Physiologic and Behavioral Effects in Mice Anesthetized with Isoflurane in a Red-tinted or a Traditional Translucent Chamber.

Isoflurane has been characterized as a distressing agent for rodents, causing both physiologic and behavioral effects. Using a "darkened home cage" has been recommended during CO2 administration for rodent euthanasia; this is arguably a similar animal experience to anesthetic induction with isoflurane. Based on the premise that rodents perceive red light as darkness via the primary optic tract, we compared physiologic and behavioral markers of stress in 2 inbred strains of mice (C57BL/6J and BALB/cJ) anesthetized with isoflurane in either a red-tinted (dark) induction chamber or a traditional translucent induction chamber. Physiologic stress was assessed based on plasma levels of norepinephrine, epinephrine, and corticosterone. Stress-related behaviors (rearing, face wiping, and jumping) were recorded on video and scored from initiation of induction to loss of consciousness. No significant correlations were found between chamber type and physiologic stress hormones. As compared with the translucent chamber, stress-related behaviors were more frequent in the red-tinted chamber, including: 1) significantly higher rearing frequencies in BALB/cJ mice; 2) higher behavioral stress scores in BALB/cJ and male C57BL/6J mice; and 3) more face wiping behavior when considering all mice combined. These findings suggest that mice do not experience significant alleviation of physiologic indices of stress when anesthetized in a red-tinted induction chamber. Furthermore, isoflurane induction in the red-tinted chamber appeared to increase the expression of stress-related behaviors, particularly in BALB/cJ mice. Based on our findings and a growing body of literature on the unintended effects of red light, we do not recommend using red-tinted chambers for induction of anesthesia in mice.

The Effectiveness of Hot Bead Sterilization in Maintaining Sterile Surgical Instrument Tips across Sequential Mouse Surgeries.

One strategy commonly employed for rodent surgeries is a "tips-only" surgical technique, which restricts the surgeon to using only the sterile working ends of the surgical instruments to manipulate the surgical field and sterilizes instrument tips with a hot bead sterilizer between consecutive rodents. Despite the common use of the "tips-only" technique, research is lacking on the number of sequential surgeries for which the same set of hot bead-sterilized instruments can be used before introducing bacterial contamination. We performed serial mouse surgeries using the "tips-only" technique under 3 different conditions (aseptic, fur contamination, or cecal contamination) and assessed aerobic bacterial growth before and after each round of hot bead sterilization. Instrument tips showed an increasing probability of contamination of at least one instrument in a series of consecutive surgeries. The probability that all surgical instrument tips in the series were sterile after hot bead sterilization fell by 4% for each surgery involving inadvertent or fur contamination and by 11.5% for each surgery with contamination for all surgical types combined (including entering the gastrointestinal tract). Based on our results, hot bead sterilization is not adequate for surgeries associated with gross contamination. Under our experimental conditions and assuming independence of outcomes between consecutive surgeries, up to 5 surgeries associated with minor or inadvertent contamination could be performed in series with a probability higher than 80% that all instrument tips were sterile for all surgeries. A case-by-case risk assessment should be conducted to derive institutional guidelines for the maximal number of surgeries that can be performed in sequence using the "tips-only" technique with hot bead sterilization of the same set of surgical instruments between surgeries. Full sterilization of instruments after every surgery provides the greatest confidence in maintaining sterility.

Effects of Acrylic Tunnel Enrichment on Anxiety-Like Behavior, Neurogenesis, and Physiology of C57BL/6J Mice.

Environmental enrichment for mice lags behind the standard enrichment offered to other laboratory rodents due to concerns about environmental variability and, in specific contexts, aggression. Our objective in this study was to evaluate concerns that the introduction of structural enrichment in the form of a single red acrylic mouse tunnel into murine housing may confound study findings. We measured effects on anxiety-like behaviors (elevated zero maze and open field activity), hippocampal neurogenesis, body weight gain, and physiologic markers of stress (adrenal gland weight, plasma corticosterone concentration, and neutrophil:lymphocyte ratio). Male and female C57BL/6J mice were randomly assigned to one of 2 groups: a standard-housed control group with enrichment consisting of paper nesting material, or an enriched group that received a single acrylic tunnel in addition to nesting material. All results fell within biologically normal ranges regardless of treatment, and variability (standard deviation) was not significantly different between groups for any measure. Mice in the enriched group showed modest differences during open field testing suggestive of decreased anxiety, traveling farther and depositing fewer fecal boli than standard-housed mice. Male mice in the tunnel-enriched group gained more body weight than standard-housed male mice. No significant effects by treatment were found in neurogenic or physiologic parameters. These results indicate that provision of simple structural enrichment is unlikely to have confounding effects on murine anxiety-like behaviors, neurogenesis, body weight gain, or physiologic parameters. We therefore recommend the inclusion of simple structural enrichment, such as an acrylic tunnel, to the standard environmental enrichment of social housing and nesting material for mice.

A Comparison of the Efficacy and Cardiopulmonary Effects of 3 Different Sedation Protocols in Otolemur garnettii.

The Northern greater galago (Otolemur garnettii) is a prosimian primate most commonly used to study the evolutionary development of vision and somatosensation. This study aimed to investigate the efficacy and cardiopulmonary effects of 3 sedation protocols commonly used in other primate species: 1) alfaxalone (Alf; 8 mg/kg IM) 2) ketamine alone (Ket; 20 mg/kg IM) and 3) ketamine + dexmedetomidine (Ket+Dex; 4 mg/kg + 25 µg/kg IM) with reversal (atipamezole; 250 µg/kg IM). A total of 34 animals were evaluated, including 11 juveniles and 23 adults. Cardiopulmonary parameters such as indirect blood pressure, heart rate, respiratory rate, and SpO2 were measured, and blood was collected for blood gas analysis and a chemistry panel. To examine the efficacy of each sedation protocol, induction time, immobilization time, and recovery time were recorded. Subjective measures of quality and efficacy included quality of induction, pedal withdrawal reflex, palpebral reflex, muscle tension, rectal temperature, and quality of recovery. All 3 protocols successfully immobilized the animals and all animals recovered from sedation. Heart rates were highest among the Ket group and the lowest for the Ket+Dex group. On average, the Alf group was immobilized for twice as long as either the Ket or Ket+Dex groups. The Ket+Dex group had the fastest average recovery time and subjectively had the best quality of recovery. Based on these results, Ket+Dex is recommended over Alf or Ket alone for brief sedation of healthy galagos.

Anesthesia and Euthanasia of Brine Shrimp (Artemia franciscana).

Invertebrates are often overlooked as laboratory animals, yet they are commonly used in toxicology, developmental, cellular and molecular biology, and radiation studies with euthanasia as an endpoint. Little is known regarding appropriate euthanasia methods for invertebrate species, particularly for Artemia. Here, we evaluated the AVMA-recommended 2-step method of euthanasia in brine shrimp (Artemia franciscana). Artemia were exposed first to anesthetic solutions of 60% alcohol, 2.5 mg/L eugenol, or 4 g/L tricaine methanesulfonate (TMS) and then were transferred to euthanasia solutions of 70% alcohol, 95% alcohol, or 10% neutral buffered formalin. We examined time to anesthesia, behavioral response to anesthesia, anesthesia recovery, and time to euthanasia. Our results show that 2.5 mg/L eugenol and 4 g/L TMS inconsistently achieved anesthesia. Although 60% alcohol produced anesthesia, the time to anesthesia varied among replicate groups, and exposure resulted in an increase in abnormal behavior. We therefore do not recommend any of the tested anesthetic solutions for use in Artemia. Although all 3 euthanasia solutions were effective, more research is needed to provide recommendations regarding euthanasia for this species.

Staphylococcus xylosus Cystitis and Struvite Urolithiasis in Nude Mice Implanted with Sustained-release Estrogen Pellets.

Female nude mice (J:NU-Foxn1nu; age, 6 wk) were injected with 1 million MCF7 human breast cancer cells in the fourth mammary fat pads and received a 21-d sustained-release estrogen pellet (0.25 mg) subcutaneously in the dorsum of the neck. All mice were maintained in sterile housing and provided sterile water and irradiated rodent chow. Approximately 6 wk after implantation, 4 of the 30 mice showed clinical signs of depression and dehydration. The 2 animals most severely affected were euthanized and presented for necropsy. The urinary bladders of these animals were distended with variable sized white, opaque uroliths. Urinalysis revealed coccal bacteria, erythrocytes, neutrophils and struvite crystals. Urine cultures from both necropsied animals grew heavy, pure growths of Staphylococcus xylosus. The organism was sensitive to all antibiotics tested except erythromycin (intermediate). Analysis of the uroliths revealed 100% struvite composition. Remaining mice in the study were evaluated clinically for hydration status, the ability to urinate, and the presence of palpable stones in the urinary bladder; one additional mouse had a firm, nonpainful bladder (urolithiasis suspected). Given the sensitivity of the organisms cultured from urine samples, the remaining mice were placed on enrofloxacin in the drinking water (0.5 mg/mL). All remaining mice completed the study without further morbidity or mortality. Previous studies have reported the association of estrogen supplementation with urinary bladder pathology, including infection and urolithiasis. Here we present a case of urolithiasis and cystitis in nude mice receiving estrogen supplementation that was associated with Staphylococcus xylosus, which previously was unreported in this context. When assessing these nude mice for urolithiasis, we found that visualizing the stones through the body wall, bladder palpation, and bladder expression were helpful in identifying affected mice.

Evaluation of Mice Undergoing Serial Oral Gavage While Awake or Anesthetized.

Although oral gavage is the most straightforward approach to achieve precise enteric administration in rodents, it is associated with potential adverse consequences. Here we compare the effects of serial oral gavage in awake compared with anesthetized mice. Female C57BL/6J mice (n = 20 per group) were assigned to 1 of 3 treatment groups (control, awake gavage, or anesthetized gavage) and gavaged daily with 0.2 mL of saline (with no manipulation on weekends) for a total of 18 treatment days. Body weight and clinical appearance were monitored throughout the treatment period, after which mice were euthanized and necropsied. Endpoints evaluated included adrenal gland weight, plasma corticosterone, lymphocyte:neutrophil ratio, and esophageal histopathology. Mean body weight did not differ between groups. Compared with other groups, the awake gavage group had more mice removed (3 of 20) prior to study completion due to body weight loss greater than 10%, with corresponding gross and histopathologic lesions attributed to the gavage procedure. Mice gavaged when awake had an over 20-fold higher incidence of incomplete retention of the administered saline than did anesthetized mice. Of the mice that completed the study, esophageal inflammation was not apparent at necropsy regardless of treatment, with the exception of a single mouse in the awake gavage group. Although WBC and lymphocyte counts were lower in mice in the anesthetized gavage group compared with other groups, none of the endpoints measured to evaluate stress (adrenal gland weight, neutrophil:lymphocyte ratio, plasma corticosterone) differed. These findings support the use of brief isoflurane anesthesia when performing serial oral gavage in mice.

A Phyllodes-like Mammary Tumor in a Breeding Galago (Otolemur garnettii).

In humans, phyllodes tumors of the breast are rare fibroepithelial tumors that are further characterized as benign, borderline, or malignant according to their histomorphologic features. Phyllodes tumors are poorly responsive to treatment other than excision. NHP have a much lower frequency of mammary neoplasia than do humans, and none of the lesions reported previously in NHP are consistent with phyllodes tumors. Here we present the case of a mammary tumor in a northern greater galago (Otolemur garnettii) that was histologically characteristic of a malignant phyllodes tumor. An 11-y-old, multiparous, pregnant galago presented with a mass in the right middle mammary gland. A fine-needle aspirate yielded neoplastic epithelial cells. Because the animal was pregnant and showed no signs of skin ulceration, pain, or distress, she was allowed to deliver and nurse the infant. At 20 wk after initial presentation, the infant was weaned and the mother was euthanized. At necropsy, the mammary mass measured 3.5 × 2.5 × 1.5 cm, a 13-fold increase in volume since initial presentation. There was no evidence of metastasis in draining lymph nodes, lungs, or any other tissue examined. The tumor was composed of neoplastic stromal, glandular, and adipose tissues and was diagnosed as a malignant phyllodes tumor in light of its high stromal cellularity, high mitotic rate, and marked atypia. This tumor also exhibited liposarcomatous differentiation, which occurs frequently in malignant phyllodes tumors. To our knowledge, this report represents the first described case involving an NHP of a mammary tumor with characteristics consistent with human phyllodes tumors.

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.

Phylogeography of the livebearer Xenophallus umbratilis (Teleostei: Poeciliidae): glacial cycles and sea level change predict diversification of a freshwater tropical fish.

The biogeography of Central America is viewed as a classic case study in understanding the impact of vicariant events on patterns of biotic dispersal. While many biogeographers have focused on community composition and geographical limits of species at broad scales across Central America, much less work has focused on post-colonization diversification patterns at finer scales. The livebearing freshwater fish Xenophallus umbratilis presents an ideal system for determining the impact of recent Earth history events on biodiversity in northern Costa Rica. Here, we test the hypotheses that marine inundation of the San Carlos and northern Limón basins during the Pliocene and Pleistocene has caused genetic fragmentation among X. umbratilis populations, despite contemporary freshwater connections. To test this idea, we collected mitochondrial (cytochrome b) sequence data in 162 individuals taken from 27 localities across northern Costa Rica. We employed a variety of analytical approaches, including: maximum parsimony and maximum likelihood, analysis of molecular variance, and demographic analysis of population size through time. We found four major clades within X. umbratilis, each geographically isolated with no shared haplotypes across drainages. Oddly, clades that occupy adjacent drainages are not always sister taxa in the phylogeny, suggesting that colonization in this species is more complex than a simple model of isolation by distance. All our results are consistent with the hypothesis that changes in sea level associated with glacial eustatic cycles have had an important effect in shaping diversification patterns in this species.