Review of Intraperitoneal Injection of Sodium Pentobarbital As a Method of Euthanasia in Laboratory Rodents

Review of Intraperitoneal Injection of Sodium Pentobarbital as a Method of Euthanasia in Laboratory Rodents

Colin A Laferriere1 and Daniel SJ Pang1,2,*

Euthanasia is one of the most commonly performed procedures in biomedical research, involving tens of millions of animals in North America and Europe every year. The use of sodium pentobarbital, injected intraperitoneally, for killing rodents is described as an acceptable technique by the AVMA and CCAC euthanasia guidelines. This drug and route are recommended over inhalant anesthetics, carbon dioxide, and physical methods for ethical and aesthetic reasons as well as efficiency. However, a growing body of evidence challenges the efficacy and utility of intraperitoneal pentobarbital. This methodology has been described as inconsistent and may induce pain and stress. With these considerations in mind, a review of the literature is needed to assess the evidence surrounding this killing method, the associated welfare implications, and potential for refinement.

Abbreviations: CCAC, Canadian Council on Animal Care; FLI, fos-like-immunoreactive; pentobarbital, sodium pentobarbital

DOI: 10.30802/AALAS-JAALAS-19-000081

There are approximately 17 million rodents (rats and mice) able with conditions’ (AVMA)5 or ‘conditionally acceptable’ used in research annually in the European Union, United (CCAC).18 This category is similarly defined by AVMA and Kingdom, and Canada.19,37,97 An overwhelming majority of CCAC and includes techniques that “may require certain condi- these rodents are euthanized either as part of a study protocol tions to be met to consistently produce humane death” (AVMA), or at the end of a research project, making euthanasia one of the “might not consistently produce humane death” (CCAC), are most commonly performed laboratory procedures. Despite the not well described or documented in the scientific literature prevalence of euthanasia procedures and its conceptual simplic- (AVMA and CCAC), may have increased safety hazards or po- ity, ensuring a ‘good death’ across a wide variety of laboratory tential for operator error (AVMA and CCAC), or “may require settings and involving such large numbers of animals is difficult. a secondary method to ensure death” (AVMA).5,18 The AVMA Killing methods in research consist of multiple—often guidelines further state that “methods acceptable with condi- conflicting—objectives: ideally, death must be rapid and pain- tions are equivalent to acceptable methods when all criteria less yet aesthetic and compatible with research protocols.49 for application of a method can be met.”5 Methods classified Furthermore, killing methods must be simple to apply and in this way remain common because acceptable methods may yield consistent results. It may be difficult, even impossible, to interfere with the collection of research data or are impractical completely satisfy all of these objectives in all instances, more for large numbers of animals. For example, overdose with CO2 27 so given the challenges in identifying important outcomes (e.g., is one of the most commonly used killing methods. CO2 is distress, pain, aesthetic appeal). practical, easy to use, inexpensive, relatively fast in action, can In North America, the AVMA and Canadian Council on Ani- be applied to multiple animals simultaneously, and requires mal Care (CCAC) provide guidance on euthanasia procedures. little or no direct handling.4,27,54 However, many studies have

Both organizations publish guidelines regularly, reflecting the demonstrated that CO2 exposure elicits an aversive response in latest evidence on killing methods in a wide range of species. In rodents and may induce distress and pain.22,27,51,61,76-79 rodents, the AVMA classifies the intraperitoneal or intravenous Inhalant anesthetics, such as isoflurane, induce aversive injection of barbituric acid derivatives (or barbiturate combina- behavior in rodents.21,61,68,69,99,110 In addition, their use re- tions), as well as injection of dissociative agent combinations, quires specialized equipment, and time to death can be slow.18 as the only acceptable methods,5 and the CCAC classifies Therefore, the CCAC guidelines rate inhalant anesthetics intraperitoneal injection of buffered and diluted barbiturate as as acceptable in rodents but recommend use of a secondary well as overdose of inhalant anesthetics (followed by another method after the animal is anesthetized to ensure death, whereas method to ensure death) as acceptable.18 euthanasia by using inhalant anesthetics is ‘acceptable with Although the above methods are categorized as acceptable, conditions’ in the AVMA guidelines. Regarding dissociative there exists a secondary category for methods termed ‘accept- agents (typically ketamine) or a combination of ketamine and an α2 adrenergic receptor agonist, such as xylazine, there is Received: 06 Jun 2019. Revision requested: 17 Jul 2019. Accepted: 23 Sep 2019. limited discussion of this euthanasia method in the current 5 1Clinical Sciences, Faculty of Veterinary Medicine, University of Montreal, Saint- AVMA guidelines, with only one supporting study cited in Hyacinthe, Québec, Canada, and 2Veterinary Clinical and Diagnostic Sciences, University which the primary objective was not qualitative evaluation of of Calgary, Calgary, Alberta, Canada dissociative agents for killing.100 *Corresponding author. Email: [email protected]

254 Review of rodent euthanasia with pentobarbital

Overdose with barbiturate is the only method classified as Intraperitoneal injection technique. Several techniques for acceptable by both the AVMA and the CCAC guidelines.5,18 intraperitoneal injection have been described, the most common Sodium pentobarbital (pentobarbital) is the most commonly variations are described here. Few studies specifically evaluate used barbiturate for killing. It has a narrow safety margin, is the outcomes of different injection techniques. Important factors potent, can be formulated as a concentrated solution so that to be considered—but seldom described—are the proficiency of relatively small volumes are needed, and has a rapid onset of the investigators, behavior of the animals (habituated compared action when given intravenously.5,25 Furthermore, pentobarbital with nonhabituated),33,86 and inherent variability associated has a long shelf life, is stable in solution, and is inexpensive.5 with drug absorption. A 2-person technique with one holder and Access and licensing requirements for purchase vary between one injector, rather than a one-person technique, is considered countries.25 When given as an intentional overdose to cause more efficient when working with mice, given that the 2-person death, general anesthesia is induced, followed by depression method led to a reduction in misinjection rate from 11% to 13% of the respiratory and cardiovascular centers of the brainstem, to 1% to 2%.7 The holder grips the upper limbs and head of the leading to cardiorespiratory arrest.25,27 animal and maintains the animal in a horizontal position. It Although the intravenous route of injection is preferred for is often recommended that the holder tilts the animal slightly pentobarbital, it is often impractical to perform on rodents, downward so that its head is lower than its abdomen. This posi- given that achieving consistently successful intravenous injec- tioning supposedly creates more space in the caudal quadrants tions requires training, restraint and often a means to induce of the abdominal cavity by moving organs cranially.103 However venodilation.39,43 As such, pentobarbital for killing rats and mice there is little evidence that the tilting of the animal has any is frequently administered via intraperitoneal injection.25,75,96 effect on the success of the injection.75 Indeed, similar misinjec- Although the onset of anesthetic effect is not as quick after tion rates have been obtained when injecting rats vertically28 intraperitoneal injection as after intravenous injection, drug or horizontally with a head down tilt.75,113 The injector, using absorption and distribution occur much more rapidly after in- one hand to hold the right hindlimb of the animal, visualizes traperitoneal dosage than after intramuscular or subcutaneous the abdomen as though divided into 4 quadrants and injects injections.75,83 Furthermore, intraperitoneal injection is relatively into the caudal left quadrant (the animal’s right side).11,28,98,113 simple and quick to perform, allows the administration of large Within the caudal left quadrant, the injection should be made volumes, and accommodates repeated injections. at the level of the coxofemoral joint, approximately halfway Thus, intraperitoneal injection of sodium pentobarbital (in- between midline and the lateral abdominal wall.98 The goal traperitoneal pentobarbital) is one of the most widely accepted of the injection is to deposit injectate into the peritoneal cavity methods of rodent euthanasia that does not have additional without piercing any of the abdominal organs. To avoid doing conditions attached to its use. This rating is shared not only so, the injection angle should be approximately 10 to 20 degrees by the CCAC and AVMA but also by animal care guidelines in relative to the body wall in mice and 20 to 45 degrees in rats, Europe,6 India58 and Australia.9 Importantly, however, despite with the needle directed cranially.98 Before injection, it is often the many advantages and widespread use of intraperitoneal suggested to aspirate the needle to assure its correct placement pentobarbital, this killing method is not without drawbacks. in the peritoneal cavity, although there is no evidence support- Here we aim to provide a narrative review of the literature ing the usefulness of this practice.13 regarding intraperitoneal pentobarbital as a killing method, Various sizes of needles and gauges can be used, but a 3/8-in. evaluate its current designation as ‘acceptable’ (by the AVMA (9.5 mm) needle is sufficiently long and may be the least likely and CCAC), identify gaps in knowledge that may be pertinent to puncture the organs in the abdominal cavity.13,28 For a suc- to the refinement of this method, and suggest potential alter- cessful intraperitoneal injection, the needle does not need to natives. exceed a depth of 4 to 5 mm beyond the skin, so a long needle is unnecessary. The maximal suggested injectable volume is 0.5 Materials and Methods to 1.0 mL in mice and 5 to 10 mL in rats.13,75 A literature search was performed by using combinations Pharmacokinetics. Sodium pentobarbital is an oxybarbiturate ligand of the γ-aminobutyric acid subtype A (GABA ) receptor. of 4 key words (euthanasia, rodents, pentobarbital, and intra- A peritoneal, creating 11 search terms) in 3 databases (PubMed, The drug increases chloride conductance through the receptor Cab Abstracts, Web of Science) in English or French from 1950 channel, causing neuronal hyperpolarization and consequent to the search date (15 May 2019). Document types were set as CNS depression. After successful administration of a lethal journal article, book, and government report. Search results dose of pentobarbital, animals become ataxic and sedated, after were screened for inclusion by reading titles and abstracts. After which they experience loss of consciousness, apnea, cardiac initial screening, full articles were obtained for review. Data, arrest, and death. The aim of an intraperitoneal injection is to results, and conclusions from these downloaded articles were deposit pentobarbital into the peritoneal cavity, from where then analyzed and reviewed for relevant content. Additional it is absorbed into the circulation. The peritoneal cavity is de- articles, books, theses, and government reports were identi- fined as the potential space between the visceral and parietal fied and included after a manual search of the reference lists of peritonea of the abdomen. The visceral peritoneum adheres to articles identified during the database search. most of the abdominal organs, whereas the parietal peritoneum adheres to the interior of the abdominal wall. The blood vessels of the visceral peritoneum connect to the mesenteric, colic, and Results intestinal veins to converge into the anterior mesenteric vein A total of 247 articles, government reports, or books passed and then carry blood to the liver via the portal system.105 Thus initial screening and were read for the purpose of this review; intraperitoneal substances may undergo first-pass metabolism if 113 of the 247 publications were used as cited references in absorbed via the visceral peritoneum.3,89 After the liver, venous the writing of the review. The complete list of all postscreen- blood flows to the heart via the caudal vena cava and then on ing articles is available in an online repository (https://doi. to the pulmonary circulation before returning to the left side of org/10.7910/DVN/DLSLDF). the heart for distribution to the systemic circulation.

255 Vol 59, No 3 Journal of the American Association for Laboratory Animal Science May 2020

Although intraperitoneal administration is considered a par- completely into the peritoneal cavity, as compared with misin- enteral route, the pharmacokinetics of substances administered jections, which fail to do so. Misinjections are a further source via intraperitoneal injections have similarities to substances of variability, as described later. Importantly, unless behavioral administered orally because of the potential for hepatic me- outcome measures (for example, loss of righting reflex) are ac- tabolism.74,96 However, the very large surface area provided companied by confirmation of a successful injection through by the peritonea and omentum, as well as the abundant blood necropsy examination, it is impossible to differentiate variability supply, allow for absorption that is more rapid than after oral resulting from partial misinjection (some, but not all, injectate or intramuscular routes.75,83 Absorption across the visceral failing to be deposited in the peritoneal cavity) compared with peritoneum and omentum is the predominant—but not the inherent variability associated with absorption routes and rates. only—route of absorption after intraperitoneal injection. Other In addition, different operational definitions of measured end- routes to the systemic circulation include absorption via the points and methodologies for their determination are apparent parietal peritoneum and lymphatic drainage. These different and contribute to variability (Table 1). absorption routes contribute variability to the pharmacokinetics Although many different doses, volumes, and concentra- and pharmacodynamics of intraperitoneal injections.23 Vessels tions of pentobarbital have been used for euthanasia, a dose draining the parietal peritoneum do not connect to the portal of 800 mg/kg IP in rats is associated with greater consistency veins but instead empty directly into systemic veins,59 thereby and speed of effect than 200 mg/kg.113 Doses in the range of providing a route to bypass first-pass metabolism. 150 to 200 mg/kg IP are commonly used and are based on be- In addition to peritoneal absorption, lymphatic absorption ing approximately 5 times the dose required to induce general across the diaphragm can affect the fate of substances deliv- anesthesia.25,43 Furthermore, at the 800-mg/kg dose, the de- ered intraperitoneally and further contribute to variability in creased variability of effect facilitates identifying misinjections intraperitoneal drug absorption and distribution.1 Access to the based on the time taken to achieve loss of righting reflex and lymphatic system occurs through stomatas (small openings on apnea. At this dose, it has been suggested that the time to loss the surface of the peritoneal mesothelium) that allow the pas- of righting reflex and apnea should not exceed approximately sage of fluids from the abdominal cavity into subperitoneal 2.5 to 4.5 min, respectively (calculated from mean + 2 SD of lacunae, which drain directly into the lymphatic system.95 study population).113 If these times are exceeded, misinjection These stomatas are located on the muscular portion of the is likely to have occurred. diaphragm in rats. Little information is available regarding optimal dose and Lymphatic drainage can be rapid and effective in delivering volume for euthanasia with pentobarbital in mice, although substances from the peritoneal space to the systemic circula- 150 mg/kg IP has long been a suggested dose.43 The anesthetic tion.62,91 Indeed, within minutes, large amounts of drained fluid dose for pentobarbital is 40 to 50 mg/kg when given intraperi- from the abdominal cavity are transported to the subclavian toneally,13,39 so by applying the same principle as described vein, which feeds directly into the cranial vena cava.91 However earlier, the minimum dose to cause death should be 200 to 250 the speed of lymphatic drainage varies and is affected by the mg/kg IP, but doses for mice described in the literature range stretching of the diaphragm during respiration.14,102 Further- from 150 to 5400 mg/kg IP (Table 1). A recent study found that more, the posture of the animal affects the rate of drainage.12 increasing the concentration of the pentobarbital solution to 390 Indeed, a slower rate of absorption was measured when rats mg/mL, which resulted in a dose greater than 1300 mg/kg for were held vertically (head up) compared with a head-down males and 1680 mg/kg for females, significantly shortened the position, whereas a sternal posture produced intermediate time to unconsciousness and death compared with a dose of absorption. 250 mg/kg (concentration of 50 mg/mL).36 Therefore, similar to Although lymphatic drainage of the peritoneal cavity has what was observed in rats, increasing the dose of pentobarbital been widely studied in multiple species, this system is often creates a more efficacious killing method when using intraperi- undescribed in discussions of intraperitoneal injection of toneal pentobarbital in mice. pentobarbital.28,75,98,109 These studies tend to focus entirely on The extent of current scientific knowledge regarding the peritoneal absorption, thereby omitting what may be a major pharmacokinetics of intraperitoneal pentobarbital is still limited component of intraperitoneal injections. Many authors have in many areas.94,96 Indeed, some of the basic principles of this suggested that lymphatic drainage is more important quanti- route of administration, such as absorption from the peritoneal tatively than absorption from either the peritoneal or visceral cavity, have yet to be fully elucidated. Determining the effect peritoneums.1,15,29,42,73 Compelling evidence demonstrating the of varying the placement of an injectate within the abdominal relative importance of lymphatic drainage is the observation cavity on absorption rates and quantifying the various absorp- that a 59% decrease in absorptive capacity from the peritoneal tion routes could lead to greater consistency and predictability space resulted after closure of the diaphragmatic stomata owing as well as improve our ability to detect misinjections. to fibrous tissue (induced by abrasion).64 In addition, ligation Disadvantages of intraperitoneal injections of sodium of the principle lymphatic ducts draining the lymphatic vessels pentobarbital. Variability in effect. A major disadvantage of in- emanating from the diaphragm markedly reduced the amount traperitoneal injections—and arguably the most significant—is of dye (delivered via intraperitoneal injection) absorbed from its variability. This variability can be divided into 2 categories: the peritoneal cavity.30,31 inherent variability and misinjection. Inherent variability refers These varied routes of absorption alter the pharmacokinetics to the existence of different pathways of absorption and distribu- of substances delivered through intraperitoneal injection by tion, as described earlier, which tend to produce a wide range providing different means of attaining the systemic circulation. of responses after successful intraperitoneal injection and thus These undoubtedly play a role in affecting the variability in create variability in euthanasia procedures. timing of physiologic responses after successful intraperito- The second source of variability is misinjection, or incorrect neal injections.23,4,113 Table 1, which presents data from various placement of administered substances. Misinjection has long euthanasia studies, illustrates this variability. Successful been described as an issue with intraperitoneal injections and intraperitoneal injections are those that administer injectate occurs when the injection fails to introduce the drug into the

256 Review of rodent euthanasia with pentobarbital

Table 1. Timing of the various effects during intraperitoneal sodium pentobarbital euthanasia at various doses in rats and mice. Dose (mg pentobarbital/ Time (s) Species kg body weight) Effects (mean [range] or mean ± 1 SD) Reference 2 Mice 5400a Loss of righting reflex 156 Rats 200a Respiratory arrest 45 Cardiac arrest 276 ± 30 Loss of righting reflex 75 Respiratory arrest 270

Reference 4 Mice 330b Ataxia 52.0 ± 8.1 Cessation of movement 80.1 ± 21.2 Cessation of breathing 317.0 ± 150.5

Reference 16 Mice 150a Ataxia 35.6 ± 11.0 Death 343.3 ± 110.3

Reference 22 Rats 200 Recumbency 174.6 ± 125.4 Loss of righting reflex 272.1 ± 204.8 Quiescent EMG 259 ± 201

Reference 36 Mice 250c Loss of righting reflex 98.5 ± 4.7 250d Cessation of heartbeat 619.6 ± 359.6 Loss of righting reflex 74.3 ± 4.9 1300–1680e Cessation of heartbeat 508.3 ± 303.4 Loss of righting reflex 66.4 ± 4.5 Cessation of heartbeat 253.8 ± 118.1

Reference 54 Rats 150 Induction of unconsciousness 152 (105–195) Mice 150 Time to respiratory arrest 676 (510–815) Induction of unconsciousness 80 (45–120) Time to respiratory arrest 482 (315-720)

Reference 88 Mice 100 Death 235

Reference 101 Rats 667 Ataxia 40.6 ± 4.4 Cessation of movement 63.0 ± 8.2 Loss of pedal reflex 175.00 ± 6.52 Heart rate < 150 bpm 444

Reference 113 Rats 200 Loss of righting reflex 111.6 ± 19.7 200f Cessation of heartbeat 485.8 ± 140.7 800 Loss of righting reflex 104.2 ± 19.3 Cessation of heartbeat 347.7 ± 72.0 Loss of Righting reflex 139.5 ± 29.6 Cessation of heartbeat 283.7 ± 38 aPentobarbital–phenytoin combination product (390 mg/mL sodium pentobarbital and 50 mg/mL sodium phenytoin) b50:50 mixture of 100 mg/mL sodium pentobarbital with 10 mg/mL of lidocaine hydrochloride cDiluted to 5 mg/mL with USP sterile water from a 50mg/mL solution d50 mg/mL e390 mg/mL with no phenytoin added fDiluted 1:3 with PBS peritoneal cavity.63,93 The principal consequence of this failure is 20 min.113 Furthermore, there is the possibility of complications a marked delay in the onset of drug action. Indeed, misinjection arising out of misinjection, which may include local irritation caused an increase in the time from intraperitoneal pentobar- and inflammation, perforation of abdominal organs, hemor- bital injection (667 mg/kg IP) to loss of the pedal reflex in rats rhage, and respiratory distress.96 from 175 s (successful injection) to 588 s (misinjection).101 In Misinjection can be difficult to identify given the inherent one study, approximately 40% of misinjections were classified variability of successful intraperitoneal injections. Aspirating as failed euthanasia procedures because time to death exceeded before injecting is often suggested, but cecal and intestinal content

257 Vol 59, No 3 Journal of the American Association for Laboratory Animal Science May 2020 may not easily be aspirated through a small-gauge needle.4 pain, including vocalization, increased locomotion, and flinch- Therefore, misinjection can only be quantified with any confi- ing, have been observed immediately after intraperitoneal dence at necropsy, through the addition of dye to the injectate. injections.2,36,104 In addition, pentobarbital is highly alkaline, The most common sites of intraperitoneal misinjection in with a pH of 11 to 12, whereas the range of pH that is said rats, ranked by frequency of occurrence, are into the cecum, to be nonirritating to local tissue at the site of intraperitoneal into the small intestine, subcutaneously, retroperitoneally, and injections is approximately 4.5 to 8.0 in rats.75,101 It follows that into the urinary bladder.59 In mice, the most frequent locations intraperitoneal injection of pentobarbital may result in irrita- of misinjections are: stomach, small intestine, uterine horn, tion to the peritoneum or surfaces of visceral organs, as well and subcutaneous.72,93 Reported rates of misinjection based as pain. Indeed, one study101 noted signs of local redness and on necropsy findings are variable, ranging from 6% to 20% in swelling after intraperitoneal pentobarbital at a dose of 667 rats and 10% to 20% in mice.2,4,11,23,28,63,87,93,103,113 Although the mg/kg with no additives in the solution. In addition, writhing application of appropriate technique and training can reduce behavior was observed, beginning approximately 11 ± 2 s after these rates,7,23,72 the documented incidence of misinjection rarely injection. Writhing was defined as “an abnormal posture in falls below 6%, particularly in rats. One study did achieve a which the rats contract their abdomen and extend their hindlegs reduction in misinjections in mice from 11% to 13% to 1% to 2% backward.”101 Writhing has been reported in both rats and mice (both values obtained after injecting 250 mice).7 This reduction after intraperitoneal injection,2,4,57,113 and it is recognized as a be- occurred after the injections were performed by using a 2-person havioral response to abdominal pain, given that it is commonly technique rather than by a single person. observed after the injection of a known irritant into the abdomi- Two studies examined the position of the cecum in rats and nal cavity92 and after abdominal surgeries, such as laparotomy mice and found that it was predominantly in the left caudal and vasectomy.85,111 The reported incidence of writhing after abdominal quadrant.28,98 This finding is consistent with the intraperitoneal pentobarbital varies widely, from approximately practice of giving injections in the right caudal quadrant to 30% to 100%.4,101,113 The reason for this discrepancy is unclear avoid penetrating the cecum. However, the lateral dominance but may stem from difficulties in assessing motor behaviors as of the organ never exceeded 80%. Moreover, the percentage pentobarbital induces general anesthesia. varied among strains, sexes, and even colonies. So the cecum, Many of these data, however, should be interpreted cau- which is the main site of intraperitoneal misinjections in rats, tiously given the difficulty in accurately quantifying behavioral lies in the middle or on the right (directly in the target of an responses dependent on gross movement in the presence of intraperitoneal injection) in as many as 20% of cases. This un- an agent that depresses motor function. Some authors have predictability appears unavoidable and is an important factor attempted to circumvent this issue through means of indirect contributing to the high misinjection rate in rats. Cecal posi- quantification of nociception. One study used the presence tion may be less variable in mice, and the organ is indeed less of electrical brain activity in anesthetized piglets to infer the frequently involved during misinjections.72,93,98 presence of nociceptive input after intraperitoneal pentobarbi- The peritoneal space is best understood as being a potential tal injection.56 Another injected pentobarbital into a hindpaw space rather than actual space.4 Therefore, the shifting and of mice and quantified the response to various pentobarbital changeable position of the abdominal organs, such as the ce- concentrations by using a paw-lick test.36 cum, within this space makes it impossible to completely avoid Additional evidence for the occurrence of pain after intraperi- unsuccessful injections. This drawback becomes a concern toneal injections can be obtained via the study of the neuronal especially when considering the number of animals killed via markers c-fos and Fos. c-fos is an early-response protooncogene this technique each year on a global scale. Misinjection prolongs that is rapidly activated and expressed in specific nociceptive the time to loss of consciousness, and injection of pentobarbital neurons of the dorsal horn after noxious or sensory stimula- into the peritoneal space may induce pain (discussed later); tion.26,48,52 The c-fos gene encodes for the protein Fos, which acts therefore, it is reasonable to consider the welfare implications as an intermediary between extracellular events and long-term of intraperitoneal pentobarbital and to develop methods to intracellular adaptations. Although much is still unknown about refine the method. the physiologic roles of c-fos and Fos, including during nocicep- Histopathologic and physiologic changes. Pentobarbital can tive processes, these markers are nonetheless commonly used damage local tissue that it contacts;8,31 significant damage to in research to measure the activity of nociceptive neurons.48 superficial cells of the organs near or at the injection site have Indeed, immunohistochemical staining is a practical and reli- been observed,45 thus suggesting the injectate was causing this able way to detect Fos activity, therefore leading to the use of damage. The same study45 also noted splenomegaly after in- FLI (fos-like immunoreactive) neurons in research.41 traperitoneal pentobarbital, most likely caused by relaxation of The quantification of FLI neurons has thus been used to infer smooth muscle, causing splenic engorgement with blood. Other pain after intraperitoneal pentobarbital. A study of FLI neurons histologic changes affecting organs farther from the injection site has shown an increase in neuronal activity after intraperitoneal may include focal congestion of intestinal serosa, congestion in pentobarbital administration in areas of the dorsal horn related pulmonary veins, necrosis in subcapsular levels of the liver and to visceral nociception.94 A 4-fold increase in the number of pancreas, lung emphysema and edema, and hyperemic kidneys FLI neurons was observed in rats administered intraperitoneal (see reference 7 for review). pentobarbital compared with rats receiving saline intraperito- In light of these physiologic disruptions, it is appropriate to neal injections, although the saline-treated group also showed consider alternative euthanasia options where blood or tissue increased FLI neurons when compared with basal levels. samples are required for a research protocol. Alternative meth- Furthermore, the addition of lidocaine (10 mg/mL) to intra- ods should be justified, weighing the balance between research peritoneal pentobarbital lowered the number of FLI neurons outcomes and the risk of failing to achieve a humane death. in the spinal cord compared with intraperitoneal pentobarbital Pain associated with intraperitoneal pentobarbital. The act administered alone. The FLI neurons were present in laminae of intraperitoneal injection, regardless of the chemical agent I, II, V, and X of the spinal dorsal horn; these laminae receive used, can induce distress and pain. Behaviors associated with visceral nociceptive input.46,71

258 Review of rodent euthanasia with pentobarbital

The relationship between FLI neurons and writhing behavior Stress. The stressful nature of intraperitoneal injections has provides further evidence that intraperitoneal pentobarbital been inferred via the measurement of hormonal markers. In produces pain. There exists a positive correlation between the one study, ACTH levels increased as much as 2-fold compared number of FLI neurons in the laminae that receive visceral with basal levels in Sprague–Dawley rats after intraperitoneal nociceptive input and the number of writhing behaviors, or injections of saline.33 However, the concentrations of ACTH stretches, observed after the intraperitoneal administration of measured varied substantially. A similar increase was observed acetic acid, a known irritant.46 The administration of analgesics in mice.10 In addition, plasma corticosterone levels in both mice (including morphine) produced dose-dependent inhibition and rats can be increased through intraperitoneal injections of of the writhing behavior and concurrently a reduction in the saline. Two studies in mice reported significant increases in number of FLI neurons. corticosterone after intraperitoneal injection of saline.10,86 In However, much debate remains surrounding the use of FLI rats, results have varied: one study reported an increased level neurons in pain studies. The relationship between FLI neuron of corticosterone in Sprague–Dawley rats after intraperitoneal activity and the conscious, cortical perception of pain is unclear. saline injections, whereas another reported no such increase.33,112 FLI neurons did not significantly increase in the ventroposte- Intraperitoneal injection of saline has been associated with rolateral nucleus of the thalamus, a region implicated in the hyperthermia and tachycardia in both rats34,47,90 and mice.24,60 perception of pain,108,109 after a noxious stimulus.3 In addi- Another study found that intraperitoneal injection of saline or tion, rats treated with morphine (as high as 10 mg/kg SC) still sham injection (needle insertion with no fluid administered), showed Fos expression in the dorsal horn yet displayed few to increased the heart rate of mice over basal levels for up to 30 no behavioral signs of pain, although Fos activity did diminish min following injection.70 The authors also showed that changes with increasing doses of morphine (from 1 to 10 mg/kg SC).81 in heart rate parallel those of plasma corticosterone and may, Therefore, the quantification of FLI neurons to infer pain is therefore, be another useful indicator of stress.71 probably oversimplistic. Moreover, stress is a confounding Stress related to laboratory procedures varies significantly factor, given that it has been reported to increase FLI neuron between strains of laboratory rodents. One study reported activity.80 Given the lack of specificity, converging lines of evi- plasma levels of corticosterone increasing from 3 to 7 fold across dence (pain-related behaviors, biomarkers, gross and histologic different strains of mice.86 If ACTH physiology is similar among tissue changes) should be used to draw inferences about the rat strains, Lewis rats may be less susceptible to stress; this strain likely presence of pain. had a minimal increase in plasma ACTH concentrations after The AVMA and CCAC euthanasia guidelines acknowledge handling or injection, compared with Sprague–Dawley rats.33 that intraperitoneal pentobarbital may cause pain.5,10,18,23 The Thus, there is evidence that the handling necessary for in- CCAC guidelines suggest the concurrent use of a local anes- traperitoneal injections can be stressful. Handling stress can be thetic, such as lidocaine, with a buffered and diluted barbiturate reduced. Repeated exposure—i.e., habituation—can reduce the and recommend that steps should be taken to ensure that the pH stress response associated with laboratory procedures such as is within a nonirritating range.18,23 However, the pH of pentobar- intraperitoneal injections. After habituation to saline intraperi- bital cannot be lowered below 10 without risk of precipitation, toneal injections, there was a reduced expression of immediate and a pH of 10 remains irritating to tissue.31,75 The addition of early response genes and corticosterone levels.33,86 lidocaine to pentobarbital (in a 50:50 mixture) reduces pain, as Characterizing the relationship between stress and distress measured by writhing behavior.4 In addition, one study noted cannot be made on hormonal markers alone. Other markers, a decrease (but not absence) in writhing when either lidocaine such as behavioral observations, should be assessed in parallel. (10 mg/mL of pentobarbital solution) or bupivacaine (2.5 mg/ Behavioral assessments of pain and stress are both challenging mL of pentobarbital solution) was added to pentobarbital (pH because they lack consistency and tend to be fairly subjective.106 10.1 and 10.2, respectively).57 This outcome suggests that the ad- However, although quantifying the effects of intraperitoneal in- dition of lidocaine represents a refinement of the intraperitoneal jections in laboratory rodents is difficult, establishing that there pentobarbital technique. Although the lidocaine–pentobarbital is an effect is less so: in the majority of studies, intraperitoneal mixture was beneficial, it still significantly increased the number injections are identified as a potential source of pain and stress. of FLI neurons 3-fold compared with a saline control injection.94 Alternatives to intraperitoneal pentobarbital. There are few, The potential for pain during intraperitoneal pentobarbital is if any, well-described alternatives to pentobarbital as an inject- compounded by the time to achieve loss of consciousness with able killing method for rats and mice. One chemical agent that this killing method, especially in rats. In addition to implications has shown promising results is ethanol. In many countries, for tissue harvesting and sample quality, this is an important government drug regulations require accounting of barbiturate concern for animal welfare. Development of a technique that drugs,4 which makes ethanol an attractive alternative. Ethanol induces a consistently shorter time to unconsciousness and is currently described in the AVMA guidelines as ‘acceptable death is desirable. with conditions,’ owing to a series of studies that explored its In light of the limited available evidence, various authors use as a killing method.65-67 Interest in this agent has recently and the AVMA guidelines both raise the need for additional resurged, and it has been determined that 100% ethanol at a dose studies.5,94 Different local anesthetic agents could be explored, of 15.3 to 15.8 g/kg results in similar rates of onset of respira- as could alternative markers of nociception. Alternative mark- tory and cardiac arrest as a similar volume (approximately 0.5 ers, such as phosphorylated extracellular signal-regulated mL) of intraperitoneal pentobarbital in mice.2,32 The injection kinases, may offer an advantage in that they are more rap- of ethanol induced pain-related behaviors such as vocalization2 idly expressed than c-fos, reaching a peak 2 to 3 min after and kicking at the needle.32 In both studies, these behaviors stimuli41,55 (c-fos induction and expression in the spinal cord were not more frequent than those seen with intraperitoneal take at least 30 min).35 Indeed, phosphorylated extracellular pentobarbital, and vocalization is not a specific indicator of signal-regulated kinases could be used to establish a timeline pain in mice.106,107 Therefore, ethanol appears at least similarly of potential nociception and pain after intraperitoneal pento- effective to pentobarbital, but with the advantage of not being barbital injection. a controlled agent. A study of the number of FLI neurons after

259 Vol 59, No 3 Journal of the American Association for Laboratory Animal Science May 2020 injection could be used to quantify neuronal activity associated tion from best practice.84 For example, times to loss of righting with ethanol injections compared with intraperitoneal pento- reflex, apnea, and cardiac arrest, and rate of failure to achieve barbital injections. In contrast to that in mice, intraperitoneal key outcomes all merit tracking. injection of ethanol in rats as a killing method was unsuccessful due to the large volumes of ethanol required (more than 7.1 mL for a dose of 20.1 g/kg) and because the time from injection to Conclusion respiratory arrest was slow (8 ± 5 min).2 In light of the data cited in this review, it is legitimate to ques- Therefore, pentobarbital remains the only practical injectable tion whether intraperitoneal pentobarbital as a killing method killing agent in rats. Alternative injectable techniques that show always meets the criterion of euthanasia. According to the potential include retroorbital and intrahepatic injection. In mice, AVMA guidelines, euthanasia procedures should be consistent, retroorbital injection of an overdose of ketamine and xylazine easy to perform, reliable, and predictable. Despite the relative resulted in rapid death (cessation of heartbeat occurring in ap- simplicity and widespread use of intraperitoneal pentobarbi- proximately 5 s), because the pharmacokinetics of retroorbital tal, there is a distinct possibility that this methodology causes injection closely resemble those of intravenous injection.50,82,88 distress and pain. The likelihood of misinjection only serves to However, neither the risks and prevalence of misinjection with exacerbate these problems. Finally, many important gaps exist this route of administration nor the potential for pain have been in scientific knowledge related to this procedure. The following studied. This technique has not been evaluated in rats. data remain to be established: Another potential route of pentobarbital administration is 1. The optimal dose of pentobarbital in mice and predictability intrahepatic. One article has reported use of this injection tech- of misinjection. nique for euthanasia of shelter cats.44 The technique proved 2. Criteria for early identification of misinjection and remedial more accurate and faster than intraperitoneal injections, given procedures to minimize the potential for pain. that most cats became recumbent almost immediately after 3. The optimal dilution of pentobarbital that minimizes pain injection of pentobarbital. A few cats did respond negatively to associated with its alkali pH yet maintains efficacy. the injection; a negative response was defined as vocalization 4. The refinement of the addition of lidocaine (dose and vol- or turning toward the injection site. However, these behav- ume) to pentobarbital. iors occurred with similar frequency during intrahepatic and 5. The potential role of other local anesthetics for mixing with intramuscular injections. The feasibility and consistency of pentobarbital. the intrahepatic injection technique remain to be evaluated in 6. The elucidation of the pharmacokinetic parameters after laboratory rodents. intraperitoneal administration, most notably the relationship These alternative methods of drug administration require between injection site and peritoneal absorption. further study to facilitate comparison with intraperitoneal injec- 7. The potential for alternative injectable routes of administration, particularly of inherent variability in effects, likelihood of tion to replace intraperitoneal injection. misinjection and speed of action. As with all proposed killing 8. The role of training in minimizing misinjection. methods, the criteria listed in the AVMA guidelines5 should be Clearly, numerous aspects of intraperitoneal pentobarbital applied to evaluate their suitability. give cause for concern. Novel approaches, such as the use Best-practice recommendations. The following recommenda- of intraperitoneal ethanol and alternative routes of injection, tions are based on the available literature and are intended as are promising but require further research to establish their suggestions to promote best practice. strengths and weaknesses before they can be proposed as suit- 1. Within an institution, establish a standard dose, volume, and able alternatives for, or improvements upon, intraperitoneal formulation of sodium pentobarbital. Standardized dosing will pentobarbital. The important limitations described for intraperi- allow for derivation of reference ranges for key outcomes, such toneal pentobarbital, current lack of suitable alternatives, and as loss of righting reflex, respiratory arrest, and cardiac arrest.113 the large number of animals killed underline the importance If dose, volume, and formulation match those in the literature of further research in this field. (Table 1), published reference ranges can be used as a benchmark for performance, provided that the methods of identifying key References outcomes is the same. Establishing reference ranges also allows 1. Abu-Hijleh MF, Habbal OA, Moqattash ST. 1995. The role of the auditing (see point 3). A backup plan should be available for diaphragm in lymphatic absorption from the peritoneal cavity. J when key outcomes are not achieved within the expected time. Anat 186:453–467. This plan may involve redosing or the use of a secondary killing 2. Allen-Worthington KA, Brice AK, Marx JO, Hankenson FC. 2015. method. When a standard drug solution volume is used for a Intraperitoneal injection of ethanol for the euthanasia of laboratory range of animal weights, the lower and upper limits of the ac- mice (Mus musculus) and rats (Rattus norvegicus). J Am Assoc Lab Anim Sci 54:769–778. ceptable body mass for each injectate volume should be available. 3. Alves HN, da Silva AL, Olsson IA, Orden JM, Antunes LM. 2. Ensure that users are proficient in the steps required to 2010. Anesthesia with intraperitoneal propofol, medetomindine, perform intraperitoneal injection. These include drug dose and fentanyl in rats. J Am Assoc Lab Anim Sci 49:454–459. calculation and preparation, handling and restraint, injection, 4. Ambrose N. 1999. Refinement of routine procedures on laboratory and identification of signs of general anesthesia and death. rodents, p 251. Faculty of Medicine and Dentistry. Birmingham Technical skills follow a learning curve, and learners achieve (United Kingdom): University of Birmingham. proficiency at different rates.17,20,58 Should time to achieve key 5. American Veterinary Medical Association. 2013. AVMA Guide- outcomes after intraperitoneal injection of pentobarbital deviate lines for the Euthanasia of Animals, 8 ed. Schaumberg (IL): American Veterinary Medical Association. substantially from reference ranges, investigating the source of 6. Animal Health and Welfare Panel. 2005. Aspects of biology and this deviation should include examining the steps involved in welfare of animals used for experimental and other scientific administering intraperitoneal injections. purposes. EFSA J 292:1–46. 3. Consider auditing key outcomes as a means of tracking 7. Arioli V, Rossi E. 1970. Errors related to different techniques of consistent practice and facilitating early identification of devia- intraperitoneal injection in mice. Appl Microbiol 19:704–705.

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