Scanning electron microscopy has proven to be a useful tool for the quantitation of three- dimensional characteristics of vascular smooth muscle. A major impediment to obtaining useful preparations of cerebral blood vessels for scanning electron microscopy is that smooth muscle in the tunica media is covered by a thin layer of basement membrane and by arachnoid tissue which contains collagen. The goal of this study was to develop a reliable, reproducible method for removing basement membrane and arachnoid tissue from cerebral arterioles, while at the same time leaving the smooth muscle intact.We compared several methods of tissue digestion in pial arterioles in adult Sprague-Dawley rats. The cerebral circulation was perfused with glutaraldehyde (2.5%) in cacodylate buffer (0.1 M) via the ascending aorta and then reinfused with packed red blood cells. In one group of rats, whole brains were immersed in osmium tetroxide (2%) for 2 hours (1 hr at 5°C and 1 hr at 40°C), followed by immersion in HC1 (8 N at 60°C) for 20-25 minutes. In another group, whole brains were dipped briefly in HC1 (8 N at 60°C) and then immersed in collagenase type II (2 mg/ml at 37° C; Sigma) for 12 hours. In a third group of rats, individual arterioles were dissected from the brain with a microsurgical knife, dipped briefly in HC1 (5-10 seconds) or KOH (5M at 60°C for 2-3minutes), and immersed in collagenase type II alone (12 hours), or in combination with collagenase type IV (2 mg/ml; 6-12 hours) and/or pepsin (2 mg/ml; 6-12 hours).
Morphological diagnosis of Alport syndrome and thin basement membrane nephropathy by low vacuum scanning electron microscopy
