Faculty Bibliography

Zebrafish are an important model in neuroscience and developmental biology and are also an emerging model in hematology and immunology. Little information is available for zebrafish regarding the physiologic impact of different euthanasia methods and whether a chosen method of euthanasia can impact serum yield. These parameters could impact the choice of euthanasia method for a study. To that end, the current study compared 3 methods of adult zebrafish euthanasia and their effects on 3 distinct criteria; time to loss of opercular movement, volume of serum obtained, and serum cortisol concentration. Blood was collected using a postmortem tail amputation and centrifugation blood collection technique. Time to loss ofopercular movement differed significantly among euthanasia methods, with animals undergoing rapid chilling displaying the shortest time (mean Rapid Chilling: 40 s; Benzocaine: 86 s; MS222: 96 s). All methods of euthanasia resulted in a comparable average serum yield (Rapid Chilling = 7.5 μL; Benzocaine = 8.5 μL; MS222 = 7.5 μL per fish). None of the euthanasia methods tested resulted in average cortisol concentrations above the reported physiologic range. Although no significant differences were observed in serum yield or serum cortisol concentration, rapid chilling remains the preferred method for painless, humane euthanasia.

There are a multitude of ways staff can incur injuries while working in a research vivarium. Many workplace injuries can be prevented by proactively identifying jobs or tasks with a higher injury risk. Workrelated musculoskeletal disorders (WMSDs) are reported by the Occupational Health and Safety Administration (OSHA) as the "most widespread occupational health hazard facing our nation today." WMSDs can occur due to lifting, standing in one position too long, repetitive movements, twisting, poor posture, and more. The creation of a work environment where WMSDs can be avoided is optimal as this category of injury often takes a long time to heal, and in severe cases, may require surgery resulting in lost workdays to turn into lost work months. In line with the aims of our institution, we set out to enhance our method of work injury reporting and tracking. Following modifications to our system of injury reporting we were able to identify the type of injury, the anatomic location of the injury, facility location where the injury occurred, the job task the employee was performing at the time of injury, and the type of equipment the employee was working with at the time of the injury. Based on injury reporting data, we determined 45.8% of the injuries reported from 2018 and 36.0% of injuries reported from the first half of 2019 could be categorized as WMSDs. Periodic workplace injury assessments using data from the modified injury reporting system helped to guide staff ergonomic and safety training, and helped to identify areas for targeted ergonomic improvement. Over a period of 2 y, we have seen a 38% reduction in the total number of injuries reported and a 34.8% reduction in the number of injuries categorized as WMSDs. We highlight changes made to our system of injury reporting while using case-based examples to illustrate how we obtained, analyzed, and used injury reporting data to help enhance workplace safety

The purpose of this study is to determine the effects of high cumulative doses of ultra-small paramagnetic iron oxide (USPIO) used in neuroimaging studies. We intravenously administered 8 mg/kg of 2 USPIO compounds daily for 4 wk to male Sprague-Dawley rats (Crl:SD). Multiecho gradient-echo MRI, serum iron levels, and histology were performed at the end of dosing and after a 7-d washout period. R2* maps and quantitative susceptibility maps (QSM) were generated from multiecho gradient-echo data. R2* maps and QSM showed iron accumulation in brain ventricles on MR images acquired at the 4- and 5-wk time points. Estimates from QSM data showed ventricular iron concentration was equal to or higher than serum iron concentration. Histologic analysis revealed choroid plexus hemosiderosis and midbrain vacuolation, without iron deposition in brain parenchyma. Serum iron levels increased with administration of both compounds, and a 7-d washout period effectively reduced serum iron levels of one but not both of the compounds. High cumulative doses from multiple, frequent administrations of USPIO can lead to iron deposition in brain ventricles, resulting in persistent signal loss on T2*-weighted images. Techniques such as QSM are helpful in quantifying iron biodistribution in this situation.

Although numerous studies have evaluated the effect of housing density on the wellbeing of laboratory mice, little is known about the effect of breeding configuration on mouse behavior. The 8th edition of the Guide for the Care and Use of Laboratory Animals lists the recommended minimal floor area per animal for a female mouse and her litter as 51 in.2 We sought to determine the effects of pair, trio, and harem breeding configurations on the maternal and weanling behavior of C57BL/6J (B6) and 129S6/SvEvTac (129) mice on the basis of nest scores and performance in pup retrieval tests, open-field test (OFT), elevated plus maze, and tail suspension test; we concurrently evaluated cage microenvironment, reproductive indices, and anatomic and clinical pathology. Harem breeding configurations enhanced B6 maternal behaviors as evidenced by significantly shorter pup retrieval times. Trio- and harem-raised B6 weanlings showed increased exploratory behaviors, as evidenced by greater time spent in the center of the OFT, when compared with pair-raised B6 mice. Conversely, breeding configuration did not alter pup retrieval times for 129 mice, and on the day of weaning trio- and harem-raised 129 mice demonstrated increased anxiety-like behavior, as evidenced by greater time spent in the periphery of the OFT, when compared with pair-raised counterparts. Behavioral differences were not noted on subsequent days for either strain. Trio- and harem-raised B6 and 129 weanling mice had significantly higher weaning weights than weanlings raised in a pair breeding configuration. Trio and harem breeding in a standard 67-in.2 shoebox cage did not detrimentally affect the evaluated welfare parameters in either C57BL/6J or 129S6/SvEvTac mice.

Environmental enrichment provides laboratory-housed species the opportunity to express natural behavior and exert control over their home environment, thereby minimizing stress. We sought to determine whether providing an artificial plant in the holding tank as enrichment influenced anxiety-like behaviors and place-preference choice in adult zebrafish. Fish were housed singly or in social groups of 5 for 3 wk in 1 of 4 experimental housing environments: single-housed enriched (n = 30), single-housed barren (n = 30), group-housed enriched (n = 30), and group-housed barren (n = 30). On week 4, individual fish were selected randomly from each of the experimental housing environments and tested by using novel-tank, light-dark, and place-preference tests. Housing fish singly in a barren environment increased anxiety-like behaviors in the novel-tank and light-dark behavioral tests. Single-housed zebrafish in barren tanks as well as zebrafish group-housed with conspecifics, both with and without plant enrichment, spent more time associating with conspecifics than with the artificial plant enrichment device during the place-preference test. Single-housed fish maintained in enriched tanks displayed no preference between a compartment with conspecifics or an artificial plant. Our results suggest the addition of an artificial plant as enrichment may benefit single-housed zebrafish when social housing is not possible.

Tolerance of adult zebrafish and efficacy of emamectin benzoate and ivermectin in eliminating Pseudocapillaria tomentosa infection were evaluated. In the tolerance study, behavioral changes, fecundity, histopathology, and mortality were evaluated for in-feed administration of emamectin (0.05, 0.10, and 0.25 mg/kg) and ivermectin (0.05 and 0.10 mg/kg). All doses of emamectin were well tolerated. Ivermectin 0.05 mg/kg administration resulted in mild behavioral changes and a transient decrease in fecundity. Ivermectin 0.10 mg/kg administration resulted in severe behavioral changes and some mortality. In the efficacy study, emamectin (0.05 and 0.25 mg/kg) and ivermectin (0.05 mg/kg) were evaluated for their efficacy in eliminating P. tomentosa infection. Emamectin reduced parasite burden in infected zebrafish, and ivermectin eliminated intestinal nematode infections. Despite a small margin of safety, ivermectin 0.05 mg/kg was effective at eliminating P. tomentosa infection in adult zebrafish. Higher doses or a longer course of treatment may be needed for complete elimination of P. tomentosa infection using emamectin. In this study, we propose two possible treatments for intestinal nematode infections in zebrafish.

Although the safety and efficacy of tricaine methanesulfonate (MS222) for anesthesia of fish are well established, other anesthetics used less commonly in fish have been less extensively evaluated. Therefore, we compared gradual cooling, lidocaine hydrochloride (300, 325, and 350 mg/L), metomidate hydrochloride (2, 4, 6, 8, and 10 mg/L), and isoflurane (0.5 mL/L) with MS222 (150 mg/L) for anesthesia of adult zebrafish. The efficacy and safety of each agent was evaluated by observing loss of equilibrium, slowing of opercular movement, response to tail-fin pinch, recovery time, and anesthesia-associated mortality rates. At 15 min after anesthetic recovery, we used a novel-tank test to evaluate whether anesthetic exposure influenced short-term anxiety-like behavior. Behavioral parameters measured included latency to enter and number of transitions to the upper half of the tank, number of erratic movements, and number of freezing bouts. Behavior after anesthesia was unaltered regardless of the anesthetic used. Efficacy and safety differed among the anesthetics evaluated. Gradual cooling was useful for short procedures requiring immobilization only, but all instrumentation and surfaces that come in contact with fish must be maintained at approximately 10 degrees C. MS222 and lidocaine hydrochloride at 325 mg/L were effective as anesthetic agents for surgical procedures in adult zebrafish, but isoflurane and high-dose lidocaine hydrochloride were unsuitable as sole anesthetic agents due to high (30%) mortality rates. Although MS222 remains the best choice for generating a surgical plane of anesthe- sia, metomidate hydrochloride and gradual cooling were useful for sedation and immobilization for nonpainful procedures.

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or agents in solutions for scientific research. Current methods for administering compounds orally to adult zebrafish are inaccurate due to variability in voluntary consumption by the fish. A gavage procedure was developed to deliver precise quantities of infectious agents to zebrafish for study in biomedical research. Adult zebrafish over 6 months of age were anesthetized with 150 mg/L of buffered MS-222 and gavaged with 5 mul of solution using flexible catheter implantation tubing attached to a cut 22-G needle tip. The flexible tubing was lowered into the oral cavity of the zebrafish until the tip of the tubing extended past the gills (approximately 1 cm). The solution was then injected slowly into the intestinal tract. This method was effective 88% of the time, with fish recovering uneventfully. This procedure is also efficient as one person can gavage 20-30 fish in one hour. This method can be used to precisely administer agents for infectious diseases studies, or studies of other compounds in adult zebrafish.

Metabolic syndrome is a condition that typically includes central obesity, insulin resistance, glucose intolerance, dyslipidemia, and hypertension. Disruption of the hypothalamic-pituitary-adrenal axis, a regulator of corticosterone secretion, occurs in some cases of metabolic syndrome and obesity, and Cushing hypercortisolemia is associated with obesity and metabolic disorders. We therefore assessed anatomic and clinical pathology in C57BL/6NCrl mice to evaluate the effects of chronic corticosterone in the drinking water at doses of 25, 50, and 100 mug/mL for 25 d. Treated mice developed obesity, glucose intolerance, electrolyte aberrations, and dyslipidemia that were dose-dependent and most severe in the 100-mu;g/mL treatment group. To evaluate return to normal function, additional C57BL/6NCrl mice received corticosterone-free water for 2 wk after the 25-d treatment period. According to results of gross examination, mice appeared to recover within days of exogenous corticosterone withdrawal; however, adrenal gland vacuolation and protein, lipid, and electrolyte abnormalities persisted. Together, these findings support chronic corticosterone exposure through the drinking water as a potentially useful, noninvasive method to induce some features of metabolic syndrome.