Pharmacokinetic Evaluation of a Topical Extended-release Analgesic in Mice.

Mice often undergo painful procedures and surgeries as part of biomedical research protocols. Buprenorphine, a partial µ-opioid receptor agonist and κ receptor antagonist, is commonly used to alleviate the pain associated with such procedures. Due to its pharmacokinetic profile, buprenorphine requires frequent dosing, resulting in handling stress that can impact animal welfare and study data. A long-acting transdermal buprenorphine formulation (LA-bup) was recently approved for use in cats to provide up to 4 d of postoperative analgesia. In this study, we characterized the pharmacokinetics of a single topical dosing of LA-bup in male and female CD-1 mice administered a 0.36-mg or 18-µL topical dose at select time points. Plasma buprenorphine concentrations were evaluated at 0.25, 0.5, 1, 1.5, 2, 4, 8, 24, 48, and 72 h (n = 3 mice/time point) and remained above the purported therapeutic threshold (1 ng/mL) from 1 to 24 h postadministration. Repeated daily dosing at 24 and 48 h demonstrated plasma levels above 1 ng/mL for up to 72 h with minimal accumulation or changes in maximal concentrations over time. Inadvertent transfer of the topical drug to nondosed mice in the same cage was evaluated by measuring plasma buprenorphine concentrations in nondosed mice cohoused with a single-dosed mouse. Male mice did not demonstrate transfer of drug via grooming or interactions, yet 2 out of 26 nondosed female mice had detectable buprenorphine plasma levels indicating a relatively low incidence of cross-ingestion in cohoused female mice. This study demonstrates that LA-bup is a promising analgesic in mice that could be used for tailored analgesia strategies, depending on the surgical model or duration of analgesic therapy.

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.

E-selectin inhibitor is superior to low-molecular-weight heparin for the treatment of experimental venous thrombosis.

BACKGROUND: This study evaluated E-selectin inhibition with GMI-1271 (Uproleselan [GMI]) alone and in combination with the standard of care low-molecular-weight heparin (LMWH) to improve vein recanalization, decrease vein wall inflammation and protect against adverse bleeding in a primate model. We sought to examine this novel treatment of venous thrombosis. METHODS: Using a well-documented primate animal model, iliac vein thrombosis was induced by balloon occlusion of the iliac vein for 6 hours. Starting on day 2 after thrombosis, animals began treatment in two phases. In phase one, nontreated controls received no treatment (n = 5) vs animals treated with the E-selectin inhibitor GMI, 25 mg/kg, subcutaneous (SC), once daily (n = 4) for 21 days (previously published data). In phase two, animals were treated with GMI plus a combination of LMWH 1.5 mg/kg or 40 mg (GMI + LMWHc) SC once daily (n = 8) for 19 days; and animals treated with LMWH 1.5 mg/kg or 40 mg (LMWHc) SC once daily (n = 6) for 19 days. Animals were evaluated by magnetic resonance venography for vein recanalization and inflammation by gadolinium extravasation, duplex ultrasound, coagulation tests (thromboelastography, bleeding time, prothrombin time, activated partial thromboplastin time, fibrinogen) and complete blood count at baseline, days 2, 7, 14, and 21 at euthanasia. Statistical analysis included using unpaired t test with Welch's correction for direct comparisons and one-way analysis of variance for comparison between the groups. RESULTS: Percent vein recanalization by magnetic resonance venography was highest in the GMI alone group followed by GMI + LMWHc, both significantly different from control. On ultrasound examination, animals treated with GMI alone had no decrease in open vein lumen by day 21, whereas decreases were observed in groups GMI + LMWHc (-26%), LMWHc (-27%), and controls (-80%). Vein wall inflammation decreased significantly in all treated groups. Intimal fibrosis and intimal thickness was best preserved in the GMI alone group. An analysis of total vein wall collagen revealed a trend in all treatment groups of decreasing vein wall collagen. No clinically significant bleeding events were noted in any group. The LMWH groups trended to have prolonged coagulation test values, whereas E-selectin inhibition with GMI did not cause clinically significant changes in coagulation measures. CONCLUSIONS: Treatment with E-selectin inhibition results in improved vein recanalization, a decrease in vein wall inflammation and vein wall intimal thickness and fibrosis, with no changes in markers of coagulation. E-selectin inhibition with GMI alone is superior to E-selectin inhibition combined with LMWH, LMWH alone, and no treatment in this deep vein thrombosis model of iliac vein thrombosis.

Compassion Fatigue in Laboratory Animal Personnel during the COVID-19 Pandemic.

Compassion Fatigue (CF) is commonly observed in professions associated with human and animal care. The COVID-19 pandemic compelled laboratory animal research institutions to implement new work practices in order to maintain essential animal care operations. These modifications ranged from shift changes to last-resort measures, such as culling animal colonies, to accommodate reduced staffing. Such changes could cause personnel to experience increased stress, isolation, and helplessness-all of which can increase CF risk. In the current study, 200 persons involved with animal research completed an online survey to gauge whether CF among laboratory animal personnel had increased during the pandemic. The survey examined professional quality of life, self-assessed levels of CF, institutional changes, perceived changes in animal welfare, and institutional measures intended to alleviate CF. A total of 86% of participants had experienced CF at some point in their career, with 41% experiencing a CF event (new or worsening symptoms of CF) during the pandemic. In addition, 90% of participants who reported a CF event also reported subsequent effects on their personal or professional lives. Health, employment, and animal-related stress that arose due to the pandemic were all found to influence CF scores significantly. Although 96% of respondents were considered essential workers, 67% did not feel as valued for their work as other essential personnel. Furthermore, 88% of personnel responsible for the euthanasia of healthy animals who experienced a CF event reported that CF also affected their personal life, professional life, or both, and 78% responded that interventions from internal CF programs or leadership did not help to alleviate symptoms of CF. The COVID-19 pandemic and resultant institutional changes will likely have lasting effects on persons and organizations. By determining and subsequently mitigating sources of CF, we can better assist the laboratory animal community during future crises.

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.

Strength and Sterility of Stock and Diluted Carprofen Over Time.

Carprofen, a nonsteroidal antiinflammatory drug, is a commonly used analgesic for laboratory animals. The manufacturer labeling indicates the stock bottle may be stored and used for up to 28 d under refrigeration. However, institutional guidelines may vary in how long diluted carprofen solutions can be stored before they must be discarded. When administered to laboratory rodents, small volumes of the stock solution are diluted to provide accurate dosing and ease of administration. Because carprofen is formulated for use in companion animals (for example, dogs) and comes in larger volume multidose vials, the majority of carprofen at our institution is discarded before it can be used. In this study, we evaluated the amount of target ingredient present (strength), sterility, and endotoxin levels of both stock and diluted carprofen when stored in a variety of containers and at multiple temperature settings for up to 180 d, mimicking facets of typical use in laboratory animal research and medicine. We demonstrated that when refrigerated and stored in sterile vials, stock and diluted carprofen can be kept and used for up to 180 d while retaining strength and sterility. For short-term use, diluted carprofen can also be stored for up to 60 d at room temperature in conical tubes. These results will help establish scientifically justified storage conditions for carprofen to ensure that these agents remain appropriately potent and sterile for therapeutic use in laboratory animals.

Development of the Nude Rabbit Model.

Loss-of-function mutations in the forkhead box N1 (FOXN1) gene lead to nude severe combined immunodeficiency, a rare inherited syndrome characterized by athymia, severe T cell immunodeficiency, congenital alopecia, and nail dystrophy. We recently produced FOXN1 mutant nude rabbits (NuRabbits) by using CRISPR-Cas9. Here we report the establishment and maintenance of the NuRabbit colony. NuRabbits, like nude mice, are hairless, lack thymic development, and are immunodeficient. To demonstrate the functional applications of NuRabbits in biomedical research, we show that they can successfully serve as the recipient animals in xenotransplantation experiments using human induced pluripotent stem cells or tissue-engineered blood vessels. Our work presents the NuRabbit as a new member of the immunodeficient animal model family. The relatively large size and long lifespan of NuRabbits offer unique applications in regenerative medicine, cancer research, and the study of a variety of other human conditions, including immunodeficiency.

Cancer Immunotherapy via Targeting Cancer Stem Cells Using Vaccine Nanodiscs.

Cancer stem cells (CSCs) proliferate extensively and drive tumor metastasis and recurrence. CSCs have been identified in over 20 cancer types to date, but it remains unknown how to target and eliminate CSCs in vivo. Aldehyde dehydrogenase (ALDH) is a marker that has been used extensively for isolating CSCs. Here we present a novel approach to target and reduce the frequency of ALDHhigh CSCs by vaccination against ALDH. We have identified ALDH1-A1 and ALDH1-A3 epitopes from CSCs and developed synthetic high-density lipoprotein nanodiscs for vaccination against ALDHhigh CSCs. Nanodiscs increased antigen trafficking to lymph nodes and generated robust ALDH-specific T cell responses. Nanodisc vaccination against ALDHhigh CSCs combined with anti-PD-L1 therapy exerted potent antitumor efficacy and prolonged animal survival in multiple murine models. Overall, this is the first demonstration of a simple nanovaccine strategy against CSCs and may lead to new avenues for cancer immunotherapy against CSCs.

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.

A new way to treat proximal deep venous thrombosis using E-selectin inhibition.

OBJECTIVE: There is an inter-relationship between thrombosis and inflammation. Previously, we have shown the importance of P-selectin in thrombogenesis and thrombus resolution in many preclinical animal models. The role of E-selectin has been explored in rodent models and in a small pilot study of clinical calf vein deep venous thrombosis. The purpose of this study was to determine the role of E-selectin in thrombosis in a primate model of proximal iliac vein thrombosis, a model close to the human condition. METHODS: Iliac vein thrombosis was induced with a well-characterized primate model. Through a transplant incision, the hypogastric vein and iliac vein branches were ligated. Thrombus was induced by balloon occlusion of the proximal and distal iliac vein for 6 hours. The balloons were then deflated, and the primates recovered. Starting on postocclusion day 2, animals were treated with the E-selectin inhibitor GMI-1271, 25 mg/kg subcutaneously, once daily until day 21 (n = 4). Nontreated control animals received no treatment (n = 5). All animals were evaluated by magnetic resonance venography (MRV); evaluation of vessel area by ultrasound, protein analysis, hematology (complete blood count), and coagulation tests (bleeding time, prothrombin time, activated partial thromboplastin time, fibrinogen, and thromboelastography) were performed at baseline, day 2, day 7, day 14, and day 21 with euthanasia. In addition, platelet function and CD44 expression on leukocytes were determined. RESULTS: E-selectin inhibition by GMI-1271 significantly increased vein recanalization by MRV vs control animals on day 14 (P < .05) and day 21 (P < .0001). GMI-1271 significantly decreased vein wall inflammation by MRV with gadolinium vein wall enhancement vs control also on day 14 (P < .0001) and day 21 (P < .0001). The thromboelastographic measure of clot strength (maximum amplitude) showed significant decreases in animals treated with GMI-1271 vs controls at day 2 (P < .05) and day 7 (P < .05). Animals treated with GMI-1271 had significant vessel area increase by day 21 vs controls (P < .05) by ultrasound. Vein wall intimal thickening (P < .001) and intimal fibrosis (P < .05) scores were significantly decreased in GMI-1271-treated animals vs controls. Importantly, no significant differences in hematology or coagulation test results were noted between all groups, suggesting that E-selectin inhibition carries no bleeding potential. GMI-1271 did not affect platelet function or aggregation or CD44 expression on leukocytes. In addition, no episodes of bleeding were noted in either group. CONCLUSIONS: This study suggests that E-selectin modulates venous thrombus progression and that its inhibition will increase thrombus recanalization and decrease vein wall inflammation, without affecting coagulation. The use of an E-selectin inhibitor such as GMI-1271 could potentially change how we treat deep venous thrombosis.

Intramuscular Administration of Alfaxalone Alone and in Combination for Sedation and Anesthesia of Rabbits (Oryctolagus cuniculus).

This study compared alfaxalone, alone and in combination with other medications, for sedative and anesthetic properties after intramuscular administration in New Zealand white rabbits. In the main portion of the study, 6 female rabbits were assigned to 5 treatment regimens in a blinded crossover design. Alfaxalone (6 mg/kg IM) was administered alone and in combination with each of the following: 0.3 mg/kg butorphanol; 1 mg/kg midazolam; 0.2 mg/kg dexmedetomidine; and both 0.3 mg/kg butorphanol and 0.2 mg/kg dexmedetomidine. An additional 6 rabbits received 0.2 mg/kg dexmedetomidine for comparison. The median time to onset of recumbency ranged from 2.0 to 5.5 min, with times significantly shorter for animals that received alfaxalone with either midazolam or dexmedetomidine than for those given dexmedetomidine only. Duration of sedation (mean ± 1 SD) was: alfaxalone only, 40 ± 7.3 min; alfaxalone with butorphanol, 47.8 ± 9.9 min; alfaxalone with midazolam, 65.2 ± 6.5 min; alfaxalone with dexmedetomidine, 157.5 ± 22.4 min; alfaxalone with butorphanol and dexmedetomidine, 157.7 ± 22.3 min, and dexmedetomidine only, 93.7 ± 11.9 min. Response to noxious stimuli was absent in 2 of the rabbits given dexmedetomidine only, 4 of those given alfaxalone with dexmedetomidine, and all 6 of the animals dosed with alfaxalone, butorphanol, and dexmedetomidine; this last group displayed the longest absence of a toe-pinch response (57 ± 3 min).

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.

Effects of subcutaneous alfaxalone alone and in combination with dexmedetomidine and buprenorphine in guinea pigs (Cavia porcellus).

OBJECTIVE: To characterize alfaxalone administered subcutaneously (SC) in guinea pigs, both alone and in combination with dexmedetomidine and buprenorphine. STUDY DESIGN: Prospective, blinded, crossover study. ANIMALS: A total of 15 healthy female guinea pigs weighing 400-600 g. METHODS: Alfaxalone (10, 20 and 40 mg kg-1) was administered SC to three guinea pigs as a pilot dose-finding study. Alfaxalone (20 mg kg-1; A20) was selected for comparison against combination protocols of alfaxalone (15 and 20 mg kg-1) with dexmedetomidine (0.25 mg kg-1) and buprenorphine (0.05 mg kg-1; A15DB, A20DB). Each protocol was randomly administered to 12 guinea pigs separated by ≥7 days. Time and quality of induction and recovery, heart rate, respiratory rate, peripheral hemoglobin oxygen saturation, rectal temperature, pedal withdrawal reflex and adverse effects were recorded. RESULTS: The median time to induction for A20, A15DB and A20DB was 6.8-8.0 minutes with no significant difference between treatments. Mean duration of recumbency for A20 was 73.6 ± 19.6 minutes. Recumbency duration for A15DB and A20DB extended to 90 minutes, at which time dexmedetomidine was antagonized using atipamezole (0.025 mg kg-1 SC). Physiological variables were within normal limits with the exception of one animal that died 45 minutes following treatment with A20DB. Pedal withdrawal reflex remained intact with all treatments. Minor side effects such as twitching or bruxism occurred sporadically with treatment A20 but not with A15DB and A20DB. CONCLUSIONS AND CLINICAL RELEVANCE: SC alfaxalone produced uncomplicated sedation that may be recommended for nonpainful procedures that do not require complete immobility. The addition of dexmedetomidine and buprenorphine increased the duration of sedation and immobility, but did not result in general anesthesia. This combination sedation protocol may be useful for nonpainful procedures requiring extended immobility.

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.

Methods of Pairing and Pair Maintenance of New Zealand White Rabbits (Oryctolagus Cuniculus) Via Behavioral Ethogram, Monitoring, and Interventions.

New Zealand White (NZW) laboratory rabbits (Oryctolagus cuniculus), as well as their ancestors the European Rabbit, are a social species that exhibit numerous benefits to being housed accordingly. Although these rabbits are innately gregarious, certain behaviors can still arise when kept in captivity, which if left unchecked, can confound research results or lead to wounding, which in extreme cases can be severe. To prevent these issues, there must be a well-structured plan for the monitoring and maintenance of paired laboratory rabbits. The purpose of this protocol is to present effective procedures for establishing newly paired NZW rabbits as well as methods for successful maintenance. Multiple methods have been tested for the creation of newly paired female rabbits from the vendor, but the most efficacious technique emphasizes capitalizing on the stress bonding from transport, urine marking, pairing in a neutral cage with no forced sharing of resources and a system of monitoring and intervention. To determine the best method of housing paired rabbits in a standard caging environment, data were collected to generate a behavioral ethogram. Behaviors were then quantified as positive, neutral or negative and were tracked across the lifespan of the pair to determine which behaviors indicated pair success or failure. With the newfound knowledge of socially housed laboratory NZW rabbit behavior, enrichment intervention was applied to alleviate aggression and prevent wounding, thus resulting in a higher percentage of successful pairs. Through several years of trialing different pairing methods, the development of the ethogram and the resulting enrichment interventions, understanding of the highly complex social constructs that dominate pair housed rabbit behavior has dramatically increased and allowed for the provision of more species-specific care and increased standards of welfare.

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.

Bacterial and Pneumocystis Infections in the Lungs of Gene-Knockout Rabbits with Severe Combined Immunodeficiency.

Using the CRISPR/Cas9 gene-editing technology, we recently produced a number of rabbits with mutations in immune function genes, including FOXN1, PRKDC, RAG1, RAG2, and IL2RG. Seven founder knockout rabbits (F0) and three male IL2RG null (-/y) F1 animals demonstrated severe combined immunodeficiency (SCID), characterized by absence or pronounced hypoplasia of the thymus and splenic white pulp, and absence of immature and mature T and B-lymphocytes in peripheral blood. Complete blood count analysis showed severe leukopenia and lymphocytopenia accompanied by severe neutrophilia. Without prophylactic antibiotics, the SCID rabbits universally succumbed to lung infections following weaning. Pathology examination revealed severe heterophilic bronchopneumonia caused by Bordetella bronchiseptica in several animals, but a consistent feature of lung lesions in all animals was a severe interstitial pneumonia caused by Pneumocystis oryctolagi, as confirmed by histological examination and PCR analysis of Pneumocystis genes. The results of this study suggest that these SCID rabbits could serve as a useful model for human SCID to investigate the disease pathogenesis and the development of gene and drug therapies.

Pharmacokinetics of a Transdermal Fentanyl Solution in Suffolk Sheep (Ovis aries).

Sheep used as surgical models require appropriate pain management, and the commonly used transdermal fentanyl patches require a long predosing period to achieve adequate plasma concentrations. The aim of this study was to assess the pharmacokinetic parameters of an FDA-approved transdermal fentanyl solution (TFS) that has yet to be tested in sheep. In this study, we compared TFS at 2.7 mg/kg (n = 2), 1.7 mg/kg (n = 3), and 0.5 mg/kg (n = 3) with the control fentanyl patch at 2 µg/kg/h (n = 1); both products were applied topically to the intrascapular region. Plasma concentrations showed significant interanimal variability. Severe adverse effects occurred at both 2.7 and 1.7 mg/kg TFS and mild to moderate adverse effects were noted at 0.5 mg/kg. At all 3 doses, TFS had greater maximal concentration, clearance rate, and volume of distribution; shorter time to maximal concentration; and similar half-lives to those of the patch. In addition, we validated the use of a commercial human fentanyl ELISA kit, which positively correlated with the liquid chromatography-mass spectroscopy data, but absolute values did not match. Overall, at all 3 dosages tested (0.5, 1.7, and 2.7 mg/kg), TFS delivered fentanyl plasma concentrations that exceeded the minimal effective concentration; however, adverse effects were noted at all 3 dosages. Caution and further study are required before the use of TFS in sheep can be recommended fully.