewise, a relatively small 2001) and dorsal root ganglia (Eberhardt et al., 2008; Harding et al., 1999; proportion (~3–15%) of uterus and colon dichotomizing neurons Huang and Neher, 1996) of rats. SP (e.g., (Mantyh, 2002)) as well as CGRP were detected in rat (Chaban et al., 2007; Li et al., 2008). The (e.g. Hill and Oliver, 2007) are nociceptive modulators/transmitters and percentage of visceral sensory neurons in a given DRG is not well provided that 1] their receptors are present and functional in DRG neurons established. However, considering that L6/S1 DRGs from young rats and/or their axons and 2] they are in close anatomical proximity contain ~16000 neurons (Mohammed and Santer, 2001), and that (including with satellite glial cells), electrical and/or chemical coupling P.R. Brumovsky, G.F. Gebhart / Autonomic Neuroscience: Basic and Clinical 153 (2010) 106–115 111

Fig. 3. Hypothetical mechanisms of peripherally mediated cross-organ sensitization — intraganglionic (A) and interaxonal coupling (B). (A) Intraganglionic release of neuropeptides and excitatory neurotransmitters such as glutamate may participate in the interaction between spatially close colon (green) and bladder (red) DRG neurons (a). This mechanism obviously requires expression of functionally active neurotransmitter receptors. Alternatively, electrical coupling (b) could have a role in cross-organ sensitization between DRG neurons. (B) Chemical (a) as well as electrical (b) coupling between injured and healthy primary afferent fibers have been described in nerves supplying skin and muscle. A similar mechanism could also be present in visceral nerves, thus allowing for cross-sensitization within nerve bundles. Images (unpublished) depicting colon (green) and bladder (red) DRG neurons (A) or biotinamide-traced pelvic nerve axons (B), were provided courtesy of Drs. Julie C. Christianson and Brian M. Davis, University of Pittsburgh and Pablo R. Brumovsky, respectively. Scale bars: 20 µm (A); 10 µm (B).

(for review, see Brumovsky et al., 2007) could contribute to cross-talk and decreased micturition thresholds (Morrison et al., 2006). between neighboring DRG neurons innervating different visceral organs. Interestingly, some of these changes were dependent on the oestrus Extensive studies on rats with spinal nerve ligation suggest that cycle of the rat, suggesting participation of gonadal hormones in the Wallerian degeneration and subsequent inflammation of injured cross-organ sensitization (Winnard et al., 2006). axons leads to the sensitization of uninjured neighboring axons Studies by Foreman and colleagues, supporting the role of central (Campbell and Meyer, 2006; Meyer and Ringkamp, 2008). Alterna- sensitization as the main player in cross-organ sensitization between the tively, bladder and colon afferents in the pelvic nerve, for example, colon and bladder, showed no detectable histological changes in the could be cross-sensitized through local, axonal release of excitatory bladder wall of rats with chronic colitis (Malykhina et al., 2004; Qin et al., neurotransmitters (Amir and Devor, 1992; Hoffmann et al., 2008) 2005). However, acute (but not chronic) colitis in rats was shown to alter (Fig. 3). Thus, noxious heat and chemical stimulation were shown to the contractility of the detrusor muscle in response to electrical field induce axonal release of CGRP from sciatic nerve fibers in rat (Sauer stimulation, cholinergic agonism with carbachol or exposure to potas- et al., 2001) and mouse (Bernardini et al., 2004), probably through sium chloride, in the absence of morphological changes or inflammatory vesicular exocytosis (Bernardini et al., 2004). Moreover, this effect was infiltration of the bladder (Noronha et al., 2007). Thus, while differences dependent on the activation of TRPV1 receptors (see Fischer and Reeh, in the outcome of several studies may relate to different methodological 2007; Leffler et al., 2008). These observations are neurophysiologically approaches, the absence of histological changes in a cross-sensitized significant. Heat stimulation of unmyelinated peripheral nerve axons organ does not imply lack of functional alterations. in mouse produces responses similar to those when the stimulus is An alternative approach to assess possible local changes during applied to the receptive field (Hoffmann et al., 2008). Accordingly, a cross-organ sensitization is altered response to drugs. For example, psychophysical study revealed transduction of thermal energy cannabinoids dose-dependently increase uterine contractions, an by the superficial radial nerve, producing pain perceived in the effect that is reduced in the presence of bladder inflammation corresponding distant receptive field (Hoffmann et al., 2009). (Dmitrieva and Berkley, 2002), suggesting alterations in the expres- sion of uterine cannabinoid receptors. Therefore, cross-organ sensi- 3.2.2. Local/inflammatory changes tization could arise not only from organ inflammation or alterations in Histological examination of bladders from rats with chronic colitis the sensitivity of afferent terminals or sensory neurons, but also in the revealed an increase in bladder mast cell density and sensory fiber number/affinity of tissue receptors and the ability to respond to drugs sensitization compared with control (Ustinova et al., 2007). Acute and endogenous mediators in different pathological conditions. signs of inflammation such as plasma extravasation have also been described in the bladder after colon or uterine inflammation, with 3.2.3. Role for the autonomic nervous system (ANS)? smaller changes in colon and uterine horn when the bladder was “The ANS is that system of neurons that controls visceral organs, inflamed (Winnard et al., 2006). Moreover, rats with experimental effectors in the skin, and the cardiovascular system” (Furness, 2006). endometriosis show increased urinary bladder plasma extravasation Earlier, Langley (1916) posited that the autonomic nerves, including 112 P.R. Brumovsky, G.F. Gebhart / Autonomic Neuroscience: Basic and Clinical 153 (2010) 106–115 the paravertebral and prevertebral ganglionic chains, coordinate the in prevertebral ganglia, innervating both healthy and pathologic activities of the organs and the sensations generated from them. organs, offers an additional means of contributing to cross-organ Prevertebral ganglia (e.g., inferior mesenteric and major pelvic sensitization. ganglia) typically consist of a mixture of cell bodies of sympathetic and parasympathetic postganglionic neurons, receiving both sympa- 3.3. An integration of concepts thetic and parasympathetic input (Furness, 2006). Interestingly, peptidergic varicosities are very abundant in autonomic ganglia We have presented different views on how cross-organ sensitiza- from different species (see de Groat, 1987), possibly derived from tion could arise and be maintained. Most studies have emphasized passing afferent fibers projecting towards their corresponding target either central or peripheral mechanisms, but it has long been organ (see Houdeau et al., 2002; Kaleczyc et al., 2003). appreciated (e.g., Sinclair et al., 1948) that neither central nor Autonomic ganglia could thus also contribute to cross-organ peripheral mechanisms alone are capable of fully explaining cross- sensitization (Fig. 4). As illustrated, collaterals in an autonomic organ sensitization. Clinical observation reveals that both peripheral ganglion from afferents innervating a pathologic organ could excite and central mechanisms are involved in the generation and ganglionic secretory and motor neurons innervating a different, non- maintenance of cross-organ sensitization, and that they possibly diseased organ. In fact, several neuropeptides have been shown to intervene in an orchestrated manner. Recently, it was indicated that exert facilitatory or inhibitory effects on cells in autonomic ganglia “the greater abundance of convergent neurons in the spinal cord likely (e.g., Cohen et al., 1996; de Groat, 1987). Moreover, activation of indicates a central amplification process in cross-organ sensitization” postganglionic parasympathetic efferents by electrical stimulation of (Qin et al., 2005). This amplification process could also be understood the sphenopalatine ganglion elicits extravasation in the ipsilateral in a much broader sense, involving a number of amplification steps dura (Asztely et al., 1998; Delepine and Aubineau, 1997). Thus, a contributing to cross-organ sensitization. Thus, taking an inflamma- primary afferent driven ‘cross-organ neurogenic inflammation’ could tory process in any given organ as an example for initiation of cross- arise, resulting in events such as plasma extravasation and inflamma- organ sensitization, one could hypothesize that the following series of tion in a non-diseased organ. events would take place (Fig. 2): One more potential peripheral mechanism of cross-organ sensiti- • Organ insult/inflammation zation should be considered: the intestinofugal afferent neuron (see • Peripheral excitation/sensitization Szurszewski et al., 2002). This unique myenteric ganglion neuron • Central sensitization. relays mechanosensory information to sympathetic prevertebral neurons, and their activation by colon distension induces acetylcho- Details contributing to the above events could include: 1) an line release in the ganglion and subsequent excitatory postsynaptic inflammatory process in an organ increases the excitability of potentials of ganglionic neurons (see Szurszewski et al., 2002). dichotomizing and non-dichotomizing sensory neurons that inner- Taken together, the important anatomical and functional relation- vate the organ and leads to cross-inflammation of a nearby organ; ship between sensory and autonomic neurons/fibers present 2) the production of inflammatory mediators AND/OR electrical and/

Fig. 4. Hypothetical mechanisms of autonomic/afferent interactions leading to cross-organ neurogenic inflammation. In this scenario, the sensitization of afferent fibers due to organ insult would not only result in the transmission of information to the central nervous system, but also in the activation of autonomic neurons in prevertebral ganglia. For example, axon collaterals of sensitized bladder primary afferent fibers would synapse onto postganglionic neurons in the major pelvic ganglion (MPG) that innervate the colon. These autonomic neurons, by release of various neurotransmitters, may in turn contribute to changes in secretory and motor function as well as generation of neurogenic inflammation in the colon. Abbreviations: IMG: inferior mesenteric ganglion; MPG: major pelvic ganglion; PN: pelvic nerve. P.R. Brumovsky, G.F. Gebhart / Autonomic Neuroscience: Basic and Clinical 153 (2010) 106–115 113 or neurochemical coupling between axons and cell bodies of sensory Bielefeldt, K., Lamb, K., Gebhart, G.F., 2006. Convergence of sensory pathways in the fi development of somatic and visceral hypersensitivity. Am. J. Physiol. Gastrointest. neurons increase the excitability of afferent bers innervating the Liver Physiol. 291, G658–G665. non-diseased organ; 3) the affected sensory neuron cell bodies Bouvier, M., Grimaud, J.C., Abysique, A., 1990. Effects of stimulation of vesical afferents increase their expression of receptors/ion channels and axonal on colonic motility in cats. Gastroenterology 98, 1148–1154. Brederson, J.D., Jarvis, M.F., 2008. Homomeric and heteromeric P2X3 receptors in transport of excitatory neurotransmitters, including neuropeptides, peripheral sensory neurons. Curr. Opin. Investig. Drugs 9, 716–725. which in turn are released in the affected organs to further promote Broad, L.M., Mogg, A.J., Beattie, R.E., Ogden, A.M., Blanco, M.J., Bleakman, D., 2009. TRP inflammation and sensitization of receptive endings in the organs; channels as emerging targets for pain therapeutics. Expert. Opin. Ther. Targets. 13, – and 4) augmented release of excitatory neurotransmitters in the 69 81. Brumovsky, P., Shi, T.S., Landry, M., Villar, M.J., Hokfelt, T., 2007. Neuropeptide tyrosine dorsal horn onto second order neurons that receive convergent input and pain. Trends Pharmacol. Sci. 28, 93–102. from more than one organ and also from somatic structures. These Campbell, J.N., Meyer, R.A., 2006. Mechanisms of neuropathic pain. Neuron 52, 77–92. central neurons would complete the process of ‘amplification’ by Cason, A.M., Samuelsen, C.L., Berkley, K.J., 2003. Estrous changes in vaginal nociception in a rat model of endometriosis. Horm. Behav. 44, 123–131. transmitting information to higher levels of the nervous system. The Chaban, V., Christensen, A., Wakamatsu, M., McDonald, M., Rapkin, A., McDonald, J., amplification process could also be aided by alterations in spinal or Micevych, P., 2007. The same dorsal root ganglion neurons innervate uterus and supraspinal glial-neuronal relations. Peripheral inflammation acti- colon in the rat. Neuroreport 18, 209–212. fl Chen, Y.,Song, B., Jin, X.Y., Xiong, E.Q., Zhang, J.H., 2005. Possible mechanism of referred pain vates microglia in the spinal cord that release proin ammatory in the perineum and pelvis associated with the prostate in rats. J. Urol.174, 2405–2408. cytokines (e.g., Milligan and Watkins, 2009) and can influence the Christianson, J.A., Liang, R., Ustinova, E.E., Davis, B.M., Fraser, M.O., Pezzone, M.A., 2007. excitability of central neurons. In such a scenario, a broad activation of Convergence of bladder and colon sensory innervation occurs at the primary afferent level. Pain 128, 235–243. glial cells in the dorsal horn could lead to the excitation of neurons Cohen, D.P., Ikeda, S.R., Lewis, D.L., 1996. Neuropeptide Y and calcitonin gene-related receiving diverse afferent input. It is appreciated that these ‘details’ peptide modulate voltage-gated Ca2+ channels in mature female rat paracervical exclude consideration of changes in descending modulatory influ- ganglion neurons. J. Soc. Gynecol. Investig. 3, 342–349. Dawson, N.J., Schmid, H., Pierau, F.K., 1992. Pre-spinal convergence between thoracic ences in what is ultimately experienced, and instead focused and visceral nerves of the rat. Neurosci. Lett. 138, 149–152. principally on peripheral events and the first central synapse. de Groat, W.C., 1987. Neuropeptides in pelvic afferent pathways. Experientia 43, 801–813. fi Acknowledgments de Groat, W.C., Kawatani, M., Houston, M.B., Rutigliano, M., and Erdman, S. Identi cation of neuropeptides in afferent pathways to the pelvic viscera of the cat. 81–90. 1987. New York, A.R. Liss. Organization of the autonomic nervous system: central and The authors are supported by the NIH awards NS 19912 and NS peripheral mechanisms. Neurology and Neurobiology. Ciriello, J., Calaresu, F., 35790 (GFG) and an IASP Early Career Research Grant (PRB). We thank Renaud, L, and Polosa, C. Ref Type: Generic. de Groat, W.C., Fraser, M.O., Yoshiyama, M., Smerin, S., Tai, C., Chancellor, M.B., Yoshimura, Drs. Julie C. Christianson and Brian M. Davis, University of Pittsburgh, N., Roppolo, J.R., 2001. Neural control of the urethra. Scand. J Urol. Nephrol. Suppl for the kind donation of photomicrographs in Figs. 2 and 3, and Mr. 35–43. Michael Burcham for the assistance in preparation of the figures. Delepine, L., Aubineau, P., 1997. Plasma protein extravasation induced in the rat dura mater by stimulation of the parasympathetic sphenopalatine ganglion. Exp. Neurol. 147, 389–400. References Denny-Brown, D., Robertson, E.G., 1933. On the physiology of micturition. Brain 56, 149–190. Alagiri, M., Chottiner, S., Ratner, V., Slade, D., Hanno, P.M., 1997. Interstitial cystitis: Dimcevski, G., Schipper, K.P., Tage-Jensen, U., Funch-Jensen, P., Kraup, A.L., Toft, E., unexplained associations with other chronic disease and pain syndromes. Urology Thorsgaard, N., Arendt-Nielsen, L., Drewes, A.M., 2006. Hypoalgesia to experimental 49, 52–57. visceral and somatic stimulation in painful chronic pancreatitis. Eur. J. Gastro- Amir, R., Devor, M., 1992. Axonal cross-excitation in nerve-end neuromas: comparison enterol. Hepatol. 18 (7), 755–764. of A- and C-fibers. J. Neurophysiol. 68, 1160–1166. Dmitrieva, N., Berkley, K.J., 2002. Contrasting effects of WIN 55212-2 on motility of the Amir, R., Devor, M., 1996. Chemically mediated cross-excitation in rat dorsal root rat bladder and uterus. J. Neurosci. 22, 7147–7153. ganglia. J. Neurosci. 16, 4733–4741. Dmitrieva, N., Johnson, O.L., Berkley, K.J., 2001. Bladder inflammation and hypogastric Amir, R., Devor, M., 2000. Functional cross-excitation between afferent A- and C- neurectomy influence uterine motility in the rat. Neurosci. Lett. 313, 49–52. neurons in dorsal root ganglia. Neuroscience 95, 189–195. Dwivedi, S., Chaturvedi, A., 2000. Angina—an Indian disease. J. Assoc. Phys. India 48 940, Amir, R., Devor, M., 2003. Electrical excitability of the soma of sensory neurons is required for 943. spike invasion of the soma, but not for through-conduction. Biophys. J. 84, 2181–2191. Dwivedi, G. and Dwivedi, S. Sushruta — the Clinician — Teacher par Excellence. Indian J Amir, R., Liu, C.N., Kocsis, J.D., Devor, M., 2002. Oscillatory mechanism in primary Chest Dis Allied Sci 49, 243–244. 2007. Ref Type: Generic. sensory neurones. Brain 125, 421–435. Eberhardt, M., Hoffmann, T., Sauer, S.K., Messlinger, K., Reeh, P.W., Fischer, M.J., 2008. Amir, R., Kocsis, J.D., Devor, M., 2005. Multiple interacting sites of ectopic spike Calcitonin gene-related peptide release from intact isolated dorsal root and electrogenesis in primary sensory neurons. J. Neurosci. 25, 2576–2585. trigeminal ganglia. Neuropeptides 42, 311–317. Ammons, W.S., Foreman, R.D., 1984. Cardiovascular and T2–T4 dorsal horn cell Euchner-Wamser, I., Sengupta, J.N., Gebhart, G.F., Meller, S.T., 1993. Characterization responses to gallbladder distention in the cat. Brain Res. 321, 267–277. of responses of T2–T4 spinal cord neurons to esophageal distension in the rat. Ammons, W.S., Blair, R.W., Foreman, R.D., 1984. Responses of T1–T5 J. Neurophysiol. 69, 868–883. spinothalamic neurons to gallbladder distension. Am. J. Physiol. 247, R995–1002. Euchner-Wamser, I., Meller, S.T., Gebhart, G.F., 1994. A model of cardiac nociception in Apkarian, A., Bruggemann, J., Shi, T., and Airapetian, L. A thalamic model for true and chronically instrumented rats: behavioral and electrophysiological effects of referred visceral pain. 217–259. 1995. Seattle, IASP Press. Visceral Pain, Progress in pericardial administration of algogenic substances. Pain 58, 117–128. Pain Research and Management. Gebhart, G. F. Ref Type: Generic. Fischer, M.J., Reeh, P.W., 2007. Sensitization to heat through G-protein-coupled receptor Asztely, A., Havel, G., Ekstrom, J., 1998. Vascular protein leakage in the rat parotid gland pathways in the isolated sciatic mouse nerve. Eur. J. Neurosci. 25, 3570–3575. elicited by reflex stimulation, parasympathetic nerve stimulation and administra- Floyd, K., McMahon, S.B., Morrison, J.F., 1978. Inhibition of the micturition reflex by tion of neuropeptides. Regul. Pept. 77, 113–120. stimulation of pelvic nerve afferents from the colon [proceedings]. J. Physiol. 284, Bahns, E., Ernsberger, U., Janig, W., Nelke, A., 1986. Functional characteristics of lumbar 39P–40P. visceral afferent fibres from the urinary bladder and the urethra in the cat. Pflugers Floyd, K., McMahon, S.B., Morrison, J.F., 1979. Inhibitory interactions between the Arch. 407, 510–518. colonic and vesical branches of the pelvic nerve in the cat [proceedings]. J. Physiol. Baranowski, A.P., Abrams, P., Berger, R.E., Buffington, C.A., de, C.W.A., Hanno, P., Loeser, J.D., 290, 50P–51P. Nickel, J.C., Wesselmann, U., 2008. Urogenital pain-time to accept a new approach to Floyd, K., McMahon, S.B., Morrison, J.F., 1982. Inhibitory interactions between colonic phenotyping and, as a consequence, management. Eur. Urol. 53, 33–36. and vesical afferents in the micturition reflex of the cat. J. Physiol. 322, 45–52. Berkley, K.J., 2005. A life of pelvic pain. Physiol. Behav. 86, 272–280. Foreman, R.D., 2007. Neurological mechanisms of chest pain and cardiac disease. Cleve. Berkley, K.J., Robbins, A., Sato, Y., 1993. Functional differences between afferent fibers in Clin. J. Med. 74 (Suppl 1), S30–S33. the hypogastric and pelvic nerves innervating female reproductive organs in the Frokjaer, J.B., Andersen, S.D., Gale, J., -Nielsen, L., Gregersen, H., Drewes, A.M., 2005. An rat. J. Neurophysiol. 69, 533–544. experimental study of viscero-visceral hyperalgesia using an ultrasound-based Berkley, K.J., Rapkin, A.J., Papka, R.E., 2005. The pains of endometriosis. Science 308, multimodal sensory testing approach. Pain 119, 191–200. 1587–1589. Furness, J.B., 2006. The organisation of the autonomic nervous system: peripheral Berkley, K.J., McAllister, S.L., Accius, B.E., Winnard, K.P., 2007. Endometriosis-induced connections. Auton. Neurosci. 130, 1–5. vaginal hyperalgesia in the rat: effect of estropause, ovariectomy, and estradiol Garrison, D.W., Chandler, M.J., Foreman, R.D., 1992. Viscerosomatic convergence onto replacement. Pain 132 (Suppl 1), S150–S159. feline spinal neurons from esophagus, heart and somatic fields: effects of Bernardini, N., Neuhuber, W., Reeh, P.W., Sauer, S.K., 2004. Morphological evidence for inflammation. Pain 49, 373–382. functional capsaicin receptor expression and calcitonin gene-related peptide exocy- Gebhart, G.F., Ness, T.J., 1991. Central mechanisms of visceral pain. Can. J. Physiol. tosis in isolated peripheral nerve axons of the mouse. Neuroscience 126, 585–590. Pharmacol. 69, 627–634. 114 P.R. Brumovsky, G.F. Gebhart / Autonomic Neuroscience: Basic and Clinical 153 (2010) 106–115

Giamberardino, M.A., De Laurentis, S., Affaitati, G., Lerza, R., Lapenna, D., Vecchiet, L., Mohammed, H.A., Santer, R.M., 2001. Total neuronal numbers of rat lumbosacral 2001. Modulation of pain and hyperalgesia from the urinary tract by algogenic primary afferent neurons do not change with age. Neurosci. Lett. 304, 149–152. conditions of the reproductive organs in women. Neurosci. Lett. 304, 61–64. Morrison, T.C., Dmitrieva, N., Winnard, K.P., Berkley, K.J., 2006. Opposing viscerovisceral Giamberardino, M.A., Berkley, K.J., Affaitati, G., Lerza, R., Centurione, L., Lapenna, D., effects of surgically induced endometriosis and a control abdominal surgery on the Vecchiet, L., 2002. Influence of endometriosis on pain behaviors and muscle rat bladder. Fertil. Steril. 86, 1067–1073. hyperalgesia induced by a ureteral calculosis in female rats. Pain 95, 247–257. Munakata, J., Naliboff, B., Harraf, F., Kodner, A., Lembo, T., Chang, L., Silverman, D.H., Grundy, D., Salih, A.A., Scratcherd, T., 1981. Modulation of vagal efferent fibre discharge Mayer, E.A., 1997. Repetitive sigmoid stimulation induces rectal hyperalgesia in by mechanoreceptors in the stomach, duodenum and colon of the ferret. J. Physiol. patients with irritable bowel syndrome. Gastroenterology 112, 55–63. 319, 43–52. Naliboff, B., Munakata, J., Fullerton, S., Gracely, R.H., Kodner, A., Harraf, F., Mayer, E.A., Harding, L.M., Beadle, D.J., Bermudez, I., 1999. Voltage-dependent calcium channel 1997. Evidence for two distinct perceptual alterations in irritable bowel syndrome. subtypes controlling somatic substance P release in the peripheral nervous system. Gut 41, 505–512. Prog. Neuropsychopharmacol. Biol. Psychiatry 23, 1103–1112. Ness, T.J., Gebhart, G.F., 1990. Visceral pain: a review of experimental studies. Pain 41, Heatley, M., Rose, K., Weston, C., 2005. The heart and the oesophagus: intimate 167–234. relations. Postgrad. Med. J. 81, 515–518. Neubert, J.K., Maidment, N.T., Matsuka, Y., Adelson, D.W., Kruger, L., Spigelman, I., 2000. Hill, R.G., Oliver, K.R., 2007. Neuropeptide and kinin antagonists. Handb. Exp. Inflammation-induced changes in primary afferent-evoked release of substance P Pharmacol. 181–216. within trigeminal ganglia in vivo. Brain Res. 871, 181–191. Hobson, A.R., Khan, R.W., Sarkar, S., Furlong, P.L., Aziz, Q., 2004. Development of Neuhuber, W.L., Appelt, M., Polak, J.M., Baier-Kustermann, W., Abelli, L., Ferri, G.L., 1993. esophageal hypersensitivity following experimental duodenal acidification. Am. J. Rectospinal neurons: cell bodies, pathways, immunocytochemistry and ultrastruc- Gastroenterol. 99, 813–820. ture. Neuroscience 56, 367–378. Hoffmann, T., Sauer, S.K., Horch, R.E., Reeh, P.W., 2008. Sensory transduction in Noronha, R., Akbarali, H., Malykhina, A., Foreman, R.D., Greenwood-Van, M.B., 2007. peripheral nerve axons elicits ectopic action potentials. J. Neurosci. 28, 6281–6284. Changes in urinary bladder smooth muscle function in response to colonic Hoffmann, T., Sauer, S.K., Horch, R.E., Reeh, P.W., 2009. Projected pain from noxious heat inflammation. Am. J. Physiol. Renal. Physiol. 293, F1461–F1467. stimulation of an exposed peripheral nerve—a case report. Eur. J. Pain 13, 35–37. Peng, H.Y., Chang, H.M., Lee, S.D., Huang, P.C., Chen, G.D., Lai, C.H., Lai, C.Y., Chiu, C.H., Houdeau, E., Barranger, E., Rossano, B., 2002. Do sensory calcitonin gene-related peptide Tung, K.C., Lin, T.B., 2008a. TRPV1 mediates the uterine capsaicin-induced NMDA nerve fibres in the rat pelvic plexus supply autonomic neurons projecting to the NR2B-dependent cross-organ reflex sensitization in anesthetized rats. Am. J. uterus and cervix? Neurosci. Lett. 332, 29–32. Physiol. Renal. Physiol. 295, F1324–F1335. Huang, L.Y., Neher, E., 1996. Ca(2+)-dependent exocytosis in the somata of dorsal root Peng, H.Y., Huang, P.C., Liao, J.M., Tung, K.C., Lee, S.D., Cheng, C.L., Shyu, J.C., Lai, C.Y., ganglion neurons. Neuron 17, 135–145. Chen, G.D., Lin, T.B., 2008b. Estrous cycle variation of TRPV1-mediated cross-organ Hubscher, C.H., Johnson, R.D., 2003. Responses of thalamic neurons to input from the sensitization between uterus and NMDA-dependent pelvic–urethra reflex activity. male genitalia. J. Neurophysiol. 89, 2–11. Am. J. Physiol. Endocrinol. Metab. 295, E559–E568. Hubscher, C.H., Kaddumi, E.G., Johnson, R.D., 2004. Brain stem convergence of pelvic Peng, H.Y., Chen, G.D., Tung, K.C., Lai, C.Y., Hsien, M.C., Chiu, C.H., Lu, H.T., Liao, J.M., Lee, viscerosomatic inputs via spinal and vagal afferents. Neuroreport 15, 1299–1302. S.D., Lin, T.B., 2009. Colon mustard oil instillation induced cross-organ reflex Hummel, T., Sengupta, J.N., Meller, S.T., Gebhart, G.F.,1997. Responses of T2–4 spinal cord sensitization on the pelvic–urethra reflex activity in rats. Pain 142, 75–88. neurons to irritation of the lower airways in the rat. Am. J. Physiol. 273, R1147–R1157. Pezzone, M.A., Liang, R., Fraser, M.O., 2005. A model of neural cross-talk and irritation in Kaleczyc, J., Sienkiewicz, W., Klimczuk, M., Czaja, K., Lakomy, M., 2003. Differences in the the pelvis: implications for the overlap of chronic pelvic pain disorders. chemical coding of nerve fibres supplying major populations of neurons between Gastroenterology 128, 1953–1964. the caudal mesenteric ganglion and anterior pelvic ganglion in the male pig. Folia Pierau, F.K., Taylor, D.C., Abel, W., Friedrich, B.,1982. Dichotomizing peripheral fibres revealed Histochem. Cytobiol. 41, 201–211. by intracellular recording from rat sensory neurones. Neurosci. Lett. 31, 123–128. Keast, J.R., de Groat, W.C., 1992. Segmental distribution and peptide content of primary Pontari, M.A., 2008. Chronic prostatitis/chronic pelvic pain syndrome. Urol. Clin. North afferent neurons innervating the urogenital organs and colon of male rats. J. Comp. Am. 35, 81–89. Neurol. 319, 615–623. Qiao, L.Y., Grider, J.R., 2007. Up-regulation of calcitonin gene-related peptide and Kock, N.G., Pompeius, R., 1963. Inhibition of vesical motor activity induced by anal receptor tyrosine kinase TrkB in rat bladder afferent neurons following TNBS colitis. stimulation. Acta Chir. Scand. 126, 244–250. Exp. Neurol. 204, 667–679. Korkmaz, A., Topal, T., Oter, S., 2007. Pathophysiological aspects of cyclophosphamide Qin, C., Chandler, M.J., Foreman, R.D., 2003. Effects of urinary bladder distension on and ifosfamide induced hemorrhagic cystitis; implication of reactive oxygen and activity of T3–T4 spinal neurons receiving cardiac and somatic noxious inputs in nitrogen species as well as PARP activation. Cell Biol. Toxicol. 23, 303–312. rats. Brain Res. 971, 210–220. Laird, J.M., Souslova, V., Wood, J.N., Cervero, F., 2002. Deficits in visceral pain and Qin, C., Chandler, M.J., Foreman, R.D., 2004. Esophagocardiac convergence onto thoracic referred hyperalgesia in Nav1.8 (SNS/PN3)-null mice. J. Neurosci. 22, 8352–8356. spinal neurons: comparison of cervical and thoracic esophagus. Brain Res. 1008, Lamb, K., Zhong, F., Gebhart, G.F., Bielefeldt, K., 2006. Experimental colitis in mice and 193–197. sensitization of converging visceral and somatic afferent pathways. Am. J. Physiol. Qin, C., Malykhina, A.P., Akbarali, H.I., Foreman, R.D., 2005. Cross-organ sensitization of Gastrointest. Liver Physiol. 290, G451–G457. lumbosacral spinal neurons receiving urinary bladder input in rats with inflamed Langley, J.N., 1916. Sketch of the progress of discovery in the eighteenth century as colon. Gastroenterology 129, 1967–1978. regards the autonomic nervous system. J. Physiol. 50, 225–258. Qin, C., Chen, J.D., Zhang, J., Foreman, R.D., 2007a. Characterization of T9–T10 spinal Lawson, S.N., 2002. Phenotype and function of somatic primary afferent nociceptive neurons with duodenal input and modulation by gastric electrical stimulation in neurones with C-, Adelta- or Aalpha/beta-fibres. Exp. Physiol. 87, 239–244. rats. Brain Res. 1152, 75–86. Leffler, A., Fischer, M.J., Rehner, D., Kienel, S., Kistner, K., Sauer, S.K., Gavva, N.R., Reeh, P.W., Qin, C., Chen, J.D., Zhang, J., Foreman, R.D., 2007b. Duodenal afferent input converges Nau, C., 2008. The vanilloid receptor TRPV1 is activated and sensitized by local onto T9–T10 spinal neurons responding to gastric distension in rats. Brain Res. 1186, anesthetics in rodent sensory neurons. J. Clin. Invest. 118, 763–776. 180–187. Lembo, T., Munakata, J., Naliboff, B., Fullerton, S., Mayer, E.A., 1997. Sigmoid afferent Qin, C., Farber, J.P., Foreman, R.D., 2007c. Gastrocardiac afferent convergence in upper mechanisms in patients with irritable bowel syndrome. Dig. Dis. Sci. 42, 1112–1120. thoracic spinal neurons: a central mechanism of postprandial angina pectoris. J. Li, J., Micevych, P., McDonald, J., Rapkin, A., Chaban, V., 2008. Inflammation in the uterus Pain 8, 522–529. induces phosphorylated extracellular signal-regulated kinase and substance P Qin, C., Foreman, R.D., Farber, J.P., 2007d. Characterization of thoracic spinal neurons immunoreactivity in dorsal root ganglia neurons innervating both uterus and colon with noxious convergent inputs from heart and lower airways in rats. Brain Res. in rats. J. Neurosci. Res. 86, 2746–2752. 1141, 84–91. Lin, T.B., 2003. Dynamic pelvic-pudendal reflex plasticity mediated by glutamate in Qin, C., Foreman, R.D., Farber, J.P., 2007e. Inhalation of a pulmonary irritant modulates anesthetized rats. Neuropharmacology 44, 163–170. activity of lumbosacral spinal neurons receiving colonic input in rats. Am. J. Physiol. Lin, T.B., 2004. Tetanization-induced pelvic-to-pudendal reflex plasticity in anesthe- Regul. Integr. Comp. Physiol. 293, R2052–R2058. tized rats. Am. J. Physiol. Renal. Physiol. 287, F245–F251. Ross, J., 1888. On the segmental distribution of sensory disorder. Brain 10, 333–361 Ref Malykhina, A.P., 2007. Neural mechanisms of pelvic organ cross-sensitization. Type: Generic. Neuroscience 149, 660–672. Rouzade-Dominguez, M.L., Miselis, R., Valentino, R.J., 2003a. Central representation of Malykhina, A.P., Qin, C., Foreman, R.D., Akbarali, H.I., 2004. Colonic inflammation bladder and colon revealed by dual transsynaptic tracing in the rat: substrates for increases Na+ currents in bladder sensory neurons. Neuroreport 15, 2601–2605. pelvic visceral coordination. Eur. J. Neurosci. 18, 3311–3324. Malykhina, A.P., Qin, C., Greenwood-Van, M.B., Foreman, R.D., Lupu, F., Akbarali, H.I., Rouzade-Dominguez, M.L., Pernar, L., Beck, S., Valentino, R.J., 2003b. Convergent 2006. Hyperexcitability of convergent colon and bladder dorsal root ganglion responses of Barrington's nucleus neurons to pelvic visceral stimuli in the rat: a neurons after colonic inflammation: mechanism for pelvic organ cross-talk. juxtacellular labelling study. Eur. J. Neurosci. 18, 3325–3334. Neurogastroenterol. Motil. 18, 936–948. Ruch, T.C. Pathophysiology of pain. 345–363. 1965. Philadelphia, Saunders. Physiology Mantyh, P.W., 2002. Neurobiology of substance P and the NK1 receptor. J Clin. Psychiatry and biophysics. Ruch, T. C. and Patton, H. D. Ref Type: Generic. 63 (Suppl 11), 6–10. Rudick, C.N., Chen, M.C., Mongiu, A.K., Klumpp, D.J., 2007. Organ cross talk modulates Mantyh, P.W., Allen, C.J., Rogers, S., DeMaster, E., Ghilardi, J.R., Mosconi, T., Kruger, L., pelvic pain. Am. J. Physiol. Regul. Integr. Comp. Physiol. 293, R1191–R1198. Mannon, P.J., Taylor, I.L., Vigna, S.R., 1994. Some sensory neurons express Saini, R., Gonzalez, R.R., Te, A.E., 2008. Chronic pelvic pain syndrome and the overactive neuropeptide Y receptors: potential paracrine inhibition of primary afferent bladder: the inflammatory link. Curr. Urol. Rep. 9, 314–319. nociceptors following peripheral nerve injury. J. Neurosci. 14, 3958–3968. Sarkar, S., Aziz, Q., Woolf, C.J., Hobson, A.R., Thompson, D.G., 2000. Contribution of central Meyer, R.A., Ringkamp, M., 2008. A role for uninjured afferents in neuropathic pain. sensitisation to the development of non-cardiac chest pain. Lancet 356, 1154–1159. Sheng Li Xue. Bao. 60, 605–609. Sarkar, S., Hobson, A.R., Furlong, P.L., Woolf, C.J., Thompson, D.G., Aziz, Q., 2001. Central Milligan, E.D., Watkins, L.R., 2009. Pathological and protective roles of glia in chronic neural mechanisms mediating human visceral hypersensitivity. Am. J. Physiol. pain. Nat. Rev. Neurosci. 10, 23–36. Gastrointest. Liver Physiol. 281, G1196–G1202. P.R. Brumovsky, G.F. Gebhart / Autonomic Neuroscience: Basic and Clinical 153 (2010) 106–115 115

Sarkar, S., Woolf, C.J., Hobson, A.R., Thompson, D.G., Aziz, Q., 2006. Perceptual wind-up rat trigeminal ganglion: evidence for intraganglionic neurotransmission. Pain 91, in the human oesophagus is enhanced by central sensitisation. Gut 55, 920–925. 219–226. Sato, Y., Hotta, H., Nakayama, H., Suzuki, H., 1996. Sympathetic and parasympathetic Ustinova, E.E., Fraser, M.O., Pezzone, M.A., 2006. Colonic irritation in the rat sensitizes regulation of the uterine blood flow and contraction in the rat. J. Auton. Nerv. Syst. urinary bladder afferents to mechanical and chemical stimuli: an afferent origin of 59, 151–158. pelvic organ cross-sensitization. Am. J. Physiol. Renal Physiol. 290, F1478–F1487. Sauer, S.K., Reeh, P.W., Bove, G.M., 2001. Noxious heat-induced CGRP release from rat Ustinova, E.E., Gutkin, D.W., Pezzone, M.A., 2007. Sensitization of pelvic nerve afferents sciatic nerve axons in vitro. Eur. J. Neurosci. 14, 1203–1208. and mast cell infiltration in the urinary bladder following chronic colonic irritation Sinclair, D.C., Weddell, G., Feindel, W.H., 1948. Referred pain and associated phenomena. is mediated by neuropeptides. Am. J. Physiol. Renal Physiol. 292, F123–F130. Brain Res. 71, 184–211 Ref Type: Generic. van de Merwe, J.P., Nordling, J., Bouchelouche, P., Bouchelouche, K., Cervigni, M., Daha, L. Stanford, E.J., Dell, J.R., Parsons, C.L., 2007. The emerging presence of interstitial cystitis K., Elneil, S., Fall, M., Hohlbrugger, G., Irwin, P., Mortensen, S., van, O.A., Osborne, J.L., in gynecologic patients with chronic pelvic pain. Urology 69, 53–59. Peeker, R., Richter, B., Riedl, C., Sairanen, J., Tinzl, M., Wyndaele, J.J., 2008. Diagnostic Sturge, W.A., 1888. The phenomena of angina pectoris and their bearing upon the theory criteria, classification, and nomenclature for painful bladder syndrome/interstitial of counter-irritation. Brain 5, 492–510 Ref Type: Generic. cystitis: an ESSIC proposal. Eur. Urol. 53, 60–67. Suckow, S.K., Caudle, R.M., 2008. Identification and immunohistochemical character- Vilensky, J.A., Bell, D.R., Gilman, S., 2004. “On the physiology of micturition” by Denny– ization of colospinal afferent neurons in the rat. Neuroscience 153, 803–813. Brown and Robertson: a classic paper revisited. Urology 64, 182–186. Szurszewski, J.H., Ermilov, L.G., Miller, S.M., 2002. Prevertebral ganglia and intestino- Warren, J.W., Langenberg, P., Greenberg, P., Diggs, C., Jacobs, S., Wesselmann, U., 2008. fugal afferent neurones. Gut 51 (Suppl 1), i6–10. Sites of pain from interstitial cystitis/painful bladder syndrome. J. Urol. 180, Taylor, D.C., Pierau, F.K., 1982. Double fluorescence labelling supports electrophysiolo- 1373–1377. gical evidence for dichotomizing peripheral sensory nerve fibres in rats. Neurosci. Whorwell, P.J., McCallum, M., Creed, F.H., Roberts, C.T., 1986. Non-colonic features of Lett. 33, 1–6. irritable bowel syndrome. Gut 27, 37–40. Taylor, D.C., Pierau, F.K., Schmid, H.,1983. The use of fluorescent tracers in the peripheral Willis Jr., W.D., 1999. Dorsal root potentials and dorsal root reflexes: a double-edged sensory nervous system. J. Neurosci. Methods 8, 211–224. sword. Exp. Brain Res. 124, 395–421. Theoharides, T.C., Whitmore, K., Stanford, E., Moldwin, R., O'Leary, M.P., 2008. Winnard, K.P., Dmitrieva, N., Berkley, K.J., 2006. Cross-organ interactions between Interstitial cystitis: bladder pain and beyond. Expert. Opin. Pharmacother. 9, reproductive, gastrointestinal, and urinary tracts: modulation by estrous stage and 2979–2994. involvement of the hypogastric nerve. Am. J. Physiol. Regul. Integr. Comp. Physiol. Ulrich-Lai, Y.M., Flores, C.M., Harding-Rose, C.A., Goodis, H.E., Hargreaves, K.M., 2001. 291, R1592–R1601. Capsaicin-evoked release of immunoreactive calcitonin gene-related peptide from Obesity Surgery (2020) 30:3570–3582 https://doi.org/10.1007/s11695-020-04762-3

REVIEW

Personalized Nutrition for Management of Micronutrient Deficiency—Literature Review in Non-bariatric Populations and Possible Utility in Bariatric Cohort

Shannon Galyean1 & Dhanashree Sawant1 & Andrew C. Shin1

Received: 31 January 2020 /Revised: 01 June 2020 /Accepted: 04 June 2020 / Published online: 20 June 2020 # The Author(s) 2020

Abstract Background Bariatric surgery can effectively treat morbid obesity; however, micronutrient deficiencies are common despite recommendations for high-dose supplements. Genetic predisposition to deficiencies underscores necessary identification of high- risk candidates. Personalized nutrition (PN) can be a tool to manage these deficiencies. Methods Medline, PubMed, and Google Scholar were searched. Articles involving genetic testing, micronutrient , and bariatric surgery were included. Results Studies show associations between genetic variants and micronutrient metabolism. Research demonstrates genetic testing to be a predictor for outcomes among obesity and bariatric surgery populations. There is limited research in bariatric surgery and micronutrient genetic variants. Conclusion Genotype-based PN is becoming feasible to provide an effective treatment of micronutrient deficiencies associated with bariatric surgery. The role of genomic technology in micronutrient recommendations needs further investigation.

Keywords Obesity . Gene . Polymorphism . Gene expression . Nutrients . Supplementation . Deficiency

Background calcium and D deficiency 25–50%, vitamin B12 defi- ciency about 33%, and deficiency as high as 45% [3]. For The prevalence of obesity is consistent with more than one third malabsorptive procedures, patients are recommended to take at of adults having the disease of obesity [1]. Obesity is the focus least double the recommended daily dose of a multivitamin for many public health efforts in the USA with one treatment plus mineral supplement and additional 1200–2400 mg calci- option being bariatric surgery [1, 2]. Achieving weight loss is a um, 3000 IU to reach levels > 30 ng/mL, and vitamin benefit from bariatric surgery; however, micronutrient deficien- B12 as needed for normal levels [5]. In a cohort of adults who cies can occur [2]. Micronutrient deficiencies are associated underwent bariatric surgery, 73% of the patients had at least with serious consequences due to the negative effects on met- one nutritional deficiency 5 years later even though they report- abolic and cellular signaling pathways. Possible causes of mi- ed taking a dietary supplement [6]. However, there are some cronutrient deficiencies after bariatric surgery are decreased patients, up to 47%, that may be non-responders to supplements food intake, food intolerance, reduced gastric secretions, bypass even with compliance rates of about 86–93% [7]. Indeed, indi- of intestinal surface area for absorption, as well as failure to viduals respond differently to dietary interventions. Genetic comply with recommended vitamin regimens [3, 4]. Multiple variation among individuals could be the root cause for varying case series have reported postoperative, malabsorptive proce- responses to the same regimen and explains why some individ- dures to increase prevalence of iron deficiency to 20–49%, uals respond better to a certain regimen than others in the same environmental conditions [8].

* Shannon Galyean [email protected] Introduction

1 Department of Nutritional Sciences, Texas Tech University, 1301 Genetic testing can be a critical tool for health and medical Akron Ave, Lubbock, TX 79409, USA diagnosis, treatment, and prevention. Predictive testing may OBES SURG (2020) 30:3570–3582 3571 be among the most useful tests regarding medical nutrition those at risk for deficiencies. This may recognize the need therapy (MNT). Genetics along with environment and behav- for increasing consumption of essential nutrients to intervene ior are the key to providing the best assessment, intervention, prior to bariatric surgery and develop strategies to prevent and tailored changes for an individual [9]. MNT should follow micronutrient deficiency postoperatively. an appropriate paradigm that encompasses prediction (early diagnosis), prevention (intervention on healthy persons), and a tailored therapy for patients [9]. Identifying ways for early Methods intervention may help develop strategies for preventing poor nutritional status and maximizing surgery-induced metabolic Due to the limited amount of literature published on micronu- benefits later. trient deficiencies, micronutrient genetic variants, and bariat- Sequencing of the and identifying gene- ric surgery, the authors conducted a narrative review. A com- nutrient interactions are the underlying concept of PN [10]. prehensive search of the literature from 1975 to 2020 was Nutrigenomics is the study of the effect of specific nutri- conducted to identify articles examining the association be- ents on gene expression [10], while nutrigenetics refers to tween genetic variants of micronutrient metabolic pathways the study of genetic variations of an individual that can and serum levels of micronutrients. Searches were conducted provide some prediction to help prevent as well as contrib- in databases that contain research related to health and meta- ute to personalized dietary management [11]. Both bolic outcomes, including PubMed, Medline, and Google nutrigenomics and nutrigenetics may be a strategy to im- Scholar. The search terms that were used included genetic prove understanding of the gene-diet interaction and deliv- variants, micronutrient metabolism, treatment of genetic de- er individualized MNT to prevent chronic nutrition-related fects of micronutrients, precision nutrition, nutrigenomics, diseases [(10], [11)]. The usefulness and validity of this and bariatric surgery. Additionally, review articles produced type of PN are in their infancy, although some studies have through the database searches were examined for further arti- shown that individuals find dietary recommendations cles that fit within the inclusion criteria and thus were included based on genetics more beneficial than general dietary ad- in the results. vice [12]. A survey conducted by the publisher Nature showed that 27% of respondents who had their genomes analyzed changed their diet, lifestyle, or medication based on their genetic information [13]. However, another study Inclusion/Exclusion Criteria reported that genetic testing led to no short-term changes in specific dietary or exercise behaviors [14]. Thus, increased Criteria for inclusion in the review were [1] peer-reviewed understanding and awareness of these tests is required to articles, [2] articles that included empirical data, [3]articles effectively use them among public and healthcare pro- published or available in English, [4] articles that included viders [12]. people with low micronutrient levels, [5] articles that included Since many micronutrients control energy metabolism, people that had micronutrient genetic variants, [6] their deficiencies can result in an array of symptoms, ranging supplementation/treatment regimens for people with genetic from anemia to neurological dysfunction [15, 16]. variants and low micronutrient levels, and [7] articles that Additionally, subclinical micronutrient deficiency can lead included genetic testing among obesity and bariatric surgery to increased risks for coronary artery disease, infections, populations and outcomes that were examined. Only two ar- age-related macular degeneration, and oxidative damage [17, ticles involving micronutrient genetic variants among bariatric 18]. Therefore, measuring nutritional status in the context of surgery patients were found. Due to this limitation, studies pathophysiology is critical, but this is a major challenge be- involving non-surgical patients and micronutrient genetic var- cause it is influenced by a number of factors including dietary iants were included. Other studies involving genetic testing consumption, physical/social stressors, and infections [19]. and bariatric surgery were included to demonstrate its poten- Furthermore, the impact of nutrition could vary among indi- tial as a tool for this patient population. There were also lim- viduals and specific population subgroups based on their mo- itations on studies involving treatment and supplementation lecular and genetic make-up [19]. Studying this complex according to genetic variants. Ideally, inclusion criteria would nutrient-gene relationship to understand the metabolic net- comprise of studies with a high number of participants, a works in context of health and disease should be a focus. It control group, and that used similar measures and procedures can provide information on potential biomarkers of nutritional across studies for comparison; however, using the search status, disease progression, and response to interventions. This methods and criteria described above, 80 articles met the in- literature review aims to summarize data from studies of clusion criteria. All the authors confirmed that the articles met involved in micronutrient metabolism. Identifying these the inclusion criteria and were appropriate for the review. The nutrient-gene pathways and their variants can help predict articles that met the inclusion criteria focused on micronutrient 3572 OBES SURG (2020) 30:3570–3582 genetic variants and genetic testing in diverse populations observational studies assessing vitamin D status in patients shifting to describe the findings. undergoing bariatric surgery, the mean (25(OH)D) level was less than 30 ng/ml (which is the minimum recommended level for optimal long-term health), before and after bariatric sur- Results gery, despite various vitamin D supplementation regimens [42]. Another review of 30 studies showed vitamin D defi- Of the 80 articles included, 2 were published between 1975 ciency prevalence to range from 13 to 90% preoperatively and 1990, 5 were published between 1991 and 2000, 25 were which was maintained after surgery [43]. published between 2001 and 2010, and 48 were published The heritability of vitamin D status is estimated to be 30% between 2011 and 2020. Twenty-two of these studies took and common variants group-specific component (GC)(also place in the USA, and the rest were conducted in other coun- known as vitamin D–binding protein); 7-dehydrocholesterol tries. The articles were divided into four categories which are reductase (DHCR7)andCYP2R1 (involved in 25-hydroxylase used to organize the results: [1] micronutrient deficiencies production) are associated with fasting plasma 25(OH) D con- prevalent among bariatric surgery patients (19 articles), [2] centrations [25, 44, 45]. Nissen and colleagues have shown micronutrient genetic variants prevalence among different that 7 prominent variants in CYP2R1 and GC genes were populations (29 articles), [3] clinical trials involving supple- significantly associated with low serum 25(OH) D concentra- mentation for micronutrient genetic variants (26 articles), and tions [26]. People who have these common genetic variations [4] genetic testing studies in persons with obesity and bariatric could be treated on a more individualized basis to correct surgery populations (6 articles). deficiencies that occur. One randomized controlled trial looked at older Australians Genetic Variants and Their Effect on Vitamin and randomly assigned to monthly doses of 30,000 IU or

Mineral Pathways and Response to Supplementation 60,000 IU vitamin D3 for 12 months and found that genetic variability is associated with response to supplementation, Genetic variations in specific genes among vitamin and min- perhaps suggesting that some people might need a higher dose eral metabolic pathways are associated with altered nutrient to reach optimal 25(OH) D levels [46]. Another study inves- homeostasis and adverse health outcomes [19]. SNPs are the tigated 41 candidate single nucleotide polymorphisms (SNPs) most common type of genetic variations among people [20]. in vitamin D and calcium pathway genes among healthy non- In the human genome, SNPs may occur at every 1000 nucle- Hispanic white participants and stated that the increase in otides, which means that a person may have 4–5millionSNPs [25(OH)D] attributable to vitamin D3 supplementation may [20]. SNPs are known to impact micronutrient status or chron- vary according to common genetic differences in CYP2R1, ic diseases related to micronutrient metabolism [19, 21–23]. 24-hydroxylase (CYP24A1), and vitamin D receptor (VDR) The ability to identify a person having genetic variants in- genes [27]. There is evidence from three randomized con- volved in vitamin and mineral metabolism may reduce the trolled trials that indicate a strong association between genetic chance of developing micronutrient deficiencies that can lead polymorphisms and levels of serum 25(OH) D in response to to various diseases [19]. GWAS have shown that several ge- 40,000 IU vitamin D/week given for 6 months [47]. However, netic variants associated with vitamin metabolism can affect there is a wide variation in the response of blood 25(OH) D to circulating vitamin levels, which could lead to abnormal vita- vitamin D supplementation that is associated with genetic var- min function [24]. Most GWAS have been conducted among iants in vitamin D metabolism [25]. healthy, Caucasian populations, which is a limitation in this research [24]. Table 1 demonstrates recent studies that associ- Vitamin B12 ate genetic variants and micronutrient metabolism. Vitamin B12 is a coenzyme, , and essential component Vitamin D in vitamin B complex. It is essential for cardiac health [48]and cognitive function [49, 50]. Deficiency of vitamin B12 can lead Vitamin D is essential for many functions of the body. to deleterious consequences including macrocytic anemia, neu- Deficiency of vitamin D is associated with many cancers, ropsychiatric symptoms [51], cardiovascular diseases [52, 53], autoimmune disorders, and cardiovascular disease as well as and onset of different forms of cancer [54, 55]. The most com- significantly affects musculoskeletal function [39–41]. mon cause of vitamin B12 deficiency is loss of intrinsic factor Obesity has been identified as a risk factor of vitamin D defi- (IF) as absorption depends on it [56]. People who have bariatric ciency, and those seeking bariatric surgery for obesity treat- surgery, short gut syndrome, long-term vegetarian, or vegan ment have an additional risk for low vitamin D levels post-op diets can potentially develop vitamin B12 deficiency [56]. [39]. One study showed that 57.4% of patients seeking bariat- While vitamin B12 level can be normal at baseline, it is often ric surgery were vitamin D-deficient preoperatively [39]. In 51 found to be lower in individuals after bariatric surgery [57, 58]. OBES SURG (2020) 30:3570–3582 3573

Table 1 Relevance of genetic variants associated with micronutrient metabolism

Micronutrients Genes identified Relevance in micronutrient status Reference with micronutrients

Vitamin D 1. GC 1. GC gene encodes Vitamin D Binding Protein (DBP) which is a glycosylated [25–28] 2. CYP2R1 alpha-globulin that transports vitamin D metabolites from gut and skin 3. DHCR7 to target end-organs. 4. CYP24A 2. CYP2R1 gene encodes 25-hydroxylase, which converts Vitamin D to 25(OH)D. 5. VDR 3. DHCR7 gene provides instructions for making 7-dehydrocholesterol reductase, an enzyme involved in the final step of cholesterol production. 4. CYP24A gene provides instructions for making 24-hydroxylase, an enzyme that controls the amount of active vitamin D in the body. 5. VDR gene provides instructions for making vitamin D receptor (VDR) protein, which allows the body to respond appropriately to vitamin D >A variation in these genes may impact body vitamin D levels. B12 1. FUT2 1. FUT2 gene encodes for fucosyltransferase 2 gene and is involved in Vit B12 [28–31] 2. CUBN absorption and transport. 3. TCN1 2. CUBN gene provides instructions for making cubilin protein which is involved 4. MTRR in the uptake of vitamin B12. 5. TCN2 3. TCN1 gene encodes B12-binding protein family which facilitates the transport 6. MTR of cobalamin into cells. 7. MMAA 4. MTRR gene is responsible for maintaining adequate levels of activated vitamin 8. MMACHC B12, which maintains methionine synthase enzyme in its active state. 5. TCN2 provides instructions for making transcobalamin. 6. MTR gene provides instructions for making methionine synthase enzyme which needs B12 and is involved in the formation of the amino acid methionine 7. The protein encoded by MMAA gene is involved in the translocation of cobalamin into the . 8. It is postulated that the protein encoded by MMACHC gene may have a role in the binding and intracellular trafficking of cobalamin. >SNP related to these genes can lead to insufficient B12 levels in the body. Folic acid 1.MTHFR 1. MTHFR gene produces Methylenetetrahydrofolate reductase (MTHFR) [28, 32–34] which is a vital enzyme for the folate pathway. >SNP related to this gene may be an important marker to identify people at risk for lower plasma folate concentrations, changes in folate form distribution, and elevated plasma homocysteine concentrations. 1.SLC19A2 SLC19A2, SLC19A3 and SLC35F3 genes code for thiamine transporter [28, 35, 36] 2. SLC19A3 protein which allow thiamine to move into the cells. 3. SLC35F3 >Mutations in these gene can cause thiamine deficiency leading to thiamine responsive megaloblastic anemia. Iron 1.TMPRSS6 1. TMPRSS6 gene codes for the protein matriptase-2 which helps in regulation [28, 37, 38] 2.TFR2 of iron balance. 3.TF 2. TFR2 gene codes for TFR2 protein which facilitates entry of iron into the cells. 4. HFE 3. TF gene codes for protein transferrin which is a transport protein for iron in the body. 4. HFE gene provides instruction for production of HFE protein which determines iron absorption from diet and iron release from body stores. >A variation in these genes together has an impact on the risk of insufficient iron levels in the body.

Nutritional parameters were compared preoperatively and at and 27% in another study among Icelandic sibling pairs [65]. similar periods postoperatively among patients undergoing Variants of the transcobalamin 1 (TCN1) gene (vitamin B12 malabsorptive procedures [59]. Vitamin B12 abnormalities pri- binding protein, transcobalamin I (TCI)) have been associated or to surgery ranged from 3.2–8.3% to 24–25% at 1 year post- with circulating B12 concentrations [29, 66]. Genetic variants op [59]. In a study of gastric bypass surgery subjects, vitamin of fucosyltransferase 2 (FUT2 gene) that codes for an enzyme B12 deficiency was observed in 33.3% at 2 years and in 27.2% in the vitamin B12 pathway are associated with B12 levels at 3 years postoperatively [60]. [29]. Transcobalamin 2 (TCN2) gene is responsible for mak- Genetic variants may impact the involved in vita- ing a B12-binding protein called transcobolamin II (TC) that min B12 absorption, cellular uptake, and intracellular metab- carries B12 from the intestine to blood and liver. Although TC olism [61–63]. Genetic influence for B12 levels is estimated to represents approximately 10–20% of circulating B12, the be 59% in a study using monozygotic and dizygotic twins [64] most common variant of this gene among Caucasian 3574 OBES SURG (2020) 30:3570–3582 populations has been associated with B12 levels [29]. In a surgery despite supplementation [82, 83]. The prevalence of study among Irish men, having this SNP and homozygous abnormalities 1 year after gastric bypass were higher com- CC genotype had lower vitamin B12 levels than those with pared to preoperative levels in 232 patients with elevated ho- GG genotype [67]. This demonstrates that different genotypes mocysteine as high as 29% and low RBC folate in 12% of 149 of transcobalamin impact the distribution of vitamin B12 and postoperative subjects [82]. Another study found similar re- shows an association between this genetic variant and B12 sults among patients undergoing bariatric surgery with 13% levels [67]. having folate deficiency postoperatively [84]. Vitamin B12 along with folate influences one-carbon me- Several studies have shown an association between SNPs tabolism. Cubulin (CUBN) is the intestinal (IF) and polymor- related to folate metabolism, folate deficiency, and elevated phisms of this gene have been associated with chronic dis- homocysteine [70]. A common genetic variant in MTHFR is eases in individuals with low B12 status [29]. A study involv- known to influence blood folate and prevalent in 10% of the ing a Canadian population found that many SNPs in genes population worldwide [85, 86]. Steluti and colleagues studied related to folate, B12, and homocysteine metabolism— polymorphism frequencies and differences in homocysteine CUBN, TCN1, TCN2, methylenetetrahydrofolate reductase concentrations even in the presence of folic acid fortification (MTHFR), MUT (methylmalonyl coenzyme A mutase), and and found that homocysteine levels increased in those carry- FUT2—are possibly correlated with B vitamin-related dis- ing genetic variants in folate metabolism, specifically in the eases [30]. Genetic polymorphisms of MTHFR, MTR, MTHFR gene [87]. The prevalence of variant MTHFR TT has MTRR, MMAA (methylmalonic aciduria (cobalamin defi- been found in 25% of Americans of Hispanic origin, 10–15% ciency) cb1A type), MMACHC (methylmalonic aciduria among white Americans, and only 0–1% for African and homocystinuria, cblC type), and MUT have been ana- Americans [77, 87–89]. A review examining the nutritional lyzed. This research has failed to show an association between deficiencies, bariatric surgery, and serum homocysteine levels MTHFR gene polymorphisms and B12 concentrations [29]. found that the mutations of the MTHFR gene can be one of the However, a study using a classic twin model found that com- reasons for persistent elevated serum homocysteine after sur- mon gene variants—MMAA, MMACHC, MTRR, and gery despite supplementation with B-group [76]. MUT—were significantly associated with B12 levels and Knowing the presence of genetic variants of folate metabolism could explain the variation in B12 levels, which might facili- would provide a critical personalized care to those that might tate the prevention and treatment of B12 insufficiency/ benefit from the methylated form of folic acid to prevent ele- deficiency in individuals at a higher risk of associated diseases vatedhomocysteinelevels[76]. [68]. A cross sectional study looking at 56 SNPs of the B12 pathway among an older female population and found TCN2 Thiamine to be significantly associated with elevated serum methylmalonic acid (MMA) levels, a marker for available Thiamine is essential for glucose, amino acid, and energy B12 [69]. When using MMA levels as a marker for B12, it [90–92]. Deficiency of thiamine can cause com- is suggested that TCN2 gene variants may lead to decreased plications including cardiovascular and neurological diseases, vitamin B12 availability [69]. This review spotlights the com- including Wernicke-Korsakoff syndrome [90, 93]. plex nature of nutrigenomics and vitamin B12. Identifying Preoperative thiamine deficiency is prevalent in about 29% these gene variants among people having bariatric surgery of patients undergoing bariatric surgery [57]. Studies have could contribute to a more personalized nutrition plan. found that preexisting thiamine deficiency can be present in 15.5% and as high as 47% of patients; however, race plays a Folate role showing Hispanic patients with the highest level of prev- alence followed by African Americans (31%) and Caucasians Folate plays a role in one-carbon metabolism, methylation and (7%) [57, 79, 94]. Similarly, a retrospective study showed DNA synthesis, and methionine regeneration [70–72]. Folate 33.6% of patients having thiamine deficiency pre-operatively, deficiency is associated with elevated homocysteine, cardio- suggesting that people with obesity, especially those with vascular diseases, neural tube defects, cleft lip and palate, late many weight loss attempts, may have different needs to main- pregnancy complications, neurodegenerative and psychiatric tain adequate thiamine levels [95]. disorders [73–75]. Elevated homocysteine levels are a risk Mutations in thiamine transporter genes, SLC19A2 and marker for dementia, Alzheimer’s disease, bone fractures, SLC19A3, have been observed in cases of thiamine deficien- cancers, and cardiovascular diseases [76–78]. Many studies cy due to decreased absorption of thiamine that leads to neu- show folate deficiency to be low due to food fortification in rological dysfunction [91]. SLC35F3 is another thiamine America [39, 79–81]. Although preoperative deficiencies are transporter gene that plays a role in cardiac health and blood not alarming, prevalence of folate deficiency and elevated pressure. Genetic variants have been associated with thiamine homocysteine have shown to persist or worsen after bariatric deficiency as well as hypertension [35]. Prevalence of OBES SURG (2020) 30:3570–3582 3575 mutations in these genes is largely unaccounted for despite maintenance of iron homeostasis [37]. Mutations in the HFE recent advances in GWA studies. However, studies show that gene can lead to hereditary hemochromatosis, an iron over- thiamine deficiency and cardiac dysfunction associated with load disorder [114]. These factors should be considered to these genetic variants are alleviated with thiamine supplemen- possibly affect iron absorption and thus response to treatment. tation [96–99]. Literature reviews have shown that treatment TMPRSS6 mutations have been associated with refractori- for thiamine deficiency vary according to the genetic defect of ness to oral iron and studies confirm the role of TMPRSS6 in thiamine metabolism and that supplementation results in ade- predicting oral iron response [114, 115]. One study evaluated quate thiamine levels and improved clinical outcomes [100, subjects with persistent IDA to poorly respond to oral iron, 101]. The best responses to thiamine therapy were associated indicating that TMPRSS6 polymorphisms are more frequent with early referral for genetic testing and early initiation of in subjects with persistent IDA [115]. Identifying mutations of thiamine treatment. This evidence demonstrates that early di- these iron-related genes can help with providing personalized agnosis of these mutations can be beneficial. It may also im- iron supplementation for a common deficiency post bariatric plicate the hereditability of thiamine deficiency and that ther- surgery. apeutic doses of thiamine vary according to the genetic defect. Association Between Genetic Defects and Iron Micronutrient Supplementation

Iron is essential for metabolic processes like oxygen transport, The vitamin and mineral supplementation studies that focus deoxyribonucleic acid (DNA) synthesis, electron transport, as on treating genetic disorders are mainly case studies. well as cellular functions can affect one’swell-being[102]. In Supplementation studies for vitamin D-related genetic vari- individuals with obesity, the chronic inflammatory state relat- ants have been conducted in populations that are overweight ed to obesity might be a possible risk factor for iron deficien- and have obesity. Limited data is available on micronutrient cy, which is also called the anemia of inflammation [57, supplementation according to genetic variants in bariatric sur- 103–105]. Studies have shown that the prevalence of iron gery populations. Table 2 shows studies involving micronu- deficiency in adults with obesity is remarkable, and a decrease trient supplementation according to genetic defect in diverse in serum iron and transferrin saturation levels is inversely populations. associated with an increase in body mass index [103, 106–108]. A study involving bariatric surgery candidates Bariatric Surgery, Genetic Testing, and Gene showed 86.2% of females and 80% of males to be iron defi- Expression Profiles cient prior to surgery [109]. A retrospective analysis of pa- tients undergoing RYGB surgery showed that 43.9% were Genetic expression patterns can be a predictive tool for re- iron-deficient pre-operatively, which may be associated with sponsiveness to nutritional treatments. Some studies have in- higher complication rates as well as worsening of iron defi- dicated that surgery-induced weight loss was associated with ciency after surgery [57, 110, 111]. These findings reaffirm remodeling of the epigenome that helps regulate metabolic the need to assess and possibly intervene to manage deficiency gene expression [125, 126]. One study found that 1366 genes in bariatric surgery candidates preoperatively. were differentially expressed after bariatric surgery and sub- Considering the results of several GWAS, there is strong sequent weight loss, which are associated with gene transcrip- evidence of genetic regulation of iron metabolism, and muta- tion and energy metabolism [127]. Knowing the impact of tions in transmembrane serine protease 6 (TMPRSS6) gene bariatric surgery on the vitamin/mineral metabolic pathways that encodes for an enzyme that regulates hepcidin involved in can lead to successful prevention and treatment of micronutri- iron homeostasis, iron carrier transferrin (TF), and transferrin ent deficiencies. A study that specifically assessed the mRNA receptor-2 (TFR2) genes have been associated with iron defi- of genes within B12 degradation pathway after gastric bypass ciency [112]. A GWAS concluded that identifying mutations found that the intestine reprogrammed its genetic phenotype in the TMPRSS6 gene has broad applications in understand- to compensate for the changes in B12 metabolism. The au- ing clinical disorders of iron metabolism, and polymorphisms thors also found decreased expression of TCN1 but an in- in TMPRSS6 gene may contribute to iron deficiency anemia creased production of CUBN, which reflects adaptive genetic (IDA) in individuals even in absence of other predisposing reprogramming [128]. However, research on the role of vita- factors for IDA [112]. Studies have shown a common min metabolism genes and their adaptation after bariatric sur- TMPRSS6 gene variant to be prevalent in 45% of the individ- gery is scarce. We do know that healthy individuals and peo- uals without iron deficiency and clinically relevant inflamma- ple with obesity have different gene expression profiles and tory conditions [104] and 36.5–41.7% in a group of non- bariatric surgery further modifies the epigenome [129, 130]. pregnant women [113]. TF and human hemochromatosis Genetic testing is a useful tool for applying personalized med- (HFE) genes are involved in genetic regulation of icine in bariatric surgery patients as demonstrated by 3576 OBES SURG (2020) 30:3570–3582

Table 2 Supplementation trials according micronutrient defect

Reference Micronutrient Defective or Dosage and monitoring No. of patients Summary mutated gene

[116] Thiamine SLC19A2 75 mg thiamine/day Case study of 1 Patients with this defect present with diabetes female patient mellitus, megaloblastic anemia, and sensorineural deafness. Thiamine supplementation improved blood glucose and insulin requirements decreased. [117] SLC19A3 100 mg thiamine 2×/day along with Case study of 1 This genetic defect causes ophthalmoplegia, 10 mg biotin 2×/day for 5 months female patient ataxia and confusion. Oral biotin and thiamine improved the symptoms dramatically the next day. [100, TPK1 500 mg thiamine/day 2 patients with Early thiamine supplementation prevented 118] homozygous encephalopathic episodes and improved TPK1mutation- developmental progression. Evidence s suggests that thiamine supplementation may rescue TPK enzyme activity. [119] Vitamin D GC 50,000 IU vitamin D3 per week for 234 participants Carriers of GC mutation showed the lowest 8 weeks, followed by daily with vitamin D baseline 25(OH)D levels and lowest maintenance of 1000 IU vitamin D3 for deficiency response to vitamin D supplementation. 4months Mutations in GC gene can predict response to vitamin D supplementation. [27]CYP2R1,Vitamin D3 (1000 IU/day) and/or calcium 1787 healthy The increase in [25(OH)D] attributable to CYP24- carbonate (1200 mg/day elemental participants vitamin D3 supplementation may vary A1, VDR calcium) according to common genetic differences in CYP2R1, CYP24A1, and VDR genes. [120] Folic acid MTHFR Each treatment taken once daily for 480 subjects with MTHFR mutation can affect homocysteine (FA) 8 weeks. mild or concentration at baseline and post-FA 1. Enalapril only (10 mg, control group) moderate treatment as well as can modify 2. Enalapril-FA tablet (10 mg enalapril essential therapeutic responses to various dosages combined with 0.4 mg of FA) hypertension of FA supplementation. 3. Enalapril-FA tablet (10 mg enalapril combined with 0.8 mg of FA) [121]MTHFR3 random dietary interventions (4 months 126 healthy The TT homozygotes tended to have low 677C → each): subjects (42 plasma folate and high plasma T 1. Exclusion diet (avoidance of TT, 42 CT, and homocysteine levels. Folate intervention genotype FA–fortified foods) 42 CC on plasma folate was observed across 2. Folate-rich diet (folate-rich foods to genotypes) genotypes. However, the TT homozygotes achieve 400 mcg folate/d) required higher supplement intervention to 3. Supplement (exclusion diet plus a folate achieve similar effects observed in other supplement of 400 mcg/day) genotypes suggesting a need for supplementation with at least 400–600 mcg/day for individuals with the TT genotype. [122] Vitamin B12 MTHFR One vitamin tablet consisting of 2 mg of 52 patients with Vitamin supplementation lowered 677C→T folic acid, 25 mg vitamin B6, and migraine with homocysteine and reduced migraine genotype 400 μgofvitaminB12dailyfor aura. disability in a subgroup of patients. In this 6months patient group the treatment effect on both homocysteine levels and migraine disability was associated with MTHFR C677T genotype; carriers of the C allele experienced a greater response compared to TT genotypes concluding that TT genotypes require a larger dosage of vitamins to exhibit the same effect as C alleles. [123] Iron HFE, Iron supplementation with autrin capsules 181 pregnant The HFE variant had a positive effect with TMPRS- (ferrous fumarate; 98.6 mg elemental women with significant improvement in hemoglobin, S6, TF iron) once a day for 20 weeks from the anemia iron and ferritin. This shows an association time of diagnosis of genetic variants and iron absorption and thus response to treatment. The TMPRSS6 OBES SURG (2020) 30:3570–3582 3577

Table 2 (continued)

Reference Micronutrient Defective or Dosage and monitoring No. of patients Summary mutated gene

mutation was significantly associated with higher serum iron and hemoglobin. The presence of variants in STEAP3, TMPRSS6, SLC11A2, SLC40A1, HAMP and TF genes indicate a probable genetic association with iron status. [124] TMPRSS6 Intravenous iron gluconate Case study of 1 A comprehensive assessment that includes (1.3 mg/kg/day) for 5 days as first female patient sequence analysis of TMPRSS6 can help course and same dose was repeated after to confirm the genotype-phenotype 5months association of genes involved in iron Followed by different supplementation metabolism and may also be useful for therapy in which the patient received predicting the patient’s response to iron liposomaloralironatadoseof treatment. 10 mg/day for 3 months

Bandstein et al. that showed presurgery vitamin D levels may responders will be required to understand this population impact the size of genotype effects of FTO rs9939609 on and area of research. Genotype along with micronutrient weight loss among gastric bypass surgery patients [131]. blood levels would be the initial step in applying PN Nutritional genomics may provide the path for precise nutri- among bariatric surgery patients. Genetic marker is only tion recommendations to provide high-risk individuals with one factor that influences improvements related to micro- personalized treatment and to prevent micronutrient nutrient status [132]. deficiencies. Future work should focus on genotyping for multiple var- iants in the micronutrient metabolic pathways and their addi- tive and interactive effects to get a complete understanding of Discussion the influence of genetic factors on micronutrient metabolism. Then, utilizing genomic technology to understand this influ- From the review, it is evident that the deficiencies of the stud- ence on the responses to micronutrient supplementation is also ied micronutrients are influenced by genetic mutations. important. This would involve micronutrient status, genetic Postbariatric surgery, patients frequently have these deficien- variations, and genetic interactions within metabolic pathways cies and knowledge of these mutations may have bearing on involving the micronutrient, its molecular targets, and envi- its management. Additional research is needed to establish ronmental stressors [133]. this association. This review confirmed the scarcity of re- Furthermore, studies should focus to understand the role of search that has been conducted in the area of bariatric surgery the gut microbiome and its influence on metabolism and phys- and micronutrient genetic variants, with only two articles be- iology. The human gut microbiota (which has its own ge- ing found in this search. This limitation should be considered nome) can modulate signaling pathways and regulate gene when interpreting the findings in this discussion. Furthermore, expression [134]. Diet, lifestyle, medications, and environ- the treatment regimen for those who have micronutrient ge- mental exposure can increase inflammation within the gut, netic variants and undergoing bariatric surgery should be an causing dysbiosis, which can contribute to chronic diseases area of future research. and other illnesses [135]. Interestingly, gut microbial contri- Personalized dietary and supplement advice derived bution to vitamin metabolism has been recognized in whole- from genetic testing should be based on appropriately de- genome metagenomic studies, suggesting microbe-mediated signed studies. Utility of data from GWAS in providing vitamin metabolism [136, 137]. Pre- and probiotics as well as dietary advice is limited because it is not known what diet diet can alter the gut microbiome in a manner that improves and supplement intakes are required to prevent and treat human health [138]. Investigating how the gut microbes can the deficiencies that might be caused by micronutrient positively influence vitamin metabolism is warranted. genetic variants. Identifying how a genetic variant mod- Techniques used for genetic testing will determine the cost. ifies the response to supplementation on the micronutrient Methods being used in healthcare and research to identify status and possibly identify responders and non- genetic variations are known as next-generation sequencing 3578 OBES SURG (2020) 30:3570–3582

(NGS) [28]. Sequencing costs have rapidly decreased, which may affect the impact of genotype-based personalized advice has increased NGS applications in the clinical setting. There [146]. The researchers reported that PN advice resulted in are 3 NGS approaches used: targeted gene panels (TGP), greater dietary changes compared with general healthy eating whole-exome sequencing (WES), and whole-genome se- advice [146]. Analyzing biochemical markers for vitamins/ quencing (WGS) [139]. WGS implies the determination of minerals as well as defining a person’s “nutrigenomic profile” the sequence of the entire genome of an individual; WES is for those undergoing bariatric surgery will open the door to a component of the genome; and TGP analyze specific muta- implement more personalized recommendations for micronu- tions in a given set of genes or gene regions [140]. Per sample trient supplementation. costs of diagnostic NGS applications, that include the total cost (in euros) of processing and analysis are estimated at Compliance with Ethical Standards €333 (TGP), €792 (WES), and €1669 (WGS) [139]. Another study showed cost analysis for cancer diagnosis using Conflict of Interest The authors declare that they have no conflicts of NGS. From the pre-analytical phase to delivery of results, cost interest. per patient for TGP ranged from €376 to €968 [141]. Costs for Ethical Approval For this type of study, ethical approval and informed NGS have declined and will continue to decline with innova- consent do not apply as it is a narrative review. tions in genome-sequencing technologies and strategies [142]. In the area of bariatric surgery research, TGP could be utilized Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adap- with the specific genes involved in micronutrient metabolic tation, distribution and reproduction in any medium or format, as long as pathways, thus facilitating a more cost-effective and easy-to- you give appropriate credit to the original author(s) and the source, pro- interpret analysis. vide a link to the Creative Commons licence, and indicate if changes were Concerns and limitations involved in genetic testing in- made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a clude [1] the rapid growth of direct-to-consumer (DTC) ge- credit line to the material. If material is not included in the article's netic testing services with non-evidenced based testing, [2] Creative Commons licence and your intended use is not permitted by potential ethical dilemmas, and [3] applications to the broader statutory regulation or exceeds the permitted use, you will need to obtain community [143]. Clinicians will be critical in providing ge- permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. netic counseling regarding health decisions based on genomic information [144]. Many issues must be addressed prior to genetic testing such as informed consent, sample and data storage, return of results, and privacy and confidentiality to References minimize these risks that can be introduced with such testing [145]. As mentioned previously, the majority of GWAS are 1. Ogden CL, Carroll MD, Kit BK, et al. Prevalence of childhood conducted among Caucasian/European subjects. However, it and adult obesity in the United States, 2011–2012. JAMA. is difficult to extrapolate from these studies to other popula- 2014;311(8):806–14. https://doi.org/10.1001/jama.2014.732. tions. Sex differences also must be considered to improve the 2. Bloomberg RD, Fleishman A, Nalle JE, et al. Nutritional deficien- cies following bariatric surgery: what have we learned? Obes application of genetic tests to the broader community [143]. Surg. 2005;15(2):145–54. https://doi.org/10.1381/ Addressing these areas and concerns are critical to implement 0960892053268264. genome sequencing in clinical practice. This type of analysis 3. Sawaya RA, Jaffe J, Friedenberg L, et al. Vitamin, mineral, and is likely to be cost-effective, especially in specific populations drug absorption following bariatric surgery. Curr Drug Metab. 2012;13(9):1345–55. such as patients with bariatric surgery. 4. Ahmad DS, Esmadi M, Hammad H. Malnutrition secondary to non-compliance with vitamin and mineral supplements after gas- tric bypass surgery: What can we do about it? Am J Case Rep. Conclusion 2012;13:209–13. https://doi.org/10.12659/AJCR.883335. 5. Mechanick JI, Apovian C, Brethauer S, et al. Clinical practice guidelines for the perioperative nutrition, metabolic, and nonsur- PN is the delivery of dietary advice at an individual level and gical support of patients undergoing bariatric procedures - 2019 future work should verify if this targeted nutrition can change update: cosponsored by American Association of Clinical behaviors and have an impact on health outcomes. Dietitians Endocrinologists/American College of Endocrinology, the currently provide PN advice based on diet and phenotype; Obesity Society, American Society for Metabolic & Bariatric Surgery, Obesity Medicine Association, and American Society however, genotype-based PN advice is not so readily avail- of Anesthesiologists - Executive Summary. Endocr Pract. able. One review, involving a variety of populations such as 2019;25(12):1346–59. https://doi.org/10.4158/GL-2019-0406. patients with a history of weight loss failures, people with 6. Lombardo M, Franchi A, Padua E, et al. Potential nutritional de- obesity, as well as healthy men and women of various ages, ficiencies in obese subjects 5 years after bariatric surgery. Bariatric Surgical Practice and Patient Care. 2019;14(3):125–30. examined the evidence for genotype-based personalized infor- 7. Mahlay NF, Verka LG, Thomsen K, et al. Vitamin D status before mation on motivating behavioral change, and factors which Roux-en-Y and efficacy of prophylactic and therapeutic doses of OBES SURG (2020) 30:3570–3582 3579

vitamin D in patients after Roux-en-Y gastric bypass surgery. interindividual variability in cholecalciferol bioavailability in Obes Surg. 2009;19(5):590–4. https://doi.org/10.1007/s11695- healthy men. J Nutr. 2016;146(12):2421–8. https://doi.org/10. 008-9698-1. 3945/jn.116.237115. 8. Ferguson LR, De Caterina R, Görman U, et al. Guide and position 26. Nissen J, Rasmussen LB, Ravn-Haren G, et al. Common variants of the International Society of Nutrigenetics/Nutrigenomics on in CYP2R1 and GC genes predict vitamin D concentrations in personalised nutrition: part 1 - fields of precision nutrition. J healthy Danish children and adults. PLoS One. 2014;9(2): Nutrigenet Nutrigenomics. 2016;9(1):12–27. https://doi.org/10. e89907. https://doi.org/10.1371/journal.pone.0089907. 1159/000445350. 27. Barry EL, Rees JR, Peacock JL, et al. Genetic variants in 9. Trovato GM. Behavior, nutrition and lifestyle in a comprehensive CYP2R1, CYP24A1, and VDR modify the efficacy of vitamin health and disease paradigm: skills and knowledge for a predic- D3 supplementation for increasing serum 25-hydroxyvitamin D tive, preventive and personalized medicine. EPMA J. 2012;3(1):8. levels in a randomized controlled trial. J Clin Endocrinol Metab. https://doi.org/10.1007/s13167-012-0141-2. 2014;99(10):E2133–7. https://doi.org/10.1210/jc.2014-1389. 10. Fallaize R, Macready AL, Butler LT, et al. An insight into the 28. NIH. Genetics Home Reference [cited 2019]. Available from: public acceptance of nutrigenomic-based personalised nutrition. https://ghr.nlm.nih.gov/. Nutr Res Rev. 2013;26(1):39–48. https://doi.org/10.1017/ 29. Surendran S, Adaikalakoteswari A, Saravanan P, et al. An update S0954422413000024. on vitamin B12-related gene polymorphisms and B12 status. 11. Crovesy L, Rosado EL. Interaction between genes involved in Genes Nutr. 2018;13:2. Epub 2018/02/06. https://doi.org/10. energy intake regulation and diet in obesity. Nutrition. 2019;67– 1186/s12263-018-0591-9. 68:110547. https://doi.org/10.1016/j.nut.2019.06.027. 30. Zinck JW, de Groh M, MacFarlane AJ. Genetic modifiers of fo- 12. Nielsen DE, El-Sohemy A. A randomized trial of genetic infor- late, vitamin B-12, and homocysteine status in a cross-sectional mation for personalized nutrition. Genes Nutr. 2012;7(4):559–66. study of the Canadian population. Am J Clin Nutr. 2015;101(6): https://doi.org/10.1007/s12263-012-0290-x. 1295–304. Epub 2015/05/06. https://doi.org/10.3945/ajcn.115. 13. Maher B. Nature readers flirt with personal genomics. Nature. 107219. 2011;478(7367):19. https://doi.org/10.1038/478019a. 31. Hazra A, Kraft P, Selhub J, et al. Common variants of FUT2 are 14. Bloss CS, Schork NJ, Topol EJ. Effect of direct-to-consumer associated with plasma vitamin B12 levels. Nat Genet. genomewide profiling to assess disease risk. N Engl J Med. 2008;40(10):1160–2. https://doi.org/10.1038/ng.210. 2011;364(6):524–34. https://doi.org/10.1056/NEJMoa1011893. 32. Tanaka T, Scheet P, Giusti B, et al. Genome-wide association 15. Brolin RE, LaMarca LB, Kenler HA, et al. Malabsorptive gastric study of vitamin B6, vitamin B12, folate, and homocysteine blood bypass in patients with superobesity. J Gastrointest Surg. concentrations. Am J Hum Genet. 2009;84(4):477–82. https://doi. 2002;6(2):195–203. discussion 4-5 org/10.1016/j.ajhg.2009.02.011. 16. Berger JR. The neurological complications of bariatric surgery. 33. Frosst P, Blom HJ, Milos R, et al. A candidate genetic risk factor Arch Neurol. 2004;61(8):1185–9. https://doi.org/10.1001/ for vascular disease: a common mutation in methylenetetrahydro- archneur.61.8.1185. folate reductase. Nat Genet. 1995;10(1):111–3. Epub 1995/05/01. 17. Shenkin A. Micronutrients in health and disease. Postgrad Med J. https://doi.org/10.1038/ng0595-111. 2006;82(971):559–67. https://doi.org/10.1136/pgmj.2006. 34. Bagley PJ, Selhub J. A common mutation in the methylenetetra- 047670. hydrofolate reductase gene is associated with an accumulation of 18. Geissler C, Powers H. Fundamentals of human nutrition E-book: formylated tetrahydrofolates in red blood cells. Proc Natl Acad Sci for students and practitioners in health sciences: Churchill U S A. 1998;95(22):13217–20. https://doi.org/10.1073/pnas.95. Livingstone Elsevier; 2009. 324 p. 22.13217. 19. Reddy VS, Palika R, Ismail A, et al. Nutrigenomics: Opportunities 35. Zhang K, Huentelman MJ, Rao F, et al. Genetic implication of a & challenges for public health nutrition. Indian J Med Res. novel thiamine transporter in human hypertension. J Am Coll 2018;148(5):632–41. https://doi.org/10.4103/ijmrIJMR_1738_ Cardiol. 2014;63(15):1542–55. Epub 2014/02/05. https://doi.org/ 18. 10.1016/j.jacc.2014.01.007. 20. Lister Hill National Center for Biomedical Communications. 36. Mikstiene V, Songailiene J, Byckova J, et al. Thiamine responsive Genetics home reference [unspecified]. Bethesda, MD: National megaloblastic anemia syndrome: a novel homozygous SLC19A2 Library of Medicine, National Institutes of Health,; 2003. gene mutation identified. Am J Med Genet A. 2015;167(7):1605– Available from: http://ghr.nlm.nih.gov/. 9. Epub 2015/02/23. https://doi.org/10.1002/ajmg.a.37015. 21. Sharp P, Srai SK. Molecular mechanisms involved in intestinal 37. Pichler I, Minelli C, Sanna S, et al. Identification of a common iron absorption. World J Gastroenterol. 2007;13(35):4716–24. variant in the TFR2 gene implicated in the physiological regula- https://doi.org/10.3748/wjg.v13.i35.4716. tion of serum iron levels. Hum Mol Genet. 2011;20(6):1232–40. 22. Kluijtmans LA, van den Heuvel LP, Boers GH, et al. Molecular https://doi.org/10.1093/hmg/ddq552. genetic analysis in mild hyperhomocysteinemia: a common muta- 38. Blanco-Rojo R, Baeza-Richer C, López-Parra AM, et al. Four tion in the methylenetetrahydrofolate reductase gene is a genetic variants in transferrin and HFE genes as potential markers of iron risk factor for cardiovascular disease. Am J Hum Genet. deficiency anaemia risk: an association study in menstruating 1996;58(1):35–41. women. Nutr Metab (Lond). 2011;8:69. https://doi.org/10.1186/ 23. Borel P, Desmarchelier C. Bioavailability of fat-soluble vitamins 1743-7075-8-69. and phytochemicals in humans: effects of genetic variation. Annu 39. Gemmel K, Santry HP, Prachand VN, et al. Vitamin D deficiency Rev Nutr. 2018;38:69–96. https://doi.org/10.1146/annurev-nutr- in preoperative bariatric surgery patients. Surg Obes Relat Dis. 082117-051628. 2009;5(1):54–9. https://doi.org/10.1016/j.soard.2008.07.008. 24. Dib MJ, Elliott R, Ahmadi KR. A critical evaluation of results 40. Bischoff-Ferrari HA, Willett WC, Wong JB, et al. Fracture pre- from genome-wide association studies of micronutrient status vention with vitamin D supplementation: a meta-analysis of ran- and their utility in the practice of precision nutrition. Br J Nutr. domized controlled trials. JAMA. 2005;293(18):2257–64. https:// 2019;122(2):121– 30. https://doi.org/10.1017/ doi.org/10.1001/jama.293.18.2257. S0007114519001119. 41. Holick MF. High prevalence of vitamin D inadequacy and impli- 25. Desmarchelier C, Borel P, Goncalves A, et al. A combination of cations for health. Mayo Clin Proc. 2006;81(3):353–73. https:// single-nucleotide polymorphisms is associated with doi.org/10.4065/81.3.353. 3580 OBES SURG (2020) 30:3570–3582

42. Chakhtoura MT, Nakhoul NN, Shawwa K, et al. Hypovitaminosis 60. Vargas-Ruiz AG, Hernández-Rivera G, Herrera MF. Prevalence D in bariatric surgery: A systematic review of observational stud- of iron, folate, and vitamin B12 deficiency anemia after laparo- ies. Metabolism. 2016;65(4):574–85. https://doi.org/10.1016/j. scopic Roux-en-Y gastric bypass. Obes Surg. 2008;18(3):288–93. metabol.2015.12.004. https://doi.org/10.1007/s11695-007-9310-0. 43. Peterson LA, Zeng X, Caufield-Noll CP, et al. Vitamin D status 61. Quadros EV. Advances in the understanding of cobalamin assimila- and supplementation before and after bariatric surgery: a compre- tion and metabolism. Br J Haematol. 2010;148(2):195–204. Epub hensive literature review. Surg Obes Relat Dis. 2016;12(3):693– 2009/10/12. https://doi.org/10.1111/j.1365-2141.2009.07937.x. 702. https://doi.org/10.1016/j.soard.2016.01.001. 62. Li N, Rosenblatt DS, Kamen BA, et al. Identification of two mutant 44. Afzal S, Brøndum-Jacobsen P, Bojesen SE, et al. Genetically low alleles of transcobalamin II in an affected family. Hum Mol Genet. vitamin D concentrations and increased mortality: Mendelian 1994;3(10):1835–40. https://doi.org/10.1093/hmg/3.10.1835. randomisation analysis in three large cohorts. BMJ. 2014;349: 63. Coelho D, Suormala T, Stucki M, et al. Gene identification for the g6330. https://doi.org/10.1136/bmj.g6330. cblD defect of vitamin B12 metabolism. N Engl J Med. 45. Shea MK, Benjamin EJ, Dupuis J, et al. Genetic and non-genetic 2008;358(14):1454–64. https://doi.org/10.1056/NEJMoa072200. correlates of vitamins K and D. Eur J Clin Nutr. 2009;63(4):458– 64. Nilsson SE, Read S, Berg S, et al. Heritabilities for fifteen routine 64. https://doi.org/10.1038/sj.ejcn.1602959. biochemical values: findings in 215 Swedish twin pairs 82 years 46. Waterhouse M, Tran B, Armstrong BK, et al. Environmental, of age or older. Scand J Clin Lab Invest. 2009;69(5):562–9. personal, and genetic determinants of response to vitamin D sup- https://doi.org/10.1080/00365510902814646. plementation in older adults. J Clin Endocrinol Metab. 65. Grarup N, Sulem P, Sandholt CH, et al. Genetic architecture of 2014;99(7):E1332–40. https://doi.org/10.1210/jc.2013-4101. vitamin B12 and folate levels uncovered applying deeply se- 47. Didriksen A, Grimnes G, Hutchinson MS, et al. The serum 25- quenced large datasets. PLoS Genet. 2013;9(6):e1003530. Epub hydroxyvitamin D response to vitamin D supplementation is related 2013/06/06. https://doi.org/10.1371/journal.pgen.1003530. to genetic factors, BMI, and baseline levels. Eur J Endocrinol. 66. Johnston J, Bollekens J, Allen RH, et al. Structure of the cDNA 2013;169(5):559–67. https://doi.org/10.1530/EJE-13-0233. encoding transcobalamin I, a neutrophil granule protein. J Biol 48. Quinlivan EP, McPartlin J, McNulty H, et al. Importance of both Chem. 1989;264(27):15754–7. folic acid and vitamin B12 in reduction of risk of vascular disease. 67. Stanisławska-Sachadyn A, Woodside JV, Sayers CM, et al. The Lancet. 2002;359(9302):227–8. https://doi.org/10.1016/s0140- transcobalamin (TCN2) 776C>G polymorphism affects homocys- 6736(02)07439-1. teine concentrations among subjects with low vitamin B(12) sta- 49. Hin H, Clarke R, Sherliker P, et al. Clinical relevance of low serum tus. Eur J Clin Nutr. 2010;64(11):1338–43. Epub 2010/09/01. vitamin B12 concentrations in older people: the Banbury B12 https://doi.org/10.1038/ejcn.2010.157. study. Age Ageing. 2006;35(4):416–22. https://doi.org/10.1093/ 68. Andrew T, Gill R, Gillham-Nasenya I, et al. Unravelling the basis ageing/afl033. of variability in cobalamin levels in the general population. Br J 50. O'Leary F, Samman S. Vitamin B12 in health and disease. Nutr. 2013;110(9):1672–9. Epub 2013/04/29. https://doi.org/10. Nutrients. 2010;2(3):299–316. https://doi.org/10.3390/ 1017/S0007114513000974. nu2030299. 69. Matteini AM, Walston JD, Bandeen-Roche K, et al. 51. Lechner K, Födinger M, Grisold W, et al. Vitamin B12 deficiency. Transcobalamin-II variants, decreased vitamin B12 availability New data on an old theme. Wien Klin Wochenschr. 2005;117(17): and increased risk of frailty. J Nutr Health Aging. 2010;14(1): 579–91. https://doi.org/10.1007/s00508-005-0406-z. 73–7. https://doi.org/10.1007/s12603-010-0013-1. 52. Collaboration HLT. Dose-dependent effects of folic acid on blood 70. Nazki FH, Sameer AS, Ganaie BA. Folate: metabolism, genes, poly- concentrations of homocysteine: a meta-analysis of the random- morphisms and the associated diseases. Gene. 2014;533(1):11–20. ized trials. Am J Clin Nutr. 2005;82(4):806–12. https://doi.org/10. Epub 2013/10/01. https://doi.org/10.1016/j.gene.2013.09.063. 1093/ajcn/82.4.806. 71. Bailey LB, Gregory JF. Folate metabolism and requirements. J 53. Spence JD, Bang H, Chambless LE, et al. Vitamin intervention for Nutr. 1999;129(4):779–82. https://doi.org/10.1093/jn/129.4.779. stroke prevention trial: an efficacy analysis. Stroke. 2005;36(11): 72. Hubner RA, Houlston RS. Folate and colorectal cancer preven- 2404–9. https://doi.org/10.1161/01.STR.0000185929.38534.f3. tion. Br J Cancer. 2009;100(2):233–9. Epub 2008/12/16. doi: 54. Arendt JF, Nexo E. Unexpected high plasma cobalamin: proposal https://doi.org/10.1038/sj.bjc.6604823. for a diagnostic strategy. Clin Chem Lab Med. 2013;51(3):489– 73. Blom HJ, Smulders Y. Overview of homocysteine and folate me- 96. https://doi.org/10.1515/cclm-2012-0545. tabolism. With special references to cardiovascular disease and 55. Arendt JF, Pedersen L, Nexo E, et al. Elevated plasma vitamin neural tube defects. J Inherit Metab Dis. 2011;34(1):75–81. B12 levels as a marker for cancer: a population-based cohort Epub 2010/09/04. https://doi.org/10.1007/s10545-010-9177-4. study. J Natl Cancer Inst. 2013;105(23):1799–805. https://doi. 74. Smithells RW, Sheppard S, Schorah CJ. Vitamin deficiencies and org/10.1093/jnci/djt315. neural tube defects. Arch Dis Child. 1976;51(12):944–50. https:// 56. Stabler SP. Vitamin B12 deficiency. N Engl J Med. 2013;368(21): doi.org/10.1136/adc.51.12.944. 2041–2. https://doi.org/10.1056/NEJMc1304350. 75. Prevention of neural tube defects: results of the Medical Research 57. Flancbaum L, Belsley S, Drake V, et al. Preoperative nutritional Council Vitamin Study. MRC Vitamin Study Research Group. status of patients undergoing Roux-en-Y gastric bypass for morbid Lancet. 1991;338(8760):131–7. obesity. J Gastrointest Surg. 2006;10(7):1033–7. https://doi.org/ 76. Komorniak N, Szczuko M, Kowalewski B, et al. Nutritional defi- 10.1016/j.gassur.2006.03.004. ciencies, bariatric surgery, and serum homocysteine level: review 58. Mehaffey JH, Mehaffey RL, Mullen MG, et al. Nutrient deficien- of current literature. Obes Surg. 2019;29(11):3735–42. https://doi. cy 10 years following Roux-en-Y gastric bypass: who’s responsi- org/10.1007/s11695-019-04100-2. ble? Obes Surg. 2017;27(5):1131–6. https://doi.org/10.1007/ 77. Eldibany MM, Caprini JA. Hyperhomocysteinemia and thrombosis: s11695-016-2364-0. an overview. Arch Pathol Lab Med. 2007;131(6):872–84. https://doi. 59. Skroubis G, Sakellaropoulos G, Pouggouras K, et al. Comparison of org/10.1043/1543-2165(2007)131[872:HATAO]2.0.CO;2. nutritional deficiencies after Roux-en-Y gastric bypass and after 78. Clarke R, Halsey J, Lewington S, et al. Effects of lowering homo- biliopancreatic diversion with Roux-en-Y gastric bypass. Obes Surg. cysteine levels with B vitamins on cardiovascular disease, cancer, 2002;12(4):551–8. https://doi.org/10.1381/096089202762252334. and cause-specific mortality: meta-analysis of 8 randomized trials OBES SURG (2020) 30:3570–3582 3581

involving 37 485 individuals. Arch Intern Med. 2010;170(18): hyperglycemia. Ann Vasc Surg. 2006;20(5):653–8. Epub 1622–31. https://doi.org/10.1001/archinternmed.2010.348. 2006/05/31. https://doi.org/10.1007/s10016-006-9055-6. 79. Xanthakos SA. Nutritional deficiencies in obesity and after bariat- 97. Tanaka T, Sohmiya K, Kono T, et al. Thiamine attenuates the ric surgery. Pediatr Clin N Am. 2009;56(5):1105–21. https://doi. hypertension and metabolic abnormalities in CD36-defective org/10.1016/j.pcl.2009.07.002. SHR: uncoupling of glucose oxidation from cellular entry accom- 80. de Luis DA, Pacheco D, Izaola O, et al. Clinical results and nutri- panied with enhanced protein O-GlcNAcylation in CD36 defi- tional consequences of biliopancreatic diversion: three years of ciency. Mol Cell Biochem. 2007;299(1–2):23–35. https://doi. follow-up. Ann Nutr Metab. 2008;53(3–4):234–9. Epub org/10.1007/s11010-005-9032-3. 2008/12/16. https://doi.org/10.1159/000185641. 98. Alaei-Shahmiri F, Soares MJ, Zhao Y, et al. The impact of thiamine 81. Mallory GN, Macgregor AM. Folate status following gastric by- supplementation on blood pressure, serum lipids and C-reactive pro- pass surgery (the great Folate mystery). Obes Surg. 1991;1(1):69– tein in individuals with hyperglycemia: a randomised, double-blind 72. https://doi.org/10.1381/096089291765561493. cross-over trial. Diabetes Metab Syndr. 2015;9(4):213–7. Epub 82. Toh SY, Zarshenas N, Jorgensen J. Prevalence of nutrient defi- 2015/04/29. https://doi.org/10.1016/j.dsx.2015.04.014. ciencies in bariatric patients. Nutrition. 2009;25(11–12):1150–6. 99. Zang XL, Han WQ, Yang FP, Ji KD, Wang JG, Gao PJ, et al. Epub 2009/05/31. https://doi.org/10.1016/j.nut.2009.03.012. Association of a SNP in SLC35F3 gene with the risk of hyperten- 83. Bal BS, Finelli FC, Shope TR, et al. Nutritional deficiencies after sion in a Chinese Han population. Front Genet. 2016;7:108. Epub bariatric surgery. Nat Rev Endocrinol. 2012;8(9):544–56. Epub 2016/06/20. doi: https://doi.org/10.3389/fgene.2016.00108. 2012/04/24. https://doi.org/10.1038/nrendo.2012.48. 100. Ortigoza-Escobar JD, Molero-Luis M, Arias A, et al. Treatment of 84. Gudzune KA, Huizinga MM, Chang HY, et al. Screening and genetic defects of thiamine transport and metabolism. Expert Rev diagnosis of micronutrient deficiencies before and after bariatric Neurother. 2016;16(7):755–63. Epub 2016/05/23. https://doi.org/ – surgery. Obes Surg. 2013;23(10):1581 9. https://doi.org/10.1007/ 10.1080/14737175.2016.1187562. s11695-013-0919-x. 101. Marcé-Grau A, Martí-Sánchez L, Baide-Mairena H, et al. Genetic de- 85. Wilcken B, Bamforth F, Li Z, et al. Geographical and ethnic var- fects of thiamine transport and metabolism: a review of clinical pheno- iation of the 677C>T allele of 5,10 methylenetetrahydrofolate re- types, genetics, and functional studies. J Inherit Metab Dis. 2019;42(4): ductase (MTHFR): findings from over 7000 newborns from 16 581–97. Epub 2019/06/24. https://doi.org/10.1002/jimd.12125. – areas world wide. J Med Genet. 2003;40(8):619 25. https://doi. 102. Troadec MB, Loréal O, Brissot P. The interaction of iron and the org/10.1136/jmg.40.8.619. genome: for better and for worse. Mutat Res. 2017;774:25–32. 86. Hiraoka M, Kagawa Y. Genetic polymorphisms and folate status. Epub 2017/09/14. https://doi.org/10.1016/j.mrrev.2017.09.002. Congenit Anom (Kyoto). 2017;57(5):142–9. Epub 2017/07/20. 103. Aigner E, Feldman A, Datz C. Obesity as an emerging risk factor https://doi.org/10.1111/cga.12232. for iron deficiency. Nutrients. 2014;6(9):3587–600. Epub 87. Steluti J, Carvalho AM, Carioca AAF, et al. Genetic variants in- 2014/09/11. https://doi.org/10.3390/nu6093587. volved in one-carbon metabolism: polymorphism frequencies and 104. Dopsaj V, Topić A, Savković M, et al. Associations of Common differences in homocysteine concentrations in the folic acid forti- Variants in. Dis Markers. 2019;2019:4864370. Epub 2019/03/07. fication era. Nutrients. 2017;9(6) Epub 2017/05/25 https://doi.org/ https://doi.org/10.1155/2019/4864370. 10.3390/nu9060539. 105. Sonnweber T, Ress C, Nairz M, et al. High-fat diet causes iron 88. Schneider JA, Rees DC, Liu YT, et al. Worldwide distribution of a deficiency via hepcidin-independent reduction of duodenal iron common methylenetetrahydrofolate reductase mutation. Am J Hum absorption. J Nutr Biochem. 2012;23(12):1600–8. Epub Genet. 1998;62(5):1258–60. https://doi.org/10.1086/301836. 2012/03/23. https://doi.org/10.1016/j.jnutbio.2011.10.013. 89. Botto LD, Yang Q. 5,10-Methylenetetrahydrofolate reductase gene variants and congenital anomalies: a HuGE review. Am J 106. Micozzi MS, Albanes D, Stevens RG. Relation of body size and Epidemiol. 2000;151(9):862–77. https://doi.org/10.1093/ composition to clinical biochemical and hematologic indices in – oxfordjournals.aje.a010290. US men and women. Am J Clin Nutr. 1989;50(6):1276 81. 90. Guerrini I, Thomson AD, Cook CC, et al. Direct genomic PCR https://doi.org/10.1093/ajcn/50.6.1276. sequencing of the high affinity thiamine transporter (SLC19A2) 107. Cepeda-Lopez AC, Osendarp SJ, Melse-Boonstra A, et al. Sharply gene identifies three genetic variants in Wernicke Korsakoff syn- higher rates of iron deficiency in obese Mexican women and children drome (WKS). Am J Med Genet B Neuropsychiatr Genet. are predicted by obesity-related inflammation rather than by differ- – 2005;137B(1):17–9. https://doi.org/10.1002/ajmg.b.30194. ences in dietary iron intake. Am J Clin Nutr. 2011;93(5):975 83. 91. Zhao R, Goldman ID. Folate and thiamine transporters mediated Epub 2011/03/16. https://doi.org/10.3945/ajcn.110.005439. by facilitative carriers (SLC19A1–3 and SLC46A1) and folate 108. Tussing-Humphreys LM, Nemeth E, Fantuzzi G, et al. Elevated receptors. Mol Asp Med. 2013;34(2–3):373–85. https://doi.org/ systemic hepcidin and iron depletion in obese premenopausal fe- – 10.1016/j.mam.2012.07.006. males. Obesity (Silver Spring). 2010;18(7):1449 56. Epub 92. Frank RA, Leeper FJ, Luisi BF. Structure, mechanism and catalytic 2009/10/08. https://doi.org/10.1038/oby.2009.319. duality of thiamine-dependent enzymes. Cell Mol Life Sci. 109. Malek M, Yousefi R, Safari S, et al. Dietary intakes and biochem- 2007;64(7–8):892–905. https://doi.org/10.1007/s00018-007-6423-5. ical parameters of morbidly obese patients prior to bariatric sur- 93. Eshak ES, Arafa AE. Thiamine deficiency and cardiovascular dis- gery. Obes Surg. 2019;29(6):1816–22. https://doi.org/10.1007/ orders. Nutr Metab Cardiovasc Dis. 2018;28(10):965–72. Epub s11695-019-03759-x. 2018/06/22. https://doi.org/10.1016/j.numecd.2018.06.013. 110. Harju E. Empty iron stores as a significant risk factor in abdominal 94. Carrodeguas L, Kaidar-Person O, Szomstein S, et al. Preoperative surgery. JPEN J Parenter Enteral Nutr. 1988;12(3):282–5. https:// thiamine deficiency in obese population undergoing laparoscopic bar- doi.org/10.1177/0148607188012003282. iatric surgery. Surg Obes Relat Dis. 2005;1(6):517–22; discussion 22. 111. Ruz M, Carrasco F, Rojas P, et al. Heme- and nonheme-iron absorp- Epub 2005/09/28 https://doi.org/10.1016/j.soard.2005.08.003. tion and iron status 12 mo after sleeve gastrectomy and Roux-en-Y 95. Costello E, Kerns J. Thiamine deficiency in people with obesity. gastric bypass in morbidly obese women. Am J Clin Nutr. Current Developments in Nutrition. June 2019;3(Supplement_1): 2012;96(4):810–7. Epub 2012/09/05. https://doi.org/10.3945/ajcn. P18–060–19. 112.039255. 96. Arora S, Lidor A, Abularrage CJ, et al. Thiamine (vitamin B1) 112. McLaren CE, McLachlan S, Garner CP, et al. Associations be- improves endothelium-dependent vasodilatation in the presence of tween single nucleotide polymorphisms in iron-related genes and 3582 OBES SURG (2020) 30:3570–3582

iron status in multiethnic populations. PLoS One. 2012;7(6): 129. Nicoletti CF, Cortes-Oliveira C, Pinhel MAS, Nonino CB. e38339. https://doi.org/10.1371/journal.pone.0038339. Bariatric surgery and precision nutrition. Nutrients. 2017;9(9). 113. Lee PL, Barton JC, Khaw PL, et al. Common TMPRSS6 muta- Epub 2017/09/06. doi: https://doi.org/10.3390/nu9090974. tions and iron, erythrocyte, and pica phenotypes in 48 women with 130. Leyvraz C, Verdumo C, Suter M, et al. Changes in gene expres- iron deficiency or depletion. Blood Cells Mol Dis. 2012;48(2): sion profile in human subcutaneous adipose tissue during signifi- 124–7. https://doi.org/10.1016/j.bcmd.2011.12.003. cant weight loss. Obes Facts. 2012;5(3):440–51. Epub 2012/06/ 114. De Falco L, Tortora R, Imperatore N, et al. The role of TMPRSS6 30. https://doi.org/10.1159/000341137. and HFE variants in iron deficiency anemia in celiac disease. Am J 131. Bandstein M, Schultes B, Ernst B, et al. The role of FTO and Hematol. 2018;93(3):383–93. Epub 2017/12/18. https://doi.org/ vitamin D for the weight loss effect of Roux-en-Y gastric bypass 10.1002/ajh.24991. surgery in obese patients. Obes Surg. 2015;25(11):2071–7. https:// 115. Poggiali E, Andreozzi F, Nava I, et al. The role of TMPRSS6 doi.org/10.1007/s11695-015-1644-4. polymorphisms in iron deficiency anemia partially responsive to 132. Guest NS, Horne J, Vanderhout SM, et al. Sport nutrigenomics: – oral iron treatment. Am J Hematol. 2015;90(4):306 9. Epub personalized nutrition for athletic performance. Front Nutr. 2015/03/02. https://doi.org/10.1002/ajh.23929. 2019;6:8. https://doi.org/10.3389/fnut.2019.00008. 116. Tahir S, Leijssen LG, Sherif M, et al. A novel homozygous SLC19A2 133. Hesketh J, Méplan C. Transcriptomics and functional genetic mutation in a Portuguese patient with diabetes mellitus and thiamine- polymorphisms as biomarkers of micronutrient function: focus responsive megaloblastic anaemia. Int J Pediatr Endocrinol. on selenium as an exemplar. Proc Nutr Soc. 2011;70:1–9. Epub 2015;2015(1):6. https://doi.org/10.1186/s13633-015-0002-6. 2011/05/03. https://doi.org/10.1017/S0029665111000115. 117. Fassone E, Wedatilake Y, DeVile CJ, et al. Treatable Leigh-like 134. Paul B, Barnes S, Demark-Wahnefried W, et al. Influences of diet encephalopathy presenting in adolescence. BMJ Case Rep. and the gut microbiome on epigenetic modulation in cancer and 2013;2013:200838. https://doi.org/10.1136/bcr-2013-200838. other diseases. Clin Epigenetics. 2015;7:112. https://doi.org/10. 118. Banka S, de Goede C, Yue WW, et al. Expanding the clinical and 1186/s13148-015-0144-7. molecular spectrum of thiamine pyrophosphokinase deficiency: a 135. Nicholson JK, Holmes E, Kinross J, et al. Host-gut microbiota treatable neurological disorder caused by TPK1 mutations. Mol – Genet Metab. 2014;113(4):301–6. Epub 2014/10/05. https://doi. metabolic interactions. Science. 2012;336(6086):1262 7. Epub org/10.1016/j.ymgme.2014.09.010. 2012/06/06. https://doi.org/10.1126/science.1223813. 119. Al-Daghri NM, Mohammed AK, Bukhari I, et al. Efficacy of 136. Lozupone CA, Stombaugh JI, Gordon JI, et al. Diversity, stability vitamin D supplementation according to vitamin D-binding pro- and resilience of the human gut microbiota. Nature. – tein polymorphisms. Nutrition. 2019;63–64:148–54. https://doi. 2012;489(7415):220 30. https://doi.org/10.1038/nature11550. org/10.1016/j.nut.2019.02.003. 137. Das P, Babaei P, Nielsen J. Metagenomic analysis of microbe- 120. Qin X, Li J, Cui Y, et al. MTHFR C677T and MTR A2756G mediated vitamin metabolism in the human gut microbiome. polymorphisms and the homocysteine lowering efficacy of differ- BMC Genomics. 2019;20(1):208. https://doi.org/10.1186/ ent doses of folic acid in hypertensive Chinese adults. Nutr J. s12864-019-5591-7. 2012;11(2) https://doi.org/10.1186/1475-2891-11-2. 138. Holmes E, Kinross J, Gibson GR, et al. Therapeutic modulation of 121. Ashfield-Watt PA, Pullin CH, Whiting JM, et al. microbiota-host metabolic interactions. Sci Transl Med. 2012;4(137): Methylenetetrahydrofolate reductase 677C–>T genotype modu- 137rv6. https://doi.org/10.1126/scitranslmed.3004244. lates homocysteine responses to a folate-rich diet or a low-dose 139. van Nimwegen KJ, van Soest RA, Veltman JA, et al. Is the $1000 folic acid supplement: a randomized controlled trial. Am J Clin genome as near as we think? A cost analysis of next-generation Nutr. 2002;76(1):180–6. https://doi.org/10.1093/ajcn/76.1.180. sequencing. Clin Chem. 2016;62(11):1458–64. Epub 2016/09/14. 122. Lea R, Colson N, Quinlan S, et al. The effects of vitamin supplemen- https://doi.org/10.1373/clinchem.2016.258632. tation and MTHFR (C677T) genotype on homocysteine-lowering 140. Adam MP, Ardinger HH, Pagon RA, Wallace SE, Bean LJH, and migraine disability. Pharmacogenet Genomics. 2009;19(6):422– Stephens K, et al. GeneReviews. 1993. 8. https://doi.org/10.1097/FPC.0b013e32832af5a3. 141. Marino P, Touzani R, Perrier L, et al. Cost of cancer diagnosis 123. Athiyarath R, Shaktivel K, Abraham V, et al. Association of genetic using next-generation sequencing targeted gene panels in routine variants with response to iron supplements in pregnancy. Genes Nutr. practice: a nationwide French study. Eur J Hum Genet. 2015;10(4):474. https://doi.org/10.1007/s12263-015-0474-2. 2018;26(3):314–23. https://doi.org/10.1038/s41431-017-0081-3. 124. Capra AP, Ferro E, Cannavò L, et al. A child with severe iron- 142. Institute NNHGR. The cost of sequencing a human genome 2019. deficiency anemia and a complex TMPRSS6 genotype. – 143. Vlahovich N, Hughes DC, Griffiths LR, et al. Genetic testing for Hematology. 2017;22(9):559 64. https://doi.org/10.1080/ exercise prescription and injury prevention: AIS-Athlome consor- 10245332.2017.1317990. tium-FIMS joint statement. BMC Genomics. 2017;18(Suppl 8): 125. Donkin I, Versteyhe S, Ingerslev LR, et al. Obesity and bariatric 818. https://doi.org/10.1186/s12864-017-4185-5. surgery drive epigenetic variation of spermatozoa in humans. Cell 144. Annas GJ, Elias S. 23andMe and the FDA. N Engl J Med. Metab. 2016;23(2):369–78. Epub 2015/12/06. https://doi.org/10. 2014;370(11):985–8. Epub 2014/02/12. https://doi.org/10.1056/ 1016/j.cmet.2015.11.004. NEJMp1316367. 126. Barres R, Kirchner H, Rasmussen M, et al. Weight loss after gastric bypass surgery in human obesity remodels promoter meth- 145. Green ED, Guyer MS, Institute NHGR. Charting a course for geno- – mic medicine from base pairs to bedside. Nature. 2011;470(7333): ylation. Cell Rep. 2013;3(4):1020 7. Epub 2013/04/11. https:// – doi.org/10.1016/j.celrep.2013.03.018. 204 213. doi: https://doi.org/10.1038/nature09764,. 127. Pinhel MAS, Noronha NY, Nicoletti CF, et al. Changes in global 146. O'Donovan CB, Walsh MC, Gibney MJ, et al. Knowing your transcriptional profiling of women following obesity surgery by- genes: does this impact behaviour change? Proc Nutr Soc. – pass. Obes Surg. 2018;28(1):176–86. https://doi.org/10.1007/ 2017;76(3):182 91. Epub 2017/01/20. https://doi.org/10.1017/ s11695-017-2828-x. S0029665116002949. 128. Sala P, Belarmino G, Torrinhas RS, et al. Gastrointestinal transcriptomic response of metabolic vitamin B12 pathways in Publisher’sNoteSpringer Nature remains neutral with regard to jurisdic- Roux-en-Y gastric bypass. Clin Transl Gastroenterol. 2017;8(1): tional claims in published maps and institutional affiliations. e212. https://doi.org/10.1038/ctg.2016.67. Kijima et al. Journal of Medical Case Reports (2019) 13:93 https://doi.org/10.1186/s13256-019-2026-7

CASEREPORT Open Access Adhesion-induced chronic abdominal pain: a case report on the diagnostic value of Carnett’s test Tsunetaka Kijima1*, Ryoji Hyakudomi2, Tatsuya Hashimoto3, Akari Kusaka4, Toshihiko Nakatani5 and Yutaka Ishibashi1

Abstract Background: Chronic abdominal pain is a common clinical problem. However, diagnosing chronic abdominal pain often requires detailed diagnostic evaluations in addition to sufficient history taking and physical examination, owing to its uncertain etiology. Case presentation: We report a case of a 36-year-old man with chronic abdominal pain originating from postoperative adhesions. Postoperative adhesions are common phenomena, and abdominal surgery can cause severe abdominal pain, the source of which can be difficult to detect. Carnett’s test is useful to detect abdominal wall tenderness and to determine the affected abdominal quadrant. Incorporating its use with a detailed chronological clinical history contributes to the improvement of diagnostic accuracy. In addition to the above- mentioned information, attention to subtle imaging findings may provide greater diagnostic accuracy. Conclusions: Abdominal pain induced by postoperative adhesions was reduced by laparoscopic adhesiolysis. Carnett’s test is an effective tool for evaluating pain and detecting its cause. Keywords: Chronic abdominal pain, Adhesion, Carnett’s test

Background thoracic spine [5]), abdominal wall pain (for example, Chronic abdominal pain is a relatively common clinical pain originating in the structure of the abdominal wall problem that requires detailed diagnostic evaluations, [4, 6–8]), visceral wall pain (for example, pain due to owing to its uncertain etiology. Chronic abdominal pain pelvic adhesions [9]), vascular conditions (for example, is associated with many causes, so a systematic approach aneurysm [10]), infectious diseases (for example, chla- that considers both anatomical and physiological factors mydial infection [11]), neoplasm (for example, pancre- can improve clinical reasoning [1]. An Ishikawa diagram atic cancer [2]), collagen/allergic conditions (for (fish bone diagram) is used to perform clinical reasoning example, immunoglobulin G4 [IgG4]-related disease (Fig. 1) and helps to identify the causes of chronic ab- [12], familial Mediterranean fever [13]), iatrogenic causes dominal pain associated with digestive system diseases (for example, chronic pain due to intraabdominal adhe- (for example, chronic pancreatitis [2], functional gastro- sion after operation [14–17]), and psychosomatic condi- intestinal disorders [3]), the skin/innervation (for ex- tions (for example, psychogenic abdominal pain [18, ample, abdominal cutaneous nerve entrapment 19]). Among these causes, we focused on abdominal syndrome [4]), musculoskeletal causes (including re- pain that is associated with the skin, nerve, abdominal ferred pain from the spine, such as nerve irritation muscle, and peritoneal wall [4]. This pain has some fea- caused by slipping rib syndrome or disorders of the tures that help differentiate it from pain associated with visceral diseases. Similar features include abdominal ten- derness, mild appetite loss, and nausea, and different * Correspondence: [email protected] features include pain unrelated to meals or bowel func- 1Department of General Medicine, Faculty of Medicine, Shimane University, 89-1, Enyacho, Izumo City, Shimane 693-8501, Japan tion, pain severity that is possibly related to posture, and Full list of author information is available at the end of the article

© The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Kijima et al. Journal of Medical Case Reports (2019) 13:93 Page 2 of 7

Fig. 1 An Ishikawa diagram (fish bone diagram) for a man presenting with a complaint of “chronic abdominal pain” tender pain that arises from a location that is only a few Case presentation centimeters in diameter. Because there are many similar- A 36-year-old man, originally from Latin America, pre- ities, abdominal wall pain is likely to be misdiagnosed sented at our outpatient department with complaints of and often results in inappropriate diagnostic testing, abdominal pain that had persisted for 2 months. The pa- leading to unsatisfactory treatment. Therefore, we fo- tient had first noticed right lateral abdominal pain 2 cused on Carnett’s test, a useful physical examination to months prior to the visit, and the pain was gradually distinguish abdominal wall pain from visceral pain [6]. worsening. The abdominal pain was localized in an area This test was reported by Carnett in 1926 [20]. It is per- ranging from the right upper to the right lateral abdo- formed by palpating a limited area of tenderness in a su- men. The patient had undergone cholecystectomy for pine, relaxed patient (described as Carnett’s test A; see acute cholecystitis as a 32-year-old in Latin America and Fig. 2a), which subsequently confirms continued tender- had moved to Japan for work approximately 3 years ness as the patient tenses the abdominal wall while the prior to his initial visit to our hospital. He had returned head and shoulders are elevated or while raising both to Latin America once about 6 to 7 months before pre- legs off the table (described as Carnett’s test B; see Fig. 2b senting at our clinic. When he went back to Japan, his and c). Carnett and Greenbaum et al. defined the posi- weight had increased from 130 kg to 145 kg. He did not tive sign as follows: If tensed abdominal muscles have al- experience abdominal pain immediately after his return most as much or more tenderness than the relaxed to Japan, but, as noted above, he started to gradually feel abdominal muscles, it is positive [8, 20]. Carnett hypoth- pain in the right lateral region about 2 months prior to esized that if that pain derives from a visceral source, the presentation. tensed abdominal muscles protect the underlying struc- He first visited another hospital emergency depart- tures, and therefore the tenderness should be reduced, ment 1 month after onset of the pain. Initially, gastro- whereas continued tenderness during muscle contrac- intestinal tract spasm was suspected, and he was treated tion indicates the abdominal wall as the origin of pain with tiquizium bromide. Though the medication par- [4, 20]. Moreover, Carnett’s test is reported to be useful tially relieved his abdominal pain, most of the pain per- for diagnosing psychogenic abdominal pain [18]. We re- sisted. The result of a workup by a urologist was port a case of chronic abdominal pain that took 2 years negative, even though nephrolithiasis was suspected. His to diagnose and that necessitated two operations. abdominal pain was exacerbated upon changing posture, Kijima et al. Journal of Medical Case Reports (2019) 13:93 Page 3 of 7

Fig. 2 Carnett’s test A (a) and Carnett’s test B (b, c). First, the examiner confirms the area of tenderness with the patient in a relaxed, supine position (a). Second, the examiner confirms whether tenderness continues, worsens, or decreases at the same point while the patient raises the legs (b) or elevates the head and shoulders (c) and thus it was suspected to be of somatic rather than analysis indicated red blood cell count < 1/high-power visceral origin. Abdominal pain persisted despite treat- field, white blood cell count 1–4/high-power field. Add- ment with loxoprofen sodium hydrate, and any cause of itionally, no abnormality was detected for Chlamydia abdominal pain was not detected on further evaluations, trachomatis IgG/IgA, and no abnormality was apparent including hematologic laboratory analysis, urine analysis, on the electrocardiogram. Enhanced CT revealed bilat- gastroscopy, or abdominal computed tomography (CT). eral renal stones and fatty liver. Finally, he was referred to our hospital for further We first considered abdominal wall pain due to nerve examination. entrapment because the Carnett’s test result was posi- The results of screening for depression were negative, tive; therefore, we scheduled a trigger point injection at and the patient did not have symptoms such as loss of the site of tenderness. About 2 weeks later, the patient interest, depressed feelings, or any specific changes of visited the emergency department of our hospital, surrounding conditions, such as family or work environ- reporting that his prior abdominal pain had decreased ment changes. He had no history of sexually transmitted but that he was experiencing right inguinal pain. Loxo- infection, and his vital signs were within normal limits. profen administration had no effect on the pain. Costo- His physical examination result was positive for Carnett’s vertebral angle pain was apparent on tapping, the result test, and a prior surgical scar of approximately 18 cm of urine analysis was positive for occult blood, and ab- was apparent at the right subcostal region. The patient dominal CT revealed a urinary stone at the right urinary experienced strong pain surrounding the surgical scar duct to the bladder. After pentazocine hydrochloride that was exacerbated by tapping. There were no skin was administered for pain relief, the urinary stone was rashes localized surrounding the pain. His pain exacer- passed the following day. However, the patient’s right lat- bated to 8 on a pain scale when he moved, such as dur- eral abdominal pain was not relieved. ing standing up or rolling over simultaneously. When he He felt that lying in the lateral position mostly relieved stopped moving, pain was partially relieved within 1 mi- his pain. He had occasional vomiting. The abdominal nute (3 on a pain scale). When he moved again, abdom- pain was exacerbated by movements, such as rolling inal pain was again exacerbated. Hence, he was over, standing up, walking, and coughing. Injection of awakened by the abdominal pain when rolling over. No 1% xylocaine 10 ml at a trigger point of the right lateral inflammation was detected (leukocyte count was 8580/ region led to about 30% relief in pain. The patient was mm3 and C-reactive protein was 0.10 mg/dl), and other referred to an anesthesiologist for further evaluation and laboratory findings were nonspecific, including liver/kid- treatment, who performed transverse abdominal plane ney function, blood glucose, and electrolytes. Urinary block and administered multiple analgesic medications Kijima et al. Journal of Medical Case Reports (2019) 13:93 Page 4 of 7

(tramadol hydrochloride, pregabalin, celecoxib, and sco- problems due to abdominal pain and then resumed his polamine butylbromide). These medications decreased job 6 months after the operation. the patient’s pain somewhat, and he reported that sco- However, his abdominal pain deteriorated within 1 polamine butylbromide was most effective when the month after he resumed working. He presented with pain worsened. Because the patient’s symptoms were not bleeding at the umbilicus, which was the laparoscopic port relieved after trigger point treatment to the abdominal site, and abdominal incisional hernia was confirmed on wall, we considered potential causes that might be asso- the basis of CT. Repair of the abdominal incisional hernias ciated with the location between the abdominal wall and and laparoscopic adhesiolysis were performed 8 months visceral wall or related to other sources, including psy- after the first operation. After the second operation, chosocial, physiological, and other anatomical factors. although it took time for some symptoms to improve We rechecked the abdominal CT scan for a suspected because of surgical site infection, the patient’s symptoms adhesion or abdominal hernia at the region of tender- were ultimately relieved, and he resumed his job again 5 ness due to the prior surgical procedure, and we asked a months after undergoing the second operation. Although radiologist to reevaluate the right upper abdomen in he reported mild abdominal pain and required analgesic more detail. The radiologist confirmed a slight abnor- medication, his weight decreased to 133 kg, and he was mality in the right upper abdomen and suggested the able to walk normally and work full-time, 2 years after he possibility of an adhesion around the surgical scar initially visited our hospital. The timeline of interventions (Fig. 3). We referred the patient to a gastrointestinal sur- and outcomes is shown in Additional file 1. geon for laparoscopic evaluation and adhesiolysis. The patient underwent additional investigations, including Discussion cholecystocholangiography and colonoscopy for sus- We report a patient with chronic abdominal pain in- pected postcholecystectomy syndrome, biliary dyskin- duced by abdominal adhesion and the great challenge esia, or colon abnormality. However, no cause of the we experienced in identifying the origin of his pain. Spe- abdominal pain was identified. On laparoscopic evalu- cific symptoms and physical examinations were particu- ation, a broad adhesion was observed. Adhesiolysis was larly valuable for determining the origin of the pain. performed 6 months after the patient first visited our First, we suspected that the abdominal pain originated hospital. Figure 4a shows adhesion between the periton- in the abdominal wall for the following three reasons: eum and omentum, liver, and ascending colon; Fig. 4b shows the condition after adhesiolysis. One month after 1. Our patient presented with sharp, dull pain at the adhesiolysis, the patient’s right abdomen pain level dur- right abdomen and demonstrated a positive Carnett’s ing movement improved from 8 to 2–3 on a pain scale. test. Although this test’s reliability has some Therefore, he was able to move with less pain, and he limitations, it has a 78% sensitivity and 88% specificity did not feel pain when rolling over. The result of Car- for pain arising from an abdominal wall source [4]. nett’s test was negative. After the patient started walking Abdominal wall pain is characterized by being around his house, he felt abdominal pain about 5 mi- initially sharp, followed by a dull persistent ache, and nutes after walking. Hence, he was afraid of recurrence is associated with a positive Carnett’stest[4]. of abdominal pain and felt a little depressed and frus- 2. With postural changes, such as standing up or trated because he was unable to return to work early. turning over in bed, the patient’s pain was We recommended a gradual increase in activity. He exacerbated. This situation also indicated abdominal went on a trip 4 months after the operation without wall pain. Srinivasan et al. [4] noted that pain may

AB

Fig. 3 The transverse abdominal muscle shows atrophy at the postoperative scar on plain abdominal coronal computed tomography (a) and plain abdominal transverse computed tomography (b). Arrows indicate adhesive region of the greater omentum Kijima et al. Journal of Medical Case Reports (2019) 13:93 Page 5 of 7

Fig. 4 Adhesion was observed between the peritoneum and greater omentum, liver, and ascending colon (a). Laparoscopic adhesiolysis was conducted at those adhesive regions (b)

radiate diffusely when abdominal wall pressure association of chronic abdominal pain with intraperito- increases or nerve traction occurs during standing, neal adhesion, particularly in the fields of obstetrics/ lifting, walking, or coughing. gynecology and gastroenterological surgery [9, 27–29]. 3. The patient’s pain was chronic abdominal pain, Kresch et al. [9] compared laparoscopic findings be- which is a feature of abdominal wall pain that can tween 100 women with chronic pelvic pain who under- easily become chronic [4]. went the procedure to identify the source of pain and 50 asymptomatic women who underwent the procedure for Some studies suggest that in approximately 10–30% of tubal ligation. They found that 83% of women with all patients with chronic abdominal pain, the abdominal chronic abdominal pain had abnormal pelvic organs, in- wall is the origin [4, 21]. More than 90% of patients with cluding adhesions, compared with 29% of asymptomatic chronic abdominal pain have a positive Carnett’s test re- women and concluded that while adhesions can cause sult, and the condition can be relieved by local pain, not all of them cause pain [9]. Adhesions were anesthetic injection in the trigger points [22, 23]. Hence, qualitatively different between the symptomatic and con- we referred the patient to an anesthesiologist. When the trol groups, such that in patients with chronic pain and anesthesiologist conducted a local injection with anes- adhesions, there was restriction of motion or expansibil- thetics, our patient’s abdominal pain was reduced but ity in one or more organs. Moreover, adhesions involv- did not change substantially. Carnett [20] also indicated ing the parietal peritoneum or bowel are more likely to that there are three sensory layers of the abdominal wall cause pain than adhesions involving other sites [9, 28]. that cause tenderness: the skin, muscles, and periton- Considering these studies, the history of abdominal sur- eum. Finally, we speculated that the pain was associated gery, severe abdominal pain during moving, positive with the abdominal wall and, in particular, considered Carnett’s test result, and ineffective local anesthesia that a peritoneal adhesion might also be involved be- made us suspect the cause of abdominal pain to be the cause local anesthetic injections were less effective. peritoneum adhesion. The patient’s pain was not only sharp but also dull, We referred the patient to the department of digestive and it was relieved by scopolamine butylbromide rather surgery, where he underwent diagnostic laparoscopy and than loxoprofen sodium hydrate. In addition, he experi- adhesiolysis 7 months after the onset of abdominal pain. enced nausea. These features are associated with visceral That pain was relieved after the first laparoscopic adhe- pain. Scopolamine butylbromide is used to treat abdom- siolysis. Mueller et al. [28] recommended that patients inal pain associated with cramps induced by gastrointes- with chronic abdominal pain lasting more than 6 tinal spasms as well as biliary acute spasm or renal colic months who have a history of laparotomy or pelvic in- [24, 25]. The pain emanated from around a postopera- flammatory disease should undergo diagnostic laparos- tive scar, and a history of former abdominal surgery is copy. The application of laparoscopic adhesiolysis for the most important predictive factor for adhesion forma- chronic abdominal pain is controversial. Some studies tion. In addition, the most common complications asso- have reported significant reduction in chronic abdominal ciated with adhesion are small bowel obstruction and pain after laparoscopic adhesiolysis [14, 30, 31]. Inter- chronic pain syndrome [26]. Chlamydial infection is mittent nausea and vomiting with colicky pain may be known to bring about right upper quadrant abdominal associated with intestinal adhesion, and in patients with pain due to intraabdominal adhesion (Fitz-Hugh–Curtis a history of endometriosis or surgery, laparoscopic adhe- syndrome); however, chlamydial antibodies were nega- siolysis is effective for the treatment of chronic pelvic tive in our patient. Many studies have addressed the pain in the long term [16]. In contrast, other studies Kijima et al. Journal of Medical Case Reports (2019) 13:93 Page 6 of 7

have reported that pain reduction does not differ be- Acknowledgements tween laparoscopic adhesiolysis and diagnostic laparos- The authors thank Professor Douglas S. Paauw, director of the medical student program, University of Washington, for insightful comments and copy alone and therefore they did not recommend suggestions. laparoscopic adhesiolysis as a treatment for adhesions in patients with chronic abdominal pain [31, 32]. In care- Funding The Department of General Medicine, Faculty of Medicine, Shimane fully selected patients with chronic abdominal pain, the University, is an endowment department supported by Oda City. positive effects of laparoscopic adhesiolysis can be sus- tained beyond 15 years after the surgery [33]. In one Availability of data and materials The data that support the findings of this report are available from the study, some patients (43%) underwent repeat adhesioly- corresponding author upon reasonable request. sis because of pain aggravation [14], and it took approxi- mately 6 months for the pain to decrease from an Authors’ contributions TK wrote the first draft of the manuscript. YI wrote and supervised the paper. average preoperative score of 8 to a postoperative score RH, TH, AK, and TN supervised the paper. All authors read and approved the of 2 [15]. This patient improved for movements such as final manuscript. standing up and walking, as well as turning over in bed Ethics approval and consent to participate after laparoscopic adhesiolysis; therefore, he could re- Not applicable. sume his work. Abdominal adhesion does not always re- quire laparoscopic adhesiolysis; however, if the adhesion Consent for publication Written informed consent was obtained from the patient for publication of causes restrictions in daily life activities, this procedure this case report and any accompanying images. A copy of the written should be considered. consent is available for review by the Editor-in-Chief of this journal. An association between obesity and ventral hernia has Competing interests been reported [34], and our patient gained weight just The authors declare that they have no competing interests. before abdominal pain was noticed. Therefore, obesity may have been one factor in the etiology of the patient’s Publisher’sNote pain and his need for a second operation. We considered Springer Nature remains neutral with regard to jurisdictional claims in the possibility that weight gain was associated with this published maps and institutional affiliations. pathology. Increased obesity increases the amount of vis- Author details ceral fat, which may cause adhesions involving the par- 1Department of General Medicine, Faculty of Medicine, Shimane University, 2 ietal peritoneum or bowel to restrict motion or 89-1, Enyacho, Izumo City, Shimane 693-8501, Japan. Department of Digestive and General Surgery, Faculty of Medicine, Shimane University, 89-1, expansibility, ultimately causing more severe pain, as Enyacho, Izumo City, Shimane 693-8501, Japan. 3Palliative Care Center, noted by Kresch et al. [9]. Therefore, we advised our pa- Shimane University Hospital, 89-1, Enyacho, Izumo City, Shimane 693-8501, 4 tient to lose weight, and his symptoms did finally im- Japan. Department of Anesthesiology, Faculty of Medicine, Shimane University, 89-1, Enyacho, Izumo City, Shimane 693-8501, Japan. 5Department prove with the restoration of his original weight. of Palliative Care, Faculty of Medicine, Shimane University, 89-1, Enyacho, Izumo City, Shimane 693-8501, Japan.

Conclusions Received: 30 October 2018 Accepted: 21 February 2019 Physicians should be aware of Carnett’s test as part of the evaluation of chronic undiagnosed abdominal pain, References because many physicians are not familiar with this test 1. Wong KC. How to apply clinical cases and medical literature in the and do not routinely perform it. It is difficult to detect framework of a modified “failure mode and effects analysis” as a clinical reasoning tool—an illustration using the human biliary system. J Med Case the cause of chronic abdominal pain. If there is evidence Rep. 2016;10:85. of adhesion-induced abdominal pain based on the pa- 2. Kaufman M, Singh G, Das S, Concha-Parra R, Erber J, Micames C, et al. tient’s history, symptoms, and physical examination, es- Efficacy of endoscopic ultrasound-guided celiac plexus block and celiac plexus neurolysis for managing abdominal pain associated with chronic pecially when the patient has a positive result for pancreatitis and pancreatic cancer. J Clin Gastroenterol. 2010;44:127–34. Carnett’s test, physicians should carefully assess the find- 3. Drossman DA. The functional gastrointestinal disorders and the Rome II ings of imaging modalities such as CT and consider re- process. Gut. 1999;45(Suppl 2):II1–5. 4. Srinivasan R, Greenbaum DS. Chronic abdominal wall pain: a frequently ferring the patient to a digestive surgeon for diagnostic overlooked problem: practical approach to diagnosis and management. Am laparoscopy and adhesiolysis. J Gastroenterol. 2002;97:824–30. 5. Sharpstone D, Colin-Jones DJG. Chronic, non-visceral abdominal pain. Gut. 1994;35:833–6. Additional file 6. Suleiman S, Johnston DE. The abdominal wall: an overlooked source of pain. Am Fam Physician. 2001;64:431–8. 7. Gallegos N, Hobsley M. Abdominal wall pain: an alternative diagnosis. Br J Additional file 1: Timeline of interventions and outcomes. (DOCX 55 kb) Surg. 1990;77:1167–70. 8. Greenbaum DS, Greenbaum RB, Joseph JG, Natale JE. Chronic abdominal wall pain. Dig Dis Sci. 1994;39:1935–41. Abbreviation 9. Kresch AJ, Seifer DB, Sachs LB, Barrese I. Laparoscopy in 100 women with CT: Computed tomography chronic pelvic pain. Obstet Gynecol. 1984;64:672–4. Kijima et al. Journal of Medical Case Reports (2019) 13:93 Page 7 of 7

10. Mousavi SR, Zirakzadeh H, Samsami M, Fallah M, Shirazi FH. Superior mesenteric artery aneurysm in a patient with chronic abdominal pain. Acta Med Iran. 2011;49:766–8. 11. Ghaly AF, Chien PF. Chronic pelvic pain: clinical dilemma or clinician’s nightmare. Sex Transm Infect. 2000;76:419–25. 12. Mannion M, Cron RQ. Successful treatment of pediatric IgG4 related systemic disease with mycophenolate mofetil: case report and a review of the pediatric autoimmune pancreatitis literature. Ped Rheumatol Online J. 2011;9:1. 13. Simon A, van der Meer JW, Drenth JP. Familial Mediterranean fever—a not so unusual cause of abdominal pain. Best Pract Res Clin Gastroenterol. 2005; 19:199–213. 14. Shayani V, Siegert C, Favia P. The role of laparoscopic adhesiolysis in the treatment of patients with chronic abdominal pain or recurrent bowel obstruction. JSLS. 2002;6:111–4. 15. McClain GD, Redan JA, McCarus SD, Caceres A, Kim J. Diagnostic laparoscopy and adhesiolysis: does it help with complex abdominal and pelvic pain syndrome (CAPPS) in general surgery? JSLS. 2011;15:1–5. 16. Nezhat FR, Crystal RA, Nezhat CH, Nezhat CR. Laparoscopic adhesiolysis and relief of chronic pelvic pain. JSLS. 2000;4:281–5. 17. Perkins FM, Kehlet H. Chronic pain as an outcome of surgery: a review of predictive factors. Anesthesiology. 2000;93:1123–33. 18. Takada T, Ikusaka M, Ohira Y, Noda K, Tsukamoto T. Diagnostic usefulness of Carnett’s test in psychogenic abdominal pain. Intern Med. 2011;50:213–7. 19. Matsunaga S, Eguchi Y. Importance of a physical examination for efficient differential diagnosis of abdominal pain: diagnostic usefulness of Carnett’s test in psychogenic abdominal pain. Intern Med. 2011;50:177–8. 20. Carnett JB. Intercostal neuralgia as a cause of abdominal pain and tenderness. Surg Gynecol Obstet. 1926;42:625–32. 21. Gray DW, Dixon JM, Seabrook G, Collin J. Is abdominal wall tenderness a useful sign in the diagnosis of non-specific abdominal pain? Ann R Coll Surg Engl. 1988;70:233–4. 22. Lindsetmo RO, Stulberg J. Chronic abdominal wall pain—a diagnostic challenge for the surgeon. Am J Surg. 2009;198:129–34. 23. van Assen T, Brouns JA, Scheltinga MR, Roumen RM. Incidence of abdominal pain due to the anterior cutaneous nerve entrapment syndrome in an emergency department. Scand J Trauma Resusc Emerg Med. 2015;23:19. 24. Tytgat GN. Hyoscine butylbromide: a review of its use in the treatment of abdominal cramping and pain. Drugs. 2007;67:1343–57. 25. Tytgat GN. Hyoscine butylbromide – a review on its parenteral use in acute abdominal spasm and as an aid in abdominal diagnostic and therapeutic procedures. Curr Med Res Opin. 2008;24:3159–73. 26. Szomstein S, Lo Menzo E, Simpfendorfer C, Zundel N, Rosenthal RJ. Laparoscopic lysis of adhesions. World J Surg. 2006;30:535–40. 27. Hermann RE. Chronic lower abdominal pain. JAMA. 1990;264:2450. 28. Mueller MD, Tschudi J, Herrmann U, Klaiber C. An evaluation of laparoscopic adhesiolysis in patients with chronic abdominal pain. Surg Endosc. 1995;9: 802–4. 29. Freys S, Fuchs K, Heimbucher J, Thiede A. Laparoscopic adhesiolysis. Surg Endosc. 1994;8:1202–7. 30. Keltz MD, Gera PS, Olive DL. Prospective randomized trial of right-sided paracolic adhesiolysis for chronic pelvic pain. JSLS. 2006;10:443. 31. Brüggmann D, Tchartchian G, Wallwiener M, Münstedt K, Tinneberg HR, Hackethal A. Intra-abdominal adhesions: definition, origin, significance in surgical practice, and treatment options. Dtsch Arztebl Int. 2010;107:769–75. 32. Swank D, Swank-Bordewijk S, Hop W, Van Erp W, Janssen I, Bonjer H, et al. Laparoscopic adhesiolysis in patients with chronic abdominal pain: a blinded randomised controlled multi-centre trial. Lancet. 2003;361:1247–51. 33. Paajanen P, Fagerstrom A, Paajanen H. Laparoscopic adhesiolysis in chronic abdominal pain: 15-year follow-up study. J Clin Gastroenterol. 2018;52:e32–6. 34. Rose M, Eliakim R, Bar-Ziv Y, Vromen A, Rachmilewitz D. Abdominal wall hernias: the value of computed tomography diagnosis in the obese patient. J Clin Gastroenterol. 1994;19:94–6. HHS Public Access Author manuscript

Author Manuscript Author ManuscriptEpigenomics Author Manuscript. Author manuscript; Author Manuscript available in PMC 2016 April 01. Published in final edited form as: Epigenomics. 2015 April ; 7(2): 235–245. doi:10.2217/epi.14.75.

Epigenetic regulation of chronic pain

Lingli Liang1, Brianna Marie Lutz2, Alex Bekker1, and Yuan-Xiang Tao1,3,* 1Department of Anesthesiology, New Jersey Medical School, Rutgers, The State University of New Jersey, Newark, NJ 07103, USA 2Rutgers Graduate School of Biomedical Sciences, New Jersey Medical School, Rutgers, The State University of New Jersey, Newark, NJ 07103, USA 3Departments of Cell Biology & Molecular Medicine, Neurology & Neuroscience, and Physiology & Pharmacology, New Jersey Medical School, Rutgers, The State University of New Jersey, Newark, NJ 07103, USA

Abstract Chronic pain arising from peripheral inflammation and tissue or nerve injury is a common clinical symptom. Although intensive research on the neurobiological mechanisms of chronic pain has been carried out during previous decades, this disorder is still poorly managed by current drugs such as and non-steroidal anti-inflammatory drugs. Inflammation-, tissue injury-, and/or nerve injury-induced changes in gene expression in sensory neurons of the dorsal root ganglion (DRG), spinal cord dorsal horn, and pain-associated brain regions are thought to participate in chronic pain genesis; however, how these changes occur is still elusive. Epigenetic modifications including DNA methylation and covalent histone modifications control gene expression. Recent studies have shown that peripheral noxious stimulation changes DNA methylation and histone modifications and that these changes may be related to the induction of pain hypersensitivity under chronic pain conditions. This review summarizes the current knowledge and progress in epigenetic research in chronic pain and discusses the potential role of epigenetic modifications as therapeutic antinociceptive targets in this disorder.

Keywords DNA methylation; Histone acetylation; Histone methylation; Dorsal root ganglion; Spinal cord; Inflammatory pain; Neuropathic pain; Chronic pain

Introduction Chronic pain is a major public health problem that affects approximately 30% of the general population in the USA. It is a cause of grave physiological and psychological distress in

*Address correspondence to Dr. Tao: Department of Anesthesiology, New Jersey Medical School, Rutgers, The State University of New Jersey, 185 S. Orange Ave., MSB, F-548, Newark, NJ 07103. Tel: +1-973-972-9812; Fax: +1-973-972-1644. [email protected]. Disclosure of Potential Conflicts of Interest No potential conflicts of interest were disclosed. Liang et al. Page 2

those affected, and it places significant pressures on the health care system. About 100 Author Manuscript Author Manuscript Author Manuscript Author Manuscript billion US dollars are spent on chronic pain related health care expenses, and many patients experience a loss of productivity [1]. Chronic pain usually arises from inflammation, or tissue and nerve injury. Although intensive research on the neurobiological mechanisms of chronic pain has been carried out during previous decades, this disorder is still poorly managed by current drugs such as opioids and non-steroidal anti-inflammatory drugs, which are ineffective and/or produce severe side effects [2]. Peripheral inflammation and nerve injury produce transcriptional and translational changes in the expression of receptors, enzymes, ion channels, neurotransmitters, neuromodulators, and structural proteins in primary sensory neurons of dorsal root ganglion (DRG), spinal cord, and other pain-related regions in the brain [2–4]. These changes contribute to the induction and maintenance of chronic pain; however, how these changes are regulated by peripheral noxious stimuli is still not fully understood.

Recent studies have suggested that the mechanism for gene regulation involves epigenetic modifications. Environmental toxins, medications, diet, and psychological stress alter epigenetic processes such as DNA methylation, covalent histone modification (e.g., acetylation and methylation), and non-coding RNA expression. Accumulating evidence demonstrates that these processes play an important role in synaptic plasticity during memory formation as epigenetic changes correlate with hippocampal activity [5–10]. Given that peripheral and central sensitization under chronic pain conditions share common mechanisms with the neuronal plasticity of memory formation, it is very likely that similar epigenetic mechanisms occur under both conditions. Indeed, peripheral inflammation and nerve injury drive changes in DNA methylation, histone modifications, and non-coding RNAs in pain-related regions [8;9;11–14]. These changes might be responsible for inflammation/nerve injury-induced alterations of some pain-associated genes in central neurons. The evidence suggests that modification of epigenetic processes participates in the mechanisms that underlie the induction and maintenance of chronic pain.

The role of non-coding RNAs including microRNAs and long non-coding RNAs in chronic pain has recently been discussed [8]. This article focuses on the evidence for the changes in DNA methylation and histone modification, mostly in DRG and spinal cord, under chronic pain conditions. We explore how these changes are induced by peripheral noxious stimuli and how these epigenetic processes regulate pain related genes. We finally deduce potential mechanisms of how the changes in DNA methylation and histone modification contribute to the development and maintenance of chronic pain.

1. Histone modification in chronic pain 1a. The Process of histone modification The nucleosome is the basic unit of chromatin, composed of about 140 base pairs of DNA wrapped around a histone octamer. Histones are small, alkaline proteins categorized into five major families: H1/H5, H2A, H2B, H3 and H4. Histones H2A, H2B, H3 and H4 are known as the core histones, while histones H1 and H5 are known as linker histones. The N- terminal histone tail protrudes from the nucleosome and can be post-translationally modified, including acetylation, methylation, phosphorylation, citrullination, SUMOylation,

Epigenomics. Author manuscript; available in PMC 2016 April 01. Liang et al. Page 3

ubiquitination, and ADP-ribosylation [15]. These modifications result in changes in the Author Manuscript Author Manuscript Author Manuscript Author Manuscript three-dimensional chromatin structure and gene expression [15].

1b. Histone acetylation and deacetylation in chronic pain Histone acetylation and deacetylation are the processes by which the histones on lysine residues within the N-terminal tail and on the surface of the nucleosome core are acetylated by histone acetyltransferase (HAT) or deacetylated by histone deacetylases (HDACs) [16]. Acetyl-Coenzyme A is the major source of the acetyl group in histone acetylation [16–19]. Conventionally, histone acetylation makes the condensed chromatin into a more relaxed structure, and consequently promotes gene transcription. In contrast, histone deacetylation tightly condenses chromatin resulting in gene silencing [16] (Figure 1).

Effect of HDAC inhibitors on inflammatory pain—Evidence from a pharmacological study has shown that HDAC inhibitors can relieve inflammatory pain [20–22]. A 5-day subcutaneous treatment with either of the two HDAC inhibitors, MS-275 and SAHA, substantially reduced nociceptive behaviors in the second phase of the formalin test and led to an increase in mGluR2 (but not mGluR1a, mGluR4 or mGluR5) in the dorsal root ganglion (DRG) [21](Table 1). This antinociception could be abrogated by an mGlu2/3 receptor antagonist. The induction of DRG mGlu2 receptors in response to SAHA was associated with increased acetylation of p65/RelA on lysine 310, a process that enhances the transcriptional activity of p65/RelA at nuclear factor-kappaB-regulated genes [21]. Given that transcription of the mGlu2 receptor gene is activated by p65/RelA in DRG neurons, HDAC inhibition may produce antinociception by up-regulating mGlu2 receptor expression in DRG. However, it is not clear whether formalin injection changes the histone acetylation conditions and HDAC expression and activity in the DRG.

It was reported that complete Freund’s adjuvant (CFA)-induced peripheral inflammation increased the levels of class IIa HDAC members (HDAC4, 5, 7, 9), but not class I HDAC members (HDAC1, 2, 3), in the spinal dorsal horn [20]. Intrathecal administration of HDAC inhibitors targeting class II (SAHA, TSA, LAQ824) or IIa (VPA, 4-PB) significantly delayed the development of thermal hyperalgesia and attenuated existing thermal hyperalgesia in a CFA-induced inflammatory pain model [20] (Table 1). It appears that class I and II or IIa HDAC members function differentially in inflammatory pain models. A recent investigation from Zhang and colleagues revealed the epigenetic mechanism of inflammatory pain in central pain-modulating neurons. CFA induced the increase of global histone H3 and H4 acetylation in brainstem nucleus raphe magnus (NRM), a crucial supraspinal site for maintenance of pain hypersensitivity [22]. However, acetylated H3 was reduced in the Gad2 gene promoter region which epigenetically suppresses the transcription of Gad2 (encoding glutamic acid decarboxylase 65) and consequently causes impaired inhibitory function. Local injection of HDAC inhibitors TSA and SAHA into NRM reversed this effect and produced a similar analgesic effect on CFA-induced inflammatory pain using systemic administration [22](Table 1).

Effect of HDAC inhibitors on visceral pain—One study on visceral pain also provided evidence to support the involvement of central epigenetic mechanisms in pain [23].

Epigenomics. Author manuscript; available in PMC 2016 April 01. Liang et al. Page 4

Intracerebroventricular administration of TSA significantly attenuated water avoidance Author Manuscript Author Manuscript Author Manuscript Author Manuscript stress induced visceral hypersensitivity in rats [23] (Table 1). However, the targets of histone acetylation were not known in this study.

Effects of HAT inhibitors and HDAC inhibitors on neuropathic pain—Several groups reported that the HDAC inhibitors had an antinociceptive effect in neuropathic pain. Oral administration of Sodium butyrate, a HDAC inhibitor, not only attenuated chronic constriction injury (CCI)-induced pain hypersensitivity but also reduced the CCI-induced increase in TNF-αin the sciatic nerve [24] (Table 1). However, how HDAC inhibition affects TNF-α expression under CCI-induced neuropathic pain conditions is unknown. Intrathecal pre-treatment with class I HDAC inhibitors (MS-275 or MGDC0103) attenuated mechanical and thermal hypersensitivity in models of traumatic nerve injury and antiretroviral drug (stavudine)-induced peripheral neuropathy [25] (Table 1). This analgesic effect may be related to the increase in global H3K9ac in the spinal cord but not in DRG, suggesting that any potential mechanism could be found in the central nervous system [25]. Interestingly, the acetylation changes at the promoters of some pain-related genes, like mu receptor, Kv4.3, Nav1.8, and brain-derived neurotrophic factor (BDNF), in DRG neurons have been reported in a neuropathic pain model [26–29] (Table 1). Nerve injury- induced reductions of histone H3 and H4 acetylation at the promoter regions of mu receptor, Nav1.8, and Kv4.3 silence their expression in DRG and may represent an underlying cause of common negative symptoms associated with neuropathic pain [27;28], whereas nerve injury-promoted increases in histone H3 and H4 acetylation at the promoter regions of BDNF in DRG and in histone H4 acetylation at the promoter regions of Cdk5 in spinal cord up-regulates the expression of BDNF and CdK5, respectively, and may contribute to the induction or maintenance of neuropathic pain [29;30] (Table 1).

Interestingly, data from other groups showed that HAT inhibitors also had an antinociceptive effect in neuropathic pain. The HAT inhibitor anacardic acid relieved spinal nerve ligation (SNL)-induced neuropathic pain by suppressing the hyperacetylation of histone H3 in the promoter region of macrophage inflammatory protein 2 (MIP-2) and its receptor chemokine CC motif receptor 2 (CXCR2), resulting in the blockade of SNL- induced up-regulation of MIP-2 and CXCR2 in the injured sciatic nerve [31;32] (Table 1). In another report, CCI increased the expression of p300, a HAT E1A binding protein, in the lumbar spinal cord [33;34] (Table 1). Intrathecal administration of p300 shRNA or an inhibitor of p300 HAT reversed CCI-induced mechanical allodynia and thermal hyperalgesia and suppressed the expression of cyclooxygenase-2 (COX-2) in spinal cord [33;34](Table 1). Consistently, the intrathecal administration of resveratrol, an activator of Sirt1 (a classic III HDAC), attenuated CCI-induced mechanical allodynia and thermal hyperalgesia, reversed the CCI-induced decrease in spinal Sirt1, and blocked the CCI- induced increase in spinal histone H3 acetylation [35]. The analgesic effect of HAT inhibition was also reported in other persistent pain conditions. In an incision model, injection of the HAT inhibitor anacardic acid intraperitoneally reduced incision-induced pain hypersensitivity [36;37]. As expected, the HDAC inhibitor suberoylanilide hydroxamic acid exacerbated mechanical hypersensitivity after incision [36;37] (Table 1). Given that neuropathic pain and opioid tolerance/opioid-induced hyperalgesia share some common

Epigenomics. Author manuscript; available in PMC 2016 April 01. Liang et al. Page 5

intracellular pathways in their mechanisms [38], the evidence suggests that histone Author Manuscript Author Manuscript Author Manuscript Author Manuscript modification is also involved in the development and maintenance of opioid tolerance and opioid-induced hyperalgesia. Indeed, daily administration of the HAT inhibitor curcumin with for 4 days reduced the development of morphine-induced mechanical allodynia, thermal hyperalgesia, tolerance, and physical dependence [39]. Conversely, the HDAC inhibitor SAHA enhanced these responses [39] (Table 1). The intrathecal injection of resveratrol suppressed the established morphine analgesic tolerance, reversed the morphine- induced decrease in spinal Sirt1, and attenuated the morphine-induced increase in spinal histone H3 acetylation [40]. Interestingly, baicalin, a flavonoid compound isolated from Huang Qin, ameliorated SNL-induced neuropathic pain by suppressing HDAC1 expression and preventing histone-H3 acetylation in the spinal cord dorsal horn [41] (Table 1).

Given that the degree of histone acetylation is controlled by the enzymes HATs and HDACs, conventionally, HAT inhibitors or HDAC activators should have opposite effects compared to HDAC inhibitors. Based on provious observations described above, how HATs and HDACs are involved in neuropathic pain is still elusive. The role of histone acetylation and deacetylation in neuropathic pain remains to be verified.

1c. Histone methylation and demethylation in chronic pain Histone methylation is another process of histone modification by which methyl groups are transferred to amino of histone proteins in (Figure 1). Histone methylation is catalyzed by S-adenosylmethionine- (SAM) dependent histone lysine methyltransferases (KMTs) and protein arginine methyltransferases (PRMTs), whereas histone demethylation is catalyzed by histone N-methylated lysine residue demethylases and the peptidyl arginine deiminases [15;42]. Histone methylation could repress or activate gene transcription depending on the sites and content being methylated. In general, methylation of histone H3 at Lys9 or Lys27 (H3K9 or H3K27) or histone H4 at Lys20 (H4K20) correlates with transcriptional repression, whereas methylation of H3K4, H3K36 and H3K79 correlates with enhanced transcription [15].

Although histone methylation has been reported to participate in the mechanism of formation of long-term memories and learning [7;43–45], the role of histone methylation in chronic pain is still unclear. Evidence indicates that histone methylation may be related to the expression of chemokine (C-C motif) ligands (CCLs), a class of small cytokines, in neuropathic pain [32;46]. The peripheral nerve injury-induced reduction in H3K27me3 in the promoter region of monocyte chemotactic protein 3 (MCP-3, known as CCL7) might be responsible for the nerve injury-induced increase in the expression of MCP-3 in spinal cord [46]. Interleukin 6 may be involved in this response as the increased MCP-3 expression was almost abolished in interleukin 6 knockout mice with partial sciatic nerve ligation [46]. Peripheral nerve injury also increased the mRNA levels of CCL2, CCL3 and their receptors (CCR2 and CCR1/CCR5, respectively) in the injured sciatic nerve. These increases could be related to the increased H3K4me3 in the promoter regions of these cytokine genes [32]. An increase in global histone methylation was also observed in spinal cord after intrathecal injection of pertussis toxin, which induced significant thermal hyperalgesia [47]. However, whether these methylation sites are really required for these changes in gene expression is

Epigenomics. Author manuscript; available in PMC 2016 April 01. Liang et al. Page 6

unknown. Furthermore, whether nerve injury-induced changes in histone methylation Author Manuscript Author Manuscript Author Manuscript Author Manuscript contribute to neuropathic pain remains to be investigated. A recent study showed that an increase in the expression of MeCP2 in mouse central nucleus of the amygdala (CeA) was caused by both CFA-induced chronic inflammatory pain and repeated morphine exposure [48]. The increased MeCP2 bound to and repressed the transcriptional repressor histone dimethyltransferase G9a, resulting in a reduction in G9a-catalyzed repressive marker H3K9me2 and an increase in the expression of brain-derived neurotrophic factor in CeA [48]. Overexpression of CeA MeCP2 or knockdown of CeA G9a facilitated behavior of morphine reward, whereas knockdown of CeA MeCP2 inhibited behavior of morphine reward [48]. Whether such epigenetic cascade occurs in neuropathic pain needs to be confirmed.

2. DNA methylation and chronic pain 2a. Process of DNA methylation In mammalian cells, DNA methylation is a biochemical process, in which a methyl group is added to the 5th carbon of cytosine residues situated adjacent to a guanine residue (CpG site). DNA sequences with a high concentration of CpG residues are referred to as CpG islands and are generally located at the start of the gene sequence within the promoter region (Figure 2). The process of DNA methylation is mediated by a group of DNA methyltransferases (DNMTs) that includes DNMT1, DNMT3a, and DNMT3b; this protein family also includes DNMT2, an inactivated isoform, and DNMT3L, which lacks the conserved catalytic domain [49–51]. DNMT1 maintains the methylation of DNA that is already established at the genome and is considered to be the primary maintenance DNMT [50;51]. Both DNMT3a and DNMT3b act as de novo methyltransferases and methylate unmethylated DNA [50;51]. Evidence has now expanded the role of DNMT1 to also include facilitation of de novo DNA methylation by DNMT3a and DNMT3b at gene promoters [6;50;52–54].

DNA methylation interferes with gene transcription by (1) physically interfering with the binding of transcription factors and (2) serving as docking sites for methyl-CpG-binding domain proteins (MBDs) [55;56] (Figure 2). MBDs contain a specific domain of ~70 residues, the methyl-CpG-binding domain, which directly binds to one or more methylated CpGs of a gene promoter. MBDs function as docking sites, in which they recruit other transcriptional co-repressors, such as histone deacetylases (HDAC), to the targeted gene for gene silencing, or co-activators, such as CREB1, for transcription activation [55–57]. The ability to recruit these proteins may be why DNA methylation has such a profound effect on gene expression.

2b. DNA methytransferases and DNA methylation in chronic pain Effect of the DNMT inhibitors on inflammatory pain—Although the function of DNA methylation has been reported in other pathological states, so far, only a few studies have demonstrated the potential role of DNA methylation and the activity and expressional levels of DNMTs in pain. Cystathionine-β-synthase (Cbs) synthesizes hydrogen sulfide, an endogenous gas molecule, which is necessary and sufficient to elicit mechanical pain

Epigenomics. Author manuscript; available in PMC 2016 April 01. Liang et al. Page 7

hypersensitivity and increased excitability of DRG neurons. Peripheral inflammation Author Manuscript Author Manuscript Author Manuscript Author Manuscript induced by CFA leads to demethylation of the cystathionine-β-synthase (cbs) gene in DRG [58]. This demethylation may be associated with the CFA-induced increase in expression of Cbs mRNA and protein in DRG and could influence the induction of inflammation-induced mechanical hypersensitivity [58]. Given the fact that peripheral inflammation did not decrease DNMT expression and activity in DRG [12], it is unclear how demethylation occurs in DRG under chronic inflammatory pain conditions. The level of DNA methylation is controlled by both DNMTs and demethylation enzymes (e.g., ten-eleven translocation dioxygenases). Whether peripheral inflammation changes the expression and activity of DNA demethylation enzymes in DRG remains to be determined. Interestingly, a recent study reported the CFA-induced hypermethylation of CpG islands in the miR-129 promoter in spinal cord neurons [59]. This methylation may regulate chronic inflammatory pain by targeting CaMKIIγ [59].

Effect of the DNMT inhibitors on neuropathic pain—In addition to peripheral inflammation, peripheral nerve injury caused by sciatic nerve chronic constriction injury (CCI) increased the level of global DNA methylation in the spinal cord [60]. Blocking spinal cord DNA methylation with intrathecal 5-azacytidine attenuated CCI-induced thermal and mechanical pain hypersensitivities [60]. CCI also increased the level of DNA methylation in the proximal promoter region of the μ gene in DRG [61]. This increase may be related to the CCI-induced decrease in the analgesic effect of opioids [61]. In the spared nerve injury-induced neuropathic pain model, DNMT1 and DNMT3a (but not DNMT3b) transcripts were up-regulated in the injured DRGs [62]. Interestingly, the level of global DNA methylation was reduced in the prefrontal cortex and amygdala (but not the visual cortex and thalamus) following spared nerve injury [63]. This reduction strongly correlated with the severity of pain behaviors [63]. It appears that nerve injury-induced changes in DNA methylation are spatially different in the central nervous system and are implicated in distinct functions in spinal and supraspinal levels under neuropathic pain conditions. However, which type of cells in the nervous system express these changes is elusive. A recent study reported that DNMT1 was found in both neurons and satellite glial cells of DRG, DNMT3a in DRG satellite glial cells, and DNMT3b in DRG neurons [62], but the conclusion remains uncertain because no specific neuronal and glial markers were used [62]. Additionally, the specificity and selectivity of the antibodies used were not addressed [62]. These earlier studies raise several unanswered questions. For example, is the expression and/or activity of DNMTs and demethylation enzymes spatially and temporally changed following peripheral inflammation or nerve injury? If so, which type of DNMT? What are the downstream targeted genes of DNMTs under chronic pain conditions? Do DNMT inhibitors lead to side effects in addition to antinociception given that they are pharmacologically non-selective for specific DNMTs? Broader future investigations are required.

DNA methylation in patients with chronic pain—Alterations of DNA methylation have also been observed in patients with painful diseases. Women with fibromyalgia showed significant differences in DNA methylation patterns compared to aged-matched healthy controls, when genomic DNA isolated from whole blood was examined [64]. Fibromyalgia

Epigenomics. Author manuscript; available in PMC 2016 April 01. Liang et al. Page 8

associated genes with differential methylation include brain-derived neurotrophic factor, Author Manuscript Author Manuscript Author Manuscript Author Manuscript histone deacetylase 4, N-Acetyltransferase 15, protein kinase C alpha, and protein kinase G1 [64]. Increased methylation at the extracellular matrix protein SPARC (Secreted Protein, Acidic, Rich in Cysteine) gene promoter was reported in patients experiencing chronic low back pain associated with disc degeneration [65]. The endothelin B receptor gene promoter was heavily methylated in human oral squamous cell carcinoma lesions, which are highly painful, whereas this promoter was not methylated in human oral dysplasia lesions, which are typically not painful [66]. A regulatory DNA methylation region in the CpG-island shore of the TRPA1 promoter was reported to have a possible impact on TRPA1 gene expression and thermal sensitivity [67]. Joint resident synovial fibroblasts from patients with rheumatoid arthritis exhibited a global hypomethylation or both hypomethylation and hypermethylation patterns compared to patients with osteoarthritis or healthy controls [68– 70]. This hypomethylation was identified in key genes relevant for rheumatoid arthritis, related to multiple pathways, and associated with increased gene expression [70]. A reduction of DNMT1 and an increase in the expression of S-adenosyl methionine decarboxylase, spermidine/spermine N1-acetyltransferase, and polya-mine-modulated factor1-binding protein1 may be associated with hypomethylation in rheumatoid arthritis [68;71]. Additionally, promoter methylation states of the death receptor 3, interleukin (IL) 6, IL10, ILR2 and chemokine ligand 12 genes were altered in blood mononuclear cells and synovial fibroblasts in rheumatoid arthritis [42;72]. It appears that DNA methylation has the potential to serve as a biomarker for some types of painful disorders (e.g., autoimmune disorders or inflammation).

2c. methyl-CpG-binding domain proteins (MBDs) in chronic pain As discussed above, DNA methylation-triggered gene transcriptional changes require a family of MBDs. The MBD family is composed of methyl-CpG-binding protein 2 (MeCP2) and MBD1–4. Each of these proteins, with the exception of MBD3, is capable of binding specifically to methylated DNA [73]. Accumulating evidence indicates that MeCp2 may be related to chronic pain. MeCP2 is associated closely with Rett syndrome, a neurodevelopmental disorder, which is primarily caused by mutations in the MeCP2 locus and patients display decreased pain sensitivity [74;75]. Preclinical studies showed that the expression of MeCP2 and the level of its phosphorylation were increased in the superficial dorsal horn under CFA-induced inflammatory pain conditions [76;77]. This phosphorylation is controlled by a descending serotonergic pathway as serotonergic depletion prevented CFA-induced MeCP2 phosphorylation [76]. It has benn demonstrated that once MeCP2 is phosphorylated, it can be dissociated from the promoter regions of genes that have been repressed [78]. Therefore, the CFA-induced increase in MeCP2 phosphorylation may facilitate gene expression in the superficial dorsal horn under inflammatory pain conditions. However, the significance of how this increased phosphorylation is related to descending inhibitory serotonergic function in this model is unclear. The changes in MeCP2 expression following peripheral nerve injury are inconsistent. An increase in MeCP2 expression was observed in the spinal cord of CCI rats [60], whereas a decrease was detected in the superficial dorsal horn of rats after spared nerve injury [77]. The role of MeCP2 in neuropathic pain remains to be further clarified.

Epigenomics. Author manuscript; available in PMC 2016 April 01. Liang et al. Page 9

Author ManuscriptFuture Author Manuscript perspectives Author Manuscript Author Manuscript The evidence described above suggests that histone modifications and DNA methylation in DRG and spinal cord are involved in chronic pain. The conclusion depends on the behavioral observations following pharmacological inhibitor administration. These inhibitors have varying degrees of specificity and selectivity for the corresponding epigenetic enzymes. They may also exert their effects through non-epigenetic mechanisms, resulting in potential side effects. For example, HATs and HDACs are not histone-specific and can also acetylate and deacetylate, respectively, other targets in the cytoplasm. These factors call for careful interpretation of current findings claiming the role of a particular epigenetic enzyme in chronic pain. Therefore, the development of pharmacologic inhibitors for isoform- or subtype-specific epigenetic enzymes and/or the use of targeted genetic inhibition of isoform- or subtype-specific epigenetic enzymes will be required. Moreover, whether these epigenetic enzymes are activated by peripheral noxious insults and how their activation contributes to chronic pain remain to be investigated. Given that chronic pain remains a challenging condition to manage and that the contribution of epigenetic mechanisms underlying this disorder is becoming increasingly recognized, it is conceivable that the significance of histone modification and DNA methylation in chronic pain will become even more apparent in the coming years.

Acknowledgments

This work was supported by grants from the NIH (NS072206, HL117684, and DA033390) and the Rita Allen Foundation.

References 1. Morley S. Psychology of pain. Br J Anaesth. 2008; 101(1):25–31. [PubMed: 18511440] 2. Latremoliere A, Woolf CJ. Central sensitization: a generator of pain hypersensitivity by central neural plasticity. J Pain. 2009; 10(9):895–926. [PubMed: 19712899] 3. Campbell JN, Meyer RA. Mechanisms of neuropathic pain. Neuron. 2006; 52(1):77–92. [PubMed: 17015228] 4. Wang W, Gu J, Li YQ, Tao YX. Are voltage-gated sodium channels on the dorsal root ganglion involved in the development of neuropathic pain? Mol Pain. 2011; 7:16. [PubMed: 21345196] 5. Bali P, Im HI, Kenny PJ. Methylation, memory and addiction. Epigenetics. 2011; 6(6):671–674. [PubMed: 21586900] 6. Feng J, Zhou Y, Campbell SL, et al. Dnmt1 and Dnmt3a maintain DNA methylation and regulate synaptic function in adult forebrain neurons. Nat Neurosci. 2010; 13(4):423–430. [PubMed: 20228804] 7. Kramer JM, Kochinke K, Oortveld MA, et al. Epigenetic regulation of learning and memory by Drosophila EHMT/G9a. PLoS Biol. 2011; 9(1):e1000569. [PubMed: 21245904] 8**. Lutz BM, Bekker A, Tao YX. Noncoding RNAs: new players in chronic pain. Anesthesiology. 2014; 121(2):409–417. More updated review regarding the role of non-coding RNAs in chronic pain. [PubMed: 24739997] 9. Wang F, Stefano GB, Kream RM. Epigenetic modification of DRG neuronal gene expression subsequent to nerve injury: etiological contribution to complex regional pain syndromes (Part II). Med Sci Monit. 2014; 20:1188–1200. [PubMed: 25027291] 10. Zovkic IB, Guzman-Karlsson MC, Sweatt JD. Epigenetic regulation of memory formation and maintenance. Learn Mem. 2013; 20(2):61–74. [PubMed: 23322554]

Epigenomics. Author manuscript; available in PMC 2016 April 01. Liang et al. Page 10

11. Mauck M, Van d V, Shaw AD. Epigenetics of chronic pain after thoracic surgery. Curr Opin Author Manuscript Author ManuscriptAnaesthesiol. Author Manuscript 2014; Author Manuscript 27(1):1–5. [PubMed: 24300461] 12. Rahn EJ, Guzman-Karlsson MC, David SJ. Cellular, molecular, and epigenetic mechanisms in non-associative conditioning: implications for pain and memory. Neurobiol Learn Mem. 2013; 105:133–150. [PubMed: 23796633] 13. Seo S, Grzenda A, Lomberk G, Ou XM, Cruciani RA, Urrutia R. Epigenetics: a promising paradigm for better understanding and managing pain. J Pain. 2013; 14(6):549–557. [PubMed: 23602266] 14. Stone LS, Szyf M. The emerging field of pain epigenetics. Pain. 2013; 154(1):1–2. [PubMed: 23159573] 15. Kouzarides T. Chromatin modifications and their function. Cell. 2007; 128(4):693–705. [PubMed: 17320507] 16. Kuo MH, Allis CD. Roles of histone acetyltransferases and deacetylases in gene regulation. Bioessays. 1998; 20(8):615–626. [PubMed: 9780836] 17. Bannister AJ, Kouzarides T. Regulation of chromatin by histone modifications. Cell Res. 2011; 21(3):381–395. [PubMed: 21321607] 18. Contestabile A, Sintoni S. Histone acetylation in neurodevelopment. Curr Pharm Des. 2013; 19(28):5043–5050. [PubMed: 23448460] 19. Gong F, Miller KM. Mammalian DNA repair: HATs and HDACs make their mark through histone acetylation. Mutat Res. 2013; 750(1–2):23–30. [PubMed: 23927873] 20. Bai G, Wei D, Zou S, Ren K, Dubner R. Inhibition of class II histone deacetylases in the spinal cord attenuates inflammatory hyperalgesia. Mol Pain. 2010; 6:51. [PubMed: 20822541] 21. Chiechio S, Zammataro M, Morales ME, et al. Epigenetic modulation of mGlu2 receptors by histone deacetylase inhibitors in the treatment of inflammatory pain. Mol Pharmacol. 2009; 75(5): 1014–1020. [PubMed: 19255242] 22*. Zhang Z, Cai YQ, Zou F, Bie B, Pan ZZ. Epigenetic suppression of GAD65 expression mediates persistent pain. Nat Med. 2011; 17(11):1448–1455. The study recognized the significant role of histone modefication in inflammatory pain. [PubMed: 21983856] 23. Tran L, Chaloner A, Sawalha AH, Greenwood Van-Meerveld B. Importance of epigenetic mechanisms in visceral pain induced by chronic water avoidance stress. Psychoneuroendocrinology. 2013; 38(6):898–906. [PubMed: 23084728] 24. Kukkar A, Singh N, Jaggi AS. Attenuation of neuropathic pain by sodium butyrate in an experimental model of chronic constriction injury in rats. J Formos Med Assoc. 2013; S0929– 6646(13):00180–0. 25. Denk F, Huang W, Sidders B, et al. HDAC inhibitors attenuate the development of hypersensitivity in models of neuropathic pain. Pain. 2013; 154(9):1668–1679. [PubMed: 23693161] 26. Matsushita Y, Araki K, Omotuyi O, Mukae T, Ueda H. HDAC inhibitors restore C-fibre sensitivity in experimental neuropathic pain model. Br J Pharmacol. 2013; 170(5):991–998. [PubMed: 24032674] 27**. Uchida H, Ma L, Ueda H. Epigenetic gene silencing underlies C-fiber dysfunctions in neuropathic pain. J Neurosci. 2010; 30(13):4806–4814. The first study to identify the role of histone modefication in neuropathic pain. [PubMed: 20357131] 28. Uchida H, Sasaki K, Ma L, Ueda H. Neuron-restrictive silencer factor causes epigenetic silencing of Kv4.3 gene after peripheral nerve injury. Neuroscience. 2010; 166(1):1–4. [PubMed: 20006971] 29. Uchida H, Matsushita Y, Ueda H. Epigenetic regulation of BDNF expression in the primary sensory neurons after peripheral nerve injury: implications in the development of neuropathic pain. Neuroscience. 2013; 240:147–154. [PubMed: 23466809] 30. Li K, Zhao GQ, Li LY, Wu GZ, Cui SS. Epigenetic upregulation of Cdk5 in the dorsal horn contributes to neuropathic pain in rats. Neuroreport. 2014; 25(14):1116–1121. [PubMed: 25055140] 31. Kiguchi N, Kobayashi Y, Maeda T, et al. Epigenetic augmentation of the macrophage inflammatory protein 2/C-X-C chemokine receptor type 2 axis through histone H3 acetylation in

Epigenomics. Author manuscript; available in PMC 2016 April 01. Liang et al. Page 11

injured peripheral nerves elicits neuropathic pain. J Pharmacol Exp Ther. 2012; 340(3):577–587. Author Manuscript Author Manuscript[PubMed: Author Manuscript 22135382] Author Manuscript 32. Kiguchi N, Kobayashi Y, Saika F, Kishioka S. Epigenetic upregulation of CCL2 and CCL3 via histone modifications in infiltrating macrophages after peripheral nerve injury. Cytokine. 2013; 64(3):666–672. [PubMed: 24135048] 33. Zhu XY, Huang CS, Li Q, et al. p300 exerts an epigenetic role in chronic neuropathic pain through its acetyltransferase activity in rats following chronic constriction injury (CCI). Mol Pain. 2012; 8:84. [PubMed: 23176208] 34. Zhu XY, Huang CS, Li Q, et al. Temporal distribution of p300/CBP immunoreactivity in the adult rat spinal dorsal horn following chronic constriction injury (CCI). Cell Mol Neurobiol. 2013; 33(2):197–204. [PubMed: 23129231] 35. Yin Q, Lu FF, Zhao Y, et al. Resveratrol facilitates pain attenuation in a rat model of neuropathic pain through the activation of spinal Sirt1. Reg Anesth Pain Med. 2013; 38(2):93–99. [PubMed: 23337935] 36. Sun Y, Liang D, Sahbaie P, Clark JD. Effects of methyl donor diets on incisional pain in mice. PLoS One. 2013; 8(10):e77881. [PubMed: 24205011] 37. Sun Y, Sahbaie P, Liang DY, et al. Epigenetic regulation of spinal CXCR2 signaling in incisional hypersensitivity in mice. Anesthesiology. 2013; 119(5):1198–1208. [PubMed: 23756451] 38. Mayer DJ, Mao J, Holt J, Price DD. Cellular mechanisms of neuropathic pain, morphine tolerance, and their interactions. Proc Natl Acad Sci U S A. 1999; 96(14):7731–7736. [PubMed: 10393889] 39. Liang DY, Li X, Clark JD. Epigenetic regulation of opioid-induced hyperalgesia, dependence, and tolerance in mice. J Pain. 2013; 14(1):36–47. [PubMed: 23273833] 40. He X, Ou P, Wu K, et al. Resveratrol attenuates morphine antinociceptive tolerance via SIRT1 regulation in the rat spinal cord. Neurosci Lett. 2014; 566:55–60. [PubMed: 24561089] 41. Cherng CH, Lee KC, Chien CC, et al. Baicalin ameliorates neuropathic pain by suppressing HDAC1 expression in the spinal cord of spinal nerve ligation rats. J Formos Med Assoc. 2014; 113(8):513–520. [PubMed: 23684218] 42. Klein K, Ospelt C, Gay S. Epigenetic contributions in the development of rheumatoid arthritis. Arthritis Res Ther. 2012; 14(6):227. [PubMed: 23164162] 43. Covington HE III, Maze I, Sun H, et al. A role for repressive histone methylation in cocaine- induced vulnerability to stress. Neuron. 2011; 71(4):656–670. [PubMed: 21867882] 44. Gupta S, Kim SY, Artis S, et al. Histone methylation regulates memory formation. J Neurosci. 2010; 30(10):3589–3599. [PubMed: 20219993] 45. Maze I, Covington HE III, Dietz DM, et al. Essential role of the histone methyltransferase G9a in cocaine-induced plasticity. Science. 2010; 327(5962):213–216. [PubMed: 20056891] 46. Imai S, Ikegami D, Yamashita A, et al. Epigenetic transcriptional activation of monocyte chemotactic protein 3 contributes to long-lasting neuropathic pain. Brain. 2013; 136(Pt 3):828– 843. [PubMed: 23364351] 47. Tsai RY, Shen CH, Feng YP, et al. Ultra-low-dose enhances the antinociceptive effect of morphine in PTX-treated rats: regulation on global histone methylation. Acta Anaesthesiol Taiwan. 2012; 50(3):106–111. [PubMed: 23026169] 48. Zhang Z, Tao W, Hou YY, Wang W, Kenny PJ, Pan ZZ. MeCP2 repression of G9a in regulation of pain and morphine reward. J Neurosci. 2014; 34(27):9076–9087. [PubMed: 24990928] 49. Chedin F, Lieber MR, Hsieh CL. The DNA methyltransferase-like protein DNMT3L stimulates de novo methylation by Dnmt3a. Proc Natl Acad Sci U S A. 2002; 99(26):16916–16921. [PubMed: 12481029] 50. Jeltsch A. Molecular enzymology of mammalian DNA methyltransferases. Curr Top Microbiol Immunol. 2006; 301:203–225. [PubMed: 16570849] 51. Siedlecki P, Zielenkiewicz P. Mammalian DNA methyltransferases. Acta Biochim Pol. 2006; 53(2):245–256. [PubMed: 16582985] 52. Fatemi M, Hermann A, Gowher H, Jeltsch A. Dnmt3a and Dnmt1 functionally cooperate during de novo methylation of DNA. Eur J Biochem. 2002; 269(20):4981–4984. [PubMed: 12383256]

Epigenomics. Author manuscript; available in PMC 2016 April 01. Liang et al. Page 12

53. Gowher H, Stockdale CJ, Goyal R, Ferreira H, Owen-Hughes T, Jeltsch A. De novo methylation of Author Manuscript Author Manuscriptnucleosomal Author Manuscript DNA by Author Manuscript the mammalian Dnmt1 and Dnmt3A DNA methyltransferases. Biochemistry. 2005; 44(29):9899–9904. [PubMed: 16026162] 54. Liang G, Chan MF, Tomigahara Y, et al. Cooperativity between DNA methyltransferases in the maintenance methylation of repetitive elements. Mol Cell Biol. 2002; 22(2):480–491. [PubMed: 11756544] 55. Lubin FD, Gupta S, Parrish RR, Grissom NM, Davis RL. Epigenetic Mechanisms: Critical Contributors to Long-Term Memory Formation. Neuroscientist. 2011 56. Mifsud KR, Gutierrez-Mecinas M, Trollope AF, Collins A, Saunderson EA, Reul JM. Epigenetic mechanisms in stress and adaptation. Brain Behav Immun. 2011; 25(7):1305–1315. [PubMed: 21704151] 57. Turek-Plewa J, Jagodzinski PP. The role of mammalian DNA methyltransferases in the regulation of gene expression. Cell Mol Biol Lett. 2005; 10(4):631–647. [PubMed: 16341272] 58. Qi F, Zhou Y, Xiao Y, et al. Promoter demethylation of cystathionine-beta-synthetase gene contributes to inflammatory pain in rats. Pain. 2013; 154(1):34–45. [PubMed: 23273102] 59. Pan Z, Zhu LJ, Li YQ, et al. Epigenetic modification of spinal miR-219 expression regulates chronic inflammation pain by targeting CaMKIIgamma. J Neurosci. 2014; 34(29):9476–9483. [PubMed: 25031391] 60*. Wang Y, Liu C, Guo QL, et al. Intrathecal 5-azacytidine inhibits global DNA methylation and methyl- CpG-binding protein 2 expression and alleviates neuropathic pain in rats following chronic constriction injury. Brain Res. 2011; 1418:64–69. The first study to demonstrate the role of DNA methylation in neuropathic pain. [PubMed: 21925646] 61. Zhou XL, Yu LN, Wang Y, et al. Increased methylation of the MOR gene proximal promoter in primary sensory neurons plays a crucial role in the decreased analgesic effect of opioids in neuropathic pain. Mol Pain. 2014; 10:51. [PubMed: 25118039] 62. Pollema-Mays SL, Centeno MV, Apkarian AV, Martina M. Expression of DNA methyltransferases in adult dorsal root ganglia is cell-type specific and up regulated in a rodent model of neuropathic pain. Front Cell Neurosci. 2014; 8:217. [PubMed: 25152711] 63. Tajerian M, Alvarado S, Millecamps M, et al. Peripheral nerve injury is associated with chronic, reversible changes in global DNA methylation in the mouse prefrontal cortex. PLoS One. 2013; 8(1):e55259. [PubMed: 23383129] 64. Menzies V, Lyon DE, Archer KJ, et al. Epigenetic alterations and an increased frequency of micronuclei in women with fibromyalgia. Nurs Res Pract. 2013; 2013:795784. [PubMed: 24058735] 65. Tajerian M, Alvarado S, Millecamps M, et al. DNA methylation of SPARC and chronic low back pain. Mol Pain. 2011; 7:65. [PubMed: 21867537] 66. Viet CT, Ye Y, Dang D, et al. Re-expression of the methylated EDNRB gene in oral squamous cell carcinoma attenuates cancer-induced pain. Pain. 2011; 152(10):2323–2332. [PubMed: 21782343] 67. Bell JT, Loomis AK, Butcher LM, et al. Differential methylation of the TRPA1 promoter in pain sensitivity. Nat Commun. 2014; 5:2978. [PubMed: 24496475] 68. Karouzakis E, Gay RE, Michel BA, Gay S, Neidhart M. DNA hypomethylation in rheumatoid arthritis synovial fibroblasts. Arthritis Rheum. 2009; 60(12):3613–3622. [PubMed: 19950268] 69. Liu CC, Fang TJ, Ou TT, et al. Global DNA methylation, DNMT1, and MBD2 in patients with rheumatoid arthritis. Immunol Lett. 2011; 135(1–2):96–99. [PubMed: 20937307] 70. Nakano K, Whitaker JW, Boyle DL, Wang W, Firestein GS. DNA methylome signature in rheumatoid arthritis. Ann Rheum Dis. 2013; 72(1):110–117. [PubMed: 22736089] 71. Karouzakis E, Gay RE, Gay S, Neidhart M. Increased recycling of polyamines is associated with global DNA hypomethylation in rheumatoid arthritis synovial fibroblasts. Arthritis Rheum. 2012; 64(6):1809–1817. [PubMed: 22170508] 72. Klein K, Gay S. Epigenetic modifications in rheumatoid arthritis, a review. Curr Opin Pharmacol. 2013; 13(3):420–425. [PubMed: 23384968] 73. Sansom OJ, Maddison K, Clarke AR. Mechanisms of disease: methyl-binding domain proteins as potential therapeutic targets in cancer. Nat Clin Pract Oncol. 2007; 4(5):305–315. [PubMed: 17464338]

Epigenomics. Author manuscript; available in PMC 2016 April 01. Liang et al. Page 13

74. Cuddapah VA, Pillai RB, Shekar KV, et al. Methyl-CpG-binding protein 2 (MECP2) mutation Author Manuscript Author Manuscripttype Author Manuscript is associated with Author Manuscript disease severity in Rett syndrome. J Med Genet. 2014; 51(3):152–158. [PubMed: 24399845] 75. Downs J, Geranton SM, Bebbington A, et al. Linking MECP2 and pain sensitivity: the example of Rett syndrome. Am J Med Genet A. 2010; 152A(5):1197–1205. [PubMed: 20425824] 76*. Geranton SM, Fratto V, Tochiki KK, Hunt SP. Descending serotonergic controls regulate inflammation-induced mechanical sensitivity and methyl-CpG-binding protein 2 phosphorylation in the rat superficial dorsal horn. Mol Pain. 2008; 4:350. First study that demonstrated the involvement of MeCP2 in inflammatory pain. 77. Tochiki KK, Cunningham J, Hunt SP, Geranton SM. The expression of spinal methyl-CpG-binding protein 2, DNA methyltransferases and histone deacetylases is modulated in persistent pain states. Mol Pain. 2012; 8:14. [PubMed: 22369085] 78. Chen WG, Chang Q, Lin Y, et al. Derepression of BDNF transcription involves calcium-dependent phosphorylation of MeCP2. Science. 2003; 302(5646):885–889. [PubMed: 14593183]

Epigenomics. Author manuscript; available in PMC 2016 April 01. Liang et al. Page 14

Author Manuscript Author Manuscript Author Manuscript Author Manuscript Exclusive summary Histone modification in chronic pain • HDAC inhibitors reduce inflammatory pain and visceral pain. • Both HAT inhibitors and HDAC inhibitors show antinociceptive effects in neuropathic pain.

• Peripheral nerve injury or inflammation alters histone methylation in the promoter regions of some pain-related genes.

DNA methylation and chronic pain • DNMT inhibitors blocks inflammatory pain. • DNMT inhibitors attenuates neuropathic pain • Alterations of DNA methylation have been found in patients with some painful diseases.

Epigenomics. Author manuscript; available in PMC 2016 April 01. Liang et al. Page 15 Author Manuscript Author Manuscript Author Manuscript Author Manuscript

Fig. 1. Histone modification regulates gene expression. (A) Methylation (Me) of histones and deacetylation of histones with HDAC results in a condensed chromatin. Under this condition, transcription factors (TF) cannot bind to the promoter region of the gene, thereby gene transcription is repressed. (B) Histone acetylation (Ac) with HAT results in loose chromatin that allows the transcription factor (TF) to bind to the promoter region of the gene. Consequently, gene transcription is activated.

Epigenomics. Author manuscript; available in PMC 2016 April 01. Liang et al. Page 16 Author Manuscript Author Manuscript Author Manuscript Author Manuscript

Fig. 2. DNA methylation represses gene transcriptional processes. (A) Without methylation at the CpG sites of the gene promoter, the transcription factor (TF) and RNA polymerase II (RNAPII) bind to the promoter region of the gene, thereby gene transcription is activated. (B) When methyl groups are added at the CpG islands by methyl-CpG-binding domain protein (MBD)-mediated DNA methyltransferases (DNMTs), the transcription factor (TF) and RNA polymerase II (RNAPII) cannot bind the promoter region of the gene, resulting in the repression of gene transcription. TSS: Transcription start site.

Epigenomics. Author manuscript; available in PMC 2016 April 01. Liang et al. Page 17 ] ] ] 25 23 ] ] 21 ] ] 24 41 22 ] ] 39 Author Manuscript Author Manuscript Author Manuscript Author Manuscript ] ] ] ] 35 20 , 2013 [ 40 , 2013 [ 29 30 32 ; – ] ] 31 27 et al. et al. 34 37 ; ; 33 36 Ref Chiechio, 2009 [ Bai, 2010 [ Zhang, 2011 [ Tran Kukkar, 2013 [ Denk Cherng, 2014 [ Uchida, 2010; 2013 [ Kiguchi, 2012; 2013 [ Zhu, 2012; 2013 [ Li, 2014 [ Yin, 2013 [ Sun, 2013; 2013 [ He, 2014 [ Liang, 2013 [ α Target genes mGluR2/3 na GAD65 na TNF- na mu receptor, Nav1.8, Kv4.3, BDNF MIP-2 and its receptor CXCR2 COX-2 Cdk5 na na na na na na ), ) ) ) ) ) ) ↓ ↓ ↓ ↓ ↓ ↓ ↓ ), thermal (-) ), thermal (-) ↓ ↓ ) ) ) ), mechanical ( ), mechanical ( ), mechanical ( ), mechanical ( ), mechanical ( ), mechanical ( ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ) ↓ phase ( nd Tolerance mechanical allodynia, thermal mechanical allodynia, thermal Nociceptive behavior response to inhibitors 2 Thermal ( Thermal ( Visceral hypersensitivity( Thermal ( Thermal ( Thermal ( na Thermal ( Thermal ( Thermal ( Mechanical ( Mechanical ( ↓ ↓ ↑ cold( hyperalgesia, tolerance, and physical dependence hyperalgesia, tolerance, and physical dependence Table 1 Inhibitors MS-275, SAHA (s.c.) SAHA, TSA, LAQ824, VPA, 4-PB (i.t.) TSA and SAHA (NRM) TSA(i.c.v.) Sodium butyrate(oral) MS-275, MS-275 or MGDC0103 (i.t.) Baicalin na Anacardic acid (i.p.) p300 shRNA or C646 (i.t.) Resveratrol anacardic acid (i.p.) SAHA Resveratrol Curcumin SAHA Tissue DRG spinal dorsal horn NRM na sciatic nerve spinal cord Spinal dorsal horn DRG injured sciatic nerve spinal cord spinal cord spinal cord na spinal cord na ) ↓ ) ) ↑ ↑ ), ↑ ) ↑ ) ↑ ) ) on promoter ↑ ) Acetyl H3 ( ↑ ↑ ) ↑ ), Acetyl H3( ), Acetyl H3( ↓ ↓ Changes of acetylation or enzyme expression na HDAC4, 5, 7, 9 ( Global histone H3 and H4 acetylation ( na na global H3K9ac( HDAC1( Acetylation changes on promoter of genes H3K9 ac( p300 ( H4 acetylation at Cdk5 promoter ( Sirt ( na Sirt ( na HDAC1, 2, 3 (-) of MIP-2 and CXCR2 Pain model Formalin CFA Water avoidance stress CCI Traumatic nerve injury and stavudine-induced peripheral neuropathy SNL CCI Incision Morphine Summary of studies on histone acetylation and deacetylation

Epigenomics. Author manuscript; available in PMC 2016 April 01. Liang et al. Page 18 Author Manuscript Author Manuscript Author Manuscript Author Manuscript na: not applicable; DRG: dorsal root ganglion; CFA: complete Freund’s adjuvant; NRM: nucleus raphe magnus; CCI: chronic constriction injury; SNL: spinal nerve ligation; COX-2: cyclooxygenase-2; MIP-2: macrophage inflammatory protein 2; CXCR2: chemokine CC motif receptor TSA: Trichostatin A; SAHA: suberoylanilide hydroxamic acid; i.t.: intrathecally; i.p.: intraperitoneal injection; s.c.: subcutaneous injection; i.c.v.: intracerebroventricular injection

Epigenomics. Author manuscript; available in PMC 2016 April 01. Open access Protocol Examination of psychological risk factors for chronic pain following cardiac surgery: protocol for a prospective observational study

Michael H McGillion,1,2 Shaunattonie Henry,1,2 Jason W Busse,3,4 Carley Ouellette,1,2 Joel Katz,5 Manon Choinière,6 Andre Lamy,2,4 Richard Whitlock,2,4 Shirley Pettit,2 Jacqueline Hare,2 Krysten Gregus,2 Katheryn Brady,2 Nazari Dvirnik,2,4 Stephen Su Yang,2,4 Joel Parlow,7 Deborah Dumerton-Shore,8 Ian Gilron,7 D Norman Buckley,3 Harsha Shanthanna,3 Sandra L Carroll,1 Peter C Coyte,9 Shanil Ebrahim,4 Wanrudee Isaranuwatchai,9 Denise N Guerriere,9 Jeffrey Hoch,10 James Khan,3 Joy MacDermid,11 Geraldine Martorella,12 J Charles Victor,9 Judy Watt-Watson,13 Kimberly Howard-Quijano,14 Aman Mahajan,14 Matthew T V Chan,15 Hance Clarke,16 P J Devereaux2,4

To cite: McGillion MH, Abstract Strengths and limitations of this study Henry S, Busse JW, et al. Introduction Approximately 400 000 Americans and Examination of psychological 36 000 Canadians undergo cardiac surgery annually, ►► This is a prospective, multisite study with a large risk factors for chronic pain and up to 56% will develop chronic postsurgical pain following cardiac surgery: cohort of cardiac surgery patients. (CPSP). The primary aim of this study is to explore protocol for a prospective ►► One-year follow-up is compliant with Initiative for the association of pain-related beliefs and gender- observational study. BMJ Open Methods, Measurement, and Pain Assessment in based pain expectations on the development of CPSP. 2019;9:e022995. doi:10.1136/ Clinical Trials recommendations to standardise tim- bmjopen-2018-022995 Secondary goals are to: (A) explore risk factors for ing of outcome assessment for prognostic studies of poor functional status and patient-level cost of illness ► Prepublication history for chronic postsurgical pain (CPSP). ► from a societal perspective up to 12 months following this paper is available online. ►► A robust analysis plan using generalised estimating To view these files, please visit cardiac surgery; and (B) determine the impact of CPSP equations will be used to model the primary analy- the journal online (http://dx.​ ​doi.​ on quality-adjusted life years (QALYs) borne by cardiac sis: the association between pain-related beliefs and org/​10.​1136/​bmjopen-​2018-​ surgery, in addition to the incremental cost for one gender-based pain expectations with the develop- 022995). additional QALY gained, among those who develop ment of CPSP at 6 months and 1 year while adjusting CPSP compared with those who do not. for prespecified covariates. Received 5 July 2018 Methods and analyses In this prospective cohort ►► Assiduous follow-up procedures will be adhered to, Revised 15 November 2018 study, 1250 adults undergoing cardiac surgery, Accepted 16 November 2018 which have been proven effective in prior prospec- including coronary artery bypass grafting and open- tive observational studies. heart procedures, will be recruited over a 3-year period. ►► There is reliance on pain and quality of life self-re- Putative risk factors for CPSP will be captured prior to port outcome measures; however, rigorous criteria surgery, at postoperative day 3 (in hospital) and day 30 to define CPSP will be applied, and valid and reliable (at home). Outcome data will be collected via telephone instruments will be used. interview at 6-month and 12-month follow-up. We will employ generalised estimating equations to model the primary (CPSP) and secondary outcomes (function and cost) while adjusting for prespecified model covariates. Introduction © Author(s) (or their QALYs will be estimated by converting data from the Approximately 400 000 Americans and employer(s)) 2019. Re-use Short Form-12 (version 2) to a utility score. 36 000 Canadians undergo cardiac surgery permitted under CC BY-NC. No commercial re-use. See rights Ethics and dissemination This protocol has been annually, and these numbers are expected 1–5 and permissions. Published by approved by the responsible bodies at each of the to rise as the population ages. Despite BMJ. hospital sites, and study enrolment began May 2015. the proven survival and symptom-related For numbered affiliations see We will disseminate our results through CardiacPain. benefits of cardiac surgeries, mounting end of article. Net, a web-based knowledge dissemination platform, evidence suggests that chronic postsurgical presentation at international conferences and pain (CPSP)—and related poor functional Correspondence to publications in scientific journals. recovery—following these procedures are Dr Michael H McGillion; Trial registration number NCT01842568. ​mmcgill@​mcmaster.​ca major clinical problems.6–31 Moreover, the

McGillion MH, et al. BMJ Open 2019;9:e022995. doi:10.1136/bmjopen-2018-022995 1 Open access economic consequences of persistent pain and dysfunc- cognitive-evaluative and affective-motivational compo- tion remain uncertain. Identification of factors associated nents.48 Like any form of chronic pain, ongoing pain after with the development of CPSP could facilitate efforts surgery can lead to pathological nervous system changes, to improve outcomes among high-risk patients, yet the collectively known as sensitisation47—a function of what majority of putative risk factors examined to date are not we now understand to be neuronal modifiability.46 Sensi- tenably modifiable in the perioperative context. Three tisation of the nervous system may lead to increased pain psychological factors that do show promise as modifi- sensitivity (hyperalgesia), augmentation of the normal able, potential risk factors for CPSP include pain-related duration (hyperpathia) amplitude of pain, perception beliefs, gender-based pain expectations and somatic of non-painful stimuli as painful (allodynia)47 49 and preoccupation and coping. The purpose of this study is abnormal, unpleasant hypersensitivity (dysesthesia).50 to examine whether these factors are associated with tran- As Katz and Seltzer argued,32 critical to understanding sition to CPSP following cardiac surgery. the nature of CPSP is appreciating that in each case, the pain was once acute and involved a transition phase. CPSP following cardiac surgery There is much work to be done to continue to develop Due to conceptual and methodological differences in our understanding of risk factors, which predispose the assessment of pain, and conflicting opinions about cardiac surgical patients to pain chronicity. the duration of ‘chronicity’, there is no one accepted definition of CPSP.32 However, there is consensus among Prevalence and consequences experts32–38 that CPSP should meet the minimum criteria, We reviewed 26 published/under review studies to date, set forth by Macrae and Davies33 and others,34–40 as across 14 countries,6–31 which have examined the prev- follows. It must: (A) have developed after the surgical alence and/or factors associated with CPSP following procedure, (B) be different from pain experienced prior cardiac surgery. On careful examination of the available to the procedure, (C) not be caused by other factors (eg, data, it is important to recognise that cross-sectional and cancer recurrence and chronic infection), (D) be present retrospective studies have generally reported higher prev- for at least 2–3 months and (E) interfere significantly with alence rates (14%–56%)%) than those investigations with health-related quality of life.34–40 prospective designs (7.5%–45%). In the recent (2013) Open cardiac surgeries involve many pain-sensitive large-scale Canadian CARDpain study (n=1010), Choin- structures, as they require a median sternotomy, retrac- ière et al28 reported CPSP prevalence rates of 40%, 22% tion of the ribs and invasion of muscles and visceral and 17% at 3, 6, and 12 months following cardiac surgery, tissues. In coronary artery bypass surgery (CABG), the respectively. Routledge et al31 found similar prevalence grafting procedure requires harvesting at several sites rates of CPSP in their prospective extension (Women’s including, most commonly, the internal mammary artery Recovery from Sternotomy-Extension (WREST-E)) of a (IMA). The manipulation and retraction of the sternum randomised clinical trial (Women’s Recovery from Ster- as well as the use of electrocautery to dissect the IMA notomy (WREST)) (n=222) to examine the impact of a from the chest wall may result in nerve damage that leads novel compression undergarment on women’s recovery to intercostal neuralgia.41–44 The greater and lesser saphe- from median sternotomy (3 months postoperative [post- nous veins are also used as grafts in CABG surgery and op]: 41%; 12 months post-op: 16.7%). In contrast to require significant leg incisions. These procedures may CARDpain and WREST-E, 1 year CPSP prevalence rates as result in pain that can last for variable periods and may be high as 39% and 45% have been reported in prospective inflammatory or neuropathic in nature. CPSP in cardiac studies of patients following CABG in Turkey27 and the surgery patients is often experienced in the thorax and Netherlands.30 Aside from differences in study design, the legs but has also been described, to a lesser degree, in observed variability in reported prevalence rates of CPSP the shoulders, back and neck.10 12 45 The pathophysiolog- after cardiac surgery may be explained by the use of point ical pathways underlying CPSP are multifactorial. Tissue prevalence versus cumulative prevalence, variability with damage leads to release of high concentrations of brady- respect to the operational definitions of CPSP, timing of kinin, adenosine, lactate and potassium in the periph- outcome measurement and duration of follow-up period. eral microenvironment, thereby causing CPSP has been associated with the development of activation.46 47 These mediators activate capsaicin-sensi- anxiety and depressive disorders,51–55 sleep disturbances tive TRVP1 receptors, which serve as the primary trans- and fatigue,56–60 as well as poor self-rated health.7 51 53 61 ducer of the .47 Other neurochemicals, For example, among those with CPSP in the CARDpain such as the neuropeptides substance P and calcitonin study, over 50% reported significant pain-related interfer- gene-related peptide, further augment pain.47 These ence with activities of daily living—including family and peripheral nociceptive processes are modulated in the home responsibilities, recreation and employment—at 3, central nervous system by mechanisms involving selec- 6 and 12 months following cardiac surgery.28 62 tion, abstraction and synthesis of information from the total sensory input.48 The amount, quality and nature Risk factors for CPSP of the pain experienced are therefore dynamic and Several studies have attempted to establish risk factors for multidimensional products of sensory-discriminative, CPSP in cardiac surgery patients.

2 McGillion MH, et al. BMJ Open 2019;9:e022995. doi:10.1136/bmjopen-2018-022995 Open access

Their limitations can be summarised63 as: (1) many study24 to date. Similar to diabetes mellitus, the majority of studies focused on univariate analyses, or were insuffi- prospective studies20 21 23 (including one RCT)20 reported ciently powered to employ multivariate modelling tech- no predictive ability of baseline chronic pain condi- niques, (2) the vast majority of risk factors examined tions in the literature (OR=1.00–1.04, where reported). to date are not tenably modifiable in the perioperative To date, CARDpain28 is the only prospective examina- context, (3) psychological risk factors (affective and tion to report that pre-existing chronic pain at baseline cognitive) are substantially understudied in comparison (non-anginal) is positively associated with CPSP (adjusted with demographic, clinical/surgical and analgesic risk OR=1.44, 95% CI 1.12 to 1.86).28 factors, constituting a major gap and (4) although retro- The evidence pertaining to the predictive value of spective and cross-sectional studies provide some insight preoperative angina is also mixed. Two cross-sectional on potential variables associated with CPSP, cross-sec- studies reported preoperative angina that was positively tional studies lack the temporal orientation to make solid associated with CPSP (OR, where reported=1.62)7 12; inferences about putative, causal relationships and retro- however, another cross-sectional17 and two additional spective studies can be limited by availability and quality large-scale prospective studies14 28 found no significant of data. In addition, even robust retrospective may be associations to infer that preoperative angina is a signif- limited in terms of risk factors explored and related data icant risk factor for CPSP. collection methods. Risk factors for CPSP can be classi- The majority of studies have reported no associ- fied into four categories: (A) demographic, (B) baseline ation6 12 13 17 22 28 50 between a range of surgical factors, clinical, technical-surgical, and hospitalisation-related including: (A) type of surgical technique, (B) number factors, (C) acute post-op pain and (D) psychological and type of bypass grafts per operation, (C) harvesting factors. technique and (D) total cross-clamp time (ie, total time aorta is clamped to separate systemic circulation from Demographic factors cardiac outflow) and the development of CPSP. There Demographic factors examined include age, sex, level is some evidence to suggest that not skeletonising the of education, body mass index (BMI) and smoking internal thoracic artery harvest (ie, harvesting it along history. Younger age has been positively associated with with its surrounding pedicle of vascular tissue) is more CPSP7 9 12 17 20 25 28 in multiple retrospective, cross-sectional likely to invoke CPSP64; those who have undergone left and prospective studies, as observational data embedded IMA harvesting may also be at higher risk.13 42 In general, within randomised controlled trials (RCTs); significant post-op complications and related adverse events (eg, ORs have ranged from 1.43 to 7.03 in cases where this reoperation for bleeding and infections) have not been outcome was dichotomised (ie, younger vs older patients). associated with CPSP,9 12 14 16 20 28 with the exception of one However, four of the more recent published studies to prospective study that identified post-op resternotomy as date (one retrospective,17one cross-sectional,18 one RCT50 a significant risk factor (OR=3.38).21 Cardiac surgeries and one prospective21) have found no positive association of longer duration (ie, total OR time) 18 20 also do not between age and the development of CPSP. Conflicting seem predictive of CPSP; in fact, the CARDpain28 study findings have also been reported for sex. Although some found that the longer the OR time, the less likely CPSP studies indicate higher risk of CPSP with women,21 29 30 was to develop. Finally, there seems to be no conclusive multiple studies with divergent designs9 12 14 18 20 28 48 have evidence to suggest that length of time in the intensive reported no significant association between sex and the care unit,18 20 28 or total duration of hospitalisation18 development of CPSP. Examination of BMI as a risk factor contribute to the development of CPSP after cardiac for CPSP has also produced mixed results. While two surgery. studies (one cross-sectional7 and one RCT [embedded observational data],20 ORs=1.34 and 9.05, respectively) Acute post-op pain provided supportive evidence, other cross- sectional17 18 Two prospective studies found that severe pain (ie, and prospective studies9 28 found no association between numeric rating scale [NRS] ≥7/10) on post-op day 3 was a CPSP and BMI (OR range: 1.02–1.1). Finally, we are aware significant risk factor for CPSP at 1-year follow-up,21 as well of two prospective studies to date that have examined the as worst and average pain ratings at 2-year follow-up.28 A association of CPSP with formal level of education28 and third prospective study found that severe pain on post-op smoking history,14 respectively; no significant association day 30 positively predicted CPSP at 3 months.23 The asso- was found in either case. ciation between analgesic therapy and CPSP is uncer- tain.10–12 18 19 21 23 27 28 Baseline clinical, surgical and hospitalisation-related factors Among baseline clinical factors, neither a history of Psychological factors diabetes mellitus9 14 17 23 24 50 or peripheral arterial disease24 Only the CARDpain28 study has examined the role of have been significantly associated with the development psychological risk factors in the development of CPSP of CPSP. and found that presurgical anxiety, as measured by the However, pre-existing peripheral arterial disease has Hospital Anxiety and Depression Scale (HADS), was a been examined as a risk factor in just one retrospective significant risk factor, with a 10% increase in the odds of

McGillion MH, et al. BMJ Open 2019;9:e022995. doi:10.1136/bmjopen-2018-022995 3 Open access developing CPSP for each unit increase in HADS-A scale surgery (n=202), a majority (83%) reported that they scores (OR=1.10, 95% CI 1.06 to 1.14). Other psycholog- would not voluntarily ask for pain medication when they ical risk factors examined (catastrophising and depres- needed it, although most reported unrelieved moder- sion) demonstrated no association. ate-to-severe pain from post-op day 2 (80%) until day 5 (69%).67 As of 2013, data indicate that this unfortu- Genetic factors nate scenario remains largely unchanged. Cogan et al82 Several members of this investigative team (eg, HC and found that among cardiac surgery patients (n=564), 36% JK) are involved in studies investigating the influence believed that ‘pain medication should be spared until the of genetic polymorphisms on the development of CPSP pain is very severe’, 20% believed that ‘good patients do after cardiac and other types of surgery. The science of not speak of their pain’ and 31% believed it is ‘very easy pain genetics is evolving; investigations of this nature are to become addicted to pain medication’ while recovering complex, requiring extensive research infrastructure for from surgery. The particular role of these beliefs per se genotyping and related proteomic methods. Controlling in the development of CPSP has yet to be examined; we for the influence of genetic factors is beyond the scope will do so in this study using the Pain Barriers Question- this study. naire (PBQ) (validated in multiple populations).

Conceptual underpinnings and study focus Gender-based pain expectations To address the above noted gap in the research to date, our As with a number of fields in the health sciences, the primary objective is to examine the potential influence of study of sex and gender, as they relate to pain, is evolving. psychological factors on the development of CPSP after Our comprehensive review of risk factors for CPSP after cardiac surgery. Clear justification for the specific puta- cardiac surgery revealed that, thus far, investigation has tive risk factors to be measured requires that we first expli- been limited to the contribution of sex only as a risk factor. cate the conceptual underpinnings of our study. Given For the purposes of this study, we employ the following the complexity of the multidimensional pain experience, distinctions between sex and gender, set forth by Lips,83 there are many ways to conceptualise CPSP.65 We are which have been adopted in a number of well-cited pain aligned with the biobehavioural view of pain, espoused by studies84–99: sex: the biological distinction of being male or international leaders in the science of the cognitive and female; gender: learnt masculinity or femininity, related learning aspects of pain.65 66 Fundamental to the biobe- to socially-constructed roles and behaviours attributed to havioural perspective is the assertion that people learn to men and women in society.83 84 predict future events based on prior learning experiences Emerging evidence suggests that gender-based pain and information processing. As such, patients’ behaviours elicit responses from significant others, including health- expectations defined as ‘Sex-related stereotypic attribu- tions about pain sensitivity, pain endurance, and willing- care professionals, which can reinforce both adaptive and 87 maladaptive modes of thinking, feeling and behaving.65 ness to report pain’ may lead to important differences With this understanding, patients’ pain-related cogni- in the experience of pain and related response. Robinson tions and behaviours are of chief concern with respect to et al were among the first to investigate gender-based pain expectations, using the Gender Role Expectations identifying factors that may contribute to the transition 87 from acute post-op pain to chronic pain. In moving the of Pain Questionnaire (GREP). Their study of pain science forward, we therefore give primacy to the cogni- cognitions in 156 men and 235 women found that men tive-behavioural side of the global biobehavioural view of were perceived to be less willing to report pain than pain, as the conceptual premise for our primary objec- women, women were perceived to be more sensitive and tive. According to the fundamental tenets of the cogni- less enduring of pain than men and that men rated their tive-behavioural perspective of pain65 66: (A) behaviour is pain endurance as higher than average. Further testing 94 reciprocally determined by the person and environment, of the GREP by Wise et al found that after controlling (B) people can learn more adaptive ways of thinking and for age, GREP scores accounted for 7%, 11% and 21% behaving and (C) people are capable of and should be of the variance in pain threshold, tolerance and pain involved as active agents in the change of maladaptive unpleasantness scores, respectively, for women (n=87) thoughts, amenable to intervention.65 Our focus there- and men (n=61) exposed to thermal testing. A recent 91 fore will be on the contribution of patients’ pain-related meta-analysis by Alabas et al, for example, examined beliefs and expectations, as follows: the role of gender-related cognitions in the experience of pain.91 Pooling the results of six trials (406 men and Pain-related beliefs 539 women), they found that those who considered Decades of work9 67–82 in the fields of post-op pain and themselves more masculine and less sensitive to pain, anaesthesia has demonstrated that surgical patients have than the typical man, exhibited higher pain thresholds beliefs about pain and pain medication, which: (A) are and tolerances in a variety of settings. Using the GREP, based on incorrect information and (B) serve to block our study will be the first we know of to examine the role effective pain assessment and management. For example, of gender-based pain expectations on the development one study found that among patients undergoing CABG of CPSP after cardiac surgery.

4 McGillion MH, et al. BMJ Open 2019;9:e022995. doi:10.1136/bmjopen-2018-022995 Open access

Health-related quality of life predictors will be collected at baseline. The total follow-up Overwhelming evidence documents the deleterious period is 12 months, with pain, functional status and cost impact of CPSP on health-related quality of life.6–31 50–62 of illness-related data being collected at 6 months and 12 months following cardiac surgery. Cost of illness We will examine the impact of CPSP on patient-level cost, Patient and public involvement calculated from a societal perspective, wherein all costs We collected patient testimonials to articulate the nature irrespective of payer are included thereby comprising of the chronic pain problem following cardiac surgery private and public costs, using the Ambulatory Home from the patient perspective and establish the need for Care Record. Data are available that indicate that from this study. Following the completion of the study, we will 20% to 30% of the occurrence of chronic pain is related to debrief the patient panel with the results of our findings. CPSP.98 99 Given the rates of cardiac surgery in Canada,4 5 literature has shown that CPSP contributes substantially Study population to the $22.2 billion in direct and indirect costs borne by The target population of 1250 cardiac surgery patients cardiovascular interventions and services annually.15 With will be recruited from participating hospital sites in a view to comprehensive examination of the impact of Canada, USA and Hong Kong. Patients eligible for our CPSP, we will: (A) estimate the extra cost, expressed in study will be undergoing a first-time cardiac surgery healthcare costs, for patients with CPSP compared with involving a median sternotomy, including CABG and all those without and (B) estimate an incremental cost-ef- open heart procedures, such as valvular repairs/replace- fectiveness ratio, that is, the incremental cost for one ment. Eligible patients will also be able to read, speak additional quality-adjusted life year (QALY) gained, and understand English and have a telephone allowing by virtue of cardiac surgery, among those who develop for follow-up. Patients will be ineligible if they: (A) have CPSP compared with those who do not. QALY is a pref- undergone previous cardiac surgery, thoracotomy or erence-based utility measure of health-related quality of mastectomy, (B) are scheduled for an isolated pericardial life as perceived by the patient.100 101 QALYs incorporate window procedure (due to malignancy), pericardectomy, both length of life and quality of life into a single measure permanent pacemaker, or defibrillator implantation, (C) and are calculated by combining health- related quality have a major cognitive disorder precluding participation, of life measures with data on health state duration. As or (D) have a hearing impairment or speech impediment such, QALY is the gold standard measure of effectiveness precluding telephone-based follow-up. recommended for economic evaluation and represents Cardiac surgery inpatients will be recruited in one of a universally comparable outcome measure. QALY will two ways: (1) from the hospital sites preoperative assess- be derived from our Short Form-12 (SF-12) version 2 ment clinic, if their surgery is prebooked, or (2) from (SF-12v2) data. the cardiac surgical ward, if they have been admitted to hospital via the hospital’s emergency department or the Study objectives heart investigation unit. A study nurse will obtain written, Our primary objective is to examine the influence of informed consent to participate among those willing and pain-related beliefs and gender-based pain expectations interested. The study enrolment period will conclude on the development of chronic pain following cardiac once the 1-year follow-up telephone interview is complete. surgery. Our secondary objectives are to: (A) examine the influence of pain-related beliefs and gender-based pain Data collection expectations on functional status and patient-level cost of Immediately following enrolment, standard baseline illness following cardiac surgery; and (B) to determine demographic, independent variable data (participants’ the impact of CPSP on the QALY borne by cardiac surgery age, sex, ethnicity, highest level of formal education, and the incremental cost for one additional QALY gained and marital and employment status) and data on base- for patients, by virtue of cardiac surgery, among those line covariates (age and sex) will be collected by the who develop CPSP compared with those who do not. study nurse via interview and chart audit. Postopera- tively, the study nurse will collect data on surgical details via chart audit, and data on post-op day 3 cumulative Methods and analysis analgesic dose and pain intensity scores via chart audit Design and participant interview, respectively. The study nurse This study is a substudy of the Vascular Events In Surgery will contact patients by phone at 30 days, and 6 and 12 patIents cOhort evaluatioN - Cardiac Surgery study months after surgery; the 30-day call will be for post-op (https://​clinicaltrials.​gov/​ct2/​show/​NCT01842568), pain monitoring, and the two subsequent calls will be for examining 30-day all-cause mortality, myocardial injury outcome assessment. Data on dependent variables will be and related complications following cardiac surgery measured at 6 months and 12 months following cardiac in 15 000 participants. In this substudy, we propose surgery. Table 1 outlines this visit schedule. The timing to prospectively follow a cohort of patients who have of this follow-up outcome measurement is in compliance undergone cardiac surgery for 1 year. Data on potential with recommendations (2013) set forth by the Initiative

McGillion MH, et al. BMJ Open 2019;9:e022995. doi:10.1136/bmjopen-2018-022995 5 Open access

Table 1 Visit schedule Baseline Postoperative day 3 Day 30 6 months 1 year Pain Barriers Questionnaire X Gender-based pain expectations X Somatic Pre-Occupation and Coping X State-Trait Anxiety Inventory X Hospital Anxiety and Depression Scale X (HADS) Short Form-12 (SF-12) X X X X CPSP-related disability X X X Analgesic chart audit X Brief Pain Inventory X X X X Ambulatory Home Care Record X X for Methods, Measurement, and Pain Assessment in Clin- Functional status ical Trials to standardise the timing of outcome assess- Functional status will be measured with the SF-12v2), an ment for prognostic studies of CPSP.102 established reliable and validated health status measure.118 It consists of 12 items taken from the Short Form 36 Dependent variables (SF-36), which is a widely accepted instrument that was Chronic postsurgical pain developed from the Medical Outcomes Study.119–121 The The development of CPSP will be measured using a tele- SF-12v2 was developed to reduce respondent burden. It phone structured interview protocol, defined as pain: can be administered by telephone interview and consists (A) that developed after the surgical procedure, (B) is of two scales that measure physical and mental health different from pain experienced prior to the procedure status. The SF-12v2 comprises eight domains, measured (eg, preopeative angina), (C) is not caused by other via eight subscales: (1) physical functioning; (2) role factors (eg, cancer recurrence and chronic infection), limitations due to physical problems; (3) role limitations (D) is present for at least 2–3 months and (E) that inter- 34–40 due to emotional problems; (4) bodily pain; (5) general feres significantly with health-related quality of life. health; (6) vitality; (7) social functioning; and (8) mental If participants answer in the affirmative to each of these health. Results may be expressed as physical component questions, it will be indicated that ‘Yes’ they have developed summary (PCS) and mental component summary scores. CPSP; otherwise, it will be indicated that ‘No’ they have These scores range from 0 (worst) to 100 (best).118 not. Among those deemed to have developed CPSP (ie, ‘yes’) pain intensity, and its related interference with usual Cost of illness daily activities, will be measured via the Brief Pain Inven- The Ambulatory and Home Care Record (AHCR)122–132 tory-Short Form (BPI-SF).103–107 The BPI-SF includes four will be used to measure patient-level cost of illness from 11-point NRSs of pain intensity, which measure ‘average’, a societal perspective. This approach gives equal consid- ‘least’ and ‘worst’ pain intensity in the past 24 hours, respec- eration to health system costs and costs borne by patients tively, as well as pain intensity ‘now’ (0=no pain, 10=pain and unpaid caregivers, such as family members and as bad as you can imagine). As is common to studies of friends. Items in the AHCR can be categorised as publicly CPSP28 29 62 67 108–113 (including cardiac surgery), participants financed care (ie, resources paid for by the public sector) will be asked for their ‘worst’ pain intensity rating both on or privately financed care (ie, all out-of-pocket payments, rest and movement in the past 24 hours. The BPI-SF inter- third party insurance payments and time costs incurred ference subscale103–107 will also be used, which measures the by caregiver). Face validity of the AHCR has been assessed degree to which pain interferes with general activity, mood, by several healthcare providers, health economists and walking, work, relations with others, sleep and enjoyment of administrators who work in the field of ambulatory and life (NRS for each item; 0=does not interfere, 10=completely home-based care.122 125 Reliability of the AHCR has been interferes). A total interference score is taken by calcu- assessed via the level of agreement between self-reports of lating the sum of these seven items. The BPI-SF has strong cost by cystic fibrosis care recipients and administrative psychometric properties with well-established reliability and data.125 Moderate to almost perfect agreement was found validity across divergent surgical groups,29 103–117 including between study participants’ responses on the AHCR and those reporting acute and chronic pain following cardiac administrative data (kappa=0.41–1.00).125 The AHCR surgery.28 29 62 67 112 113 The BPI-SF also contains supple- has since been used to evaluate various conditions,124–132 mental items,103–106 for optional use (pain treatment and including chronic cardiology patients who were inter- body diagram). Of these, only the body diagram will be viewed over the phone131 132 Additionally, the AHCR has used for descriptive purposes. been used to assess costs for an array of patients, including

6 McGillion MH, et al. BMJ Open 2019;9:e022995. doi:10.1136/bmjopen-2018-022995 Open access the elderly, middle-aged adults and children.122–132 The well-validated anxiety measure.139 140 The STAI has 40 AHCR has been used in telephone and face-to-face inter- items that comprise two domains: the State (STAI-S) and views as well as in mailed form; it has been translated into Trait (STAI-T) score, both ranging from 20 to 80, with several languages.122–132 higher scores representing higher levels of anxiety. The STAI-S measures the transitional emotional status evoked Independent variables by a stressful situation, such as surgery. The STAI-T score Pain-related beliefs reflects enduring individual differences in the likelihood Pain-related beliefs will be examined at baseline using of anxiety.141 The STAI has been found reliable and valid 76 77 74 76–79 133 134 the PBQ version II (PBQ-II) The PBQ-II among patients undergoing cardiac surgery (Cronbach’s includes 27 items divided into four subscales: erroneous alpha=0.94)142 and is commonly applied in studies capturing beliefs regarding secondary effects of medication (12 preoperative anxiety among cardiac surgery patients.143 144 items) and their harmful effects (six items), fatalism about the control of pain (three items) and attitudes regarding Acute post-op pain reporting pain to health professionals (six items). Each Pain on post-op days 3 and 30 will be measured with the item is rated on a 0–5 scale (0: totally disagree; 5: totally BPI. Cumulative 24 hours analgesic on post-op day 3, agree). A total score and scores for each subscale can be as an indication of analgesic dosing in hospital during calculated by taking the sum of the items. The PBQ-II has recovery, will be determined via chart audit using a tool established validity, internal consistency and sensitivity to we have used in previous cardiac studies.28 62 67 Opioid 113 135 136 change and has recently been adapted and vali- dosage will be converted into parenteral morphine equiv- 113 dated for use with cardiac surgical patients. alents per day using standard dosage tables.28 62 67

Gender-based pain expectations Sample size Gender-based pain expectations will be measured at base- 87 The primary analysis for this study is the association of line using the GREP. The GREP measures stereotypic pain-related beliefs and gender-based pain expectations attributions regarding three constructs: pain endurance, with CPSP at 6 months and 12 months while adjusting for pain sensitivity and willingness to report of pain. Each a number of prespecified covariates. Therefore, sample construct includes four 100 mm visual analogue scales size was calculated based on the methods used by Hsieh regarding how women and men perceive themselves and and colleagues145 for multivariable logistic regression. In the opposite sex, relative to: (A) their own sex and (B) the this validated method, the sample size for a simple logistic opposite sex with respect to how much pain can males/ regression modelling a single independent variable X1 on females endure, how sensitive to pain males/females are the outcome is inflated by a variance inflation factor equal and how willing males/females are to report pain; respon- to 1 / (1-ρ2×2…xp), where ρ2×2…xp is equal to the propor- dents indicate their views on a 100 mm line anchored by 0 tion of the variance of X1 explained by the regression (far less) and 100 (far more). An average score is derived relationship with X2…Xp.145 Additionally, sample size was for each construct; greater scores indicate more stereo- inflated to account for the clustered nature of the data (ie, typical views. The GREP has now been used in multiple 6-month and 12-month measurements) by incorporating pain investigations.87 89 91–93 137 138 Test–retest reliability 87 an additional design effect equivalent to 1+ (m−1)*ρICC, is acceptable across items (0.53 to 0.93), and internal where m is the number of measurements per cluster (ie, consistency reliability testing has demonstrated high two time points) and ρICC represents the correlation correlations (−0.71 to −0.81) between individual items of responses within clusters. A conservative scenario was which assess opposite perceived gender roles (eg, typical 87 assumed in which the correlation between the two follow-up masculine vs feminine orientation to pain endurance). measurements could be as high as 0.60, and the variance of 2 Covariates the independent variables explained by covariates (ie, R ) We will control for the following demographic, clinical was 0.16, resulting in a requirement of 1250 participants to and surgical covariates: sex, age, BMI, diabetes mellitus, detect a significant change in the odds of post-op pain of peripheral arterial disease, preoperative chronic pain 5% (ie, OR of 1.05). This calculation allows the prevalence and angina (Canadian Cardiovascular Society class), of CPSP to be as low as 10% (as found in some previous non-skeletonised internal throacic artery harvest, re-ster- studies). Should the prevalence of CPSP be higher, the notomy and operating time. Additional covariates include correlation between measurements be smaller, or the vari- baseline functional status, anxiety and acute post-op pain. ance explained in the independent variables be smaller, 1250 participants will provide >80% power.145 Functional status We will control for baseline functional status using the Data analyses SF-12v2 PCS score.118 Categorical data (eg, presence or absence of CPSP at 6 months and 1 year) will be summarised with frequen- Baseline anxiety cies and proportions. Continuous data (eg, functional We will control for anxiety at baseline using the Spiel- disability scores) will be evaluated for normality using berger State-Trait Anxiety Inventory (STAI), a widely used, Shapiro-Wilk tests of normality and summarised using

McGillion MH, et al. BMJ Open 2019;9:e022995. doi:10.1136/bmjopen-2018-022995 7 Open access measures of central tendency and dispersion (eg, means presented at international conferences and published in and SD for normally distributed factors and medians and scientific journals. IQRs for non-normally distributed data). Generalised estimating equations (GEEs) will be used to model the Implications primary analysis: the association between pain-related CPSP is an important socioeconomic problem with beliefs and gender-based pain expectations with the devel- well-documented deleterious consequences on func- opment of CPSP at 6 months and 1 year while adjusting tional status for cardiac patients. We aim to investigate for prespecified covariates. GEE models account for putative psychological risk factors that could be targeted the lack of independence in outcome measurements for preventative intervention. We will also examine the 146 introduced by multiple measurements. We will enter economic consequences of CPSP comprehensively, all prespecified variables in the model and retain them including the impact on QALYs, with no additional data throughout the analysis. For each model, the inclusion collection required. This study may contribute towards of an interaction term between the two independent vari- reducing the risk and impact of CPSP after cardiac ables of interest (pain belief scale and gender- based pain surgery. expectations) will be guided by 95% CIs and likelihood ratio significance tests. Model diagnostics will consist of Author affiliations influential observation examination and Breslow-Day 1School of Nursing, McMaster University, Faculty of Health Sciences, Hamilton, 147 148 Ontario, Canada tests for goodness of fit. We will also assess for multi- 2 collinearity in our model via assessment of condition Anesthesiology, Perioperative Medicine and Surgical Research Unit, Population 147 148 Health Research Institute, Hamilton, Ontario, Canada indices. 3Department of Anesthesia, McMaster University, Hamilton, Ontario, Canada 100 101 QALYs will be estimated by converting SF-12v2 4Department of Health Research Methods, Evidence and Impact, McMaster data collected in the study to utility score using a vali- University, Hamilton, Ontario, Canada 149 5Department of Psychology, York University, Toronto, Ontario, Canada dated algorithm. After estimating QALYs, we will 6 analyse it as a dependent variable using regression to esti- Centre de recherche de Centre hospitalier de l’Université de Montreal, Montreal, Quebec, Canada mate the difference in expected QALYs between the two 7Department of Anesthesiology and Perioperative Medicine, Queen’s University, groups (ie, those with CPSP vs those without). In addi- Kingston, Ontario, Canada tion, after calculating total cost from the AHCR, we will 8Department of Anesthesiology, Kingston General Hospital, Kingston, Ontario, Canada analyse it as a dependent variable using regression to esti- 9 mate the difference in expected healthcare cost between Institute of Health Policy, Management and Evaluation, University of Toronto, Toronto, Ontario, Canada the two groups (ie, patients with CPSP vs those without). 10Department of Public Health Sciences, University of California, Davis, Davis, Employing regression will allow for the adjustment of California, USA potential confounders. With a variety of different types 11School of Physical Therapy, Western University, London, Ontario, Canada of regression (ie, ordinary least squares and generalised 12College of Nursing, Florida State University, Tallahassee, Florida, USA 13 linear models), we will explore the impact of various Lawrence S. Bloomberg Faculty of Nursing, University of Toronto, Toronto, Ontario, Canada modelling assumptions. In addition, we will compare 14Department of Anesthesiology, David Geffen School of Medicine at University of parametric and non-parametric CIs using bootstrapping. California, Los Angeles, California, USA In theory, an ordinary least squares model produces 15Department of Anesthesia and Intensive Care, The Chinese University of Hong unbiased estimates even if the data are skewed; however, Kong, Shatin, Hong Kong 16 different estimation methods (eg, generalised linear Department of Anesthesia, University of Toronto, Toronto, Ontario, Canada models) and different uncertainty methods (eg, non-para- Contributors MHM, PJD, JWB, JCV, JKa, AL, RW, SP, HC, SLC, ND, HS and DNB metric bootstrapping) will facilitate careful investiga- contributed to the conception and design of the study. KB, JH, KG, DD-S and SH tion of the impact that various assumptions have on our contribute to the acquisition of data; data analyses and interpretation will be conclusions.150–153 The regression models will provide conducted by MHM, JWB, PJD, SH, CO, PCC, SE, DNG, JH, WI, JKa, SI, JM, HC, estimates of differences in QALYs and costs for partici- GM, JCV, SSY, JP, IG, MTVC, MC, JW-W, KH-Q, AM. MHM, JB, JKh and PJD wrote the first draft of the protocol. JCV, JKa, AL, MC, RW, SP, KB, HS and JW-W revised pants who develop CPSP versus those who do not develop the protocol critically for important intellectual content. All authors have read and CPSP, which will allow us to calculate incremental cost for approved the final version of the manuscript to be published. The authors wish to one QALY gained. A cost-effectiveness acceptability curve thank our patient advisors for their testimonials, which served to establish the need and 95% CI will be used to characterise the uncertainty for this study. of our findings.153 Funding This work is supported with funding from the Canadian Institutes of Health Research, #MOP–133714, The Hamilton Academic Health Sciences Organization, and the General Research Fund 14101414, Research Grant Council, Ethics and dissemination Hong Kong SAR. Both integrated and end-of-grant dissemination strat- Competing interests M­­­­HM and PJD are members of a research group with a egies will be implemented. Study progress and results policy of not accepting honorariums or other payments from industry for their own will be disseminated on CardiacPain.Net,154 a web-based personal financial gain. They do accept honorariums/payments from industry to pain resource centre (http://​cardiacpain.​onlinecjc.​ca/) support research endeavours and costs to participate in meetings. Based on study questions, PJD has originated and grants he has written, he has received grants linked to Elsevier’s global online readership, featuring from Abbott Diagnostics, Boehringer Ingelheim, Covidien, Octapharma, Philips active knowledge ‘push’ mechanisms including e-banner Healthcare, Roche Diagnostics and Stryker. PJD has participated in an consultancy advertising and opt-in email blasts. Final results will be advisory board meeting for Boehringer Ingelheim.

8 McGillion MH, et al. BMJ Open 2019;9:e022995. doi:10.1136/bmjopen-2018-022995 Open access

Patient consent Obtained. 22. Wiklund L, Johansson M, Bugge M, et al. Early outcome and graft patency in mammary artery grafting of left anterior descending Ethics approval This protocol has been approved by the responsible bodies at artery with sternotomy or anterior minithoracotomy. Ann Thorac each of the hospital sites. Hamilton Integrated Research Ethics Board. Surg 2000;70:79–83. Provenance and peer review Not commissioned; peer reviewed for ethical and 23. Lee W, Yan YY, Jensen MP, et al. Predictors and patterns of chronic funding approval prior to submission. pain three months after cardiac surgery in Taiwan. Pain Med 2010;11:1849–58. Open access This is an open access article distributed in accordance with the 24. Garland R, Frizelle FA, Dobbs BR, et al. A retrospective audit of Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which long-term lower limb complications following leg vein harvesting permits others to distribute, remix, adapt, build upon this work non-commercially, for coronary artery bypass grafting. Eur J Cardiothorac Surg and license their derivative works on different terms, provided the original work is 2003;23:950–5. 25. Herlitz J, Brandrup-Wognsen G, Evander MH, et al. Symptoms of properly cited, appropriate credit is given, any changes made indicated, and the use chest pain and dyspnoea during a period of 15 years after coronary is non-commercial. See: http://​creativecommons.​org/​licenses/​by-​nc/​4.​0/. artery bypass grafting. Eur J Cardiothorac Surg 2010;37:112–8. 26. Momeni M, De Kock M, Lavand'homme P, et al. Abnormal sensations evoked over the chest and persistent peri-incisional References chest pain after cardiac surgery. Acta Anaesthesiol Belg 1. Public Health Agency of Canada. Tracking heart disease and stroke 2010;61:55–62. in canada 2009. http://www.​phac-​aspc.​gc.​ca/​publicat/​2009/​cvd-​ 27. Onan B, Onan IS, Kilickan L, et al. Effects of epidural anesthesia on avc/​index-​eng.​php (accessed Nov 2017). acute and chronic pain after coronary artery bypass grafting. J Card 2. Yusuf S, Reddy S, Ounpuu S, et al. Global burden of cardiovascular Surg 2013;28:248–53. diseases: Part II: variations in cardiovascular disease by specific 28. Choinière M, Watt-Watson J, Victor JC, et al. Prevalence of and risk ethnic groups and geographic regions and prevention strategies. factors for persistent postoperative nonanginal pain after cardiac Circulation 2001;104:2855–64. surgery: a 2-year prospective multicentre study. CMAJ 2014;186:E2 3. Svendsen A. The current status of cardiovascular disease in 13–E223. Canada--a call to action. Can J Cardiovasc Nurs 2004;14:5–7. 29. Parry M, Watt-Watson J, Hodnett E, et al. Pain experiences of men 4. Roger VL, Go AS, Lloyd-Jones DM, et al. Heart disease and and women after coronary artery bypass graft surgery. J Cardiovasc stroke statistics--2012 update: a report from the american heart Nurs 2010;25:E9–E15. association. Circulation 2012;125:e2–e220. 30. van Leersum NJ, van Leersum RL, Verwey HF, et al. Pain symptoms 5. Feindel CM. The current status of cardiac surgery workforce in accompanying chronic poststernotomy pain: a pilot study. Pain Canada. 2010 http://www.​royalcollege.​ca/​portal/​pls/​portal/!​PWEB_​ Med 2010;11:1628–34. PORTAL.​wwpob_​page.​show?_​docname=​509027.​PDF. 31. Routledge FS, Tsuyuki RT, Hervas-Malo M, et al. The influence 6. Bar-El Y, Gilboa B, Unger N, et al. Skeletonized versus pedicled of coronary artery bypass graft harvest site on women’s pain, internal mammary artery: impact of surgical technique on post functional status, and health services utilization throughout the CABG surgery pain. Eur J Cardiothorac Surg 2005;27:1065–9. first post-operative year: a longitudinal study. Int J Nurs Stud 7. Bruce J, Drury N, Poobalan SA, et al. The prevalence of chronic 2009;46:1054–60. chest and leg pain following cardiac surgery: a historical cohort 32. Katz J, Seltzer Z. Transition from acute to chronic postsurgical study. Pain 2003;104:265–73. pain: risk factors and protective factors. Expert Rev Neurother 8. Eisenberg E, Pultorak Y, Pud D, et al. Prevalence and characteristics 2009;9:723–44. of post coronary artery bypass graft surgery pain (PCP). Pain 33. Macrae WA, Davies HTO, et alChronic postsurgical pain. In: 2001;92:11–17. Crombie IK, Croft PR, Linton SJ, LeResche L, Von Korf M, . eds. 9. Gjeilo KH, Klepstad P, Wahba A, et al. Chronic pain after Epidemiology of pain. Seattle: IASP Press, 1999:125–42. cardiac surgery: a prospective study. Acta Anaesthesiol Scand 34. Kalso E, Perttunen K, Kaasinen S. Pain after thoracic surgery. Acta 2010;54:70–8. Anaesthesiol Scand 1992;36:96–100. 10. Ho SC, Royse CF, Royse AG, et al. Persistent pain after cardiac 35. Perttunen K, Tasmuth T, Kalso E. Chronic pain after thoracic surgery: an audit of high thoracic epidural and primary opioid surgery: a follow-up study. Acta Anaesthesiol Scand 1999;43:563–7. analgesia therapies. Anesth Analg 2002;95:820–3. 36. Jung BF, Ahrendt GM, Oaklander AL, et al. Neuropathic pain 11. Jensen MK, Andersen C. Can chronic poststernotomy pain after following breast cancer surgery: proposed classification and cardiac valve replacement be reduced using thoracic epidural research update. Pain 2003;104:1–13. analgesia? Acta Anaesthesiol Scand 2004;48:871–4. 37. Callesen T, Bech K, Kehlet H. Prospective study of chronic pain 12. Kalso E, Mennander S, Tasmuth T, et al. Chronic post-sternotomy after groin hernia repair. Br J Surg 1999;86:1528–31. pain. Acta Anaesthesiol Scand 2001;45:935–9. 38. Perkins FM, Kehlet H. Chronic pain as an outcome of surgery. A 13. King KM, Parry M, Southern D, et al. Women’s Recovery from review of predictive factors. Anesthesiology 2000;93:1123–33. Sternotomy-Extension (WREST-E) study: examining long-term pain 39. Werner MU, Kongsgaard UE. I. Defining persistent post-surgical and discomfort following sternotomy and their predictors. Heart pain: is an update required? Br J Anaesth 2014;113:1–4. 2008;94:493–7. 40. Weinrib AZ, Azam MA, Birnie KA, et al. The psychology of chronic 14. Lahtinen P, Kokki H, Hynynen M. Pain after cardiac surgery: post-surgical pain: new frontiers in risk factor identification, a prospective cohort study of 1-year incidence and intensity. prevention and management. Br J Pain 2017;11:169–77. Anesthesiology 2006;105:794–800. 41. Moore R, Follette DM, Berkoff HA. Poststernotomy fractures 15. Mailis A, Umana M, Feindel CM. Anterior intercostal nerve damage and pain management in open cardiac surgery. Chest after coronary artery bypass graft surgery with use of internal 1994;106:1339–42. thoracic artery graft. Ann Thorac Surg 2000;69:1455–8. 42. Eng J, Wells FC. Morbidity following coronary artery 16. Meyerson J, Thelin S, Gordh T, et al. The incidence of chronic post- revascularisation with the internal mammary artery. Int J Cardiol sternotomy pain after cardiac surgery--a prospective study. Acta 1991;30:55–9. Anaesthesiol Scand 2001;45:940–4. 43. Conacher ID, Doig JC, Rivas L, et al. Intercostal neuralgia 17. Steegers MA, van de Luijtgaarden A, Noyez L, et al. The role of associated with internal mammary artery grafting. Anaesthesia angina pectoris in chronic pain after coronary artery bypass graft 1993;48:1070–1. surgery. J Pain 2007;8:667–73. 44. Defalque RJ, Bromley JJ. Poststernotomy neuralgia: a new pain 18. Taillefer MC, Carrier M, Bélisle S, et al. Prevalence, characteristics, syndrome. Anesth Analg 1989;69:81–2. and predictors of chronic nonanginal postoperative pain after a 45. Watts R, Davies R, Treasure T. Internal mammary artery grafting cardiac operation: a cross-sectional study. J Thorac Cardiovasc increases the incidence of shoulder girdle pain after cardiac surgery. Surg 2006;131:1274–80. J Brit Heart 1988;59:105–6. 19. Ucak A, Onan B, Sen H, et al. The effects of gabapentin on acute 46. Wolf CJ, Salter M. Plasticity and pain. In: MacMahon SB, and chronic postoperative pain after coronary artery bypass graft Koltzenburg M, eds. Wall and Melzack’s Textbook of Pain. 5 edn. surgery. J Cardiothorac Vasc Anesth 2011;25:824–9. Philadelphia: Elsevier Churchill Livingstone, 2006:91–105. 20. van Gulik L, Ahlers SJ, van de Garde EM, et al. Remifentanil during 47. Basbaum A, Bushnell MC, Devor M. Pain: basic mechanisms. cardiac surgery is associated with chronic thoracic pain 1 yr after In: Castro-Lopes J, Raja S, Schmelz M, Pain 2008. An Updated sternotomy. Br J Anaesth 2012;109:616–22. Review Refresher Course Syllabus. Glasgow: IASP Press, 21. van Gulik L, Janssen LI, Ahlers SJ, et al. Risk factors for chronic 2008:3–10. thoracic pain after cardiac surgery via sternotomy. Eur J 48. Melzack R, Wall PD. Pain mechanisms: a new theory. Science Cardiothorac Surg 2011;40:1309–13. 1965;150:971–8.

McGillion MH, et al. BMJ Open 2019;9:e022995. doi:10.1136/bmjopen-2018-022995 9 Open access

49. Katz J, Rosenbloom BN, Fashler S. Chronic pain, psychopathology, 76. Ward SE, Berry PE, Misiewicz H. Concerns about among and dsm-5 somatic symptom disorder. Can J Psychiatry patients and family caregivers in a hospice setting. Res Nurs Health 2015;60:160–7. 1996;19:205–11. 50. Markman PL, Rowland MA, Leong JY, et al. Skeletonized internal 77. Ward S, Donovan HS, Owen B, et al. An individualized intervention thoracic artery harvesting reduces chest wall dysesthesia to overcome patient-related barriers to pain management in women after coronary bypass surgery. J Thorac Cardiovasc Surg with gynecologic cancers. Res Nurs Health 2000;23:393–405. 2010;139:674–9. 78. Ward SE, Goldberg N, Miller-McCauley V, et al. Patient-related 51. Gureje O, Von Korff M, Simon GE, et al. Persistent pain and well- barriers to management of cancer pain. Pain 1993;52:319–24. being: a world health organization study in primary care. JAMA 79. Ward SE, Hernandez L. Patient-related barriers to management of 1998;280:147–51. cancer pain in Puerto Rico. Pain 1994;58:233–8. 52. Gureje O, Simon GE, Von Korff M. A cross-national study 80. Watt-Watson J, Garfinkel P, Gallop R, et al. The impact of nurses’ of the course of persistent pain in primary care. Pain empathic responses on patients’ pain management in acute care. 2001;92:195–200. Nurs Res 2000;49:191–200. 53. Blyth FM, March LM, Brnabic AJ, et al. Chronic pain in Australia: a 81. Watt-Watson J, Pain MA. In: Watt-Watson J, Donovan M, Pain prevalence study. Pain 2001;89:127–34. management: Nursing perspective. St, Louis: Mosby, 1992:36–58. 54. McWilliams LA, Cox BJ, Enns MW. Mood and anxiety disorders 82. Cogan J, Ouimette MF, Vargas-Schaffer G, et al. Patient attitudes associated with chronic pain: an examination in a nationally and beliefs regarding pain medication after cardiac surgery: representative sample. Pain 2003;106:127–33. barriers to adequate pain management. Pain Manag Nurs 55. Ohayon MM, Schatzberg AF. Using chronic pain to predict 2014;15:574–9. depressive morbidity in the general population. Arch Gen Psychiatry 83. Lips HM. Sex and Gender: An Introduction. Mountain View, CA: 2003;60:39–47. Mayfield, 1993. 56. Zigmond AS, Snaith RP. The hospital anxiety and depression scale. 84. Tolver MA, Strandfelt P, Rosenberg J, et al. Female gender is a risk Acta Psychiatr Scand 1983;67:361–70. factor for pain, discomfort, and fatigue after laparoscopic groin 57. Ashburn MA, Staats PS. Management of chronic pain. The Lancet hernia repair. Hernia 2013;17:321–7. 1999;353:1865–9. 85. Uchiyama K, Kawai M, Tani M, et al. Gender differences in 58. Fricker J. Pain in Europe: Mundipharma International Ltd. www.​ postoperative pain after laparoscopic cholecystectomy. Surg painineurope.​com, 1-23. 2003. Endosc 2006;20:448–51. 59. Becker N, Sjøgren P, Bech P, et al. Treatment outcome of chronic 86. Campesi I, Fois M, Franconi F. Sex and gender aspects in non-malignant pain patients managed in a danish multidisciplinary anesthetics and pain medication. Handb Exp Pharmacol pain centre compared to general practice: a randomised controlled 2012;214:265–78. trial. Pain 2000;84:203–11. 87. Robinson ME, Riley JL, Myers CD, et al. Gender role expectations 60. Wilson KG, Mikail SF, D'Eon JL, et al. Alternative diagnostic criteria of pain: relationship to sex differences in pain. J Pain 2001;2:251–7. for major depressive disorder in patients with chronic pain. Pain 88. Hunt K, Adamson J, Hewitt C, et al. Do women consult more 2001;91:227–34. than men? A review of gender and consultation for back pain and headache. J Health Serv Res Policy 2011;16:108–17. 61. Mäntyselkä PT, Turunen JH, Ahonen RS, et al. Chronic pain and 89. Robinson ME, Wise EA, Gagnon C, et al. Influences of gender role poor self-rated health. JAMA 2003;290:2435–42. and anxiety on sex differences in temporal summation of pain. J 62. Watt-Watson J, Choinière CJ. Prevalence characteristics and risk Pain 2004;5:77–82. factors of persistent post-operative pain after cardiac surgery. 90. Soetanto AL, Chung JW, Wong TK. Gender differences in pain Montreal, Canada: The 13th International Association for the Study perception: a signal detection theory approach. Acta Anaesthesiol of Pain World Congress Proceedings, 2010:2956. Taiwan 2004;42:15–22. 63. Gilron I, Vandenkerkhof E, Katz J, et al. Evaluating the association 91. Alabas OA, Tashani OA, Tabasam G, et al. Gender role affects between acute and chronic pain after surgery: impact of pain experimental pain responses: a systematic review with meta- measurement methods. Clin J Pain 2017;33:588–94. analysis. Eur J Pain 2012;16:1211–23. 64. Boodhwani M, Lam BK, Nathan HJ, et al. Skeletonized internal 92. Defrin R, Shramm L, Eli I. Gender role expectations of pain is thoracic artery harvest reduces pain and dysesthesia and associated with pain tolerance limit but not with pain threshold. improves sternal perfusion after coronary artery bypass surgery: a Pain 2009;145:230–6. randomized, double-blind, within-patient comparison. Circulation 93. Defrin R, Eli I, Pud D. Interactions among sex, ethnicity, religion, and 2006;114:766–73. gender role expectations of pain. Gend Med 2011;8:172–83. 65. Flor H, Turk DC. Cognitive and learning aspects. In: MacMahon SB, 94. Wise EA, Price DD, Myers CD, et al. Gender role expectations Koltzenburg M, eds. Wall and Melzack’s Textbook of Pain. 5 edn. of pain: relationship to experimental pain perception. Pain Philadelphia: Elsevier Churchill Livingstone, 2006:241–58. 2002;96:335–42. 66. Turk DC, Flor H. The cognitive-behavioural approach to pain 95. Wandner LD, Scipio CD, Hirsh AT, et al. The perception of pain in management. In: McMahon S, Koltzenburg M, eds. Wall & Melzack’s others: how gender, race, and age influence pain expectations. J textbook of pain. London: Elsevier, 2006:339–48. Pain 2012;13:220–7. 67. Watt-Watson J, Stevens B, Katz J, et al. Impact of preoperative 96. Alabas OA, Tashani OA, Johnson MI. Gender role expectations education on pain outcomes after coronary artery bypass graft of pain mediate sex differences in cold pain responses in healthy surgery. Pain 2004;109:73–85. Libyans. Eur J Pain 2012;16:300–11. 68. Gunnarsdottir S, Serlin RC, Ward S. Patient-related barriers to 97. Robinson ME, Gagnon CM, Riley JL, et al. Altering gender role pain management: the icelandic Barriers Questionnaire II. J Pain expectations: effects on pain tolerance, pain threshold, and pain Symptom Manage 2005;29:273–85. ratings. J Pain 2003;4:284–8. 69. Leegaard M, Nåden D, Fagermoen MS. Postoperative pain and self- 98. Semple T. Post-surgical pain syndromes the medical insurance management: women’s experiences after cardiac surgery. J Adv group. 2010 www.​miga.​com.​au/​riskresources/​library/​10RRAR07.​ Nurs 2008;63:476–85. pdf. 70. Lorentzen V, Hermansen IL, Botti M. A prospective analysis of pain 99. Davies HTO, Crombie IK, Macrae WA, et al. Pain clinic patients in experience, beliefs and attitudes, and pain management of a cohort northern britain. Pain Clin 1992;5:129–35. of Danish surgical patients. Eur J Pain 2012;16:278–88. 100. Gold M, Siegel J, Russell L, et al. Cost-effectiveness in health and 71. Macrae WA. Chronic post-surgical pain: 10 years on. Br J Anaesth medicine. New York: Oxford University Press, 1996. 2008;101:77–86. 101. Drummond M, Sculpher M, Torrance G, et al. Methods for the 72. McDonald DD, McNulty J, Erickson K, et al. Communicating economic evaluation of health care programmes. 3rd ed. New York: pain and pain management needs after surgery. Appl Nurs Res Oxford University Press, 2005. 2000;13:70–5. 102. VanDenKerkhof EG, Peters ML, Bruce J. Chronic pain after surgery: 73. Murnion BP, Gnjidic D, Hilmer SN. Prescription and administration time for standardization? A framework to establish core risk factor of opioids to hospital in-patients, and barriers to effective use. Pain and outcome domains for epidemiological studies. Clin J Pain Med 2010;11:58–66. 2013;29:2–8. 74. Ward S, Gatwood J. Concerns about reporting pain and using 103. Cleeland C. Pain assessment in cancer. Osoba D, Effect of cancer analgesics. A comparison of persons with and without cancer. on quality of life. Florida: CRC Press, 1999:293–305. Cancer Nurs 1994;17:200–6. 104. Cleeland C. The Brief Pain Inventory user guide. Houston, Texas: M. 75. Watt-Watson J, Stevens B, Garfinkel P, et al. Relationship between D Anderson 2009, Cancer Centre. nurses’ pain knowledge and pain management outcomes for their 105. Cleeland CS, Ryan KM. Pain assessment: global use of the brief postoperative cardiac patients. J Adv Nurs 2001;36:535–45. pain inventory. Ann Acad Med Singapore 1994;23:129–38.

10 McGillion MH, et al. BMJ Open 2019;9:e022995. doi:10.1136/bmjopen-2018-022995 Open access

106. Cleeland CS, Syrjala KL. How to assess cancer pain. In: Turk DC, caregivers) of in-home publicly financed care. Healthc Policy Melzack R, eds. Handbook of pain assessment. New York, NY: 2007;3:141–e159. Guilford, 1992. 131. McGillion MH, Watt-Watson J, Stevens B, et al. Randomized 107. Tittle MB, McMillan SC, Hagan S. Validating the brief pain inventory controlled trial of a psychoeducation program for the self- for use with surgical patients with cancer. Oncol Nurs Forum management of chronic cardiac pain. J Pain Symptom Manage 2003;30:325–30. 2008;36:126–40. 108. Watt-Watson J, Chung F, Chan VW, et al. Pain management 132. McGillion MH, Croxford R, Watt-Watson J, et al. Cost of illness following discharge after ambulatory same-day surgery. J Nurs for chronic stable angina patients enrolled in a self-management Manag 2004;12:153–61. education trial. Can J Cardiol 2008;24:759–64. 109. Wilson R. A randomized control trial of an individualized 133. Ward SE, Carlson-Dakes K, Hughes SH, et al. The impact on quality preoperative education intervention for symptom management of life of patient-related barriers to pain management. Res Nurs following total knee arthroplasty (Doctoral dissertation: University of Health 1998;21:405–13. Toronto, 2011. 134. Gunnarsdottir S, Donovan HS, Serlin RC, et al. Patient-related 110. Mendoza T, Mayne T, Rublee D, et al. Reliability and validity barriers to pain management: the Barriers Questionnaire II (BQ-II). of a modified brief pain inventory short form in patients with Pain 2002;99:385–96. osteoarthritis. Eur J Pain 2006;10:353–61. 135. Griffee D. Questionnaire translation and questionnaire validation: 111. Ochroch EA, Gottschalk A, Augoustides JG, et al. Pain and are they the same? Proceedings of the Annual Meeting of the physical function are similar following axillary, muscle-sparing vs American Association of Applied Linguistics. St Louis: MO URL, posterolateral thoracotomy. Chest 2005;128:2664–70. 2001. 112. Gjeilo KH, Stenseth R, Wahba A, et al. Validation of the brief pain 136. Racine M, Tousignant-Laflamme Y, Kloda LA, et al. A systematic inventory in patients six months after cardiac surgery. J Pain literature review of 10 years of research on sex/gender and pain Symptom Manage 2007;34:648–56. perception - part 2: do biopsychosocial factors alter pain sensitivity 113. Martorella G, Côté J, Racine M, et al. Web-based nursing differently in women and men? Pain 2012;153:619–35. intervention for self-management of pain after cardiac surgery: pilot 137. Chapman CR. Psychological aspects of pain patient treatment. randomized controlled trial. J Med Internet Res 2012;14:e177. Arch Surg 1977;112:767–72. 114. Klepstad P, Loge JH, Borchgrevink PC, et al. The Norwegian brief 138. Scott LE, Clum GA, Peoples JB. Preoperative predictors of pain inventory questionnaire: translation and validation in cancer postoperative pain. Pain 1983;15:283–93. pain patients. J Pain Symptom Manage 2002;24:517–25. 139. Spielberger CD, Grambow SC, Machado C, et al. Manual for 115. Ochroch EA, Gottschalk A, Troxel AB, et al. Women suffer more the State-Trait Anxiety Inventory. Palo Alto, CA: Consulting short and long-term pain than men after major thoracotomy. Clin J Psychologists Press, 1983. Pain 2006;22:491–8. 140. Spielberger CD, Gorsuch A, Lushane R, et al. Manual for the 116. Dworkin RH, Turk DC, Farrar JT, et al. Core outcome measures State-Trait Anxiety Inventory. Palo Alto, California: Consulting for chronic pain clinical trials: IMMPACT recommendations. Pain Psychologists Press, 1970. 2005;113(1-2):9–19. 141. Cserép Z, Losoncz E, Balog P, et al. The impact of preoperative 117. Larue F, Colleau SM, Brasseur L, et al. Multicentre study of cancer anxiety and education level on long-term mortality after cardiac pain and its treatment in France. BMJ 1995;310:1034–7. surgery. J Cardiothorac Surg 2012;7:86. 118. Ware J, Kosinski M, Keller SD. A 12-Item Short-Form Health 142. Cserép Z, Balog P, Székely J, et al. Psychosocial factors and major Survey: construction of scales and preliminary tests of reliability and adverse cardiac and cerebrovascular events after cardiac surgery. validity. Med Care 1996;34:220–33. Interact Cardiovasc Thorac Surg 2010;11:567–72. 119. Ware JE, Sherbourne CD. The MOS 36-item short-form health 143. Detroyer E, Dobbels F, Verfaillie E, et al. Is preoperative anxiety and survey (SF-36). I. Conceptual framework and item selection. Med depression associated with onset of delirium after cardiac surgery Care 1992;30:473–83. in older patients? A prospective cohort study. J Am Geriatr Soc 120. McHorney CA, Ware JE, Lu JF, et al. The MOS 36-item Short- 2008;56:2278–84. Form Health Survey (SF-36): III. Tests of data quality, scaling 144. Székely A, Balog P, Benkö E, et al. Anxiety predicts mortality and assumptions, and reliability across diverse patient groups. Med Care 1994;32:40–66. morbidity after coronary artery and valve surgery--a 4-year follow- 121. Ware JE, SF-36 Physical & Mental Health Summary Scales: A User’s up study. Psychosom Med 2007;69:625–31. Manual. Lincoln, RI: Quality Metric Inc, 1997. 145. Hsieh FY, Bloch DA, Larsen MD. A simple method of sample 122. Guerriere DN, Ungar WJ, Corey M, et al. Evaluation of the size calculation for linear and logistic regression. Stat Med ambulatory and home care record: Agreement between self- 1998;17:1623–34. reports and administrative data. Int J Technol Assess Health Care 146. Hubbard AE, Ahern J, Fleischer NL, et al. To GEE or not to GEE: 2006;22:203–10. comparing population average and mixed models for estimating 123. Stevens B, Guerriere D, McKeever P, et al. Economics of home the associations between neighborhood risk factors and health. vs. hospital breastfeeding support for newborns. J Adv Nurs Epidemiology 2010;21:467–74. 2006;53:233–43. 147. Harrell FE. Regression modeling Strategie. New York: Springer, 124. Drummond MF, O’Brien B, Stoddart GL, et al. Methods for the 2006. Economic Evaluation of Health Care Programmes. Toronto: Oxford 148. Babyak MA. What you see may not be what you get: a brief, University Press, 1997. nontechnical introduction to overfitting in regression-type models. 125. Guerriere DN, Tullis E, Ungar WJ, et al. Economic burden of Psychosom Med 2004;66:411–21. ambulatory and home-based care for adults with cystic fibrosis. 149. Brazier J, Rowen D, Hanmer J. Revised SF-6D scoring Treat Respir Med 2006;5:351–9. programmes: A summary of improvements. PRO newsletter 126. Guerriere DN, Wong AY, Croxford R, et al. Costs and determinants 2008;40:14–15. of privately financed home-based health care in Ontario, Canada. 150. Briggs A, Nixon R, Dixon S, et al. Parametric modelling of cost data: Health Soc Care Community 2008;16:126–36. some simulation evidence. Health Econ 2005;14:421–8. 127. Guerriere DN, Tranmer JE, Ungar WJ, et al. Valuing care recipient 151. Barber J, Thompson S. Multiple regression of cost data: and family caregiver time: a comparison of methods. Int J Technol use of generalised linear models. J Health Serv Res Policy Assess Health Care 2008;24:52–9. 2004;9:197–204. 128. Guerriere DN, Choinière M, Dion D, et al. The Canadian STOP- 152. Glick HA, Doshi JA, Sonnad SS, et al. Economic evaluation in PAIN project - Part 2: What is the cost of pain for patients on clinical trials: OUP Oxford, 2014. waitlists of multidisciplinary pain treatment facilities? Can J Anaesth 153. Hoch JS, Rockx MA, Krahn AD. Using the net benefit regression 2010;57:549–58. framework to construct cost-effectiveness acceptability curves: 129. Guerriere DN, Zagorski B, Fassbender K, et al. Cost variations an example using data from a trial of external loop recorders in ambulatory and home-based palliative care. Palliat Med versus Holter monitoring for ambulatory monitoring of “community 2010;24:523–32. acquired” syncope. BMC Health Serv Res 2006;6:68. 130. Leong VW, Guerriere DN, Croxford R, et al. The magnitude, share 154. Arthur MM. No amount of pain is satisfactory: New perspectives on and determinants of private costs incurred by clients (and their persistent cardiac pain. Can J Cardiol 2012;28:2.

McGillion MH, et al. BMJ Open 2019;9:e022995. doi:10.1136/bmjopen-2018-022995 11 EDITORIAL published: 29 September 2020 doi: 10.3389/fphar.2020.600037

Editorial: Mechanisms and New Targets for the Treatment of Chronic Pain

Francisco Rafael Nieto 1,2,3*, Sonja Maksim Vuckovic 4 and Milica S. Prostran 4

1 Department of Pharmacology, School of Medicine, University of Granada, Granada, Spain, 2 Institute of Neuroscience, Biomedical Research Center, University of Granada, Granada, Spain, 3 Biosanitary Research Institute, University Hospital Complex of Granada, Granada, Spain, 4 Department of Pharmacology, Clinical Pharmacology and Toxicology, Faculty of Medicine, University of Belgrade, Belgrade, Serbia

Keywords: chronic pain, persistent pain, neuropathic pain, musculoskeletal pain, visceral pain, cancer-related pain, analgesics, pain

Editorial on the Research Topic

Mechanisms and New Targets for the Treatment of Chronic Pain

BACKGROUND

Nociceptive pain has a physiological protective role in preventing tissue injury. However, pain can become chronic due to a multitude of pathophysiological states, such as: inflammation, nerve injury, tumors, infections, autoimmune diseases, and vascular and metabolic disorders. These pathological states can trigger alterations of the pain pathways that can lead to painful hypersensitivity, and in such circumstances, pain loses its protective role and instead, becomes chronic, pathologic, and it Edited and reviewed by: can be extremely debilitating for people who suffer from it (Basbaum et al., 2009). Chronic pain has Nicholas M. Barnes, fi University of Birmingham, recently been de ned as pain that persists or recurs for more than 3 months (Treede et al., 2019) fi United Kingdom Chronic pain is a very signi cant health problem around the world. Although its prevalence is fi *Correspondence: dif cult to calculate, it has been estimated that a 10% of adults are diagnosed with chronic pain each Francisco Rafael Nieto year (Goldberg and McGee, 2011), and in a very recently meta-analysis, the prevalence of chronic ́ [email protected] pain in the general population of developing countries was estimated at 18% (Sa et al., 2019). However, the management of chronic pain is still no fully satisfactory probably due to the variety of Specialty section: persistent pain conditions with different etiologies, such as musculoskeletal (McWilliams and This article was submitted to Walsh, 2017), neuropathic (Alles and Smith, 2018), visceral (Gebhart and Bielefeldt, 2016) and Neuropharmacology, cancer-related (Lam, 2016) pain, whose pathophysiological mechanisms are only partially known. a section of the journal In addition, current analgesics for pathologic pain are not totally effective in all patients and are Frontiers in Pharmacology associated with serious side effects and/or abuse and dependence problems, in fact, opioids still keep Received: 28 August 2020 an important role in the treatment of certain chronic pain conditions (Finnerup et al., 2015; Noori Accepted: 16 September 2020 et al., 2019). Non-pharmacological treatments for chronic pain relief, such as acupuncture and Published: 29 September 2020 different neurostimulation techniques might have some therapeutic benefits, although the clinical Citation: efficacy of these complementary therapeutic approaches are difficult to prove (Coutaux, 2017; Nieto FR, Vuckovic SM and Lefaucheur, 2019). Therefore, there is a huge need for new effective therapies with less adverse Prostran MS (2020) Editorial: effects for the control and prevention of the different types of chronic pain. Mechanisms and New Targets for the Treatment of Chronic Pain. Chronic pain can emerge as a consequence of dysfunction of the nociceptive circuits at any level Front. Pharmacol. 11:600037. of the nervous system, resulting in an increased perception of pain (hyperalgesia and allodynia), and doi: 10.3389/fphar.2020.600037 even spontaneous pain. Tissue damage and inflammation lead to a reduced threshold and increased

Frontiers in Pharmacology | www.frontiersin.org1 September 2020 | Volume 11 | Article 600037 Nieto et al. Editorial: Mechanisms and Targets for the Treatment of Chronic Pain sensitivity of the peripheral terminals of the nociceptors, induced painful neuropathies, although the scientificevidenceto resulting in pain hypersensitivity – a process known as support its efficacy in these neuropathies is still limited (Ibrahim and peripheral sensitization (Costigan et al., 2009). On the other Ehrlich, 2020). Meng et al. provided important evidence on the hand, central sensitization represents a phenomenon of mechanisms involved in the ameliorative effects of duloxetine in enhanced function of neurons and circuits of central pain chemotherapy-induced neuropathic pain that point to an inhibition pathways, caused by increased membrane excitability and of the activation of p38 MAPK and NF-kB induced by synaptic efficacy and by reduced inhibition (Latremoliere chemotherapy, thus reducing the inflammatory response in the and Woolf, 2009). Peripheral and central sensitization DRG. In addition, this study showed that duloxetine did not affect is a manifestation of the extraordinary plasticity of the the antitumor activity of oxaliplatin and paclitaxel, which is a key somatosensory nervous system in response to inflammation or issue for the pharmacological management of chemotherapy- injury and they are major contributor of chronic pain (Woolf induced peripheral neuropathies. Starobova et al. established a and Salter, 2000). Neuronal functional changes, including new model of peripheral neuropathy induced by vincristine based alterations in neurotransmitter synthesis and signaling, and on the local administration of this drug into the hind paw of mice. changes in expression and trafficking of receptors and ion They compared the resulting pain phenotypes from this new model channels can trigger the sensitization of the pain pathways to that of a conventional model based on systemic administration of (Costigan et al., 2009; Latremoliere and Woolf, 2009). In vincristine. They found that mechanical allodynia was decreased in addition, accumulating evidence suggests that non-neuronal mice lacking Toll-like receptor 4, as well as in mice treated with the cells mainly immune and glial cells play active roles in the antibiotic minocycline, which has been shown to modulate the pathogenesis and resolution of chronic pain by releasing activation of microglia and immune cells. neuroactive mediators that modulate pain (Hore and Denk, Two studies evaluated the analgesic effects of natural compounds 2019). Among these non-neuronal cells, satellite glial cells, on models of neuropathic pain induced by chronic constriction macrophages and T cells in the peripheral nervous system, and injury (CCI) of the sciatic nerve. Jin et al. demonstrated that microglia and astrocytes in the central nervous system, are the systemic administration of koumine (an indole alkaloid presents most studied by far. Interestingly, nociceptive neurons can also in Gelsemium elegans) reduced CCI-induced mechanical allodynia release active substances on glial and immune cells, which can in rats by reducing astrocyte-mediated neuroinflammation. In the also contribute to the neuroinflammatory process that is study conducted by Fotio et al. was shown that a combination of involved in chronic pain (Ji et al., 2016). Therefore, there is an several natural products, including N-Palmitoylethanolamide authentic cross-talk between neurons and immune and glial cells (PEA), beta-caryophyllene, carnosic acid and myrrh extract which has attracted the attention of numerous pain scientists in administered orally, was as effective as gabapentin, a first line the last years to increase our knowledge of how neuroimmune drug for treating neuropathic pain (Finnerup et al., 2015), and neuroglial mechanisms contribute to chronic pain states. reducing pain behaviors induced by CCI in mice. Two other articles evaluate the effects of natural plant-derived products on different pain models. Uddin et al. described the OVERVIEW OF THE ARTICLES INCLUDED antinociceptive effects of the methanol extract of Anisomeles IN THIS RESEARCH TOPIC indica on two classical model of chemical-induced pain such as, -induced writhing test and formalin-induced licking test. This Research Topic includes twenty four articles that address Huang et al. found that Bulleyaconitine A, a diterpenoid alkaloid different aspect of the pathophysiology and treatment of chronic isolated from Aconitum Bulleyanum, produced antinociceptive pain. Most of the studies are basic research with experimental effect in the acetic acid-induced writhing test in rats. In addition, animals, but our Research Topic also includes clinical research. this compound showed anxiolytic effects and improved In addition, the reader will also find several reviews and meta- gastrointestinal function in rats with chronic visceral pain. analysis addressing very interesting subjects. Other basic research studies collected in this Research Topic Among the basic research studies, three articles focused on evaluated the analgesic effects of several drugs on different pain neuropathic pain induced by chemotherapy, one of the most models. Thus, Zhang et al. described that duloxetine had analgesic prevalent adverse effects of cancer patients treated with effects on formalin-induced pain hypersensitivity through antineoplastic drugs (Sisignano et al., 2014). Kim et al. showed increasing the concentration of serotonin in the central nucleus of that a selective phosphodiesterase (PDE)-4 inhibitor called amygdala (CeA). Hu et al. described that the local administration of Rolipram attenuated mechanical hypersensitivity induced by the TRPV1 antagonist had analgesic effects on pain hypersensitivity antitumour drug paclitaxel in rats. The mechanism involved seems associated to a rat model of complex regional pain syndrome to be related to the decreased on the expression of inflammatory (CRPS) induced by ischemia. This study suggests that TRPV1 cytokines in the DRG induced by paclitaxel. In another article channel might have an important role on this pain syndrome, conducted by Meng et al., it was shown that the antidepressant drug whose pathophysiology remain poorly understood (Eldufani et al., duloxetine (a serotonin-norepinephrine reuptake Inhibitor) was 2020). Idris et al. showed that two synthetic benzimidazole effective in reducing neuropathic pain induced by two derivatives attenuated morphine-induced paradoxical pain in antineoplastic drugs (paclitaxel or oxaliplatin) in mice. Duloxetine mice. Srebro et al. showed that systemic administration of is considered a first line drug for the treatment of chemotherapy- tramadol had both prophylactic and therapeutic analgesic effects

Frontiers in Pharmacology | www.frontiersin.org2 September 2020 | Volume 11 | Article 600037 Nieto et al. Editorial: Mechanisms and Targets for the Treatment of Chronic Pain in inflammatory pain and edema induced by intraplantar injection Finally, the present Research Topic also offers to the reader a of carrageenan. Interestingly, they found that the co-administration good number of review articles that provide an update on the of a fixed dose of tramadol with different doses of magnesium pathophysiology and treatment of different type of chronic pain. sulfate led to a dose-dependent enhancement of the analgesic effect Valente performed a mini review about the pharmacology of of tramadol, opening the door to the use of magnesium as adjuvant pain associated with the monoiodoacetate (MIA) model of therapy along with tramadol to counteract inflammatory pain. osteoarthritis. This model consists of intra-articular injection Magnesium ion acts as a blocker of the N-methyl-D-aspartate of MIA to rodents and results in histopathological changes and (NMDA) receptor in the central nervous system and it is well functional disability that mimic to some extend some of the known that NMDA receptor activation is key in the central features of patients with osteoarthritis. Vuckovič ́et al. conducted sensitization process (Latremoliere and Woolf, 2009). an extensive narrative review about the role of cannabinoids in the Another study conducted by Metcalf et al. used a non- treatment of pain. This article includes preclinical and clinical pharmacological approach to demonstrate that music can research about the pharmacodynamics, pharmacokinetics, enhance analgesia and antiseizure effects of several drugs in efficacy, safety and tolerability of these compounds. Joksimovic animal models of pain and epilepsy. The playlist used in the et al. addressed the role of neurosteroids in the management of experiments comprised several Mozart`s compositions. Although pain with an interesting review that focuses on their involvement the analgesic effects of music “per se” (Lunde et al., 2019)oras in the pain pathway, their potential use as analgesics in different adjunct to analgesic drugs (Chai et al., 2017) have been previously models of pain, as well as the future therapeutic perspectives. described in patients, Metcalf et al. presented the first animal study Llorián-Salvador and González-Rodrıgueź carried out an opinion on music-enhanced antinociceptive activity of analgesic drugs. article about the involvement of the vascular endothelial growth This Research Topic also includes a technology report factor (VEGF) in several types of pain. VEGF is an important performed by González-Cano et al. which described a software to mediator with pro-angiogenic properties, being an important simplify the process of calculating 50% mechanical threshold values, regulatory factor in tumor angiogenesis what explains the use of when using calibrated von Frey filaments with the up–down testing anti-VEGF therapies for the treatment of cancer. However, VEGF paradigm. This article can be of interest for scientists working on has been proposed as a potential key factor in the establishment basic pain research with rodents, as von Frey filamentsarebyfarthe and maintenance of chronic pain. tool used most frequently to assess tactile allodynia in mice and rats There are also three review articles with a more clinical (González-Cano et al., 2020). They present an open-source perspective. Vigano et al. performed an update on the role of computer program that can read handwritten von Frey result neurophysiological abnormalities and maladaptive plasticity on sheets and translate these measurements into mechanical the treatment of migraine with neuromodulation. They reviewed hypersensitivity threshold values, eliminating the chance of a the available evidence from therapeutic and physiological studies manual calculation error. using neuromodulation in chronic migraine. Stamenkovic et al. There are also several clinical reports in this Research Topic. carried out a narrative review to analyze preventive measures to Thibautetal.presented a pilot crossover randomized controlled avoid the development of chronic pain in intensive care unit study to evaluate the effects of transcranial direct current stimulation (ICU) patients. In addition, they outlined possible management and transcranial pulsed current stimulation on brain oscillations in options to minimize both chronic pain and long term opioid use patients with chronic visceral pain. Knezevic et al. conducted an following ICU discharge. Knezevic et al. performed a review open-labeled pilot study to determine the efficacy and safety of a about the role of corticosteroids in the treatment of chronic pain. new formulation of gabapentin-extended release in patients with These drugs have been widely used in the treatment of back pain postamputation pain. Gabapentin is a first-line drug for the and osteoarthritis over the past decades. However, their use has treatment of neuropathic pain (Finnerup et al., 2015)and been also questioned due to the serious adverse events of Knezevic et al. demonstrated for the first time, that once-daily corticosteroids, and the emergence of new therapeutic options. dosing of gabapentin-extended release shows significant improvement in pain severity and functional status in these patients, with rates of side effects similar to previously published AUTHOR CONTRIBUTIONS literature. Shan et al. assessed the current availableevidenceofa traditional Chinese medicine herb from the rhizome of Ligusticum All authors contributed to the article and approved the chuanxiong, for the treatment of migraine. The results of this submitted version. systematic review article indicated that this herb can reduce frequency, duration and pain severity of migraine, with a low rate of side effects. Another systematic review article conducted by ACKNOWLEDGMENTS Garcıa-Henareś et al. showed that low doses of intravenous ketamine (a NMDA receptor antagonistin) reduced morphine We thank all the authors of the articles collected in this Research consumption and pain intensity scores after remifentanil-based Topic. We also thank all the reviewers who have provided the general anesthesia for major and minor surgery in adults. This authors with very useful suggestions. Finally, we would also like report also showed that ketamine did not increase the incidence of to thank to all the staff of the Editorial Offices of Frontiers for the adverse effects. great help offered in handling this Research Topic.

Frontiers in Pharmacology | www.frontiersin.org3 September 2020 | Volume 11 | Article 600037 Nieto et al. Editorial: Mechanisms and Targets for the Treatment of Chronic Pain

Lam, D. K. (2016). Emerging Factors in the Progression of Cancer-Related Pain. REFERENCES Pain Manag. 6, 487–496. doi: 10.2217/pmt-2015-0003 Latremoliere, A., and Woolf, C. J. (2009). Central sensitization: a generator of pain Alles, S. R. A., and Smith, P. A. (2018). Etiology and Pharmacology of Neuropathic hypersensitivity by central neural plasticity. J. Pain. 10 (9), 895–926. – Pain. Pharmacol. Rev. 70, 315 347. doi: 10.1124/pr.117.014399 doi: 10.1016/j.jpain.2009.06.012 Basbaum, A. I., Bautista, D. M., Scherrer, G., and Julius, D. (2009). Cellular and Lefaucheur, J. P. (2019). Transcranial magnetic stimulation. Handb. Clin. Neurol. – molecular mechanisms of pain. Cell 139, 267 284. doi: 10.1016/ 160, 559–580. doi: 10.1016/B978-0-444-64032-1.00037-0 j.cell.2009.09.028 Lunde, S. J., Vuust, P., Garza-Villarreal, E. A., and Vase, L. (2019). Music-induced Chai, P. R., Carreiro, S., Ranney, M. L., Karanam, K., Ahtisaari, M., Edwards, R., analgesia: how does music relieve pain? Pain 160, 989–993. doi: 10.1097/ et al. (2017). Music as an Adjunct to Opioid-Based Analgesia. J. Med. Toxicol. j.pain.0000000000001452 – 13, 249 254. doi: 10.1007/s13181-017-0621-9 McWilliams, D. F., and Walsh, D. A. (2017). Pain mechanisms in rheumatoid Costigan, M., Scholz, J., and Woolf, C. J. (2009). Neuropathic pain: a maladaptive arthritis. Clin. Exp. Rheumatol. 35 Suppl 107, 94–101. – response of the nervous system to damage. Annu. Rev. Neurosci. 32, 1 32. Noori, S. A., Aiyer, R., Yu, J., White, R. S., Mehta, N., and Gulati, A. (2019). doi: 10.1146/annurev.neuro.051508.135531 Nonopioid versus opioid agents for chronic neuropathic pain, rheumatoid Coutaux, A. (2017). Non-pharmacological treatments for pain relief: TENS and arthritis pain, cancer pain and low back pain. Pain Manag. 9, 205–216. – acupuncture. Joint Bone Spine 84, 657 661. doi: 10.1016/j.jbspin.2017.02.005 doi: 10.2217/pmt-2018-0052 Eldufani, J., Elahmer, N., and Blaise, G. (2020). A medical mystery of complex Sá, K. N., Moreira, L., Baptista, A. F., Yeng, L. T., Teixeira, M. J., Galhardoni, R., regional pain syndrome. Heliyon 6:e03329. doi: 10.1016/j.heliyon.2020.e03329 et al. (2019). Prevalence of chronic pain in developing countries: systematic Finnerup, N. B., Attal, N., Haroutounian, S., McNicol, E., Baron, R., Dworkin, R. review and meta-analysis. Pain Rep. 4, e779. doi: 10.1097/PR9.00000 H., et al. (2015). Pharmacotherapy for neuropathic pain in adults: a systematic 00000000779 – review and meta-analysis. Lancet Neurol. 14, 162 173. doi: 10.1016/S1474- Sisignano, M., Baron, R., Scholich, K., and Geisslinger, G. (2014). Mechanism- 4422(14)70251-0 based treatment for chemotherapy-induced peripheral neuropathic pain. Nat. Gebhart, G. F., and Bielefeldt, K. (2016). Physiology of Visceral Pain. Compr. Rev. Neurol. 10, 694–707. doi: 10.1038/nrneurol.2014.211 – Physiol. 6, 1609 1633. doi: 10.1002/cphy.c150049 Treede, R. D., Rief, W., Barke, A., Aziz, Q., Bennett, M. I., Benoliel, R., et al. (2019). Goldberg, D. S., and McGee, S. J. (2011). Pain as a global public health priority. Chronic pain as a symptom or a disease: the IASP Classification of Chronic BMC Public Health 11:770. doi: 10.1186/1471-2458-11-770 fi – ́ Pain for the International Classi cation of Diseases (ICD-11). Pain 160, 19 27. González-Cano, R., Montilla-Garcıa,́ A, Ruiz-Cantero, M. C., Bravo-Caparrós, I., ́ doi: 10.1097/j.pain.0000000000001384 Tejada, MA, Nieto, F. R., et al. (2020). The search for translational pain Woolf, C. J., and Salter, M. W. (2000). Neuronal plasticity: increasing the gain in fi outcomes to re ne analgesic development: Where did we come from and pain. Science 288, 1765–1769. doi: 10.1126/science.288.5472.1765 where are we going? Neurosci. Biobehav. Rev. 113, 238–261. doi: 10.1016/ j.neubiorev.2020.03.004 Conflict of Interest: The authors declare that the research was conducted in the Hore, Z., and Denk, F. (2019). Neuroimmune interactions in chronic pain - An absence of any commercial or financial relationships that could be construed as a interdisciplinary perspective. Brain Behav. Immun. 79, 56–62. doi: 10.1016/ potential conflict of interest. j.bbi.2019.04.033 Ibrahim, E. Y., and Ehrlich, B. E. (2020). Prevention of Chemotherapy-Induced Copyright © 2020 Nieto, Vuckovic and Prostran. This is an open-access article Peripheral Neuropathy: A Review of Recent Findings. Crit. Rev. Oncol. distributed under the terms of the Creative Commons Attribution License (CC BY). Hematol. 145:102831. doi: 10.1016/j.critrevonc.2019.102831 The use, distribution or reproduction in other forums is permitted, provided the Ji, R. R., Chamessian, A., and Zhang, Y. Q. (2016). Pain regulation by non- original author(s) and the copyright owner(s) are credited and that the original neuronal cells and inflammation. Science 354, 572–577. doi: 10.1126/ publication in this journal is cited, in accordance with accepted academic practice. No science.aaf8924 use, distribution or reproduction is permitted which does not comply with these terms.

Frontiers in Pharmacology | www.frontiersin.org4 September 2020 | Volume 11 | Article 600037 NMC Case Report Journal 2020; 7: 161–165 DOI: 10.2176/nmccrj.cr.2019-0247 NMCCRJ Case Report Thoracic Disc Herniation Manifesting as Abdominal Pain Alone Associated with Thoracic Radiculopathy NMC Case Report Journal Motonori Ishii,1 Yusuke Nishimura,1 Masahito Hara,2 Kaoru Eguchi,1 Yoshitaka Nagashima,1 Takayuki Awaya,1 Ryo Ando,1 Shoichi Haimoto,1 and Toshihiko Wakabayashi1

0470-8105 Case Report Symptomatic thoracic disc herniation (TDH) with tho- racic radiculopathy alone is an extremely rare condition. A 52-year-old male was referred to us with medically 1349-8029 Here, we report a rare case of TDH in a 52-year-old man refractory severe right flank pain lasting for 1 year. The pain who presented with medically refractory severe right radiated from the back to the umbilics on the right side in the The Japan Neurosurgical Society flank pain. Based on pain distribution, pain intensity T9 dermatomal distribution, which was relieved by truncal changes according to truncal position, ineffectiveness of extension and deteriorated by truncal flexion. He was intercostal nerve block, and radiological findings, his working as a security guard and had to put a lumbar brace on pain was determined to be caused by TDH at T9-10 level. to maintain the truncal extension posture and avoid daily Symptomatic TDH often requires invasive surgery. How- excruciating pain. He had visited multiple medical institutions 10.2176/nmccrj.cr.2019-0247 ever, TDH with radiculopathy alone can be treated via a and was unsuccessfully prescribed with various type of pain posterior approach without spinal cord manipulation or killers. He also underwent several intercostal nerve block pro- nmccrj.2019-0247 spinal fusion. We could eliminate the pain by removing TDH with hemilaminectomy and microdiscectomy using cedures performed by anesthetists with little effect. Other- an O-arm-based navigation system. wise, he was neurologically intact, without weakness and XX sensory disturbance in the four limbs. His past medical his- Keywords: thoracic disc herniation, thoracic tory was unremarkable, and there was no history of trauma, radiculopathy, intercostal neuralgia, abdominal wall pain, prior surgery or skin infection. Thoracic spine magnetic reso- posterior approach nance imaging (MRI) showed a centrolateral to foraminal TDH at the T9-10 level (Fig. 1). Thoracic spine computed Introduction XX tomography (CT) showed intraforaminal calcification, indi- Symptomatic thoracic disc herniation (TDH) is estimated to cating the presence of calcified disc herniation (Fig. 2). We 1,2) occur with a frequency of 1 per million each year. The com- evaluated that his symptoms were compatible with T9 tho- monest location of TDH is central or centrolateral to the spinal racic radiculopathy caused by TDH, based on his pain distri- canal below the T7 level. When TDH occurs in these positions, it bution, characteristic pain intensity changes in accordance 1) XX2020 is extremely rare that radicular pain is the only symptom. A with his truncal position, and the corresponding radiological patient who only complains of radiculopathy, such as abdominal imaging findings. We also judged that the prior intercostal XX pain, might be mistakenly diagnosed with psychogenic pain, nerve block procedures had been ineffective, since the intercostal nerve entrapment caused by muscle stiffness and pathology was located far proximal to the injection site for the truncal position or visceral disease without history of trauma, XX nerve block. The patient declined to undergo additional nerve prior surgery or skin infection, thereby resulting in the risk of root block, such as T9 nerve root block, so we proceeded to downgrading the severity of the disease or undergoing unneces- perform a thoracic hemilaminectomy and microdiscectomy 3) 6November2019 sary invasive procedures. We report here a rare case of a at T9-10 level via a posterior approach, following the patient’s 52-year-old male, who presented with unilateral severe flank wish. We used an O-arm-based navigation system to control pain with a previous diagnosis of intercostal neuralgia after vis- the scope of facet joint resection up to 50%. The herniated iting different hospitals. We evaluated that his pain derived from disc was successfully removed, taking the pressure off from thoracic radiculopathy caused by TDH and successfully treated the affected T9 nerve root (Figs. 3D and 3E). The surgery him surgically. In the discussion section, we also review the liter- was successful, without any neurological complications and ature of TDH presenting with thoracic radiculopathy to discuss his pain disappeared immediately after surgery. Postoperative 2020 how to make a correct diagnosis and provide adequate treatment. CT showed that more than half of the facet joint was pre- served (Figs. 3A–3C). At his postoperative 1-year follow-up, 1Department of Neurosurgery, Nagoya University School of Medi- he is completely free from the pain. cine, Nagoya, Aichi, Japan 2Department of Neurosurgery, Aichi Medical University, Naga- Discussion kute, Aichi, Japan There is a wide variety of signs and symptoms caused by Received: November 6, 2019; Accepted: February 3, 2020 TDH. Quint et al.4) reported that 92% of TDH patients com- Online September 15, 2020 plained of pain, and more than half of them complained of Copyright© 2020 by The Japan Neurosurgical Society 5) This work is licensed under a Creative Commons Attribution- radicular pain. In their review of literature, Elhadi et al. 3February2020 NonCommercial-NoDerivatives International License. reported that 39.5% of the 488 TDH patients showed 161 M. Ishii et al.

Fig. 1 Sagittal (A, B) and axial (C) section of T2-weighted MRI showing centrolateral to foraminal disc herniation compressing the T9 nerve root. Arrows indicate the herniated disc. MRI: magnetic resonance imaging.

Fig. 2 Sagittal (A and B) and axial section (C) of CT showing the calcified TDH and narrowing of the intervertebral foramen (B). Arrows indicate the calcified TDH. CT: computed tomography, TDH: thoracic disc herniation.

radiculopathy. Radicular pain itself is not uncommon for thoracic spinal dermatomal distribution, occasionally accom- TDH. However, it is extremely rare that radiculopathy is the panied by nausea and vomiting, which may easily be misdi- only symptom.1) To the best of our knowledge, there are only agnosed with intra-abdominal or pelvic pathology.15,16) Some 11 cases in the literature reporting a TDH with radiculopathy of the reported cases underwent unnecessary invasive proce- alone, such as chest, abdominal pain, and abdominal muscle dures, such as laparoscopy, before the correct diagnosis of weakness (Table 1).3,6–14) Radiculopathy of TDH could result TDH was made.3) While a thorough visceral test should be in abdominal muscle weakness and pain along with the required in these cases, when a diagnosis of visceral disease

162 Thoracic Disc Herniation with Thoracic Radiculopathy

Fig. 3 Postoperative CT images showing the location of laminectomy (arrow) and decompressed intervertebral foramen (A). Axial CT images show that more than half of the facet joint was preserved (B, C). D showed T9 disc herniation. T9 nerve root (arrow) was severely compressed by the disc TDH (arrowhead) and displaced rostrally, looking congested (E). The pressure was successfully taken off from the T9 root (arrow) and TDH was nicely removed (arrowhead). C: caudal side, CT: computed tomography, M: medial side, L: lateral side, R: rostral side, TDH: thoracic disc herniation. is ruled out, we need to differentiate abdominal wall pain. prior surgery and deteriorates on coughing, sneezing, and Abdominal wall pain includes intercostal neuralgia, myofas- deep inspiration.18) It is sometimes difficult to identify the cial pain, and visceral disease in which inflammation thoracic radiculopathy as a cause of intercostal neuralgia. In spreads to the peritoneum other than thoracic radiculopathy. the present case, the intercostal nerve block had almost no Srinivasan et al.17) reported the usefulness of Carnett's test effect on the pain, indicating far more proximal lesion to the (judged as positive if the tenderness exacerbates when the site of nerve block. Furthermore, the pain distribution corre- abdominal wall is tensed) in differentiating abdominal wall sponded to a dermatomal distribution at the index level and pain from visceral diseases. They described that chronic the intensity of pain varied in accordance with spinal move- abdominal wall pain can be diagnosed with 85% sensitivity ments. Several reports described that the radiculopathy and 97% specificity if any of the following criteria are satis- caused by TDH is aggravated with lateral bending, rotation, fied with pain lasting more than 1 month: (1) very localized flexion of the trunk,19) and supine or side-lying position.14,20) pain that can be covered with a fingertip and (2) fulfillment The patient in this report exhibited pain relief only when the of any of the following criteria: fixed location of tenderness trunk was extended when standing with a rigid lumbar brace. with superficial tenderness; point tenderness less than 2.5 cm; We think that reduction of intervertebral disc pressure by or positive Carnett's test. Routine needle EMG is also extension and/or stabilization of functional motion unit of helpful for the assessment of nerve root impairment, dis- T9-10 by rigid lumbar brace led to temporary pain relief. closing positive sharp waves or fibrillation potentials in mus- Furthermore, we observed a clearly calcified TDH squeezing cles on the radiculopathy level.12) Carnett's test was positive the T9 nerve root on MRI, which was consistent with his in the present case, while invasive tests such as EMG was not pain distribution. These findings prompted us to consider performed since the diagnosis could be made based on radi- that his pain originated from the thoracic spine. ological findings and clinical symptoms. Intercostal neu- The surgical indications and surgery of choice for treating ralgia is the most common in the abdominal wall pain, which TDH should be determined on a case-by-case basis. Surgical is usually caused by herpes zoster, direct injury to the nerve treatment is indicated in the case of medically refractory pain or nerve entrapment associated with iatrogenic neuroma or or progressive myelopathy.21,22) Several surgical approaches

163 M. Ishii et al.

Table 1 Summary of thoracic disc herniation cases with radiculopathy alone Age (years)/ Wrong site surgery or Authors and year Level Location Symptoms Treatment sex invasive examination Whitcomb DC et al., NS/F T7-8 NS Epigastric pain Pancreatico-jejunostomy Microdiscectomy 1995 Abdominal and pelvic Laminectomy and Xiong Y et al., 2001 32/F T9-10 Centrolateral Laparoscopy pain discectomy Stetkarova I et al., Flank pain and muscle 50/F T12-L1 Centrolateral NA Medication 2007 weakness Stetkarova I et al., T10-11, 34/M Centrolateral Lower abdominal pain NA Root nerve injection 2007 T11-12 Abdominal muscle Split-table intermittent LaBan MM et al., 75/M T12-L1 Centrolateral weakness, and flank and NA pelvic tractionthermal 2007 abdominal pain therapy Laparoscopy, Papadakos N et al., Left loin pain radiating to 52/F T11-12 Centrolateral colonoscopy, and Microdiscectomy 2009 groin cystoscopy T2-3, Baranto A et al., 2009 24/M Centrolateral Chest pain NA Physiotherapy T3-4 Hafsteinsdottir B 59/M T11-12 Centrolateral Right flank pain NA Conservating therapy et al., 2012 Srinivasan V et al., 72/M T9-10 Centrolateral Left-sided chest pain NA Operation 2012 Piccirilli M et al., Subscapular right-sided 36/F T7-8 Centrolateral NA Medication 2012 dorsal pain Subscapular left-sided dorsal radiating pain, and Medication and Sari H et al., 2016 38/M T7-8 Centrolateral NA paresthesia radiating to physical therapy left chest wall Present case 52/M T9-10 Centrolateral Right flank pain NA Microdiscectomy NA: not applicable, NS: not specified.

have been suggested for the removal of TDH without manipu- bone resection had been performed as planned, resulting in lating the spinal cord. These procedures are classified into no indication of spinal instability. posterolateral, posterior, lateral transthoracic approach, and less invasive techniques using image-guided thoracoscopy, Conclusion endoscope, and novel retractor system.23–26) Because the her- We report here a patient manifesting abdominal pain due to niated disc was not centrally located in our case, we could radiculopathy caused by TDH at the T9-10 level. If abdom- successfully remove TDH without spinal cord manipulation inal pain alone is observed, it is necessary to investigate the via the posterior approach without spinal fusion. Fusion may characteristics of pain and considering the possible presence be avoided if the lateral portion of the facet joint is pre- of TDH. TDH with only thoracic radiculopathy can be treated served.21,22) As reported by Kim et al.27) and Schlenzka with less invasive surgical procedure, without spinal fusion. et al.,28) an O-arm-based navigation system helped to firmly identify the spinal anatomy. We could determine the extent of Conflicts of Interest Disclosure the bone resection and muscles dissection with the image The authors report no conflict of interest concerning the guidance technique resulting in preservation of more than materials or methods used in this study or the findings speci- half of the facet joint. O-arm-based navigation system allows fied in this paper. The authors have no personal, financial, or us to minimize muscle invasion and damage to medial branch institutional interest in any of the drugs, materials, or devices of posterior ramus of the spinal nerves. Furthermore, we described in this manuscript. could easily identify the index level with this system even for References hard-to-localize middle to upper thoracic levels. We could 1) Arce CA, Dohrmann GJ: Thoracic disc herniation. Improved diagno- eventually preserve more than half of the facet joint with the sis with computed tomographic scanning and a review of the literature. image guidance technique. Postoperative CT showed that the Surg Neurol 23: 356–361, 1985

164 Thoracic Disc Herniation with Thoracic Radiculopathy

2) Stillerman CB, Chen TC, Couldwell WT, Zhang W, Weiss MH: 15) Rohde RS, Kang JD: Thoracic disc herniation presenting with Experience in the surgical management of 82 symptomatic herniated chronic nausea and abdominal pain. J Bone Joint Surg Am 86: 379– thoracic discs and review of the literature. J Neurosurg 88: 623–633, 381, 2004 1998 16) Shirzadi A, Drazin D, Jeswani S, Lovely L, Liu J: Atypical presentation 3) Papadakos N, Georges H, Sibtain N, Tolias CM: Thoracic disc pro- of thoracic disc herniation: Case series and review of the literature. Case lapse presenting with abdominal pain: case report and review of the Rep Orthop 2013: 621476, 2013 literature. Ann R Coll Surg Engl 91: W4–W6, 2009 17) Srinivasan R, Greenbaum DS: Chronic abdominal wall pain: a fre- 4) Quint U, Bordon G, Preissl I, Sanner C, Rosenthal D: Thoracoscopic quently overlooked problem. Practical approach to diagnosis and man- treatment for single level symptomatic thoracic disc herniation: a pro- agement. Am J Gastroenterol 97: 824–830, 2002 spective followed cohort study in a group of 167 consecutive cases. 18) Dureja GP: Intercostal Neuralgia: A review. J Neurol Transl Neurosci 5: Eur Spine J 21: 637–645, 2012 1076–1076, 2017 5) Elhadi AM, Zehri AH, Zaidi HA, et al.: Surgical efficacy of mini- 19) Kato K, Yabuki S, Otani K, et al.: Unusual chest wall pain caused by mally invasive thoracic discectomy. J Clin Neurosci 22: 1708–1713, thoracic disc herniation in a professional baseball pitcher. Fukushima J 2015 Med Sci 62: 64–67, 2016 6) Baranto A, Börjesson M, Danielsson B, Hellström M, Swärd L: Acute 20) Ozturk C, Tezer M, Sirvanci M, Sarier M, Aydogan M, Hamzaoglu A: chest pain in a top soccer player due to thoracic disc herniation. Spine Far lateral thoracic disc herniation presenting with flank pain. Spine J 6: 34: E359–362, 2009 201–203, 2006 7) Hafsteinsdottir B, Olafsson E: Thoracal radiculopathy owing to disc 21) Bouthors C, Benzakour A, Court C: Surgical treatment of thoracic disc herniation. Arch Neurol 69: 1080–1081, 2012 herniation: an overview. Int Orthop 43: 807–816, 2019 8) LaBan MM, Gorin G: A thoracic disc herniation presenting as an 22) Court C, Mansour E, Bouthors C: Thoracic disc herniation: Surgical abdominal hernia. Am J Phys Med Rehabil 86: 601, 2007 treatment. Orthop Traumatol Surg Res 104: S31–S40, 2018 9) Piccirilli M, Lapadula G, Caporlingua F, Martini S, Santoro A: Spon- 23) Bordon G, Burguet Girona S: Experience in the treatment of thoracic taneous regression of a thoracic calcified disc herniation in a young herniated disc using image-guided thorascopy. Rev Esp Cir Ortop Trau- female: a case report and literature review. Clin Neurol Neurosurg 114: matol 61: 124–129, 2017 779–781, 2012 24) Cho JY, Lee SH, Jang SH, Lee HY: Oblique paraspinal approach for 10) Sari H, Misirlioglu TO, Palamar D: Regression of a symptomatic tho- thoracic disc herniations using tubular retractor with robotic holder: a racic disc herniation with a calcified intervertebral disc component. technical note. Eur Spine J 21: 2620–2625, 2012 Acta Orthop Traumatol Turc 50: 698–701, 2016 25) Deviren V, Kuelling FA, Poulter G, Pekmezci M: Minimal invasive 11) Srinivasan V, Kesler H, Johnson M, Dorfman H, Walter K: Tophaceous anterolateral transthoracic transpleural approach: a novel technique for pseudogout of the thoracic spine. Acta Neurochir (Wien) 154: 747–750; thoracic disc herniation. A review of the literature, description of a new discussion 750, 2012 surgical technique and experience with first 12 consecutive patients. 12) Stetkarova I, Chrobok J, Ehler E, Kofler M: Segmental abdominal J Spinal Disord Tech 24: E40–48, 2011 wall paresis caused by lateral low thoracic disc herniation. Spine 32: 26) Wagner R, Telfeian AE, Iprenburg M, et al.: Transforaminal Endoscopic E635–639, 2007 Foraminoplasty and Discectomy for the Treatment of a Thoracic Disc 13) Whitcomb DC, Martin SP, Schoen RE, Jho HD: Chronic abdominal Herniation. World Neurosurg 90: 194–198, 2016 pain caused by thoracic disc herniation. Am J Gastroenterol 90: 27) Kim JS, Eun SS, Prada N, Choi G, Lee SH: Modified transcorporeal 835–837, 1995 anterior cervical microforaminotomy assisted by O-arm-based naviga- 14) Xiong Y, Lachmann E, Marini S, Nagler W: Thoracic disk herniation tion: a technical case report. Eur Spine J 20 Suppl 2: S147–152, 2011 presenting as abdominal and pelvic pain: a case report. Arch Phys Med 28) Schlenzka D, Laine T, Lund T: Computer-assisted spine surgery. Eur Rehabil 82: 1142–1144, 2001 Spine J 9 Suppl 1: S57–64, 2000

Corresponding author: Yusuke Nishimura, MD, PhD, Department of Neurosurgery, Nagoya University School of Medicine, 65 Tsurumaicho, Showa-ku, Nagoya, Aichi 466-8550, Japan. * [email protected]

165 Research Article Molecular Pain Volume 13: 1–12 ! The Author(s) 2017 Analgesia induced by the epigenetic drug, Reprints and permissions: sagepub.com/journalsPermissions.nav L-acetylcarnitine, outlasts the end of DOI: 10.1177/1744806917697009 journals.sagepub.com/home/mpx treatment in mouse models of chronic inflammatory and neuropathic pain

Serena Notartomaso1, Giada Mascio1, Matteo Bernabucci1, Cristina Zappulla1, Pamela Scarselli1, Milena Cannella1, Tiziana Imbriglio1, Roberto Gradini1,2, Giuseppe Battaglia1, Valeria Bruno1,3 and Ferdinando Nicoletti1,3

Abstract Background: L-acetylcarnitine, a drug marketed for the treatment of chronic pain, causes analgesia by epigenetically up-regulating type-2 metabotropic glutamate (mGlu2) receptors in the spinal cord. Because the epigenetic mechanisms are typically long-lasting, we hypothesized that analgesia could outlast the duration of L-acetylcarnitine treatment in models of inflammatory and neuropathic pain. Results: A seven-day treatment with L-acetylcarnitine (100 mg/kg, once a day, i.p.) produced an antiallodynic effect in the complete Freund adjuvant mouse model of chronic inflammatory pain. L-Acetylcarnitine-induced analgesia persisted for at least 14 days after drug withdrawal. In contrast, the analgesic effect of pregabalin, amitryptiline, ceftriaxone, and N-acet- ylcysteine disappeared seven days after drug withdrawal. L-acetylcarnitine treatment enhanced mGlu2/3 receptor protein levels in the dorsal region of the spinal cord. This effect also persisted for two weeks after drug withdrawal and was associated with increased levels of acetylated histone H3 bound to the Grm2 gene promoter in the dorsal root ganglia. A long-lasting analgesic effect of L-acetylcarnitine was also observed in mice subjected to chronic constriction injury of the sciatic nerve. In these animals, a 14-day treatment with pregabalin, amitryptiline, tramadol, or L-acetylcarnitine produced a significant antiallodynic effect, with pregabalin displaying the greatest efficacy. In mice treated with pregabalin, tramadol or L-acetylcarnitine the analgesic effect was still visible 15 days after the end of drug treatment. However, only in mice treated with L-acetylcarnitine analgesia persisted 37 days after drug withdrawal. This effect was associated with an increase in mGlu2/3 receptor protein levels in the dorsal horns of the spinal cord. Conclusions: Our findings suggest that L-acetylcarnitine has the unique property to cause a long-lasting analgesic effect that might reduce relapses in patients suffering from chronic pain.

Keywords Pain, L-acetylcarnitine, metabotropic glutamate receptors, long-lasting analgesia

Date received: 20 September 2016; revised: 3 January 2017; accepted: 18 January 2017

Introduction 1I.R.C.C.S. Neuromed, Pozzilli, Italy A large body of evidence indicates that L-acetylcarnitine 2Department of Experimental Medicine, Sapienza University, Rome, Italy (LAC) is clinically effective in the treatment of neuro- 3Department of Physiology and Pharmacology, Sapienza University, Rome, pathic pain, fibromyalgia, and other types of chronic Italy pain, with an excellent profile of safety and tolerabil- 1–10 Corresponding author: ity. LAC treatment may also cause antidepressant- Ferdinando Nicoletti, Department of Physiology and Pharmacology, like effects that may be advantageous in the management University Sapienza, Piazzale Aldo Moro, 5, 00185 Roma, Italy. 11–14 of patients suffering from chronic pain. To our Email: [email protected]

Creative Commons Non Commercial CC-BY-NC: This article is distributed under the terms of the Creative Commons Attribution- NonCommercial 3.0 License (http://www.creativecommons.org/licenses/by-nc/3.0/) which permits non-commercial use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the SAGE and Open Access pages (https:// us.sagepub.com/en-us/nam/open-access-at-sage). 2 Molecular Pain knowledge, LAC is the only drug that causes analgesia and ceftriaxone were purchased from Sigma Aldrich via an epigenetic mechanism mediated by acetylation of (St. Louis, MO); pregabalin was purchased from Tocris p65/RelA, a member of the NFkB family of transcrip- Cookson (Avonmouth, Bristol, UK). All these drugs tion factors. Acetylation of p65/RelA leads to an were dissolved into saline. We used a clinical injectable enhanced expression of type-2 metabotropic glutamate formulation of tramadol HCl (Contramal, 50 mg/ml), (mGlu2) receptors in the dorsal root ganglia and dorsal which was also dissolved into saline. horns of the spinal cord with an ensuing reduction of glutamate release from primary afferent sensory Animals fibres.15 In rats subjected to chronic constriction injury (CCI) of the sciatic nerve, repeated administrations of We used adult male C57BL/6 J mice (20–25 g, b.w.) pur- LAC reduced mechanical allodynia after five to seven chased from Charles River (Calco, Italy). All mice were days, and a single injection of an mGlu2/3 receptor housed five per cage, under a standard 12/12 h light/dark antagonist at the end of LAC treatment was sufficient cycle with food and water ad libitum for at least two to abrogate LAC-induced analgesia.16 Acetylation of weeks prior to the experiments. All experiments were p65/RelA and induction of mGlu2 receptors by LAC carried out according to the European (86/609/EEC) could also be demonstrated in cultured dorsal root gang- and Italian (D: Lgs. 116/92) guidelines of animal care. lia neurons, and the induction of mGlu2 receptors was The experimental protocol was approved by the Italian abrogated by pharmacological inhibition of NFkB.17 Ministry of Health. All efforts were made to minimize Interestingly, drugs that inhibit histone deacetylases animal suffering and the number of animals. (HDACs) mimicked the analgesic action of LAC and were also able to enhance p65/RelA acetylation and Induction of chronic inflammatory pain and drug mGlu2 receptor expression in the dorsal horns of the treatment design spinal cord.18 That epigenetic regulation of mGlu2 expression is critically involved in the control of pain is Chronic inflammatory pain was induced by intraplantar also demonstrated by the evidence that pharmacological (i.pl.) injections of 20 ml of CFA (F5881 Sigma-Aldrich; inhibition of histone acetyl transferase (HAT) causes a 1 mg/ml) in the right hind paw. Control mice received an marked down-regulation of mGlu2 receptors in the i.pl. injection of saline. In a first experiment, six groups spinal cord and a hypoacetylation of histones H3 and of six CFA-injected mice were treated intraperitoneally H4 in the dorsal root ganglia, thereby preventing the (i.p.) as follows: two groups of mice received a single analgesic activity of the mGlu2/3 receptor agonist, injection of saline or LAC (100 mg/kg) and pain thresh- LY379268.19 LAC treatment was shown to enhance the olds were assessed after 1 h; the four remaining groups of amount of acetylated histone H3 acetylation bound to mice were treated daily with saline or LAC for either the promoter of the mGlu2 receptor-encoding gene three or seven days, and pain thresholds were assessed (Grm2 gene) in the prefrontal cortex and hippocampus 1 h after the last injection. Mice of the first two groups of spontaneously depressed rats.12 Whether LAC were killed 24 h after a single injection of saline or LAC enhances histone acetylation in the spinal cord of mice for the analysis of mGlu2/3 receptor protein levels in the with chronic pain is unknown. spinal cord. Mice of all the other groups were killed On the basis of these findings, we predicted that LAC immediately after the assessment of pain thresholds. In treatment could induce long-lasting changes in the expres- a second experiment, four groups of mice were treated as sion of mGlu2 receptors in the spinal cord resulting into a follows: (1) i.pl. injection of saline followed, 1 h later, by persisting analgesic effect. We now demonstrate that i.p. injection of saline (once a day for seven days); (2) LAC-induced analgesia persists for at least two weeks i.pl. injection of saline followed by i.p. injection of LAC after drug withdrawal in the complete Freund adjuvant (100 mg/kg, once a day for seven days); (3) i.pl. injection (CFA) mouse model of inflammatory pain and for more of CFA followed by i.p. injection of saline (once a day than one month in the CCI mouse model of neuropathic for seven days); and (4) i.pl. injection of CFA followed pain. In addition, we offer the first evidence that LAC by i.p. injection of LAC (once a day for seven days). treatment enhances the amount of acetylated histone H3 Mechanical pain thresholds were assessed under basal bound to the Grm2 promoter in the dorsal root ganglia. conditions (i.e. before i.pl. injection of CFA or saline) after seven days (i.e. at the end of systemic treatment with LAC or saline), and seven days after drug with- Materials and methods drawal (corresponding to 14 days after i.pl. injection of Drugs CFA or saline). Parallel groups of mice injected i.pl. with CFA and treated with saline or LAC for seven days were LAC was a generous gift from Sigma Tau Laboratories killed at the end of saline or LAC treatment or seven (Pomezia, Italy); N-acetylcysteine (NAC), amitriptyline days later for measurements of mGlu2/3 receptor protein Notartomaso et al. 3 levels in the spinal cord. In a third experiment, two a modified version of the up-down paradigm, as reported groups of mice were injected i.pl. with CFA and treated by Chaplan et al.21 The filaments were applied five times for seven days with either saline or LAC. One week after each, in order of increasing forces, and pressed perpen- drug withdrawal (i.e. 14 days after CFA injection), mice dicularly to the plantar surface of the hind paw until they were killed 1 h after measurements of pain thresholds, bent. The first filament that evoked at least three and the dorsal root ganglia were used for chromatin responses was assigned as the pain threshold in grams. immunoprecipitation analysis. In a third experiment, For the assessment of mechanical sensitivity, mice were six groups of mice were injected i.pl. with CFA and trea- placed in individual cages with elevated mesh floor 1 h ted i.p. once a day for seven days with saline, LAC before all behavioral testing. (100 mg/kg), pregabalin (30 mg/kg), amitryptiline (10 mg/kg), ceftriaxone (200 mg/kg), or NAC (100 mg/kg). Western blot analysis Pain thresholds were assessed prior to CFA injection (basal), 1 h after the end of drug treatment, and then at Tissues (dorsal regions of the lumbar segments of the seven and 14 days after drug withdrawal (corresponding spinal cord ipsilateral to i.pl. injection of CFA or to 14 and 21 days after CFA injection). One hour after saline) were homogenized on ice with RIPA buffer the last injection, three to four mice selected from the containing protease inhibitors cocktail tablet (Santa groups treated with i.p. with saline, LAC, or pregabalin Cruz Biotechnology, Inc., Temecula, CA, USA). were killed for measurements of mGlu2/3 receptor pro- Homogenates were centrifuged at 14,000 rpmin for tein in the spinal cord. 10 min and an aliquot was used for protein determin- ations. Equal amounts of proteins (20 mg) from super- Induction of neuropathic pain and drug natants were separated by 8% SDS polyacrilamide gel treatment design and transferred on Immuno PVDF membranes (Bio- Rad, Milan, Italy) for 7 min using Trans Blot Turbo Neuropathic pain was induced by CCI of the sciatic System (Bio-Rad, Milan, Italy) for the detection of nerve using the method described by Bennet and Xie20 mGlu2/3 receptor. Filters were blocked overnight in modified as follows: mice were anesthetized with isoflur- blocking buffer (TBS, 0.05% Tween-20 and 5% ane (5% for induction and 2% for maintenance), the non-fat milk) at 4C. Membrane were incubated for biceps femoris, and gluteus superficialis were separated 1 h with rabbit polyclonal anti-mGlu2/3 receptors by blunt dissection, and the left sciatic nerve was exposed (1:500, Sigma-Aldrich), and secondary peroxidase- for ligation. One ligature was tied loosely around the coupled anti-rabbit antibodies (1:7000 Calbiochem, nerve, until it elicited a brief twitch in the respective Milano, Italy). The blots were re-probed with anti-b- hind limb, taking care to preserve epineural circulation. actin monoclonal antibody (1:50,000 Sigma-Aldrich). The incision was cleaned and the skin was closed with Immunostaining was revealed by enhanced chemilumin- two to three ligatures of 5-0 dermalon. Mice were then escence luminosity (Amersham Pharmacia Biotech, placed on a warmed surface and, following recovery, Arlington Height, IL). they were returned to their home cages and checked rou- tinely for 72 h. Five groups of mice (n ¼ 7–8 per group) Chromatin immunoprecipitation assay were treated i.p. daily for 14 days with saline, LAC (100 mg/kg), pregabalin (30 mg/kg), amitryptiline Chromatin immunoprecipitation assay was performed (10 mg/kg), or tramadol (100 mg/kg). Mechanical by standard procedures. Briefly, dorsal root ganglia pain thresholds were measured prior to CCI, and at were cross-linked using 1% at room the end of drug treatment, and then at 7 and 37 days temperature for 10 min. The cross-linking reaction was after drug withdrawal. mGlu2/3 Receptor protein levels stopped by adding glycine to a final concentration of were measured exclusively in CCI mice treated with 0.125 M. Tissue was then washed three times with saline or LAC. ice-cold PBS supplemented with protease inhibitors (Sigma-Aldrich), homogenized in lysis buffer (5 mM Measurements of mechanical allodynia PIPES pH 8.0, 85 mM KCl, 0.5% NP40, protease inhibi- tors), and nuclei lysis buffer (1% SDS, 10 mM EDTA, Mechanical allodynia was quantified by measuring the 50 mM Tris-HCl pH 8.0, protease inhibitors), and then hind paw withdrawal response to von Frey filament sonicated on ice using a Covaris S220 ultrasonicator. stimulation. In brief, mice were placed in a Plexiglas Following sonication, chromatin fragments of 0.2–0.6 kb box (20 cm high, 9 cm diameter) with a wire grid in length were obtained. Ten percent of the sonicated bottom through which the von Frey filaments (North lysate was saved and successively used to quantify Coast Medical, Inc., San Jose, CA, USA; bending the total amount of DNA present in different samples force range from 0.008 to 3.5 g) were applied by using before immunoprecipitation (inputs). The chromatin 4 Molecular Pain solution was precleared with salmon sperm DNA/ Results protein A-agarose 50% gel slurry for 1 h at 4C and LAC produced long-lasting analgesia and a long-lasting immunoprecipitated overnight at 4 C with antibodies increase in mGlu2/3 receptor protein levels in the CFA against pan-acetylated-H3 histone (6 mg/ml, Upstate model of chronic inflammatory pain Biotechnology, Billerica, MA, USA) or the respective isotype matched control Ig. After precipitation, the chro- LAC treatment is known to produce analgesia in the matin-antibody complexes were collected using Protein CCI model of neuropathic pain after at least five days A Sepharose beads and washed. Samples were then of daily injections.16 First, we examined whether LAC eluted with 1% SDS, 100 mM NaHCO3 at room was also able to produce analgesia in the CFA model temperature for 15 min, reverse cross-linked with NaCl with a similar temporal profile. i.pl. CFA injection 100 mM at 65C overnight, and treated with proteinase-K. induced a large reduction in pain thresholds, which per- Levels of histone acetylation at the Grm2 gene sisted for at least two weeks. LAC failed to induce promoter were expressed as percentage of the input changes in mechanical pain thresholds after a single DNA that was immunoprecipitated by the anti-acetyl- administration or three days of injections, but produced histone H3 antibody using the following equation: substantial analgesia after seven days of daily injections % (acetDNA-IP/total input) ¼ 2 [(Ct(10%input) 3.32) (Figure 1(a)). To examine whether the effect of LAC Ct (acetDNAIP)] 100%. outlasted the end of treatment, we performed a second experiment in which LAC or saline were administered Statistical analysis daily for seven days. Pain thresholds were assessed at the end of the treatment and, again, seven days later. Statistical analysis was performed by one-way ANOVA LAC treatment produced substantial analgesia in CFA- or two-way ANOVA for repeated measures followed by injected mice at both time points (Figure 1(b)). LAC Fisher’s LSD or, alternatively, by Student’s t-test, as treatment did not significantly affect mechanical thresh- indicated. olds in control mice that received i.pl. injection of saline

(a) Single administration (b) Basal 3 days of treatment 7 days of treatment + 1 hr of withdrawal 7 days of treatment 7 days of treatment + 7 days of withdrawal 0.4 0.90 # * 0.75 0.3 # 0.60

0.2 0.45

0.30 0.1 * Mechanical threshold (g) Mechanical Mechanical threshold (g) Mechanical 0.15 *

0 0 Saline LAC Saline, i.pl., Saline, i.pl., CFA, i.pl., CFA, i.pl., saline, i.p. LAC, i.p. Saline, i.p. LAC, i.p. CFA, i.pl.

Figure 1. Effect of LAC on mechanical pain thresholds in the CFA mouse model of chronic inflammatory pain. (a) Six groups of six CFA- injected mice were treated intraperitoneally (i.p.) as follows: two groups of mice received a single injection of saline or LAC (100 mg/kg) and pain thresholds were assessed after 1 h; the four remaining groups of mice were treated daily with saline or LAC for either three or seven days, and pain thresholds were assessed 1 h after the last injection. Values are means S.E.M. of six determinations per group. *p < 0.05 (two-way ANOVA þ Fisher’s LSD; F(5,25) ¼ 11.13). Mechanical pain thresholds in mice injected i.pl. with saline or CFA and then treated i.p. with either LAC (100 mg/kg, once a day for seven days) or saline are shown in (b). Thresholds were measured under basal conditions (i.e. prior to i.pl. injections of saline or CFA), at the end of the seven days of treatment with saline or LAC (1 h of withdrawal), and again after seven days of withdrawal. Values are means S.E.M. of five to seven determinations. p < 0.01 vs. the corresponding values obtained in mice injected i.pl. and i.p. with saline (*) or vs. the corresponding values obtained in mice injected i.pl. with CFA and i.p. with saline (#) (two-way ANOVA for repeated measures þ Fisher’s LSD; treatment x time F(8,24) ¼ 4.975). CFA: complete Freund adjuvant; LAC: L-acetylcarnitine. Notartomaso et al. 5

(a)mGlu2/3 receptors (b) Single administration Dimer- - 220 kDa mGlu2/3 receptors Monomer- - 110 kDa 7 days of treatment Dimer- - 220 kDa β-Actin- - 42 kDa Monomer- - 110 kDa 3 days of treatment β-Actin- Dimer- - 220 kDa ______- 42 kDa Saline______1 hr wtd 7 days wtd Monomer- - 110 kDa ______LAC, 100 mg/kg, i.p. β-Actin- - 42 kDa ______CFA, i.pl. Saline LAC, 100 mg/kg, i.p. CFA, i.pl. Saline Saline LAC, 100 mg/kg, i.p. LAC, 100 mg/kg, i.p., 7 days of treatment 1.6 0.35 400

0.30 * -actin -actin β β

1.2 imer 300 0.25

0.20 days) 7 eptor d eptor * 0.8 200

0.15

line at line /3 rec /3

0.10

0.4 100

(% sa (% mGlu2 0.05 mGlu2/3 receptor dimer/ mGlu2/3 receptor dimer/ 0 0 0 Single 3 days of 1 hr wtd 7 days wtd administration treatment CFA, i.pl. CFA, i.pl.

Figure 2. Repeated injections of LAC induced an increase in mGlu2/3 receptor protein levels that outlasted the end of drug treatment. mGlu2/3 protein levels were measured in the dorsal regions of the lumbar spinal cord ipsilateral to CFA injection. Data in (a) and (b) were obtained by the same groups of mice as in Figure 1(a) and (b), respectively. Mice treated with a single injection of saline or LAC were killed after 24 h. Mice treated with saline or LAC for three or seven days were killed pain assessment 1 h following the last injection. Data at seven days from animals used in Figure 1(a) are not shown. Values are means þ S.E.M. of four to five and 5–10 determinations in (a) and (b), respectively. *p < 0.05 vs. the corresponding values of mice treated i.p. with saline (one-way ANOVA þ Fisher’s LSD; F(3,23) ¼ 5.884). Wdt: withdrawal; CFA: complete Freund adjuvant; mGlu2: type-2 metabotropic glutamate. instead of CFA (Figure 1(b)). Moving from the evidence Increased levels of acetylated histone H3 bound to the that induction of mGlu2 receptors mediates the analgesic 16 Grm2 gene promoter in the dorsal root ganglia one action of LAC, we measured mGlu2/3 receptor protein week after LAC treatment in CFA mice levels in the dorsal regions of the lumbar spinal cord ipsilateral to the site of CFA injection. Western blot ana- Induction of mGlu2 receptors by LAC in the dorsal root lysis showed two bands at 100–110 KDa, of which the ganglia and spinal cord has been related to an increased upper band corresponds to mGlu2/3 receptor mono- acetylation of p65/RelA.17 However, acetylation of a mers, and an additional band at >200 kDa correspond- transcription factor may not be sufficient to explain the ing to the active, dimeric form of mGlu2/3 receptor. long-lasting increase in mGlu2/3 receptor protein we LAC had no effect on mGlu2/3 receptor protein levels have seen in mice treated with LAC. Thus, we examined after a single administration or three days of treatment whether LAC treatment was also able to enhance histone (Figure 2(a)). In contrast, seven days of treatment with acetylation on the Grm2 gene promoter in the dorsal root LAC enhanced mGlu2/3 receptor protein levels in the ganglia as already observed in other brain regions.12 Two dorsal region of the lumbar spinal cord. This effect per- additional groups of mice injected with CFA were trea- sisted after seven days of drug withdrawal (data of the ted with saline or LAC for seven days, and killed seven two experiments are similar, and only data of the second days later. We could confirm in these mice that LAC experiment are shown in Figure 2(b)). induced an analgesic effect that outlasted the end of 6 Molecular Pain

observed in the group of mice treated with pregabalin (a)0.4 (b) 2.5 Saline * LAC, 100 mg/kg, i.p. (Figure 4(c)). * 2.0 0.3 Long-lasting analgesic effect of LAC in the CCI mouse model of neuropathic pain 1.5 Mice subjected to CCI of the sciatic nerve were treated 0.2 daily for 14 days, i.p., with either saline, LAC (100 mg/ 1.0 (% control) (% kg), pregabalin (30 mg/kg), amitriptyline (10 mg/kg), or tramadol (100 mg/kg), and mechanical pain thresholds 0.1

Mechanical threshold (g) threshold Mechanical 0.5 were measured 1 h after the end of treatments and at 7

GRM2 promoter H3 acetylation H3 promoter GRM2 and 37 days after drug withdrawal. CCI mice treated

0 0 with saline showed the expected reduction in mechanical pain threshold as compared to sham-operated mice. At 7 days of treatment + 7 days of withdrawal least under our conditions, mechanical allodynia CFA, i.pl. remained stable up to 51 days after CCI in saline-treated mice (Figure 5(a)). At the end of systemic treatments Figure 3. Increased levels of acetylated histone H3 bound to the (i.e. 14 days after CCI), a significant increase in pain Grm2 gene promoter in the dorsal root ganglia of mice with thresholds was seen in all groups of mice treated with inflammatory pain treated with LAC. Mice were injected i.pl. with drugs, with pregabalin displaying the greatest analgesic CFA and then treated with either saline or LAC for seven days. effect (Figure 5(a)). Seven days after the end of treat- Pain thresholds and levels of acetylated histone H3 bound to the ments, analgesia was still observed in mice treated with Grm2 gene promoter in the dorsal root ganglia were measured pregabalin, tramadol, or LAC, but not in mice treated seven days after the end of drug treatment and shown in (a) and with amitryptiline (Figure 5(a)). In contrast, only mice (b), respectively. Values are means S.E.M. of six to nine deter- minations for mechanical threshold and five to seven determin- treated with LAC still showed a significant increase in ations for Grm2 gene promoter. *p < 0.05 (Student’s t-test) vs. the pain thresholds 37 days after drug withdrawal corresponding values obtained in mice treated i.p. with saline (Figure 5(a)). An increase in mGlu2/3 receptor protein (t ¼3.384 for mechanical threshold and t ¼2.301 for Grm2 levels was observed in the ipsilateral dorsal horns of the gene promoter). spinal cord of the group of mice treated with LAC after CFA: complete Freund adjuvant. 37 days of withdrawal as compared to mice treated with saline (Figure 5(b)). treatment (Figure 3(a)). ChIP analysis showed an increased acetylation of histone H3 bound to the Grm2 Discussion promoter in the dorsal root ganglia of LAC-treated mice after seven days of withdrawal, as compared to mice A number of recent reviews highlight the role of epigen- treated with saline (Figure 3(b)). etic mechanisms in the pathophysiology and treatment of chronic pain.22–26 Changes in histone acetylation have LAC was the only tested analgesic drug that produced been consistently found in the pain neuraxis of mice a long-lasting increase in pain thresholds in the CFA developing chronic inflammatory or neuropathic pain. model of chronic inflammatory pain CFA-induced inflammatory pain is associated with an increased expression of class-II HDACs in the dorsal Mice injected i.pl. with CFA were treated for seven days horns of the spinal cord.27 Spinal nerve ligation causes with either saline, LAC (100 mg/kg), pregabalin (30 mg/kg), a reduced acetylation of histone H3 bound to the pro- amitriptyline (10 mg/kg), ceftriaxone (200 mg/kg), or moter of the gene encoding for the GABA-synthesizing N-acetylcysteine (NAC, 100 mg/kg). All drugs produced enzyme, glutamate decarboxylase-65, in the nucleus similar analgesic effects at the end of treatment, but only raphe magnus, resulting into a disinhibition of serotoner- mice treated with LAC retained the analgesic at seven or gic neurons projecting to the spinal cord.28 In models of 14 days after drug withdrawal (Figure 4(a)). Selected neuropathic pain, changes in acetylation mechanisms mice treated with saline, LAC, or pregabalin were also affect the expression of genes encoding for proteins used for measurements of mGlu2/3 receptor protein that critically regulate pain thresholds, such as m opiate levels in the dorsal region of the spinal cord after the receptors, Kv4.3 potassium channels, Nav1.8 voltage- last determination of pain thresholds. LAC-treated sensitive sodium channels, and brain-derived neuro- mice showed increased mGlu2/3 receptor protein levels trophic factor.28–31 Drugs that enhance acetylation of after 14 days of drug withdrawal (Figure 4(b)), whereas a histones or transcription factors, such as HDAC inhibi- small reduction in mGlu2/3 receptor protein levels was tors, consistently cause analgesia in models of oatms tal. et Notartomaso n ftetet,adte n 4dy fe rgwtdaa r hw n() hr ausaemeans are values where (a), the in amitryptiline at shown mg/kg), (30 conditions, are pregabalin withdrawal basal drug mg/kg), under (100 after measured LAC days thresholds saline, 14 Pain either and mg/kg). with 7 (100 days NAC then *p seven and or for treatments, mg/kg) (200 day of ceftriaxone a end mg/kg), once (30 i.p. pregabalin treated mg/kg), and (10 CFA with i.p. injected 4. Figure F F:cmlt rudajvn;LC -ctlantn;ml2 ye2mtbtoi glutamate. metabotropic type-2 mGlu2: means t L-acetylcarnitine; are t-test; LAC: values (Student’s adjuvant; where saline Freund (b), with complete in treated CFA: shown mice are in pregabalin obtained or values LAC saline, with treated (23,161) < .5v.tecrepnigvle bandi ietetdip ihsln oewyAOAfrrpae measures repeated for ANOVA (one-way saline with i.p. treated mice in obtained values corresponding the vs. 0.05 ¼ .9.ml23rcpo rti eesi h oslrgo fteisltrllma pnlcr ftesm ruso mice of groups same the of cord spinal lumbar ipsilateral the of region dorsal the in levels protein receptor mGlu2/3 6.399. A a h nqepoet oidc oglsigaagsai h F oe fcrncifamtr an iewere Mice pain. inflammatory chronic of model CFA the in analgesia long-lasting a induce to property unique the has LAC

Mechanical threshold (g) (a) 0.5 1.0 1.5 2.0 2.5 3.0 Monomer- Monomer- (b) 0 β β Dimer- Dimer- -actin- -actin- Basal (pre-CFAinjection) 7 daysoftreatment+1hrwithdrawal 7 daysoftreatment+14withdrawal 7 daysoftreatment+14withdrawal Saline t Saline Ctx t Saline Ctx mGlu2/3 receptors 100 mg/kg * LAC, * * Pregabalin Pregabalin, 30 mg/kg ¼ * .9 o rgblnadt and pregabalin for 3.199 LAC CFA, i.pl. - 110kDa - 42kDa Amitryptiline, - 220kDa 7 daysoftreatment+withdrawal 7 daysoftreatment+14withdrawal 10 mg/kg * - 110kDa - 42kDa - 220kDa ...o he ofu eemntos *p determinations. four to three of S.E.M. Ceftriaxone, Ceftriaxone, 200 mg/kg

¼ mGlu2/3 receptor dimer/β-actin mGlu2/3 receptor dimer/β-actin * 0.5 1.0 1.5 2.0 2.5 0.5 1.0 1.5 2.0 3.0 2.5 0 0 40 o LAC). for 24.07 Saline Pregabalin Pregabalin Saline LAC 100 mg/kg * * * NAC, ...o determinations. 8 of S.E.M. þ ihrsLSD). Fisher’s < .5vs. 0.05 7 8 Molecular Pain

(a) 14 days of treatment + 1 hr withdrawal 14 days of treatment + 14 days of withdrawal 0.7 14 days of treatment + 37 days of withdrawal ** 0.6

0.5

0.4 * * 0.3 * * * 0.2 * Mechanical threshold (g) Mechanical

0.1

0 Saline LAC, Pregabalin, Tramadol, Amitryptiline, 100 mg/kg 30 mg/kg 100 mg/kg 10 mg/kg CCI

(b) 1.2 Saline mGlu2/3 receptor LAC, 100 mg/kg, i.p. Dimer - - 220 kDa -actin 1.0 * β Monomer - - 110 kDa 0.8

β-actin - - 42 kDa 0.6 ______SO Saline LAC 0.4 100 mg/kg, i.p. ______0.2

CCI mGlu2/3 receptor dimer/ ______0 14 days of treatment + 37 days of withdrawal

Figure 5. Comparative effects of LAC and other analgesic drugs on pain thresholds in the CCI model of neuropathic pain. Mice were subjected to CCI of the sciatic nerve and then treated i.p. once a day for 14 days with either saline, LAC (100 mg/kg), pregabalin (30 mg/kg), tramadol (100 mg/kg), or amitryptiline (10 mg/kg). Pain thresholds were measured under basal conditions, at the end of drug treatments, and then after 7 or 37 days. Data are shown in (a). Basal pain thresholds are not shown and were similar in all experimental groups. Values are means S.E.M. of seven to eight determinations. *p < 0.05 (one-way ANOVA for repeated measures þ Fisher’s LSD; F(14,89) ¼ 8.984). mGlu2/3 protein levels in the ipsilateral dorsal region of the lumbar spinal cord of mice treated with saline or LAC at the end of experiment (i.e. after 37 days of withdrawal) are shown in (b). Values are means S.E.M. of seven determinations. Two samples from sham-operated rats (S.O., 51 days after surgery) are also shown in one of the blots. *p < 0.05 (Student’s t-test; t ¼3.89) if compared to CCI mice treated with saline. CCI: chronic constriction injury; LAC: L-acetylcarnitine; mGlu2: type-2 metabotropic glutamate. inflammatory and neuropathic pain, although histone transcriptional activity at the Grm2 gene promoter.17 acetyl-transferase inhibitors may also relieve pain.24 This mechanism is shared by at least two HDAC inhibi- The acetylating agent, LAC, causes analgesia by enhan- tors, which were shown to cause analgesia in the second cing the expression of mGlu2 receptors in the dorsal root phase of the formalin test by up-regulating mGlu2 ganglia and dorsal horns of the spinal cord.15–17 At least receptors.18 a component of the analgesic action of LAC is mediated LAC is a safe drug that is currently marketed for the by acetylation of NFkB p65/RelA, which enhances its treatment of chronic pain (see Introduction section and Notartomaso et al. 9 references section therein). However, LAC is not con- end of treatment in mice with neuropathic pain but not sidered as a first-line drug in the treatment of inflamma- in mice with chronic inflammatory pain. In mice with tory, mixed, or neuropathic pain32 in spite of its neuropathic pain, pregabalin may cause a prolonged widespread use in the medical practice. Our data analgesia by preventing the increased trafficking of the demonstrate that LAC has the unique property to pro- a2d subunit from the dorsal root ganglia to the spinal duce an analgesic effect that outlasts by several days or cord.34 LAC was less efficacious than pregabalin in weeks the end of treatment in models of chronic inflam- enhancing mechanical pain thresholds in CCI mice, but matory and neuropathic pain. This strengthens the value its effect persisted after five weeks of drug withdrawal, of LAC as an analgesic drug and supports the hypothesis when the effect of all other drugs was no longer visible. that epigenetic mechanisms are valuable candidate drug The long-lasting effects of LAC in the CFA and CCI targets in the treatment of chronic pain. As active com- models of chronic pain suggest that the drug causes per- parators, we used drugs that are known to cause anal- sistent changes in the molecular machinery that controls gesia by targeting mechanisms that regulate pain pain threshold in the CNS. LAC is known to induce the threshold in the spinal cord and other region of the expression of mGlu2 receptors by enhancing the acetyl- 15–17 pain neuraxis. Pregabalin binds to the a2d subunit of ation of the transcription factor, p65/RelA. This voltage-sensitive Ca2þ channels, thereby restraining mechanism has been clearly demonstrated in in vitro neurotransmitter release at the synapses between pri- and in vivo models,11,12,17 but it is insufficient to explain afferent fibres and spinal cord neurons.33 the induction of mGlu2 receptors we have seen in the Pregabalin also prevents the increased transport of the spinal cord of CFA and CCI mice after weeks of drug a2d subunit from dorsal root ganglia to the spinal cord withdrawal. At least in CFA mice, we have demonstrated associated with neuropathic pain,34 thus weakening the that levels of acetylated histone H3 bound to Grm2 pro- molecular machinery that allows Ca2þ entry in the pre- moter were elevated in LAC-treated animals after two synaptic terminals of primary sensory fibres. Although weeks of withdrawal. This suggests that LAC-induced pregabalin is a first-line drug in the treatment of neuro- acetylation is not restricted to transcription factors, pathic pain,32 it also shows efficacy in the CFA model of and that durable changes in chromatin structure caused chronic inflammatory pain.35 Amiptryptiline is a tricyclic by histone acetylation may underlie an analgesic effect antidepressant that is also used as a first-line drug in the that outlasts by several days or weeks the end of LAC treatment of neuropathic pain32 and acts primarily by treatment. It is likely that LAC enhanced histone acetyl- inhibiting noradrenaline and serotonin uptake from pre- ation acting as an acetyl donor at the Grm2 promoter. synaptic terminals. However, amitryptiline exerts pleio- However, we cannot exclude that LAC acts indirectly to tropic effects that widen its action to different types of enhance H3 histone acetylation at the Grm2 promoter. pain, such as inhibition of TrpV1 channels36 and induc- For example, LAC might acetylate a transcription factor tion of endogenous opioid release.37 Tramadol is a m that represses HDAC expression, thereby enhancing his- opioid receptor agonist that can also inhibit serotonin tone acetylation. and noradrenaline uptake and is widely used in the treat- An increased histone acetylation by LAC has already ment of different types of pain including neuropathic been found in the hippocampus and prefrontal cortex of pain.32 Ceftriaxone is a b-lactam antibiotic that induces spontaneously depressed Flinders Sensitive Line rats, the expression of the glial glutamate transporter, GLT-1, where LAC was shown to induce a long-lasting antide- thereby causing analgesia in experimental animal models pressant-like effect.12 It is therefore possible that acetyl- and humans.38–40 NAC is an activator of the cystine- ation of both p65/Rel and histones contributes to Grm2 glutamate antiporter that causes analgesia by enhancing induction in response to LAC, and that histone acetyl- the endogenous activation of mGlu2 receptors.41,42 We ation in particular accounts for the long-lasting increase used all these drugs at a single dose, selected in conform- in mGlu2 receptor expression and analgesia in models of ity with previous reports.16,35,40–44 In the CFA model of chronic pain. A direct correlation between histone acetyl- chronic inflammatory pain, all drugs were equally effect- ation and mGlu2/3 receptor expression in the spinal cord ive in increasing mechanical pain thresholds, but only in has been demonstrated in a model of stress-induced irrit- mice treated with LAC the analgesic effect was still able bowel disorder, in which HDAC inhibition attenu- observed after one or two weeks of drug withdrawal. ates visceral hypersensitivity by enhancing the levels of In the CCI model, pregabalin and amitryptiline dis- acetylated histone H3 bound to Grm2 and Grm3 played the greatest efficacy in causing analgesia, as promoters.45 expected by their first-line indication in the treatment mGlu2 and mGu3 receptors are expressed in all sta- of neuropathic pain.32 Interestingly, the analgesic effect tions of the pain neuraxis, and their activation consist- of pregabalin and tramadol persisted after one week of ently produces analgesia in a variety of pain models.46–56 drug withdrawal, whereas the effect of amitryptiline was At least in the formalin model of inflammatory pain, not durable. Pregabalin-induced analgesia outlasted the mGlu2 rather than mGlu3 receptors appear to regulate 10 Molecular Pain pain threshold and mediate the analgesic activity of References 57 mGlu2/3 receptor agonists. Thus, our findings support 1. Onofrj M, Fulgente T, Melchionda D, et al. L-acetylcarni- 15 the hypothesis that targeting the epigenetic mechan- tine as a new therapeutic approach for peripheral neuro- isms that regulate mGlu2 receptor expression may be a pathies with pain. Int J Clin Pharmacol Res 1995; 15: 9–15. valuable strategy for an efficient and durable treatment 2. Sima AA, Calvani M, Mehra M, et al. Acetyl-L-carnitine of chronic pain. improves pain, nerve regeneration, and vibratory percep- tion in patients with chronic diabetic neuropathy: an ana- lysis of two randomized placebo-controlled trials. Diabetes Conclusions Care 2005; 28: 89–94. We have shown that LAC, a drug marketed for the treat- 3. Flatters SJ, Xiao WH and Bennett GJ. Acetyl-L-carnitine prevents and reduces paclitaxel-induced painful peripheral ment of chronic pain, produces a long-lasting analgesia in neuropathy. Neurosci Lett 2006; 397: 219–223. mouse models of inflammatory and neuropathic pain. 4. Evans JD, Jacobs TF and Evans EW. Role of acetyl-L- Analgesia outlasted by several days or weeks the end of carnitine in the treatment of diabetic peripheral neur- treatment. This property was unique to LAC in mice with opathy. Ann Pharmacother 2008; 42: 1686–1691. chronic inflammatory pain, and was also shared by preg- 5. Memeo A and Loiero M. Thioctic acid and acetyl-L-carni- abalin and tramadol in mice with neuropathic pain. tine in the treatment of sciatic pain caused by a herniated However, at a late time point after drug withdrawal, disc: a randomized, double-blind, comparative study. Clin only mice with neuropathic pain treated with LAC Drug Investig 2008; 28: 495–500. retained the analgesic effect. An increase in histone acetyl- 6. Xiao WH and Bennett GJ. Chemotherapy-evoked neuro- ation at Grm2 gene promoter may contribute to the long- pathic pain: abnormal spontaneous discharge in A-fiber lasting analgesic effect of LAC. These findings suggest and C-fiber primary afferent neurons and its suppression that LAC may relieve pain for a long time causing a per- by acetyl-L-carnitine. Pain 2008; 135: 262–270. 7. Janiri L, Martinotti G, Tonioni F, et al. Acetyl-L-carnitine sistent up-regulation of mGlu2 receptors, which are key in the management of pain during methadone withdrawal players in the regulation of pain threshold. These findings syndrome. Clin Neuropharmacol 2009; 32: 35–40. strengthen the value of mGlu receptors as drug targets for 58–62 8. Valcour V, Yeh TM, Bartt R, Clifford D, et al. Acetyl-l- the treatment of pain. The peculiar mechanism of carnitine and nucleoside reverse transcriptase inhibitor- action of LAC is promising from a clinical standpoint associated neuropathy in HIV infection. HIV Med 2009; considering that the benefit achieved with current anal- 10: 103–110. gesic drugs in patients suffering of neuropathic pain and 9. Leombruni P, Miniotti M, Colonna F, et al. A randomised other types of chronic pain is suboptimal and a large per- controlled trial comparing duloxetine and acetyl L-carni- centage of patients is resistant to medication.63 Our data tine in fibromyalgic patients: preliminary data. Clin Exp encourage clinical studies with LAC in patients suffering Rheumatol 2015; 33: S82–S85. from chronic inflammatory or neuropathic pain in which 10. Li S, Li Q, Li Y, et al. Acetyl-L-carnitine in the treatment the relapse rate at different time intervals after drug with- of peripheral neuropathic pain: a systematic review and drawal is included as a primary endpoint. meta-analysis of randomized controlled trials. PLoS One 2015; 10: e0119479. 11. Cuccurazzu B, Bortolotto V, Valente MM, et al. Author Contributions Upregulation of mGlu2 receptors via NF-kB p65 acetyl- ation is involved in the Proneurogenic and antidepressant SN and GM contributed equally to this article. SN, GM, effects of acetyl-L-carnitine. Neuropsychopharmacology MB, CZ, PS, MC, TI: performed experiments and ana- 2013; 38: 2220–2230. lyzed data; RG, GB, VB, FN: designed experiments, 12. Nasca C, Xenos D, Barone Y, et al. L-acetylcarnitine supervised research and wrote the manuscript. All causes rapid antidepressant effects through the epigenetic authors read and approved the final manuscript. induction of mGlu2 receptors. Proc Natl Acad Sci USA 2013; 110: 4804–4809. 13. Zanardi R and Smeraldi E. A double-blind, randomised, Declaration of Conflicting Interests controlled clinical trial of acetyl-L-carnitine vs. amisulpride The author(s) declared no potential conflicts of interest in the treatment of dysthymia. Eur Neuropsychopharmacol with respect to the research, authorship, and/or publica- 2006; 16: 281–287. 14. Bersani G, Meco G, Denaro A, et al. L-Acetylcarnitine in tion of this article. dysthymic disorder in elderly patients: a double-blind, mul- ticenter, controlled randomized study vs. fluoxetine. Eur Funding Neuropsychopharmacol 2013; 23: 1219–1225. The author(s) disclosed receipt of the following financial sup- 15. Chiechio S, Copani A, Zammataro M, et al. port for the research, authorship, and/or publication of this Transcriptional regulation of type-2 metabotropic glutam- article: This work was supported by the Italian Ministry of ate receptors: an epigenetic path to novel treatments for Health (project code: RF-2011-02352582). chronic pain. Trends Pharmacol Sci 2010; 31: 153–160. Notartomaso et al. 11

16. Chiechio S, Caricasole A, Barletta E, et al. 33. Dooley DJ, Taylor CP, Donevan S, et al. Ca2þ channel L-Acetylcarnitine induces analgesia by selectively up- alpha2delta ligands: novel modulators of neurotransmis- regulating mGlu2 metabotropic glutamate receptors. sion. Trends Pharmacol Sci 2007; 28: 75–82. Mol Pharmacol 2002; 61: 989–996. 34. Bauer CS, Nieto-Rostro M, Rahman W, et al. The 17. Chiechio S, Copani A, De Petris L, et al. Transcriptional increased trafficking of the calcium channel subunit regulation of metabotropic glutamate receptor 2/3 expres- alpha2delta-1 to presynaptic terminals in neuropathic sion by the NF-kappaB pathway in primary dorsal root pain is inhibited by the alpha2 delta ligand pregabalin. ganglia neurons: a possible mechanism for the analgesic J Neurosci 2009; 29: 4076–4088. effect of L-acetylcarnitine. Mol Pain 2006; 2: 20. 35. Maciel IS, Silva RB, Morrone FB, et al. Synergistic effects 18. Chiechio S, Zammataro M, Morales ME, et al. Epigenetic of celecoxib and bupropion in a model of chronic inflam- modulation of mGlu2 receptors by histone deacetylase mation-related depression in mice. PLoS One 2013; 8: inhibitors in the treatment of inflammatory pain. Mol e77227. Pharmacol 2009; 75: 1014–1020. 36. Ola´hZ,Jo´svay K, Pecze L, et al. Anti-calmodulins and 19. Zammataro M, Sortino MA, Parenti C, et al. HDAC and tricyclic adjuvants in pain therapy block the TRPV1 chan- HAT inhibitors differently affect analgesia mediated by nel. PLoS One 2007; 2: e545. group II metabotropic glutamate receptors. Mol Pain 37. Gray AM, Spencer PS and Sewell RD. The involvement of 2014; 10: 68. the opioidergic system in the antinociceptive mechanism of 20. Bennett GJ and Xie YK. A peripheral mononeuropathy in action of antidepressant compounds. Br J Pharmacol 1998; rat that produces disorders of pain sensation like those 124: 669–674. seen in man. Pain 1988; 33: 87–107. 38. Lin Y, Tian G, Roman K, et al. Increased glial glutamate 21. Chaplan SR, Bach FW, Pogrel JW, et al. Quantitative transporter EAAT2 expression reduces visceral nociceptive assessment of tactile allodynia in the rat paw. J Neurosci response in mice. Am J Physiol Gastrointest Liver Physiol Methods 1994; 53: 55–63. 2009; 296: G129–G134. 22. Kurita M, Moreno JL, Holloway T, et al. Repressive epi- 39. Chen Z, He Y and Wang ZJ. The beta-lactam antibiotic, genetic changes at the mGlu2 promoter in frontal cortex of ceftriaxone, inhibits the development of opioid-induced hyperalgesia in mice. Neurosci Lett 2012; 509: 69–71. 5-HT2A knockout mice. Mol Pharmacol 2013; 83: 40. Macaluso A, Bernabucci M, Trabucco A, et al. Analgesic 1166–1175. effect of a single preoperative dose of the antibiotic cef- 23. Descalzi G, Ikegami D, Ushijima T, et al. Epigenetic triaxone in humans. J Pain 2013; 14: 604–612. mechanisms of chronic pain. Trends Neurosci 2015; 38: 41. Bernabucci M1, Notartomaso S, Zappulla C, et al. 237–246. N-Acetyl-cysteine causes analgesia by reinforcing the 24. Ge´ranton SM and Tochiki KK. Could targeting epigenetic endogenous activation of type-2 metabotropic glutamate processes relieve chronic pain states? Curr Opin Support receptors. Mol Pain 2012; 8: 77. Palliat Care 2015; 9: 138–146. 42. Truini A, Piroso S, Pasquale E, et al. N-acetyl-cysteine, a 25. Ge´ranton SM and Tochiki KK. Regulation of gene expres- drug that enhances the endogenous activation of group-II sion and pain states by epigenetic mechanisms. Prog Mol metabotropic glutamate receptors, inhibits nociceptive Biol Transl Sci 2015; 131: 147–183. transmission in humans. Mol Pain 2015; 11: 14. 26. Liang L, Lutz BM, Bekker A, et al. Epigenetic regulation 43. Jesse CR, Wilhelm EA and Nogueira CW. Depression-like of chronic pain. Epigenomics 2015; 7: 235–245. behavior and mechanical allodynia are reduced by bis sel- 27. Bai G, Wei D, Zou S, et al. Inhibition of class II histone enide treatment in mice with chronic constriction injury: a deacetylases in the spinal cord attenuates inflammatory comparison with fluoxetine, amitriptyline, and bupropion. hyperalgesia. Mol Pain 2010; 6: 51. Psychopharmacology 2010; 212: 513–522. 28. Zhang Z, Cai YQ, Zou F, et al. Epigenetic suppression of 44. Mika J, Jurga AM, Starnowska J, et al. Effects of chronic GAD65 expression mediates persistent pain. Nat Med doxepin and amitriptyline administration in naı¨ve mice and 2011; 17: 1448–1455. in neuropathic pain mice model. Neuroscience 2015; 294: 29. Uchida H, Ma L and Ueda H. Epigenetic gene silencing 38–50. underlies C-fiber dysfunctions in neuropathic pain. 45. Cao DY, Bai G, Ji Y, et al. Histone hyperacetylation J Neurosci 2010; 30: 4806–4814. modulates spinal type II metabotropic glutamate receptor 30. Uchida H, Matsushita Y and Ueda H. Epigenetic regula- alleviating stress-induced visceral hypersensitivity in female tion of BDNF expression in the primary sensory neurons rats. Mol Pain 2016; 12: 1–12. after peripheral nerve injury: implications in the develop- 46. Neugebauer V, Chen PS and Willis WD. Groups II and III ment of neuropathic pain. Neuroscience 2013; 240: metabotropic glutamate receptors differentially modulate 147–154. brief and prolonged nociception in primate STT cells. 31. Uchida H, Sasaki K, Ma L, et al. Neuron-restrictive silen- J Neurophysiol 2000; 84: 2998–3009. cer factor causes epigenetic silencing of Kv4.3 gene after 47. Popik P, Kozela E and Pilc A. Selective agonist of group II peripheral nerve injury. Neuroscience 2010; 166: 1–4. glutamate metabotropic receptors, LY354740, inhibits 32. Attal N, Cruccu G, Baron R, et al. EFNS guidelines on the tolerance to analgesic effects of morphine in mice. Br J pharmacological treatment of neuropathic pain: 2010 revi- Pharmacol 2000; 130: 1425–1431. sion. European Federation of Neurological Societies. Eur J 48. Simmons RM, Webster AA, Kalra AB, et al. Group II Neurol 2010; 17: 1113–e88. mGluR receptor agonists are effective in persistent and 12 Molecular Pain

neuropathic pain models in rats. Pharmacol Biochem Behav antiallodynic effects of R-isovaline. Neuroscience 2015; 2002; 73: 419–427. 293: 15–156. 49. Yang D and Gereau RW 4th. Peripheral group II metabo- 56. Kiritoshi T and Neugebauer V. Group II mGluRs modu- tropic glutamate receptors mediate endogenous anti-allo- late baseline and arthritis pain-related synaptic transmis- dynia in inflammation. Pain 2003; 106: 411–417. sion in the rat medial prefrontal cortex. 50. Yang D and Gereau RW 4th. Peripheral group II Neuropharmacology 2015; 95: 388–394. metabotropic glutamate receptors (mGluR2/3) regulate 57. Zammataro M, Chiechio S, Montana MC, et al. mGlu2 prostaglandin E2-mediated sensitization of capsaicin metabotropic glutamate receptors restrain inflammatory responses and thermal nociception. J Neurosci 2002; 22: pain and mediate the analgesic activity of dual mGlu2/ 6388–6393. mGlu3 receptor agonists. Mol Pain 2011; 7: 6. 51. Jones CK, Eberle EL, Peters SC, et al. Analgesic effects of 58. Neugebauer V. Peripheral metabotropic glutamate recep- the selective group II (mGlu2/3) metabotropic glutamate tors: fight the pain where it hurts. Trends Neurosci 2001; receptor agonists LY379268 and LY389795 in persistent 24: 550–552. and inflammatory pain models after acute and repeated 59. Neugebauer V. Metabotropic glutamate receptors – dosing. Neuropharmacology 2005; 49: 206–218. important modulators of nociception and pain behavior. 52. Du J, Zhou S and Carlton SM. Group II metabotropic Pain 2002; 98: 1–8. glutamate receptor activation attenuates peripheral sensi- 60. Goudet C, Magnaghi V, Landry M, et al. Metabotropic tization in inflammatory states. Neuroscience 2008; 154: receptors for glutamate and GABA in pain. Brain Res Rev 754–766. 2009; 60: 43–56. 53. Carlton SM, Du J and Zhou S. Group II metabotropic 61. Montana MC and Gereau RW. Metabotropic glutamate glutamate receptor activation on peripheral nociceptors receptors as targets for analgesia: antagonism, activation, modulates TRPV1 function. Brain Res 2009; 1248: 86–95. and allosteric modulation. Curr Pharm Biotechnol 2011; 12: 54. Zhou HY, Chen SR, Chen H, et al. Functional plasticity 1681–1688. of group II metabotropic glutamate receptors in 62. Brumfield S, Korakas P, Silverman LS, et al. Synthesis and regulating spinal excitatory and inhibitory synaptic input SAR development of novel mGluR1 antagonists for the in neuropathic pain. J Pharmacol Exp Ther 2011; 336: treatment of chronic pain. Bioorg Med Chem Lett 2012; 254–264. 22: 7223–7226. 55. Asseri KA, Puil E, Schwarz SK, et al. Group II metabo- 63. Eisenstein M. Neuropathy: a name for their pain. Nature tropic glutamate receptor antagonism prevents the 2016; 535: S10–S11. NIH Public Access Author Manuscript Front Biol (Beijing). Author manuscript; available in PMC 2014 December 03.

NIH-PA Author ManuscriptPublished NIH-PA Author Manuscript in final edited NIH-PA Author Manuscript form as: Front Biol (Beijing). 2014 June ; 9(3): 216–224. doi:10.1007/s11515-014-1304-4.

Neurotrophin signaling and visceral hypersensitivity

Li-Ya Qiao Department of Physiology and Biophysics, School of Medicine, Virginia Commonwealth University, Richmond, VA 23298, USA

Abstract Neurotrophin family are traditionally recognized for their nerve growth promoting function and are recently identified as crucial factors in regulating neuronal activity in the central and peripheral nervous systems. The family members including nerve growth factor (NGF), brain- derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) are reported to have distinct roles in the development and maintenance of sensory phenotypes in normal states and in the modulation of sensory activity in disease. This paper highlights receptor tyrosine kinase (Trk) -mediated signal transduction by which neurotrophins regulate neuronal activity in the visceral sensory reflex pathways with emphasis on the distinct roles of NGF and BDNF signaling in physiologic and pathophysiological processes. Viscero-visceral cross-organ sensitization exists widely in human diseases. The role of neurotrophins in mediating neural cross talk and interaction in primary afferent neurons in the dorsal root ganglia (DRG) and neurotrophin signal transduction in the context of cross-organ sensitization are also discussed.

Keywords neurotrophin; signal transduction; visceral hypersensitivity; cross-sensitization

Introduction Visceral hypersensitivity refers to increased sensation of stimuli to the visceral organs. It is a major source of abdominal pain which is attributable to abnormal responses of the sensory reflex pathways that govern the viscera. Information arising from the visceral organ project to extrinsic sensory neurons located in the dorsal root ganglia (DRG) and/or the nodose ganglia where information are organized and passed along to the central nervous system (CNS) in the spinal cord and brainstem. Descending nerves carrying excitatory or inhibitory neurotransmission in turn regulate the functionality of the organs. Neuronal tracing dye techniques have allowed identifying the spinal segmental distribution patterns of visceral sensory pathways and provide a powerful tool in study of the phenotypes of specifically labeled primary afferent neurons that innervate a particular organ. For example, neuronal

© Higher Education Press and Springer-Verlag Berlin Heidelberg 2014 Correspondence: Li-Ya QIAO, [email protected]. Compliance with ethics guidelines Li-Ya Qiao declares that she has no conflict of interest. This manuscript is a review article and does not involve a research protocol requiring approval by the relevant insitutional review board or ethics committee. Qiao Page 2

tracing studies demonstrate that primary afferent neurons with projection to the distal colon, the urinary bladder or a small number of neurons with dichotomizing projection to both

NIH-PA Author Manuscript NIH-PA Author Manuscriptbladder NIH-PA Author Manuscript and colon are located in thoracolumbar (T10-L2) and lumbosacral DRG (L6-S1) (Applebaum et al., 1980; Morgan et al., 1981; Keast and Degroat, 1992; Christianson et al., 2007; Qiao and Grider, 2007). DRG neurons are of the pseudo-unipolar type. They have an axon with two branches referred to as a distal process in the periphery and a proximal process at the terminals in the dorsal horn of the spinal cord. Primary afferents synapse at the dorsal gray of the spinal cord with the efferent neurons located in the lateral horn of the spinal cord, and lead to changes in the efferent pathways and ultimately in the function of visceral organs.

The primary afferent neurons exhibit extensive plasticity in response to a variety of conditions and innocuous/noxious stimuli of the viscera, resulting in alterations in neurochemical, structural, organizational and electrophysiological properties of the neurons. One of the driving forces that lead to sensory neuronal plasticity under pathophysiologic conditions arises from the visceral organs which demonstrate increased levels of endogenous factors including growth factors, cytokines, chemokines, cannabinoids, adenosines, etc. (Dinis et al., 2004; Theiss et al., 2004; Nazif et al., 2007; Saini et al., 2008). The large number of mediators that are identified in the viscera during organ inflammation or injury not only play significant roles in mediating inflammatory process in the organs but also can lead to increases in the excitability of the axonal terminals located in the organ, resulting in sensory hypersensitivity (Nazif et al., 2007). The increases in the axonal terminal excitability in turn lead to neuropeptide expression in and release from primary afferent neurons at the peripheral terminals through multiple pathways and ion channel activation, which result in an increase in local blood flow exacerbating the inflammatory process and dysfunction of the visceral organ (Donnerer et al., 1992; Donnerer and Stein, 1992; Tonra et al., 1998; Roza and Reeh, 2001). Among the numerous factors generated by the viscera, nerve growth factor (NGF) has a prominent role in regulating sensory sensitivity. Exogenous NGF injected into the normal rat bladder (Lamb et al., 2004; Zvara and Vizzard, 2007) or intrathecal NGF to rat spinal cord (Yoshimura et al., 2006) result in bladder hyperactivity; conversely treatment with NGF inhibitor attenuates visceral hypersensitivity and organ hypertrophy as results of inflammation (Tyagi et al., 2006; Chung et al., 2010; Matricon et al., 2013). NGF is produced in and released from the epithelial cells and mast cells during visceral inflammation, where it acts in a paracrine manner to regulate the cytological changes and the sensitivity of the viscera (Skaper et al., 2001; Stanzel et al., 2008). Within the NGF family, other members including brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) also have critical roles in modulating sensory activity in diseases (Obata and Noguchi, 2006; Tender et al., 2011). Distinct receptors and signal transduction mediated by these neurotrophins endow them unique roles in the sensory reflex pathway that govern the visceral organs.

Neurotrophin signal transduction The mammalian neurotrophins consist of four secreted small proteins, NGF, BDNF, NT-3, and NT-4/5. The high affinity receptors for each distinct neurotrophin are tyrosine kinase A (TrkA) binding to NGF, TrkB binding to BDNF or NT-4, and TrkC binding to NT-3. Under

Front Biol (Beijing). Author manuscript; available in PMC 2014 December 03. Qiao Page 3

some circumstances, NT-3 can also bind to TrkA and TrkB. The general neurotrophin receptor (NTR) p75 that was originally cloned and identified as a receptor for NGF binds to

NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript −9 each of the neurotrophins with low affinity (Kd ~ 10 M) when present in cells alone or with −11 high affinity (Kd ~ 10 M) when co-expressed with Trk by which the cell responsiveness to neurotrophins is further enhanced (Zampieri and Chao, 2004; Wehrman et al., 2007). The unprocessed precursor of the neurotrophins, the proneurotrophins, can also bind to p75NTR in the present of sortilin. Sortilin recognizes the pro-domain of the proneurotrophins, which upon dimerization with p75NTR, transmits signals through the intracellular docking protein to the intracellular domain of p75NTR. The cellular responses to Trk/p75NTR complex verse sortilin/p75NTR complex are often leading to opposite and antagonistic effects, with the former enhancing survival and growth promoting signals and the latter causing apoptosis (Wehrman et al., 2007; Skeldal et al., 2012). These are due to that interaction of p75NTR and Trk can increase the specificity and affinity of Trk for specific ligands thereby enhancing cell growth and survival through activation of the phosphoinositide 3-kinase (PI3K)/Akt pathway or the mitogen-activated protein kinases (MAPK)/extracellular signal- regulated kinases (ERK) pathway, while interaction of p75NTR with sortilin often activates the c-Jun N-terminal kinases (JNK), nuclear factor κ-B (NFκB), or Rho pathways which contribute to inflammatory responses and/or apoptosis. Neurotrophins influence cell biologic function through two mechanisms: 1) activation of signal transduction cascades at the nerve terminals, and 2) retrogradely transport of signaling molecules or signal from the nerve terminals to the neuronal cell body. The PI3K/Akt and ERK5 pathways are involved in the target-derived neurotrophin retrograde signaling cascades (Watson et al., 2001). The phospholipase C gamma (PLCγ) pathway activated by neurotrophins leads to Ca2+ and Na+ influx through the activation of ion channels and contributes prominently to long-term potentiation (LTP) and neuronal plasticity in CNS (Gruart et al., 2007). Another feature of neurotrophin transport involves anterograde transport of neurotrophins and receptors away from the neuronal cell body toward axonal terminals (Tonra et al., 1998; Wang et al., 2002; Ng et al., 2007; Ha et al., 2008).

Neurotrophin signaling in the viscera NGF was discovered by Rita Levi-Montalcini, 1986 Nobel Laureate in Medicine, who found that NGF released from malignant tumors caused nerve fibers to grow rapidly. Subsequent studies have revealed that NGF is a target-derived trophic factor that promotes neuronal innervation and axonal terminal branching during development and has roles in the maintenance of neural homeostasis in adult. In the visceral organs, NGF and its receptor TrkA are found for their expression in neuronal and non-neuronal structures of the gut (Lin et al., 2005; Barada et al., 2007; Stanzel et al., 2008; Qiao and Grider, 2010), and NGF and TrkA are also expressed by the urinary bladder epithelial cells and detrusor muscle (see review (Cruz, 2014)). Pancreatic β cells are able to synthesize and secrete NGF (Rosenbaum et al., 1998) which can lead to selective hyperinnervation of the islets when NGF is overexpressed (Edwards et al., 1989). When the visceral organs are inflamed or dysfunctional by diseases, NGF levels are often elevated in the viscera which then become a strong chemical cue acting on cells within the organ via a paracrine manner and/or act on the nerve terminals and sensitize the nervous system (Nazif et al., 2007; Yu et al., 2012; Qiao et

Front Biol (Beijing). Author manuscript; available in PMC 2014 December 03. Qiao Page 4

al., 2013). In inflammatory states, NGF is a critical contributor in promoting excessive collagen production and deposition thereby resulting in poor compliance and stiffness of the

NIH-PA Author Manuscript NIH-PA Author Manuscriptorgan NIH-PA Author Manuscript (Chung et al., 2010; Kilic et al., 2011). In the urinary bladder with inflammation, endogenous NGF promotes type I collagen production through the MAPK and PI3K/Akt pathways (Chung et al., 2010). In chronic allergic airway inflammation, the increased NGF production contributes to type III collagen expression and deposition in the subepithelial compartment of the airways (Kilic et al., 2011). The novel role of NGF in regulating non- neuronal structure morphology is also suggested by transgenic overexpression of NGF in the organ which leads to excessive collagen deposition resulting in altered visceral physiology (Allen and Saban, 2010; Schnegelsberg et al., 2010; Kilic et al., 2011). NGF-regulated collagen production is mediated by the MAPK pathways involving the activation of ERK and p38, however, is independent of the traditional pathway involving the transforming growth factor beta (TGF-β)1/mothers against decapentaplegic homolog (SMAD) pathway (Kilic et al., 2011; Zhang and Qiao, 2012).

In addition to the non-neuronal tissue, overexpression of NGF in the smooth muscle cells under the control of the smooth muscle alpha-actin promoter results in robust sprouting of sympathetic axons in the colon and bladder (Elliott et al., 2009). Overexpression of NGF in the urothelium under the control of the uroplakin II promoter results in a marked increase in the density of sensory afferent fibers positive to calcitonin gene-related peptide (CGRP), substance P, and neurofilament 200, as well as sympathetic nerve fibers positive to tyrosine hydroxylase (Schnegelsberg et al., 2010). Inhibition of NGF action in vivo by NGF antiserum or Trk inhibitor K252a reverses peripheral mechanical hypersensitivity as a result of bladder inflammation suggesting Trk-mediated NGF action in the regulation of sensory activity (Guerios et al., 2006). This may be due to an action of NGF on receptors localized at the sensory nerve terminals in the viscera. NGF receptors TrkA and p75NTR are both expressed in the neuronal and non-neuronal structures in the primary afferent pathways. These transmembrane proteins are generally embedded in the plasma membrane of the cells in the visceral organ or nerve terminals of innervating neurons. TrkA is also found to be expressed in the mitochondrial compartment (Carito et al., 2012) and is likely to be involved in the process of oxidation stress (Podratz and Windebank, 2005; Ersahin et al., 2012; Valdovinos-Flores and Gonsebatt, 2013).

BDNF, which has about 50% amino acid identity with NGF, was first isolated from pig brain (Barde et al., 1982). BDNF is also found to express in non-neuronal tissues such as gut mucosa, adipocytes, liver, lung, pancreas and the urinary bladder (Lommatzsch et al., 1999; Bonini et al., 2001; Lucini et al., 2003; Lommatzsch et al., 2005; Grider et al., 2006; Steinkamp et al., 2012; Yu et al., 2012; Cruz, 2014). Its role in peripheral tissues is less studied. In addition to its ability in affecting neuronal function via retrograde fashion, BDNF and receptor TrkB more likely undergo antergrade transport away from neuronal cell bodies (Tonra et al., 1998; Ng et al., 2007; Ha et al., 2008). NT-4, which is often interchangeable with BDNF in initiating TrkB-mediated signal transduction, also has a role in the periphery in sensitizing peripheral innervation and sensory sensitivity (Krimm et al., 2006; Aven et al., 2014; Huang and Krimm, 2014).

Front Biol (Beijing). Author manuscript; available in PMC 2014 December 03. Qiao Page 5

Neurotrophin signaling in primary afferent neurons

NIH-PA Author Manuscript NIH-PA Author ManuscriptThe NIH-PA Author Manuscript primary afferent pathways such as the vagal pathway and the spinal pathway contain primary afferent neurons that convey normal and aberrant sensation of the visceral organs. Primary afferent neurons are composed of a variety of cells in terms of their size, genotype and function. The spinal afferent pathways projecting via DRG at different levels of the spinal cord support the visceral reflexes including both nociceptive and nonnociceptive signals. Nociceptive signals from primary afferents enter the spinal cord through Lissauer’s tract, a thin tract of small axons capping the dorsal horn, and terminate in laminae I of the dorsal horn gray matter, where ascend one or two segments before crossing to the contralateral side.

The distribution and expression level of each neurotrophin or Trk in primary afferent pathways are affected by many factors and dependent on the region, cell type and developmental stage. In adult DRG, TrkA, TrkB and TrkC mRNA are specifically expressed in functionally distinct neurons at a similar level ranging from 10% to 35% of the total number of neurons (Kashiba et al., 2003), with TrkA in nociceptive and thermoceptive small sensory neurons sensing temperature and noxious stimuli, TrkB in less specifically characterized touch neurons, and TrkC in proprioceptive neurons that sense body position (Huang and Reichardt, 2003). The phenotypic formation of DRG sensory neurons is largely dependent on the expression of certain Trks in these neurons (Wright and Snider, 1995). During development, all nociceptive neurons initially expressed TrkA, which mediates target-dependent cell survival. In mice lack of TrkA activation, 70% – 85% of sensory neurons are lost (Silossantiago et al., 1995); however, when mice are engineered to express TrkC protein from the TrkA genomic locus, overexpression of TrkC rescues the DRG neurons from apoptosis, but turns these DRG neurons from TrkA-containing nociceptive and thermoceptive phenotype to TrkC-containing proprioceptive-like neurons (Moqrich et al., 2004). TrkA also plays a role in sensory neuron diversification and maturation. During the first 2 to 3 postnatal weeks, a fraction of nociceptive neurons switch their neurotrophic factor dependence by downregulating expression of TrkA and upregulating the expression of Ret, the GDNF receptor, turning the nociceptive sensory neurons from TrkA-containing peptidergic to Ret-containing non-peptidergic subtypes (Molliver et al., 1997; Chen et al., 2006; Lopes et al., 2012). Activation of Trks in sensory neurons is mediated by retrograde or autocrine/paracrine neurotrophin signals (Korsching, 1993; Delcroix et al., 2003). NGF synthesis in the peripheral tissue and TrkA expression in the sensory neuronal soma do not begin until the fibers reach their peripheral targets (Davies et al., 1987). BDNF, on the other hand, is synthesized in the afferent neuronal cell body and released synaptically or extrasynaptically to induce pre- or post-synaptic efficacy, or maintain sensory neuronal survival (Swanwick et al., 2004; Obata and Noguchi, 2006; Qiao et al., 2008; Vaz et al., 2011; Xia et al., 2012). BDNF protein is expressed in small- and medium-sized sensory neurons that also express TrkA but not TrkB (Kashiba et al., 1997; Michael et al., 1997; Mannion et al., 1999; Qiao et al., 2013). Within the DRG, BDNF acts on TrkB-expressing neurons via a paracrine manner (Obata and Noguchi, 2006; Qiao and Grider, 2007; Xia et al., 2012).

Front Biol (Beijing). Author manuscript; available in PMC 2014 December 03. Qiao Page 6

Neurotrophin signaling in visceral sensory activity

NIH-PA Author Manuscript NIH-PA Author ManuscriptThe NIH-PA Author Manuscript expression of neurotrophins in sensory neurons at physiologic state keeps at a low level. In normal healthy rats, less than 10% of DRG neurons express BDNF (Qiao et al., 2008; Lin et al., 2011; Yu et al., 2012). The level of BDNF dramatically increases with inflammation (Mannion et al., 1999; Obata and Noguchi, 2006; Qiao et al., 2008; Lin et al., 2011; Yu et al., 2012). The mechanism underlying BDNF upregulation in sensory neurons has been suggested showing an involvement of NGF-mediated ERK and PI3K/Akt pathways (Michael et al., 1997; Obata et al., 2003; Qiao and Grider, 2010; Yu et al., 2012; Qiao et al., 2013). Changes in the level of neurotrophins in sensory neurons with inflammation also contribute to the upregulation of a series of sensory markers thereby enhancing the activity of pronociceptive receptors and ion channels. CGRP is one of the most prominent sensory markers that labels peptidergic nociceptors and has a key role in mediating visceral sensory hypersensitivity (Plourde et al., 1997; Delafoy et al., 2006). In DRG, CGRP is co-expressed with TrkA and partially TrkB and its expression is regulated by NGF and BDNF (Patel et al., 2000; Ruiz and Banos, 2005; Qiao and Grider, 2007; Yu et al., 2012). In cultured DRG neurons, NGF, BDNF and NT-3 regulate CGRP expression with a different magnitude and time course (Mulderry 1994; Sterne et al., 1998; Qiao and Grider, 2007). NGF induces CGRP expression with an acute to prolonged effects (Ruiz and Banos, 2005); while acute BDNF treatment has no effects on CGRP expression but prolonged BDNF treatment is able to enhance CGRP expression (Qiao and Grider, 2007); in contrast, CGRP is unable to be regulated by NT-3 (Sterne et al., 1998). In an animal model of bladder inflammation, NGF- induced CGRP expression in DRG is regulated by the ERK5 but not the PI3K/Akt pathway (Yu et al., 2012). It is suggested that NGF-induced Akt activation participates in the expression, activation and trafficking of vaniloid receptor TRPV1 (Zhang et al., 2005; Stein et al., 2006; Zhu and Oxford, 2007). In DRG, TRPV1 is expressed in both peptidergic and non-peptidergic neurons and 60% of CGRP DRG neurons contain TRPV1 immunoreactivity, however, there is scarce overlap of TRPV1 and CGRP fibers in the dorsal horn of the spinal cord (Guo et al., 1999). NGF-induced TRPV1 expression also involves the activation of Ras-mediated MAPK (Bron et al., 2003; Zhu and Oxford, 2007), and Rac- mediated p38 MAPK (Puntambekar et al., 2005), which upregulates TRPV1 expression and increases pain perception (Ji et al., 2002). Other ion channels that participate in visceral pain perception, e.g. the tetrodotoxin-resistant (TTX-R) sodium channel α subunits Nav1.8 and Nav1.9, are also co-expressed with TrkA in sensory neurons and is regulated by NGF (Benn et al., 2001). Conversely, intrathecal infusion of NT-3 reduces the levels of Nav1.8 and Nav1.9 mRNA and proteins in DRG neurons (Wilson-Gerwing et al., 2008). This is consistent with an antagonistic role of NT-3 in suppressing thermal hyperalgesia associated with neuropathic pain (Wilson-Gerwing et al., 2005).

The axon terminals of primary afferent neurons are located in the dorsal horn of the spinal cord. Afferent input received by the sensory cell bodies in DRG pass along to the spinal cord where the signals are organized and in turn affect the physiological function of peripheral organ through efferent output. The spinal plasticity is initiated by the neurotransmitters produced in the afferent cell body and released at the terminal. These neuroactive compounds include glutamate, substance P, somatostatin, VIP, CGRP, neurotrophins, etc.

Front Biol (Beijing). Author manuscript; available in PMC 2014 December 03. Qiao Page 7

Electrophysiological recording of C-fiber evoked field potentials in spinal dorsal horn shows that exogenous BDNF is able to induce a LTP-like activity, which is blocked by antagonists

NIH-PA Author Manuscript NIH-PA Author Manuscriptof TrkB, NIH-PA Author Manuscript N-methyl-D-aspartate (NMDA) receptor, ERK, p38MAPK, NF-κB, but not by JNK inhibitor (Zhou et al., 2008). Upregulation of BDNF in the DRG during visceral inflammation may also release at the central axonal terminals located in the spinal cord and regulates spinal central sensitization by activating the MEK/ERK pathway (Qiao et al., 2008). CGRP is able to induce the activation of cAMP-responsive element binding protein (CREB), a molecular switch of neuronal plasticity, in the spinal cord through signal convergent of PI3K/Akt and NMDA receptor-mediated pathways (Kay et al., 2013) and contribute to cystitis-induced bladder hyperactivity (Kay et al., 2013).

Neurotrophin signaling in cross-organ sensitization Inter-organ cross-talk has been identified between visceral organs, such as the distal colon and the urinary bladder. Clinical evidence has shown sensory cross-sensitization between the urinary bladder and the distal colon (Alagiri et al., 1997; Ben-Ami et al., 2002). Patients with bowel dysfunction such as inflammatory bowel disease (IBD) are more likely to experience nocturia and some forms of urinary urge incontinence compared to the non-IBD population (Ben-Ami et al., 2002). Case studies show that 50% of patients with bowel dysfunction have evidence of bladder dysfunction including detrusor instability (Whorwell et al., 1986), suggesting the necessity of periodic urologic evaluation in the management of patients with bowel problems. Vice versa, individuals with interstitial cystitis are 100 times more likely to have IBD in comparison to the general population (Alagiri et al., 1997). In the past years, rigorous studies have been initiated to increase the understanding of the underlying mechanisms and pathways by which cross-organ sensitization is regulated (Malykhina, 2007; Brumovsky and Gebhart, 2010; Daly et al., 2013). In regard to the role of neurotrophins, strong evidence suggests that NGF elevated in one visceral organ such as the distal colon may lead to activation of the primary afferent neurons projecting to this organ and cross-activation of the nearby afferent neurons projecting to a different viscus such as the urinary bladder (Qiao and Grider, 2010; Xia et al., 2012). In an animal model involving visceral-somatic cross-sensitization, injection of NGF to the urinary bladder causes enhanced sensitivity to mechanical and thermal stimulation of both hind paws (Bielefeldt et al., 2006). Overexpression of NGF in the urinary bladder also triggers hypersensitivity of the distal colon (Bielefeldt et al., 2006). This suggests that excess of NGF at the nerve terminals may induce neuronal alterations in the primary afferent pathway that receives afferent input from multiple organs. NGF signaling can undergo retrograde transport to the DRG and regulate a paracrine effect within the DRG to facilitate neuron-neuron interaction (Qiao and Grider, 2010; Xia et al., 2012), or may act on dichotomizing DRG neurons that project to both organs (Christianson et al., 2007; Qiao and Grider, 2007). NGF signal can also activate the ERK pathway and lead to neuropeptide expression (Li et al., 2008; Berger, 2009). As for BDNF, its paracrine action on nearby TrkB-containing DRG neurons or perineuronal satellite cells may lead to inter-cell interaction and neuronal cross-sensitization (Lee et al., 1999; Qiao and Grider, 2007; Xia et al., 2012). Cross-organ sensitization can also be regulated at the spinal level (Qin et al., 2005). It is not known whether BDNF release to the spinal cord regulates this process. The increases in mast cells are suggested to have a role in

Front Biol (Beijing). Author manuscript; available in PMC 2014 December 03. Qiao Page 8

cross-organ sensitization (Fitzgerald et al., 2013). In the periphery, TrkB/BDNF binding can acutely sensitize nocireceptive pathway that require the presence of mast cells (Huang and

NIH-PA Author Manuscript NIH-PA Author ManuscriptReichardt, NIH-PA Author Manuscript 2001). Activation of ion channels such as TRPV1 and TTX-R sodium channels in the DRG are involved in viscera-visceral cross-organ sensitization (Malykhina et al., 2006; Chaban, 2008; Pan et al., 2010; Lei and Malykhina, 2012). The ability of neurotrophins in regulating the activity of these channels (Benn et al., 2001; Natura et al., 2005; Zhang et al., 2005; Stein et al., 2006; Zhu and Oxford, 2007; Zhang et al., 2008) may underlie the mechanisms of DRG neuronal cross - activation.

Conclusions Neurotrophins are traditionally known for their roles in inducing the survival, differentiation, and growth of neurons. With each neurotrophin binding to a specific receptor and facilitate unique signaling pathways, the family members, NGF, BDNF and NT-3 as mostly studied, have distinct roles in mediating neuronal physiology especially in visceral hypersensitivity. Target-derived NGF acts on sensory nerve terminals leading to sensory activation and neuropeptide production in the primary afferent neurons that innervate the visceral organ. BDNF generated by the primary afferent neurons acts in a paracrine manner within the DRG leading to neuron-neuron cross-activation, and also releases to the nerve terminals peripherally to the target organ where reinforcing terminal sensitization in the presence of mast cells and/or centrally to the spinal cord leading to central sensitization. NT-3 often has antagonistic role in sensory hypersensitivity. Although supporting documents suggest the role of neurotrophins in regulating cross-organ sensitization, the underlying mechanisms and signaling pathways by which neurotrophins regulating neuropeptide expression and ion channel activity is unknown. Tanezumab, a humanized monoclonal antibody against NGF, has been tested in reducing pain arising from inflammation of the urinary bladder (Evans et al., 2011), however it shows unfavorable side effects. In March 2012 the FDA Committee voted in favor of a continuation of Anti-NGF testing as long as certain safety precautions were made. A better understanding of neurotrophin action and signal transduction in visceral hypersensitivity and pain may provide strategies for an alternative target in the neurotrophin cascades.

Acknowledgments

This work was supported by National Institutes of Health (Grant suppot: NIH DK077917).

References Alagiri M, Chottiner S, Ratner V, Slade D, Hanno PM. Interstitial cystitis: Unexplained associations with other chronic disease and pain syndromes. Urology. 1997; 49(5 5A):52–57. [PubMed: 9146002] Allen AM, Saban R. Model for chronic overexpression of NGF challenges old paradigms: focus on “overexpression of NGF in mouse urothelium leads to neuronal hyperinnervation, pelvic sensitivity, and changes in urinary bladder function”. Am J Physiol Regul Integr Comp Physiol. 2010; 298(3):R532–R533. [PubMed: 20089712] Applebaum AE, Vance WH, Coggeshall RE. Segmental Localization of Sensory Cells That Innervate the Bladder. J Comp Neurol. 1980; 192(2):203–209. [PubMed: 7400394]

Front Biol (Beijing). Author manuscript; available in PMC 2014 December 03. Qiao Page 9

Aven L, Paez-Cortez J, Achey R, Krishnan R, Ram-Mohan S, Cruikshank WW, Fine A, Ai X. An NT4/TrkB-dependent increase in innervation links early-life allergen exposure to persistent airway hyperreactivity. FASEB J. 2014; 28(2):897–907. [PubMed: 24221086] NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript Barada KA, Mourad FH, Sawah SI, Khoury C, Safieh-Garabedian B, Nassar CF, Tawil A, Jurjus A, Saadé NE. Up-regulation of nerve growth factor and interleukin-10 in inflamed and non-inflamed intestinal segments in rats with experimental colitis. Cytokine. 2007; 37(3):236–245. [PubMed: 17517520] Barde YA, Edgar D, Thoenen H. Purification of a new neurotrophic factor from mammalian brain. EMBO J. 1982; 1(5):549–553. [PubMed: 7188352] Ben-Ami H, Ginesin Y, Behar DM, Fischer D. Diagnosis and treatment of urinary tract complications in Crohn’s disease: An experience over 15 years. Can J Gastroenterol. 2002; 16(4):225–229. [PubMed: 11981574] Benn SC, Costigan M, Tate S, Fitzgerald M, Woolf CJ. Developmental expression of the TTX- resistant voltage-gated sodium channels Nav1.8 (SNS) and Nav1.9 (SNS2) in primary sensory neurons. J Neurosci. 2001; 21(16):6077–6085. [PubMed: 11487631] Berger RE. Inflammation in the uterus induces phosphorylated extracellular signal-regulated kinase and substance P immunoreactivity in dorsal root ganglia neurons innervating both uterus and colon in rats comment. J Urol. 2009; 181(3):1136–1136. Bielefeldt K, Lamb K, Gebhart GF. Convergence of sensory pathways in the development of somatic and visceral hypersensitivity. Am J Physiol Gastrointest Liver Physiol. 2006; 291(4):G658–G665. [PubMed: 16500917] Bonini P, Pierucci D, Cicconi S, Porzio O, Lauro R, Marlier LNJL, Borboni P. Neurotrophins and neurotrophin receptors mRNAs expression in pancreatic islets and insulinoma cell lines. JOP. 2001; 2(3):105–111. [PubMed: 11870332] Bron R, Klesse LJ, Shah K, Parada LF, Winter J. Activation of Ras is necessary and sufficient for upregulation of vanilloid receptor type 1 in sensory neurons by neurotrophic factors. Mol Cell Neurosci. 2003; 22(1):118–132. [PubMed: 12595244] Brumovsky PR, Gebhart GF. Visceral organ cross-sensitization - an integrated perspective. Auton Neurosci. 2010; 153(1–2):106–115. [PubMed: 19679518] Carito V, Pingitore A, Cione E, Perrotta I, Mancuso D, Russo A, Genchi G, Caroleo MC. Localization of nerve growth factor (NGF) receptors in the mitochondrial compartment: characterization and putative role. Biochim Biophys Acta. 2012; 1820(2):96–103. [PubMed: 22138126] Chaban VV. Visceral sensory neurons that innervate both uterus and colon express nociceptive TRPv1 and P2X3 receptors in rats. Ethn Dis. 2008; 18 Suppl 2(2) S2–20–24. Chen CL, Broom DC, Liu Y, de Nooij JC, Li Z, Cen C, Samad OA, Jessell TM, Woolf CJ, Ma Q. Runx1 determines nociceptive sensory neuron phenotype and is required for thermal and neuropathic pain. Neuron. 2006; 49(3):365–377. [PubMed: 16446141] Christianson JA, Liang RM, Ustinova EE, Davis BM, Fraser MO, Pezzone MA. Convergence of bladder and colon sensory innervation occurs at the primary afferent level. Pain. 2007; 128(3): 235–243. [PubMed: 17070995] Chung CW, Zhang QL, Qiao LY. Endogenous nerve growth factor regulates collagen expression and bladder hypertrophy through Akt and MAPK pathways during cystitis. J Biol Chem. 2010; 285(6): 4206–4212. [PubMed: 19996110] Cruz CD. Neurotrophins in bladder function: what do we know and where do we go from here? Neurourol Urodyn. 2014; 33(1):39–45. [PubMed: 23775873] Daly DM, Nocchi L, Grundy D. Highlights in basic autonomic neurosciences: cross-organ sensitization between the bladder and bowel. Auton Neurosci. 2013; 179(1–2):1–4. [PubMed: 23810481] Davies AM, Bandtlow C, Heumann R, Korsching S, Rohrer H, Thoenen H. Timing and site of nerve growth-factor synthesis in developing skin in relation to innervation and expression of the receptor. Nature. 1987; 326(6111):353–358. [PubMed: 3031505] Delafoy L, Gelot A, Eschalier A, Bertrand C, Doherty AM, Diop L. Interactive involvement of brain derived neurotrophic factor, nerve growth factor, and calcitonin gene related peptide in colonic hypersensitivity in the rat. Gut. 2006; 55(7):940–945. [PubMed: 16401692]

Front Biol (Beijing). Author manuscript; available in PMC 2014 December 03. Qiao Page 10

Delcroix JD, Valletta JS, Wu C, Hunt SJ, Kowal AS, Mobley WC. NGF signaling in sensory neurons: Evidence that early endosomes carry NGF retrograde signals. Neuron. 2003; 39(1):69–84. [PubMed: 12848933] NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript Dinis P, Charrua A, Avelino A, Yaqoob M, Bevan S, Nagy I, Cruz F. Anandamide-evoked activation of vanilloid receptor 1 contributes to the development of bladder hyperreflexia and nociceptive transmission to spinal dorsal horn neurons in cystitis. J Neurosci. 2004; 24(50):11253–11263. [PubMed: 15601931] Donnerer J, Schuligoi R, Stein C. Increased content and transport of substance P and calcitonin gene- related peptide in sensory nerves innervating inflamed tissue: evidence for a regulatory function of nerve growth factor in vivo. Neuroscience. 1992; 49(3):693–698. [PubMed: 1380138] Donnerer J, Stein C. Evidence for an increase in the release of CGRP from sensory nerves during inflammation. Ann N YAcad Sci. 1992; 657:505–506. 1 Calcitonin Ge. Edwards RH, Rutter WJ, Hanahan D. Directed expression of NGF to pancreatic beta cells in transgenic mice leads to selective hyperinnervation of the islets. Cell. 1989; 58(1):161–170. [PubMed: 2665941] Elliott J, MacLellan A, Saini JK, Chan J, Scott S, Kawaja MD. Transgenic mice expressing nerve growth factor in smooth muscle cells. Neuroreport. 2009; 20(3):223–227. [PubMed: 19444944] Ersahin M, Ozdemir Z, Özsavcı D, Akakın D, Yeğen BÇ, Reiter RJ, Şener G. Melatonin treatment protects against spinal cord injury induced functional and biochemical changes in rat urinary bladder. J Pineal Res. 2012; 52(3):340–348. [PubMed: 22220508] Evans RJ, Moldwin RM, Cossons N, Darekar A, Mills IW, Scholfield D. Proof of concept trial of tanezumab for the treatment of symptoms associated with interstitial cystitis. J Urol. 2011; 185(5): 1716–1721. [PubMed: 21420111] Fitzgerald JJ, Ustinova E, Koronowski KB, de Groat WC, Pezzone MA. Evidence for the role of mast cells in colon-bladder cross organ sensitization. Autonomic Neuroscience-Basic & Clinical. 2013; 173(1–2):6–13. [PubMed: 23182915] Grider JR, Piland BE, Gulick MA, Qiao LY. Brain-derived neurotrophic factor augments peristalsis by augmenting 5-HT and calcitonin gene-related peptide release. Gastroenterology. 2006; 130(3): 771–780. [PubMed: 16530518] Gruart A, Sciarretta C, Valenzuela-Harrington M, Delgado-Garcia JM, Minichiello L. Mutation at the TrkB PLC gamma-docking site affects hippocampal LTP and associative learning in conscious mice. Learn Mem. 2007; 14(1–2):54–62. [PubMed: 17272652] Guerios SD, Wang ZY, Bjorling DE. Nerve growth factor mediates peripheral mechanical hypersensitivity that accompanies experimental cystitis in mice. Neurosci Lett. 2006; 392(3):193– 197. [PubMed: 16203088] Guo A, Vulchanova L, Wang J, Li X, Elde R. Immunocyto-chemical localization of the vanilloid receptor 1 (VR1): relationship to neuropeptides, the P2X(3) purinoceptor and IB4 binding sites. Eur J Neurosci. 1999; 11(3):946–958. [PubMed: 10103088] Ha J, Lo KW, Myers KR, Carr TM, Humsi MK, Rasoul BA, Segal RA, Pfister KK. A neuron-specific cytoplasmic dynein isoform preferentially transports TrkB signaling endosomes. J Cell Biol. 2008; 181(6):1027–1039. [PubMed: 18559670] Huang EJ, Reichardt LF. Neurotrophins: roles in neuronal development and function. Annu Rev Neurosci. 2001; 24(1):677–736. [PubMed: 11520916] Huang EJ, Reichardt LF. Trk receptors: roles in neuronal signal transduction. Annu Rev Biochem. 2003; 72(1):609–642. [PubMed: 12676795] Huang T, Krimm RE. BDNF and NT4 play interchangeable roles in gustatory development. Dev Biol. 2014; 386(2):308–320. [PubMed: 24378336] Ji RR, Samad TA, Jin SX, Schmoll R, Woolf CJ. p38 MAPK activation by NGF in primary sensory neurons after inflammation increases TRPV1 levels and maintains heat hyperalgesia. Neuron. 2002; 36(1):57–68. [PubMed: 12367506] Kashiba H, Uchida Y, Senba E. Distribution and colocalization of NGF and GDNF family ligand receptor mRNAs in dorsal root and nodose ganglion neurons of adult rats. Brain Res Mol Brain Res. 2003; 110(1):52–62. [PubMed: 12573533]

Front Biol (Beijing). Author manuscript; available in PMC 2014 December 03. Qiao Page 11

Kashiba H, Ueda Y, Ueyama T, Nemoto K, Senba E. Relationship between BDNF- and trk-expressing neurones in rat dorsal root ganglion: An analysis by in situ hybridization. Neuroreport. 1997; 8(5): 1229–1234. [PubMed: 9175119] NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript Kay JC, Xia CM, Liu M, Shen S, Yu SJ, Chung C, Qiao LY. Endogenous PI3K/Akt and NMDAR act independently in the regulation of CREB activity in lumbosacral spinal cord in cystitis. Exp Neurol. 2013; 250:366–375. [PubMed: 24184018] Keast JR, Degroat WC. Segmental distribution and peptide content of primary afferent neurons innervating the urogenital organs and colon of male-rats. J Comp Neurol. 1992; 319(4):615–623. [PubMed: 1619047] Kilic A, Sonar SS, Yildirim AO, Fehrenbach H, Nockher WA, Renz H. Nerve growth factor induces type III collagen production in chronic allergic airway inflammation. J Allergy Clin Immunol. 2011; 128(5):1058–1066. e1051–e1054. [PubMed: 21816457] Korsching S. The Neurotrophic Factor Concept - a Reexamination. J Neurosci. 1993; 13(7):2739– 2748. [PubMed: 8331370] Krimm RF, Davis BM, Noel T, Albers KM. Overexpression of neurotrophin 4 in skin enhances myelinated sensory endings but does not influence sensory neuron number. J Comp Neurol. 2006; 498(4):455–465. [PubMed: 16937395] Lamb K, Gebhart GF, Bielefeldt K. Increased nerve growth factor expression triggers bladder overactivity. J Pain. 2004; 5(3):150–156. [PubMed: 15106127] Lee SL, Kim JK, Kim DS, Cho HJ. Expression of mRNAs encoding full-length and truncated TrkB receptors in rat dorsal root ganglia and spinal cord following peripheral inflammation. Neuroreport. 1999; 10(13):2847–2851. [PubMed: 10511451] Lei Q, Malykhina AP. Colonic inflammation up-regulates voltage-gated sodium channels in bladder sensory neurons via activation of peripheral transient potential vanilloid 1 receptors. Neurogastroenterol Motil. 2012; 24(6) 575–e257. Li JC, Micevych P, McDonald J, Rapkin A, Chaban V. Inflammation in the uterus induces phosphorylated extracellular signal-regulated kinase and substance P immunoreactivity in dorsal root ganglia neurons innervating both uterus and colon in rats. J Neurosci Res. 2008; 86(12):2746– 2752. [PubMed: 18478547] Lin A, Lourenssen S, Stanzel RDP, Blennerhassett MG. Nerve growth factor sensitivity is broadly distributed among myenteric neurons of the rat colon. J Comp Neurol. 2005; 490(2):194–206. [PubMed: 16052501] Lin YT, Ro LS, Wang HL, Chen JC. Up-regulation of dorsal root ganglia BDNF and trkB receptor in inflammatory pain: an in vivo and in vitro study. J Neuroinflammation. 2011; 8(1):126. [PubMed: 21958434] Lommatzsch M, Braun A, Mannsfeldt A, Botchkarev VA, Botchkareva NV, Paus R, Fischer A, Lewin GR, Renz H. Abundant production of brain-derived neurotrophic factor by adult visceral epithelia - Implications for paracrine and target-derived neurotrophic functions. Am J Pathol. 1999; 155(4): 1183–1193. [PubMed: 10514401] Lommatzsch M, Quarcoo D, Schulte-Herbrüggen O, Weber H, Virchow JC, Renz H, Braun A. Neurotrophins in murine viscera: a dynamic pattern from birth to adulthood. Int J Dev Neurosci. 2005; 23(6):495–500. [PubMed: 15978771] Lopes C, Liu ZJ, Xu Y, Ma Q. Tlx3 and runx1 Act in combination to coordinate the development of a cohort of nociceptors, thermoceptors, and pruriceptors. J Neurosci. 2012; 32(28):9706–9715. [PubMed: 22787056] Lucini C, Maruccio L, Girolamo P, Castaldo L. Brain-derived neurotrophic factor in higher pancreas: immunolocalization in glucagon cells. Anat Embryol (Berl). 2003; 206(4):311–318. [PubMed: 12649729] Malykhina AP. Neural mechanisms of pelvic organ cross-sensitization. Neuroscience. 2007; 149(3): 660–672. [PubMed: 17920206] Malykhina AP, Qin C. greenwood-van meerveld, foreman, lupu, akbarali. Hyperexcitability of convergent colon and bladder dorsal root ganglion neurons after colonic inflammation: mechanism for pelvic organ cross-talk. Neurogastroenterol Motil. 2006; 18(10):936–948. [PubMed: 16961697]

Front Biol (Beijing). Author manuscript; available in PMC 2014 December 03. Qiao Page 12

Mannion RJ, Costigan M, Decosterd I, Amaya F, Ma QP, Holstege JC, Ji RR, Acheson A, Lindsay RM, Wilkinson GA, Woolf CJ. Neurotrophins: peripherally and centrally acting modulators of tactile stimulus-induced inflammatory pain hypersensitivity. Proc Natl Acad Sci USA. 1999; NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript 96(16):9385–9390. [PubMed: 10430952] Matricon J, Muller E, Accarie A, Meleine M, Etienne M, Voilley N, Busserolles J, Eschalier A, Lazdunski M, Bourdu S, Gelot A, Ardid D. Peripheral contribution of NGF and ASIC1a to colonic hypersensitivity in a rat model of irritable bowel syndrome. Neurogastroenterol Motil. 2013; 25(11):e740–e754. [PubMed: 23902154] Michael GJ, Averill S, Nitkunan A, Rattray M, Bennett DLH, Yan Q, Priestley JV. Nerve growth factor treatment increases brain-derived neurotrophic factor selectively in TrkA-expressing dorsal root ganglion cells and in their central terminations within the spinal cord. J Neurosci. 1997; 17(21):8476–8490. [PubMed: 9334420] Molliver DC, Wright DE, Leitner ML, Parsadanian AS, Doster K, Wen D, Yan Q, Snider WD. IB4- binding DRG neurons switch from NGF to GDNF dependence in early postnatal life. Neuron. 1997; 19(4):849–861. [PubMed: 9354331] Moqrich A, Earley TJ, Watson J, Andahazy M, Backus C, Martin-Zanca D, Wright DE, Reichardt LF, Patapoutian A. Expressing TrkC from the TrkA locus causes a subset of dorsal root ganglia neurons to switch fate. Nat Neurosci. 2004; 7(8):812–818. [PubMed: 15247919] Morgan C, Nadelhaft I, de Groat WC. The distribution of visceral primary afferents from the pelvic nerve to Lissauer's tract and the spinal gray matter and its relationship to the sacral parasympathetic nucleus. J Comp Neurol. 1981; 201(3):415–440. [PubMed: 7276258] Mulderry PK. Neuropeptide expression by newborn and adult-rat sensory neurons in culture – effects of nerve growth-factor and other neurotrophic factors. Neuroscience. 1994; 59(3):673–688. [PubMed: 7516508] Natura G, von Banchet GS, Schaible HG. Calcitonin gene-related peptide enhances TTX-resistant sodium currents in cultured dorsal root ganglion neurons from adult rats. Pain. 2005; 116(3):194– 204. [PubMed: 15927395] Nazif O, Teichman JM, Gebhart GF. Neural upregulation in interstitial cystitis. Urology. 2007; 69(4 Suppl):24–33. [PubMed: 17462476] Ng BK, Chen L, Mandemakers W, Cosgaya JM, Chan JR. Anterograde transport and secretion of brain-derived neurotrophic factor along sensory axons promote Schwann cell myelination. J Neurosci. 2007; 27(28):7597–7603. [PubMed: 17626221] Obata K, Noguchi K. BDNF in sensory neurons and chronic pain. Neurosci Res. 2006; 55(1):1–10. [PubMed: 16516994] Obata K, Yamanaka H, Dai Y, Tachibana T, Fukuoka T, Tokunaga A, Yoshikawa H, Noguchi K. Differential activation of extracellular signal-regulated protein kinase in primary afferent neurons regulates brain-derived neurotrophic factor expression after peripheral inflammation and nerve injury. J Neurosci. 2003; 23(10):4117–4126. [PubMed: 12764099] Pan XQ, Gonzalez JA, Chang S, Chacko S, Wein AJ, Malykhina AP. Experimental colitis triggers the release of substance P and calcitonin gene-related peptide in the urinary bladder via TRPV1 signaling pathways. Exp Neurol. 2010; 225(2):262–273. [PubMed: 20501335] Patel TD, Jackman A, Rice FL, Kucera J, Snider WD. Development of sensory neurons in the absence of NGF/TrkA signaling in vivo. Neuron. 2000; 25(2):345–357. [PubMed: 10719890] Plourde V, St-Pierre S, Quirion R. Calcitonin gene-related peptide in viscerosensitive response to colorectal distension in rats. Am J Physiol. 1997; 273(1 Pt 1):G191–G196. [PubMed: 9252526] Podratz JL, Windebank AJ. NGF rescues DRG neurons in vitro from oxidative damage produced by hemodialyzers. Neurotoxicology. 2005; 26(3):343–350. [PubMed: 15935206] Puntambekar P, Mukherjea D, Jajoo S, Ramkumar V. Essential role of Rac1/NADPH oxidase in nerve growth factor induction of TRPV1 expression. J Neurochem. 2005; 95(6):1689–1703. [PubMed: 16283857] Qiao LY, Grider JR. Up-regulation of calcitonin gene-related peptide and receptor tyrosine kinase TrkB in rat bladder afferent neurons following TNBS colitis. Exp Neurol. 2007; 204(2):667–679. [PubMed: 17303123]

Front Biol (Beijing). Author manuscript; available in PMC 2014 December 03. Qiao Page 13

Qiao LY, Grider JR. Colitis elicits differential changes in the expression levels of receptor tyrosine kinase TrkA and TrkB in colonic afferent neurons: a possible involvement of axonal transport. Pain. 2010; 151(1):117–127. [PubMed: 20638179] NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript Qiao LY, Gulick MA. bowers kuemmerle grider. Differential changes in brain-derived neurotrophic factor and extracellular signal-regulated kinase in rat primary afferent pathways with colitis. Neurogastroenterol Motil. 2008; 20(8):928–938. [PubMed: 18373519] Qiao LY, Yu SJ, Kay JC, Xia CM. In Vivo Regulation of Brain-Derived Neurotrophic Factor in Dorsal Root Ganglia Is Mediated by Nerve Growth Factor-Triggered Akt Activation during Cystitis. PLoS ONE. 2013; 8(11):e81547. [PubMed: 24303055] Qin C, Malykhina AP, Akbarali HI, Foreman RD. Cross-organ sensitization of lumbosacral spinal neurons receiving urinary bladder input in rats with inflamed colon. Gastroenterology. 2005; 129(6):1967–1978. [PubMed: 16344065] Rosenbaum T, Vidaltamayo R, Sanchez-Soto MC, Zentella A, Hiriart M. Pancreatic beta cells synthesize and secrete nerve growth factor. Proc Natl Acad Sci USA. 1998; 95(13):7784–7788. [PubMed: 9636228] Roza C, Reeh PW. Substance P, calcitonin gene related peptide and PGE2 co-released from the mouse colon: a new model to study nociceptive and inflammatory responses in viscera, in vitro. Pain. 2001; 93(3):213–219. [PubMed: 11514080] Ruiz G, Banos JE. The effect of endoneurial nerve growth factor on calcitonin gene-related peptide expression in primary sensory neurons. Brain Res. 2005; 1042(1):44–52. [PubMed: 15823252] Saini R, Gonzalez RR, Te AE. Chronic pelvic pain syndrome and the overactive bladder: the inflammatory link. Curr Urol Rep. 2008; 9(4):314–319. [PubMed: 18765131] Schnegelsberg B, Sun TT, Cain G, Bhattacharya A, Nunn PA, Ford APDW, Vizzard MA, Cockayne DA. Overexpression of NGF in mouse urothelium leads to neuronal hyperinnervation, pelvic sensitivity, and changes in urinary bladder function. Am J Physiol Regul Integr Comp Physiol. 2010; 298(3):R534–R547. [PubMed: 20032263] Silossantiago I, Molliver DC, Ozaki S, Smeyne RJ, Fagan AM, Barbacid M, Snider WD. Non-TrkA- expressing small DRG neurons are lost in TrkA deficient mice. J Neurosci. 1995; 15(9):5929– 5942. [PubMed: 7666178] Skaper SD, Pollock M, Facci L. Mast cells differentially express and release active high molecular weight neurotrophins. Brain Res Mol Brain Res. 2001; 97(2):177–185. [PubMed: 11750074] Skeldal S, Sykes AM, Glerup S, Matusica D, Palstra N, Autio H, Boskovic Z, Madsen P, Castren E, Nykjaer A, Coulson EJ. Mapping of the Interaction Site between Sortilin and the p75 Neurotrophin Receptor Reveals a Regulatory Role for the Sortilin Intracellular Domain in p75 Neurotrophin Receptor Shedding and Apoptosis. J Biol Chem. 2012; 287(52):43798–43809. [PubMed: 23105113] Stanzel RD, Lourenssen S, Blennerhassett MG. Inflammation causes expression of NGF in epithelial cells of the rat colon. Exp Neurol. 2008; 211(1):203–213. [PubMed: 18377896] Stein AT, Ufret-Vincenty CA, Hua L, Santana LF, Gordon SE. Phosphoinositide 3-kinase binds to TRPV1 and mediates NGF-stimulated TRPV1 trafficking to the plasma membrane. J Gen Physiol. 2006; 128(5):509–522. [PubMed: 17074976] Steinkamp M, Schulte N, Spaniol U, Pfluger C, Hartmann C, Kirsch J, Von Boyen G. Brain derived neurotrophic factor inhibits apoptosis in enteric glia during gut inflammation. Med Sci Monit. 2012; 18(4):Br117–Br122. [PubMed: 22460084] Sterne GD, Brown RA, Green CJ, Terenghi G. NT-3 modulates NPY expression in primary sensory neurons following peripheral nerve injury. J Anat. 1998; 193(2):273–281. [PubMed: 9827642] Swanwick CC, Harrison MB, Kapur J. Synaptic and extrasynaptic localization of brain-derived neurotrophic factor and the tyrosine kinase B receptor in cultured hippocampal neurons. J Comp Neurol. 2004; 478(4):405–417. [PubMed: 15384067] Tender GC, Kaye AD, Li YY, Cui JG. Neurotrophin-3 and tyrosine kinase C have modulatory effects on neuropathic pain in the rat dorsal root ganglia. Neurosurgery. 2011; 68(4):1048–1055. [PubMed: 21221027]

Front Biol (Beijing). Author manuscript; available in PMC 2014 December 03. Qiao Page 14

Theiss AL, Fruchtman S, Lund PK. Growth factors in inflammatory bowel disease: the actions and interactions of growth hormone and insulin-like growth factor-I. Inflamm Bowel Dis. 2004; 10(6): 871–880. [PubMed: 15626905] NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript Tonra JR, Curtis R, Wang V, Cliffer KD, Park JS, Timmers A, Nguyen T, Lindsay RM, Acheson A, Distefano PS. Axotomy upregulates the anterograde transport and expression of brain-derived neurotrophic factor by sensory neurons. J Neurosci. 1998; 18(11):4374–4383. [PubMed: 9592114] Tyagi P, Banerjee R, Basu S, Yoshimura N, Chancellor M, Huang L. Intravesical antisense therapy for cystitis using TAT-peptide nucleic acid conjugates. Mol Pharm. 2006; 3(4):398–406. [PubMed: 16889433] Valdovinos-Flores C, Gonsebatt ME. Nerve growth factor exhibits an antioxidant and an autocrine activity in mouse liver that is modulated by buthionine sulfoximine, arsenic, and acetaminophen. Free Radic Res. 2013; 47(5):404–412. [PubMed: 23472850] Vaz SH, Jorgensen TN, Cristovao-Ferreira S, Duflot S, Ribeiro JA, Gether U, Sebastiao AM. Brain- derived Neurotrophic Factor (BDNF) Enhances GABA Transport by Modulating the Trafficking of GABA Transporter-1 (GAT-1) from the Plasma Membrane of Rat Cortical Astrocytes. J Biol Chem. 2011; 286(47):40464–40476. [PubMed: 21969376] Wang X, Butowt R, Vasko MR, von Bartheld CS. Mechanisms of the release of anterogradely transported neurotrophin-3 from axon terminals. J Neurosci. 2002; 22(3):931–945. [PubMed: 11826122] Watson FL, Heerssen HM, Bhattacharyya A, Klesse L, Lin MZ, Segal RA. Neurotrophins use the Erk5 pathway to mediate a retrograde survival response. Nat Neurosci. 2001; 4(10):981–988. [PubMed: 11544482] Wehrman T, He XL, Raab B, Dukipatti A, Blau H, Garcia KC. Structural and mechanistic insights into nerve growth factor interactions with the TrkA and p75 receptors. Neuron. 2007; 53(1):25–38. [PubMed: 17196528] Whorwell PJ, Lupton EW, Erduran D, Wilson K. Bladder Smooth-Muscle Dysfunction in Patients with Irritable-Bowel-Syndrome. Gut. 1986; 27(9):1014–1017. [PubMed: 3758813] Wilson-Gerwing TD, Dmyterko MV, Zochodne DW, Johnston JW, Verge VM. Neurotrophin-3 suppresses thermal hyperalgesia associated with neuropathic pain and attenuates transient receptor potential vanilloid receptor-1 expression in adult sensory neurons. J Neurosci. 2005; 25(3):758–767. [PubMed: 15659614] Wilson-Gerwing TD, Stucky CL, McComb GW, Verge VMK. Neurotrophin-3 significantly reduces sodium channel expression linked to neuropathic pain states. Exp Neurol. 2008; 213(2):303–314. [PubMed: 18601922] Wright DE, Snider WD. Neurotrophin receptor mRNA expression defines distinct populations of neurons in rat dorsal root ganglia. J Comp Neurol. 1995; 351(3):329–338. [PubMed: 7706545] Xia CM, Gulick MA, Yu SJ, Grider JR, Murthy KS, Kuemmerle JF, Akbarali HI, Qiao LY. Up- regulation of brain-derived neurotrophic factor in primary afferent pathway regulates colon-to- bladder cross-sensitization in rat. J Neuroinflammation. 2012; 9(1):30. [PubMed: 22335898] Yoshimura N, Bennett NE, Hayashi Y, Ogawa T, Nishizawa O, Chancellor MB, de Groat WC, Seki S. Bladder overactivity and hyperexcitability of bladder afferent neurons after intrathecal delivery of nerve growth factor in rats. J Neurosci. 2006; 26(42):10847–10855. [PubMed: 17050722] Yu SJ, Grider JR, Gulick MA, Xia C, Shen S, Qiao LY. Up-regulation of brain-derived neurotrophic factor is regulated by extracellular signal-regulated protein kinase 5 and by nerve growth factor retrograde signaling in colonic afferent neurons in colitis. Exp Neurol. 2012; 238(2):209–217. [PubMed: 22921460] Yu SJ, Xia CM, Kay JC, Qiao LY. Activation of extracellular signal-regulated protein kinase 5 is essential for cystitis- and nerve growth factor-induced calcitonin gene-related peptide expression in sensory neurons. Mol Pain. 2012 Yu YB, Zuo XL, Zhao QJ, Chen FX, Yang J, Dong YY, Wang P, Li YQ. Brain-derived neurotrophic factor contributes to abdominal pain in irritable bowel syndrome. Gut. 2012; 61(5):685–694. [PubMed: 21997550] Zampieri N, Chao MV. Structural biology. The p75 NGF receptor exposed. Science. 2004; 304(5672): 833–834. [PubMed: 15131296]

Front Biol (Beijing). Author manuscript; available in PMC 2014 December 03. Qiao Page 15

Zhang QL, Qiao LY. Regulation of IGF-1 but not TGF-beta 1 by NGF in the smooth muscle of the inflamed urinary bladder. Regul Pept. 2012; 177(1–3):73–78. [PubMed: 22579999]

NIH-PA Author Manuscript NIH-PA Author ManuscriptZhang NIH-PA Author Manuscript XM, Huang JH, McNaughton PA. NGF rapidly increases membrane expression of TRPV1 heat-gated ion channels. EMBO J. 2005; 24(24):4211–4223. [PubMed: 16319926] Zhang YH, Chi XX, Nicol GD. Brain-derived neurotrophic factor enhances the excitability of rat sensory neurons through activation of the p75 neurotrophin receptor and the sphingomyelin pathway. Journal of Physiology-London. 2008; 586(13):3113–3127. Zhou LJ, Zhong Y, Ren WJ, Li YY, Zhang T, Liu XG. BDNF induces late-phase LTP of C-fiber evoked field potentials in rat spinal dorsal horn. Exp Neurol. 2008; 212(2):507–514. [PubMed: 18565512] Zhu WG, Oxford GS. Phosphoinositide-3-kinase and mitogen activated protein kinase signaling pathways mediate acute NGF sensitization of TRPV1. Mol Cell Neurosci. 2007; 34(4):689–700. [PubMed: 17324588] Zvara P, Vizzard MA. Exogenous overexpression of nerve growth factor in the urinary bladder produces bladder overactivity and altered micturition circuitry in the lumbosacral spinal cord. BMC Physiol. 2007; 7(1):9. [PubMed: 17725832]

Front Biol (Beijing). Author manuscript; available in PMC 2014 December 03. diagnostics

Review Visceral Origin: An Underestimated Source of Neck Pain. A Systematic Scoping Review

Ángel Oliva-Pascual-Vaca 1 , Carlos González-González 1, Jesús Oliva-Pascual-Vaca 1,2,3,*, Fernando Piña-Pozo 3 , Alejandro Ferragut-Garcías 4, Juan Carlos Fernández-Domínguez 4 and Alberto Marcos Heredia-Rizo 1 1 Department of Physiotherapy, Faculty of Nursing, Physiotherapy and Podiatry, University of Sevilla, 41009 Sevilla, Spain; [email protected] (Á.O.-P.-V.); [email protected] (C.G.-G.); [email protected] (A.M.H.-R.) 2 Escuela de Osteopatía de Madrid, 28002 Madrid, Spain 3 Department of Physiotherapy, Universitary School of Osuna, University of Sevilla, 41640 Sevilla, Spain; [email protected] 4 Department of Nursing and Physiotherapy, University of the Balearic Islands, 07112 Palma de Mallorca, Spain; [email protected] (A.F.-G.); [email protected] (J.C.F.-D.) * Correspondence: [email protected]

 Received: 3 October 2019; Accepted: 6 November 2019; Published: 12 November 2019 

Abstract: The diagnosis of neck pain is challenging. Many visceral disorders are known to cause it, and clinical practice guidelines recommend to rule them out during neck pain diagnosis. However, the absence of suspicion of any cause impedes one from establishing that specific aetiology as the final diagnosis. To investigate the degree of consideration given to visceral aetiology, a systematic search of trials about neck pain was carried out to evaluate their selection criteria. The search yielded 309 eligible articles, which were screened by two independent reviewers. The PEDro scale score was used to assess the methodological quality of the studies. The following information was retrieved: number of authors affiliated to a clinical or non-clinical institution, number of citations in the Web of Science, study aims, characteristics of participants, and eligibility criteria. The top 15 most cited trials, and the 15 most recent studies about treatment efficacy in neck pain, published in first quartile journals of the Journal Citation Reports, were selected. Females represented 67.5% of participants. A single study was of poor methodological quality (4/10). Based on the eligibility criteria of the articles that were systematically reviewed, it would appear that visceral aetiology was not considered in eighty percent of the trials on neck pain, showing a low level of suspicion both in research and clinical settings.

Keywords: referred pain; visceral pain; diagnosis; phrenic nerve; neck pain

1. Introduction Neck pain (NP) constitutes a major health problem. Its prevalence varies from 4.8% to 79.5%, and is more common in females and in high-incomes countries [1]. It is ranked the 4th most disabling condition as measured by years lived with disability [2]; hence, it poses a substantial economic burden due to extended periods of sick leave and high use of health services [3]. Those individuals with a precise pathoanatomical cause for their NP, e.g., radiculopathy [4,5], facet joint pain [6], chronic rheumatic diseases [7], or cancer, are categorized as having specific NP. Yet, patients without a well identified source for their NP are labelled as having idiopathic, mechanical, or non-specific NP, which is the most common type [8]. As a sign of visceral suffering, pain originating in internal organs is amongst the most frequent forms of pain experienced by individuals in the course of life, and pain involving internal organs

Diagnostics 2019, 9, 186; doi:10.3390/diagnostics9040186 www.mdpi.com/journal/diagnostics Diagnostics 2019, 9, 186 2 of 23

isDiagnostics a major 2019 occurrence, 9, x FOR PEER in theREVIEW clinical setting [9,10]. The rule in visceral nociception is that pain2 of 24 is referred to somatic tissues, being felt at a site other than the affected viscera [10]. Visceral referred painto somatic (VRP) tissues, occurs, being as secondary felt at a site hyperalgesia, other than the in somatic affected areas viscera neuromerically [10]. Visceral referred connected pain with (VRP) the aoccurs,ffected as organs secondary [10]. Thehyperalgesia, overlap of somaticin somatic and ar visceraleas neuromerically afferent information connected into with a shared the affected neural pathwayorgans [10]. seems The to overlap be related of somatic to a misinterpretation and visceral afferent at peripheral, information spinal, into or supraspinala shared neural levels pathway [10,11]. Theseems precise to be substrate related to underlying a misinterpretation this phenomenon at peripheral, remains spinal, unknown or supraspi [12]; itnal has levels been stated[10,11]. that The it mightprecise explain substrate the underlying strong association this phenomenon between back remain pains unknown and digestive [12]; disordersit has been [13 stated]. that it might explainUnderstanding the strong association and awareness between of ba referredck pain and pain digestive is key fordisorders a precise [13]. diagnosis of the pain sourceUnderstanding [14]. Previous and evidenceawareness shows of referred that gastrointestinal,pain is key for a precise biliary, diagnosis renal, hepatic, of the pain heart, source and pulmonary[14]. Previous disorders evidence may shows evoke that referred gastrointestinal pain to the, biliary, upper renal, quadrant hepatic, of the heart, body, and including pulmonary the neckdisorders region may [15 ].evoke The discriminationreferred pain to between the upper visceral quadra andnt somatic of the body, sources including of pain isthe not neck always region evident, [15]. andThe discrimination if it is not achieved, between it mayvisceral lead and to extensivesomatic sources diagnostic of pain procedures is not always and evident, ineffective and treatment if it is not approachesachieved, it may [16]. lead Visceral to extensive disorders diagnostic may evoke proc referrededures and altered ineffective sensitivity, treatment e.g., approaches hyperalgesia [16]. or allodyniaVisceral disorders [17]. For instance,may evoke the radiationreferred altered of pain tosensit theivity, neck ande.g.,/or hyperalgesia upper extremity or allodynia that occurs [17]. during For acuteinstance, coronary the radiation syndromes of pain [18] to is the experienced neck and/or in more upper than extremity 65% of that cases occurs [19]. Eighty-eightduring acute percent coronary of patientssyndromes with [18] colonoscopy-induced is experienced in more splenic than injury 65% complainof cases [19]. of pain Eighty-eight along the percent C3–C4 dermatomes of patients with due tocolonoscopy-induced irritation of the diaphragm splenic injury or distention complain of of the pain splenic along capsule the C3-C4 [20] (Figuredermatomes1)[ 21 ].due That to irritation happens duringof the diaphragm attempts at or sheath distention insertion of intothe splenic the right ca orpsule middle [20] hepatic (Figure vein 1) [21]. in liver That biopsy happens as well during [22]. Further,attempts it at can sheath also insertion be caused into by the more right common, or middle frequently hepatic long-lasting,vein in liver biopsy and not as so well life-threatening [22]. Further, conditions,it can also be such caused as hiatal by more hernias common, and gastroesophageal frequently long-lasting, reflux diseaseand not [ so23 ].life-threatening conditions, such as hiatal hernias and gastroesophageal reflux disease [23].

Figure 1. PatternPattern of of visceral visceral referred referred pain pain along along the the C2–C3–C4 C2–C3–C4 dermatomes. dermatomes. (A) (AnteriorA) Anterior view; view; (B) (posteriorB) posterior view. view.

When routinely evaluating patients with NP, itit isis easyeasy toto missmiss manifestationsmanifestations ofof anan underlyingunderlying disease, and misdiagnose neck disorders ofof visceralvisceral originorigin [[15,24].15,24]. Clinical practice guidelines for the management of NP NP recommend recommend a a detailed detailed physical physical examination examination to to rule rule out out the the possibility possibility of ofVRP VRP in inindividuals individuals with with NP NP [25]. [25 ].Hence, Hence, clinical clinical trials trials assessing assessing treatment treatment efficacy efficacy in in NP NP should excludeexclude participants with suspectedsuspected VRPVRP afterafter aa comprehensivecomprehensive evaluation.evaluation. Otherwise, this selection bias would show an underconsideration of that source of NP, and in addition, result in a likely incorrect estimation of the treatment’s effect size. Therefore, the aim of the systematic review was to investigate to what extent the top 15 most cited and the 15 most recent clinical trials published in high impact

Diagnostics 2019, 9, 186 3 of 23 would show an underconsideration of that source of NP, and in addition, result in a likely incorrect estimation of the treatment’s effect size. Therefore, the aim of the systematic review was to investigate to what extent the top 15 most cited and the 15 most recent clinical trials published in high impact journals, by November 2018, that assessed treatment outcomes in patients with NP, took into account VRP when establishing their eligibility criteria.

2. Materials and Methods The present systematic review was performed according to the Preferred Reporting Item for Systematic Reviews and Meta-Analyses extension for Scoping Reviews guidelines [26]. It has been registered in the International Prospective Register of Systematic Reviews (PROSPERO), with registration number CRD42018101987.

2.1. Data Sources and Search Strategy One author (C.G.-G.) conducted a systematic computerized search between November and December 2018 using the Web of Science database. The search used the key terms neck pain and trial, and considered the following limitations: both key terms being included in the title of the article; language—English/Spanish/Italian/French; and having a publication date between January 1995 and November 2018.

2.2. Study Selection In order to obtain the information from high-quality studies, eligible articles were the top 15 most cited clinical trials published between 1995 and 2018, and the 15 most recent studies included in high impact journals (first quartile of the Journal Citation Reports in the year of publication of the study), which assessed any therapy for subjects suffering from NP. Those articles with any of the following characteristics were excluded: NP patients with only a traumatic, surgical, or neurological origin for the condition; study protocols for clinical trials; studies including only elderly adults (older than 65 years), or including adolescents or children (younger than 18 years); or a lack of a clear description of the eligibility criteria. All relevant titles were saved in a reference manager (EndNote®, version X8.2, Thomson Reuters). Two researchers (C.G.-G. and Á.O.-P.-V.) independently performed the assessment of the studies in a blinded and standardized manner, taking into account the eligibility criteria previously set out. In the case of a disagreement, the issue was discussed together with a third member of the research team (A.M.H.-R.) until a final consensus was reached.

2.3. Assessment of the Methodological Quality The Physiotherapy Evidence Database (PEDro) scale score was used to assess the methodological quality of the clinical trials. The PEDro scale is an 11-item tool where items are scored as either absent (0) or present (1), except for item 1 that refers to external validity of the study. A final score from 0 to 10 is given. The PEDro scale is a valid [27] and reliable [28] tool to rate the methodological quality of clinical trials. A cut-off of at least 5 or 6 points is required for a study to be of adequate quality [29]. PEDro scores were extracted from the PEDro database. Two independent raters (J.C.F.-D. and A.F.-G.) evaluated, with the PEDro scale, those trials not included in the PEDro database. A final consensus about the final score was reached, together with a third member of the research team (A.M.H.-R.), whenever necessary.

2.4. Data Extraction Once the studies were selected, two authors (C.G.-G. and J.O.-P.-V.) independently retrieved the following information from each article following a standardized form: the number of authors affiliated with a clinical institution, e.g., hospital, private practice, or health-center, and the number affiliated with a non-clinical institution, e.g., a university or research center; total number of citations Diagnostics 2019, 9, 186 4 of 23 in the Web of Science; the PEDro scale score; aims of the study; sample size and characteristics of participants (distribution by sex, mean age and pain duration); and eligibility criteria (inclusion and exclusion criteria). Data collected from the studies we included were pooled into tables. Diagnostics 2019, 9, x FOR PEER REVIEW 4 of 24 3. Results 3. Results 3.1. Study Selection 3.1. Study Selection The search strategy resulted in a total of 309 relevant articles that were retrieved through the Web of ScienceThe search database. strategy Then, resulted 94 studies in werea total excluded of 309 re forlevant not matching articles that the eligibilitywere retrieved criteria. through From thethe remainingWeb of Science 215 articles, database. 30 of Then, them 94 were studies finally were included. excluded All selectedfor not matching studies were the writteneligibility in English.criteria. TwoFrom of the the remaining top 15 most 215 cited articles, articles 30 were of them excluded were andfinally replaced included. by the All next selected most studies cited clinical were trialswritten in thein English. list. The Two reason of wasthe top that 15 the most sample cited population articles we andre excluded the eligibility and criteriareplaced used by werethe next the samemost ascited in otherclinical studies trials within the a higherlist. The number reason ofwas citations that the that sample were publishedpopulation by and the the same eligibility research criteria groups used and thatwere had the beensame already as in other included studies for with further a higher analysis. number Figure of citations2 shows that the flowwere diagrampublished for by the the study same selectionresearch process.groups and that had been already included for further analysis. Figure 2 shows the flow diagram for the study selection process.

FigureFigure 2.2. FlowchartFlowchart diagramdiagram ofof thethe studystudy selectionselection processprocess (Preferred(Preferred ReportingReporting ItemItem forfor SystematicSystematic ReviewsReviews andand Meta-Analyses,Meta-Analyses, PRISMA,PRISMA, guidelines).guidelines). Q1,Q1, firstfirst quartile;quartile; JCR,JCR, JournalJournal CitationCitation Reports.Reports. 3.2. Study Characteristics 3.2. Study Characteristics All of the clinical trials which were included were randomized and controlled. Detailed All of the clinical trials which were included were randomized and controlled. Detailed descriptions of articles included in this systematic review are presented in Table1; Table2. Researchers descriptions of articles included in this systematic review are presented in Table 1; Table 2. from non-clinical institutions authored more than 90% of the studies (28 out of 30), while authors Researchers from non-clinical institutions authored more than 90% of the studies (28 out of 30), while from clinical institutions, e.g., hospitals, health-centers, and private practices, participated in 79% of authors from clinical institutions, e.g., hospitals, health-centers, and private practices, participated in trials (21 out of 30). The studies included a total of 4467 participants, with females representing 67.5% 79% of trials (21 out of 30). The studies included a total of 4467 participants, with females representing of the total (3017 females). One clinical trial did not clearly specify the sex distribution of the study 67.5% of the total (3017 females). One clinical trial did not clearly specify the sex distribution of the sample [30]. Two studies recruited exclusively females [31,32], and only two of them selected more study sample [30]. Two studies recruited exclusively females [31,32], and only two of them selected male than female individuals [33,34]. The mean age of participants was between 35 and 53 years, more male than female individuals [33,34]. The mean age of participants was between 35 and 53 with one study including younger participants (mean age of 21 years) [35]. years, with one study including younger participants (mean age of 21 years) [35].

3.3. Methodological Quality of Studies The assessment of the methodological quality by means of the PEDro scale revealed that, in general, the top 15 most cited clinical trials denoted adequate to good methodological quality, with a final score of six points or higher, except for one study that scored five out of 10 points (Table 1) [36].

Diagnostics 2019, 9, 186 5 of 23

3.3. Methodological Quality of Studies The assessment of the methodological quality by means of the PEDro scale revealed that, in general, the top 15 most cited clinical trials denoted adequate to good methodological quality, with a final score of six points or higher, except for one study that scored five out of 10 points (Table1)[ 36]. Similar findings were observed amongst the 15 most recent articles published in high impact journals (Table2), although one study denoted poor methodological quality (four out of 10) [ 37]. One of all studies included achieved excellent methodological quality (10 out of 10) [38]. The reliability between coders for those studies whose scores were not available in the PEDro database was almost perfect (Kappa = 0.84) [39]. Diagnostics 2019, 9, 186 6 of 23

Table 1. Top 15 most cited clinical trials about treatment efficacy in neck pain published between 1995 and 2018. NP, neck pain; Clinical, number of authors belonging to clinical institutions; Non-Clinical, number of authors belonging to academic institutions; SM, spinal manipulation; PT, physical therapy; GP, general practitioner.

Study PEDro Aim Participants Inclusion Criteria Exclusion Criteria Authors’ Institutions Score (Sex and Mean Age) Number of Citations Ylinen et al., 2003 [32] 7/10 Assess the efficacy of intensive N = 180 Females; aged 25 to 53 years; office Severe neck disorders, e.g., disk prolapse Clinical: 5 isometric training and light All females worker; permanently employed; and spinal stenosis; postoperative Non-Clinical: 4 endurance training in chronic NP 46 years motivated to continue working and for conditions in the neck-shoulder; severe Citations: 285 rehabilitation; constant or frequently trauma; instability; spasmodic torticollis; occurring NP > than 6 months frequent migraine; peripheral nerve entrapment; fibromyalgia; shoulder tendonitis, bursitis, or capsulitis; inflammatory rheumatic disease; severe psychiatric illness; diseases that prevent physical loading; pregnancy Cleland et al., 2005 [40] 8/10 Evaluate the immediate effects of N = 36 Aged 18 to 60 years; primary complaint Red flags for serious spinal conditions, e.g., Clinical: 5 thoracic SM in chronic NP 27 females of mechanical NP, defined as infection, tumors, osteoporosis, fracture; Non-Clinical: 0 9 males non-specific pain in the cervicothoracic positive signs or symptoms suggestive of Citations: 156 35 years region and exacerbated by neck nerve root involvement, e.g., altered upper movements limb reflexes, sensation, or strength; cervical or thoracic surgery; prior SM treatment; thoracic spine hypermobility; pregnancy Jordan et al., 1998 [36] 5/10 Assess the effectiveness of intensive N = 119 Aged 20 to 60 years; NP > 3 months Acute NP with no freedom of movement; Clinical: 3 cervical training vs. PT vs. 88 females 31 males with or without non-radicular pain; to PT, SM, or training for the neck-upper Non-Clinical: 3 chiropractic treatment in chronic NP 39 years live within a close distance to the extremity within 6 months; neuropathy; Citations: 152 hospital; X-ray examination of the systemic disease; inflammatory joint or cervical spine; to be able to speak and muscle disease; headache dominating over read Danish NP; migraine; hypermobility; whiplash; primary shoulder or upper extremity problems; previous neck surgery Irnich et al., 2001 [41] 7/10 Compare the efficacy of acupuncture N = 177 Aged 18 to 85 years; chronic NP; Previous surgery, dislocation or fracture; Clinical: 0 vs. massage combined with “sham” 117 females 60 males painful restriction of neck mobility > 1 neurological deficits; systemic disorders; Non-Clinical: 10 laser acupuncture in chronic NP 52 years month; had not received any treatment contraindications to any of the applied Citations: 146 in the previous 2 weeks treatments Diagnostics 2019, 9, 186 7 of 23

Table 1. Cont.

Study PEDro Aim Participants Inclusion Criteria Exclusion Criteria Authors’ Institutions Score (Sex and Mean Age) Number of Citations Korthals-de Bos et al., 6/10 Evaluate the cost effectiveness of PT, N = 183 Aged 18 to 70 years; NP > 2 weeks PT or manual therapy for NP in the 2003 [42] manual therapy, and GP care for 121 females 62 males (confirmed during physical previous 6 months; neck surgery; a specific Clinical: 0 acute, subacute and chronic NP 45 years examination); willingness to comply cause for the NP (for example, malignancy, Non-Clinical: 9 with treatment and follow up fracture, or inflammation) Citations: 137 Cleland et al., 2007 [43] 7/10 Compare the effect of thoracic N = 60 Aged 18 to 60 years; a primary Signs suggestive of a non-musculoskeletal Clinical: 4 nonthrust vs. mobilization/SM, and 33 females 27 males complaint of NP with or without aetiology; whiplash within 6 weeks; Non-Clinical: 2 compare frequencies, side effects, and 43 years unilateral upper-extremity symptoms; cervical spinal stenosis; signs of nerve root Citations: 133 durations in acute and subacute NP a baseline Neck Disability Index score compression (decrease of at least 2 of the 10% following: myotomal strength, sensation, ≥ or reflexes); central nervous system involvement; previous cervicothoracic surgery; pending legal action Irnich et al., 2002 [44] 6/10 Evaluate immediate effects of 2 N = 34 NP > 2 months; diagnosis of Radicular cervical syndrome; segmental Clinical: 0 different modes of acupuncture vs. 25 females myofascial syndrome or irritation instability; fracture or surgery of the Non-Clinical: 8 sham procedure in chronic NP 9 males syndrome based on history, pain cervical spine; contradictions to Citations: 127 52 years characteristics, radiological findings acupuncture; drug, PT or manual and manual examination treatment in the last 4 weeks Viljanen et al., 2003 [31] 8/10 Assess the effectiveness of dynamic N = 393 Female sex; aged 30 to 60 years; suffer Cancer; major trauma; rheumatic disease; Clinical: 6 muscle training and relaxation All females from chronic non-specific NP > 12 neural entrapment; major rehabilitation in Non-Clinical: 0 training in chronic NP 45 years weeks the previous 3 months Citations: 117 Hurwitz et al., 2002 [45] 7/10 Compare the relative effectiveness of N = 336 Aged 18 to 70 years; NP, defined as NP due to fracture, severe Clinical: 0 cervical SM and mobilization in 231 females 105 pain within the upper thoracic spine to spondyloarthropathy, tumor, infection, or Non-Clinical: 6 acute, subacute and chronic NP males the occiput and the surrounding other non-mechanical cause; progressive Citations: 112 46 years musculature; members of health neurological deficit, myelopathy, herniated maintenance organization; had sought nucleus pulposus, or severe incapacitating care at one of the study sites; had not pain; severe coexisting disease; previous received NP treatment in the past electrotherapy treatment; blood month coagulation disorder; use of anticoagulant or corticosteroids; stroke or transient ischemic attacks; inability to read English; pain involving third-party liability or compensation Diagnostics 2019, 9, 186 8 of 23

Table 1. Cont.

Study PEDro Aim Participants Inclusion Criteria Exclusion Criteria Authors’ Institutions Score (Sex and Mean Age) Number of Citations White et al., 2004 [46] 7/10 Compare acupuncture and placebo in N = 135 Aged 18 to 80 years; mechanical NP > 2 Previous neck fracture or surgery; cervical Clinical: 2 chronic NP 87 females 48 males months; pain score > 30 mm on a congenital abnormality; uncontrolled low Non-Clinical: 2 53 years Visual Analogue Scale for 5 of 7 days back pain; contraindication to Citations: 104 before treatment acetaminophen; systemic illness, e.g., rheumatoid arthritis; ongoing litigation or disability claims; current or recent manual neck treatment or steroid use (oral or local injection); or pregnancy Evans et al., 2002 [47] 7/10 Compare the effects of SM combined N = 191 Aged 20 to 65 years; mechanical NP > NP referred from peripheral joints or Clinical: 1 with low-tech rehabilitative exercise, 113 females 78 males 12 weeks; no specific, identifiable viscera; progressive neurologic deficits; Non-Clinical: 3 MedX rehabilitative exercise, or SM 44 years aetiology (i.e., inflammatory disease, severe osteopenia; vascular disease of the Citations: 102 alone in chronic NP infection); pain reproduced by neck neck or upper extremity; significant movement or provocation tests and infectious disease or other severe disabling localized between the most inferior health conditions; previous neck surgery; part of the occipital bone and T1 inability to work because of NP; current or spinous process pending litigation, SM or exercise therapy within 3 months; concurrent treatment for NP by other health care providers Manchikanti et al., 2010 10/10 Evaluate the clinical outcomes of N = 120 Function-limiting NP > 6 months; 18 Disc herniation with radicular pain; [38] therapeutic cervical medial branch 89 females 31 males years or older; to provide written symptomatic spinal stenosis; neck surgery Clinical: 3 blocks with local anesthetic with or 45 years informed consent; positive results with within 3 months; uncontrolled major Non-Clinical: 1 without steroids in chronic NP of controlled diagnostic cervical facet depression or psychiatric disorders; heavy Citations: 96 facet joint origin joint nerve blocks (80% pain relief and opioid usage; acute or uncontrolled the ability to perform previously medical illness; chronic severe conditions; painful movements) inability to stay in a prone position; history of adverse reactions to local anesthetics or steroids; or pregnant or lactating women Hoving et al., 2006 [48] 8/10 Compare the effectiveness of manual N = 183 Aged 18 to 70 years; pain and/or No specific cause for the pain, e.g., Clinical: 0 therapy, PT and continued care by 111 females 72 males stiffness in the neck > 2 weeks; systemic disease, fracture, or organic Non-Clinical: 11 the GP over a 1 year period 45 years nonspecific neck complaints disorders; a history of trauma or additional Citations: 92 reproducible during active or passive dominant complaints, such as headache or range of motion; willingness to nonradicular pain; previous neck surgery; participate manual or physiotherapy in the previous 6 months Diagnostics 2019, 9, 186 9 of 23

Table 1. Cont.

Study PEDro Aim Participants Inclusion Criteria Exclusion Criteria Authors’ Institutions Score (Sex and Mean Age) Number of Citations Chiu et al., 2005 [49] 7/10 Evaluate the efficacy of a neck N = 145 Aged 20 to 70 years; NP (of various Previous neck or upper back (T1-T6) injury; Clinical: 0 exercise program in chronic NP 100 females intensity of pain) > 3 months; able to inflammatory condition, e.g., rheumatoid Non-clinical: 3 45 males read Chinese arthritis; former neck surgery; a Citations: 86 44 years malignancy or congenital spinal abnormality; parallel NP treatment; contraindication for infrared irradiation; neurologic symptoms, e.g., muscle weakness or changes in spinal reflex jerks; other musculoskeletal problems; acute NP with no freedom of movement; training or SM for NP within 6 months; work-related injuries Bronfort et al., 2012 7/10 Determine the relative efficacy of SM, N = 272 Aged 18 to 65 years; primary symptom Cervical spine instability; fracture; NP [50] medication, and home exercise with 178 females 94 males of mechanical, nonspecific NP referred from peripheral joints or viscera; Clinical: 3 advice for acute and subacute NP in 48 years equivalent to grades I or II of the Bone progressive neurologic deficits: cardiac Non-clinical: 3 the short and long term. and Joint Decade 2000–2010 Task Force disease requiring medical treatment; blood Citations: 81 on NP and Its Associated Disorders clotting disorders; diffuse idiopathic classification; NP between 2–12 weeks hyperostosis; inflammatory or destructive duration; NP 3 on a 0 to 10 scale; not tissue changes of the cervical spine; ≥ seeking additional NP treatment infectious disease; substance abuse; cervical spine surgery; severe disabling health problems; pending or current litigation; having received any of the study treatments within 3 months; pregnancy or breastfeeding Diagnostics 2019, 9, 186 10 of 23

Table 2. List of the fifteen most recent clinical trials, by November 2018, about treatment efficacy in neck pain published in high impact journals. NP, neck pain; Clinical, number of authors belonging to clinical institutions; Non-Clinical, number of authors belonging to academic institutions; NDI, neck disability index; PT, physical therapy; VAS, visual analogue scale; SM, spinal manipulation; N/S, non-clearly specified.

PEDro Study Aim Participants Inclusion Criteria Exclusion Criteria Score Authors’ Institutions Number of Citations Celenay et al., 2016 [51] 6/10 Assess the effect of neck stabilization N = 60 Aged 18 to 65 years; NP > 3 months; Stenosis; traumatic injury history; previous Clinical: 0 and scapulo-thoracic treatment with 39 females baseline NDI 20% neck surgery; cancer; hypermobility; ≥ Non-Clinical: 3 and without connective tissue 21 males inflammatory rheumatologic diseases; Citations: 13 massage in chronic NP 48 years severe psychological disorders; exercise or PT intervention in the last 3 months; pregnancy Celenay et al., 2016 [52] 7/10 Compare the effect of stabilization N = 102 Aged 18 to 65 years; NP > 3 months, Inflammatory rheumatologic diseases, Clinical: 0 exercises with or without manual 74 females with symptoms provoked by structural deformity, or malignity; Non-Clinical: 3 therapy in patients with mechanical 28 males postures, movements, or palpation previous cervical surgery; spinal stenosis; Citations: 8 chronic NP 46 years bilateral upper extremity symptoms; 2 ≥ positive radicular signs of nerve root compression; referred pain > than 7 on a 0-10 VAS in the related dermatome in the upper extremities; capsular pattern of arthritis; severe psychological disorder; pregnancy; any intervention including exercise or PT within 3 months Cerezo et al., 2016 [30] 6/10 Assess the effect of deep dry needling N = 128 NP (with or without radiation) > 6 Major trauma; widespread pain; Clinical: 4 of myofascial trigger points in Sex distribution: N/S months, with no known pathological inflammatory, hormonal, or neurological Non-Clinical: 4 chronic nonspecific NP 50 years basis (neurological, trauma); having disorders; upper limbs tendinopathy; Citations: 10 myofascial pain syndrome severe psychiatric illness; inability to speak or write Spanish; use of muscle relaxant, analgesic, antidepressant, or anticoagulant medication in the last week; fibromyalgia; any contraindication to PT (infection, fever, hypothyroidism, wounds, metal allergy, cancer or systemic disease, fear of needles); or pregnancy De Araujo et al., 2018 [34] 8/10 Assess the effectiveness of the Pilates N = 64 Aged 18 to 65 years; non-specific NP Fibromyalgia; spine trauma, infection or Clinical: 0 method in chronic NP 14 females according to the Neck Pain Task inflammation; NP radiating to the upper Non-Clinical: 5 50 males Force; pain > 3 months; and pain limbs; having started or changed physical Citations: 0 49 years intensity between 3–8 cm on a 0 to activity > 2/week within 3 months; visual 10 cm rating scale impairments and no use of glasses; new or changed pain medication, or injections in the last 3 months; neurological diseases; musculoskeletal diseases hindering the practice of Pilates; pregnancy Diagnostics 2019, 9, 186 11 of 23

Table 2. Cont.

PEDro Study Aim Participants Inclusion Criteria Exclusion Criteria Score Authors’ Institutions Number of Citations Essex et al., 4/10 Assess the cost-effectiveness of usual N = 517 NP > 3 months; score > 28% on the Current acupuncture treatment for NP or 2017 [37] care vs. acupuncture and usual care 347 females Northwick Park Neck Pain attended Alexander lessons in the last 2 Clinical: 1 vs. Alexander Technique and usual 170 males Questionnaire years; litigation; serious underlying Non-Clinical: 16 care for chronic NP 53 years pathology; prior neck surgery; alcohol or Citations: 0 drug dependency; involvement in other trial; history of psychosis, rheumatoid arthritis, osteoporosis, hemophilia, ankylosing spondylitis, cancer, HIV or hepatitis; inability to speak English; pregnancy Fernández-Carnero et al., 8/10 Assess the immediate effect of neural N = 54 Aged 18 to 65 years; NP within the Systemic or degenerative diseases; 2018 [35] tension technique in chronic NP 41 females nuchal line and T1 spinous process > headache and/or low back pain within 9 Clinical: 3 13 males 12 weeks; no radicular symptoms to months; NP linked with whiplash; Non-Clinical: 2 21 years head, trunk, or upper limbs; ability to moderate or severe depression; red flags Citations: 0 write and speak Spanish (metabolic diseases, tumor, fracture, rheumatoid arthritis, osteoporosis); fibromyalgia; neck surgery; cervical radiculopathy; disc herniation; neck or face pain within 6 months; NP with vertigo caused by vertebrobasilar insufficiency; non-cervicogenic headache after trauma within last year Griswold et al., 2018 [53] 7/10 Compare the effect of concordant N = 103 Aged 18 to 70 years; having Contraindications to manual therapy Clinical: 1 cervical and thoracic non-thrust vs. 76 females mechanical NP; NDI 20%; and NP (fracture, malignancy, rheumatoid arthritis, ≥ Non-Clinical: 4 thrust SM for chronic mechanical NP 27 males > 2 on a 0 to 10 rating scale in the last myelopathy, osteoporosis); prior cervical Citations: 0 47 years 24 h or thoracic spine surgery; seeking litigation; nerve root compression (at least 2 or more neurological signs); receiving other nonsurgical care; inability to reproduce the concordant sign in the cervical or thoracic spine during testing Krøll et al., 5/10 Evaluate the efficacy of aerobic N = 70 A minimum of 2 attacks of migraine; Whiplash; significant neck trauma, 2018 [54] exercise in migraine and coexisting 62 females a minimum of 1 day with (fracture, distortion, or violent attack); Clinical: 4 tension-type headache and chronic 8 males tension-type headache; a minimum neck nerve root compression; persistent Non-Clinical: 1 NP 37 years of 1 day with NP per month headache linked with trauma; medication Citations: 1 overuse; severe physical and/or mental illness; trigeminal neuralgia; cluster headache; alcohol and drugs abuse; breastfeeding; inability to speak Danish; pregnancy Diagnostics 2019, 9, 186 12 of 23

Table 2. Cont.

PEDro Study Aim Participants Inclusion Criteria Exclusion Criteria Score Authors’ Institutions Number of Citations Lauche et al., 2016 [55] 7/10 Evaluate the efficacy of Tai Chi for N = 114 Age > 18 years; nonspecific NP > 3 NP caused by trauma, disc protrusion, Clinical: 1 treating chronic NP 91 females consecutive months for at least 5 whiplash, spinal deformity, stenosis, Non-Clinical: 8 23 males days a week; NP > 45 mm on a 0 to neoplasm, neurological disorder, Citations: 7 49 years 100 mm VAS rheumatic or active severe affective disorder, addiction, psychosis, or oncologic disease; invasive spinal treatment within 4 weeks; spinal surgery in the last year; new or modified drug regimen; opioids intake; regular practice of Tai Chi, Qigong, or Yoga within 6 months; any disability precluding exercise; pregnancy Lauche et al., 2016 [56] 6/10 Assess the efficacy of the Alexander N = 72 Aged 18 to 50 years; non-specific NP NP caused by disc protrusion or prolapse; Clinical: 3 Technique, local heat and guided 65 females > 3 months; NP intensity > 40 mm on spinal congenital deformity; spinal Non-Clinical: 5 imagery in patients with chronic 7 males a 100 mm VAS stenosis; whiplash; neoplasm, Citations: 3 non-specific NP 41 years inflammatory rheumatic disease; neurological disorder; active oncologic disease; affective disorder; addiction; psychosis; previous spinal surgery or invasive spinal treatment within 3 weeks; ongoing application for disability pension; previous Alexander technique experience; participation in other clinical trials; pregnancy Monticone et al., 2017 [57] 8/10 Evaluate the effect of a group based N = 170 Age >18 years; documented history Acute and subacute NP; cognitive Clinical: 6 multidisciplinary rehabilitation 121 females of non-specific NP >3 months; a good impairment; clear aetiology for their NP, Non-Clinical: 1 programme in chronic NP 49 males understanding of Italian e.g., previous spinal surgery, deformity, Citations: 3 53 years disc herniation, infection, fracture, myelopathy or malignancy, whiplash, and systemic or neuromuscular diseases; having received cognitive-behavioral therapy Pillastrini et al., 2016 [58] 8/10 Evaluate the effectiveness of global N = 96 Nonspecific NP > 3 months; aged 18 Acute or subacute NP; specific cause of NP, Clinical: 1 postural reeducation vs. manual 72 females to 80 years; ability to read and speak e.g., systemic, rheumatic, neuromuscular Non-Clinical: 7 therapy in chronic NP 22 males Italian diseases; central or peripheral neurological Citations: 6 48 years signs; cognitive impairment, spinal surgery; or PT treatments in the prior 6 months Diagnostics 2019, 9, 186 13 of 23

Table 2. Cont.

PEDro Study Aim Participants Inclusion Criteria Exclusion Criteria Score Authors’ Institutions Number of Citations Ris et al., 6/10 Assess the effect of pain education, N = 200 Aged >18 years; traumatic or Clinically confirmed radiculopathies; 2016 [59] exercises and graded physical 149 females non-traumatic NP > half a year; NDI progressive medical treatment; unstable Clinical: 1 activity vs. pain education alone in 51 males >10; NP, primary pain; complete social/working conditions; current Non-Clinical: 5 chronic NP 45 years medical diagnostic procedures fractures; score > 29 in the Beck Depression Citations: 7 Inventory-II; conditions limiting participation; pregnancy Thompson et al., 2016 [33] 5/10 Evaluate the effect of physiotherapist N = 57 Non-specific NP > 3 months; fluency Serious pathology (fracture, dislocation, Clinical: 2 led cognitive—behavioral 27 females in English; have not received PT for carcinoma or infection); radiculopathy; Non-Clinical: 1 intervention plus exercise in chronic 28 males NP in the past 3 months myelopathy; rheumatological disorder; Citations: 4 NP 48 years diagnosed major psychiatric illness Tunwattanapong et al., 8/10 Determine the effect of neck and N = 96 Office workers who rated themselves Performing regular stretching exercise; a 2016 [60] shoulder stretching exercises for 87 females with moderate to severe neck or history of severe neck injury, or neck or Clinical: 1 chronic NP among office workers 9 males shoulder pain (VAS 5 of 10 cm) for shoulder contracture (defined by a ≥ Non-clinical: 2 35 years more than 3 months limitation range of motion in all Citations: 10 directions); previous neck or shoulder surgery; abnormal neurological signs Diagnostics 2019, 9, 186 14 of 23

3.4. Eligibility Criteria (Inclusion and Exclusion Criteria) Used by Trials Of all trials analyzed, a single study [38] defined stringent inclusion criteria to avoid the recruitment of participants with possible VRP. This study included patients with a positive response to cervical facet joint nerve blockers; e.g., 80% pain relief and the ability to perform previously painful movements. With regard to the exclusion criteria, five clinical trials listed them to avoid the selection of individuals with suspected VRP as the cause of their NP. Two studies explicitly excluded participants with “NP referred from peripheral joints or viscera” [47,50]. The other three studies excluded individuals who suffered from NP with a “non-mechanical cause” [45], reported “any medical sign suggestive of a non-musculoskeletal aetiology” for their NP [43], or were diagnosed with a “specific cause for the neck pain”; e.g., organic disorders or systemic diseases [48]. A “clear aetiology” [57] or a “specific cause” [57,58] for the NP was also listed as an exclusion criterion in three other trials. Yet, none of the latter studies mentioned visceral or organic disorders as possible specific causes for the NP. Some other visceral sources of NP were enumerated in other clinical trials: (a) hepatitis [37]; (b) systemic disorders, including metabolic disease [30,35,36,41,46,57,58]; (c) abuse of alcohol and drugs [37,54]; (d) rheumatic disease [31–33,35,37,46,49,51–53,55,56,58]; (e) cancer [30,31,35,37,42,51–53,55–57]; (f) HIV [37]; and (g) infection [30,33,34,40,45,47,57]. Pregnancy was also included as exclusion criteria in almost half of the trials [30,32,34,37,38,40,46,50–52,54–56,59]. Two clinical trials did not refer to any possible visceral aetiology of neck pain in their inclusion and exclusion criteria [44,60].

4. Discussion The present findings suggest that amongst the most cited and the most recently published clinical trials assessing treatment efficacy in NP there is a lack of consideration for VRP as a plausible source of NP. This appears to be the case when the eligibility criteria for recruiting participants are analyzed. Only 20% of all selected trials (six out of 30) defined stringent enough criteria to avoid the recruitment of individuals with a suspected visceral referred NP. All these studies were amongst the top 15 most cited articles. Three other trials excluded patients with a specific aetiology or cause for their NP, although authors did not even mention the visceral area. This implies that most of the assessed trials might have included patients with a visceral source of NP despite the fact that the visceral disorder would not be the target of the treatment. Therefore, it might show an underconsideration of this neck pain aetiology; in addition, incorrect estimations of the effects or efficacies of the interventions could have occurred.

4.1. Eligibility Criteria Used by Trials to Select NP Patients Manchikanti et al. [38] considered as eligible, those patients with a positive response to cervical facet joint nerve blockers, which excluded individuals with visceral referred NP. The rest of the clinical trials, however, established general inclusion criteria; e.g., neck stiffness; mechanical pain with reproducibility of symptoms during physical examination, neck movement, or posture maintenance; and myofascial pain syndrome, among others. All these symptoms mainly refer to increased local sensitization and muscle tension, which can be due to a visceral issue. A primary visceral disorder may also be accompanied by hyperalgesia of the painful area, and is often associated with sustained muscle contraction [61], and it may extend to subcutaneous tissues when the visceral disorder is persistent [62]. Furthermore, the increased muscle tone may explain mechanical symptoms and lead, in the long-term, to the presence of myofascial trigger points and myofascial pain syndrome [63,64]. Hence, visceral pain can evoke many different neck symptoms, including muscle spasms in addition to pain [15], and when sustained, may help to develop central sensitization and cortical changes [61]. Despite all this, surprisingly, the visceral aetiology of NP was only properly considered in six clinical trials [38,43,45,47,48,50]. These findings may imply a general misdiagnosis of NP in research and clinical scopes, thus patients with visceral referred NP might not receive the most accurate therapeutic approach. Visceral pain shares many features with pain from deep somatic structures and requires well-developed Diagnostics 2019, 9, 186 15 of 23 propedeutics to avoid inadequate diagnosis and treatment [65]. The test for cutaneous allodynia appears to have the greatest likelihood of identifying a visceral source of pain compared to somatic sources of pain [16]. Therefore, a detailed clinical history, physical examination, and supplemental laboratory and imaging examination is needed to diagnose the primary source of pain [61,64], because the somatic manifestation will persist until the visceral disorder resolves by itself or has been discovered and treated [23,66]. This discovery is more likely to happen in subjects with constant neck pain and/or aggravation of visceral symptoms, but is less likely in recurrent, episodic NP and/or subtle visceral symptoms [15]. Additionally, in cases of chronic NP, the right diagnosis can be favored along the course of the disease by the obtaining of abnormalities in blood, urine, digestive, heart ... tests, either developed ad hoc to diagnose the origin of NP or during medical assessments for other reasons. Since many of the visceral disorders which may trigger NP are chronic, they enable the presence of either episodic (due to episodic aggravation of the visceral disorder), recurrent (due to frequent aggravation of the visceral disorder), or chronic NP.

4.2. Somatic Consequences of Visceral Disorders Visceral referred NP is linked to the involvement of the vagus and/or phrenic nerves. The nociceptive input from any of the organs innervated by the vagus nerve sensitizes the trigeminocervical nuclear complex that descends to C3 or C4 levels, and may trigger a headache [67] and/or NP [14]. The phrenic nerve is a motor and sensitive nerve formed by C3–C4 roots, with C5 as an accessory root. Either directly or through celiac connections, it supplies the diaphragm, pleura, right atrium [68] pericardium [68,69], esophagus [70], peritoneum [68,71], stomach [15], falciform and coronary ligaments of the liver [72], the Glisson capsule [72,73], the hepatic vein [74], the inferior vena cava [68,70,74], the liver [68,72,75] (parenchyma) [74], the gallbladder [72,76,77] and the rest of the biliary tract [71,72,77]—including the duodenal papilla and the sphincter of Oddi [77,78] —, the pancreas [15], the small intestine [15], and the suprarenal glands [68,70,71]. Hence, disorders of many of these structures, such as the pancreas, or even the spleen or kidneys, can evoke referred pain along the C3–C4 dermatomes either due to the autonomic connections, diaphragmatic pressure, or peritoneal irritation [15]. This has been described as “phrenic pain” [23]. To date, there is no data about the prevalence of NP of visceral origin in general practice or musculoskeletal settings. Nevertheless, in a previous study [79] that seemed not to consider the visceral aetiology, a well identified cause of the NP was not found in 32% of patients receiving a complete evaluation in a private pain clinic, where, probably, those NP patients with the most severe symptoms are a majority. As well, a history of previous trauma was present in most of the patients. However, the authors do not clarify if the inability to achieve a specific cause was more frequent in patients who had previous trauma or in those who did not. It is important to consider the prevalence of the causes of any condition, because that determines the pretest probability, the order of the investigations, and it can also affect to prognosis. The prevalence of visceral disorders that may trigger pain in the neck-shoulder area is high. It is estimated that the one-year prevalence of gastroesophageal reflux with weekly symptoms is 14% [80], and 15% of Americans have silent gallstones, 10–18% of whom develop biliary pain [81]. Besides, non-alcoholic fatty liver is present in around 30% of the population in western countries [82]. Females are more prone than males to have widespread hyperalgesia from recurrent visceral pain [61], and also NP is more prevalent in females. Further, NP of high intensity/low disability or high disability is strongly related to cardiovascular and digestive disorders [83]. That may suggest, eventually, a visceral origin for the pain, which together with cervical spasms have been observed in animal models [84] and in humans [23] with gastric or esophageal disorders. It is also known that NP is highly associated with obesity [85], LDL cholesterol [86], and metabolic syndrome [87]. For instance, the prevalence of NP in those with metabolic syndrome ranges between 16% for males and 25% for females [87]. This is remarkably important because fatty liver, obesity, and metabolic syndromes entail hepatic suffering; e.g., increased pressure, swelling, and hepatomegaly. Phrenic afferents in the hepatic parenchyma, hepatic veins, Diagnostics 2019, 9, 186 16 of 23 and the inferior vena cava just need light pressure to respond [74,88]. Further, all this can stretch and sensitize the Glisson capsule, which is known to evoke phrenic pain [72]. However, most patients do not relate their NP and the concomitant visceral disorder, or fail to report gastrointestinal or hepatic/biliary symptoms [15], which contributes to the misdiagnosis of NP as mechanical or non-specific. It is interesting to note that experimental research of gastric sensitivity is performed in rats by means of gastric distension, which is very common in obesity, and is related to dyspepsia. This gastric distension triggers an increase in the electromyographic activity of the neck muscles and also affects to the neck posture [89]. The addition of substances which increase the insult to the stomach enhances this visceromotor response [84]. The same mechanisms have been used to experimentally study the gastric hypersensitivity frequently observed in patients with long-standing diabetes [90]. The increase of muscle tone in the area of referred hyperalgesia does not appear only when the stomach is injured, since it has also been demonstrated by artificial ureteric stones [91]. Moreover, the neck muscles’ tone decreased in these models when the viscera was treated by means of electrical stimulation [92,93]. Likewise, manual visceral treatment has also been shown to improve NP and electromyographic recordings of the upper trapezius muscles of subjects suffering from chronic non-specific NP and dyspepsia [94], and has improved neck mobility and NP thresholds in subjects suffering from gastroesophageal reflux disease [95]. On the contrary, the likely participation of patients with NP of visceral origin might contribute to explaining the scarce success of usual treatments for NP, achieving at most moderate effects in the short-term [96]. Therefore, the visceral origin of NP might be more easily diagnosed if it only triggers VRP, because there will be no modification of pain related to activity or posture. However, as previously exposed, mechanical consequences can be also elicited in case of visceral aetiology of NP [15,24,61,63,64,66,84, 89–93], hindering the correct diagnosis. The presence of muscle hypertonus, myofascial trigger points, and/or myofascial pain syndrome may increase symptoms during musculoskeletal activity. Similarly, pain modification related to movement and/or postures has also been described during the affectation of the spleen [97], gallbladder [72,98], kidney [99,100], and heart [15,101].

4.3. Needs for the Future Our results suggest that clinical trials about NP fail to suspect a visceral origin of NP. This poor consideration seems to be shared in the research and clinical settings, considering that authors from clinical institutions participated in nearly 80% of the studies reviewed. Thus, our study points out the need to further develop the knowledge of somatic consequences of visceral disorders, at least when related to NP. Additionally, it shows the need for more research to get to know the prevalence of NP of visceral origin in different settings (primary care, clinics of rehabilitation, chiropractic, osteopathy, and physiotherapy).

4.4. Limitations The present findings should be carefully interpreted for several reasons. First, the search strategy was conducted in a single database. The aim was to select those trials with higher impacts, based on the number of citations or on the publication in first quartile journals of the Journal Citation Reports. This strategy was set in order to select those trials which could be representative of the best research about neck pain; i.e., the most cited (showing that they are used as a reference by many researchers) and those published in the best quality journals (which are supposed to publish the best studies). The Web of Science database provides the number of citations and the quartile, and it is considered of high prestige in the Health Sciences field. Other databases such as Medline do not provide numbers of citations nor journal impact factors. Other reviews used different strategies to show a general overview about a subject, such as random selection of studies and/or selection of specific major journals [102–105]. Second, with respect to sample size, it could be argued that our sample size (30 studies) is not big enough to be representative. The right sample size to perform this kind of study has not been established. In the literature, systematic reviews about research bias can be found Diagnostics 2019, 9, 186 17 of 23 with sample sizes ranging between 10 and 44 studies [106–111]. Third, despite the high prevalence of visceral disorders, it is not possible to conclude that these studies actually included patients with VRP to the neck area. Therefore, the influence of this issue on the results of each trial remains unknown.

5. Conclusions In conclusion, it seems that most of the top cited and most recent clinical trials assessing treatments in NP lacked the consideration of visceral referred NP according to their eligibility criteria, showing that neck pain of visceral origin is underestimated. Although NP referred from viscera is difficult to diagnose, more stringent inclusion and exclusion criteria may be required in clinical trials. Otherwise, this may imply an incorrect estimation of the usefulness of the interventions.

Author Contributions: Conceptualization, Á.O.-P.-V. and F.P.-P.; methodology, J.C.F.-D. and A.M.H.-R.; formal analysis, C.G.-G. and A.F.-G.; investigation, C.G.-G., A.F.-G., J.C.F.-D., J.O.-P.-V., Á.O.-P.-V., and A.M.H.-R.; data curation, C.G.-G.; writing—original draft preparation, Á.O.-P.-V. and A.M.H.-R.; writing—review and editing, F.P.-P. and J.O.-P.-V.; supervision, Á.O.-P.-V. Funding: This research received no external funding. Conflicts of Interest: The authors declare no conflict of interest.

References

1. Hoy, D.G.; Protani, M.; De, R.; Buchbinder, R. The epidemiology of neck pain. Best Pract. Res. Clin. Rheumatol. 2010, 24, 783–792. [CrossRef][PubMed] 2. Hoy, D.; March, L.; Woolf, A.; Blyth, F.; Brooks, P.; Smith, E.; Vos, T.; Barendregt, J.; Blore, J.; Murray, C.; et al. The global burden of neck pain: Estimates from the global burden of disease 2010 study. Ann. Rheum. Dis. 2014, 73, 1309–1315. [CrossRef][PubMed] 3. Driessen, M.T.; Lin, C.W.; van Tulder, M.W. Cost-effectiveness of conservative treatments for neck pain: A systematic review on economic evaluations. Eur. Spine J. 2012, 21, 1441–1450. [CrossRef][PubMed] 4. Bono, C.M.; Ghiselli, G.; Gilbert, T.J.; Kreiner, D.D.; Reitman, C.; Summers, J.T.; Baisden, J.T.; Easa, J.; Fernand, R.; Lamer, T.; et al. An evidence-based clinical guideline for the diagnosis and treatment of cervical radiculopathy from degenerative disorders. Spine J. 2011, 11, 64–72. [CrossRef][PubMed] 5. Kjaer, P.; Kongsted, A.; Hartvigsen, J.; Isenberg-Jorgensen, A.; Schiottz-Christensen, B.; Soborg, B.; Krog, C.; Moller, C.M.; Halling, C.M.B.; Lauridsen, H.H.; et al. National clinical guidelines for non-surgical treatment of patients with recent onset neck pain or cervical radiculopathy. Eur. Spine J. 2017, 26, 2242–2257. [CrossRef] [PubMed] 6. Falco, F.J.; Erhart, S.; Wargo, B.W.; Bryce, D.A.; Atluri, S.; Datta, S.; Hayek, S.M. Systematic review of diagnostic utility and therapeutic effectiveness of cervical facet joint interventions. Pain Physician 2009, 12, 323–344. [PubMed] 7. Ambrose, N.L.; Cunnane, G. Importance of full evaluation in patients who complain of neck pain. Ir. J. Med. Sci. 2009, 178, 209–210. [CrossRef][PubMed] 8. Childs, J.D.; Cleland, J.A.; Elliott, J.M.; Teyhen, D.S.; Wainner, R.S.; Whitman, J.M.; Sopky, B.J.; Godges, J.J.; Flynn, T.W. Neck pain: Clinical practice guidelines linked to the International Classification of Functioning, Disability, and Health from the Orthopaedic Section of the American Physical Therapy Association. J. Orthop. Sports Phys. Ther. 2008, 38, A1–A34. [CrossRef][PubMed] 9. Giamberardino, M.A.; Costantini, R.; Affaitati, G.; Fabrizio, A.; Lapenna, D.; Tafuri, E.; Mezzetti, A. Viscero-visceral hyperalgesia: Characterization in different clinical models. Pain 2010, 151, 307–322. [CrossRef][PubMed] 10. Giamberardino, M.; Affaitati, G.; Costantini, R. Visceral referred pain. J. Musculoskelet. Pain 2010, 18, 403–410. [CrossRef] 11. Ruch, T.; Patton, H. Pathophysiology of Pain: Physiology and Biophysics; Saunders: Philadelpia, PA, USA, 1965. 12. Luz, L.L.; Fernandes, E.C.; Sivado, M.; Kokai, E.; Szucs, P.; Safronov, B.V. Monosynaptic convergence of somatic and visceral C-fiber afferents on projection and local circuit neurons in lamina I: A substrate for referred pain. Pain 2015, 156, 2042–2051. [CrossRef][PubMed] Diagnostics 2019, 9, 186 18 of 23

13. Smith, M.D.; Russell, A.; Hodges, P.W. How common is back pain in women with gastrointestinal problems? Clin. J. Pain 2008, 24, 199–203. [CrossRef][PubMed] 14. Lee, D.G.; Chang, M.C. Neck-to-shoulder pain as an unusual presentation of pulmonary embolism in a patient with cervical spinal cord injury: A case report. Medicine 2017, 96, e8288. [CrossRef][PubMed] 15. Goodman, C.C. Screening for gastrointestinal, hepatic/biliary, and renal/urologic disease. J. Hand Ther. 2010, 23, 140–156. [CrossRef][PubMed] 16. Jarrell, J.; Giamberardino, M.A.; Robert, M.; Nasr-Esfahani, M. Bedside testing for chronic pelvic pain: Discriminating visceral from somatic pain. Pain Res. Treat. 2011, 2011, 692102. [CrossRef][PubMed] 17. Head, H. On disturbances of sensation with especial reference to the pain of visceral disease. Brain 1893, 16, 1–133. [CrossRef] 18. Amsterdam, E.A.; Wenger, N.K.; Brindis, R.G.; Casey, D.E.; Ganiats, T.G.; Holmes, D.R.; Jaffe, A.S.; Jneid, H.; Kelly, R.F.; Kontos, M.C.; et al. 2014 AHA/ACC Guideline for the management of patients with non-st-elevation acute coronary syndromes: A report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines. J. Am. Coll. Cardiol. 2014, 64, e139–e228. [CrossRef][PubMed] 19. Horne, R.; James, D.; Petrie, K.; Weinman, J.; Vincent, R. Patients’ interpretation of symptoms as a cause of delay in reaching hospital during acute myocardial infarction. Heart 2000, 83, 388–393. [CrossRef][PubMed] 20. Saad, A.; Rex, D.K. Colonoscopy-induced splenic injury: Report of 3 cases and literature review. Dig. Dis. Sci. 2008, 53, 892–898. [CrossRef][PubMed] 21. Lee, M.W.L.; McPhee, R.W.; Stringer, M.D. An evidence-based approach to human dermatomes. Clin. Anat. 2008, 21, 363–373. [CrossRef][PubMed] 22. Khosa, F.; McNulty, J.G.; Hickey, N.; O’Brien, P.; Tobin, A.; Noonan, N.; Ryan, B.; Keeling, P.W.; Kelleher, D.P.; McDonald, G.S. Transvenous liver biopsy via the femoral vein. Clin. Radiol. 2003, 58, 487–491. [CrossRef] 23. Flanagin, B.A.; Mitchell, M.T.; Thistlethwaite, W.A.; Alverdy, J.C. Diagnosis and treatment of atypical presentations of hiatal hernia following bariatric surgery. Obes. Surg. 2010, 20, 386–392. [CrossRef][PubMed] 24. Lehwald, N.; Krausch, M.; Franke, C.; Assmann, B.; Adam, R.; Knoefel, W.T. Sandifer syndrome: A multidisciplinary diagnostic and therapeutic challenge. Eur. J. Pediatr. Surg. 2007, 17, 203–206. [CrossRef][PubMed] 25. Monticone, M.; Iovine, R.; de Sena, G.; Rovere, G.; Uliano, D.; Arioli, G.; Bonaiuti, D.; Brugnoni, G.; Ceravolo, G.; Cerri, C.; et al. The Italian Society of Physical and Rehabilitation Medicine (SIMFER) recommendations for neck pain. G. Ital. Med. Lav. Ergon. 2013, 35, 36–50. [PubMed] 26. Tricco, A.C.; Lillie, E.; Zarin, W.; O´Brien, K.K.; Colquhoun, H.; Levac, D.; Moher, D.; Peters, M.D.J.; Horsley, T.; Weeks, L.; et al. PRISMA extension for Scoping Reviews (PRISMA-ScR): Checklist and Explanation. Ann. Int. Med. 2018, 169, 467–473. [CrossRef][PubMed] 27. De Morton, N.A. The PEDro scale is a valid measure of the methodological quality of clinical trials: A demographic study. Aust. J. Physiother. 2009, 55, 129–133. [CrossRef] 28. Maher, C.G.; Sherrington, C.; Herbert, R.D.; Moseley, A.M.; Elkins, M. Reliability of the PEDro scale for rating quality of randomized controlled trials. Phys. Ther. 2003, 83, 713–721. [PubMed] 29. Armijo-Olivo, S.; da Costa, B.R.; Cummings, G.G.; Ha, C.; Fuentes, J.; Saltaji, H.; Egger, M. PEDro or Cochrane to assess the quality of clinical trials? A Meta-Epidemiological Study. PLoS ONE 2015, 10, e0132634. [CrossRef][PubMed] 30. Cerezo-Téllez, E.; Torres-Lacomba, M.; Fuentes-Gallardo, I.; Perez-Munoz, M.; Mayoral-del-Moral, O.; Lluch-Girbes, E.; Prieto-Valiente, L.; Falla, D. Effectiveness of dry needling for chronic nonspecific neck pain: A randomized, single-blinded, clinical trial. Pain 2016, 157, 1905–1917. [CrossRef][PubMed] 31. Viljanen, M.; Malmivaara, A.; Uitti, J.; Rinne, M.; Palmroos, R.; Laippala, P. Effectiveness of dynamic muscle training, relaxation training, or ordinary activity for chronic neck pain: Randomised controlled trial. BMJ 2003, 327, 475. [CrossRef][PubMed] 32. Ylinen, J.; Takala, E.P.; Nykänen, M.; hakkinen, A.; Malkia, E.; Pohjolainen, T.; Karppi, S.-L.; Kautiainen, H.; Airaksinen, O. Active neck muscle training in the treatment of chronic neck pain in women: A randomized controlled trial. JAMA 2003, 289, 2509–2516. [CrossRef][PubMed] 33. Thompson, D.P.; Oldham, J.A.; Woby, S.R. Does adding cognitive-behavioural physiotherapy to exercise improve outcome in patients with chronic neck pain? A randomised controlled trial. Physiotherapy 2016, 102, 170–177. [CrossRef][PubMed] Diagnostics 2019, 9, 186 19 of 23

34. De Araujo Cazotti, L.; Jones, A.; Roger-Silva, D.; Ribeiro, L.H.C.; Natour, J. Effectiveness of the Pilates method in the treatment of chronic mechanical neck pain: A randomized controlled trial. Arch. Phys. Med. Rehabil. 2018, 99, 1740–1746. [CrossRef][PubMed] 35. Fernández-Carnero, J.; Sierra-Silvestre, E.; Beltran-Alacreu, H.; Gil-Martinez, A.; La Touche, R. Neural tension technique improves immediate conditioned pain modulation in patients with chronic neck pain: A randomized clinical trial. Pain Med. 2019, 20, 1227–1235. [CrossRef][PubMed] 36. Jordan, A.; Bendix, T.; Nielsen, H.; Hansen, F.R.; Host, D.; Winkel, A. Intensive training, physiotherapy, or manipulation for patients with chronic neck pain. A prospective, single-blinded, randomized clinical trial. Spine 1998, 23, 311–319. [CrossRef][PubMed] 37. Essex, H.; Parrott, S.; Atkin, K.; Ballard, K.; Blanda, M.; Eldred, J.; Hewitt, C.; Hopton, A.; Keding, A.; Lansdown, H.; et al. An economic evaluation of Alexander Technique lessons or acupuncture sessions for patients with chronic neck pain: A randomized trial (ATLAS). PLoS ONE 2017, 12, e0178918. [CrossRef] [PubMed] 38. Manchikanti, L.; Singh, V.; Falco, F.J.; Cash, K.A.; Fellows, B. Comparative outcomes of a 2-year follow-up of cervical medial branch blocks in management of chronic neck pain: A randomized, double-blind controlled trial. Pain Physician 2010, 13, 437–450. [PubMed] 39. Viera, A.J.; Garrett, J.M. Understanding interobserver agreement: The kappa statistic. Fam. Med. 2005, 37, 360–363. [PubMed] 40. Cleland, J.A.; Childs, J.D.; McRae, M.; palmer, J.A.; Stowell, T. Immediate effects of thoracic manipulation in patients with neck pain: A randomized clinical trial. Man. Ther. 2005, 10, 127–135. [CrossRef][PubMed] 41. Irnich, D.; Behrens, N.; Molzen, H.; Konig, A.; Gleditsch, J.; Krauss, M.; Natalis, M.; Senn, E.; Beyer, A.; Schops, P. Randomised trial of acupuncture compared with conventional massage and “sham” laser acupuncture for treatment of chronic neck pain. BMJ 2001, 322, 1574–1578. [CrossRef][PubMed] 42. Korthals-de Bos, I.B.; Hoving, J.L.; van Tulder, M.W.; Rutten-van-Molken, M.P.M.H.; Ader, H.J.; de Vet, H.C.M.; Koes, B.W.; Vondeling, H.; Bouter, L.M. Cost effectiveness of physiotherapy, manual therapy, and general practitioner care for neck pain: Economic evaluation alongside a randomised controlled trial. BMJ 2003, 326, 911. [PubMed] 43. Cleland, J.A.; Glynn, P.; Whitman, J.M.; Eberhart, S.L.; MacDonald, C.; Childs, J.D. Short-term effects of thrust versus nonthrust mobilization/manipulation directed at the thoracic spine in patients with neck pain: A randomized clinical trial. Phys. Ther. 2007, 87, 431–440. [CrossRef][PubMed] 44. Irnich, D.; Behrens, N.; Gleditsch, J.M.; Stor, W.; Schreiber, M.A.; Schops, P.; Vickers, A.J.; Beyer, A. Immediate effects of dry needling and acupuncture at distant points in chronic neck pain: Results of a randomized, double-blind, sham-controlled crossover trial. Pain 2002, 99, 83–89. [CrossRef] 45. Hurwitz, E.L.; Morgenstern, H.; Harber, P.; Koniski, G.F.; Yu, F.; Adams, A.H. A randomized trial of chiropractic manipulation and mobilization for patients with neck pain: Clinical outcomes from the UCLA neck-pain study. Am. J. Public Health 2002, 92, 1634–1641. [CrossRef][PubMed] 46. White, P.; Lewith, G.; Prescott, P.; Conway, J. Acupuncture versus placebo for the treatment of chronic mechanical neck pain: A randomized, controlled trial. Ann. Intern. Med. 2004, 141, 911–919. [CrossRef] [PubMed] 47. Evans, R.; Bronfort, G.; Nelson, B.; Goldsmith, C.H. Two-year follow-up of a randomized clinical trial of spinal manipulation and two types of exercise for patients with chronic neck pain. Spine 2002, 27, 2383–2389. [CrossRef][PubMed] 48. Hoving, J.L.; de Vet, H.C.; Koes, B.W.; van Mameren, H.; Deville, W.L.J.M.; van der Windt, D.A.W.M.; Assendelft, W.J.J.; Pool, J.J.M.; Scholten, R.J.P.M.; Korthals-deBos, I.B.C.; et al. Manual therapy, physical therapy, or continued care by the general practitioner for patients with neck pain: Long-term results from a pragmatic randomized clinical trial. Clin. J. Pain 2006, 22, 370–377. [CrossRef][PubMed] 49. Chiu, T.T.; Lam, T.H.; Hedley, A.J. A randomized controlled trial on the efficacy of exercise for patients with chronic neck pain. Spine 2005, 30, E1–E7. [CrossRef][PubMed] 50. Bronfort, G.; Evans, R.; Anderson, A.V.; Svendsen, K.H.; Bracha, Y.; Grimm, R.H. Spinal manipulation, medication, or home exercise with advice for acute and subacute neck pain: A randomized trial. Ann. Intern. Med. 2012, 156, 1–10. [CrossRef][PubMed] Diagnostics 2019, 9, 186 20 of 23

51. Celenay, S.T.; Kaya, D.O.; Akbayrak, T. Cervical and scapulothoracic stabilization exercises with and without connective tissue massage for chronic mechanical neck pain: A prospective, randomised controlled trial. Man. Ther. 2016, 21, 144–150. [CrossRef][PubMed] 52. Celenay, S.T.; Akbayrak, T.; Kaya, D.O. A comparison of the effects of stabilization exercises plus manual therapy to those of stabilization exercises alone in patients with nonspecific mechanical neck pain: A randomized clinical trial. J. Orthop. Sports Phys. Ther. 2016, 46, 44–55. [CrossRef][PubMed] 53. Griswold, D.; Learman, K.; Kolber, M.J.; O’Halloran, B.; Cleland, J.A. Pragmatically applied cervical and thoracic nonthrust manipulation versus thrust manipulation for patients with mechanical neck pain: A multicenter randomized clinical trial. J. Orthop. Sports Phys. Ther. 2018, 48, 137–145. [CrossRef][PubMed] 54. Krøll, L.S.; Hammarlund, C.S.; Linde, M.; Gard, G.; Jensen, R.H. The effects of aerobic exercise for persons with migraine and co-existing tension-type headache and neck pain. A randomized, controlled, clinical trial. Cephalalgia 2018, 38, 1805–1816. [CrossRef][PubMed] 55. Lauche, R.; Stumpe, C.; Fehr, J.; Cramer, H.; Cheng, Y.W.; Wayne, P.M.; Rampp, T.; Langhorst, J.; Dobos, G. The effects of Tai Chi and neck exercises in the treatment of chronic nonspecific neck pain: A randomized controlled trial. J. Pain. 2016, 17, 1013–1027. [CrossRef][PubMed] 56. Lauche, R.; Schuth, M.; Schwickert, M.; Ludtke, R.; Musial, F.; Michalsen, A.; Dobos, G.; Choi, K.E. Efficacy of the Alexander Technique in treating chronic non-specific neck pain: A randomized controlled trial. Clin. Rehabil. 2016, 30, 247–258. [CrossRef][PubMed] 57. Monticone, M.; Ambrosini, E.; Rocca, B.; Cazzaniga, D.; Liquori, V.; Pedrocchi, A.; Vernon, H. Group-based multimodal exercises integrated with cognitive-behavioural therapy improve disability, pain and quality of life of subjects with chronic neck pain: A randomized controlled trial with one-year follow-up. Clin. Rehabil. 2017, 31, 742–752. [CrossRef][PubMed] 58. Pillastrini, P.; de Lima E Sá Resende, F.; Banchelli, F.; Burioli, A.; Di CIaccio, E.; Guccione, A.A.; Villafañe, J.H.; Vanti, C. Effectiveness of Global Postural Re-education in patients with chronic nonspecific neck pain: Randomized controlled trial. Phys. Ther. 2016, 96, 1408–1416. [CrossRef][PubMed] 59. Ris, I.; Søgaard, K.; Gram, B.; Agerbo, K.; Boyle, E.; Juul-Kristensen, B. Does a combination of physical training, specific exercises and pain education improve health-related quality of life in patients with chronic neck pain? A randomised control trial with a 4-month follow up. Man. Ther. 2016, 26, 132–140. [CrossRef] [PubMed] 60. Tunwattanapong, P.; Kongkasuwan, R.; Kuptniratsaikul, V.The effectiveness of a neck and shoulder stretching exercise program among office workers with neck pain: A randomized controlled trial. Clin. Rehabil. 2016, 30, 64–72. [CrossRef][PubMed] 61. Giamberardino, M.A. Clinical Updates. Visceral Pain. Pain 2005, 12, 1–6. 62. Giamberardino, M.A. Recent and forgotten aspects of visceral pain. Eur. J. Pain 1999, 3, 77–92. [CrossRef] [PubMed] 63. Gerwin, R. Myofascial and visceral pain syndromes: Visceral-somatic pain representations. J. Musculoskelet. Pain 2002, 10, 65–75. [CrossRef] 64. Srbely, J.Z. New trends in the treatment and management of myofascial pain syndrome. Curr. Pain Headache Rep. 2010, 14, 346–352. [CrossRef][PubMed] 65. Campos Kraychete, D.; Tesseroli de Siqueira, J.; Batista García, J.; Kimiko Sakata, R.; Sousa, A.M.; de Andrade, D.C.; Mariotto Zakka, T.R.; Jacobsen Teixeira, M. Clinical evidence on visceral pain. Systematic review. Rev. Dor 2017, 18, 65–71. [CrossRef] 66. Rana, A.Q.; Yousuf, M.S.; Joian, S. A chronic case of adult-onset Sandifer syndrome. Neurol. Sci. 2013, 34, 405–406. [CrossRef][PubMed] 67. Han, D.G.; Lee, C.J. Headache associated with visceral disorders is “parasympathetic referred pain”. Med. Hypotheses 2009, 73, 561–563. [CrossRef][PubMed] 68. Williams, P.; Bannister, L.; Berry, M. Gray’s Anatomy, 38th ed.; Churchill Livingstone: New York, NY, USA, 1995. 69. Kostreva, D.R.; Pontus, S.P. Pericardial mechanoreceptors with phrenic afferents. Am. J. Physiol. 1993, 264, H1836–H1846. [CrossRef][PubMed] 70. Loukas, M.; Du Plessis, M.; Louis, R.G.; Tubbs, R.S.; Wartmann, C.T.; Apaydin, N. The subdiaphragmatic part of the phrenic nerve—Morphometry and connections to autonomic ganglia. Clin. Anat. 2016, 29, 120–128. [CrossRef][PubMed] Diagnostics 2019, 9, 186 21 of 23

71. Rusu, M.C. Considerations on the phrenic ganglia. Ann. Anat. 2006, 188, 85–92. [CrossRef][PubMed] 72. Netter, F. Atlas of Human Anatomy; Saunders/Elsevier: Philadelphia, PA, USA, 2011. 73. Hartmann, H.; Beckh, K. Nerve supply and nervous control of liver function. In Textbook of Clinical Hematology; McIntyre, N., Benhamou, J., Bircher, J., Eds.; Oxford University Press: Oxford, UK, 1992; p. 93. 74. Kostreva, D.R.; Pontus, S.P. Hepatic vein, hepatic parenchymal, and inferior vena caval mechanoreceptors with phrenic afferents. Am. J. Physiol. 1993, 265, 15–20. [CrossRef][PubMed] 75. Abdel-Misih, S.R.; Bloomston, M. Liver anatomy. Surg. Clin. N. Am. 2010, 90, 643–653. [CrossRef][PubMed] 76. Iwahashi, K.; Matsuda, R.; Tsunekawa, K. Afferent innervation of the gallbladder in the cat, studied by the horseradish peroxidase method. J. Auton. Nerv. Syst. 1991, 32, 145–151. [CrossRef] 77. Yi, S.Q.; Ohta, T.; Tsuchida, A.; Terayama, H.; Naito, M.; Li, J.; Wang, H.X.; Yi, N.; Tanaka, S.; Itoh, M. Surgical anatomy of innervation of the gallbladder in humans and Suncus murinus with special reference to morphological understanding of gallstone formation after gastrectomy. World J. Gastroenterol. 2007, 13, 2066–2071. [CrossRef][PubMed] 78. Yi, S.Q.; Ren, K.; Kinoshita, M.; Takano, M.; Itoh, M.; Ozaki, N. Innervation of extrahepatic biliary tract, with special reference to the direct bidirectional neural connections of the gall bladder, sphincter of Oddi and duodenum in Suncus murinus, in whole-mount immunohistochemical study. Anat. Histol. Embryol. 2016, 45, 184–188. [CrossRef][PubMed] 79. Yin, W.; Bogduk, N. The nature of neck pain in a private pain clinic in the United States. Pain. Med. 2008, 9, 196–203. [CrossRef][PubMed] 80. Eusebi, L.H.; Ratnakumaran, R.; Yuan, Y.; Solaymani-DOdaran, M.; Bazzoli, F.; Ford, A.C. Global prevalence of, and risk factors for, gastro-oesophageal reflux symptoms: A meta-analysis. Gut 2018, 67, 430–440. [CrossRef][PubMed] 81. Knab, L.M.; Boller, A.M.; Mahvi, D.M. Cholecystitis. Surg. Clin. N. Am. 2014, 94, 455–470. [CrossRef] [PubMed] 82. Bedogni, G.; Miglioli, L.; Masutti, F.; Tiribelli, C.; Marchesini, G.; Bellentani, S. Prevalence of and risk factors for nonalcoholic fatty liver disease: The Dionysos nutrition and liver study. Hepatology 2005, 42, 44–52. [CrossRef][PubMed] 83. Côté, P.; Cassidy, J.D.; Carroll, L. The factors associated with neck pain and its related disability in the Saskatchewan population. Spine 2000, 25, 1109–1117. [CrossRef][PubMed] 84. Ozaki, N.; Bielefeldt, K.; Sengupta, J.N.; Gebhart, G.F. Models of gastric hyperalgesia in the rat. Am. J. Physiol. Gastrointest. Liver Physiol. 2002, 283, G666–G676. [CrossRef][PubMed] 85. Palacios-Ceña, D.; Alonso-Blanco, C.; Hernández-Barrera, V.; Carrasco-Garrido, P.; Jimenez-Garcia, R.; Fernandez-de-las-Penas, C. Prevalence of neck and low back pain in community-dwelling adults in Spain: An updated population-based national study (2009/10–2011/12). Eur. Spine J. 2015, 24, 482–492. [CrossRef] [PubMed] 86. Kumagai, G.; Wada, K.; Tanaka, T.; Kudo, H.; Asari, T.; Chiba, D.; Ota, S.; Nakaji, S.; Ishibayi, I. Associations between neck symptoms and LDL cholesterol in a cross-sectional population-based study. J. Orthop. Sci. 2018, 23, 277–281. [CrossRef][PubMed] 87. Mäntyselkä, P.; Kautiainen, H.; Vanhala, M. Prevalence of neck pain in subjects with metabolic syndrome: A cross-sectional population-based study. BMC Musculoskelet. Disord. 2010, 11, 171. [CrossRef][PubMed] 88. Nair, J.; Streeter, K.A.; Turner, S.M.F.; Sunshine, M.D.; Bolser, D.C.; Fox, E.J.; Davenport, P.W.; Fuller, D.D. Anatomy and physiology of phrenic afferent neurons. J. Neurophysiol. 2017, 118, 2975–2990. [CrossRef] [PubMed] 89. Rouzade, M.L.; Fioramonti, J.; Bueno, L. A model for evaluation of gastric sensitivity in awake rats. Neurogastroenterol. Motil. 1998, 10, 157–163. [CrossRef][PubMed] 90. Zhang, H.H.; Hu, J.; Zhou, Y.L.; Hu, S.F.; Wang, Y.M.; Chen, W.; Xiao, Y.; Huang, L.Y.M.; Jiang, X.H.; Xu, G.Y. Promoted interaction of nuclear factor -kB with demethylated cystathione-β-synthetase gene contributes to gastric hypersensitivity in diabetic rats. J. Neurosci. 2013, 33, 9028–9038. [CrossRef][PubMed] 91. Giamberardino, M.A.; Affaitati, G.; Lerza, R.; fano, G.; Fulle, S.; Belia, S.; Lapenna, D.; Vecchiet, L. Evaluation of indices of skeletal muscle contraction in areas of referred hyperalgesia from an artificial ureteric stone in rats. Neurosci. Lett. 2003, 338, 213–216. [CrossRef] Diagnostics 2019, 9, 186 22 of 23

92. Sun, Y.; Tan, Y.; Song, G. Effects and mechanisms of gastric electrical stimulation on visceral pain in a rodent model of gastric hyperalgesia secondary to chemically induced mucosal ulceration. Neurogastroenterol. Motil. 2014, 26, 176–186. [CrossRef][PubMed] 93. Dong, Y.; Li, S.; Yin, J.; Chen, J.D.Z. Ameliorating effects of optimized gastric electrical stimulation and mechanisms involving nerve growth factor opioids in a rodent model of gastric hypersensitivity. Neurogastroenterol. Motil. 2019, 31, e13551. [CrossRef][PubMed] 94. Silva, A.C.O.; Biasotto-Gonzalez, D.A.; Oliveira, F.H.M.; Andrade, A.O.; Gomes, C.A.F.D.; Lanza, F.D.; Amorim, C.F.; Politti, F. Effect of osteopathic visceral manipulation on pain, cervical range of motion, and upper trapezius muscle activity in patients with chronic nonspecific neck pain and functional dispepsia: A randomized, doublé-blind, placebo-controlled pilot study. Evid. Based Complement. Alternat. Med. 2018, 2018, 4929271. [CrossRef][PubMed] 95. Eguaras, N.; Rodriguez-Lopez, E.S.; Lopez-Dicastillo, O.; Franco-Sierra, M.A.; Ricard, F.; Oliva-Pascual-Vaca, A. Effects of osteopathic visceral treatment in patients with gastroesophageal reflux: A randomized controlled trial. J. Clin. Med. 2019, 8, 1738. [CrossRef][PubMed] 96. Sterling, M.; de Zoete, R.M.J.; Coppieters, I.; Farrell, S.C. Best evidence rehabilitation for chronic pain Part 4: Neck pain. J. Clin. Med. 2019, 8, 1219. [CrossRef][PubMed] 97. Sergent, S.R.; Johnson, S.M.; Ashurst, J.; Johnston, G. Epstein-Barr virus associated atraumatic spleen laceration presenting with neck and shoulder pain. Am. J. Case Rep. 2015, 16, 774–777. [CrossRef][PubMed] 98. Carter, C.T. Acute thoracolumbar pain due to pholecystitis: A case study. Chiropr. Man. Ther. 2015, 23, 34. [CrossRef][PubMed] 99. Srirangam, S.J.; Pollard, A.J.; Adeyoju, A.A.; O’Reilly, P.H. Nephroptosis: Seriously misunderstood? Bju Int. 2009, 103, 296–300. [CrossRef][PubMed] 100. Hoenig, D.M.; Hemal, A.K.; Shalhav, A.L.; Clayman, R.V. Nephroptosis: A “disparaged” condition revisited. Urology 1999, 54, 590–596. [CrossRef] 101. Luong, C.; Starovoytov, A.; Heydari, M.; Sedlak, T.; Aymong, E.; Saw, J. Clinical presentation of patients with spontaneous coronary artery dissection. Catheter. Cardiovasc. Interv. 2017, 89, 1149–1154. [CrossRef] [PubMed] 102. Li, L.; Xu, C.; Deng, K.; Zhou, X.; Liu, Z.B.; Busse, J.W.; Ren, Y.; Zou, K.; Sun, X. The reporting of safety among drug systematic reviews was poor before the implementation of the PRISMA harms checklist. J. Clin. Epidemiol. 2019, 105, 125–135. [CrossRef][PubMed] 103. Prasad, V.; Gall, V.; Cifu, A. The frequency of medical reversal. Arch. Intern. Med. 2011, 171, 1675–1676. [CrossRef][PubMed] 104. Crutzen, R.; Viechtbauer, W.; Spigt, M.; Kotz, D. Differential attrition in health behaviour change trials: A systematic review and meta-analysis. Psychol. Health. 2015, 30, 122–134. [CrossRef][PubMed] 105. Mbuagbaw, L.; Thabane, M.; Vanniyasingam, T.; Debono, V.B.; Kosa, S.; Zhang, S.Y.; Ye, C.L.; Parpia, S.; Dennis, B.B.; Thabane, L. Improvement in the quality of abstracts in major clinical journals since CONSORT extension for abstracts: A systematic review. Contemp. Clin. Trials 2014, 38, 245–250. [CrossRef][PubMed] 106. Kho, M.E.; Duffet, M.; Willison, D.J.; Cook, D.J.; Brouwers, M.C. Written informed consent and selection bias in observational studies using medical records: Systematic review. BMJ 2009, 338, b866. [CrossRef] [PubMed] 107. Hrobjartsson, A.; Skou, A.S.; Emanuelsson, F.; Tendal, B.; Hilden, J.; Boutron, I.; Ravaud, P.; Brorson, S. Observer bias in randomised clinical trials with binary outcomes: Systematic review of trials with both blinded and non-blinded outcome assessors. BMJ 2012, 334, e1119. [CrossRef][PubMed] 108. Hrobjartsson, A.; Skou, A.S.; Emanuelsson, F.; Tendal, B.; Hilden, J.; Boutron, I.; Ravaud, P.; Brorson, S. Observer bias in randomized clinical trials with measurement scale outcomes: A systematic review of trials with both blinded and nonblinded outcome assessors. CMAJ 2013, 185, E201–E211. [CrossRef][PubMed] 109. Grimm, N.L.; Shea, K.G.; Leaver, R.W.; Aoki, S.K.; Carey, J.L. Efficacy and degree of bias in knee injury prevention studies: A systematic review of RCTs. Clin. Orthop. Relat. Res. 2013, 471, 308–316. [CrossRef] [PubMed] Diagnostics 2019, 9, 186 23 of 23

110. Thaler, K.; Kien, C.; Nussbaumer, B.; Van Noord, M.G.; Griebler, U.; Klerings, I.; Gartlehner, G. Inadequate use and regulation of intervention against publication bias decreases their effectiveness: A systematic review. J. Clin. Epidemiol. 2015, 68, 792–802. [CrossRef][PubMed] 111. Farzanfar, D.; Abumuamar, A.; Kim, J.; Sirotich, E.; Wang, Y.; Pullenayegum, E. Longitudinal studies that use data collected as part of usual care risk reporting biased results: A systematic review. BMC Med. Res. Methodol. 2017, 17, 133. [CrossRef][PubMed]

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). Evolution of mechanisms and behaviour important for pain royalsocietypublishing.org/journal/rstb Edgar T. Walters1 and Amanda C. de C. Williams2

1Department of Integrative Biology and Pharmacology, McGovern Medical School at UTHealth, 6431 Fannin Street, Houston, TX 77030, USA 2Research Department of Clinical, Educational and Health Psychology, University College London, Gower Street, Introduction London WC1E 6BT, UK ETW, 0000-0003-0718-102X; ACCW, 0000-0003-3761-8704 Cite this article: Walters ET, Williams ACdeC. 2019 Evolution of mechanisms and behaviour Our understanding of the biology of pain is limited by our ignorance about important for pain. Phil. Trans. R. Soc. B 374: its evolution. We know little about how states in other species showing 20190275. various degrees of apparent similarity to human pain states are related to http://dx.doi.org/10.1098/rstb.2019.0275 human pain, or how the mechanisms essential for pain-related states evolved. Nevertheless, insights into the evolution of mechanisms and behav- iour important for pain are beginning to emerge from wide-ranging Accepted: 8 August 2019 investigations of cellular mechanisms and behavioural responses linked to nociceptor activation, tissue injury, inflammation and the environmental One contribution of 19 to a Theo Murphy context of these responses in diverse species. In February 2019, an unprece- meeting issue ‘Evolution of mechanisms and dented meeting on the evolution of pain hosted by the Royal Society brought behaviour important for pain’. together scientists from disparate fields who investigate nociception and pain-related behaviour in , insects, leeches, gastropod and cepha- lopod molluscs, fish and mammals (primarily rodents and humans). Subject Areas: Here, we identify evolutionary themes that connect these research efforts, behaviour, evolution, neuroscience including adaptive and maladaptive features of pain-related behavioural and neuronal alterations—some of which are quite general, and some that may apply primarily to humans. We also highlight major questions, includ- Keywords: ing how pain should be defined, that need to be answered as we seek to nociception, evolutionary medicine, understand the evolution of pain. phylogenetic, subjective pain, chronic pain, This article is part of the Theo Murphy meeting issue ‘Evolution of mech- social modulation anisms and behaviour important for pain’.

Authors for correspondence: Edgar T. Walters 1. Introduction e-mail: [email protected] No human experience is more compelling than that of intense pain. It seems Amanda C. de C. Williams safe to assume that pain did not appear de novo in humans—that the functions e-mail: [email protected] and mechanisms of human pain are products of prior evolution. While the con- trol of human pain has long been a medical priority, and experimental animals have played a large role in preclinical efforts to develop new analgesics, remark- ably little is known about the evolution of pain. This reflects, in part, relatively little interest in evolution among most clinically oriented pain researchers, and little attention paid to pain by most evolutionary biologists. It also reflects the complexity of human pain and problems in applying the most widely accepted definition of human pain to other species. Nevertheless, a small but growing number of scientists are seeking a better understanding of the biology of human pain by considering the mechanisms and functions of pain from an evolutionary perspective. This theme issue resulted from the first international meeting on the evolution of pain, which brought together experts in various areas of neuro- science, psychology, medicine and evolutionary biology. The focus was on the evolution of pain behaviour and associated mechanisms as revealed by comparisons of pain-related phenomena across diverse invertebrate and vertebrate species, including humans. The single meeting and resulting theme issue could not accommodate all the relevant topics. For example, we have not included articles focused on the evolution of neuroanatomical structures important for pain or the evolution of endogenous opioids and their receptors.

© 2019 The Author(s) Published by the Royal Society. All rights reserved. However, we are confident the following articles will stimu- also [9]). Given the enormous scale and economic impact of 2 late much-needed discussion and research on the topics the human use of fish and crustaceans, it is not surprising royalsocietypublishing.org/journal/rstb explicitly covered as well as related topics on the evolution that such arguments have met with strong resistance from of pain that could not be addressed directly in this pioneering some quarters. theme issue. A second approach for defining animal pain has been favoured (often implicitly) by many investigators of pain- like phenomena in invertebrates, for which argument by analogy is necessarily much weaker than for mammals. 2. How should pain be defined by biologists? This is to define pain on the basis of functional rather than In trying to understand the broader biology of pain, a major subjective properties, as advocated by Elwood [3]. For problem immediately arises. The most influential definition example, researchers studying injury-induced sensitization of pain neglects all species but our own. The vast majority of of defensive behaviour in Drosophila assume the functions pain researchers around the globe have accepted the following of such sensitization are the same as for mammalian allo-

definition of pain from the International Association for the dynia and hyperalgesia (types of conscious evoked pain as B Soc. R. Trans. Phil. Study of Pain (IASP): ‘An unpleasant sensory and emotional defined by the IASP) and use these terms and sometimes experience associated with actual or potential tissue damage, the word ‘pain’ to refer to injury-related states in this or described in terms of such damage’ (https://www.iasp- insect—without explicitly arguing that flies might have pain.org/terminology?navItemNumber=576#Pain). This defi- conscious pain experience. This approach is illustrated in nition was developed by clinicians and clinically oriented the articles by Lopez-Bellido et al. [10] and by Khuong et al.

pain researchers. It has proven useful for defining human [11]. By contrast, investigators of molluscs such as Howard 374 pain, albeit with some limitations, such as the lack of reference et al. [12] and annelids such as Paulsen & Burrell [13] have 20190275 : to cognitive and social components of pain that are important generally preferred to use terms such as nociception and clinically [1]. The IASP is in the process of revising its definition nociceptive sensitization that do not imply conscious pain, of pain. The version posted for comments from the public rather than stating that these invertebrates exhibit pain, (https://www.iasp-pain.org/PublicationsNews/NewsDetail. allodynia or hyperalgesia [6]. Nonetheless, virtually all inves- aspx?ItemNumber=9218&navItemNumber=643) is similar in tigators of invertebrates, including all of those represented wording to the previous definition, but among other changes in this theme issue [3,10–16] assume that some of the mech- has substituted ‘aversive’ for ‘unpleasant’ and dropped the anisms and functions under investigation in invertebrates phrase ‘or described in terms of such damage,’ making it are also likely to be involved in mammalian pathways that more appropriate for animal pain. However, the revised defi- promote or suppress conscious pain. nition continues to define pain as an ‘emotional experience,’ Central states linked to nociceptive plasticity in many a phrase that in everyday English and for many researchers species meet many of the criteria of an early functional defi- (e.g., [2]) is closely linked to conscious, subjective feeling. nition by Manfred Zimmermann in which pain is ‘an aversive That this is the intended implication is emphasized by the sensory experience caused by actual or potential injury that first note appended to the revised IASP definition, which elicits protective motor and vegetative reactions, results in states that ’pain is always a subjective experience’. While the learned avoidance and may modify species-specific behav- revised IASP definition allows for the possibility that nonver- iour, including social behaviour’ [17, p. 1]. This definition is bal behaviours can express pain, the most (and perhaps only) consistent with behavioural observations described in this conclusive evidence available for subjective experience is issue for crustaceans [3] and fish [5], as well as insects verbal report. Thus, the IASP definitions are useful for identify- [10,11,14] and molluscs ([12] see also [6]). It ties pain to a ing human pain, but perhaps not for a vast number of species complex aversive state that (1) is induced by noxious stimu- for which inferences of subjective pain experience must remain lation during injury or imminent injury, (2) has a presumed weak at best. Further, the focus on subjective experience protective function involving overt defensive behaviour, eclipses the motivational functions of pain that are key to an internal physiological alterations, and an aversive motiva- evolutionary understanding [3–7]. tional state that can promote avoidance learning, and (3) Two definitional approaches have been used to address can be revealed by operant tests such as avoidance learning pain in non-verbal animals. One is to accept definitions that that demonstrate the aversive state. Assuming that the emphasize conscious experience, such as the IASP definitions, word ‘experience’ encompasses unconscious as well as con- and to argue on the basis of sufficient neuroanatomical, scious experience, all of these features are amenable to physiological/pharmacological and behavioural analogies experimental investigation in non-human animals. In a few that certain species are likely to experience pain similarly to non-mammalian species, operant tests have provided some humans (while admitting that compelling proof of the exist- evidence for the existence of pain-like aversive states, as ence of conscious pain in other species may be impossible). reviewed here by Elwood [3] and Sneddon [5] as well as by This approach has profound ethical and legal implications, Walters [6], but this evidence would be strengthened by the as persuasive evidence for conscious pain (and thus for poten- addition of conventional conditioned place avoidance and tial suffering) is the basis for including selected species for conditioned place preference tests, which increasingly are protection under laws. Argument by lists of used in rodent studies to reveal the affective–motivational apparent analogies has been used to conclude that mammals component of pain. One part of Zimmermann’s pain defi- and other large-brained animals are likely to experience con- nition that will not apply to many animals (vertebrate and scious pain (e.g. [8]). As explained in review articles in this invertebrate) is ‘including social behaviour’ because function- issue by Elwood [3] and by Sneddon [5], this approach has ally defined pain also is likely to occur in asocial animals. provided systematic arguments for the likelihood of pain While functional definitions of pain encourage comparative and the possibility of suffering in crustaceans and fish (see studies of pain-related processes across diverse taxa, providing valuable evolutionary insights, purely functional nociceptors to evoke defensive responses. The receptors 3 definitions of pain do not include what for many is the core involved are ion channels in the TRPM and TRPA families, royalsocietypublishing.org/journal/rstb feature of human pain—its distressing subjective component. and their phylogenetic analysis suggests the TRPM family In sum, at least two types of pain definition can serve as was present in ancestral Precambrian bilaterians. foundations for enquiries into the evolution of pain. One type A notable feature of nociception and pain compared with emphasizes the distressing subjective experience that defines other senses is the complex modulation that can occur at all human pain but is not directly accessible for study in other stages of sensory transmission. That this feature is not species. The other emphasizes the protective and motiva- unique to mammalian pain pathways is emphasized by two tional functions of aversive pain states induced by bodily articles in this theme issue. In a broad review of cannabinoid injury without reference to subjective experience. These signalling related to nociception in mammals and invert- views of pain should be complementary, but they have ebrates, Paulsen & Burrell [13] provide evidence of been difficult to integrate [6,8,9]. This discordance has con- molecular conservation across chordates and several other tributed to the contentious debate about which species feel phyla at the level of cannabinoid receptors (both GPCRs

pain (e.g. see the target article by Key [18] and accompanying and TRP channels) and the enzymes that synthesize and B Soc. R. Trans. Phil. commentary on whether fish feel pain). degrade endocannabinoids. Unexpected functional parallels have been found between rodents and a leech species in the opposing effects of endocannabinoids at different neural 3. Evolution of mechanisms important for loci; these can depress transmission at nociceptor synapses while potentiating (by disinhibition) transmission at synapses

nociception and pain of other primary sensory neurons. This suggests that a com- 374 Whether their emphasis is on the subjective experience of plex pattern of endocannabinoid modulation of nociceptive 20190275 : pain or pain-related functions, clinicians and researchers responses has been conserved for over half a billion years, agree that pain is closely (although not always) linked to and/or that this pattern is a product of convergent evolution. nociceptive activity in the nervous system that normally is Among the types of mammalian pain that respond to produced by bodily injury and inflammation. Thus, while endocannabinoids (and many other intracellular signals) is nociception is distinct from the experience of pain [19], it is inflammatory pain. While chemicals released by peripheral clear that activity in nociceptive pathways usually drives and central inflammatory and immune cells are known to pain, and that enhanced nociceptive activity increases pain be important for many types of pain in mammals [23–25], [20,21]. This makes comparisons of the mechanisms of noci- little is known about inflammatory and immune modulation ception and of nociceptive alterations across diverse species of nociception and pain-like behaviour in invertebrates. useful for enquiries into the evolution of pain. For example, it was not known whether any invertebrate Nociception begins when energy that produces or threat- exhibits generalized allodynia-like hypersensitivity to tactile ens to produce imminent injury (a noxious stimulus) is stimulation during a systemic immune challenge. Adamo & transduced into neural activity, leading to organized McMillan [14] report that larvae of the hawkmoth, Manduca responses that defend the tissue under threat and aid in its sexta, display a reduction in threshold for defensive responses repair. In addition to strong mechanical and thermal stimuli, to punctate mechanical stimuli 2 h after ingestion or injection caustic chemical stimuli such as acids can threaten tissue of heat-killed bacteria. This effect resembles the allodynia that integrity and homeostasis. In their review article, Pattison often accompanies sickness behaviour in mammals, and it et al. [22] show that acid nociception is a feature of many pri- may represent a widespread adaptation to maintain anti- mary nociceptors, and it has been documented in several predator vigilance when an animal is weakened by infection. phyla. Prominent proton-sensing molecules in diverse mam- The large larvae of Manduca offer many advantages for mals include acid-sensing ion channels (ASICs), transient neurophysiological investigations into mechanisms of pain- receptor potential vanilloid 1 (TRPV1), and specific two- like behavioural plasticity [26], which could be especially pore-domain K+ channels and G protein-coupled receptors, useful when combined with molecular predictions obtained which are often expressed in polymodal nociceptors that from genetic manipulations of Drosophila larvae (e.g. also are sensitive to other noxious stimuli and/or inflamma- [10,11,15]). tory signals. As reviewed by Sneddon [5], ASICs occur in fish Equally notable is the remarkable capacity of nociception and vigorous defensive behaviour is evoked in teleost fish by and pain to become chronically enhanced after injury, inflam- either injection or bath application of weak acids. Interest- mation, toxin exposure or other bodily stresses. Long-term ingly, while the nociceptor molecules that detect protons in sensitization (lasting a week or longer) of defensive responses phyla other than Chordata have yet to be identified, Lopez- in an invertebrate was first demonstrated in the large marine Bellido et al. [10] demonstrate in Drosophila that multiple snail Aplysia after noxious shock [27] or peripheral injury classes of primary sensory neurons, including nociceptors, [28]. Transection or strong artificial depolarization (mimicking are specifically required for defensive responses evoked by a major consequence of cellular injury) of peripheral axons of cutaneous application of strong acid. The availability of primary nociceptors caused persistent hyperexcitability of powerful genetic tools in Drosophila coupled with access to nociceptor somata [29] and axonal segments that had been known proton-sensing sensory neurons in this fly should transiently depolarized [30]. Mihail et al. [16] have now begin to answer the question of whether arthropods share demonstrated that the hyperexcitability of an axonal segment ancient acid-nociception mechanisms with , or persisting after transient depolarization depends upon a core they have independently evolved different mechanisms. As signalling pathway that has also been shown to drive hyperex- described by Himmel et al. [15], these genetic tools combined citability in mouse nociceptors. Previous evidence indicated with neurophysiological and behavioural tests in Drosophila that local protein synthesis dependent on mechanistic target revealed that another chemical stimulus, menthol, activates of rapamycin (mTOR) signalling was required for long-lasting hyperexcitability of axons in Aplysia nociceptors [30] and for epigenetic mechanisms important for persistent pain-like 4 various injury-related responses of primary afferent neurons alterations are across different phyla. royalsocietypublishing.org/journal/rstb in mammals [31]. Here, Mihail et al. [16] describe a further As reviewed by Mogil [39], there has been substantial requirement in Aplysia axons for signalling by mitogen- evolutionary divergence in some traits related to pain— activated protein kinase interacting kinase (MNK) to eukaryotic documented within closely related mammals, such as rats translation initiation factor (eIF) 4E, which is known to regulate and mice, and even between different strains of the same mTOR. Their findings indicate that phosphorylation of eIF4E species of mouse. These differences may have contributed to by MNK at a highly conserved molecular site is an ancient the limited translational success in human clinical trials of mechanism for maintaining sensory hyperexcitability. candidate analgesics developed largely on the basis of efficacy Chronic pain in humans is often caused by peripheral tests in rodents. On the other hand, many of the papers in nerve injury, which also is used experimentally in rodents to this theme issue add to accumulating evidence of strong con- investigate chronic pain mechanisms. Khuong et al. [32] servation of fundamental molecular mechanisms of neuronal recently reported that amputation of a leg in adult Drosophila plasticity that induce and maintain pain-like alterations

produces allodynia-like mechanical hypersensitivity that across the animal kingdom [6]. To optimize the chances of B Soc. R. Trans. Phil. lasts at least three weeks, far longer than the allodynia-like discovering drug targets important for pain that have been alterations previously described in larval Drosophila. Khuong conserved in both humans and rodents, Mogil [39] provides et al. [11] now show that this hypersensitivity requires the strong arguments for coordinated, essentially identical studies expression of an α2δ3 voltage-gated Ca2+ channel auxiliary on humans and rodents, with the results from each species subunit in primary nociceptors. In mammals, closely related being used to adjust the design of the experiments for both

subunits are targeted by gabapentinoid analgesics, which species. He provides three successful examples of this com- 374 are among the most effective treatments available for neuro- bined experimental strategy, involving a stress-induced 20190275 : pathic pain. Remarkably, both the amputation-induced analgesia gene, emotional contagion of pain (see §5) and hypersensitivity and an associated apparent delayed loss of context-dependent pain hypersensitivity. central inhibitory neurons (which should produce permanent hypersensitivity) were attenuated by early treatment with gabapentin or pregabalin. These findings encourage the use 4. Adaptive and maladaptive features of pain of Drosophila to help discover drugs that target fundamental mechanisms important for persistent pain. behaviour and its mechanisms Unlike other sensory systems, nociceptive systems can Understanding how evolution shaped the mechanisms and sometimes undergo very long-lasting, even permanent, behaviours important for pain requires that the evolutionarily enhancement of function following sufficiently intense acti- adaptive, neutral and maladaptive aspects of pain be ident- vation. This feature has long been assumed to contribute to ified. This is not straightforward. For example, the protective chronic pain conditions and to involve transcriptionally functions of acute nociception and pain have always seemed dependent neuronal alterations [33,34]. Until now, the long- obvious, an assumption supported by the high morbidity est-lasting sensitizing effects of noxious stimulation in any of humans with congenital insensitivity to pain [40] and by invertebrate had been reported for Aplysia, where nociceptor direct experimental support in flies [41]. However, the general sensitization after nerve injury persisted for over a month assumption of pain researchers and evolutionary psycholo- [35], a duration similar to that of behavioural alterations gists that chronic pain is necessarily maladaptive is being recently reported after leg amputation in Drosophila [11,32]. questioned. Several papers in this theme issue show that com- For the first time in any invertebrate, Howard et al. [12] bining an evolutionary perspective with sophisticated pain describe lifelong (13 weeks) sensitization of probable primary studies can modify entrenched assumptions about adaptive afferent neurons. This was produced by mechanical trauma and maladaptive pain behaviour. to the fins of newly hatched squid (using the small, relatively While pain is often associated with pathology in the body, short-lived Hawaiian bobtail squid). Interestingly, the pri- this certainly does not mean that pain itself is pathological. As mary afferent sensitization did not correlate directly with Nesse & Schulkin write [42], ‘Pain always seems like a problem, complex alterations observed in defensive behaviour or aver- but usually it is part of the solution’, and they emphasize that it sive learning, suggesting that potent effects of injury also is not pain but the capacity for pain that is subject to natural occurred within the central nervous system that could over- selection. They discuss Tinbergen’s four questions that need ride the permanent peripheral sensitization. Some of the to be answered to explain any behaviour, including pain: effects of early-life injury in this squid resemble lifelong What is its adaptive significance, its phylogeny,its mechanisms effects of early-life stress in mammals. Work reviewed by and its ontogeny? Each question demands different methods to Geranton [36] has shown that early-life stress in mammals answer, and the answers are largely independent. For example, produces a lifelong predisposition to chronic pain that despite the enormous progress made in identifying mechan- involves the increased expression of a regulatory protein isms related to pain, this knowledge provides limited insight within the stress axis, FKBP51. Importantly, this molecular into adaptive functions of pain-related behaviour, Moreover, response depends upon a persistent epigenetic change, mechanistic investigators tend to assume dysfunction rather reduced methylation of the FKBP5 gene, which encodes than adaptation when investigating pain-related phenomena FKB51. While epigenetic regulation has been implicated in in which protective benefits are not immediately obvious. allodynia-like alterations in Aplysia [37], different roles of Nesse & Schulkin [42] are concerned with explanations methylation in this example compared with known examples based in evolutionary medicine for why pain is often expressed in mammalian chronic pain models [36], as well as differ- inappropriately. One explanation, common to many defensive ences in DNA methylation in Drosophila compared with traits, is the ‘smoke detector principle’, in which evolution has mammals [38], raise questions about how conserved the traded off the relatively small cost of numerous false alarms against the very high cost of failing to respond to a threat of an adaptation to what is likely to be a highly dangerous 5 serious injury or infection. Another is that a system that sensi- environment, extending previous evidence and suggestions royalsocietypublishing.org/journal/rstb tizes readily has a cost in the inherent vulnerability of such for the adaptiveness of long-lasting nociceptive sensitization systems to runaway positive feedback that may lead to chronic induced in adults [34,43,47]. A similar suggestion for the adap- pain. A third explanation is the mismatch between the modern tiveness of amputation-induced allodynia mediated by loss of human lifestyle and the environment to which the pain system inhibitory interneurons in flies has been made by Khuong et al. was adapted (e.g. lower back and joint pain resulting from [32], although it will be important to show that large-scale, sedentary habits, chronic pelvic pain from more numerous permanent neuronal loss [11] does not also have major menstrual cycles in the modern world). In her review, Williams maladaptive consequences in this species. For extremely long- [7] finds unexpected evidence that chronic pain results from a lasting alterations, Geranton [36] even considers the possibility mismatch of the pain system with the modern environment. that trans-generational effects of stress mediated by changes in She notes that virtually all documented observations of chronic DNA methylation can increase the resilience of offspring to pain have come either from reports of humans or observations stress and pain.

of their dependent farm and companion animals, or from The adaptiveness of persistent pain is also consistent with B Soc. R. Trans. Phil. dependent laboratory animals. She suggests that chronic pain evidence from fossils, behavioural observations in the field expressed in the wild is probably maladaptive for most species and distinctive specializations of nociceptors. Dangers from because it interferes with necessary physical activity (whereas predators and aggressive conspecifics have probably been a timely return to normal activity appears to suppress persis- major selection pressures for persistent alterations in nociceptive tent pain), and that it may occur only under conditions systems since the Precambrian era. Presenting a systematic

where inactive individuals experiencing chronic pain can be meta-analysis of fossil evidence from the Mesozoic Era, Hearn 374 ministered to by human caretakers. & Williams [48] conclude that dinosaurs could survive long 20190275 : Finlay [4] also seeks evolutionary explanations for pain, par- after severe injuries, during which time guarding behaviour ticularly for apparently inappropriate degrees of pain appears to have been present, possibly accompanied by persist- experienced by humans. By analogy to other perceptual sys- ent pain. Walters [45] argues on the basis of such evidence and tems (especially vision), she suggests that pain evolved to from field and laboratory observations of living species that guide adaptive behaviour, and that this involves complex pro- some forms of chronic pain and persistent nociceptor hyperac- cessing in the brain to assess contingent relationships between tivity are adaptations that promote survival after injuries noxious stimuli and behavioural actions. This predictive proces- severe enough to cause permanent disfigurement and impair- sing enables pain to be minimized during voluntary activities ment of motor function. Such injuries greatly increase the risk such as extreme exercise, cosmetic procedures and self-harm of, and vulnerability to, subsequent attack. Walters also shows in humans. Conversely, Finlay suggests that evolution has that the ‘ectopic activity’ of primary sensory neurons caused amplified the experience of pain in women beginning labour by neural injury in mammals, which always was assumed to (beyond the degree of pain expected from the amount of be a purely pathological effect, instead exhibits properties tissue damage early in labour), because the resulting pain be- expected of an evolutionary adaptation to promote ongoing haviour provides an ‘honest signal’ that effectively solicits pain and hypervigilance (anxiety) under the conditions of heigh- help and protection from partners and relatives during child- tened vulnerability that follow severe injury. In particular, the birth, thereby enhancing the survival of mother and child. complex, functionally coherent set of mechanisms that enable Whetherchronic pain and its mechanisms can be adaptive is persistent ongoing activity in nociceptors after severe injury addressed by several papers in the theme issue. As mentioned, shows non-random organization and coordination that satisfy Williams [7] argues that chronic pain, or at least the behaviour the ‘design criterion’ for an evolutionary adaptation [49,50]. that is taken to indicate ongoing pain, can be maladaptive and may be much less common in the wild than has been suggested by human clinical, preclinical and veterinary experi- 5. Evolutionary aspects of pain-related social ence. On the other hand, enhanced survival during predatory encounters resulting from the induction of potentially long- behaviour lasting sensitization and hypervigilance induced by injury or While true social behaviour is found in a minority of all animal electric shock has been shown in squid [43] and amphipod crus- species [51], it appears important for nociceptive function and taceans [44] (see also [3,12]). Long-lasting, pain-related pain in humans and in the rodents that are employed for most hypervigilance is also likely to influence estimate of risk and preclinical studies of pain [1]. Furthermore, the environmental thus behavioural decisions in mammals [7,45]. The opposite context of any animal contains challenges [52]: predators seen effect, discounting of potentially painful experience in or unseen, behaving in a threatening or non-threatening way; humans pursuing a valued goal, is discussed by Finlay [4] in a potential mate or competitor for a mate, to be impressed or human behaviours rarely subject to scientific research, includ- deterred by appearing healthy and strong. Several papers ing self-harm and cosmetic procedures. She recommends in this issue address questions about pain-related social further exploration of habituation to pain [46], and of the var- behaviour that involve evolutionary considerations. Hearn & iance in human pain attributable to expectation and to agency. Williams [48] discuss evidence from fossils (including fossilized Nesse & Schulkin [42] and Walters [45] also emphasize the track patterns) and from the behaviour of contemporary archo- importance of mechanisms that turn off pain when not needed saurs ( and crocodiles) that is consistent with parental and so that chronic pain does not occur. On the other hand, several family care by dinosaurs. This may have extended to help with articles consider evidence that chronic pain-like alterations can injured family members expressing pain. Mogil [39] describes a be adaptive under appropriate conditions. Howard et al. [12] rudimentary form of empathy in mice, emotional contagion, in suggest that permanent sensitization of primary afferent neur- which a mouse receiving painful stimulation displays synchro- ons in bobtail squid after traumatic early-life injury represents nized levels of pain behaviour with familiar mice tested alongside it, but not with strangers. He describes the results of experience, human pain is most familiar and most important, 6 coordinated experiments on mice and humans that have so the most relevant comparisons are between other species royalsocietypublishing.org/journal/rstb revealed for the first time in either species that the lack of and our own. Such comparisons bring up a second obstacle, emotional contagion of pain between strangers is caused by which is the special status of the human species. This reflects stress, as shown by analogous procedures that reduce stress in the enormous complexity of pain-linked behaviour (including each species and enable pain contagion between strangers. uniquely complex social behaviour in humans) and of pain’s Whether emotional contagion has adaptive functions, and intricate substrates involving large parts of the human nervous whether these functions could be similar between mice and system, plus the ethical impermissibility of controlled studies humans is a very interesting question. As mentioned, Finlay on severe and/or chronic pain induced experimentally in [4,53], building upon the capability of expressed pain to elicit healthy human volunteers. Despite impressive recent progress helping behaviour in humans [54], has proposed that evolution with human imaging and neural stimulation methods, identify- has enhanced the pain experienced by women during the initial ing the neuronal populations critical for even transient pain stages of labour in order to more effectively obtain help and experience in the human nervous system is incomplete at

protection from others during childbirth. best. Compounding these obstacles is the fact that pain is an B Soc. R. Trans. Phil. Social context is likely to determine whether pain is inferred internal state, rather than an obvious external behav- expressed overtly and by which behaviours. For example, iour, and thus is extremely difficult in any species to assess some behaviours are detectable at long range, including by accurately using behavioural or neural activity measures. predators and competitors, while others require proximity, Consequently,there is considerable uncertainty about which be- which is more likely for kin and allies. This prediction has havioural features, neural circuits, cell types and molecules to

important implications for questions about the adaptive sig- compare across taxa when defining evolutionary relationships 374 nificance of pain expression [54], and it has been supported (homologous and analogous traits). In addition, behavioural 20190275 : by observations in rodents (e.g. [39]), in companion and farm states leave very little fossil evidence (although some has been animals [7] and in humans [55]. However, conclusive exper- found) for making inferences about when, and in which types imental evidence for pain expression altering the behaviour of animal, pain states appeared during evolution. of conspecifics is surprisingly sparse for any species, including In spite of these obstacles, substantive insights into the evol- humans [39]. This is illustrated by Kappesser’s review [55] of ution of pain are emerging, as illustrated by the following findings on social threat and facial expression of pain in articles. Regarding the evolution of mechanisms important humans, which reveals inconsistent findings that may be for pain, there is no doubt that human pain is usually initia- attributable to differing theories and models (most of which ted and often maintained by electrical activity in primary do not include evolutionary premises), different method- nociceptors. In contrast to other cell types involved in nocicep- ologies, limits on the degree of pain and social threat tive and pain-related processing, the basic functions and permissible in ethically acceptable experimental paradigms, anatomical locations of nociceptive primary afferents are and experimentally uncontrolled complexities in social known in humans, other mammals, and in some representa- relationships that may influence the expression of pain and tives from other major taxa, including some of the fish, related anxiety. How the social context influences pain behav- annelids, arthropods and molluscs discussed in this issue, as iour raises questions about what is socially threatening in well as nematodes. This has enabled direct comparisons of cel- humans, which have potentially important implications—for lular and molecular traits important for nociception and example, for more effectively treating patients for whom non- nociceptive plasticity in nociceptors across invertebrate and verbal behaviour is the only route of communication. Most of mammalian species. These comparisons have revealed similar the studies reviewed by Kappesser [55] are experimental roles of conserved proteins, including TRPA, TRPV, TRPM studies using healthy subjects and brief evoked pain. Manipu- and ASIC ion channels, as well as α2δ auxiliary subunits of vol- lation of threat value, and of social relationship between tage-gated Ca2+ channels; many protein kinases, including participants, as is possible in some common clinical procedures MNK and mTOR for regulating local protein synthesis; and that are painful or that exacerbate significant pain, may well be transcription factor families such as PRDM and CREB (see more informative for revealing the nature and functions of also [6]). Divergent mechanisms are certain to be involved as differences in the facial expression of pain in different social well, including possible differences across phyla in the roles contexts. of epigenetic mechanisms, and various differences even within closely related mammalian taxa in pain-related effects (e.g. on gene expression). At the functional level, some primary afferent neurons in different phyla have been found to exhibit 6. Conclusion similar hyperactivity long after strong noxious stimulation, A biological understanding of pain in humans and other contributing to hypersensitive states resembling allodynia and species requires knowledge of pain’s evolutionary context. hyperalgesia in mammals. The degree to which conserved This requires extensive comparisons of the behavioural func- and convergent mechanisms in nociceptors contribute to tions, cellular mechanisms, and sequences of involved genes these functional similarities in pain-like states across diverse and/or gene products important for pain across diverse taxa is a fascinating question. living species representing the major phyla. Two major Given our limited knowledge about the evolution of pain, obstacles stand in the way of such enquiries into the evolution particularly important questions concern the evolutionary of pain. One is the difficulty in defining pain in a way that adaptiveness, or lack thereof, of various forms of pain. allows pain and its possible evolutionary antecedents to be Whereas acute pain is universally agreed to be adaptive, recognized and compared across species, a task that is only recently have possible benefits been recognized for especially challenging for attempted comparisons of the some forms of chronic pain or very severe pains that seem conscious component of pain. Because of our personal out of proportion to the existing state of tissue damage. Some of these pains are likely to represent mismatches to be strongly modulated by social context. In humans, 7 between evolved pain systems and the modern environment, evolution may have amplified the experience of pain under royalsocietypublishing.org/journal/rstb or trade-offs with other adaptations, such as effective conditions, such as labour, in which an ‘honest signal’ can immune function. However, plausible arguments also suggest solicit aid that enhances reproductive success. While adaptive that evolution selected mechanisms in diverse species that arguments are speculative, they and their alternatives have can persistently maintain pain-related hypervigilance as adap- scientific and clinical implications that can be tested rigorously tations to especially dangerous environments and to enhanced by behavioural and mechanistic pain research that is informed vulnerability persisting long after disfiguring injury. Identifi- by evolutionary principles. cation of evolutionarily adaptive and maladaptive features of pain behaviour, in relation to environmental (including Data accessibility. This article does not contain any additional data. social) variables and the state of the organism, may require Authors’ contributions. This Introduction was written jointly by the two investigatory frameworks and experimental paradigms that authors. are novel for the pain research field. For some species, evol- Competing interests. We declare we have no competing interests.

ution has selected processes that allow the expression of pain Funding. We received no funding for this study. B Soc. R. Trans. Phil.

References

1. Williams AC, Craig KD. 2016 Updating the definition hyperexcitability, cognitive deficit and altered 23. Cook AD, Christensen AD, Tewari D, McMahon SB, of pain. Pain 157, 2420–2423. (doi:10.1097/j.pain. defensive behaviour in the squid Euprymna Hamilton JA. 2018 Immune cytokines and their 374

0000000000000613) scolopes. Phil. Trans. R. Soc. B 374, 20190281. receptors in inflammatory pain. Trends Immunol. 20190275 : 2. Izard CE. 2009 Emotion theory and research: (doi:10.1098/rstb.2019.0281) 39, 240–255. (doi:10.1016/j.it.2017.12.003) highlights, unanswered questions, and emerging 13. Paulsen RT, Burrell BD. 2019 Comparative studies of 24. Grace PM, Hutchinson MR, Maier SF, Watkins LR. issues. Annu. Rev. Psychol. 60,1–25. (doi:10.1146/ endocannabinoid modulation of pain. Phil. 2014 Pathological pain and the neuroimmune annurev.psych.60.110707.163539) Trans. R. Soc. B 374, 20190279. (doi:10.1098/rstb. interface. Nat. Rev. Immunol. 14, 217–231. (doi:10. 3. Elwood RW. 2019 Discrimination between 2019.0279) 1038/nri3621) nociceptive reflexes and more complex responses 14. Adamo SA, McMillan LE. 2019 Listening to your gut: 25. Ji RR, Chamessian A, Zhang YQ. 2016 Pain consistent with pain in crustaceans. Phil. Trans. R. immune challenge to the gut sensitizes body wall regulation by non-neuronal cells and inflammation. Soc. B 374, 20190368. (doi:10.1098/rstb.2019.0368) nociception in the caterpillar . Phil. Science 354, 572–577. (doi:10.1126/science. 4. Finlay BL. 2019 The neuroscience of vision and Trans. R. Soc. B 374, 20190278. (doi:10.1098/rstb. aaf8924) pain: evolution of two disciplines. Phil. Trans. R. Soc. 2019.0278) 26. Tabuena DR, Solis A, Geraldi K, Moffatt CA, Fuse M. B 374, 20190292. (doi:10.1098/rstb.2019.0292) 15. Himmel NJ, Letcher JM, Sakurai A, Gray TR, Benson 2017 Central neural alterations predominate in 5. Sneddon LU. 2019 Evolution of nociception MN, Cox DN. 2019 Drosophila menthol sensitivity an insect model of nociceptive sensitization. and pain: evidence from fish models. Phil. and the Precambrian origins of TRP-dependent J. Comp. Neurol. 525, 1176–1191. (doi:10.1002/ Trans. R. Soc. B 374, 20190290. (doi:10.1098/rstb. chemosensation. Phil. Trans. R. Soc. B 374, cne.24124) 2019.0290) 20190369. (doi:10.1098/rstb.2019.0369) 27. Pinsker HM, Hening WA, Carew TJ, Kandel ER. 1973 6. Walters ET. 2018 Nociceptive biology of molluscs 16. Mihail SM, Wangzhou A, Kunjilwar KK, Moy JK, Long-term sensitization of a defensive withdrawal and arthropods: evolutionary clues about functions Dussor G, Walters ET, Price TJ. 2019 MNK-eIF4E reflex in Aplysia. Science 182, 1039–1042. (doi:10. and mechanisms potentially related to pain. Front. signalling is a highly conserved mechanism for 1126/science.182.4116.1039) Physiol. 9, 1049. (doi:10.3389/fphys.2018.01049) sensory neuron axonal plasticity: evidence from 28. Walters ET. 1987 Site-specific sensitization of 7. Williams ACdeC. 2019 Persistence of pain in humans Aplysia californica. Phil. Trans. R. Soc. B 374, defensive reflexes in Aplysia: a simple model of and other mammals. Phil. Trans. R. Soc. B 374, 20190289. (doi:10.1098/rstb.2019.0289) long-term hyperalgesia. J. Neurosci. 7, 400–407. 20190276. (doi:10.1098/rstb.2019.0276) 17. Zimmermann M. 1986 [Physiological mechanisms of (doi:10.1523/JNEUROSCI.07-02-00400.1987) 8. Bateson P. 1991 Assessment of . pain and its treatment]. In Schmerztherapie 29. Walters ET, Alizadeh H, Castro GA. 1991 Similar Anim. Behav. 42, 827–839. (doi:10.1016/S0003- (Klinische Anästhesiologie und Intensivtherapie, vol. neuronal alterations induced by axonal injury and 3472(05)80127-7) 32) (ed. H Bergman), pp. 1–19 (in German). learning in Aplysia. Science 253, 797–799. (doi:10. 9. Sneddon LU, Elwood RW, Adamo SA, Leach MC. (doi:10.1007/978-3-642-71333-0_1) 1126/science.1652154) 2014 Defining and assessing animal pain. Anim. 18. Key B. 2016 Why fish do not feel pain. Anim. Sent. 30. Weragoda RM, Ferrer E, Walters ET. 2004 Behav. 97, 201–212. (doi:10.1016/j.anbehav.2014. 2016, no. 3. Memory-like alterations in Aplysia axons after 09.007) 19. Sherrington CS. 1906 The integrative action of the nerve injury or localized depolarization. J. Neurosci. 10. Lopez-Bellido R, Himmel NJ, Gutstein HB, Cox DN, nervous system. New York, NY: C. Scribner’s Sons. 24, 10 393–10 401. (doi:10.1523/JNEUROSCI.2329- Galko MJ. 2019 An assay for chemical nociception in 20. Basbaum AI, Bautista DM, Scherrer G, Julius D. 04.2004) Drosophila larvae. Phil. Trans. R. Soc. B 374, 2009 Cellular and molecular mechanisms 31. Price TJ, Géranton SM. 2009 Translating nociceptor 20190282. (doi:10.1098/rstb.2019.0282) of pain. Cell 139, 267–284. (doi:10.1016/j.cell. sensitivity: the role of axonal protein synthesis in 11. Khuong TM, Hamoudi Z, Manion J, Loo L, 2009.09.028) nociceptor physiology. Eur. J. Neurosci. 29, Muralidharan A, Neely GG. 2019 Peripheral 21. Gold MS, Gebhart GF. 2010 Nociceptor sensitization 2253–2263. (doi:10.1111/j.1460-9568.2009. straightjacket (α2δ Ca2+ channel subunit) in pain pathogenesis. Nat. Med. 16, 1248–1257. 06786.x) expression is required for neuropathic sensitization (doi:10.1038/nm.2235) 32. Khuong TM, Wang QP, Manion J, Oyston LJ, Lau MT, in Drosophila. Phil. Trans. R. Soc. B 374, 20190287. 22. Pattison LA, Callejo G, St John Smith E. 2019 Towler H, Lin YQ, Neely GG. 2019 Nerve injury (doi:10.1098/rstb.2019.0287) Evolution of acid nociception: ion channels and drives a heightened state of vigilance and 12. Howard RB, Lopes LN, Lardie CR, Perez PP, Crook RJ. receptors for detecting acid. Phil. Trans. R. Soc. B neuropathic sensitization in Drosophila. Sci. Adv. 5, 2019 Early-life injury produces lifelong neural 374, 20190291. (doi:10.1098/rstb.2019.0291) eaaw4099. (doi:10.1126/sciadv.aaw4099) 33. Ji RR, Woolf CJ. 2001 Neuronal plasticity and signal 39. Mogil JS. 2019 The translatability of pain across 47. Crook RJ, Hanlon RT, Walters ET. 2013 Squid have 8 transduction in nociceptive neurons: implications for species. Phil. Trans. R. Soc. B 374, 20190286. nociceptors that display widespread long-term royalsocietypublishing.org/journal/rstb the initiation and maintenance of pathological pain. (doi:10.1098/rstb.2019.0286) sensitization and spontaneous activity after bodily Neurobiol. Dis. 8,1–10. (doi:10.1006/nbdi.2000. 40. Nagasako EM, Oaklander AL, Dworkin RH. 2003 injury. J. Neurosci. 33, 10 021–10 026. (doi:10. 0360) Congenital insensitivity to pain: an update. 1523/JNEUROSCI.0646-13.2013) 34. Walters ET. 1994 Injury-related behavior and Pain 101, 213–219. (doi:10.1016/S0304- 48. Hearn L, Williams ACdC. 2019 Pain in dinosaurs: neuronal plasticity: an evolutionary perspective on 3959(02)00482-7) what is the evidence? Phil. Trans. R. Soc. B 374, sensitization, hyperalgesia, and analgesia. Int. Rev. 41. Robertson JL, Tsubouchi A, Tracey WD. 2013 Larval 20190370. (doi:10.1098/rstb.2019.0370) Neurobiol. 36, 325–427. (doi:10.1016/S0074- defense against attack from parasitoid wasps 49. Andrews PW, Gangestad SW, Matthews D. 7742(08)60307-4) requires nociceptive neurons. PLoS ONE 8, e78704. 2002 Adaptationism—how to carry out an 35. Gasull X, Liao X, Dulin MF, Phelps C, Walters ET. (doi:10.1371/journal.pone.0078704) exaptationist program. Behav. Brain Sci. 25, 2005 Evidence that long-term hyperexcitability of 42. Nesse RM, Schulkin J. 2019 An evolutionary medicine 489–504. (doi:10.1017/S0140525X02000092) the sensory neuron soma induced by nerve injury in perspective on pain and its disorders. Phil. Trans. R. Soc. 50. Stearns SC, Medzhitov R. 2016 Evolutionary – Aplysia is adaptive. J. Neurophysiol. 94, 2218 2230. B 374, 20190288. (doi:10.1098/rstb.2019.0288) medicine. Sunderland, MA: Sinauer Associates. B Soc. R. Trans. Phil. (doi:10.1152/jn.00169.2005) 43. Crook RJ, Dickson K, Hanlon RT, Walters ET. 2014 51. Wilson EO. 2012 The social conquest of Earth. 36. Géranton SM. 2019 Does epigenetic ‘memory’ of Nociceptive sensitization reduces predation risk. Curr. New York, NY: W. W. Norton. early-life stress predispose to chronic pain in later Biol. 24, 1121–1125. (doi:10.1016/j.cub.2014.03.043) 52. Mobbs D, Kim JJ. 2015 Neuroethological life? A potential role for the stress regulator FKBP5. 44. Perrot-Minnot MJ, Banchetry L, Cézilly F. 2017 studies of fear, anxiety, and risky decision- Phil. Trans. R. Soc. B 374, 20190283. (doi:10.1098/ Anxiety-like behaviour increases safety from fish making in rodents and humans. Curr. Opin. Behav. –

rstb.2019.0283) predation in an amphipod crustacea. R. Soc. open Sci. 5,8 15. (doi:10.1016/j.cobeha.2015.06.005) 374 37. Bédécarrats A, Chen S, Pearce K, Cai D, Glanzman sci. 4, 171558. (doi:10.1098/rsos.171558) 53. Finlay BL, Syal S. 2014 The pain of altruism. 20190275 : DL. 2018 RNA from trained Aplysia can induce an 45. Walters ET. 2019 Adaptive mechanisms driving Trends Cogn. Sci. 18, 615–617. (doi:10.1016/j.tics. epigenetic engram for long-term sensitization in maladaptive pain: how chronic ongoing activity in 2014.08.002) untrained Aplysia. eNeuro 5, ENEURO.0038-18.2018. primary nociceptors can enhance evolutionary 54. Williams AC. 2002 Facial expression of pain: an (doi:10.1523/ENEURO.0038-18.2018) fitness after severe injury. Phil. Trans. R. Soc. B 374, evolutionary account. Behav. Brain Sci. 25, 38. Deobagkar D. 2018 Epigenetics with special 20190277. (doi:10.1098/rstb.2019.0277) 439–455. reference to the human X inactivation 46. De Paepe AL, Williams ACdC, Crombez G. 2019 55. Kappesser J. 2019 The facial expression of pain in and the enigma of Drosophila DNA methylation. Habituation to pain: a motivational-ethological humans considered from a social perspective. Phil. J. Genet. 97, 371–378. (doi:10.1007/s12041-018- perspective. Pain 160, 1693–1697. (doi:10.1097/j. Trans. R. Soc. B 374, 20190284. (doi:10.1098/rstb. 0937-5) pain.0000000000001533) 2019.0284)