Congruence of total and perfused capillary network in rat brains

In awake normocapnic rats, the density of the total and of the perfused capillary network was determined in 10 brain areas. The density of perfused capillaries was measured by using fluorescein isothiocyanate (FITC) globulin or Evans blue as intravenous marker and by fluorescent microscopy. The density of morphologically existing capillaries was determined according to either the histochemical alkaline phosphatase method or a newly developed immunohistochemical fluorescent method that allows marking of the capillary wall constituent fibronectin with a primary antibody directed against fibronectin. This antibody is made visible by a second FITC-coupled antibody (indirect immunofluorescence). Comparison of perfused and existing capillary counts revealed high congruence when fluorescent results were compared. In contrast, the alkaline phosphatase technique yielded capillary counts that were consistently 30% lower than the fibronectin and the FITC globulin counts. The identity of the perfused and the morphologically existing capillary network could be confirmed by a newly developed double-staining technique. First, the perfused capillaries were quantified by intravascular Evans blue. Then, the existing capillaries were relocated in the same measuring field by the fibronectin technique. Such double staining resulted in identical capillary counts in 97% of all cases. The following conclusions have been reached: 1) Fluorescent methods show a perfusion of virtually all capillaries in the brain of the awake normocapnic rat. 2) The alkaline phosphatase technique appears to underestimate the capillary density in the rat brain.