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Stroke is a leading cause of death and invalidism worldwide. This study investigated the effect of post-stroke, direct AT-2 receptor stimulation with the non-peptide AT2R-agonist compound 21 (C21) on infarct size, survival and neurological outcome after middle cerebral artery occlusion (MCAO) in mice and looked for potential underlying mechanisms . MCAO is
one of the most frequently used animal models for stroke and provides opportunities for implementing refinement and reduction. First step is the study design and statistical planning. C57/BL6J or AT2R-knockout mice (AT2-KO) underwent MCAO for 30 min followed by reperfusion . Starting 45 min after MCAO, mice were treated once daily for 4 days with either vehicle or C21 (0,03mg/kg i.p.) . Neurological deficits were scored daily in accordance to Garcia et al. . This provides the opportunity to define humane endpoints on behalf of refinement. Furthermore, infarct volumes were measured 96 h post-stroke by MRI. Calculation of infarct volume by MRI is mild and related to reduction, because it offers the possibility to measure, infarct volume at different time points without killing
animals and save the brain for further investigations. C21 significantly improved survival after MCAO when compared to vehicle treated mice . C21 treatment had no impact on infarct size, but significantly attenuated neurological deficits . Expression of brain-derived neurotrophic factor (BDNF), tyrosine kinase receptor B (TrkB) (receptor for BDNF) and growth–associated protein 43 (GAP-43) were significantly increased in the periinfarct cortex of C21-treated mice when compared to vehicle-treated mice . Furthermore, the number of apoptotic neurons was significantly decreased in the peri-infarct cortex in mice treated with C21 compared to controls . There were no effects of C21 on neurological outcome, infarct size and expression of BDNF or GAP-43 in AT2-KO mice . From these data, it can be concluded that AT2R stimulation attenuates early mortality and neurological deficits after experimental stroke through neuroprotective mechanisms in an AT2R-specific way . By using refinement and reduction strategies like good statistical planning and study design as well as using MRI and Garcia Score the number of animals would be reduced by half minimum and welfare of animals could be improved and led to valid scientific data.