A Novel Scoring System for Humane Endpoints in Mice with Cecal Ligation and Puncture-Induced Sepsis.

Animal-based research is essential to the study of sepsis pathophysiology, diagnostics, and therapeutics. However, animal models of sepsis are often associated with high mortality because of the difficulty in predicting imminent death based on premortem assessment of the animals. The use of validated visual scoring would allow researchers to systematically identify humane endpoints but visual approaches require high interobserver agreement for accurate results. The objective of this study was to establish a scoring system for mice undergoing cecal ligation and puncture (CLP)-induced sepsis based on 3 visual parameters: respiratory status, activity and response to stimulus (ASR), and eye appearance, with scores ranging from 0 to 3. In the first study, we evaluated interobserver agreement. Veterinary and investigative staff assessed 283 mice with CLP and had substantial to near-perfect agreement for all 3 parameters as evaluated using weighted Cohen κ statistic. The second study assessed the ability of the scoring system and temperature to predict death. The scoring system and subcutaneous transpond- ers were used to monitor C57BL/6J mice (n = 80, male and female) until death or for 7 days after CLP. Results showed that the scoring system discriminates between surviving (n = 26) and nonsurviving (n = 54) septic mice. The scoring system was accurate in predicting death, with an AUC of 0.8997. The sensitivity and specificity of the ASR parameter were 96% and 92%, respectively, and for the eye parameter were 94% and 73%. A sum of the ASR and eye scores that was 5 or more was also predictive of death. Temperature was a quantitative predictor, with sensitivity and specificity of 93% and 92%, respectively. This scoring system refines the CLP model by allowing identification of humane endpoints and avoidance of spontaneous death.

Sterility of Sustained-release Buprenorphine.

Sustained-release formulations of controlled substances are commonly used to provide analgesia in research animals. These formulations represent refinements that offer the advantage of prolonged, multiday pain relief with a single injection, thereby decreasing handling stress in animals and saving time for scientists. Compounding pharmacies produce sustainedrelease buprenorphine for veterinary use (i. e., buprenorphine SR-LAB); one of these pharmacies has shortened the original 6-mo shelf-life to 28 d to comply with United States Pharmacopeia standards for ensuring sterility. This limitation risks increasing the waste of controlled substances, which require an expensive destruction process that is legally enforced in our state. To assess whether the sterility of buprenorphine SR-LAB is preserved for at least 6 mo in a general laboratory setting, we tested 5 bottles for the presence of endotoxin and bacterial and fungal contamination monthly for 6 mo. Overall, results of the study showed that the bottles remained sterile over the 6-mo duration as no endotoxin was detected and the bottles did not become contaminated with bacteria or fungi. In conclusion, when stored securely and used with aseptic handling techniques, buprenorphine SR-LAB can be maintained in a sterile state for 6 mo in a general laboratory setting.

Micro- and Macroenvironmental Conditions and Stability of Terrestrial Models.

Environmental variables can have profound effects on the biological responses of research animals and the outcomes of experiments dependent on them. Some of these influences are both predictable and unpredictable in effect, many are challenging to standardize, and all are influenced by the planning and conduct of experiments and the design and operation of the vivarium. Others are not yet known. Within the immediate environment where the research animal resides, in the vivarium and in transit, the most notable of these factors are ambient temperature, relative humidity, gaseous pollutant by-products of animal metabolism and physiology, dust and particulates, barometric pressure, electromagnetic fields, and illumination. Ambient temperatures in the animal housing environment, in particular those experienced by rodents below the thermoneutral zone, may introduce degrees of stress and thermoregulatory compensative responses that may complicate or invalidate study measurements across a broad array of disciplines. Other factors may have more subtle and specific effects. It is incumbent on scientists designing and executing experiments and staff responsible for animal husbandry to be aware of, understand, measure, systematically record, control, and account for the impact of these factors on sensitive animal model systems to ensure the quality and reproducibility of scientific studies.

Comparative Effects of 1/4-inch and 1/8-inch Corncob Bedding on Cage Ammonia Levels, Behavior, and Respiratory Pathology of Male C57BL/6 and 129S1/Svlm Mice.

Corncob is a common bedding material used in laboratory rodents, but little is known about differences in the effects of the 2 available sizes on rodent models and health. This study compared the effects of these 2 corncob bedding sizes on cage ammonia levels, behavior, and respiratory pathology in mice. We hypothesized that the beddings would not differ significantly in their effects on these parameters. Two strains of male mice (C57BL/6 and 129S1/Svlm) were housed in static, filter-top cages containing 1 of the 2 bedding types for the duration of the study (12 wk). Intracage ammonia was measured during 1 wk of the study on days 0, 3, 5, and 7. Behavior was evaluated by using circadian rhythm, open field, and Morris water-maze tests. Animals were euthanized with injectable euthanasia solution to collect respiratory and ocular tissues for histopathologic lesion scoring. Animals that were euthanized immediately upon arrival from the vendor served as negative controls. Bedding size did not significantly affect behavior or ammonia levels. Average intracage ammonia levels on day 7 were 525 ppm for 1/4-in. bedding and 533 ppm for 1/8-in. bedding. Regardless of the bedding size, lesions noted in both strains of mice were of similar incidence and severity, were limited to the nose, and consisted of minimal to mild suppurative rhinitis. The eyes, trachea, and lungs were not affected. In conclusion, 1/4-in. and 1/8-in. corncob beddings have comparable effects on cage ammonia levels and the behavior and respiratory pathology in male mice of the strains tested.

A Comparison of Ketamine or Etomidate Combined with Xylazine for Intraperitoneal Anesthesia in Four Mouse Strains.

Intraperitoneal (IP) injection is a common route of anesthetic administration in mice. Ketamine-xylazine (KX) anesthesia is one of the most widely used IP protocols, but has limitations. Etomidate is an alternative to ketamine that has been used in both human and veterinary medicine yet has not been widely studied in mice. The purpose of this study was to evaluate etomidate-xylazine (EX) anesthesia as an alternative to KX. We hypothesized that EX would be as safe and effective as KX, with both sex- and strain-dependent differences. Male and female Crl:CD1(ICR), C57BL/6NCrl, BALB/cJ and NU/J mice were given a single IP dose of ketamine 100 mg/kg and xylazine 10 mg/kg or etomidate 20 mg/kg and xylazine 10 mg/kg. Sedation times were similar between KX and EX, with CD1 mice exhibiting shorter sedation times. Surgical anesthesia was achieved in 44% of EX mice, compared with 4% of KX mice. C57BL/6NCrl mice were significantly more likely to achieve surgical anesthesia when given EX (94%) or KX (18%) than were other strains. In all strains except C57BL/6NCrl mice, females were more likely to reach surgical anesthesia than males. Several mice experienced an adverse hyperexcitement response during induction, with BALB/cJ (79%) and NU/J (87%) mice given EX significantly more likely than other strains to experience hyperexcitement. EX and KX protocols had no overall differences in lowest respiration rate, lowest systolic blood pressure, lowest rectal temperature, or levels of acidosis, although the lowest heart rates were significantly higher with EX, indicating that EX and KX have similar safety profiles. Thus, EX and KX administration were associated with several significant physiologic differences when comparing sexes or individual strains. Our results indicate that EX is an equally effective sedative and a more effective surgical anesthetic than KX; however, EX is only recommended for invasive procedures in C57BL/6 mice due to the high rate of hyper-excitement and inconsistent surgical depth seen in other strains. Further study is needed to optimize EX for use in multiple mouse strains.

Brief anesthesia by isoflurane alters plasma corticosterone levels distinctly in male and female rats: Implications for tissue collection methods.

Euthanasia by anesthetic agents is commonly performed prior to tissue collection in order to minimize pain and distress to the animal. However, depending on their mechanism of action as well as administration regimen, different methods of anesthesia may trigger an acute stress response through engaging the hypothalamic-pituitary-adrenal (HPA) axis, which can impact numerous other physiological processes that the researcher may wish to examine as endpoints. We investigated the effects of the commonly used anesthetic agent isoflurane on two different endpoints related to the stress response: plasma corticosterone levels and gene expression of the glucocorticoid receptor (GR) as well as several of its regulators including FK506-binding protein 51 (Fkbp5) in the hippocampus of male and female rats. Our results indicate that brief exposure to anesthesia by isoflurane prior to decapitation can alter plasma corticosterone levels differentially in male and female rats within minutes without impacting gene expression in the hippocampus. We conclude that collection methods can influence stress-related physiological endpoints in female rats and the potential influence of even brief anesthesia as well as sex differences in response to anesthesia should be evaluated during the experimental design process and data interpretation. This finding is particularly important in light of new NIH standards regarding sex and reproducibility, and care should be taken to be certain that sex differences in endpoints of interest are not an artifact of sex differences in response to collection paradigms.

Safety and efficacy of topical lime sulfur in mice infested with Myocoptes musculinus.

Current treatment options for murine fur mites have limitations in safety and efficacy. This study evaluated whether topical lime sulfur (LS) is an adjunct or alternative to traditional treatment options for Myocoptes musculinus. To evaluate the safety of topical LS, mice were dipped in a 3% LS solution at 34 and 41 d of age. Mice were observed daily for side effects and mortality, with blood work and necropsy at 42 d of age to evaluate for pathologic changes. To determine the efficacy of topical LS, postweanling mice infested with M. musculinus were treated with LS once weekly for 2 wk and then housed with uninfested sentinel mice for 4 wk. Weekly tape tests and postmortem tape tests and skin scrapings were performed on all mice. Treated postweanling mice had significantly lower Hgb levels and higher BUN levels than did control animals. In mite-infested mice, the number of positive cages at euthanasia was the same between treated and control animals. Although topical LS did not cause gross or microscopic changes to organ systems, it may cause clinicopathologic changes, and topical LS is not effective as a sole treatment for M. musculinus infestation of postweanling mice.

Comparison of the effects of ketamine, ketamine-medetomidine, and ketamine-midazolam on physiologic parameters and anesthesia-induced stress in rhesus (Macaca mulatta) and cynomolgus (Macaca fascicularis) macaques.

This study compared the cardiovascular, respiratory, anesthetic, and glucocorticoid effects of ketamine alone with ketamine-medetomidine and ketamine-midazolam in rhesus and cynomolgus macaques. Macaques were given either intramuscular ketamine (10 mg/kg), intramuscular ketamine-medetomidine (3 mg/kg; 0.15 mg/kg), or oral midazolam (1 mg/kg) followed by intramuscular ketamine (8 mg/kg). The addition of medetomidine, but not midazolam, provided muscle relaxation and abolishment of reflexes that was superior to ketamine alone. In addition, medetomidine did not cause clinically relevant effects on cardiovascular and respiratory parameters when compared with ketamine. These 3 protocols did not have significantly different effects on fecal glucocorticoid metabolites. These results suggest that medetomidine can be a valuable addition to ketamine for healthy patients, whereas oral midazolam at the tested dose does not provide additional benefits.

Moxidectin toxicity in senescence-accelerated prone and resistant mice.

Moxidectin has been used safely as an antiparasitic in many animal species, including for the eradication of the mouse fur mite, Mycoptes musculinus. Although no side effects of moxidectin have previously been reported to occur in mice, 2 strains of the senescence-accelerated mouse (SAMP8 and SAMR1) sustained considerable mortality after routine prophylactic treatment. To investigate the mechanism underlying this effect, moxidectin toxicosis in these mice was evaluated in a controlled study. Moxidectin was applied topically (0.015 mg), and drug concentrations in both brain and serum were analyzed by using HPLC coupled with mass spectrometry. The moxidectin concentration in brain of SAMP8 mice was 18 times that in controls, and that in brain of SAMR1 mice was 14 times higher than in controls, whereas serum moxidectin concentrations did not differ significantly among the 3 strains. Because deficiency of the blood-brain barrier protein P-glycoprotein leads to sensitivity to this class of drugs in other SAM mice, Pgp immunohistochemistry of brain sections from a subset of mice was performed to determine whether this commercially available analysis could predict sensitivity to this class of drug. The staining analysis showed no difference among the strains of mice, indicating that this test does not correlate with sensitivity. In addition, no gross or histologic evidence of organ toxicity was found in brain, liver, lung, or kidney. This report shows that topically applied moxidectin at a standard dose accumulates in the CNS causing toxicosis in both SAMP8 and SAMR1 mice.