Cytochemical study of various phosphatase--activities on the capillary walls of rat cerebral cortex.

Abstract

Normal adult Sprague-Dawley rats were used to demonstrate enzymatic activity on capillaries of the cortex. Fixation was performed by intracardiac perfusion of 2% glutaraldehyde in a 0.1 M cacodylate buffer of pH 7.2 with 8% sucrose. After perfusion for 2 min from 1.5 m in height, the brain was removed. Coronal slices (1.0 mm thickness) of the frontal cerebral cortex were fixed for an additional period of 10 to 30 min by immersion in the same fixative and then washed overnight in the same buffer solution. Frozen sections of 40 μm in thickness were obtained from the washed tissues. The sections were immersed at 37°C for 30 min in the incubation media for acid phosphatase, alkaline phosphatase (ALPase), glucose-6-phosphatase, thiamine pyrophosphatase (TPPase), Mg++-ATPase, Na+, K+-ATPase substituted for measuring K+-dependent p-nitrophenylphosphatase, 5'-nucleotidase (5'-Nase), and nucleoside diphosphatase (NDPase). Among them, ALPase, Mg++-ATPase, TPPase, and NDPase demonstrated positive staining on the capillary walls when viewed with light microscope. The ultrastructural localization of these four enzymes on the capillary walls was investigated. The cytochemical findings gave the following results. All four enzymatic activities were found on the abluminal surfaces of the endothelial cells and on the surfaces of the pericytes. Electron dense precipitations on the luminal surfaces of endothelial cells were infrequently seen and were considered an artefact. Intense reaction products of ALPase and Mg++-ATPase were evenly distributed on the surfaces of the astrocytic processes in contact with the thick basal lamina. Heavy, dotted reaction products of TPPase and NDPase were found in the basal lamina. The ultrastructural localization of ALPase, TPPase, Mg++-ATPase, and NDPase were also briefly discussed in connection with cerebral microcirculation and the blood-brain barrier.