Andre Rex

Assessing Post-Stroke Behavior in Mouse Models of Focal Ischemia:

Experimental treatment strategies and neuroprotective drugs that showed therapeutic promise in animal models of stroke have failed to produce beneficial effects in human stroke patients. The difficulty in translating preclinical findings to humans represents a major challenge in cerebrovascular research. The reasons behind this translational road block might be explained by a number of factors, including poor quality control in various stages of the research process, the validity of experimental stroke models, and differences in drug administration and pharmacokinetics. Another major difference between animal studies and clinical trials is the choice of end point or outcome measures. Here, we discuss the necessity of poststroke behavioral testing to bridge the gap between clinical and experimental end points. We review established sensory-motor tests for outcome determination after focal ischemia based on the published literature as well as our own personal experience. Selected tests are described in more detail and good laboratory practice standards for behavioral testing are discussed. This review is intended for stroke researchers planning to use behavioral testing in mice.

Catabolic Signaling and Muscle Wasting After Acute Ischemic Stroke in Mice Indication for a Stroke-Specific Sarcopenia

Background and Purpose— Muscle wasting is a common complication accompanying stroke. Although it is known to impair poststroke recovery, the mechanisms of subacute catabolism after stroke have not been investigated in detail. The aim of this study is to investigate mechanisms of local and systemic catabolism and muscle wasting (sarcopenia) in a model of ischemic stroke systematically. Methods— Changes in body composition and catabolic activation in muscle tissue were studied in a mouse model of acute cerebral ischemia (temporal occlusion of the middle cerebral artery). Tissue wasting (nuclear magnetic resonance spectroscopy), tissue catabolism (caspases-3 and -6, myostatin), and proteasome activity were assessed. Food intake, activity levels, and energy expenditure were assessed, and putative mechanisms of postischemic wasting were tested with appropriate interventions. Results— Severe weight loss in stroke animals (day 3: weight loss, –21.7%) encompassed wasting of muscle (–12%; skeletal and myocardium) and fat tissue (–27%). Catabolic signaling and proteasome activity were higher in stroke animals in the contralateral and in the ipsilateral leg. Cerebral infarct severity correlated with catabolic activity only in the contralateral leg but not in the ipsilateral leg. Lower energy expenditure in stroke animals together with normal food intake and activity levels suggests compensatory mechanisms to regain weight. Interventions (high caloric feeding, &bgr;-receptor blockade, and antibiotic treatment) failed to prevent proteolytic activation and muscle wasting. Conclusions— Catabolic pathways of muscle tissue are activated after stroke. Impaired feeding, sympathetic overactivation, or infection cannot fully explain this catabolic activation. Wasting of the target muscle of the disrupted innervation correlated to severity of brain injury. Our data indicate the presence of a stroke-specific sarcopenia.

5-HT1A-receptor over-expressing mice: Genotype and sex dependent responses to antidepressants in the forced swim-test

Deficiencies in serotonergic neurotransmission are involved in the pathophysiology of depression. Due to its modulatory effect on serotonin (5-HT) release, the 5-HT(1A)-receptor is thought to play a decisive role in the therapy of this mood disorder. However, it is not fully understood how antidepressant effects are mediated by pre- and postsynaptic receptor sites. In this study we examined the impact of postsynaptic 5-HT(1A)-receptor over-expression in corticolimbic areas of male and female mice on the performance in the forced swim-test (FST). Furthermore, we investigated their response to the serotonin selective reuptake inhibitor (SSRI) citalopram in comparison to the selective noradrenaline reuptake inhibitor reboxetine, as well as the partial 5-HT(1A)-receptor agonists, buspirone and S 15535. Additionally, these drugs were evaluated in the open field-test in order to observe effects on motor activity. The density of 5-HT(1A)-receptors in discrete corticolimbic regions was determined in detail by quantitative autoradiography with [(3)H]8-OH-DPAT to investigate genotype as well as sex dependent differences in the expression pattern. [(3)H]8-OH-DPAT binding differed depending on sex with female mice of both genotypes displaying higher receptor binding in distinct brain areas. In the FST untreated male but not female over-expressing (OE) mice showed an antidepressant-like behaviour compared to wild-type (WT) mice. Citalopram yielded an antidepressant effect without influencing locomotor activity in OE mice but not in WT mice. Reboxetine had no antidepressant-like effect in OE mice, but sex-dependently in WT mice. The two partial agonists, buspirone and S 15535 produced no antidepressant-like activity in both genotypes and sexes, but aberrant motor effects. The antidepressant-like phenotype of male transgenic mice accounts for an involvement of postsynaptic 5-HT(1A)-receptors in the FST behaviour. In addition, the selective over-expression of postsynaptic 5-HT(1A)-receptors in mice contributes to the antidepressant response to citalopram in the FST. Although further pharmacological analysis is required, the data provide novel support for a role of postsynaptic 5-HT(1A)-receptors in the effects of SSRIs.

Vascular Signal Transducer and Activator of Transcription-3 Promotes Angiogenesis and Neuroplasticity Long-Term After Stroke

Center for Stroke Research Berlin, Charite-Universitatsmedizin Berlin, Germany; Dept of Neurology, Charite-Universitatsmedizin Berlin, Germany; Max-Delbruck Center for Molecular Medicine, Berlin, Germany; Institute of Clinical Neurobiology, University Hospital, University of Wurzburg, Germany; Cluster of Excellence NeuroCure, Charite-Universitatsmedizin Berlin, Germany; German Center for Neurodegenerative Diseases (DZNE), Partner Site, Berlin, Germany; German Center for Cardiovascular Diseases (DZHK), Partner Site, Berlin, GermanyBackground— Poststroke angiogenesis contributes to long-term recovery after stroke. Signal transducer and activator of transcription-3 (Stat3) is a key regulator for various inflammatory signals and angiogenesis. It was the aim of this study to determine its function in poststroke outcome. Methods and Results— We generated a tamoxifen-inducible and endothelial-specific Stat3 knockout mouse model by crossbreeding Stat3floxed/KO and Tie2-CreERT2 mice. Cerebral ischemia was induced by 30 minutes of middle cerebral artery occlusion. We demonstrated that endothelial Stat3 ablation did not alter lesion size 2 days after ischemia but did worsen functional outcome at 14 days and increase lesion size at 28 days. At this late time point vascular Stat3 expression and phosphorylation were still increased in wild-type mice. Gene array analysis of a CD31-enriched cell population of the neurovascular niche showed that endothelial Stat3 ablation led to a shift toward an antiangiogenic and axon growth-inhibiting micromilieu after stroke, with an increased expression of Adamts9. Remodeling and glycosylation of the extracellular matrix and microglia proliferation were increased, whereas angiogenesis was reduced. Conclusions— Endothelial Stat3 regulates angiogenesis, axon growth, and extracellular matrix remodeling and is essential for long-term recovery after stroke. It might serve as a potent target for stroke treatment after the acute phase by fostering angiogenesis and neuroregeneration.

Vascular Stat3 Promotes Angiogenesis and Neuroplasticity Long-Term After Stroke

Center for Stroke Research Berlin, Charite-Universitatsmedizin Berlin, Germany; Dept of Neurology, Charite-Universitatsmedizin Berlin, Germany; Max-Delbruck Center for Molecular Medicine, Berlin, Germany; Institute of Clinical Neurobiology, University Hospital, University of Wurzburg, Germany; Cluster of Excellence NeuroCure, Charite-Universitatsmedizin Berlin, Germany; German Center for Neurodegenerative Diseases (DZNE), Partner Site, Berlin, Germany; German Center for Cardiovascular Diseases (DZHK), Partner Site, Berlin, GermanyBackground— Poststroke angiogenesis contributes to long-term recovery after stroke. Signal transducer and activator of transcription-3 (Stat3) is a key regulator for various inflammatory signals and angiogenesis. It was the aim of this study to determine its function in poststroke outcome. Methods and Results— We generated a tamoxifen-inducible and endothelial-specific Stat3 knockout mouse model by crossbreeding Stat3floxed/KO and Tie2-CreERT2 mice. Cerebral ischemia was induced by 30 minutes of middle cerebral artery occlusion. We demonstrated that endothelial Stat3 ablation did not alter lesion size 2 days after ischemia but did worsen functional outcome at 14 days and increase lesion size at 28 days. At this late time point vascular Stat3 expression and phosphorylation were still increased in wild-type mice. Gene array analysis of a CD31-enriched cell population of the neurovascular niche showed that endothelial Stat3 ablation led to a shift toward an antiangiogenic and axon growth-inhibiting micromilieu after stroke, with an increased expression of Adamts9. Remodeling and glycosylation of the extracellular matrix and microglia proliferation were increased, whereas angiogenesis was reduced. Conclusions— Endothelial Stat3 regulates angiogenesis, axon growth, and extracellular matrix remodeling and is essential for long-term recovery after stroke. It might serve as a potent target for stroke treatment after the acute phase by fostering angiogenesis and neuroregeneration.

Increasing efficiency of preclinical research by group sequential designs

Despite the potential benefits of sequential designs, studies evaluating treatments or experimental manipulations in preclinical experimental biomedicine almost exclusively use classical block designs. Our aim with this article is to bring the existing methodology of group sequential designs to the attention of researchers in the preclinical field and to clearly illustrate its potential utility. Group sequential designs can offer higher efficiency than traditional methods and are increasingly used in clinical trials. Using simulation of data, we demonstrate that group sequential designs have the potential to improve the efficiency of experimental studies, even when sample sizes are very small, as is currently prevalent in preclinical experimental biomedicine. When simulating data with a large effect size of d = 1 and a sample size of n = 18 per group, sequential frequentist analysis consumes in the long run only around 80% of the planned number of experimental units. In larger trials (n = 36 per group), additional stopping rules for futility lead to the saving of resources of up to 30% compared to block designs. We argue that these savings should be invested to increase sample sizes and hence power, since the currently underpowered experiments in preclinical biomedicine are a major threat to the value and predictiveness in this research domain.

Evaluation of cage leaving behaviour in rats as a free choice paradigm.

INTRODUCTIONnThe free exploratory paradigm is regarded as a reliable test for trait anxiety in mice but it may also be useful in rats. Previously, we showed that rat strains differ in their free exploration of novel areas, i.e. the surroundings of their familiar home cage when the lid was removed.nnnAIMnTherefore, the purpose of the present study was to further examine strain, sex, and age differences in animals from different breeders in combination with pharmacological treatment designed to modify anxiety.nnnMETHODSnIn the present study free exploratory behaviour test was evaluated in Sprague Dawley and Wistar rats from different breeders. We assessed seasonal variation, habituation to the test, and the impact of gender and age on exploration. Furthermore, we monitored exploration following intraperitoneal diazepam, 8-OH-DPAT and caffeine administration. Parameters measured were latency to start exploring the outside of the cage, the percentage of rats that explored the outside, as well as the number of visits.nnnRESULTSnThere was no seasonal variability in free exploratory behaviour. However, strains and sexes differed in the test results, though age-related differences had less impact. Diazepam (2mg/kg) and 8-OH-DPAT (30, 100 and 300μg/kg) decreased neophobia while caffeine (50mg/kg) increased the latency to explore the outside the next day.nnnDISCUSSIONnThe free exploratory behaviour test can be used as a simple and complementary test to study trait anxiety-related behaviour in rats.

Anxiety-related behaviour of low brain angiotensinogen transgenic rats in the canopy test

This study investigated risk assessment and anxiolytic/anxiogenic drug effects in low brain angiotensinogen transgenic rats (TGR) in comparison to wild-type Sprague-Dawley rats (SD) in the canopy test of anxiety-related behaviour. TGR showed a higher frequency of the risk assessment behaviour as indicated by performance of stretched attend posture (SAP) compared to SD. Diazepam (0.25mg/kg) reduced SAP in both strains, whereas FG-7142 had no significant effect. The 5-HT(1B/2C) agonist mCPP (0.5-2mg/kg) reduced SAP in both strains. Diazepam (0.25-1mg/kg) increased head dips and decreased the time spent under the canopy in SD rats. There were significant anxiogenic effects of both FG-7142 (3-6 mg/kg) and mCPP (0.5-2mg/kg) on these parameters for SD but not TGR. Diazepam (1mg/kg) increased the number of entries into the open zone in both strains. mCPP reduced this parameter in SD (2mg/kg) and TGR (0.5-2mg/kg). FG-7142 had a similar effect in SD (3-6 mg/kg) and TGR (6 mg/kg). This study showed a significant transgenic effect on SAP. The increased number of SAP seen in TGR could be reduced with diazepam. Although both FG-7142 and mCPP are generally anxiogenic, no significant effects of FG-7142 on SAP were observed and mCPP even reduced SAP.

Atlas registration for edema-corrected MRI lesion volume in mouse stroke models:

Lesion volume measurements with magnetic resonance imaging are widely used to assess outcome in rodent models of stroke. In this study, we improved a mathematical framework to correct lesion size for edema which is based on manual delineation of the lesion and hemispheres. Furthermore, a novel MATLAB toolbox to register mouse brain MR images to the Allen brain atlas is presented. Its capability to calculate edema-corrected lesion size was compared to the manual approach. Automated image registration performed equally well in in a mouse middle cerebral artery occlusion model (Pearson ru2009=u20090.976, pu2009=u20092.265e-11). Information encapsulated in the registration was used to generate maps of edema induced tissue volume changes. These showed discrepancies to simplified tissue models underlying the manual approach. The presented techniques provide biologically more meaningful, voxel-wise biomarkers of vasogenic edema after stroke.