Identification of Rodent Husbandry Refinement Opportunities through Benchmarking and Collaboration.

Expanding the use of methods that refine, reduce, and replace (3Rs) the use of animals in research is fundamental for both ethical and scientific reasons. The mission of the 3Rs Translational and Predictive Sciences Leadership Group (3Rs TPS LG) of the International Consortium for Innovation and Quality in Pharmaceutical Development (IQ Consortium) is to promote sharing and integration of science and technology to advance the 3Rs in the discovery and development of new medicines, vaccines, medical devices, and health care products for humans and animals. The 3Rs TPS LG is dedicated to identifying opportunities for member companies to share practices, enhance learning, promote discussions, and advance the 3Rs across the industry. One such opportunity was a benchmarking survey, conducted by the Contract Research Organization (CRO) Outreach Working Group, designed to share practices in rodent husbandry for drug safety research and to identify potential opportunities for refinement. IQ member companies and CROs in Asia, North America, and Europe were surveyed. Areas identified for potential alignment included provision of corncob bedding and wire-grid flooring, management of the nest at cage change, approaches to social housing for male mice, evidence-based enrichment strategies, and evaluating the effects of the timing of studies in relation to the animals' circadian rhythm and light-cycle, with consideration for how such extrinsic factors influence animal welfare and scientific outcomes. This manuscript presents the results of the benchmarking survey, including general trends in mouse and rat husbandry practices in toxicology studies, considerations for social housing, enrichment selection, and potential effects of bedding substrate, emphasizing opportunities for collaboration that can help to identify refinements to rodent husbandry practices.

Selective Neuronal Uptake and Distribution of AAVrh8, AAV9, and AAVrh10 in Sheep After Intra-Striatal Administration.

BACKGROUND: Transgenic sheep are currently the only large animal model of Huntington's disease expressing full-length mutant human huntingtin. These transgenic sheep provide an opportunity to test adeno associated virus (AAV) therapies directly targeting the huntingtin gene. A recent study demonstrated that self-complementary (sc) AAV with artificial miRNA against human huntingtin reduced mutant human huntingtin in caudate and putamen after a single injection near the internal capsule. OBJECTIVE: To identify an AAV serotype among AAVrh8, AAV9 and AAVrh10 with the highest neuronal uptake and distribution, with no obvious cell loss in the neostriatum of the sheep. METHODS: We tested AAVrh8, AAV9 and AAVrh10 by stereotactic direct unilateral injection into the neostriatum of sheep, near the internal capsule. Four weeks after administration, we examined the viral spread and neuronal uptake of each serotype of AAV containing GFP. We compared single stranded (ss) and scAAVs. Further, we measured the distribution of AAVrh8 and AAV9 to a variety of tissues outside the brain. RESULTS: Sc AAV9 had the best combination of neuronal uptake and distribution throughout the neostriatum. scAAVrh10 demonstrated good spread, but was not taken up by neurons. scAAVrh8 demonstrated good spread, but had less neuronal uptake than AAV9. Six hours after convection-enhanced administration to the neostriatum, both AAVrh8 and AAV9 viral genomes were detected in blood, saliva, urine, feces and wool. By four weeks, viral genomes were detected in wool only. Administration of AAVrh8, AAV9 and AAVrh10 was not associated with loss of neostriatal, medium spiny neuron number as measured by DARPP32 immunohistochemistry. CONCLUSIONS: Altogether, we found scAAV9 had the best neuronal uptake and spread, showed no loss of neurons at one-month post-injection, and was not measurable in body fluids one month after injection. This information will guide future clinical experiments requiring brain injection of AAV for therapeutics for gene or miRNA deliveries in sheep transgenic for the human huntingtin gene.

Xenopus laevis oocytes infected with multi-drug-resistant bacteria: implications for electrical recordings.

The Xenopus laevis oocyte has been the workhorse for the investigation of ion transport proteins. These large cells have spawned a multitude of novel techniques that are unfathomable in mammalian cells, yet the fickleness of the oocyte has driven many researchers to use other membrane protein expression systems. Here, we show that some colonies of Xenopus laevis are infected with three multi-drug-resistant bacteria: Pseudomonas fluorescens, Pseudomonas putida, and Stenotrophomonas maltophilia. Oocytes extracted from infected frogs quickly (3-4 d) develop multiple black foci on the animal pole, similar to microinjection scars, which render the extracted eggs useless for electrical recordings. Although multi-drug resistant, the bacteria were susceptible to amikacin and ciprofloxacin in growth assays. Supplementing the oocyte storage media with these two antibiotics prevented the appearance of the black foci and afforded oocytes suitable for whole-cell recordings. Given that P. fluorescens associated with X. laevis has become rapidly drug resistant, it is imperative that researchers store the extracted oocytes in the antibiotic cocktail and not treat the animals harboring the multi-drug-resistant bacteria.