Intramicrovascular behavior of platelets in rat brain observed by high-speed laser confocal fluorescence microscopy

Abstract

Although abundant data are available on in vitro platelet behavior, in situ studies in the brain microvasculature, especially in the intraparenchymal vessels, have not previously been possible because of methodological difficulties. We used our newly developed high-speed (125-1000 frames per sec) confocal fluorescence microscopy and image analysis system employing Matlab and our own KEIO-IS1 and KEIO-IS2 software to visualize platelet flow, tracking individual platelets in the microvasculature in living rat cerebral cortex. The program exploits the light intensity difference between the dark background and the light platelets in the visual field of the microscope to detect the platelets automatically. A small cranial window was trephined in the parietal region of urethane-anesthetized Wistar rats (n=10). Fluorescein isothiocyanate (FITC)-dextran (70 kDa) was injected intravenously to stain the microvessels. Subsequently, carboxylfluorescein succinimidyl ester (CFSE) was also injected intravenously to label platelets. Platelets in circulating blood became visible as bright particles in the intraparenchymal microvessels. In the veins, platelets were seen flowing slowly along endothelial cell walls (rolling) with a standard video camera at 30 frames per sec for a couple of minutes after the CSFE injection. In the arteries/arterioles, flowing platelets were detected only at high speed settings (125-1000 frames per sec). Contrary to expectation, platelet flow was well mixed in the circulating blood, and did not occur preferentially in the peripheral layer. However, transient contact of platelets with endothelial cells and detachment ( gtouch and go h phenomenon) were occasionally observed in the pial arteries. Flowing platelets in the capillaries were by no means stationary. For documentation of platelet tracking, video clips taken at 250-500 frames per sec for a 10 sec interval were analyzed with KEIO-IS2 (see Schiszler et al. Brain'05). The software automatically assigned individual numbers to platelets, calculated their velocities, and presented the results on a spreadsheet (Excel). The velocities were 2.72 }0.52 mm/sec for arterioles, 0.75 }0.3 mm/sec for single capillaries and 0.21 }0.07 mm/sec for venules. In the later stage of experiments (5-10 min after CSFE injection), platelets began to adhere to the wall of venules, occasionally forming large platelet aggregates, probably owing to some toxic effect of CFSE itself on the endothelial cells. The high-speed laser confocal fluorescence microscope is a promising tool for investigating hemodynamic changes at the vessel wall, including formation of platelet clots and their resolution with therapeutic agents.