Evaluation of Efficacy of 2 Extended-release Bupivacaine Products in a Porcine Model of Incisional Pain.

Extended-release (ER) local anesthetics are often incorporated in multi-modal analgesia or as an alternative when the effect of systemic analgesics may confound research. In this study, we compared the analgesic efficacy of 2 ER bupivacaine anesthetics with different ER mechanisms, a slow-release bupivacaine-meloxicam polymer (BMP) and a sucrose acetate isobutyrate bupivacaine (SABER-B) system. We used a full-thickness unilateral skin incision porcine model to evaluate the efficacy of these 2 ER bupivacaine analgesics. Eighteen male swine were randomized into 3 groups: control (saline; n = 6), bupivacaine:meloxicam (10 mg/kg, 0.3 mg/kg; n = 6), and SABER-B (10 mg/kg; n = 6). After surgery, pigs were assessed for changes in body weight, salivary cortisol level, and response to von Frey testing at 1, 3, 6, 24, 48, 72, 96, 120, and 168 h. Body weight and salivary cortisol levels were not significantly different between groups. Based on the von Frey testing, the pigs that received analgesics showed a significantly higher withdrawal threshold of nociceptive stimulus than those that received saline at 1, 3, 6, and 24 h after the surgery. At 48 h after surgery, the SABER-B group had a significantly higher withdrawal threshold than the saline group. The withdrawal threshold was not significantly different from the baseline measurement on intact skin at 3 and 6 h after surgery in the BMP group or 1 and 3 h for the SABERB group. The analgesic effects of BMP were greatest at 3 and 6 h after surgery and that of SABER-B as 1 and 3 h SABER-B provided an earlier onset of analgesia and longer analgesia duration than did BMP. This study demonstrates that ER bupivacaine can provide pigs with 24 to 48 h of analgesia for incisional pain.

Comparison of Systemic Extended-release Buprenorphine and Local Extended-release Bupivacaine-Meloxicam as Analgesics for Laparotomy in Mice.

Extended-release (ER) local anesthetics can be used in multi-modal analgesia or in situations in which systemic analgesics may alter animal physiology and thus introduce interpretational confounds. In this study, we compared the analgesic efficacy of an ER buprenorphine formulation with that of a synergistic combination of ER bupivacaine and meloxicam. Female and male CD1 mice were randomly assigned to receive subcutaneous buprenorphine (3.25mg/kg) preemptively, subcutaneous infiltration of bupivacaine???meloxicam (0.03mL at incision closure (bupivacaine, 35mg/kg; meloxicam, 1mg/kg), or saline (10mL/kg SC) after induction of anesthesia. After laparotomy, mice were assessed for changes in daily body weight, rearing frequency, nest consolidation scores, time-to-integrate-nest test (TINT), and response to von Frey testing at 4, 8, 24, 48, and 72h after surgery. Daily weight, nest consolidation scores and rearing frequency were not significantly different among the 3 groups. TINT had fallen significantly response at 24 and 48h after injection in the ER buprenorphine group as compared with the saline and ER bupivacaine-meloxicam groups. Nociceptive thresholds, as assessed with von Frey testing, differed between saline controls and both analgesic groups at 4, 8, 24, 48, and 72 h after surgery. None of the mice in the bupivacaine???meloxicam group developed signs of neurotoxicity, a potential side effect of high-dose local anesthetics. This study demonstrates that local ER bupivacaine???meloxicam may be a useful alternative to systemic, ER buprenorphine for the relief of pain after laparotomy in mice.

Evaluation of alfaxalone total intravenous anesthesia in rabbits (Oryctolagus cuniculus) premedicated with dexmedetomidine or dexmedetomidine and buprenorphine.

OBJECTIVE: To evaluate alfaxalone for total intravenous anesthesia (TIVA) in rabbits premedicated with dexmedetomidine or dexmedetomidine and buprenorphine. STUDY DESIGN: Crossover study (part 1) with observational study (part 2). ANIMALS: A total of eight New Zealand White rabbits (Oryctolagus cuniculus), four female and four male, aged 12-16 weeks and weighing 2.8-3.5 kg in part 1. Separately, four additional rabbits in part 2. METHODS: Crossover study design with eight rabbits per treatment. Rabbits were administered treatment D, dexmedetomidine (0.2 mg kg-1), or treatment DB, dexmedetomidine (0.1 mg kg-1) and buprenorphine (0.05 mg kg-1) intramuscularly. Anesthesia was induced with alfaxalone intravenously until a supraglottic airway device was placed to deliver 100% oxygen. Anesthesia was maintained with alfaxalone (TIVA). Infusion rates were adjusted to achieve an absent pedal withdrawal reflex. Heart rate, respiratory rate, noninvasive blood pressure, end-tidal carbon dioxide partial pressure and peripheral hemoglobin oxygen saturation (SpO2) were recorded every 5 minutes. Subsequently, four rabbits underwent ovariohysterectomy using treatment DB and alfaxalone TIVA. RESULTS: The mean ± standard deviation alfaxalone infusion rate was 9.6 ± 2.6 and 4.5 ± 1.3 mg kg-1 hour-1 for treatments D and DB, respectively. In both treatments, blood pressure remained within acceptable range and SpO2 was > 95%. Postinduction apnea and respiratory depression were observed in both treatments and managed with manual positive pressure ventilation. Four separate rabbits underwent successful ovariohysterectomy with treatment DB and alfaxalone TIVA. One rabbit required supplementation with inhalant anesthesia; three rabbits were successfully maintained using alfaxalone TIVA alone. CONCLUSIONS AND CLINICAL RELEVANCE: Premedication with dexmedetomidine-buprenorphine combined with alfaxalone TIVA may be a viable alternative for performing abdominal surgery in the rabbit. The use of supplemental oxygen and ability to provide respiratory support are advised.

Vaccine nanodiscs plus polyICLC elicit robust CD8+ T cell responses in mice and non-human primates.

Conventional cancer vaccines based on soluble vaccines and traditional adjuvants have produced suboptimal therapeutic efficacy in clinical trials. Thus, there is an urgent need for vaccine technologies that can generate potent T cell responses with strong anti-tumor efficacy. We have previously reported the development of synthetic high-density protein (sHDL) nanodiscs for efficient lymph node (LN)-targeted co-delivery of antigen peptides and CpG oligonucleotides (a Toll-like receptor-9 agonist). Here, we performed a comparative study in mice and non-human primates (NHPs) to identify an ideal vaccine platform for induction of CD8+ T cell responses. In particular, we compared the efficacy of CpG class B, CpG class C, and polyICLC (a synthetic double-stranded RNA analog, a TLR-3 agonist), each formulated with antigen-carrying sHDL nanodiscs. Here, we report that sHDL-Ag admixed with polyICLC elicited robust Ag-specific CD8+ T cell responses in mice, and when used in combination with α-PD-1 immune checkpoint inhibitor, sHDL-Ag + polyICLC eliminated large established (~100 mm3) MC-38 tumors in mice. Moreover, sHDL-Gag + polyICLC induced robust Simian immunodeficiency virus Gag-specific, polyfunctional CD8+ T cell responses in rhesus macaques and could further amplify the efficacy of recombinant adenovirus-based vaccine. Notably, while both sHDL-Ag-CpG-B and sHDL-Ag-CpG-C generated strong Ag-specific CD8+ T cell responses in mice, their results were mixed in NHPs. Overall, sHDL combined with polyICLC offers a strong platform to induce CD8+ T cells for vaccine applications.

Anesthetic agents affect urodynamic parameters and anesthetic depth at doses necessary to facilitate preclinical testing in felines.

Urodynamic studies, used to understand bladder function, diagnose bladder disease, and develop treatments for dysfunctions, are ideally performed with awake subjects. However, in small and medium-sized animal models, anesthesia is often required for these procedures and can be a research confounder. This study compared the effects of select survival agents (dexmedetomidine, alfaxalone, and propofol) on urodynamic (Δpressure, bladder capacity, bladder compliance, non-voiding contractions, bladder pressure slopes) and anesthetic (change in heart rate [∆HR], average heart rate [HR], reflexes, induction/recovery times) parameters in repeated cystometrograms across five adult male cats. The urodynamic parameters under isoflurane and α-chloralose were also examined in terminal procedures for four cats. Δpressure was greatest with propofol, bladder capacity was highest with α-chloralose, non-voiding contractions were greatest with α-chloralose. Propofol and dexmedetomidine had the highest bladder pressure slopes during the initial and final portions of the cystometrograms respectively. Cats progressed to a deeper plane of anesthesia (lower HR, smaller ΔHR, decreased reflexes) under dexmedetomidine, compared to propofol and alfaxalone. Time to induction was shortest with propofol, and time to recovery was shortest with dexmedetomidine. These agent-specific differences in urodynamic and anesthetic parameters in cats will facilitate appropriate study-specific anesthetic choices.

Safety Considerations When Working with Humanized Animals.

Research using laboratory animals has been revolutionized by the creation of humanized animal models, which are immunodeficient animals engrafted with human cells, tissues, or organs. These animal models provide the research community a unique and promising opportunity to mimic a wide variety of disease conditions in humans, from infectious disease to cancer. A vast majority of these models are humanized mice like those injected with human CD34+ hematopoietic stem cells and patient-derived xenografts. With this technology comes the need for the animal research enterprise to understand the inherent and potential risks, such as exposure to bloodborne pathogens, associated with the model development and research applications. Here, we review existing humanized animal models and provide recommendations for their safe use based on regulatory framework and literature. A risk assessment program-from handling the human material to its administration to animals and animal housing-is a necessary initial step in mitigating risks associated with the use of humanized animals in research. Ultimately, establishing institutional policies and guidelines to ensure personnel safety is a legal and ethical responsibility of the research institution as part of the occupational health and safety program and overall animal care and use program.

Analgesic Efficacy and Hematologic Effects of Robenacoxib in Mice.

NSAID analgesics may confound models that require inflammation to mimic disease development in humans. This effect presents a challenge for veterinary staff and investigators, because surgery is often necessary to create mouse models of disease and NSAID are first-line analgesics used to treat postoperative pain. We evaluated robenacoxib, a NSAID highly selective for cyclooxygenase 2, in a carrageenan paw edema (CPE) assay and surgical model of venous thrombosis (VT). We generated a mouse-specific dose-response curve by using the CPE assay for robenacoxib doses of 3.2, 10, 32 and 100 mg/kg SC. Electronic von Frey assay, calipers, and novel software for measuring open-field activity revealed that all robenacoxib doses provided, identified effective analgesia at 3 and 6 h, compared with saline. In addition, the 100-mg/kg dose had measurable antiinflammatory effects but yielded adverse clinical side effects. Because the 32-mg/kg dose was the highest analgesic dose that did not decrease paw swelling, we evaluated it further by using the same nociceptive and behavioral assays in addition to a novel nest-consolidation test, and assessment of blood clotting and hematologic parameters in the surgical VT model. A single preemptive dose of either 32 mg/kg SC robenacoxib or 5 mg/kg SC carprofen protected against secondary hyperalgesia at 24 and 48 h. Neither drug altered clot formation or hematology values in the VT model. The open-field activity software and our novel nest consolidation test both identified significant postoperative discomfort but did not differentiate between saline and analgesia groups. In light of these data, a single preemptive subcutaneous dose of 32 mg/kg of robenacoxib or 5 mg/kg of carprofen did not impede this VT mode but also failed to provide sufficient postoperative analgesia.

Refinement of Perioperative Feeding in a Mouse Model of Vertical Sleeve Gastrectomy.

Provision of liquid enteral nutrition (LEN) during the perioperative period is standard practice for rodents undergoing bariatric surgery, yet these diets are associated with several challenges, including coagulation of the liquid diet within the delivery system and decreased postoperative consumption. We investigated the use of a commercially available high-calorie dietary gel supplement (DG) as an alternative food source for mice during the perioperative period. C57BL/6J male mice were fed high-fat diet for 8 to 10 wk prior to surgery. The study groups were: vertical sleeve gastrectomy (VSG) +DG, VSG+LEN, sham surgery+DG, and sham+LEN. Food and water intakes, body weight, and body fat composition was monitored throughout the study. Mice that received DG lost significantly more weight preoperatively than those fed LEN. However, during the postoperative period, body weight, body fat composition, and water and caloric intake were similar among all experimental diet groups. Three mice in the VSG+LEN group were euthanized due to clinical illness during the course of the study. In summary, feeding a high-calorie DG to mice undergoing VSG surgery is a viable alternative to LEN, given that DG does not significantly affect the surgical model of weight loss or result in adverse clinical outcomes. We recommend additional metabolic characterization of DG supplementation to ensure that this novel diet does not confound specific research goals in the murine VSG model.

Apixaban Versus Warfarin for Mechanical Heart Valve Thromboprophylaxis in a Swine Aortic Heterotopic Valve Model.

OBJECTIVE: Warfarin is the current standard for oral anticoagulation therapy in patients with mechanical heart valves, yet optimal therapy to maximize anticoagulation and minimize bleeding complications requires routine coagulation monitoring, possible dietary restrictions, and drug interaction monitoring. As alternatives to warfarin, oral direct acting factor Xa inhibitors are currently approved for the prophylaxis and treatment of venous thromboembolism and reduction of stroke and systemic embolization. However, no in vivo preclinical or clinical studies have been performed directly comparing oral factor Xa inhibitors such as apixaban to warfarin, the current standard of therapy. APPROACH AND RESULTS: A well-documented heterotopic aortic valve porcine model was used to test the hypothesis that apixaban has comparable efficacy to warfarin for thromboprophylaxis of mechanical heart valves. Sixteen swine were implanted with a bileaflet mechanical aortic valve that bypassed the ligated descending thoracic aorta. Animals were randomized to 4 groups: control (no anticoagulation; n=4), apixaban oral 1 mg/kg twice a day (n=5), warfarin oral 0.04 to 0.08 mg/kg daily (international normalized ratio 2-3; n=3), and apixaban infusion (n=4). Postmortem valve thrombus was measured 30 days post-surgery for control-oral groups and 14 days post-surgery for the apixaban infusion group. Control thrombus weight (mean) was significantly different (1422.9 mg) compared with apixaban oral (357.5 mg), warfarin (247.1 mg), and apixiban 14-day infusion (61.1 mg; P<0.05). CONCLUSIONS: Apixaban is a promising candidate and may be a useful alternative to warfarin for thromboprophylaxis of mechanical heart valves. Unlike warfarin, no adverse bleeding events were observed in any apixaban groups.

Effects of analgesic use on inflammation and hematology in a murine model of venous thrombosis.

Venous thrombosis (VT) is a significant cause of morbidity and mortality in humans. Surgical animal models are crucial in studies investigating the pathogenesis of this disease and evaluating VT therapies. Because inflammation is critical to both the development and resolution of VT, analgesic medications have the potential to adversely affect multiple parameters of interest in VT research. The objective of this study was to determine how several common analgesics affect key variables in a murine ligation model of deep vein thrombosis. Male C57BL/6 mice were randomly assigned to receive either local (bupivacaine) or systemic parenteral analgesia (buprenorphine, tramadol, or carprofen) or 0.9% NaCl (control). All mice underwent laparotomy and ligation of the inferior vena cava, and treatment was continued until euthanasia at 6 or 48 h after surgery. Analysis of harvested tissues and blood included: hematology, thrombus weight, serum and vein-wall cytokines (IL1ß, IL6, IL10, TNFα), soluble P-selectin, and vein-wall leukocyte infiltration. Compared with 0.9% NaCl, all of the analgesics affected multiple parameters important to VT research. Carprofen and tramadol affected the most parameters and should not be used in murine models of VT. Although they affected fewer parameters, a single dose of bupivacaine increased thrombus weight at 6 h, and buprenorphine was associated with reduced vein wall macrophages at 48 h. Although we cannot recommend the use of any of the evaluated analgesic dosages in this mouse model of VT, buprenorphine merits additional investigation to ensure the highest level of laboratory animal care and welfare.

Renal transitional cell carcinoma and choristoma in a degu (Octodon degus).

A 4.5-year-old female degu (Octodon degus) was minimally responsive with a poor body condition, a rough haircoat, and moderate dehydration. Blood was present around its urethral orifice and on the cage bedding. Laboratory analyses revealed leukocytosis with neutrophilia and anemia; hypoproteinemia and hypoalbuminemia; hyperglycemia, hyperphosphatemia, and elevated alanine aminotransferase, blood urea nitrogen, and creatinine; and hematuria and pyuria with occasional squamous and transitional epithelial cells. A urine culture was positive for coagulase-negative Staphylococcus sp. On gross necropsy, the right kidney was enlarged, cystic, and greenish-brown, with a 10-mm, hemorrhagic, granular mass extending from the renal pelvis into the cranial cortex. Only a small amount of renal cortex appeared normal. The urinary bladder had focal areas of hemorrhage and contained frank blood. Histologically, the papillary mass in the right renal pelvis comprised basophilic, moderately anaplastic, clustered epithelial transition cells consistent with a transitional cell carcinoma. Internally, the tumor showed squamous metaplasia and moderate multifocal interstitial fibrosis. The right kidney cortex contained a choristoma comprising trabecular bone, mature adipocytes, and cellular infiltrates suggestive of osteocytes, lymphocytes, and plasma cells. The urinary bladder had mild to moderate, focal, hemorrhage with neutrophilic inflammation and contained focal areas of mild transitional cell epithelial hyperplasia; these changes may have been secondary to irritation by hemorrhage in the renal pelvis. There was no evidence of metastasis. Renal transitional cell tumors are rare in rodents. This is the first report of both a renal transitional cell carcinoma and a renal choristoma in a degu.

The sedative and behavioral effects of nalbuphine in dogs.

We compared the degree of sedation and frequency and intensity of adverse behaviors in dogs associated with nalbuphine when combined with acepromazine or xylazine compared with those of acepromazine or xylazine alone. Twenty-four dogs (13 female, 11 male) undergoing routine ovariohysterectomy or castration were randomly assigned to one of four groups. Group NX received 0.5 mg/kg nalbuphine and 0.5 mg/kg xylazine subcutaneously (s.c.). Group X received 0.5 mg/kg xylazine s.c. Group NA received 0.5 mg/kg nalbuphine and 0.05 mg/kg acepromazine s.c. Group A received 0.05 mg/kg acepromazine s.c. All dogs received 0.01 mg/kg glycopyrrolate s.c. All doses were administered preoperatively. Preoperative resting measurements of heart rate, respiratory rate, rectal temperature, and body weight were obtained. Sedation was scored both inside and outside a kennel prior to drug administration and at 10, 20, and 30 min after drug administration. Dogs were assessed for behavioral responses (leg withdrawal, shivering, rigidity, orienting, panting, struggling, vocalization, wide-eyed facial expression, breath holding, salivating, hiding, biting, or requiring a muzzle) during three time periods: placing the dog on the table, clipping and prepping of forelimb, and intravenous catheterization. Postoperative recovery behaviors were scored. Expired halothane concentrations were recorded at 15, 30, and 45 min postinduction. Significant differences occurred in the level of sedation at 30 min between dogs receiving nalbuphine and xylazine or xylazine only compared with dogs receiving acepromazine. There was a significant difference in behavioral scores with respect to leg withdrawal and orienting during clipping/prepping between dogs receiving nalbuphine and xylazine compared with dogs receiving xylazine. The combination of nalbuphine and xylazine is a useful premedicant which provided greater sedation than acepromazine and reduced some anxiety behaviors more than did xylazine alone. Nalbuphine is an inexpensive opioid and currently is not a controlled substance in the U.S.