Measurement of cerebral capillary perfusion with a fluorescent label

The aim of this study was to evaluate methods of labeling the perfused cerebral capillary bed using fluorescein isothiocyanate (FITC)-dextran or FITC-globulin. An alkaline phosphatase stain was used to identify the total capillary bed. All experiments were conducted on anesthetized rats. FITC labels were injected and the rats were sacrificed either 20 sec or 6 min after injection. Heads were frozen in liquid N2 or dry ice-cooled methanol. An additional group was perfused with India ink. Comparisons were made between FITC labels and methods of slide analysis. No significant differences in cerebral capillary number per square millimeter or volume per cubic millimeter were found in animals in which the heads were frozen in liquid N2 or dry ice-cooled methanol. in FITC labels, or in light sources. When sections were air dried, 56 +/- 4% of the alkaline phosphatase stained vessels were marked with FITC label in brains frozen 20 sec after FITC injection. India ink labeled 89 +/- 7% of the alkaline phosphatase stained capillaries. When sections were air dried, 86 +/- 7% of the alkaline phosphatase stained vessels were labeled in brains frozen 6 min after injection of the FITC label. Using a technique of absolute alcohol impregnation of the sections, 130 +/- 25% and 144 +/- 28% of the alkaline phosphatase stained sections were FITC labeled. It can be concluded that the absolute alcohol impregnation technique significantly overestimates the number of perfused cerebral capillaries.