Duration of Action of Sustained-Release Buprenorphine in 2 Strains of Mice

Buprenorphine HCl is a common analgesic for laboratory mice undergoing surgical procedures. The documented duration of action of buprenorphine HCl is as short as 3 to 5 h in mice, potentially necessitating readministration for continued analgesia. A long-acting buprenorphine formulation would reduce handling-associated stress and provide uninterrupted analgesia. This study used the hot-plate assay to assess the antinociceptive effects of a single injection of sustained-release buprenorphine (bup-SR), buprenorphine-HCl (bup-HCl), and saline over 72 h in young adult male BALB/cJ and SWR/J mice. SWR/J mice had shorter baseline latencies than did BALB/cJ mice, possibly reflecting greater sensitivity to thermal nociception. Relative increase from baseline latency (% maximal possible effect) was significant for buprenorphine-SR at 2, 6, and 12 h compared with saline. According to results from a hot-plate assay, the analgesic efficacy of buprenorphine-SR appears to last at least 12 h in male BALB/cJ and SWR/J mice.Abbreviation: %MPE, % maximal possible effect; bup-HCl, buprenorphine hydrochloride; bup-SR, sustained-release buprenorphineAnalgesia of adequate efficacy and duration is necessary for laboratory animals undergoing painful procedures. In a survey of experiments reported in biomedical journals, buprenorphine was the analgesic used most often in rodents that underwent surgery.¹⁶ Buprenorphine is advantageous because it is relatively long-acting, has low potential for abuse, and has been studied extensively in mice and rats.⁹ Although longer-acting than other opioids, buprenorphine still can require repeated restraint and reinjection for full postsurgical analgesia, creating stress for animals and an inconvenience for personnel. A longer-acting buprenorphine formulation would overcome these drawbacks.Buprenorphine HCl (bup-HCl) is an opioid and acts as a partial agonist at the μ-opioid receptor and as an antagonist at the κ-opioid receptor. Although its maximal analgesic effect is not as great as that of the complete μ-opioid agonist morphine,⁸ buprenorphine''s slow dissociation from the μ-opioid receptor prolongs its activity.¹⁰ However, estimates of the buprenorphine''s duration of action vary among studies, perhaps due to differences in dosage, nociceptive stimuli, and measured indicators of efficacy. One study concluded from tail-flick and hot-plate assays that buprenorphine''s duration of action at a dosage of 2.0 mg/kg SC is 3 to 5 h in mice.⁸ Other investigators found that buprenorphine (3.0 mg/kg IP) provided marked antinociception in the tail-flick test for as long as 8 h in mice.¹¹ Both studies prompt the concern that a single dose of buprenorphine at the end of the workday might not provide sufficient overnight analgesia for mice that have undergone major surgery.A sustained-release formulation of buprenorphine would benefit both laboratory animals and researchers by providing consistent, long-lasting analgesia without the need for redosing. A veterinary compounding pharmacy has developed an injectable, patent-pending formulation of sustained-release buprenorphine (bup-SR). In laboratory rats, bup-SR appears to remain at therapeutic plasma levels for 72 h and to provide analgesia for the same duration in thermal nociception and surgical postoperative pain models.⁷ Similar duration and efficacy have been reported in a study of feline clinical patients.³ However, no published studies that compare bup-SR with bup-HCl in laboratory mice are available currently.Opioids decrease thermal sensitivity in animals. Compare with untreated mice, mice treated with opioids have greater latency on thermal sensitivity tests, including the hot-plate and tail-flick tests, that is, opioid-treated mice are slower to perceive heat stimuli as unpleasant and to move away from them. As the opioid effect subsides, mice return to their normal preopioid thermal sensitivity. Although latency during the hot-plate assay may not translate directly into precise dosage recommendations for postsurgical analgesia in mice, these data are appropriate for preliminary comparisons of duration-of-action of bup-SR and conventional bup-HCl.The current study examined thermal latencies in 2 inbred strains of mice, BALB/cJ and SWR/J, treated with 2 formulations of buprenorphine or with saline placebo. Two strains were used because mice exhibit interstrain differences in sensitivity to noxious stimuli and opioid analgesics.¹³ BALB/cJ mice are moderately responsive to opioids, whereas SWR/J mice are exceptionally sensitive.¹² We hypothesized that bup-SR would have a longer duration of action than would bup-HCl or saline in both strains.     

Efficacy of Sustained-Release Buprenorphine in an Experimental Laparotomy Model in Female Mice

Mice purportedly require dosing with the opioid buprenorphine (Bup-HCl) at least every 8 to 12 h to maintain an adequate plane of analgesia. Here we used an experimental laparotomy model to determine the clinical efficacy of sustained-release formulations of buprenorphine (Bup-SR) after surgery in mice. Female CD1 mice underwent laparotomy and received either Bup-SR (0.6 mg/kg), Bup-HCl (0.1 mg/kg every 12 h), or saline (every 12 h). Pain was assessed at 1, 3, 6, 12, 24, 48, and 72 h according to the frequency of several behaviors (general activity, wheel-running activity, rearing, grooming, wound licking, orbital tightening, and percentage of integrated nest material) and daily body weight. Over time, wheel running was increased and wound licking was decreased in Bup-SR–treated mice compared with Bup-HCl– and saline-treated mice. Compared with Bup-HCl– and saline-treated mice, Bup-SR–treated mice had increased general activity and percentage of integrated nest material and decreased orbital tightening for 1 to 6 h after surgery. The Bup-HCl– and saline-treated mice had similar general activity, orbital tightening scores, and wheel running activity. Rearing activity and body weight did not differ throughout the study, and none of the observed behaviors differed between groups at 24, 48, and 72 h after surgery. These results suggest that Bup-SR at 0.6 mg/kg provides adequate analgesia after laparotomy in mice and can be used as an alternative analgesic in this context. Furthermore, Bup-HCl at 0.1 mg/kg every 12 h may be inadequate in providing analgesia for abdominal procedures in mice.Abbreviations: Bup-HCl, buprenorphine hydrochloride; Bup-SR, sustained-release buprenorphine; TINT, time-to-integrate-to-nest testPostoperative or postprocedural analgesia is imperative to eliminate undue pain or distress in murine models. Opioids are common analgesics used to treat postoperative pain in laboratory mice. The μ and κ opioid receptors are the most frequently targeted for analgesic activity, and opioid compounds are classified as either agonists, partial agonists, or agonist–antagonists. Buprenorphine (Bup-HCl) is a partial μ agonist commonly used to reduce pain in laboratory mice in a variety of models.^3,10,15,23 The most common dosing regimens for Bup-HCl are at least every 8 to 12 h.⁹ This frequency requires additional handling, and according to previous efficacy studies¹¹ and our most recent pharmacokinetic studies,²² Bup-HCl may not retain therapeutic concentrations during the entire dosing interval. Sustained-release formulations of buprenorphine (Bup-SR) reduce the amount of animal handling and were shown to retain therapeutic concentrations for as long as 48 h.²²Bup-SR was first shown to provide sustained plasma concentrations for 72 h in rats and demonstrated efficacious analgesia in a rat tibial defect model for 72 h¹⁰ and a rat incisional pain model.⁴ We performed a pharmacokinetic analysis of Bup-SR in female CD1 mice and found the formulation, when given at 0.6 mg/kg, resulted in plasma concentrations that exceeded the reported therapeutic concentration for 24 to 48 h.²² A similar pharmacokinetic analysis was performed in male C57BL/6 mice and demonstrated that Bup-SR at 1.2 mg/kg resulted in therapeutic concentrations that lasted as long as 12 h.⁶ Several studies recently have evaluated the clinical efficacy of Bup-SR in mouse models, and 3 studies demonstrated varying clinical efficacy in mice by using a thermal nociception model. Specifically, one study demonstrated the efficacy of Bup-SR for 12 h in male BALB/cJ and SWR/J mice when given at 1.0 mg/kg,² whereas another demonstrated antinociceptive effects in male Swiss–Webster mice for as long as 48 h when given at 1.5 mg/kg.¹⁷ In addition, Bup-SR at 2.2 mg/kg was effective against thermal nociception in female C57BL/6 mice for 24 h; clinical efficacy was further assessed in an experimental embryo transfer model and persisted for at least 24 h postoperatively.¹⁹The current study sought to determine the clinical efficacy of Bup-SR at 0.6 mg/kg for 72 h after experimental laparotomy in female CD1 mice. We found that Bup-SR provided adequate analgesia, greater than that in Bup-HCl– or saline-treated mice, for the first 12 h after surgery, whereas there was no clinically discernable difference in the analgesic response after the 12-h time point. These results suggest Bup-SR is a suitable alternative to Bup-HCl for abdominal surgical procedures in mice.     

Pharmacokinetics of Sustained-Release Analgesics in Mice

Buprenorphine and carprofen, 2 of the most commonly used analgesics in mice, must be administered every 8 to 12 h to provide sustained analgesia. Sustained-release (SR) formulations of analgesics maintain plasma levels that should be sufficient to provide sustained analgesia yet require less frequent dosing and thus less handling of and stress to the animals. The pharmacokinetics of SR formulations of buprenorphine (Bup-SR), butorphanol (Butp-SR), fentanyl (Fent-SR), carprofen (Carp-SR), and meloxicam (Melox-SR) were evaluated in mice over 72 h and compared with those of traditional, nonSR formulations. Bup-SR provided plasma drug levels greater than the therapeutic level for the first 24 to 48 h after administration, but plasma levels of Bup-HCl fell below the therapeutic level by 4 h. Fent-SR maintained plasma levels greater than reported therapeutic levels for 12 h. Therapeutic levels of the remaining drugs are unknown, but Carp-SR provided plasma drug levels similar to those of Carp for the first 24 h after administration, whereas Melox-SR had greater plasma levels than did Melox for the first 8 h. Butp-SR provided detectable plasma drug levels for the first 24 h, with a dramatic decrease over the first 4 h. These results indicate that Bup-SR provides a stable plasma drug level adequate for analgesia for 24 to 48 h after administration, whereas Carp-SR, Melox-SR, Fent-SR, and Butp-SR would require additional doses to provide analgesic plasma levels beyond 24 h in mice.Abbreviations: Bup-HCl, buprenorphine hydrochloride; Butp, butorphanol; Carp, carprofen; Fent, fentanyl; Melox, meloxicam; SR, sustained releasePostoperative or postprocedural analgesia is imperative to eliminate undue pain or distress in murine models. The 2 classes of analgesics typically used to treat postoperative pain in laboratory mice are opioids and NSAID. Each of these has very different mechanisms of actions and side effects. Opioid analgesics bind receptors, of which µ and κ are the most frequently targeted for analgesic activity, are classified as either agonists or partial agonist-antagonists. Fentanyl is a µ receptor agonists, whereas butorphanol has primary affinity to κ receptors and buprenorphine is a partial μ agonist. Whereas µ receptors are primarily located in the cerebral cortex, κ receptors are primarily located in the spinal cord.^9,16 Fentanyl and butorphanol are not routinely used in the laboratory animal setting because of their short half-lives. Effective plasma concentrations of fentanyl persist less than an hour,²¹ and the antinociceptive activity of butorphanol lasts only 1 to 2 h in mice,¹¹ requiring very frequent dosing to provide prolonged analgesia. In comparison, buprenorphine offers the advantage of having a longer duration of effect. Its duration of analgesic efficacy was determined to be 3 to 5 h by using a hot plate and tail flick assay,¹¹ whereas satisfactory analgesia was achieved with 0.1 mg/kg twice daily in a partial hepatectomy model.³⁶ NSAID offer an alternative or adjunct to opioid analgesics. Tissue damage results in the production of prostaglandins, which increase the sensitivity of nociceptors. Prostaglandin synthesis is mediated by the cyclooxygenase enzymes COX1 and COX2. Both are constitutively expressed in tissues, with COX2 induced during the inflammatory process. NSAID inhibit the prostaglandin pathway, thereby providing analgesia. NSAID usage has some unwanted side effects including gastrointestinal toxicity, altered platelet function, and renal toxicity.^4,16Analgesia treatments for rodents continue to be developed and modified to provide optimal analgesia after surgical procedures. One of the most common analgesics used in mice is buprenorphine, which has been shown to be effective in a variety of pain models,^3,5,8 exhibits a wide safety margin as a partial µ agonist,⁴³ and has few side effects when administered appropriately.¹⁴ Depending on the severity of the procedure, many institutions, including ours, requires the provision of analgesics for the first 72 h after the procedure,^6,27,37,40 thus requiring buprenorphine to be dosed every 8 to 12 h.^8,11 This frequency of drug administration requires repeated handling, which can stress mice,¹ and can cause waxing and waning of plasma concentrations, which may result in subtherapeutic concentrations. Sustained-release (SR) formulations that deliver a constant amount of drug over time have been developed for use in rodent models and yield adequate plasma concentrations, achieve satisfactory analgesia, and reduce the amount of handling. The efficacy of a SR formulation of buprenorphine (Bup-SR)¹⁰ was evaluated in a rat model and found to provide satisfactory analgesia for 72 h after administration. More recently, Bup-SR was evaluated in mice by using a thermal nociception model, with promising results.²Other analgesics such as NSAID may be more appropriate for pain management depending on the nature of the procedure; NSAID can also be used in combination with opioids. Carprofen and meloxicam are 2 commonly used NSAID that provide analgesia by inhibiting the COX2 enzyme, with some activity on COX1.⁴ Commonly used dosing regimens for carprofen and meloxicam in mice are 5 mg/kg every 12 to 24 h and 1 to 2 mg/kg every 12 h, respectively.^7,8 In addition, an SR release formulation of an NSAID would be beneficial for the management of pain in mice.This study sought to determine the pharmacokinetics of SR formulations of buprenorphine (Bup-SR), butorphanol (Butp-SR), fentanyl (Fent-SR), carprofen (Carp-SR), and meloxicam (Melox-SR) compared with those of the nonSR formulations of buprenorphine (Bup-HCl), carprofen (Carp) and meloxicam (Melox). Our findings indicated that Bup-SR maintained plasma drug levels above therapeutic levels for the first 24 to 48 h. Fent-SR maintained plasma levels above therapeutic levels for the first 12 h. Therapeutic levels for the remaining drugs are not known in mice; however, Carp-SR provided plasma drug levels similar to those of Carp for the first 24 h, Melox-SR concentrations were greater than those of Melox for the first 8 h after administration, and Butp-SR provided measurable plasma drug levels for the first 24 h.     

The Effects of Perioperative Analgesia on Litter Size in Crl:CD1(ICR) Mice Undergoing Embryo Transfer

The objective of this study was to evaluate the effect on litter size of 2 analgesics used perioperatively during mouse embryo transfer surgery. Day 2.5 pseudopregnant CD1 mice (n = 96) were divided equally into 2 analgesic treatment groups and a saline control group. Each mouse received a single, subcutaneous dose of buprenorphine hydrochloride (0.1 mg/kg), flunixin meglumine (2.5 mg/kg), or saline immediately after induction of anesthesia with 2.5% isoflurane. Each mouse then was prepared for aseptic surgery. Blastocysts had previously been collected from C57BL/6NCrl female mice that were synchronized and superovulated by using pregnant mare serum gonadotropin and human chorionic gonadotropin and mated with C57BL/6NTac male mice 3.5 d before collection. Viable blastocysts were pooled, and 8 were selected arbitrarily and transplanted into the right uterine horn of each pseudopregnant CD1 mouse. Mice were monitored throughout pregnancy, and the number of pups at birth was documented. No statistically significant difference was found between the 3 groups. These results indicate that perioperative analgesic treatment with buprenorphine or flunixin in the CD1 mouse undergoing embryo transfer is not associated with increased embryonic loss.Abbreviation: NSAID, nonsteroidal antiinflammatory drugsEmbryo transfer in the mouse is widely used in the production of genetically engineered mice, in vitro fertilization, intracytoplasmic sperm injection, rederivation, and reconstitution of mouse lines after embryo cryopreservation.^2,5,14,30 Embryo transfer has become a principal surgical procedure in many animal care programs involving mice and is routinely performed in our program.The Guide for the Care and Use of Laboratory Animals²¹ defines major survival surgery as a procedure that penetrates and exposes a body cavity or produces substantial impairment of physical or physiologic functions. According to this definition, the laparotomy performed for embryo transfer constitutes a major survival surgery.^1,21 The attending veterinarian must provide oversight to surgical programs, and part of that responsibility includes preventing or alleviating pain associated with surgical procedures with the use of appropriate analgesics.^1,21Several classes of analgesics are used perioperatively in rodent surgical procedures, including opioids and nonsteroidal antiinflammatory drugs (NSAID).⁷ Buprenorphine is a commonly used opioid analgesic in rodent surgery.²⁵ NSAID including flunixin, aspirin, and phenylbutazone have been used extensively in veterinary medicine.¹⁰Some investigators are concerned that the administration of analgesics perioperatively during embryo transfer adversely affects embryonic survival. However, only one study has examined the effects of perioperative analgesics on litter size in mice.¹⁵ The purpose of the present study was to evaluate the effect of perioperatively administered analgesics, buprenorphine (Buprenex, Reckitt Benckiser Pharmaceuticals, Richmond, VA) and flunixin (Banamine, Schering-Plough, Union, NJ), on embryonic survival after embryo transfer in CD1 mice.     

Comparison of Side Effects between Buprenorphine and Meloxicam Used Postoperatively in Dutch Belted Rabbits (Oryctolagus cuniculus)

One of the challenges facing veterinarians and investigators who use rabbits (Oryctolagus cuniculus) as a surgical model in biomedical research is choosing an appropriate and efficacious postoperative analgesic without systemic complications and side effects. The objective of this study was to evaluate the gastrointestinal side effects associated with the postoperative use of buprenorphine in Dutch Belted rabbits. We also evaluated the analgesic meloxicam as an alternative to opioid administration during the postoperative period. Rabbits were assigned to 1 of 3 treatment groups during the postoperative period after routine ovariohysterectomy: buprenorphine (n = 10), meloxicam (n = 10), and incisional infiltration with bupivicaine (no treatment control; n = 10). Feed intake, fecal production, weight loss, urine output, and other physiologic parameters were monitored and behavior and pain assessments were performed for 7 d after surgery and compared with baseline values collected before surgery. All rabbits showed decreased pellet consumption, fecal production, and weight on day 1 after surgery. This effect was severe in some rabbits that received bupivicaine; therefore treatment of this entire group with metoclopramide, fluids, and hay was instituted to reverse gut stasis. No significant difference in feed consumption and fecal production was present between the buprenorphine- and meloxicam-treated groups. On the basis of these results, meloxicam appears to be a suitable alternative or adjunct to buprenorphine for alleviating postoperative pain with minimal risk of anorexia and gastrointestinal ileus.Abbreviation: COX, cyclooxygenase; NSAID, nonsteroidal antiinflammatory drugBecause of its small size and amiable temperament, the Dutch Belted Rabbit (Oryctolagus cuniculus) is gaining popularity as a companion animal and a model for biomedical research. A common circumstance when working with rabbits is the clear lack of reliable data regarding appropriate and effective postoperative analgesia. As a result, practicing veterinarians and researchers are forced to rely on analgesic protocols extrapolated from other small mammals. Due to a variety of physiologic, anatomic, and behavioral differences among rabbits, this practice is suboptimal. Further, the response in rabbits to treatments commonly used in private practice and laboratory animal medicine can be difficult to evaluate.A common analgesic for use in rabbits postoperatively is buprenorphine. Buprenorphine is a partial agonist with very high affinity for the mu opioid receptor but with only partial activity.³ Despite its partial activity, buprenorphine has been shown to be effective at controlling postoperative pain; side effects are primarily gastrointestinal and include nausea, anorexia, and disruption of gut peristalsis (ileus) in a variety of mammalian species.¹¹ Studies have shown that morphine, a related narcotic, has both centrally mediated and gut-specific inhibition of small intestinal transit and motility.^1,7 In mice treated with multiple doses of buprenorphine, decreased food consumption and weight loss occurred during the postoperative period.⁸ In rats, multiple doses of buprenorphine given beyond postoperative day 1 resulted in sustained weight loss despite increased food consumption.⁹ In addition rats receiving high doses of buprenorphine (0.5 mg/kg SC) exhibited signs of pica and developed gastric distension.⁵ Although these gastrointestinal side effects can be mild and easily reversed in other mammalian species, similar gastrointestinal signs in rabbits potentially could progress to overt gut stasis. Therefore, it is important to seek analgesic alternatives to prolonged buprenorphine (and opioid) use in rabbits.A substitute for buprenorphine is a long-acting nonsteroidal antiinflammatory drug (NSAID) preferential for cyclooxygenase (COX) 2, such as meloxicam, which has analgesic, antiinflammatory, and antipyretic properties.¹⁰ Meloxicam blocks COX2-related prostaglandins that are considered to be nonphysiologic and incite various responses associated with inflammation, such as vasodilation, change in capillary permeability, and potentiation of other chemical mediators of inflammation and chemotaxis.¹⁰ Because of its preferential inhibition of COX2-related prostaglandins, meloxicam has fewer gastrointestinal side effects than do other less specific NSAIDs. Further, a drug that inhibits COX2 at a lower concentration than that necessary to inhibit COX1 is safer and more desirable.² The benefits associated with the use of meloxicam in the perioperative period are well-documented. A 2003 study in which dogs given 0.1 mg/kg meloxicam or a placebo showed no significant differences in development of gastric erosions between meloxicam and control groups.¹¹ Meloxicam has also been shown to be especially effective in controlling postoperative visceral pain. In 1 study, meloxicam given during routine ovariohysterectomy of cats provided adequate analgesia.¹⁷ Finally, although the elimination half-life of meloxicam is species-specific, the drug generally is considered to be long-acting, averaging 24 h in dogs.¹⁴ The prolonged half-life of meloxicam leads to fewer administrations and potentially fewer stressful human–rabbit interactions.The objective of the present study is to assess whether the known side effects of buprenorphine occur in Dutch Belted rabbits (Oryctolagus cuniculus), through careful monitoring and data collection of eating and voiding behaviors, physical well being, and physiologic body functions. We evaluated the NSAID meloxicam as an equal or better alternative to opioid use during the postoperative period. We expect that meloxicam administration, when used as a postoperative analgesic for ovariohysterectomy, would result in clinically normal surgical recovery without unwanted gastrointestinal side effects, such as anorexia or gut stasis.     

Comparison of Nociceptive Effects of Buprenorphine,Firocoxib, and Meloxicam in a Plantar Incision Model in Sprague–Dawley Rats

Due to their reduced frequency of dosing and ease of availability, NSAIDs are generally preferred over opioids for rodent analgesia. We evaluated the efficacy of the highly COX2-selective NSAID firocoxib as compared with meloxicam and buprenorphine for reducing allodynia and hyperalgesia in rats in a plantar incision model of surgical pain. After a preliminary pharmacokinetic study using firocoxib, Sprague–Dawley rats (n = 12 per group, 6 of each sex) were divided into 6 groups: no surgery (anesthesia only), saline (surgery but no analgesia), buprenorphine (0.05 mg/kg SC every 8 h), meloxicam (2 mg/kg SC every 24 h), and 2 dosages of firocoxib (10 and 20 mg/kg SC every 24 h). The nociception assays were performed by using von Frey and Hargreaves methodology to test mechanical allodynia and thermal hyperalgesia. These assays were performed at 24 h before and at 20, 28, 44, and 52 h after start of surgery. None of the analgesics used in this study produced significantly different responses in allodynia or hyperalgesia from those of saline-treated rats. In the Hargreaves assay, female saline-treated rats experienced significantly greater hyperalgesia than did males. These findings add to a growing body of literature suggesting that commonly used dosages of analgesics may not provide sufficient analgesia in rats experiencing incisional pain.

Institutions performing studies using animals have both an ethical responsibility and a legal requirement to administer appropriate analgesia to research subjects.²⁵ Controlling postoperative pain can hasten return to normal function, minimize healing time, and prevent the development of chronic pain. In humans, postoperative pain is prevalent, with 80% of patients reporting acute pain, ranging from moderate to extreme, after surgery.⁴ Postoperative pain is not restricted to human medicine but also extends to veterinary analgesia. Therefore, we must continue to scrutinize analgesic protocols and strive to implement novel therapies.During a surgical incision, peripheral tissue injury leads to central sensitization. This process results in hyperalgesia, an increased response to a painful stimulus, and allodynia, a painful response to a normally benign stimulus.⁶⁰ Several models that are used to test nociception in rodents have demonstrated repeatability, including plantar incision, antigen-induced inflammation, and capsaicin injection.^7,8 Among these, the plantar incision model is minimally invasive and allows for objective measurements of acute postoperative pain.^11,12 In addition, this model may be particularly advantageous, because surgical incision pain in humans appears to be better controlled in terms of persistency, duration, and pain management than are other causes of nerve injury and inflammation.⁶⁰ In the plantar incision model, allodynia is assessed by using the von Frey assay of mechanical stimuli,⁴⁷ whereas hyperalgesia is assessed by using the Hargreaves assay of thermal stimuli.²³When making decisions regarding analgesia, choosing an appropriate medication that maximizes pain control and minimizes side effects is important. The 2 most commonly used classes of analgesics in rodents are NSAID and opioids. In human and veterinary medicine, the use of cyclooxygenase (COX) 2-selective NSAID is preferred because of lower potential for the adverse gastrointestinal and kidney side effects that result from COX1 inhibition.²⁹ However, the most commonly used NSAID in laboratory rodents are ketoprofen (COX nonselective), meloxicam (minimally COX2-seletive), and carprofen (variably COX selectivity by species).^31,37,43 Although newer NSAID such as firocoxib have an extremely high selectivity for the COX2 pathway (approximately 400-fold more selective for COX2)³⁷ and are being used more frequently in companion and large animals, little is known about the efficacy of firocoxib in laboratory rodents. A recent study has assessed the use of this medication in mice,⁴⁹ but no published dose determination or efficacy studies are available for rats.Buprenorphine, widely considered the ‘gold standard’ opioid analgesic for rodents,^5,18,52 is a Schedule III controlled substance under the Controlled Substances Act.⁴² Many researchers do not hold a DEA license, thus creating a barrier to use of this medication. Slow-release formulations of buprenorphine cannot be obtained in New York due to state restrictions against compounded controlled drugs for research use in animals. Standard-formulation buprenorphine must be dosed fairly frequently (every 6 to 8 h in mice and rats) to maintain efficacy, thus increasing the need for handling and injections.^5,6 In addition, potentially detrimental side effects of buprenorphine have been reported in rodents and humans, including increased activity, reduced food intake, respiratory depression, and pica.^35,46,56 These considerations must be weighed against the potential analgesic relief that the medication is intended to provide.In the current study, we first conducted a pharmacokinetic analysis of firocoxib in Sprague–Dawley rat plasma to determine an appropriate dosing frequency. We then evaluated the use of firocoxib for reducing allodynia and hyperalgesia in Sprague–Dawley rats by using a plantar incisional model of postoperative pain, comparing the efficacy of firocoxib with those of meloxicam and buprenorphine. In light of previous studies in our lab using firocoxib in a similar mouse model of plantar incisional pain,⁴⁹ we hypothesized that firocoxib and buprenorphine would produce comparable antinociception in rats.     

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.     

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.     

Respiratory Rates and Arterial Blood-Gas Tensions in Healthy Rabbits Given Buprenorphine, Butorphanol, Midazolam, or Their Combinations

The objective of this study was to evaluate the respiratory effects of buprenorphine, butorphanol, midazolam, and their combinations in healthy conscious rabbits. Six adult female New Zealand white rabbits were anesthetized briefly with isoflurane by mask to allow placement of a catheter into the central ear artery. After a 60-min recovery period, a baseline arterial sample was obtained. Animals then were injected intramuscularly with either 0.9% NaCl (1 mL), buprenorphine (0.03 mg/kg), butorphanol (0.3 mg/kg), midazolam (2 mg/kg), buprenorphine + midazolam (0.03 mg/kg, 2 mg/kg), or butorphanol + midazolam (0.3 mg/kg, 2 mg/kg). Arterial blood gases were evaluated at 30, 60, 90, 120, 180, 240, and 360 min after drug administration. All drug treatments caused significant decreases in respiratory rate, compared with saline. Buprenorphine and the combinations of midazolam–butorphanol and midazolam–buprenorphine resulted in statistically significant decreases in pO₂. No significant changes in pCO₂ pressure were recorded for any treatment. Increases in blood pH were associated with administration of butorphanol, midazolam, and the combinations of midazolam–butorphanol and midazolam–buprenorphine. In light of these results, buprenorphine and the combinations of midazolam–buprenorphine and midazolam–butorphanol result in statistically significant hypoxemia in rabbits that breathe room air. The degree of hypoxemia is of questionable clinical importance in these healthy subjects. Hypoxemia resulting from these drug combinations may be amplified in rabbits with underlying pulmonary or systemic disease.Companion animal practitioners and academic anesthesiologists advocate many different combinations of sedatives and opioids to achieve balanced anesthesia. The benefits of premedicating with an opioid–sedative combination is that patient stress is reduced, animal handling is easier, preemptive analgesia can be attained, and inhalant anesthetic requirements are reduced, resulting in less cardiovascular and respiratory depression. In laboratory animals and pet exotic animals, such as rabbits, balanced anesthesia is often foregone in preference to simple mask induction with inhalant agents. In addition, analgesia in these species is often inadequate due to concern about adverse respiratory effects of opioids. Because obtaining a controlled airway in these species can be challenging, pronounced respiratory depression from opioids is an even greater concern. As such, the mixed agonist–antagonist opioids (for example, butorphanol) or partial-agonist opioids (for example, buprenorphine) are often chosen as analgesics or premedications in laboratory animal species, because these classes of opioids may induce less respiratory depression than others.The majority of the medical literature, both human and veterinary, points to a ‘ceiling effect’ in respiratory depression with agonist–antagonist or partial-agonist opioid drugs as compared with the dose-dependent respiratory depression that may be observed with pure μ-agonist opioids, such as hydromorphone.^6,7,24,26,27 There are conflicting data in the literature, however, regarding the clinical significance of respiratory depression associated with the partial-agonist and mixed agonist–antagonist opioids. One study that examined the respiratory and cardiovascular effects of buprenorphine in rabbits reported mild hypoxemia associated with its administration.²² Another study similarly found that administration of buprenorphine to anesthetized rabbits resulted in hypoventilation but no change in the ventilatory response to hypoxia.⁸ Buprenorphine caused a moderate decrease in respiratory rate in rabbits, but significant respiratory depression as evidenced by respiratory rate was apparent only at dosages higher than those used clinically.¹¹ Butorphanol has not been as extensively studied as a sole agent in rabbits. However, butorphanol and butorphanol combinations are known to result in statistically significant respiratory depression in rabbits, dogs, sheep, horses, and humans.^{15,23-25,27,28} In another study, dogs given butorphanol postoperatively had no change in pO₂ or pCO₂.⁵ Discrepancies in the literature regarding buprenorphine''s and butorphanol''s effects on respiratory drive likely relate to differences in methodology, sampling, and doses studied. The clinical significance of any changes in blood-gas tensions that occur after buprenorphine or butorphanol in most species is questionable in healthy animals, and most studies conclude that these 2 opioid drugs do not result in clinically unacceptable respiratory depression in most species.To our knowledge, no studies have examined the concurrent use of opioids and benzodiazepines and their effects on arterial blood gases in rabbits, despite the fact that, when premedication is used, benzodiazepines and butorphanol or buprenorphine are often combined for use in these animals.⁹ Various studies show that benzodiazepines, such as midazolam, cause mild to moderate hypoxemia in rabbits and humans,^5,13,18 but the clinical significance of any additive effect of benzodiazepine-induced respiratory depression when these drugs are combined with butorphanol or buprenorphine is unknown. The combination of benzodiazepines and opioids in rabbits can cause significant sedation.¹⁷ The purpose of the current study was to assess changes in arterial blood-gas values in healthy rabbits given midazolam, butorphanol, or buprenorphine, alone or in combination. We hypothesized that administration of butorphanol, buprenorphine, midazolam, or midazolam–opioid combinations would cause statistically significant changes in respiratory rate and arterial blood gases.     

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.     

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.     

Pharmacokinetics of Single-Dose Intramuscular and Subcutaneous Injections of Buprenorphine in Common Marmosets (Callithrix jacchus)

Although buprenorphine is the most frequently used opioid analgesic in common marmosets (Callithrix jacchus), there is limited information in the literature supporting current dosing regimens used for this species. The purpose of this study was to determine the pharmacokinetic profiles of single-dose buprenorphine HCl administered intramuscularly (IM) at 0.01 mg/kg in 6 adult marmosets (1.8 to 12.8 y old; 2 males, 4 females) and subcutaneously (SQ) at 0.01 mg/kg in 6 adult marmosets (2.3-4.4 y old; 3 males, 3 females) by mass spectrometry. Blood was collected at multiple time points from 0.25 to 24 h from unsedated animals following a hybrid sparse-serial sampling design. The maximal observed plasma concentration of buprenorphine (C_max) administered IM (2.57 ± 0.95 ng/mL) was significantly higher than administered SQ (1.47 ± 0.61 ng/mL). However, the time to C_max (T_max) was not statistically different between routes (17.4 ± 6 min for IM and 19.8 ± 7.8 min for SQ). The time of the last quantifiable concentration of buprenorphine was 5 ± 1.67 h for IM compared with 6.33 ± 1.51 h for SQ, which was not statistically different. The mean buprenorphine plasma concentration-time curves were used to propose a dosing frequency of 4 to 6 h for buprenorphine at 0.01 mg/kg IM or SQ based on a theoretical therapeutic plasma concentration threshold of 0.1 ng/mL. Based on the mean pharmacokinetic parameters and plasma-concentration time curves_, both IM and SQ routes of buprenorphine at this dose provide a rapid increase in the plasma concentration of buprenorphine above the therapeutic threshold, and may be more effective for acute rather than long-lasting analgesia. Further studies are needed to examine repeated dosing regimens and the efficacy of buprenorphine in common marmosets.

The common marmoset (Callithrix jacchus), a New World (platyrrhine) monkey belonging to the Callitrichidae family, is an important experimental model used in diverse areas of study, including neuroscience, behavior, drug metabolism and toxicology, infectious diseases, and in the development of genetically modified NHP models for biomedical research.^11,28,32,33 Marmosets often undergo minor and major survival surgical procedures as part of experimental protocols, including implantations, laparotomies, and obstetric procedures. However, biomedical research and veterinary procedures performed on marmosets have the potential to cause pain and distress, requiring effective analgesic regimens.⁵ The provision of appropriate analgesia to counteract painful stimuli is a humane necessity fundamental to multiple animal research regulatory policies.^3,14,24Buprenorphine, a Schedule III opioid analgesic, is a partial μ-agonist, δ- and κ-antagonist, and nociceptin receptor agonist.^2,8,48 This drug is the most commonly used opioid analgesic in NHPs, including laboratory marmosets.^{7,18,31,33,35} Dosages recommended for marmosets range from 0.005 to 0.02 mg/kg, either alone for mild to moderate pain or as part of a multimodal analgesic plan for moderate-to-severe pain.^6,18,30,33 These dosage recommendations have been primarily based on anecdotal information and limited evidence related to use and efficacy present in the literature. However, a recent study by Fitz and colleagues evaluated the pharmacokinetics of single-dose intramuscular (IM) buprenorphine at 0.02 mg/kg and subcutaneous (SQ) sustained-release buprenorphine (Bup SR) at 0.2 mg/kg in marmosets. Based on a therapeutic plasma concentration threshold of 0.1 ng/mL, the authors recommended dosing frequencies of 6 to 8 h for buprenorphine and 3.0 to 3.5 d for Bup SR.¹⁸New World monkey species are more sensitive to opioid-induced adverse side effects such as profound respiratory depression at doses considered safe in other NHPs, rodents, and other laboratory animal species.^{6,10,18,27,35,46} For example, in one study, buprenorphine, administered to C. jacchus at 0.02 mg/kg IM as a premedication before alfaxalone induction, resulted in apnea in 8 out of 9 study animals.⁶ In unsedated marmosets, administration of buprenorphine at 0.02 mg/kg IM has resulted in mild to moderate ataxia and moderate sedation.¹⁸At the Massachusetts Institute of Technology (MIT, Cambridge, MA), buprenorphine HCl is administered to marmosets at 0.005–0.01 mg/kg IM and SQ, and rarely elicits adverse effects. However, analgesic efficacy at these doses is unknown and redosing schedules have been empirically based. In addition, absorption of SQ buprenorphine in some species such as cats can be highly variable and unreliable.⁴³ Although IM and SQ routes are commonly used in marmosets, how the route of administration affects buprenorphine absorption and plasma clearance is unknown. These issues underscore the importance of experimentally determining PK data and developing optimal dosing strategies for the use of this drug in marmosets.We performed a study using healthy adult marmosets to evaluate and compare the IM and SQ pharmacokinetics of single-dose buprenorphine HCl, administered at a clinically relevant dosage of 0.01 mg/kg. Liquid chromatography–electrospray ionization–tandem mass spectrometry was used to demonstrate that buprenorphine reached quantifiable plasma concentrations after administration, and to determine the interval for which buprenorphine was quantifiable in the plasma. We hypothesized that a single-dose of buprenorphine administered SQ would have slower absorption (longer T_max) and longer duration (longer T_last), when compared with IM administration.     

Pharmacokinetics of 2 Formulations of Buprenorphine in Macaques (Macaca mulatta and Macaca fascicularis)

Buprenorphine is the cornerstone of pain management in nonhuman primates, but the pharmacokinetics of this widely used drug are unknown. The purpose of this study was to evaluate the pharmacokinetic profiles of buprenorphine (0.01 and 0.03 mg/kg IM) and sustained-release buprenorphine (0.2 mg/kg SC) in 2 macaque species (M. mulatta and M. fascicularis) by using mass spectrometry. The pharmacokinetics did not differ significantly between species, and buprenorphine was dose-proportional at the tested doses. The low and high doses of buprenorphine had elimination half-lives of 2.6 ± 0.7 and 5.3 ± 2.0 h, respectively, but the low-dose data were constrained by the sensitivity of the analytical method. Sustained-release buprenorphine had an elimination half-life of 42.6 ± 26.2 h. The AUC_0-Tlast of buprenorphine were 9.1 ± 4.3 and 39.0 ± 25.1 ng×h/mL for the low and high doses, respectively, and sustained-release buprenorphine had an AUC_0-Tlast of 177 ± 74 ng×h/mL. Assuming a hypothesized therapeutic buprenorphine plasma concentration threshold of 0.1 ng/mL in macaques, these results suggest that buprenorphine doses of 0.01 mg/kg IM should be administered every 6 to 8 h, whereas doses of 0.03 mg/kg IM can be administered every 12 h. These results further demonstrate that a single 0.2-mg/kg SC injection of sustained-release buprenorphine maintains plasma concentrations above 0.1 ng/mL for 5 d in macaques. These findings support a new dosing strategy using sustained-release buprenorphine to improve pain management, decrease animal stress, improve animal welfare, and simplify the postoperative management of nonhuman primates in laboratory animal and zoological settings.Abbreviation: λ_z, elimination constant; C_max, maximal observed plasma concentration; HDB, high-dose buprenorphine; LDB, low-dose buprenorphine; MRT, mean residence time; SRB, sustained-release buprenorphine; T_last, time of last quantifiable plasma analyte concentration; T_max, time to C_max; V, volume of distributionBuprenorphine is a key component of veterinary multimodal pain management, especially in nonhuman primates. The long duration of action, low risk of respiratory depression, and negligible cardiovascular effects in healthy animals make it an advantageous opioid analgesic agent.⁴³ The widely accepted dosage range for buprenorphine in nonhuman primates is 0.01 to 0.03 mg/kg IM twice daily.^13,14,20 This dosage is based on the canine dose and anecdotal evidence, because few studies in the primary literature address therapeutic dosages in laboratory animal species.^24,38,42 The premise that the current recommended dosing regimen of buprenorphine provides appropriate analgesia is unsubstantiated, introducing the possibility that nonhuman primates do not gain sufficient pain control from the opioid component of the pain management plan.A new formulation of buprenorphine is reported to have analgesic activity for up to 72 h in cats and rats.^7,15 The manufacturer reports that, when administered at 0.27 mg/kg SC, this sustained-release buprenorphine (SRB; ZooPharm, Fort Collins, CO) reaches maximal plasma concentration within 1 h and remains above 1.0 ng/mL for 72 h after injection in dogs. In light of the prolonged duration attained in dogs, this new formulation warrants further evaluation in nonhuman primates.Drugs with prolonged durations of action are preferred in veterinary medicine and are typically developed for either production or companion animals rather than laboratory animal species. Recently, 2 studies evaluated cefovecin sodium in nonhuman primates, with the expectation that this third-generation cephalosporin antibiotic would have an extended duration of activity in nonhuman primates as it does in dogs and cats. Unfortunately, both studies concluded that the plasma clearance of the antibiotic was 20-fold higher in nonhuman primates than in dogs, providing only 12 to 24 h of antibiotic activity and therefore no dosing advantage over other cephalosporin antibiotics in nonhuman primates.^37,39 One study further demonstrated differences in the metabolism of the drug between nonhuman primates species.³⁹ Collectively, these findings highlight the importance of determining optimal dosing strategies for any drug in targeted nonhuman primates species, rather than simply using a published dose for a different species without further evaluation.The purpose of this study was to evaluate the plasma concentrations and elimination kinetics of buprenorphine and SRB at clinically relevant dosages and administration routes in the 2 most common Old World nonhuman primate species used in research. Specifically, we used liquid chromatography–electrospray ionization–tandem mass spectrometry to confirm that buprenorphine and SRB achieved quantifiable plasma concentrations after injection and to verify how long buprenorphine and individual metabolites remained detectable in the plasma.     

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.     

Evaluation of Common Anesthetic and Analgesic Techniques for Tail Biopsy in Mice

Tail biopsy in mice is a common procedure in genetically modified mouse colonies. We evaluated the anesthetic and analgesic effects of various agents commonly used to mitigate pain after tail biopsy. We used a hot-water immersion assay to evaluate the analgesic effects of isoflurane, ice-cold ethanol, ethyl chloride, buprenorphine, and 2-point local nerve blocks before studying their effects on mice receiving tail biopsies. Mice treated with ethyl chloride spray, isoflurane and buprenorphine, and 2-point local nerve blocks demonstrated increased tail-flick latency compared with that of untreated mice. When we evaluated the behavior of adult and preweanling mice after tail biopsy, untreated mice demonstrated behavioral changes immediately after tail biopsy that lasted 30 to 60 min before returning to normal. The use of isoflurane, isoflurane and buprenorphine, buprenorphine, 2-point nerve block, or ethyl chloride spray in adult mice did not significantly improve their behavioral response to tail biopsy. Similarly, the use of buprenorphine and ethyl chloride spray in preweanling mice did not improve their behavioral response to tail biopsy compared with that of the untreated group. However, immersion in bupivacaine for 30 s after tail biopsy decreased tail grooming behavior during the first 30 min after tail biopsy. The anesthetic and analgesic regimens tested provide little benefit in adult and preweanling mice. Given that tail biopsy results in pain that lasts 30 to 60 min, investigators should carefully consider the appropriate anesthetic or analgesic regimen to incorporate into tail-biopsy procedures for mice.Abbreviation: HWI, Hot water immersionGenetically modified mice continue to advance the study of human disease. To obtain DNA for verification of genotype, mice may undergo tissues sample collection from multiple sites, including blood, saliva, hair, and stool. However, tail biopsy of the distal 5 mm continues to be the preferred location of obtaining sufficient DNA for genotyping.^14,19 Tail biopsy involves transecting multiple tissue types, including skin, nervous tissue, muscle, tendons, vasculature, cartilage, and bone,¹⁴ and this technique has the potential to cause short-term pain and distress. Several studies have demonstrated that tail biopsy without anesthesia causes short-term pain or distress that is greatest within the first 1 to 2 h but that may last as long as 5 h.^1,4,20,26 Similar physiologic changes associated with pain and distress were apparent and prolonged in anesthetized control mice.¹Analgesic protocols at the time of biopsy vary among institutions. IACUC policies from several institutions^{3,10,18,28,29} recommend but do not require anesthesia or analgesia for tail biopsy of mice 10 to 21 d old but do require anesthesia or analgesia for older mice. The anesthetic regimens to mitigate the temporary pain associated with tail biopsy at these institutions include immersion in ice-cold ethanol, topical ethyl chloride spray, and isoflurane inhalant anesthesia. Because of their short duration of action, we hypothesized that these agents provide poor management of the postprocedural pain associated with tail biopsy, and we decided to explore other practical options. The use of traditional drugs such as lidocaine, bupivacaine, or buprenorphine may provide more appropriate analgesia for tail biopsy. Here we examined the anesthetic and analgesic effects on mouse behavior after tail biopsy in adult and preweanling outbred NSA mice that had been treated with various analgesics and anesthetics.     

Transcervical Embryo Transfer in Mice

Although efficient transcervical transfer of embryos in mice would provide many advantages over a surgical method, the low success rate of transcervical transfer has hampered its acceptance and use. Here, we describe a novel device and protocol for transcervical embryo transfer in mice. Blastocysts from CD1 female mice were transferred into the uteri of 2.5-d pseudopregnant CD1 mice by using this method resulted in the successful development of 66.7% to 73.5% of the transferred blastocysts into live-born fetuses. Our method is as efficient as surgical embryo transfer yet is much simpler, easier, and markedly less traumatic to the recipient. In addition, our method provides hygienic and economic advantages and conforms to the principles of humane experimental technique. More importantly, our method provides a model for studying transcervical embryo transfer in cattle, other large animals, and humans.The vast numbers of embryos produced, manipulated, and cryopreserved for research involving genetically modified animals require the completion of numerous embryo transfer procedures in mice. The rapid transcervical transfer of embryos from donors into the uteri of recipient mice is preferable to use of surgical techniques, but the low success rate has dissuaded researchers from adopting this method,^1-4,6 and effective procedures are not yet available. We recently developed a method for the transcervical transfer of embryos in mice that we believe will assist many researchers.     

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.     

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     

Hemodynamic and Behavioral Differences after Administration of Meloxicam,Buprenorphine, or Tramadol as Analgesics for Telemeter Implantation in Mice

Cannulation of the common carotid artery for chronic, continuous radiotelemetric recording of aortic hemodynamic properties in mice is a highly invasive recovery surgery. Radiotelemetric recording, by its continuous nature, gives the most accurate measurements of hemodynamic variables in experimental animals, and is widely used in the study of cardiovascular diseases including hypertension. The American Heart Association has recommended data acquisition by radiotelemetric recording but did not provide guidelines regarding postoperative analgesic support. We assessed hemodynamic parameters, locomotor activity, food intake, and weight loss in radiotransmitter-implanted CD1 female mice receiving analgesic support during the first 48 h after surgery. The efficacy of analgesic support from the NSAID meloxicam was compared with that of the widely used opioid agonist buprenorphine and the related compound, tramadol. Meloxicam-treated mice recovered lost body weight more rapidly than did tramadol- or buprenorphine-treated mice. Furthermore, meloxicam-treated mice maintained circadian rhythm after surgery and had tighter regulation of mean arterial pressure than did tramadol- or buprenorphine-treated mice. Meloxicam was also superior with regard to food intake, locomotor activity, and limiting variance in hemodynamic parameters. This study indicates that when compared with buprenorphine and tramadol, meloxicam should be the postoperative analgesic of choice for radiotelemeter implantation in mice.Abbreviation: HR, heart rate; MAP, mean arterial pressureMeasurement of arterial blood pressure in experimental animals is tremendously important for the study of numerous diseases, including hypertension. Currently, the most accurate and highly validated method for measuring arterial pressure in experimental animals is radiotelemetry.³² Implantation of the devices requires a skilled surgeon, and rodents need at least 7 d to recover fully after surgery.²² The immense stress placed on animals by radiotelemeter implantation surgery has prompted numerous evaluations of novel implantation procedures.^{9,22,29,30,34,42} In addition, limited evidence is available regarding which postoperative analgesic provides the greatest benefit to recovering animals. When describing the use of radiotelemetry, American Heart Association guidelines covering the measurement of blood pressure in experimental animals make no mention of analgesia.³² Buprenorphine is currently the most commonly used postoperative analgesic after telemeter implantation surgery.^9,19,30 However, its efficacy in providing postoperative pain management in the absence of adverse side effects has been questioned.^3,4,28,46Buprenorphine is a centrally acting partial μ-opioid receptor agonist and a δ- and κ-receptor antagonist that exhibits approximately 75 to 100 times the antinociceptive effect of morphine.⁴⁶ Because buprenorphine is an opioid analgesic, its use is legislatively controlled in most jurisdictions. Tramadol has similar effects to buprenorphine, also acting as a μ-receptor agonist.³⁵ Tramadol has a lower affinity for the μ receptor than does buprenorphine and therefore displays a significantly shorter half-life.³⁵ In addition to being a μ-receptor agonist, tramadol''s analgesic activity is mediated through inhibiting norepinephrine and serotonin reuptake, thus blocking nociceptive impulses at the spinal level.²³ Meloxicam, another commonly used analgesic, is a NSAID and selective inhibitor of cyclooxygenase 2 inhibitor.¹⁸ Meloxicam''s mechanism of analgesia is via inhibition of synthesis of prostaglandin H₂, a precursor of mediators that elicit pain and inflammation.¹⁸The postoperative effects of analgesics involve more than just pain relief. Commonly used analgesics are known to modify locomotor activity,²⁸ food consumption,³ fluctuations in body weight,⁵ and hemodynamic factors.^4,28 Effective analgesia minimizes changes to animal behavior over this array of experimental paradigms as it reduces pain and distress. The purpose of the current study was to compare the effects of standard doses of commonly used analgesics in mice recovering from cannulation of the common carotid artery for placement of a radiotelemetric catheter tip into the aortic arch. This surgery is accompanied by subcutaneous placement of an approximately 1 cm × 1 cm × 1 cm radiotelemeter in the lateral flank and is classed as a chronic, recovery intervention category D animal care protocol in Canada.⁸ We found that, compared with tramadol and buprenorphine, meloxicam provided superior postoperative recovery, supporting full recovery of mice in less time and with fewer disruptions to normal homeostasis.