The Vascular Biology Core will coordinate a standardized set of measurements across brains and cerebral arteries from all three projects. The main endpoints evaluated by will be smooth muscle phenotype in both large surface arteries and parenchymal vessels imaged in coronal brain slices. Phenotype will be assessed functionally in surface arteries through measurements of myogenic contractility, and structurally in all arteries through measurements of confocal colocalization of contracrile proteins. Each project will prepare their rats, manage the in vivo measurements of behavior and MRI, and then will sacrifice their animals. All brains from these sacrificed animals will be processed in the Vascular Biology Core. Two main categories of analysis will be employed: fresh tissue and fixed tissue. For fresh tissue, surface arteries will be harvested from the brain and used for myography measurements or frozen for immunoblotting measurements of contractile proteins, cell-type specific markers, and other project-specific proteins. For brains fixed in situ, the surface arteries will be removed after which the brains will be embedded, sectioned, and studied using fluorescent immunohistochemistry. The extent and character of changes in smooth muscle phenotype and myogenic function will be compared across arteries obtained from all three hemorrhage models to test our """"""""Gradient Hypothesis"""""""" that all types of cerebral hemorrhage induce cerebrovascular phenotypic transformation whose magnitude varies in relation to distance from the core of injury. In general, one fresh brain will be processed each day, but multiple fixed brains can be processed each day and then analyzed later. For immunohistochemistry and confocal microscopy studies, our current staff processes approximately 20 slides per day, which is more than sufficient to handle the anticipated workload of 6-8 fresh and 18-20 fixed brains each week. Schedules for animal harvesting will be carefully coordinated among the individual projects. This will standardize reagent and resource utilization across all projects, enable volume purchase savings, and eliminate redundancies. All methods to be used in the core have been in routine use for multiple years.
The Vascular Biology Core will employ a variety of immunoblotting, confocal microscopic, and in vitro tissue bath methods to both structurally and functionally assess changes in the expression of brain proteins and cerebrovascular smooth muscle phenotype in response to hemorrhagic injury and treatment in brain samples produced by the three main projects in this PPG.
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