In vivo staining of astrocytic elements surrounding microvessels with flowing RBCS in mice

Background and aims: We have previously visualized the astrocytic-vascular interface with sulforhodamine 101 1) in rats (unpublished observation). This study aims to examine the structural relationship of astrocytic endfeet stained with sulforhodamine 101, and endothelial cells stained with FITC dextran in the microvasculature of mouse brain in vivo during continuous recording of FITC-labeled red blood cells (RBCs) flowing through the microvasculature, as observed with a confocal fluorescence microscope. Methods: The head of a C57BL/6J mouse (n=5) was fixed to a stereotaxic apparatus under isoflurane anesthesia, a skull window was opened over the left parietal cortex 2), and the dura mater was removed carefully. A tail vein was catheterized for injection of FITC-labeled RBCs and FITC-dextran. Images of the movements of individual FITC-labelled RBCs in intraparenchymal single capillaries at a depth of approximately 50μm in the cortical region of interest (ROI) were acquired employing either a conventional video camera (30 frames/s) or a high-speed camera (500 frames/s) through a laser scanning confocal fluorescence microscope. Single capillaries were stained with a small amount of FITC-dextran injected intravenously, and astrocytes were stained with direct application of sulforhodamine 101 on the brain surface in vivo 1). Astrocytic attachment to the intraparenchymal microvasculature and its morphological changes were examined in vivo. Results: We observed no toxic effects following sulforhodamine application, and the physiological parameters remained unchanged throughout the dye-staining experiments (ca. 3 h). Circulating FITC-labeled RBCs, which had been introduced from the tail vein, were observed like fireflies on the dark background of the cerebral cortex. The astrocytes and their endfeet were visualized in brown, in good contrast to FITC-dextran-stained endothelial cells and flowing, brightly FITC-labeled RBCs in the microvasculature. Arterioles were found to be enveloped densely by a thin endfeet sheath, like a sleeve. The endfeet locally saddled capillaries with occasional protrusions, which pushed the capillary wall towards the lumen and caused marked elongation of RBCs flowing through the narrowed gap. The endfeet tended to cluster at the branching site of capillaries, forming a structure like an ants' nest tunneled with capillaries for RBC traffic. This astrocytic structure changed its morphology slowly but dynamically. In contrast, the astrocytic endfeet had no contact with the venous system. These findings are similar to the results obtained previously in SD rats, except that the capillaries and astrocytic elements appeared to be more clearly visualized in mice. Conclusions: We successfully visualized astrocytic elements and capillaries of the mouse microvasculature in which FITC-labeled RBCs were actually flowing in vivo. This technique provides a promising tool to investigate neuro-astrocytic control of capillary blood flow.