Peer Reviewed CE Article #1 and : When Cells Almost Die

Amy N. Breton, CVT, VTS (ECC) Veterinary Emergency and Specialty Center of New England Waltham, Massachusetts

schemia is defined as inadequate blood supply to a part ing energy for metabolism within cells.3 One of the fastest of the body, usually caused by partial or total block- ways that ATP is produced is by oxidative phosphorylation, age of an artery. Reperfusion injury occurs when tissue which, as the name implies, requires oxygen.3 Despite the I 3 perfusion and oxygenation are restored to an area that has importance of ATP, cells do not stockpile ATP. They only been affected by an ischemic event. make what they need for a particular time. When ischemia Ischemia/reperfusion (I/R) injury is a complex cascade occurs, oxygenation of cells ceases, resulting in anaerobic of events resulting in devastating effects on the body, ATP production, which is less efficient.3 sometimes including death. Despite more than 70 years of When oxygen becomes unavailable, cells begin anaero- research, I/R injury is not fully understood. Events such as bic glycolysis. This process can be a lifesaving way for cells gastric dilatation–volvulus (GDV), mesenteric torsion, or to obtain energy; however, it is extremely wasteful. During strangulation of a limb can lead to I/R injury. It is important anaerobic glycolysis, pyruvic acid and hydrogen atoms for all veterinary personnel to understand I/R injury so that combine with nicotinamide adenine dinucleotide (NAD) treatment and prevention can begin as early as possible. to form NADH and H+.3 If the buildup of NADH and H+ within cells becomes too great, the anaerobic process stops, The Ischemic Cascade terminating energy production.3 However, NADH and H+ The chain of events involved in I/R injury can be broken combine to form , which diffuses from cells rap- down into the ischemic cascade (BOX 1) and reperfusion idly so that the process can continue.3 Although this is not injury (BOX 2). An ischemic episode involves a series of ideal, the body can safely continue anaerobic glycolysis for events called the ischemic cascade. Within 5 minutes of the several minutes. If the process continues for too long, as development of ischemia, the electrolyte balance within in ischemia, lactic acid can build up (lactic acidosis), indi- cells becomes disturbed.1 The ischemic cascade usually cating worsening illness. As a consequence of lactic acido- continues for 2 or 3 hours but can last for days, even after sis, pH decreases, injuring and inactivating mitochondria. perfusion is restored to the affected area.2 Although the These mitochondria then break down, releasing toxins that term cascade suggests that events always follow a sequential cause . Some researchers think that lactic acid pattern, in the ischemic cascade, events can occur linearly may also interfere with the recovery of aerobic ATP pro- or simultaneously.1 duction after ischemia.3 A lactate level should be obtained To fully understand the ischemic cascade, it is important for all ischemic patients. Values <2 mmol/L are normal. In to understand the role of (ATP) in patients with GDV, a level >6 mmol/L is associated with the body. ATP is a multifunctional nucleotide (a structural increased gastric . In 1999, a study of 102 dogs component of DNA and RNA) that is considered to be with GDV found that 58% of dogs with a blood lactate level the most important nucleotide responsible for transport- >6 mmol/L survived, whereas 99% of dogs with a level <6

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BOX 1 by neutrophils.7 Even after reperfusion, the redistribu- tion of blood to affected areas may not produce enough The Ischemic Cascade1,5–9,12 force to clear clogs.7 The full pathway of NFκB is still not • Lack of oxygen causes failure of the normal aerobic understood.1 process for making ATP. • Cells switch to anaerobic respiration, creating lactic Reperfusion Injury acid. It would seem that simply reintroducing oxygen into an • Potassium leaks from cells, while sodium and calcium ischemic area would be beneficial. In patients with GDV, enter cells. oxygen is restored when the stomach is decompressed or • Calcium converts XDH to XO. untwisted, allowing oxygen and blood to flow back into the stomach wall. However, the reintroduction of oxygen • XO builds up. into affected areas initiates a complex chain of events. The • NFκB is activated, activating inflammatory mediators harsh effects of ischemia alone do not cause nearly as much and platelet-activating factor. damage as reperfusion does.1 The longer the duration of • Damaging free radicals are formed. the ischemic event, the greater the insult from reperfusion injury.1 An ischemic event may not be long enough to pro- • Mitochondria break down and start apoptosis. duce a reperfusion injury. • Neutrophils start to overwhelm the affected areas. One of the first events in reperfusion is that oxygen binds • Cells die. with XO that has built up during ischemia. The combina- tion of XO, oxygen, and hypoxanthine forms superoxide - 5 (O2 ), a . Superoxide is not that damaging but can mmol/L survived.4 Death is an ischemic event because it inactivate iron–sulfur–containing , liberating free causes oxygen deprivation at the tissue level. Lactic acid iron and generating highly reactive hydroxyl (-OH) radi- buildup is the major cause of rigor mortis.3 cals. Hydroxyl is considered to be a When ATP fails to form, cells become depolarized, (ROS). allowing calcium and sodium (normal extracellular electro- An ROS is an oxygen-containing molecule that is very lytes) to enter cells.5 Potassium, which is normally found in chemically reactive. ROS molecules react quickly with cells, leaks rapidly into the extracellular space.5 Excessive other molecules. If present in high levels, they can damage intracellular calcium overexcites cells, creating free radi- cellular macromolecules such as DNA and RNA or cause cals and many enzymes, such as xanthine dehydrogenase endothelial injury, microvascular dysfunction, and apopto- (XDH) and xanthine oxidase (XO). The extent of ischemic sis.8 ROS molecules can form within 10 to 30 seconds after damage is related to the amount of calcium that enters cells the onset of reperfusion.5 and the duration for which the intracellular calcium level During ischemia, neutrophils leak into the endothelium remains elevated.6 The longer calcium stays in cells, the because of the activation of NFκB and XO. The inflam- more harmful compounds it can create. matory response to reperfusion accelerates the influx of One of the most important events involving calcium is neutrophils to the affected area.9 Neutrophil activation the conversion of XDH to XO.4 XO requires oxygen for alone can lead to even more ROS formation.5 The inflam- activation. During ischemia, oxygen is not present, so XO matory cascade accelerates during reperfusion. In short, accumulates without getting used. Later, during reperfu- neutrophils and macrophages attack reperfused tissues. sion, XO can damage cells. Inflammatory cytokines are released as neutrophils are Another important event during ischemia is the activa- activated.1 When the body becomes overwhelmed with tion of nuclear factor–κB (NFκB), leading to the produc- inflammatory cells, cytokines can be overproduced, result- tion of inflammatory mediators.1 NFκB becomes activated ing in massive cytokine influx (hypercytokinemia) into the during stress.3 NFκB activates inflammatory cytokines and affected tissue.1 The exact mechanism behind this phenom- their receptors as well as platelet-activating factor.1 This enon is not fully understood.1 allows neutrophils to pass through the vascular endothe- lium. Activated neutrophils are generally rigid because of Complications hypoxia and acidosis, which accompany ischemia. Because The ischemic insult sets up the body for a damaging chain of the alteration of the membrane and the high number of events that is initiated by reperfusion. Although some of neutrophils, capillaries may become plugged or clogged researchers debate the exact relationship between I/R injury

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and these events, the end result can be the death of the BOX 2 patient. Reperfusion Injury1,5–9,12 Disseminated Intravascular Coagulation • Oxygen is reintroduced into the affected area. In patients with I/R injury, damaged endothelial cells release • XO forms superoxide radicals. substances that activate the clotting cascade. Eventually, the • Superoxide liberates free iron, causing creation of balance between clotting and bleeding becomes disturbed, hydroxyl ROS. resulting in disseminated intravascular coagulation (DIC). DIC is a pathologic process in which blood coagulates • ROS damage macromolecules (i.e., DNA, RNA) and cause endothelial injury, microvascular dysfunction, throughout the body. The result is depletion of platelets and apoptosis. and coagulation factors, creating a risk for increased bleed- ing.1 Petechiae, ecchymoses, and excessive bleeding are • Neutrophils overwhelm the affected areas. often noted in patients with DIC. DIC is generally triggered • The inflammatory process accelerates. when there is a major disruption in the intravascular sys- • Excessive cytokines are produced. tem.4 Anything that causes ischemia creates a major change in the intravascular system. DIC can also occur because of a low pH, which can result when lactate accumulates. kidneys, where it blocks the structures of the kidneys, caus- ing acute tubular necrosis or kidney failure. Ischemia of the Systemic Inflammatory Response Syndrome muscles can predispose the body to rhabdomyolysis.1 In Cytokines produced during reperfusion injury act as medi- children and adolescents, hypoxia associated with propofol ators of systemic inflammatory response syndrome (SIRS), administration has been shown to cause rhabdomyolysis.11 which is an inflammatory response by the entire body that Treatment must be aggressive to help the kidneys eliminate can result in death. SIRS can be diagnosed if the patient has myoglobin. two or more of the following clinical criteria10: Treatment • Heart rate: >160 bpm in dogs; <140 or >250 bpm in There is no known definitive cure for I/R injury. Many doc- cats tors and scientists agree that stopping the key components • Respiratory rate: >20 breaths/min in dogs; >40 breaths/ of the ischemic cascade would likely stop the effects of I/R min in cats injury. Most also agree that stopping the cascade at the ear- • Body temperature: <100°F (37.8°C) or >103.5°F liest possible point would produce the best results. (39.7°C) in dogs and cats It is difficult to test whether an ischemic episode has • White blood cell count: >12,000 or <4000 cells/µL or occurred.5 One of the greatest hindrances to testing is that >10% bands in dogs and cats ischemia occurs when there is a lack of oxygen, and most test samples are exposed to air at some point. This makes Multiple-Organ Dysfunction Syndrome obtaining an accurate sample very difficult. As the name implies, multiple-organ dysfunction syndrome Scientists have tested thousands of treatment methods, (MODS) is altered function of two or more organ systems. drugs, and other interventions to help prevent I/R injury in MODS usually results from endothelial cell damage caused humans.5 Very few studies have been performed in animals. by overwhelming numbers of cytokines. MODS is usually Treatment has focused on blocking ROS formation, block- a complication of sepsis or SIRS.10 If MODS occurs in con- ing calcium production, and stopping neutrophil activa- junction with SIRS, the prognosis is very poor.10 As the tion.5 Much of the research into limiting the activation of number of affected organs increases, the patient’s chance neutrophils has had poor results.5 The use of multimodal of survival decreases.10 treatments directed at limiting damage during reperfusion appears to be most effective.12 Specifically, the main focus Rhabdomyolysis is on how to either stop ROS production or scavenge the Rhabdomyolysis is the rapid breakdown of muscle fibers ROS that are formed.5 resulting from traumatic injury to skeletal muscles. The principal result is the release of muscle fiber contents, such Focusing on Reactive Oxygen Species as myoglobin, into the bloodstream. Myoglobin then circu- Dimethyl sulfoxide (DMSO) is an effective scavenger of the lates through the bloodstream and eventually through the hydroxyl radical.5,12,13 DMSO can penetrate membranes,

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showed that the use of N-acetylcysteine failed to decrease Glossary I/R injury of the liver in a canine model.14 It is clear that Aerobic—pertaining to a process that requires more research is needed. oxygen Deferoxamine has been studied for more than 20 years in humans and is now being studied in animals. The Anaerobic—pertaining to a process that does not research in humans and animals has been promising; in require oxygen a study in 2009, deferoxamine significantly protected rats Apoptosis—the body’s way of safely disposing of with ischemic .15,16 This is because deferoxamine is dead cell parts by autolysis (self-destruction) of cells an iron chelator and, therefore, inhibits hydroxyl radical Coagulation—a process in which blood becomes formation.12 sticky, forming clots Allopurinol can inhibit XO formation and neutrophil Cytokine—small, secreted proteins that help infiltration during reperfusion.17 The best results have been to mediate an inflammatory response; they are obtained when allopurinol has been used as a pretreatment released by activated monocytes, macrophages, against I/R injury. Because predicting I/R injury is almost and neutrophils impossible, it would be difficult to obtain optimal results Ecchymosis—a bruiselike spot, larger than a on the use of allopurinol in a clinical setting.17 petechia, caused by bleeding from broken blood vessels under the skin into surrounding tissue Other Options Free radicals—radicals that move from where Lidocaine may prove to be a cost-effective treatment option they were created; they are highly reactive and are for I/R injury in animals. Lidocaine may act as a sodium usually involved in chemical reactions and , ROS scavenger, and inflam- 12,17 —a small body in the cytoplasm matory mediator. Because lidocaine also has beneficial of most cells that is responsible for metabolic analgesic properties, some literature supports administer- conversion of energy ing lidocaine to all at-risk postoperative veterinary patients Petechia—a tiny red or purple spot on the skin until they no longer have signs of pain, shock, or ileus (as 12 caused by broken blood vessels in GDV). Ketamine may also prove to be a cost-effective treatment for I/R injury. Ketamine inhibits N-methyl-d-aspartate such as mitochondrial membranes, to act at intracellular receptors, reduces neutrophil adhesion, and decreases sites of free radical production.13 Animal studies have had cytokine production.18,19 A study in 2009 concluded that mixed conclusions regarding the efficacy of DMSO. In cat the use of ketamine as an anesthetic reduced intestinal I/R and rat models, pretreatment with DMSO resulted in a injury in rats.20 As with lidocaine, ketamine has analgesic decrease in neutrophil infiltration during reperfusion.5,12 properties and is frequently used in veterinary in Conversely, in a study in 2008, the use of intravenous postsurgical constant-rate infusion analgesic combinations DMSO at the start of reperfusion injury of equine jejunal such as morphine–lidocaine–ketamine.21 Perhaps ketamine mucosa caused by arteriovenous or venous obstruction did could be used similarly to lidocaine in postsurgical patients not significantly change the severity of the injury after 1 at risk for I/R injury.21 hour of reperfusion.13 This is because DMSO can create The benefit of steroids in treating I/R injury has long methyl radicals and methyl peroxy radicals when it scav- been debated. A study in 2008 suggested that predniso- enges hydroxyl radicals.12 While these other radicals are lone may suppress hepatic I/R injury.22 Steroid has not as potent as hydroxyl radicals, they can still injure cell proven to help decrease liver injury by increasing tissue membranes.12 Conclusions about the efficacy of DMSO for blood flow and suppressing production of oxygen free radi- treating I/R injury will remain elusive until more is known cals and cytokines.22 This study showed that prednisolone about these other radicals. can help to prevent I/R injury in rat livers. In the past several years, human and veterinary The use of colloids may have some benefit in reducing researchers have conducted several promising studies the effects of I/R injury.12 This is likely because high-molec- using N-acetylcysteine, which is a powerful scavenger of ular-weight colloids can help to decrease microvascular the hydroxyl radical.5 While most studies involving rats, permeability.12 rabbits, and mice have produced promising results with Therapeutic hypothermia has been shown to help this drug, the most recent (2008) veterinary-related study minimize harmful effects of the inflammatory cascade and

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decrease ROS production during reperfusion.23,24 While 10. Campbell V. SIRS, sepsis, MODS demystified. IVECCS Proc 2005. therapeutic hypothermia has not yet been used in veteri- 11. Hannah J, Ramundo M. Rhabdomyolysis and hypoxia associ- nary medicine, it is a promising possibility. One of the most ated with prolonged propofol infusion in children. Neurol J common I/R injuries in people follows , which 1998;50:301-303. 12. White N. Are we making any progress with ischemia-reperfusion cuts off oxygen to the heart. In 2007, Dr. Lance Becker injury? IVECCS Proc 2007. at the University of Pennsylvania showed that cooling the 13. Carpenter R, Angel M, Morgan R. Dimethyl sulfoxide increases body after cardiac arrest increases the chance of survival by the survival of primarily ischemic island skin flaps. Accessed February 2010 at www.dmso.org/articles/skin/ischem.htm. 16%.23 This prompted the American Heart Association to 14. Baumann J, Ghosh S, Szakmany T, et al. Short-term effects recommend cooling of every cardiac arrest patient.25 Since of N-acetylcysteine and ischemic preconditioning in a canine model of hepatic ischemia-reperfusion injury. Eur Surg Res 2007, an injectable ice–salt mixture has allowed emergency 2008;41(2):226-230. personnel to quickly cool people to help slow or even pre- 15. Marin P, Im M, Girotto J, et al. Effects of hydroxyethyl-starch- vent I/R injury. bound deferoxamine on ischemia/reperfusion injury in chronic nerve compression. J Reconstr Microsurg 1998;14(7):485-490. 16. Hanson L, Roeytenberg A, Martinez P, et al. Intranasal deferox- Conclusion amine provides increased exposure and significant protec- tion in rat ischemic stroke. J Pharmacol Exp Ther 2009;330(3): The of I/R injury is extensive and not fully under- 679-686. stood, even in people. More research is needed to help 17. Hammon T. Ischemia reperfusion injury in small animal: part II, develop tests and treatments for I/R injury. To improve diagnosis and treatment. IVECCS Proc 2007. 18. Ersek R. Dissociative anesthesia for safety’s sake: ketamine and medical and veterinary knowledge, it is important to iden- diazepam—a 35-year personal experience. Plast Reconstr Surg tify I/R injury and record treatments used. 2004;113(7):1955-1959. 19. Szekely A, Heindl B, Zahler S, et al. S(+)-ketamine, but not R(-)- ketamine, reduces postischemic adherence of neutrophils in References the coronary system of isolated guinea pig hearts. Anesth Analg 1. Grace P, Mathie R. Ischaemia-Reperfusion Injury. London: 1999;88:1017-1024. Blackwell Science; 1999. 20. Cámara C, Guzmán F, Barrera E, et al. Ketamine anesthesia 2. Sege J. Concise Dictionary of Modern Medicine. New York: reduces intestinal ischemia/reperfusion injury in rats. World J McGraw-Hill; 2006. Gastroenterol 2008;14(33):5192-5196. 3. Guyton A, Hall J. Textbook of Medical Physiology. Philadelphia: 21. Shaffran N. Leaps & bounds in with CRIS. WB Saunders; 1996. IVECCS Proc 2009. 4. Wingfield W, Raffe M. The Veterinary ICU Book. Jackson, WY: 22. Wang M, Shen F, Shi L, et al. Protective effect of prednisolone Teton NewMedia; 2002. on ischemia-induced liver injury in rats. World J Gastroenterol 5. McMichael M. Ischemia-reperfusion injury: pathophysiology, 2008;14(27):4332-4337. assessment and treatment. Proc IVECCS 2006. 23. Adler J. Back from the dead. Newsweek July 23, 2007. Accessed 6. Kamada T, Shiga T, McCuskey R. Tissue Perfusion and Organ June 2010 at www.accessmylibrary.com/article-1G1-166481709/ Function. Tokyo: Elsevier; 1996. back-dead-doctors-reinventing.html. 7. Ambrosio G, Tritto I. Myocardial reperfusion injury. Eur Heart J 24. Koran Z. Therapeutic hypothermia in the postresuscitation Suppl 2002;4(suppl B):B28-B30. patient: the development and implementation of an evidence- 8. McMichael M, Moore R. Ischemia-reperfusion injury pathophys- based protocol for the emergency department. J Trauma Nurs iology, part 1. J Vet Emerg Crit Care 2004;14(4):231-241. 2009;16(1):48-57. 9. Das D. Cellular, biochemical and molecular aspects of reperfusion 25. American Heart Association. Advanced life support. Circulation injury. New York: The New York Academy of Sciences; 1994. 2005;112:III-25–III-54.

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Article #1 FREE CE Test The article you have read qualifies for 1.0 credit hour. To receive credit from Alfred State College, choose the best an- swer to each of the following questions. Take the test online at Vetlearn.com.

1. ATP is produced by 6. ______is a highly reactive radical that causes a. an anaerobic process. damage during I/R injury. b. phosphorylation. a. Hypoxanthine c. an aerobic process. b. XO d. all of the above c. Hydroxyl d. Superoxide 2. An increase in lactic acid a. decreases pH. 7. ROS can b. activates mitochondria. a. directly cause DIC and SIRS. c. increases pH. b. damage DNA and RNA. d. causes neutrophil infiltration. c. increase lactic acid. d. decrease ATP formation. 3. When ATP fails to form and cells become depolar- ized, which two electrolytes enter cells? 8. The rapid breakdown of muscle fibers resulting from a. sodium and potassium traumatic injury to the muscles is known as b. calcium and potassium a. MODS. c. calcium and magnesium b. SIRS. d. sodium and calcium c. DIC. d. rhabdomyolysis. 4. Calcium plays a role in the conversion of a. XDH to XO. 9. Most for I/R injury have focused on b. XDH to XA. a. decreasing neutrophil infiltration. c. XO to XDH. b. increasing ATP production. d. XO to XA. c. scavenging or blocking the formation of ROS. d. increasing hydroxyl radical production. 5. Inflammatory mediators are produced because of a. ATP production. 10. ______is an effective scavenger of the hydroxyl b. XO production. radical. c. sodium. a. DMSO d. NFκB activation. b. Allopurinol c. Ketamine d. Hydroxyethyl starch

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