Analgesic Activity of Tramadol and Buprenorphine after Voluntary Ingestion by Rats (Rattus norvegicus)

Effective pain management for rats and mice is crucial due to the continuing increase in the use of these species in biomedical research. Here we used a recently validated operant orofacial pain assay to determine dose–response curves for buprenorphine and tramadol when mixed in nut paste and administered to male and female rats. Statistically significant analgesic doses of tramadol in nut paste included doses of 20, 30, and 40 mg/kg for female rats but only 40 mg/kg for male rats. For male rats receiving buprenorphine mixed in nut paste, a significant analgesic response was observed at 0.5 and 0.6 mg/kg. None of the doses tested produced a significant analgesic response in female rats. Our results indicate that at the doses tested, tramadol and buprenorphine produced an analgesic response in male rats. In female rats, tramadol shows a higher analgesic effect than buprenorphine. The analgesic effects observed 60 min after administration of the statistically significant oral doses of both drugs were similar to the analgesic effects of 0.03 mg/kg subcutaneous buprenorphine 30 min after administration. The method of voluntary ingestion could be effective, is easy to use, and would minimize stress to the rats during the immediate postoperative period.Abbreviations: VI, voluntary ingestion; LFR, lick:facial contact ratio; OPAD, orofacial pain assessment deviceEffective pain management for rats and mice is crucial due to the continuing increase in the use of these species in biomedical research. It is necessary not only to satisfy ethical and legal standards, but providing effective pain management may also reduce distress, decrease mortality, and overall eliminate many of the negative postsurgical physiologic consequences that may be confounding factors in research.⁵² Effective pain management entails providing analgesics at the optimal dosing regimen (dose, frequency, and duration). Continual refinement of the optimal analgesic dosing regimen is possible due to the availability of new methods for evaluating pain (that is, mouse and rat grimace scales),^47,69 the increasing knowledge of pain mechanisms and pathways,^56,71 and the development of new analgesic formulations.^14,68Tramadol is a centrally acting analgesic with both opioid and nonopioid mechanisms of action.⁶⁰ Its analgesic activity is due to a high affinity for μ-opioid receptors and both serotonin and norepinephrine reuptake inhibition.² In vitro, tramadol has been shown to inhibit the activity of voltage-operated Na⁺ channels, delayed rectifier K⁺ channels, N-methyl-d-aspartate receptors, and substance P receptors.^27,28,49,75 Tramadol also exhibits relatively few of the adverse effects typically associated with classic opioids, including respiratory depression and ileus.⁶⁰ The most commonly reported tramadol-associated adverse events in humans include nausea, dizziness, and drowsiness.²⁶ In the United States, oral formulations of tramadol are widely used in both human medicine and in companion animals.^59,64 Despite the potential benefits, very few studies have evaluated the efficacy of tramadol after oral administration in laboratory rats.⁵⁵In comparison, buprenorphine is one of the most common analgesics used for mild to moderate pain in rats, and its use is considered a standard of care for postoperative pain.¹⁴ Buprenorphine acts as a partial μ-opioid receptor agonist, and its slow dissociation kinetics allows for a longer duration of action compared with that of classic μ-opioid agonists, such as morphine.^45,76,79 Additional benefits of buprenorphine include a ceiling effect on respiratory depression and a lack of immunosuppression at doses relevant for analgesia.^30,57,61 Side effects in rats are usually limited but include sedation, cardiovascular depression, decreased appetite, and gastrointestinal distress, which may or may not be accompanied by pica.^15,19,63 Administration of buprenorphine by the oral route in rats is limited by a lack of information regarding its pharmacokinetics and conflicting reports of its efficacy.^{1,5,23,24,36,46,74}Providing analgesics mixed in the food or water of rats and mice is one of the least stressful methods of administration. This method eliminates postoperative manual restraint and parenteral injections, which have been shown to induce stress-like responses in mice and rats.^6,66,67 Providing analgesics by this method has several drawbacks. First, the neophobic behavior of rats and mice may lead to significant underdosing when a period of habituation to the drug is not observed.⁷⁰ Second, some drugs are unpalatable and so may not be consumed in sufficient quantities to provide analgesia.³³ Third, animals undergoing surgery typically have reduced food and water intake during the immediate postoperative period, and this behavior may limit the dose administered.²⁹ Fourth, overdosing may occur when analgesics are provided with a palatable vehicle, such as a cherry-flavored solution.⁸ Finally, when opioids are administered in the food or water, tolerance may develop, leading to a decrease in analgesic efficacy.³⁵To mitigate these issues, sufficient dosages of analgesics can be offered for voluntary ingestion (VI) by mixing the drug in a highly palatable vehicle without needing to add it to food or water. Several recent studies using a sweet nut paste for the administration of buprenorphine have shown its promise as an effective vehicle for administration.^1,23,24 Providing analgesics in this manner allows for a fixed dosage of the drug to be administered consistently, with the added assurance that the animals are consuming an effective dose and are not under- or overdosed.The goal of the current study was to evaluate the analgesic effects of a range of oral doses of tramadol or buprenorphine mixed with nut paste and provided to rats by VI. Rats were evaluated by using a recently described, nontraumatic, reversible, operant-based, thermal orofacial pain assessment device (OPAD).^53,62 In this model, a low concentration of capsaicin is applied topically to the test area for 5 min and then is removed. Capsaicin sensitizes the transient receptor potential vanilloid 1 receptor to heat.⁵¹ This receptor is a key channel for signaling and modulating heat and inflammatory pain,^12,13,58 and previous studies in mice have documented the importance of the receptor in the development of incisional pain.^40,58 Heat is then applied to the sensitized test area, thereby activating these receptors and eliminating the need for surgical procedures. Our hypothesis was that OPAD evaluation would be effective at establishing clinically relevant doses for tramadol and buprenorphine in both male and female rats.     

Analgesic Efficacy of Firocoxib,a Selective Inhibitor of Cyclooxygenase 2, in a Mouse Model of Incisional Pain

Pain management in laboratory animals is generally accomplished by using opioids and NSAIDs. However, opioid use is hindered by controlled substance requirements and a relatively short duration of action. In this study, we compared the analgesic efficacy of firocoxib (a cyclooxygenase-2-selective NSAID) with that of buprenorphine in the mouse model of plantar incisional pain by objective measurement of mechanical allodynia and thermal hyperalgesia using von Frey and Hargreaves equipment, respectively. Our experimental design included 5 treatment groups: firocoxib at 10 mg/kg IP every 24 h (F10 group); firocoxib at 20 mg/kg IP every 24 h (F20); buprenorphine at 0.2 mg/kg SC every 8 h; intraperitoneal normal saline every 24 h; and sham group (anesthesia, no incision) treated with firocoxib at 20 mg/kg IP every 24 h (sham+F20). All mice underwent nociceptive assays at 24 h before and 4, 24, 48, and 72 h after surgery. Buprenorphine alleviated allodynia at all time points after incision. The F10 treatment alleviated allodynia at 4, 24, and 48 h, whereas F20 alleviated allodynia at 24, 48, and 72 h. None of the treatments alleviated thermal hyperalgesia at 4h. Except for F10 and buprenorphine at 24 h, all treatments alleviated thermal hyperalgesia at 24, 48, and 72 h. No significant differences were noted between the 2 doses of firocoxib and buprenorphine regarding mechanical allodynia and thermal hyperalgesia at all time points. In conclusion, the analgesic efficacy of firocoxib is comparable to that of buprenorphine in this mouse pain model.Abbreviations: COX, cyclooxygenase; PWL, paw withdrawal latency; PWT, paw withdrawal thresholdEthical, scientific, and regulatory considerations compel effective pain management in laboratory animals, as is emphasized in the 8th edition of The Guide for the Care and Use of Laboratory Animals.³¹ Ineffective pain management in research animals is not only a concern for animal wellbeing but can also lead to pathophysiologic perturbation that may confound research findings. Postoperative pain is one of the most common types of acute pain in the laboratory animal setting and is a complex process that involves hypersensitivity to many stimuli,³³ including mechanical and thermal stimuli.^8,9,22,63To evaluate new analgesic therapies for acute pain, several pain models (for example, plantar incision, antigen-induced inflammation, formalin injection, capsaicin injection) have been developed. Surgical incisions in animals generally result in pain and hypersensitivity to a variety of nonnoxious (allodynia) and noxious (hyperalgesia) stimuli and are a result of peripheral^26,47 and central sensitization.^61,64 The mouse model of plantar incision-induced pain is widely used for analgesic efficacy studies, because this model has demonstrated reproducible, quantifiable allodynia and hyperalgesia after the incision.^1,48Postoperative pain management in laboratory rodents is largely centered on the use of opioids and NSAIDs. The analgesic selected depends on the experimental model and the potential influence of the analgesic on research. Buprenorphine, a centrally acting partial μ opioid agonist and a κ and δ antagonist,⁴³ is one of the most commonly used opioid analgesics in rodents.^15,51 Despite many research findings demonstrating buprenorphine''s efficacy in pain assays, its use remains controversial. Many studies cite unfavorable characteristics associated with its use, including pica (strain-dependent),⁵⁸ decreased gastrointestinal motility, respiratory and cardiovascular depression, rebound hyperalgesia, immunomodulation, and opioid tolerance when used for an extended period.^{16,25,38,46,55} Furthermore, buprenorphine''s duration of action is shorter (6 to 8 h in rats and 3 to 5 h in mice) than that of other nonopioid drugs, thus requiring an increased frequency of administration.²³ In addition, as a schedule III narcotic (as defined in the Controlled Substance Act), buprenorphine is regulated by federal and state agencies, thereby complicating its use by research staff.⁶An alternative therapeutic approach for treatment of postsurgical pain is the blockade of cyclooxygenase (COX) pathways via NSAID. Traditional NSAID such as aspirin (preferentially COX1 pathway), ketoprofen, carprofen (nonselective COX), and meloxicam (preferentially COX2 pathway) inhibit both COX1 and COX2.^3,4,19,37 The therapeutic efficacy of COX-blocking NSAID stems from the inhibition of the COX2 pathway for prostaglandin production. In contrast to the beneficial blockade of COX2-mediated inflammation, blockade of COX1 prostaglandins commonly results in adverse effects (for example, gastric irritation and hepatic damage) due to blockade of noninflammatory physiologic processes.^18,28,36 The current trend in NSAID treatment is to use a COX2-selective NSAID because inhibition of COX1 can result in harmful effects such as gastric ulceration and renal toxicity.⁵⁰ A recent study found that a single therapeutic dose of ketoprofen combined with anesthesia in rats can cause significant gastrointestinal bleeding, erosions, and ulcers in the small intestine and fatality in rats within 24 h of administration.⁵⁴ The use of COX2-preferential NSAID such as meloxicam has a lower rate of such adverse effects, but significant COX1 deactivation still occurs and leads to untoward effects. These complications may be exacerbated with repeat dosing or at increased dosage levels.¹⁴Firocoxib, a relatively new cyclooxygenase-inhibitor NSAID approved for veterinary use, is labeled for the control of postoperative pain and inflammation associated with soft tissue and orthopedic surgery.³⁶ In dogs, the ratio of the COX1 IC₅₀ to COX2 IC₅₀ for firocoxib is 350 to 430,³⁹ which is many fold higher than those reported for other commonly used cyclooxygenase inhibitors or nonselective NSAID. Because of its high selectivity, firocoxib''s COX1 inhibition is negligible at therapeutic levels.^39,57 Recent studies in dogs and horses have demonstrated the equal or greater analgesic efficacy (with negligible adverse effects) of firocoxib as compared with traditional NSAID.^{27,29,32,39,45} Because of the lack of literature and information about the analgesic efficacy of firocoxib in rodents, it has not been used for pain control in these species.In the present study, we used the mouse plantar incision model to evaluate the analgesic efficacy of firocoxib compared with buprenorphine. Although the mechanism of action differs for these 2 drugs, the clinical standard of buprenorphine analgesia is well established for the control of pain in numerous laboratory animal species.^11,24,44 We hypothesized that the analgesic efficacy of firocoxib is comparable to that of buprenorphine in the plantar incisional pain model in mice⁴⁸ and thereby provides a suitable therapeutic alternative to buprenorphine. Furthermore, we hypothesized that therapeutic administration of firocoxib does not cause gastrointestinal complications.     

Pharmacokinetics and Antinociceptive Activity of Sustained-Release Buprenorphine in Sheep

Buprenorphine is a potent analgesic commonly administered to alleviate pain in sheep used in research. Sustained-release buprenorphine (SRB) is an alternative to conventional buprenorphine hydrochloride (which must be injected repeatedly). To compare SRB with a typical conventional buprenorphine regimen (0.03 mg/kg every 8 h for 72 h), we used a simple 1:1 conversion to calculate a total SRB dose of 0.27 mg/kg per injection. The pharmacokinetics and thermal nociceptive effects of SRB were analyzed in 4 healthy adult sheep after a single intramuscular injection plus a washout period then a single subcutaneous injection. For both routes in all 4 sheep, plasma buprenorphine concentrations exceeded 0.1 ng/mL, considered the minimal threshold for therapeutic benefit, after 12 h and maintained a steady state for at least 72 h Likewise, for both routes in all sheep, thermal thresholds increased significantly between baseline and 12 h; lack of response persisted for at least 72 h. The average maximal plasma buprenorphine concentrations and bioavailability were similar for both routes. No clinical adverse effects occurred. Using a dose equivalent to the total course of conventional buprenorphine, this pilot study suggests that SRB is a well-tolerated, effective, and long-acting analgesic that can be administered as a single intramuscular or subcutaneous injection. SRB confers steady plasma concentrations and continuous analgesia in thermal nociception for at least 72 h. When compared with conventional buprenorphine, SRB has considerable advantages in improving wellbeing by minimizing handling-associated stress of repeated injection and limiting the likelihood of end-of-dose breakthrough pain.Abbreviation: SRB, sustained-release buprenorphineTo study disease and to develop and evaluate therapeutic interventions, researchers often heavily rely on animal models. Avoiding or minimizing pain in laboratory animals is of central importance to protect their wellbeing.^2,37 The administration of analgesics, which block or reduce sensitization of central and peripheral pain pathways, is a key antinociceptive method.Sheep are used to model interventions targeting airway disease, cardiovascular disease, orthopedic injury, emergency resuscitation, and vaccination.^{9,18,22,23,25,26,34,36,38,39} The development of effective analgesic regimens in sheep requires consideration of the source of pain, its anticipated duration, and the advantages and disadvantages of various methods.Buprenorphine, a partial μ opiate agonist, is among the most commonly used analgesics in laboratory animals, including sheep.^1,19,28,42 It is used primarily as an injectable and sublingual analgesic to alleviate mild to severe pain.³² Favored for its relatively long half-life and exceptional safety profile, buprenorphine has several other advantageous properties: rapid onset, higher potency than morphine, and a ceiling in terms of its respiratory effect but not its analgesic effect.^6,31 Its analgesic effect usually lasts for 6 to 8 h, but may persist for as long as 12 h, depending on the type of pain.³² The duration of effect and the response to buprenorphine are also related to species-specific metabolism and are dose-dependent.¹²Sustained-release buprenorphine (SRB) is approved for veterinary analgesic use. It offers prolonged pain relief, minimizing the need for repeated injections. Its efficacy has been demonstrated in mice, rats, cats, and NHP under different applications, including surgical models.^3,4,8,29 Similarly, extended-release opioid formulations are used clinically in humans, prolonging pain relief, lengthening dose intervals, reducing end-of-dose pain, and enhancing ease of compliance.^16,17,27,41 In sheep, limiting the need for repeated injections (which typically must occur every 4 to 12 h) is especially appealing, considering their susceptibility to stress during flock separation and under restraint when the prey flight response is blocked.Because SRB is a relatively new formulation, the dose basis and ideal route of administration have yet to be established in sheep. The purpose of this study was to characterize the pharmacokinetic profile of SRB, in relation to its analgesic efficacy, as measured by thermal threshold and safety parameters. Identifying the expected plasma concentrations of buprenorphine after SRB administration allows these data to be compared with previous reported findings for conventional buprenorphine.^28,42 Our hypothesis, based on assumptions from similar veterinary applications, was that parenteral injection of SRB every 72 h would result in therapeutic concentrations and provide continuous analgesia. Using a crossover study design, we administered SRB through a single intramuscular and then a single subcutaneous injection; we measured the response to thermal nociception and the plasma concentrations at serial time points for 7 d. In addition, we monitored sheep for side effects associated with the SRB dose and the route of administration, particularly cardiovascular or respiratory suppression and inappetence.Our hope was that the pharmacokinetic profile of SRB would provide advantages such as steady-state plasma concentrations, decreased end-of-dose failure, and minimization of injection-related handling stress. We believe that a preliminary characterization of the pharmacokinetic profile of SRB and of the response to this drug is essential to develop effective dose regimens for practical application.     

Comparison of Buprenorphine and Butorphanol Analgesia in the Eastern Red-Spotted Newt (Notophthalmus viridescens)

The experimental use of amphibian models in biomedical research increases yearly, but there is a paucity of reports concerning analgesic use in many of these species. In this study, buprenorphine given by intracoelomic injection and butorphanol added to the tank water were compared for analgesic effect in the eastern red-spotted newt after bilateral forelimb amputations. Newts undergoing anesthesia but not surgery and newts having surgery but not given analgesia postoperatively were used as control groups. Animals were tested for food consumption, spontaneous movement, response to tapping on the tank, response to being touched, and body posture. Both buprenorphine by intracoelomic injection and butorphanol in tank water significantly promoted resumption of normal behavior after bilateral surgical amputation of the forelimbs. The difference between analgesic treatment and no analgesic treatment was maintained until 72 h after surgery.Abbreviations: NS, anesthesia but no surgery; Sx, Surgery but no analgesia; Sx-Bp, surgery with buprenorphine analgesia; Sx-Bt, surgery with butorphanol analgesiaAmphibians are increasingly being used in biomedical research, especially in areas of pain research,^{2,8,11,12,15,18,20,21,23,25,28,­ 29,33,35,37-40,42,43,46} developmental biology,^{7,9,12,14,16,19,22,26,30,44,45,48} and toxicology.^1,5,6,42,47 Advances in laboratory animal care have improved scientific quality by providing healthier animals, standardizing husbandry, and decreasing confounding factors such as the effects of animal pain on studies. The ethical and legal call for refinement have made the research community more cognizant of alleviating or minimizing pain and distress in animals while performing valuable research.The Animal Welfare Act⁴ defines a painful procedure as any procedure that would reasonably be expected to cause more than slight or momentary pain or distress in a human being to which that procedure was applied, that is, pain in excess of that caused by injections or other minor procedures. Under this definition, surgical amputation of a limb would qualify and thereby require the use of analgesics. Although the Animal Welfare Act⁴ does not cover amphibians, as pointed out in a recent article,³ the Public Health Service Policy on Humane Animal Care³² does. Therefore, if an animal undergoes a painful surgical procedure, the animal should receive postoperative analgesics. Analgesic use can be withheld if the investigator provides scientific justification and the institutional animal care and use committee approves.The term ‘nociception’ refers to the perception of noxious stimuli, whereas definitions of pain include “an unpleasant sensory and emotional experience associated with actual or potential tissue damage, or described in terms of such damage”¹⁹ and “the emotional and complex perceptual experience in humans which requires a developed limbic and cortical system.”³⁴ Human pain is subjective in that it varies based on individual perception, even though essentially all people possess the components necessary to feel painful stimuli and experience pain emotionally. In response to the fact that pain differs for each individual within the human species, a recent article asked, “Why (then) should we accept that in animals, pain is what we say it is?”²⁷ Animals including fish, amphibians, birds, and mammals possess the components necessary to feel painful stimuli but may vary in how they experience it emotionally. In the case of amphibians, the strictest emotional definition of pain may not apply. However, for the purposes of the present study, ‘pain’ will be defined behaviorally to include both nociception and the emotional (reactive) response to pain, whereas ‘analgesia’ will include antinociception and the relief of the emotional response to pain.The objectives of this study were to determine whether 1) newts manifest observable changes in behavior that can be used to assess pain or distress³⁶; 2) buprenorphine given by intracoelomic injection or butorphanol in the tank water provides adequate analgesia; and 3) injected buprenorphine provides more effective analgesia than does butorphanol in the tank water.     

Pharmacokinetic Comparison of Sustained-Release and Standard Buprenorphine in Mice

Effective pain medication is important for animal stewardship and valid research results. We compared the pharmacokinetic assessments of standard, immediate-release buprenorphine (Bup IR) and a sustained-release buprenorphine formulation (Bup SR Lab) in male C57BL/6J mice, a mouse strain commonly used in biomedical research. We postulated that the administration of Bup SR Lab would achieve a more persistent blood drug concentration (>1 ng/mL) compared with single-dose Bup IR. The study assumed a blood buprenorphine concentration of 1 ng/mL as the minimum that may result in adequate analgesia, as previously reported. The 7 experimental groups included Bup IR (0.03, 0.05, 0.1, and 2 mg/kg), Bup SR Lab (0.3 and 1.2 mg/kg), and saline placebo (0.7 mL/100 g). Blood sampling occurred at 0.5, 1, 3, 6, 12, 24, 48, and 72 h for evaluation by using a forensic ELISA. Bup IR at 0.03 and 0.05 mg/kg and Bup SR Lab at 0.3 mg/kg failed to obtain maximal blood concentrations (C_max) above 1 ng/mL. All other doses (0.1 and 2 mg/kg Bup IR and 1.2 mg/kg Bup SR Lab) reached a C_max above 1 ng/mL within 3 h after injection. In addition, 1.2 mg/kg Bup SR Lab and 2 mg/kg Bup IR provided blood concentrations above 1 ng/mL for up to 12 h, and 0.1 mg/kg Bup IR achieved this criterion for as long as 3 h. In conclusion, Bup SR Lab at 1.2 mg/kg and Bup IR at 0.1 or 2.0 mg/kg achieve or surpass the published threshold for adequate analgesia in mice.Abbreviations: Bup IR, immediate-release buprenorphine; Bup SR Lab, sustained-released buprenorphine; C_max, maximal observed blood concentration; T_ThE, time to therapeutic effectiveness threshold; T_LAST, time at last quantifiable blood concentration; T_max, time to C_maxMice are the most widely used research animal, and providing them with adequate veterinary care during research studies is a vital component of implementing the basic tenets of ethical animal stewardship and of complying with animal welfare regulations and recommendations. Fundamental to this care is recognizing, preventing, assessing, and managing clinical pain.^2,13,15 Uncontrolled pain and distress can negatively affect animals’ quality of life and adversely influence research results.¹⁵ The use of an appropriate and effective pain medication is an important tool that is often used to minimize these effects.In the clinical setting, buprenorphine, a synthetic opiate classified as a partial μ agonist and κ antagonist, is a common systemic analgesic administered to rodents.^{5,7,8,11,21-23} Compared with other opioids, buprenorphine produces full analgesic effects, reduces respiratory depression, and minimally affects immune responses.^5,7,18,21,25 However, previously reported buprenorphine dose ranges and administration frequencies for different mice strains vary widely, making it a challenge to determine appropriate and effective analgesic dosages.^8,10,12,22 Recommended doses range from 0.05 to 0.1 mg/kg and frequencies from 1 to 8 doses daily.^8,12,16 The wide variations in daily administration recommendations may lead to fluctuations in bloodstream drug concentration and inconsistent analgesic control.¹⁷In an effort to provide consistent analgesia, sustained-release formulations of buprenorphine, including injectable forms and transdermal patches, have been developed and subsequently evaluated in mice and rats.^1,9,20,26 A United States veterinary compounding pharmacy has developed an injectable sustained-release buprenorphine that provides as much as 72 h of continuous analgesia in several species after a single injection.^3,9 This single injection in species other than mice results in a sustained plasma concentration over 1.0 ng/mL, the concentration associated with providing pain relief in humans, for as long as 72 h.^{3,4,9,11,19,24} However, a recent study in BALB/cJ and SWR/J mice specifically investigated the efficacy of a sustained-release buprenorphine formulation and of buprenorphine HCl by using thermal-contact response time methodology (hotplate test).¹ The authors concluded that the sustained-release formulation at 1.0 mg/kg provided an effective analgesic period of 12 h and that the clinical buprenorphine dose of 0.1 mg/kg provided little to no analgesic effect.¹ Comparing these recent findings with vendor-provided literature may lead to uncertainty regarding the appropriate dose of buprenorphine to achieve analgesia and the response of different mouse strains to similar dose–drug combinations. These findings also suggest that buprenorphine pharmacokinetics, in multidose and sustained-release formulations, are not clearly understood. Therefore, additional study is warranted to elucidate buprenorphine dosages appropriate for use in the clinical research environment.In this study, we postulated that a single dose of sustained-release buprenorphine would achieve more persistent blood drug concentration (>1 ng/mL) in mice than would a single dose of the typically used immediate-release buprenorphine (Bup IR). To determine whether these expected differences were significant, we used a pharmacokinetic study to evaluate the performance of standard Bup IR and of a recently available sustained-released buprenorphine (Bup SR Lab). The pharmacokinetic study examined the relationship between drug dose and the transient postinjection blood concentration of buprenorphine. Using a forensic ELISA method, we determined the time course of blood drug concentration after administration.¹⁴     

Effects of Buprenorphine, Meloxicam, and Flunixin Meglumine as Postoperative Analgesia in Mice

C57BL/6NCrl male mice (n = 60; age, 6 to 7 wk) underwent partial hepatectomy or no surgery and were given 1 of 3 analgesics pre- and postoperatively. Food and water consumption, body weight, running wheel activity, locomotor activity, and serum corticosterone concentrations were measured before and after surgery. Mice that were surgically manipulated weighed significantly less on days 1 through 3 after surgery than did mice not manipulated surgically. On the day of surgery, the surgery groups consumed significantly less feed (–1.5 ± 0.35 g) than did nonsurgery groups. There were no differences in water consumption on any day between surgery and nonsurgery groups or among the 3 analgesic groups. For running wheel activity, significant decreases in the surgery groups were seen at day 1 after surgery compared with baseline. Surgery groups that received buprenorphine and meloxicam returned to baseline activity levels on day 2 after surgery. Open-field testing revealed no significant differences in locomotor activity in any groups; however, posttreatment locomotor activity in the buprenorphine nonsurgery group was increased compared with baseline, and posttreatment locomotor activity in the flunixin meglumine surgery group was decreased compared with baseline. Serum corticosterone concentrations were within normal limits regardless of treatment in all groups. Comparison of the overall results indicated that meloxicam and buprenorphine, at the dose given, appear to be suitable postoperative analgesics for partial hepatectomy in mice. Flunixin meglumine at the given dosage (2.5 mg/kg) may not provide adequate analgesia for partial hepatectomy.Abbreviation: NSAID, nonsteroidal antiinflammatory drugLaboratory animals commonly undergo procedures that may cause pain and distress. The Guide for the Care and Use of Laboratory Animals states, “An integral component of veterinary medical care is prevention or alleviation of pain associated with procedural and surgical protocols. Pain is a stressor and, if not relieved, can lead to unacceptable levels of stress and distress in animals.”²⁰ Such deleterious effects, while adversely affecting an animal''s wellbeing, may also confound research data and impede research study outcome. As a result, prevention or recognition and minimization or alleviation of pain and distress are integral components of an animal care and use program. In rats, unrelieved pain and distress after a surgical procedure has been shown to affect locomotor activity, body weight, and food and water intake.^12,31,32 Although mice comprise the majority of research animals, few studies have evaluated analgesia effectiveness in this species. Studies in mice have shown that the lack of postoperative analgesia can affect locomotor activity¹⁵ and food and water intake.¹⁹Locomotor activity testing involving an open field has been used as a method to assess exploratory and locomotor activity in mice.^{17,21,37,38,41,46,49,55} The assessment of unconditioned locomotor behavior in rodents has become one of the most widely used behavioral paradigms to determine the effects of various experimental manipulations ranging from genetic changes to pharmacologic challenges.^41,46 Levels of locomotor activity are measured by using a variety of methods based on the frequency of photocell beam breaks or distance traveled as assessed by video tracking.Running wheel activity has been used in research for a variety of experiments.^8,30,48 Running wheels have been used to study the hypothalamic–pituitary–adrenocortical axis, circadian rhythms, and social-stress-affected sleep in mice.^28,53 This parameter has also been used to monitor voluntary activity levels of mice by measuring distance traveled.^8,48Commonly used analgesics include buprenorphine hydrochloride, flunixin meglumine, and meloxicam. Buprenorphine hydrochloride is used in rodent surgical models^{13,15,22,26,32,45} primarily due to its longer duration of action over other opioid drugs. It is a partial µ opiate agonist and has a maximum biologic effect achieved regardless of the maximal dose given (ceiling effect).^7,34 The duration of effect in most species is 6 to 12 h.⁴⁴ The duration of buprenorphine in mice has been documented as 3 to 5 h.¹⁴ As a controlled substance, buprenorphine is regulated by the Drug Enforcement Administration, which requires strict storage and record keeping.Flunixin meglumine and meloxicam are nonsteroidal antiinflammatory drugs (NSAID) that reduce inflammatory pain by the inhibition of prostaglandin synthesis.^10,42 Because NSAID are not controlled substances, federal licensure and other restrictive measures are not necessary. Although the drugs are considered safe, NSAID-associated gastrointestinal bleeding is listed as a side effect reported with chronic use.⁴² A recent study by our laboratory found that flunixin meglumine did not interfere with embryo implantation when used in mice undergoing embryo transfer.¹⁸ We wanted to further investigate the properties of flunixin meglumine as an analgesic agent in a different mouse surgical model. Because meloxicam is a long-acting NSAID requiring once-daily dosing, we were interested in evaluating the effects of meloxicam in a mouse surgical model.Evaluations of physiologic parameters and results of behavioral tests are often used to assess pain in rodents; however, there are very few reports on assessing pain relief in mice. Various parameters have been used to monitor rodents during the postoperative period, including ethographic scoring,⁶ body weight changes, corticosterone concentrations, and food and water consumption.^{3,12,16,31,32} Our laboratory previously evaluated whether isoflurane anesthesia would alter the body weight, voluntary exercise or open-field locomotor activity of mice administered either flunixin, meloxicam, or buprenorphine. We found that isoflurane anesthesia had no effect on these parameters with any of the 3 analgesics.The purpose of the current study was to evaluate the effects of buprenorphine hydrochloride, flunixin meglumine, and meloxicam in mice after partial hepatectomy. We hypothesized that there would be no differences in physiologic parameters in mice with the use of the 3 analgesics. Partial hepatectomy is a surgical procedure performed frequently in our facility for research studies, and we were interested in investigating the effect of the 3 analgesics in this animal model. These mice were used specifically for this study to refine the analgesic protocol for this surgical procedure. Voluntary running-wheel activity, open-field locomotor activity, and serum corticosterone concentrations were used in conjunction with body weight and food and water consumption as objective parameters for assessment of analgesic effectiveness.     

Efficacy of Various Analgesics on Shoulder Function and Rotator Cuff Tendon-to-Bone Healing in a Rat (Rattus norvegicus) Model

Although relief of postoperative pain is an imperative aspect of animal welfare, analgesics that do not interfere with the scientific goals of the study must be used. Here we compared the efficacy of different analgesic agents by using an established rat model of supraspinatus tendon healing and a novel gait-analysis system. We hypothesized that different analgesic agents would all provide pain relief in this model but would cause differences in tendon-to-bone healing and gait parameters. Buprenorphine, ibuprofen, tramadol–gabapentin, and acetaminophen were compared with a no-analgesia control group. Gait measures (stride length and vertical force) on the operative forelimb differed between the control group and both the buprenorphine (2 and 4 d postsurgery) and ibuprofen (2 d postsurgery) groups. Step length was different in the control group as compared with the tramadol–gabapentin (2 d after surgery), buprenorphine (2 and 4 d after surgery), and ibuprofen (2 d after surgery) groups. Regarding tendon-to-bone healing, the ibuprofen group showed less stiffness at the insertion site; no other differences in tendon-to-bone healing were detected. In summary, the analgesics evaluated were associated with differences in both animal gait and tendon-to-bone healing. This information will be useful for improving the management of postsurgical pain without adversely affecting tissue healing. Given its ability to improve gait without impeding healing, we recommend use of buprenorphine for postsurgical pain management in rats. In addition, our gait-analysis system can be used to evaluate new analgesics.The relief of postprocedural pain and distress is an imperative aspect of animal welfare. However adequate analgesia must be achieved without adverse effects on the goals of the study. Therefore, the detection and management of pain in animal research models is continually being studied and refined. Postprocedural pain is a complex process that involves hypersensitivity and hyperalgesia to several stimuli.^8,9,19,32,68 Furthermore, surgical procedures can cause pain through inflammation and the manipulation and damage of tissues.¹ Several methods for evaluating postprocedural pain in rodents involve variably subjective scoring systems and assessment of in-cage locomotor and behavior activity, hypersensitivity to stimuli, or observing food and water intake.^{37,39,43,53-55,61,63} An objective functional assessment test may provide a more reliable and quantifiable way to measure postoperative pain.Several rodent models to study musculoskeletal injuries are currently being used in biomedical research,^6,29,34 including a well-established rat model for rotator cuff injury.^49,51,56,60 This surgical model involves considerable injury to and manipulation of both bone and soft tissues. Because “it should be considered that procedures that cause pain in humans may also cause pain in vertebrate species,” it is clear that this model would also serve as a good model for significant postprocedural pain.^27,40,65 The objective of the current study was to compare the efficacy of different analgesic agents by using an established rat model for supraspinatus tendon healing and a novel gait-analysis system.⁵⁶We assessed different classes of analgesics, which we chose to represent common recommendations for postprocedural care. Buprenorphine is one of the most commonly used analgesics in laboratory animal medicine due to its proven analgesic qualities in rodents and other species.^{13,17,25,26,58} However, its status as a controlled substance may limit its use, and other options may be desirable. NSAID are often chosen for the management of postprocedural analgesia in both rodents and humans.^16,25,26,38 Because ibuprofen is used frequently after tendon repair in human medicine, we selected it for analysis in the current study. Due to the ease of administration, putting analgesics like acetaminophen in the drinking water of rodents has been a popular suggestion recently.^4,17,62 However, numerous studies have found variation in analgesic efficacy in rodents using acetaminophen in the drinking water.^{11,33,45,50,64} Finally, a tramadol–gabapentin combination was recently reported to have some analgesic effects in rats, but additional research is required.⁴⁴A secondary goal of the current study was to determine whether these commonly used analgesics affect tendon-to-bone healing. NSAID may have adverse effects on tendon healing,^10,14 but these are far less studied than are their effects on the healing of bone.^21,41,59 In addition, pain may influence cage activity levels, which consequently could change with the application of analgesics.^35,39,53 Increased activity may alter loads on the healing tissue as well as joint mobility, thus affecting tendon healing.^7,24,47,69In the current study, we used various spatial, temporal, and force parameters to analyze gait in the rat model of rotator cuff healing. In other species, pain in a forelimb decreases stride length, limb speed, and gait forces.^28,31,36,46 We expected to find similar changes in the gait of rats after surgery when analgesia is inadequate. Our custom gait-analysis system allowed us to measure several parameters, which were compared between treated and control groups to determine whether significant differences occurred. We used biomechanical testing procedures to determine how changes in tendon-to-bone healing after repair differed among the various analgesics. The weakest point of the tendon is the healing site, because of the development of new immature tissue, and changes in the mechanical properties of the repaired tendon indicate alterations in healing. Therefore, poor healing leads to decreases in the mechanical properties of the repaired tendon.^22,23 We hypothesized that the different analgesics evaluated all would provide pain relief in this model but would demonstrated differences in tendon-to-bone healing and in gait parameters compared with those of a no-analgesia control group.     

Analgesic Effects of Tramadol, Tramadol�CGabapentin, and Buprenorphine in an Incisional Model of Pain in Rats (Rattus norvegicus)

Postoperative pain management in laboratory animals relies heavily on a limited number of drug classes, such as opioids and nonsteroidal antiinflammatory drugs. Here we evaluated the effects of saline, tramadol, tramadol with gabapentin, and buprenorphine (n = 6 per group) in a rat model of incisional pain by examining thermal hyperalgesia and weight-bearing daily for 6 d after surgery. All drugs were administered preemptively and continued for 2 consecutive days after surgery. Rats treated with saline or with tramadol only showed thermal hyperalgesia on days 1 through 4 and 1 through 3 after surgery, respectively. In contrast, buprenorphine-treated rats showed no thermal hyperalgesia on days 1 and 2 after surgery, and rats given tramadol with gabapentin showed reduced thermal hyperalgesia on days 2 and 4. For tests of weight-bearing, rats treated with saline or with tramadol only showed significantly less ipsilateral weight-bearing on day 1 after surgery, whereas rats given either buprenorphine or tramadol with gabapentin showed no significant change in ipsilateral weight-bearing after surgery. These data suggest that tramadol alone provides insufficient analgesia in this model of incisional pain; buprenorphine and, to a lesser extent, tramadol with gabapentin provide relief of thermal hyperalgesia and normalize weight-bearing.Abbreviation: GABA, γ-aminobutyric acidPain management poses considerable challenges within the field of veterinary medicine. Administration of many analgesic drugs may cause unwanted side effects, creating an inappropriate model for the researcher. Opioids and nonsteroidal antiinflammatory drugs both may interfere with immunologic studies and behavioral testing.^11,40 Effective pain control improves the recovery process, helps animals to return to normal behaviors, and maintains the physiologic status of animals.²⁵ Preemptive and multimodal analgesic approaches are recommended to obtain optimal pain control. When doubt exists as to whether an animal is in pain, analgesic drugs should be administered.Postoperative pain is one of the most common types of pain in the laboratory animal setting. It is a complex multisystemic response, with hypersensitivity to many stimuli,²⁵ including hyperalgesia to thermal and mechanical^6,7,18,44 stimuli. Although many opiates blunt this sensitization, other classes of drugs (N-methyl-D-aspartate, antidepressants, and others) have various effects, depending on the modality of pain studied.^33,45Buprenorphine, a partial μ-opioid receptor agonist, is a commonly used postoperative analgesic drug for many laboratory animal species due to its prolonged plasma half-life.¹⁶ Prior studies showing decreased incidence of ceiling effects or side effects, such as respiratory depression,¹⁴ in addition to well-established analgesia, make buprenorphine an appropriate choice for alleviation of mild to moderate pain.^31,37 Recent evidence suggests that the submaximal response to buprenorphine at high doses may be due to interaction with the non-μ class of opioid-receptor-like (ORL1) receptors which, when upregulated, may attenuate the analgesic effects of buprenorphine.^27,28 These data, coupled with the resistance of buprenorphine to naloxone reversal,¹⁹ add to the complexity of its analgesic mechanism and subsequent potential side effects.Tramadol, a centrally acting analgesic, is known to provide pain relief by means of its primary metabolite, O-desmethyl-tramadol (approximate 600-fold higher μ-opioid receptor affinity than that of tramadol and 10-fold lower than that of morphine) through interaction with opioid receptors and the blockade of serotonin.^4,21,43 Tramadol also acts on the descending pain pathway through norepinephrine and seratonin uptake inhibition for antinociceptive effects at the level of the medulla through the α-adrenergic pathway. These early studies may explain in part tramadol''s lack of complete naloxone-induced reversal and withdrawal.²¹ Gabapentin is an antiepileptic drug which is a structural analog of γ-aminobutyric acid (GABA). Despite the suggestive name, gabapentin lacks demonstrable interaction with either GABA_A or GABA_B receptors within the CNS. Like other anticonvulsant drugs, gabapentin has been studied for the additional potential benefit of analgesia in models of neuropathic pain.²⁹ More recently, evidence has been collected supporting the potential efficacy of gabapentin in blocking other pain pathways, surgical and inflammatory pain being 2 such examples.^9,10 Although the mechanism of action contributing to these analgesic effects has yet to be elucidated fully, several studies have documented the utility of gabapentin, especially when combined with morphine.^15,30 In 1996, several authors proposed a novel mechanism of action, showing high affinity of gabapentin for the α₂δ subunit of a centrally located voltage-dependent calcium channel.²⁰ Other groups have examined further potential analgesic qualities of gabapentin and suggest its utility as an antihyperalgesic rather than an antinociceptive agent.¹⁷The potential nonopioid analgesic action of both tramadol and gabapentin, coupled with gabapentin''s lack of cross tolerance when administered chronically in combination with morphine¹⁵ and the lack of a withdrawal response for after discontinuation of tramadol,²¹ make these drugs ideal candidates for study in adjunctive therapies in analgesia. An additional benefit is that neither tramadol nor gabapentin is a federally controlled substance category, thereby enhancing their practicality in the laboratory setting. Our goal in the current study was to test the hypothesis that the analgesic effects of tramadol used alone or in combination with gabapentin in a rat model of incisional pain are similar to or better than those of buprenorphine alone.     

Eliminating Animal Facility Light-at-Night Contamination and Its Effect on Circadian Regulation of Rodent Physiology, Tumor Growth, and Metabolism: A Challenge in the Relocation of a Cancer Research Laboratory

Appropriate laboratory animal facility lighting and lighting protocols are essential for maintaining the health and wellbeing of laboratory animals and ensuring the credible outcome of scientific investigations. Our recent experience in relocating to a new laboratory facility illustrates the importance of these considerations. Previous studies in our laboratory demonstrated that animal room contamination with light-at-night (LAN) of as little as 0.2 lx at rodent eye level during an otherwise normal dark-phase disrupted host circadian rhythms and stimulated the metabolism and proliferation of human cancer xenografts in rats. Here we examined how simple improvements in facility design at our new location completely eliminated dark-phase LAN contamination and restored normal circadian rhythms in nontumor-bearing rats and normal tumor metabolism and growth in host rats bearing tissue-isolated MCF7(SR^–) human breast tumor xenografts or 7288CTC rodent hepatomas. Reducing LAN contamination in the animal quarters from 24.5 ± 2.5 lx to nondetectable levels (complete darkness) restored normal circadian regulation of rodent arterial blood melatonin, glucose, total fatty and linoleic acid concentrations, tumor uptake of O₂, glucose, total fatty acid and CO₂ production and tumor levels of cAMP, triglycerides, free fatty acids, phospholipids, and cholesterol esters, as well as extracellular-signal-regulated kinase, mitogen-activated protein kinase, serine–threonine protein kinase, glycogen synthase kinase 3β, γ-histone 2AX, and proliferating cell nuclear antigen.Abbreviation: 13-HODE, 13-hydroxyoctadecadienoic acid; γH2AX, histone 2AX; AKT, serine–threonine protein kinase; ERK1/2, extracellular signal-regulated kinase p44/46; GSK3β, glycogen synthase kinase 3β: LAN light at night; MEK, mitogen-activated protein kinase kinase, PCNA, proliferating cell nuclear antigen; SR^–, steroid-receptor–negativeRelocating laboratory animal research from one institution to another can be a daunting task for both scientists and animal care personnel with regard to control of lighting and elimination of light-at-night (LAN) contamination. Appropriate facility lighting and lighting protocols, as outlined in the Guide for the Care and Use of Laboratory Animals,³⁰ are essential for maintaining the health and wellbeing of laboratory animals and ensuring the credible outcome of scientific investigations.^16-18,22 The profound effect of light on circadian behavior and physiology is well established.^{2,3,5,9,11,12,16-18,22,29,32,44,46,49,52,55-58,64}Minor alterations in light intensity,¹¹ spectral quality,¹² and duration⁹ at any given time of day can alter or disrupt chronobiologic rhythms markedly in all mammals.^{6,17,26,44,55-59} Light information, which initially is detected by a small group of intrinsically photosensitive retinal ganglion cells containing the blue light-sensitive photopigment melanopsin,^6,26 is transmitted through the retinohypothalamic tract⁵⁹ to a central molecular clock located in the suprachiasmatic nucleus of the hypothalamus.^32,57 The suprachiasmatic nucleus, the activity of which is entrained by the light:dark cycle,^32,57 sends projections over a polysynaptic pathway to the pineal gland driving a series of molecular events leading to the production of the pineal neurohormone melatonin (N-acetyl-5 methoxytryptamine), primarily during the night.^29,46 The daily rhythmic melatonin signal contributes to the temporal coordination of normal behavioral and physiologic functions including the sleep–wake^23,46,66 and reproductive cycles,^51,55 immune function,^38,41,56 hormone levels,^{19,31,45,47,68} temperature regulation,²³ electrolyte balance,⁶⁹ neural protein synthesis,⁶⁵ and redox states.^24,53Dark-phase LAN exposure suppresses endogenous melatonin concentrations and may lead to various disease states,^42,58 including carcinogenesis,^7,8,16,18,33 and metabolic syndrome.^{17,34-37,39,70} Previous in vivo studies in our former laboratory (at the Bassett Research Institute, Cooperstown, NY) demonstrated that animal room LAN of as little as 0.2 lx (0.08 µW/cm²; rodent eye level) during an otherwise normal dark-phase suppressed normal physiologic nighttime melatonin levels, leading to markedly disrupted circadian regulation of physiology and metabolism in nontumor-bearing host animals^16,18 and a stimulation in metabolism and proliferation of both tissue-isolated MCF7 steroid-receptor–negative (SR^–) human breast cancer xenografts and syngeneic grafts of rodent hepatoma 7288CTC in rats.^7,17 This effect was mediated by melatonin receptor-mediated suppression of cAMP, leading to inhibition of tumor linoleic acid uptake and its metabolism to the mitogenic signaling molecule 13-hydroxyoctadecadienoic acid (13-HODE). These events culminated in downregulation of epidermal growth factor and insulin-like growth factor 1 pathways.^7,8,16-19,62Exposure to LAN is likely to exert pervasive and problematic effects on mammalian behavior and physiology in laboratory animal facilities around the world. During the past decade, improved facility design and better adherence to animal room lighting protocols certainly has helped to reduce the problem. In moving to our laboratory animal facility at Tulane University School of Medicine (New Orleans, LA), we discovered considerable preexisting LAN contamination that had to be eliminated before we could resume our human cancer research.The current study was performed to monitor the effects of the elimination of animal room LAN contamination over time on animal health and wellbeing, tumor growth, and metabolic profiles by assessing well-established circadian parameters in physiology and metabolism.^7,8,16-18 We measured light-induced suppression of melatonin, an accepted and sensitive marker of the effects of light on the circadian system in all mammals,^{2,3,5,9,11,12,15,16,18,20,21,29,44,46,49,52,55-58,64} before and after tumor implantation and growth. Tissue-isolated MCF7(SR^–) human breast cancer xenografts and 7288CTC rat hepatomas have been well-characterized over the years in our former light-tight facilities^7,8,16-18 and provided us with unique markers and measures of the extent to which LAN contaminated our new animal quarters. In tumor-bearing animals exposed to even minimal LAN, the latency-to-onset of tumor growth and proliferation rates of these tumors increase markedly in direct proportion to LAN intensity. As improvements were made to eliminate LAN contamination in the new location over the course of more than 20 generations of tumor passages, we measured the changes and reestablishment of normal rat and host–tumor circadian regulation. The information from this study may assist investigators and animal care personnel in addressing this important influence on the health and wellbeing of laboratory animals and consequent effects on the outcome of scientific investigations.     

Clinical Efficacy of Sustained-Release Buprenorphine with Meloxicam for Postoperative Analgesia in Beagle Dogs Undergoing Ovariohysterectomy

The goal of the current study was to compare the efficacy, adverse effects, and plasma buprenorphine concentrations of sustained-release buprenorphine (SRB) and buprenorphine after subcutaneous administration in dogs undergoing ovariohysterectomy. In a prospective, randomized, blinded design, 20 healthy adult female Beagle dogs underwent routine ovariohysterectomy and received multimodal analgesia consisting of meloxicam and one of two buprenorphine formulations. Dogs were randomly assigned to receive either SRB (0.2 mg/kg SC, once) or buprenorphine (0.02 mg/kg SC every 12 h for 3 d). Blinded observers assessed all dogs by using sedation scores, pain scores, temperature, HR, RR, and general wellbeing. Dogs were provided rescue analgesia with 0.02 mg/kg buprenorphine SC if the postoperative pain score exceeded a predetermined threshold. Blood samples were collected, and mass spectrometry was used to determine plasma buprenorphine concentrations. Data were analyzed with a linear mixed model and Tukey–Kramer multiple comparison. Age, body weight, anesthetic duration, surgical duration, sevoflurane concentration, and cardiorespiratory variables did not differ significantly between groups. Dogs in both formulation groups had comparable postoperative sedation and pain scores. One dog from each formulation group had breakthrough pain requiring rescue analgesia. Plasma buprenorphine concentrations remained above a hypothesized therapeutic concentration of 0.6 ng/mL for 136.0 ± 11.3 and 10.67 ± 0.84 h for SRB and buprenorphine, respectively. Based on the results of this study, multimodal analgesic regimens consisting of meloxicam and either buprenorphine or SRB are equally efficacious in managing pain associated with an ovariohysterectomy and show comparable side effects.Abbreviations: HR, heart rate; RR, respiratory rate; SRB, sustained-released buprenorphineBuprenorphine, a semisynthetic opioid analgesic, is a common component of veterinary multimodal pain management. It has a strong affinity for the μ opioid receptor and slow dissociation kinetics, resulting in a longer duration of action than that of other opioid analgesics. The combination of the long duration of action, low risk of respiratory depression,²² and negligible cardiovascular effects^8,23 in healthy dogs make buprenorphine an advantageous opioid analgesic agent for use with procedures associated with mild to moderate pain, including ovariohysterectomy surgery in companion animals. Numerous administration routes, including intravenous,²⁵ intramuscular,^21,29,30 subcutaneous,²⁴ oral transmucosal,¹⁹ and transdermal²⁴ have been reported in dogs with a high level of success in managing postoperative pain associated with ovariohysterectomy. Due to buprenorphine''s slow onset of peak effect (45 to 60 min^2,27), it is generally given preoperatively to provide sufficient time for onset of action. Buprenorphine may have a ceiling effect, and one study demonstrated that increasing the dose from 0.02 to 0.04 mg/kg in dogs undergoing ovariohysterectomy did not increase the analgesic effect.³⁰Recently, a new sterile, compounded sustained-release formulation of buprenorphine became available, and the compounder suggests that a single dose of the formulation can provide as long as 72 h of analgesia in dogs, on the basis of unpublished plasma buprenorphine concentrations. To date, clinical efficacy has been demonstrated for a maximum of 72 h in cats undergoing ovariohysterectomy and in rats undergoing surgical production of a tibial defect¹⁰ and 12 h in a hot-plate assay in male mice.⁶ Pharmacokinetic studies, performed in rats¹⁰ and macaques,²⁶ have confirmed sustained, high plasma buprenorphine concentrations in those species. Although the efficacy of this buprenorphine formulation is largely untested, it has potential to decrease the number of postoperative analgesic injections and improve animal welfare by further minimizing pain and distress.Previous studies have demonstrated the importance of multimodal analgesia^28,29 in postoperative dogs, given that breakthrough pain can occur when buprenorphine^19,29 or a NSAID^20,29 is administered alone. As a result, multimodal analgesia has become common clinical practice. A downside of multimodal analgesia is the potential number of medications that are required and their respective dosing frequencies. Analgesic plans have become increasingly complex, leading to potential misdosing (incorrect or missed dose) in animals in the postprocedural period. To address this problem, there is a need to identify effective multimodal analgesia strategies that are easy for staff to follow and thus minimize misdosing, provide adequate analgesia, and promote animal welfare.The objective of the current study was to evaluate the clinical efficacy and pharmacokinetics of a simplified multimodal analgesic regimen for healthy adult dogs undergoing routine ovariohysterectomy. The clinical efficacy and pharmacokinetics of sustained-release buprenorphine (SRB) and buprenorphine were directly compared in dogs undergoing ovariohysterectomy and receiving meloxicam. Dogs received 0.2 mg/kg meloxicam IV and 0.2 mg/kg SRB SC or 0.02 mg/kg buprenorphine SC prior to surgery. Postoperatively, all dogs received 0.1 mg/kg meloxicam PO once daily for 4 d. Dogs that received buprenorphine were dosed at 0.02 mg/kg SC every 12 h for 3 d, and dogs that received SRB were dosed with saline subcutaneously every 12 h for 3 d at volumes comparable to that of the buprenorphine dose. Clinical efficacy was assessed by using sedation scoring, behavioral pain scoring, temperature, heart rate (HR), respiratory rate (RR), gastrointestinal side effects, injection site reactions and the need for rescue analgesia during the postoperative monitoring period. In addition, blood was collected at regular intervals over the 7-d postoperative period, for pharmacokinetic purposes and assessment of the therapeutic plasma buprenorphine concentration.     

Comparison of Buprenorphine and Meloxicam for Postsurgical Analgesia in Rats: Effects on Body Weight, Locomotor Activity, and Hemodynamic Parameters

Buprenorphine is administered to humans and animals for postoperative pain management, although its use is associated with complications. Alternative analgesics, including the nonsteroidal antiinflammatory meloxicam, are available, but information on their postoperative effects is limited. The objective of the present study was to compare buprenorphine (0.03 mg/kg SC twice daily for 3 d) with meloxicam (2 mg/kg SC initial dose followed by 1 mg/kg SC once daily for 2 d) by assessing parameters relating to postsurgical recovery in rats that underwent surgical implantation of radiotelemetric transducers. Rats treated after surgery with buprenorphine showed greater reductions in body weight, food consumption, locomotor activity, and nighttime heart rates than did meloxicam-treated rats. Buprenorphine and meloxicam treatments both had stimulatory effects on mean arterial pressure and daytime heart rate measurements, although effects on nighttime mean arterial pressure were greater in the buprenorphine-treated rats. In summary, the lesser physiologic changes associated with meloxicam, as compared with buprenorphine, suggest that meloxicam offers advantages for use as a postoperative analgesic after laparotomy and radiotelemetric transducer implantation in rats.Abbreviation: HR, heart rate; MAP, mean arterial pressure; WD, Western dietIn collaboration with facility veterinarians, researchers who perform invasive surgical techniques must decide on an appropriate postoperative pain management regimen. From an ethical perspective, minimizing an animal''s suffering should be a primary concern. Moreover, inadequate pain management can compromise a study''s outcome, because activation of the pain cascade can influence physiologic function.¹⁰ In addition, the full pharmacodynamic spectrum of the analgesic drug should be considered, because any of its activities could affect a study''s endpoints. Therefore, appropriate selection of drugs for management of postoperative pain is not a trivial decision.Implantation of radiotelemetric transducers is an invasive but necessary surgical procedure for direct and chronic measurement of hemodynamic parameters in animals. Although noninvasive techniques for blood pressure assessments are available, direct assessment by implantation of arterial catheters is generally considered to yield far superior data,¹³ notwithstanding the cost and surgical expertise required. In rats, implantation of transducers involves an abdominal incision of approximately 4 to 5 cm, visceral manipulation, implantation of a small catheter into the abdominal aorta, suturing the transducer to the inner muscular layer, and finally suturing the abdominal incision.Postoperative analgesia for telemetry implantation, like many other surgeries, often consists of opioid analgesics such as buprenorphine,^9,11,20 a partial µ-opioid receptor agonist. Buprenorphine treatment has been shown to improve overall indications of pain and surgical stress, including postoperative food and water intake, changes in body weight, and ambient locomotor activity, compared with those of untreated controls.²⁰ Nevertheless, there can be unwanted effects associated with buprenorphine use, including rebound hyperalgesia,²³ variability in potency between sexes and strains of rodents,⁷ and issues with food intake (such as anorexia and pica).^6,15 Furthermore, assessment of postoperative pain and recovery are complicated, given that buprenorphine treatment affects growth rate, feeding, and locomotor activity in naive rats;^4,15,19 thus, discriminating between behavioral cues caused by pain compared with those due to buprenorphine treatment per se is problematic. Finally, as an opioid analgesic, buprenorphine use is controlled in most jurisdictions, thereby complicating its availability.Alternative therapeutics for treating postoperative pain include nonsteroidal antiinflammatory drugs such as meloxicam.²¹ Meloxicam acts by inhibiting cyclooxygenases and thus preventing the synthesis of prostaglandin H₂, which is a precursor to mediators that elicit pain and inflammation.²⁴ Although meloxicam is a potential alternative treatment for surgery in rats, knowledge of its effects on recovery and cardiovascular parameters after surgery is incomplete. The objective of this study was to compare the postoperative recovery in rats treated with either buprenorphine or meloxicam for management of pain after surgical implantation of telemetric transducers.     

Progression of Ulcerative Dermatitis Lesions in C57BL/6Crl Mice and the Development of a Scoring System for Dermatitis Lesions

Ulcerative dermatitis (UD) is a common, spontaneous condition in mice with a C57BL/6 background. Although initial lesions may be mild, UD is a progressive disease that often results in ulcerations or debilitating fibrotic contractures. In addition, lesions typically are unresponsive to treatment. Euthanasia is often warranted in severe cases, thereby affecting study outcomes through the loss of research subjects. Because the clinical assessment of UD can be subjective, a quantitative scoring method and documentation of the likely time-frame of progression may be helpful in predicting when animals that develop dermatitis should be removed from a study. Such a system may also be helpful in quantitatively assessing success of various treatment strategies and be valuable to clinical laboratory animal veterinarians. In this 1.5-y, prospective cohort study, we followed 200 mice to monitor the development and course of UD. Mice were examined every 2 wk. A clinical sign (alopecia, pruritus, or peripheral lymphadenopathy) was not identified that predicted development of UD lesions in the subsequent 2-wk period. Once UD developed, pruritus, the character of the lesion (single or multiple crust, coalescing crust, erosion, or ulceration), and the size of the lesion were the only parameters that changed (increased) over the course of the disease. Pruritus was a factor in the rapid progression of UD lesions. We used these findings to develop a quantitative scoring system for the severity of UD. This enhanced understanding of the progression of UD and the quantitative scoring system will enhance the monitoring of UD.Abbreviation: UD, ulcerative dermatitis; S number, scratching number; COL, character of lesionsUlcerative dermatitis (UD) is an idiopathic, spontaneous, debilitating syndrome of laboratory mice that is typically a disease of aged^1,19,43,46 C57BL/6 mice or genetically engineered mice on a C57BL/6 background.^1,19,43,44 Some reports discuss a similar condition in young, weanling mice that presents initially as alopecia.^24,42,44,45 Prevalence rates of UD between 4.1% to 21% have been reported.^1,6,19 Although no etiology has been identified, environmental factors,^{6,19,41,42,44} diet,^{5,29,41,42,46} season,^19,41,43,44 age at weaning,⁴² alopecia,^24,42,44,45 sex,^19,39,41,43 immune complex vasculitis,¹ follicular dysplasia,⁴⁴ lesion location,²⁰ and deficiencies in vitamin A metabolism⁴⁴ have all been implicated as predisposing factors for disease development. In addition, oronasal pain and chronic inflammation may lead to self-mutilation as a result of, initiating an “itch” response.¹⁰ UD is diagnosed by ruling out other causes of dermatitis in laboratory mice, such as fur mites,⁹ infections, fight wounds,¹⁷ strain phenotype,^15,35,40,49 and experimentally induced dermatitis.^4,50 Other diagnostic criteria are based on professional judgment and may include strain (C57BL/6 background),^1,19,44 lesion location (head and dorsal thorax),^1,19,43,44 intense pruritus,^1,19,44 peripheral lymphadenopathy,^6,19,39 and failure to respond to treatment.¹⁹ The rapid progression of UD lesions results in significant morbidity in laboratory mice.^6,19,44 Typically, the lesions progress to large, irregularly shaped, confluent ulcerations on the dorsal cervical and thoracic region.^1,19,39,44 As the lesions heal, contracted scar tissue forms, which can impair species-typical behaviors and mobility.^39,41,43,44 The presence of large dermal ulcerations or debilitating contractures affect animal welfare and typically necessitate euthanasia of affected mice. Although reports on the later stages of UD have been consistent,^{1,19,39,41,43,44} information on the initiation and progression of UD lesions is conflicting. Pruritus,^1,19,44 pain,¹⁰ and genetic predisposition^1,19,43,44 have been implicated as initiators of the disease. Alopecia, pruritus, erythema, and single or multiple(s) crust have all been reported as early signs of the disease.^{1,19,39,42-44} However, the majority of this information has been collected retrospectively, at timed necropsies, or based on anecdotal reports.Scoring systems are useful tools to evaluate clinical diseases in laboratory animals. For example, scoring systems have been published for tumors,^14,28 body condition,^14,28,47 and neurologic phenotype¹³ in mice to aid in assessment of clinical disease severity. Even though the progressive and severe nature of UD typically warrants eventual euthanasia, determining the severity of disease has typically been based on professional judgment,^1,39 subjective scoring,^{12,15,19,40,43,48,49} or postmortem histology.^40,44 A quantitative scoring system for UD in live mice has not been described and could greatly aid laboratory animal veterinarians and researchers in determining the severity of the disease and response to treatment.The purpose of the current study is to investigate clinical parameters that reflect the progression of UD to facilitate management and veterinary care of mice with UD. We followed 200 mice from 3 wk of age until the development of UD to determine the initial signs and progression of UD lesions. We hypothesized that mice will first develop signs of pruritus prior to any clinical lesion. From there, we predicted that clinical lesions will progress stepwise though the following stages of severity, with or without alopecia: (1) excoriations; (2) a single, small punctuate crust; (3) multiple, small punctuate crusts; (4) coalescing crust; (5) erosion; and (6) ulceration. In addition, we hypothesized that a quantitative, validated, and reliable UD scoring system can be created that is based on physical examination parameters that do, in fact, predict development and progression of UD. Having a more thorough understanding of the initiation and progression of ulcerative lesions likely will enhance our ability to predict the outcome for a given mouse and develop earlier end-points for that mouse. Furthermore, use of this scoring system will enable accurate monitoring of UD lesions.     

Postoperative Analgesia Due to Sustained-Release Buprenorphine,Sustained-Release Meloxicam,and Carprofen Gel in a Model of Incisional Pain in Rats (Rattus norvegicus)

Postoperative analgesia in laboratory rats is complicated by the frequent handling associated with common analgesic dosing requirements. Here, we evaluated sustained-release buprenorphine (Bup-SR), sustained-release meloxicam (Melox-SR), and carprofen gel (CG) as refinements for postoperative analgesia. The aim of this study was to investigate whether postoperative administration of Bup-SR, Melox-SR, or CG effectively controls behavioral mechanical and thermal hypersensitivity in a rat model of incisional pain. Rats were randomly assigned to 1 of 5 treatment groups: saline, 1 mL/kg SC BID; buprenorphine HCl (Bup HCl), 0.05 mg/kg SC BID; Bup-SR, 1.2 mg/kg SC once; Melox-SR, 4 mg/kg SC once; and CG, 2 oz PO daily. Mechanical and thermal hypersensitivity were tested daily from day–1 through 4. Bup HCl and Bup-SR attenuated mechanical and thermal hypersensitivity on days 1 through 4. Melox-SR and CG attenuated mechanical hypersensitivity–but not thermal hypersensitivity–on days 1 through 4. Plasma concentrations, measured by using UPLC with mass spectrometry, were consistent between both buprenorphine formulations. Gross pathologic examination revealed no signs of toxicity in any group. These findings suggest that postoperative administration of Bup HCl and Bup-SR—but not Melox-SR or CG—effectively attenuates mechanical and thermal hypersensitivity in a rat model of incisional pain.Abbreviations: Bup HCl, buprenorphine HCl; Bup-SR, sustained-release buprenorphine; CG, carprofen gel; Melox-SR, sustained-release meloxicamPostoperative analgesia is a vital aspect of laboratory animal medicine. Investigators have a responsibility to follow an effective and safe pain management protocol for research animals that have undergone surgical procedures. Pain and distress are serious animal welfare concerns that directly affect animal physiology and can result in altered research data.^1,17,30 Continued refinement of pre-, intra-, and postoperative pain management in rodents is necessary to improve animal wellbeing, obtain high-quality research data, and ensure compliance with standards set forth by the Guide for the Care and Use of Laboratory Animals.²¹Many classes of analgesics are available to veterinary practitioners, but in the laboratory setting, the options tend to be simpler and typically involve 1 of 2 drug classes, opioids and NSAID. Buprenorphine HCl (Bup HCl), a partial μ-opioid receptor agonist, has long been the ‘gold standard’ for postoperative analgesia in laboratory animals due to the drug''s prolonged plasma half-life and effective analgesic properties.^15,28 Buprenorphine effectively controls mild to moderate postoperative pain in rodents for 6 to 12 h.¹⁶ Because many rodent surgical procedures might cause pain for at least 48 h, researchers must handle these animals at least twice daily during this time period to readminister buprenorphine. Repeated dosing requires frequent handling of surgically manipulated animals, resulting in handling-associated stress.¹ In addition, handling an animal frequently likely is disruptive to its cagemates and potentially to animals in the same room. Because of their analgesic and antiinflammatory properties, NSAID are often used either in conjunction with or as an alternative to opioids to control pain in laboratory animals.^11,33 Meloxicam and carprofen are 2 NSAID that preferentially inhibit cyclooxygenase 2 and thus prostaglandin synthesis.^10,11 Although generally considered safe, reported side effects of NSAID include gastrointestinal ulceration, altered platelet function, and renal dysfunction.¹¹Novel formulations of opioid and NSAID analgesics have recently been introduced to the veterinary market and include sustained-release injectables,^2,5,14,22 gel-based oral compounds,^6,19 and transdermal patches.^13,18,25,37 Our group previously demonstrated the effectiveness of sustained-release buprenorphine (Bup-SR) in controlling mild to moderate incisional pain in rats.⁷ Another study found that Bup-SR successfully controlled orthopedic surgical pain in rats.¹⁴ These alternative formulations show great potential in decreasing the stress associated with frequent handling and dosing requirements. Many of these products are still considered new in the veterinary market, and few evidence-based recommendations for their use in laboratory animal species are available. The main goal of the current study was to refine postoperative analgesia by using longer-lasting or gel-formulation products. To this end, we investigated whether Bup-SR, sustained-release meloxicam (Melox-SR), or carprofen gel (CG) provided postoperative analgesia in the rat plantar incisional model according to results of behavioral testing. We hypothesized that Bup-SR, Melox-SR, and CG would provide effective postoperative analgesia as evidenced by reduced pain responses in this model.     

Comparison of the Effects of Ketamine, Ketamine�CMedetomidine, and Ketamine�CMidazolam on Physiologic Parameters and Anesthesia-Induced Stress in Rhesus (Macaca mulatta) and Cynomolgus (Macaca fascicularis) Macaques

This study compared the cardiovascular, respiratory, anesthetic, and glucocorticoid effects of ketamine alone with ketamine–medetomidine and ketamine–midazolam in rhesus and cynomolgus macaques. Macaques were given either intramuscular ketamine (10 mg/kg), intramuscular ketamine–medetomidine (3 mg/kg; 0.15 mg/kg), or oral midazolam (1 mg/kg) followed by intramuscular ketamine (8 mg/kg). The addition of medetomidine, but not midazolam, provided muscle relaxation and abolishment of reflexes that was superior to ketamine alone. In addition, medetomidine did not cause clinically relevant effects on cardiovascular and respiratory parameters when compared with ketamine. These 3 protocols did not have significantly different effects on fecal glucocorticoid metabolites. These results suggest that medetomidine can be a valuable addition to ketamine for healthy patients, whereas oral midazolam at the tested dose does not provide additional benefits.Abbreviations: KetMed, ketamine–medetomidine, KetMid, ketamine–midazolam, NHP, nonhuman primates, PaCO₂, arterial partial pressure of carbon dioxide, PaO₂, arterial partial pressure of oxygen, SO₂, oxygen saturation of hemoglobin, SaO₂, arterial oxygen saturation, SpO₂, peripheral oxygen saturation, T, timeKetamine hydrochloride is commonly used as a sole anesthetic agent in nonhuman primates (NHP) but has several drawbacks that could be ameliorated by combining it with other agents. First, animals sedated with dissociative anesthetics retain their reflexes and can exhibit varying amounts of skeletal muscle movement and, rarely, seizure activity.⁴⁶ This propensity raises concerns about personnel safety when working with animals carrying zoonotic infectious agents. In addition, excessive movement creates challenges when performing certain tasks such as tuberculosis testing or when monitoring animals for appropriate anesthetic depth during minor surgical procedures.⁵⁵ Second, the analgesic properties of ketamine in NHP are unknown. Ketamine generally is considered to be satisfactory for somatic analgesia but inadequate for deep or visceral pain. However, analgesic effects can differ widely between species, and the few studies available show variable results even for minor procedures in NHP.^21,28,53 In addition, ketamine is associated with pain on injection and volume-dependent tissue damage in many species^{7,17,18,46,55} and can cause severe psychomimetic side effects such as hallucinations and delirium in people.^29,44 Although we cannot know whether ketamine causes excessive psychological stress in NHP, policies of the Public Health Service and Animal and Plant Health Inspection Services establish a precedent for assuming that procedures affect animals as they do human beings unless there is evidence to the contrary.^3,43Medetomidine and midazolam are 2 agents that are often combined with ketamine to improve anesthesia.^{15,24,27,33,40,49,50,54,58,59} Medetomidine is a selective α2-adrenergic agonist that is a racemic mixture of 2 optical enantiomers, the active dexmedetomidine and the inactive levomedetomidine. Although the primary benefit of α2 agonists is improved muscle relaxation, they also provide documented analgesic effects in many species, including humans and NHP.^{10,11,23,35,46,57} In addition, reversal of the medetomidine with atipamezole can decrease recovery time and reverse sedation if unacceptable side effects occur.⁵⁵ As a benzodiazepine, midazolam can be expected to offer additional sedation and muscle relaxation to ketamine anesthesia.⁴⁵ In addition, the use of medetomidine or midazolam may accommodate a decreased dose of ketamine, thus reducing tissue necrosis and inflammation.⁵⁵In addition to the physical benefits of medetomidine and midazolam, these drugs may help attenuate anesthesia-related stress responses. In dogs, medetomidine decreases the stress-related hormonal changes that occur with ketamine alone.¹ Human physicians often use benzodiazepines to decrease psychotic events, anxiety, and nausea, particularly when ketamine is used.^{8,9,19,22,29,44} In NHP, premedication with midazolam may decrease the stress associated with injection and the possible psychologic effects associated with ketamine. These potential benefits are difficult to measure in animals, but various methods have been used in an effort to quantify the stress associated with anesthesia in NHP.^2,14,51,63 Food intake and urinary cortisol are affected significantly by ketamine anesthesia in macaques.^14,51 Fecal cortisol is gaining popularity as a noninvasive way to evaluate the hormonal impact of a variety of situations, including restraint and sedation.^{20,38,41,48,56,61,63} For example, fecal cortisol increased significantly in chimpanzees 2 d after sedation.⁶³ Although cortisol was the first hormone measured in fecal samples, a fecal corticosterone assay has been developed that measures glucocorticoid metabolites in primate fecal samples with less variable results than those of fecal cortisol assays.^60,62 Although any anesthetic episode may create stress, whether particular drugs or injections in an unsedated animal are exacerbating factors is unclear. We therefore used the glucocorticoid assay to compare 3 different anesthesia protocols.The purpose of this study was to determine whether medetomidine or oral midazolam could provide a deeper level of anesthesia in macaques than ketamine alone with comparable safety. In addition, we sought to determine whether either of these medications, particularly premedication with midazolam, attenuated the glucocorticoid response associated with anesthesia.     

Effects of Multimodal Analgesia with Low-Dose Buprenorphine and Meloxicam on Fecal Glucocorticoid Metabolites after Surgery in New Zealand White Rabbits (Oryctolagus cuniculus)

Despite the increasing use of rabbits as companion animals and models for biomedical research, rabbits have not been extensively studied to identify an efficacious postsurgical analgesic that does not cause systemic complications. The synergy of NSAID and systemic opioids is well-documented, and their combined use reduces the amount of either drug required for adequate analgesia. We measured fecal corticosterone metabolites (FCM) in rabbits after a minimally invasive vascular cut-down procedure. Rabbits received buprenorphine (0.03 mg/kg SC every 12 h for 3 d), meloxicam (0.2 mg/kg SC every 24 h for 3 d), buprenorphine–meloxicam (0.01 mg/kg–0.1 mg/kg SC every 24 h for 3 d), or a single dose of 0.5% bupivacaine (0.5 mL) infused locally at the incision site. By day 3 after surgery, buprenorphine, meloxicam, and bupivacaine groups showed elevated FCM levels, which continued to rise until day 7 and then gradually returned to baseline by day 28. In the buprenorphine–meloxicam group, FCM was relatively unchanged until day 3, when treatment was discontinued, and then began to rise. Rabbits in the buprenorphine–meloxicam group gained more weight over the 28-d study than did those in the other 3 treatment groups. This study shows that in rabbits low-dose buprenorphine administered with meloxicam effectively mitigates the FCM response that develops after surgery without the adverse effects associated with higher doses.Abbreviations: COX, cyclooxygenase; FCM, fecal corticosterone metabolitesRabbits have gained popularity as companion animals and models for biomedical research because of their small size and easy temperament. They are widely used in research because of their physiologic similarity to humans and have been used extensively for studies in transgenics, immunology, cardiovascular and metabolic disorders, cancer, and development of new surgical techniques.^5,30 Companion rabbits frequently present for a number of surgical procedures including ovariohysterectomy or castration, gastrotomy or enterotomy for foreign-body removal, long-bone fracture repair, soft-tissue injuries, and dental or cutaneous abscesses.^12,26,30 Pain management in companion and laboratory animals is an ethical imperative for veterinarians, investigators, and animal caretakers. The Animal Welfare Act, Public Health Service policy, and the Guide for the Care and Use of Laboratory Animals mandate that procedures expected to cause more than slight or momentary pain require the appropriate use of pain-relieving measures, unless the withholding of analgesia is scientifically justified in an approved animal care and use protocol.^2,13,23 Furthermore, pain elicits an endocrine response cascade, including release of glucocorticoids, catecholamines and other stress-associated hormones that cause a variety of physiologic, metabolic, and inflammatory changes that may lead to organ dysfunction and confound research.^3,6,14 However, little primary literature documents an effective postsurgical analgesic regimen that is not associated with systemic complications or side effects, and veterinarians must rely on analgesic protocols extrapolated from other small mammals. This practice is not ideal because of the many physiologic, anatomic, and behavioral differences among species.The use of opioids remains a major component of analgesic therapy, particularly in the treatment of moderate to severe postsurgical pain. Opioids exert their effect by inhibiting the transmission of nociceptive stimulation in the dorsal horn of the spinal cord, activating descending inhibitory pathways, inhibiting supraspinal afferents, and causing a decrease in the release of neurotransmitters in the spinal cord. Opioids commonly used in rabbits include butorphanol, buprenorphine, morphine, hydromorphone, oxymorphone, and fentanyl.¹⁴ The most commonly used mixed agonist–antagonists are butorphanol and buprenorphine. The analgesic effect of butorphanol lasts about 3 h and is suitable for mild to moderate pain in rabbits, but the necessary frequency of administration makes it impractical for many situations. The analgesic effects of buprenorphine seem to last quite a bit longer than does butorphanol, persisting for 6 to 10 h after subcutaneous injection.¹⁴ Although opioids are effective at controlling postoperative pain, side effects associated with their use can be significant. Buprenorphine in rabbits causes a marked decrease in arterial blood pressure, increased arterial carbon dioxide tension, and significant drop in respiratory rate and arterial oxygen tension resulting in mild hypoxemia.^14,27 This effect can be clinically important in animals at risk for developing hypotension or respiratory depression. Gastrointestinal adverse effects associated with opiate use in rabbits include nausea, anorexia, and disruption of gut peristalsis (ileus).^7,10 Gastrointestinal stasis can become a medical emergency if not detected promptly. Usually, the institution of forced feedings and fluid therapy is sufficient to counteract the reduction in motility that is observed after the administration of an opioid.¹⁴A substitute for buprenorphine is a long-acting NSAID such as meloxicam, which is the NSAID used most often for analgesia in rabbits currently.^10,14,30 The popularity of meloxicam is primarily a result of its relative safety, ease of administration, prolonged half-life, and apparent effectiveness.¹⁰ Meloxicam is a cyclooxygenase-2-selective NSAID with antipyretic, analgesic, and antiinflammatory mechanisms of action.^7,14,18 Such agents have fewer side effects than other NSAID that are usually gastrointestinal in origin.^{10,12,14,18,30} Meloxicam produces antiinflammatory effects through inhibition of the cyclooxygenase pathway and therefore decreases the production of downstream mediators of inflammation, including arachidonic acid, leukotrienes, and prostaglandins. Meloxicam undergoes extensive hepatic metabolism into nonbiologically active metabolites that are eliminated largely through the kidney. Safety and efficacy studies have not been conducted for NSAID in rabbits.^12,14 Meloxicam pharmacokinetic studies in rabbits have only recently been initiated.³⁰ Most of our knowledge of these drugs is based on clinical experience and extrapolation of knowledge gained from other species. NSAID are known to affect prostaglandin synthesis in rabbits.¹² Prostaglandins stimulate elimination of soft feces or cecotropes by inhibiting motility of the proximal colon and stimulating motility in the distal colon.¹² NSAID, in general, are contraindicated in animals that are pregnant or have hepatic or renal dysfunction, increased risk of bleeding, hypotension or conditions which may limit organ perfusion or known gastrointestinal ulceration.^{12,14,15,18,22}Many clinical studies involving human patients strongly suggest that total or optimal pain relief cannot be achieved with a single drug or method without significant side effects.¹⁵ Combined analgesic regimens (balanced analgesia) or a multimodal approach to treatment of pain has strongly been recommended.¹⁴ The rationale is that achievement of sufficient analgesia results from additive or synergistic effects between different analgesics,^11,15 with a concomitant reduction of side effects due to using lower doses of analgesics. Although the value of NSAID in minor to severe postoperative pain is well documented, their effect is too small for their use as the sole analgesic in more severe pain states.^6,17,30 However, NSAID do represent an ideal alternative component in the multimodal approach to postoperative pain. The additive or synergistic effects of combining NSAID with systemic opioids is well documented and has demonstrated the ability to reduce the amount of opioid required for adequate analgesia. Studies comparing postsurgical human patients receiving opiates, NSAID, or both report that the combination of opiate with NSAID decreased the occurrence of opioid-related side effects due to a reduction in opioid requirements.^9,11,16,22When animals are under stress, glucocorticoids and catecholamines are secreted by the adrenal glands. Traditionally, the concentrations of these hormones in blood have been used to evaluate the physiologic effects of many types of stressors.²⁸ A problem with this approach is that blood sample collection alone disturbs an animal, increasing its stress level and artificially raising plasma glucocorticoid levels.^25,28 Noninvasive methods for the determination of glucocorticoids or their metabolites are therefore a prerequisite for assessing stress in animals.¹ After being extensively metabolized in the liver, glucocorticoid metabolites subsequently are excreted as conjugates (sulfates or glucuronides) via urine and with bile into the gastrointestinal tract.²⁵ As a result, cortisol or corticosterone itself is virtually absent in the feces. Thus the use of enzyme immunoassays relying on group-specific antibodies has proven advantageous to measure fecal glucocorticoid metabolites.^21,25 Fecal samples offer the advantage that they can be collected easily without any need to handle the animal. Plasma glucocorticoid levels fluctuate widely as a result of their pulsatile secretion and circadian rhythms.^25,28 Fecal glucocorticoid metabolite levels represent pooled quantities of glucocorticoids that are an aggregation of glucocorticoid metabolites.^25,28In the present study, we investigated whether the concurrent administration of buprenorphine and meloxicam potentiated their individual effects sufficiently that their combination provides adequate analgesia when used at amounts recommended in literature as the lowest dose for either drug alone. Measurements of fecal corticosterone metabolites (FCM) after use of the combination were compared with single-drug analgesic regimens in rabbits that experienced a minimally invasive vascular cut-down procedure to gauge the level of physiologic stress.     

Multiple Extrauterine Pregnancy with Early and Near Full-Term Mummified Fetuses in a New Zealand White Rabbit (Oryctolagus cuniculus)

Extrauterine pregnancy (EP) is infrequent in mammalian species and occurs when fertilized ova implant and develop outside the uterus. A common outcome is abdominal pregnancy resulting in mummified fetuses (lithopedia). Here we describe an unusual case of abdominal pregnancy with early and near full-term lithopedia. Macroscopic findings supported the diagnosis of lithopedia with distinct age differences and facilitated further characterization of primary ectopia and risk factors leading to this occurrence.Abbreviation: EP, extrauterine pregnancyExtrauterine pregnancy (EP) occurs infrequently in most mammalian species.¹² The term derives from the Latin prefix meaning ‘outside’ or ‘beyond’ and refers to the implantation of a fertilized ovum outside the uterine cavity. Extrauterine pregnancy was first recognized more than 900 y ago² among other discoveries with a hereditary nature.¹³ Early reports compared EP in women, cats, dogs, and rabbits⁷ and described the presence of mummified fetuses in laboratory rabbits.^16,35EP is a serious obstetric complication that occurs asymptomatically in most cases.¹⁷ There are 4 classifications of EP: tubal, ovarian, abdominal–peritoneal, and cervical. The fallopian tube is the most common location and leads to tubal pregnancy. When gestation occurs in the abdominal–peritoneal cavity, abdominal pregnancy results and is subdivided as primary, when fertilization occurs outside the uterus after an oocyte is accidentally released from the fimbria, and secondary, when an oocyte is released due to direct tubal trauma.⁴⁵ A rare form of EP associated with high maternal morbidity and fetal mortality is called heterotopic (or combined) pregnancy, which occurs when 2 fertilized eggs coexist, one outside the uterus and the other inside.^18,33,44Undetected EP is frequently associated with fatal outcomes to the dam and offspring, including the formation of mummified fetuses, which may eventually become calcified and are called lithopedia (from the Greek: lithos, stone; paidion, child).^11,51,53 The condition is infrequent, and the factors that influence the unexpected outcomes of this pathology are not well understood.^26,34,35 Epizootiologic investigations are few,¹² although a recent report outlined the prevalence of EP in large NZW rabbit breeding colonies.⁵¹Examples of EP have been documented in dogs,¹⁷ cats,^14,39,42,49 rabbits,^20,29,45,51 hamsters,^9,46 rats,²⁶ mice,^8,12 guinea pigs,^3,30 lambs,⁴⁰ nonhuman primates,^10,34,38,50 and other species including humans.^11,12 However, despite the number of documented species, the majority of reports failed to note detailed clinical symptoms that interfered with reproduction even in instances that led to the formation of lithopedions.⁴²Experimentally, mouse embryos have successfully been transferred to a variety of sites including the peritoneal cavity, kidney, spleen, muscles, testis, and the anterior chamber of the eye.^1,6,21,31 The aim of the current report is to describe a rare case of abdominal pregnancy in which lithopedia developed clinically silently and coexisted with multiple pregnancies in a healthy doe rabbit.     

Pharmacokinetics of Sustained-Release and Transdermal Buprenorphine in G?ttingen Minipigs (Sus scrofa domestica)

The opioid buprenorphine has been shown to provide adequate postoperative analgesia in both companion and laboratory animals. However, its use is still hindered by the need for multiple parenteral injections to achieve continuous analgesia. The purpose of the current study was to conduct a pharmacokinetic analysis of 2 new long-acting formulations of buprenorphine—an injectable sustained-release buprenorphine (SRB) and a transdermal buprenorphine (TDB) patch—in healthy Göttingen minipigs by using liquid chromatography–electrospray ionization–tandem mass spectrometry. Administration of 0.18 mg/kg SC SRB and 30 μg/h TDB achieved AUC_0-Tlast of 221.6 ± 26.8 and 25.2 ± 3.9 ng × h/mL, respectively, compared with 9.7 ± 1.4 ng*h/mL for 0.02 mg/kg IV buprenorphine. By using a hypothesized therapeutic plasma buprenorphine concentration threshold of 0.1 ng/mL, therapeutic concentrations were achieved at the first study time point (5 to 30 min) and lasted an average of 8.0 ± 1.3 h for intravenous buprenorphine and 264.0 ± 32.2 h for SRB. TDB achieved therapeutic concentrations in 12 to 24 h after patch application, which lasted until the patch was removed at 72 h. The results of this study suggest that SRB and TDB are long-acting alternatives for pain management, and their use could decrease animal handling and stress, thereby simplifying pain management and improving welfare in laboratory swine.Abbreviation: AUC_0-Tlast, AUC to last quantifiable plasma concentration; C_max, peak plasma concentration; SRB, sustained-release buprenorphine; TDB, transdermal buprenorphine; T_max, time of peak plasma concentration; VAP, vascular access portBuprenorphine is a semisynthetic, partial µ-opioid receptor agonist used for analgesia in many companion and laboratory animal species.^7,29 Its popularity is due to a lower risk of respiratory depression and prolonged analgesia in comparison to pure µ-opioid agonists, such as fentanyl and hydromorphone.^7,29 Buprenorphine is often the analgesic of choice in swine because a single 0.01- to 0.1-mg/kg dose can provide analgesia for as long as 8 to 12 h with minimal adverse effects.^7,33Swine are valuable animal models for cardiovascular, digestive, urinary, and integumentary research.^32,33 As a result, laboratory swine often undergo major surgeries resulting in considerable postoperative pain. To provide analgesia of adequate efficacy and duration throughout the postoperative period, multiple injections of buprenorphine are necessary. Repeated injections and the associated handling and momentary pain can become increasingly stressful to swine with each subsequent injection. In addition, the recurrent peak and trough plasma concentrations associated with multiple parenteral injections potentially result in periods of inadequate pain relief at trough levels. A buprenorphine formulation that can be administered less frequently or in a noninjectable formulation but that can deliver a controlled and constant amount of drug over time potentially would eliminate the disadvantages of repeated dosing.Recently 2 new formulations of buprenorphine have become available: an injectable sustained-release buprenorphine (SRB) and a transdermal buprenorphine (TDB) patch. Both formulations are designed to provide a consistent, controlled release of buprenorphine over the course of several days after a single administration. Recent studies in mice,⁴ rats,⁸ cats,⁵ and dogs²⁴ support the ability of SRB to provide an extended duration of analgesia compared with that of the standard buprenorphine formulation. In addition, therapeutic plasma concentrations have been shown to last as long as 72 h in rats⁸ and 5 d in both macaques²³ and dogs.²⁴ Although SRB has been studied in several animal species, it has yet to be evaluated in swine.TDB has been extensively used in human medicine however, unlike SRB, there are relatively few studies evaluating the efficacy of TDB in animals. High-dose (35, 52.5, and 70 µg/h) and low-dose (5, 7.5, 10, 15, and 20 µg/h) TDB patches, lasting 3 and 7 d, respectively, have been used effectively in humans to treat moderate to severe, chronic pain such as lower back pain and that due to osteoarthritis or cancer.^{6,9,14,20,26,30,31,35} Currently, TDB studies in animals have only been performed by using high-dose patches and have been limited to dogs^1,21,25 and cats.²² In dogs, detectable plasma buprenorphine concentrations have been shown to last 72 h after the application of a single 52.5-µg/h TDB patch²⁵ and 108 h with a 70-µg/h patch.¹ In addition, a 70-µg/h TDB patch has been reported to provide equal postoperative analgesia in dogs that underwent an ovariohysterectomy when compared with 0.2 mg/kg of the standard formulation of buprenorphine administered subcutaneously every 6 h during the postoperative period. ²¹ To date, TDB has not been evaluated in swine.Because of the success seen with both SRB and TDB in other species, further investigation into the pharmacokinetics and clinical efficacy of SRB and TDB in swine is warranted. These buprenorphine formulations have the potential for extended drug delivery with a single administration and thus the potential to positively affect animal welfare by minimizing animal stress. The purpose of this study was to evaluate the pharmacokinetics of these 2 new formulations of buprenorphine compared with a standard dose of intravenous buprenorphine in Göttingen minipigs. We hypothesized that both SRB and TDB would achieve quantifiable plasma buprenorphine concentrations above an estimated therapeutic threshold of 0.1 ng/mL for a longer duration than that of a standard dose of intravenous buprenorphine, thus supporting the use of these new formulations as long-acting analgesics for pain management in swine.     

Assessment of Routine Procedure Effect on Breathing Parameters in Mice by Using Whole-Body Plethysmography

We used whole-body plethysmography to investigate the effect of restraint, ear marking, tail vein and retroorbital blood sampling, and tail clipping on respiration in Balb/c × TCR-HA^+/– F1 hybrid mice (F1h). Baseline values of breathing parameters were determined. During the experiment, mice experienced a procedure and then plethysmographic recordings were obtained immediately and at 4, 24, and 48 h afterward. Baseline breathing parameters showed significant differences between sexes. Restraint affected minute volume differently than did handling in male mice and to a lesser extent in female mice. Ear marking significantly changed minute volume compared with handling but not restraint in male mice and in the opposite manner in female mice. Tail vein blood sampling changed minute volume in a significant manner compared with restraint but not compared with handling in both sexes. Retroorbital blood sampling significantly changed minute volume compared with values for both handling and restraint in male mice but only compared with handling in female mice. Tail clipping modified minute volume significantly compared with handling in male mice and compared with restraint in both sexes. Analysis of data showed that routine procedures affect minute volume in mice depending on invasiveness of maneuver and in a sex-biased manner for as long as 24 h after the procedure. Our experiment shows that procedures performed on laboratory mice can change respiratory parameters and can be investigated by plethysmography.Abbreviation: F1h, Balb/c × TCR-HA^+/– F1 hybrid miceHandling, restraint, identification methods, and blood or tissue sampling are regarded as routine procedures in animal experiments. This aspect is particularly important when working with transgenic animals, where the need for genotyping demands the frequent use of these procedures, which can readily yield DNA samples.⁵ The effect of routine procedures on physiologic parameters varies depending on method, frequency, and duration, but these procedures generally are considered to be acute stressors.^2,5,26,27 A large body of research has focused on the effect of routine procedures on animals’ physiology. These effects were assessed by studying changes in behavior,^1,23,36,37 body weight,^29,37 food and water intake,^29,37 body temperature³⁵ and heart rate by radiotelemetry,^{5,10,19,26,27} corticosterone^{11,12,24,25,29} and glucose levels,³³ and blood variables.^29,36 Several differences in the response of laboratory animals to stress have been correlated to breed,^2,33 strain,^11,14,37 age,²⁴ and sex.^12,23,24 Some authors²⁴ have noted that female mice show a lower overall stress level due to the modest effect of social competition. Another study¹² showed a different, sex-specific response to hypoxic ventilation in rats, which was higher in male than female rats. In addition, these authors¹² suggested that female ovarian hormones are prime candidates for stress regulation.Whole-body plethysmography is a noninvasive, indirect method of studying respiratory function in conscious, unrestrained animals. This method has been used particularly in pharmacologic and toxicologic studies in diverse animal species including mice,^3,9,14,15,38 rats,^8,20 cats,²² dogs,^16,34 and pigs.¹³ The present study was designed to investigate the extent to which routine procedures affect breathing parameters in a transgenic mouse colony. Male and female mice were exposed to 4 routine procedures that are used for DNA sampling and identification of transgenic mice (ear marking, tail clipping, and tail vein and retroorbital blood sampling). Respiratory parameters were recorded by whole-body plethysmography immediately and at 4, 24, and 48 h after the procedure. Handling and restraint were used as control procedures.