Effect of Continuous Trio Breeding Compared with Continuous Pair Breeding in 'Shoebox' Caging on Measures of Reproductive Performance in Estrogen Receptor Knockout Mice.

Some performance standards for continuous trio breeding in 'shoebox' cages for inbred stocks and outbred strains of mice challenge the minimum floor space recommendations in the 8th edition of the Guide for the Care and Use of Laboratory Animals. In our study, we evaluated whether continuous trio breeding could be successfully applied to a breeding colony of genetically engineered mice housed in shoebox cages with a floor area of 67.6 in2. Mice heterozygous for genetically engineered mutations to estrogen receptors and their wildtype counterparts were continuously bred as trios or pairs. Confounding environmental factors were controlled through standardized husbandry practices and husbandry, and all mice were bred simultaneously to control for temporal factors. Several measures of reproductive performance-including number of litters per female, production index, interlitter interval, litter size at birth, litter size at weaning, weaning rate, and body weight of pups at weaning- were evaluated over approximately 6 mo. Regardless of genotype, interlitter interval, litter size at birth, and litter size at weaning were significantly lower for trio-bred mice than for pair-bred mice. In addition, significant interactions emerged between genotype and breeding strategy for these reproductive measures. Furthermore, significant differences between genotypes occurred for interlitter interval and weaning rate, regardless of breeding strategy. Underlying mechanisms to account for effects of genotype on interlitter interval and the interaction of genotype with breeding strategy were unclear but may reflect effects of overcrowding and reproductive suppression.

Partial MHC Constructs Treat Thromboembolic Ischemic Stroke Characterized by Early Immune Expansion.

Inflammation and thrombosis are tightly linked, with inflammation contributing to thromboembolism and to stroke outcome. Thromboembolism is a frequent cause of ischemic stroke; yet, the most used occlusion mouse models of experimental stroke do not effectively replicate thromboembolism. Our group recently described a novel thromboembolic mouse model of stroke that successfully occludes the middle cerebral artery with high reproducibility. In the current study, we characterize the peripheral and local immune outcomes as well as the ischemic response to immune therapy in a clinically relevant mouse model of thromboembolic stroke. Brain and spleen tissues were harvested 24 h after thromboembolic stroke and cells immunophenotyped by flow cytometry. We observed a significant increase in neutrophils and early activated T cells in the spleen and an increase in neutrophils and activated monocytes/microglia in the ischemic cortex after thromboembolic stroke. Moreover, as was shown previously for transient MCAO models, treatment of thromboembolic stroke with partial MHC constructs significantly reduced ischemic damage indicating an equivalent effect of this immune-based therapy in the thromboembolic model that better mimics the pathophysiology of human stroke.

A novel mouse model of thromboembolic stroke.

BACKGROUND: We previously demonstrated that tissue plasminogen activator (tPA) reduces infarct size after mechanical middle cerebral artery occlusion (MCAO) in wild-type (WT) mice and transgenic mice expressing human leukocyte antigen DR2 (DR2-Tg). Clinically, tPA limits ischemic damage by dissolving the clot blocking blood flow through a cerebral artery. To mimic the clinical situation, we developed a new mouse model of thromboembolic stroke, and tested the efficacy of tPA in WT and DR2-Tg mice. New Method Autologous blood is withdrawn into a PE-8 catheter filled with 2 IU α-thrombin. After exposing the catheter briefly to air, the catheter is reintroduced into the external (ECA) and advanced into the internal carotid artery (ICA) to allow for intravascular injection of thrombin at the MCA bifurcation. To validate the model, we tested the effect of tPA on laser-Doppler perfusion (LDP) over the MCA territory and infarct size in WT and DR2-Tg mice. RESULTS: The procedure results in a consistent drop in LDP, and leads to a highly reproducible ischemic lesion. When administered at 15min after thrombosis, tPA restored LDP and resulted in a significant reduction in infarct size at 24h after thrombosis in both WT and DR2-Tg. COMPARISON WITH EXISTING METHODS: Our model significantly reduces surgery time, requires a single anesthesia exposure, and produces a consistent and predictable infarction, with low variability and mortality. CONCLUSION: We validated the efficacy of tPA in restoring blood flow and reducing infarct in a new model of endovascular thromboembolic stroke in the mouse.

Preclinical evaluation of recombinant T cell receptor ligand RTL1000 as a therapeutic agent in ischemic stroke.

Recombinant T cell Receptor Ligand 1000 (RTL1000), a partial human major histocompatibility complex (MHC) molecule coupled to a human myelin peptide, reduces infarct size after experimental stroke in HLA-DRB1*1502 transgenic (DR2-Tg) mice. In this study, we characterized the therapeutic time window of opportunity for RTL1000; we explored the efficacy of a single dose of RTL1000 administration and determined if RTL1000 affords long-term neurobehavioral functional improvement after ischemic stroke. Male DR2-Tg mice underwent 60 min of intraluminal reversible middle cerebral artery occlusion (MCAO). RTL1000 or vehicle was injected 4, 6, or 8 h after MCAO, followed by three daily injections. In the single-dose study, one-time injection of RTL1000 was applied 4 h after MCAO. Cortical, striatal, and hemispheric infarct sizes were measured 24 or 96 h after stroke. Behavioral testing, including neuroscore evaluation, open field, paw preference, and novel object recognition, was performed up to 28 days after stroke. Our data showed that RTL1000 significantly reduced the infarct size 96 h after MCAO when the first injection was given at 4 and 6 h, but not 8 h, after the onset of stroke. A single dose of 400 or 100 µg RTL1000 also significantly reduced the infarct size 24 h after MCAO. Behavioral testing showed that RTL1000 treatment used 4 h after MCAO improved long-term cognitive outcome 28 days after stroke. Taken together, RTL1000 protects against acute injury if applied within a 6-h time window and improves long-term functional recovery after experimental stroke in DR2-Tg mice.

Different immunological mechanisms govern protection from experimental stroke in young and older mice with recombinant TCR ligand therapy.

Stroke is a leading cause of death and disability in the United States. The lack of clinical success in stroke therapies can be attributed, in part, to inadequate basic research on aging rodents. The current study demonstrates that recombinant TCR ligand therapy uses different immunological mechanisms to protect young and older mice from experimental stroke. In young mice, RTL1000 therapy inhibited splenocyte efflux while reducing frequency of T cells and macrophages in the spleen. Older mice treated with RTL1000 exhibited a significant reduction in inflammatory cells in the brain and inhibition of splenic atrophy. Our data suggest age specific differences in immune response to stroke that allow unique targeting of stroke immunotherapies.

Recombinant T cell receptor ligand treatment improves neurological outcome in the presence of tissue plasminogen activator in experimental ischemic stroke.

RTL1000 is a partial human MHC molecule coupled to a human myelin peptide. We previously demonstrated that RTL1000 was protective against experimental ischemic stroke in HLA-DR2 transgenic (DR2-Tg) mice. Since thrombolysis with recombinant tissue plasminogen activator (t-PA) is a standard therapy for stroke, we determined if RTL1000 efficacy is altered when combined with t-PA in experimental stroke. Male DR2-Tg mice underwent 60 min of intraluminal middle cerebral artery occlusion (MCAO). t-PA or vehicle was infused intravenously followed by either a single or four daily subcutaneous injections of RTL1000 or vehicle. Infarct size was measured by 2, 3, 5-triphenyltetrazolium chloride staining at 24 or 96 h of reperfusion. Our data showed that t-PA alone reduced infarct size when measured at 24 h but not at 96 h after MCAO. RTL1000 alone reduced infarct size both at 24 and 96 h after MCAO. Combining RTL1000 with t-PA did not alter its ability to reduce infarct size at either 24 or 96 h after MCAO and provides additional protection in t-PA treated mice at 24 h after ischemic stroke. Taken together, RTL1000 treatment alone improves outcome and provides additional protection in t-PA-treated mice in experimental ischemic stroke.

A novel HLA-DRα1-MOG-35-55 construct treats experimental stroke.

Chemoattraction of leukocytes into the brain after induction of middle cerebral artery occlusion (MCAO) increases the lesion size and worsens disease outcome. Our previous studies demonstrated that partial MHC class II constructs can reverse this process. However, the potential application of pMHC to human stroke is limited by the need to rapidly match recipient MHC class II with the ß1 domain of the pMHC construct. We designed a novel recombinant protein comprised of the HLA-DRα1 domain linked to MOG-35-55 peptide but lacking the ß1 domain found in pMHC and treated MCAO after 4 h reperfusion in humanized DR2 mice. Infarct volumes were quantified after 96 h reperfusion and immune cells from the periphery and CNS were evaluated for expression of CD74 and other cell surface, cytokine and pathway markers. This study demonstrates that four daily treatments with DRα1-MOG-35-55 reduced infarct size by 40 % in the cortex, striatum and hemisphere, inhibited the migration of activated CD11b+CD45high cells from the periphery to the brain and reversed splenic atrophy. Furthermore, DRα1-MOG-35-55 bound to CD74 on monocytes and blocked both binding and downstream signaling of macrophage migration inhibition factor (MIF) that may play a key role in infarct development. The novel DRα1-MOG-35-55 construct is highly therapeutic in experimental stroke and could be given to all patients at least 4 h after stroke onset without the need for tissue typing due to universal expression of DRα1 in humans.

Refining timed pregnancies in two strains of genetically engineered mice.

In order to efficiently generate genetically engineered mouse (GEM) fetuses or neonates of a specified age range, researchers must develop strain-specific strategies, including reliable early pregnancy detection. The authors evaluated pregnancy indices (pregnancy rate, plug rate, pregnant plugged rate, first litter size and body weight) in two GEM breeding colonies: homozygous soluble epoxide hydrolase knockout (sEHKO) mice (n=164 females) and L7-tau-green fluorescent protein (GFP) transgenic mice (n=61 females). The goals of the study were to determine the most accurate early pregnancy indicator and to reliably and cost-effectively produce timed pregnant females that were between gestation days 16 and 18. The authors set up each timed mating by placing two naturally synchronized females with a male for 48 h. When males were present, personnel checked each female daily for a vaginal plug. They then weighed the females immediately, 1 week and 2 weeks after removing the males. In both sEHKO and GFP colonies, increases in body weight at 1 and 2 weeks after timed male exposure more reliably and consistently indicated pregnancy than did plug detection. Further evaluations and protocol refinements are planned based on litter size and litter number in these colonies.