Resumo

Investigating the substrate of the interactions between neural, glial and vascular elements is essential for understanding activation dynamics in the healthy brain, as well as dysfunctions in neurodegenerative diseases, strokes and tumors. Available methods for vascular labeling, like alkaline phosphatase, FITC-dextran or corrosion casting, are limited by a number of critical factors, such as: maximum slice thickness, difficulty to distinguish between branches, need to perfuse the animal with markers, variable efficiency and high costs. Recently, we developed a new protocol that allows complete visualization of blood vessels in thick brain slices, and is further compatible with other markers. Morpho-functional modules can be readily identified in the rat primary somatosensory cortex, where a single barrel is activated by the mechanical stimulation of each whisker, providing an ideal model to investigate neurovascular coupling. Here, we apply our new protocol to characterize the geometrical structure of vascular networks in the normal barrel cortex. Our objectives were to capture, describe and quantify the morphology of the vascular tree, and then to compare the laminar and radial profiles of morphometric parameters across cortical layers and barrels. Male adult rat were perfused with phosphate buffer followed by 4% formaldehyde and their brain was frozen cut into serial, coronal 120mm sections. These were incubated in avidin-biotin-peroxidase, processed for nickel-intensified diaminobenzidine reaction, and wet-mounted on glass slides, before virtualization with the aid of the Neurolucida system (MBF Bioscience): Z-stacks of images were acquired throughout the extent of the barrel field in S1 area. Optical density was analyzed on ImageJ. After reconstruction, the three-dimensional structure of the vascular tree was quantified using NeuroExplorer. Barrels were characterized by a denser microvascular network (higher values for optical density, vascular length and tortuosity) when compared to neighboring laminar and columnar compartments. Besides its relevance for modeling the dynamics of sensory processing in the barrel cortex, this work provides bases for further studies aimed at understanding of how neurovascular coupling is affected in acute or chronic brain diseases, and how physical exercise and training reflect structurally on cerebral hemodynamics.