Capillary recruitment means opening and closing of capillaries in tissues depending on regional blood flow demand. In the lung for example it has been shown that only 39% of capillary segments are permanently perfused. In the brain the question of capillary recruitment has been discussed controversely, because so far it has not been possible to visualize perfused capillaries in the cerebral cortex in vivo. Until now capillary recruitment - an in vivo phenomenon - has been investigated in an ex vivo approach by killing the animals. We were now able to establish a relatively new microscopy system in vivo to investigate brain microcirculation: the technique of confocal laser scanning microscopy (CLSM) in the rat.
The skin, the skull and the dura mater of the cerebral cortex are an optically dense barrier. Therefore a so called closed cranial window is implanted over the parietal cerebral cortex in the anesthetized and physiological fully controlled rat. For this the skull and the underlying dura mater are removed (area 3 x 4 mm) and the window is closed by a cover slip with a continuous superfusion of the space between brain surface and cover slip with artificial cerebrospinal fluid. For visualization of the microcirculation the blood plasma is labelled by intravenous injection of the fluorescent dye sodium-fluorescein. Blood cells (erythrocytes and leukocytes) appear in negative contrast to the labelled plasma. Figure 1 shows the setup of the confocal laser scanning microscope with the positioning of the animal under the microscope.
In CLSM a single point in a defined optical section is illuminated by a laser light source and by using pin holes light reflected or emitted only from this point is detected. By scanning the whole area a two dimensional picture can be produced (768 x 512 pixels = 317 x 211µm, using a x 40 objective). To obtain a picture with high spatial resolution 1 second is needed. Improving time resolution (up to 1/16 seconds is possible) always means a loss in spatial resolution (48 x 32 pixels in the same size of picture). To determine the velocity of erythrocytes, which is far beyond time resolution of the two dimensional approach, a space - time picture is produced. This is achieved by scanning only one single line (2ms) (single line approach) and putting these single lines under one another. Blood cell velocity can be calculated by the distance (horizontal length) in time (vertical length). With CLSM it is possible to investigate the microcirculation of the rat brain to a depth of 300 mm under the brain surface.
A global increase of resting blood flow in the whole brain can be achieved by ventilating the animal with high CO2 tensions (10%) in the inspiratory air.
We found that even under resting conditions all capillaries in the investigated area of the cortex are perfused with plasma, and 91% of capillary segments show moving blood cells. In the unstimulated brain the fluxrate and the velocity of blood cells are mainly in the lower part of the distribution curve. During global stimulation this distribution curve is shifted to the right to higher values of velocity and fluxrates (fig. 2).
We are now interested in the question of capillary recruitment under physiological somatosensory stimulation. Deflection of the whisker hairs of the rat (2-3 x / sec) leads to an increase of the regional cerebral blood flow in the whisker-barrel-cortex. The characteristic blood flow response to 60 seconds of somatosensory stimulation, measured with a laser-Doppler probe, is shown in figure 3.
In summary, we have shown that under resting conditions as well as under global stimulation of cerebral blood flow there is no anatomical recruitment of capillaries in the microcirculation of the cerebral cortex. An increase in blood flow demand is followed by a change in the dynamic behavior with increased fluxrate and velocity of blood cells.