Patterns of Capillary Plasma Perfusion in Brains of Conscious Rats During Normocapnia and Hypercapnia
Abstract The present study aimed to investigate the distribution pattern of plasma flow velocities in brain capillaries. We tested the hypothesis that plasma flow velocities are heterogeneous in the brain capillaries of normocapnic conscious rats and become more homogeneous during increased cerebral blood flow induced by hypercapnia. We developed a method that makes it possible to detect the distribution pattern of plasma flow velocities from the intravascular dye concentrations measured in different capillaries. Evans blue was injected intravenously as a bolus, and 3 to 4 seconds later the rats were decapitated. During this period, a steep increase in arterial dye concentration was verified by frequent arterial blood sampling. Under such conditions, divergent plasma flow velocities in different capillaries yield unequal intravascular dye concentrations. Dye concentrations were measured in several hundred capillaries of brain cryosections using quantitative fluorescence microscopy based on calibration curves obtained from anesthetized rats. The results show a high degree of variation in the intravascular dye concentration during normocapnia. During increasing stages of hypercapnia, the variation was gradually reduced. The coefficient of variation (SD/mean · 100) of intracapillary dye concentration decreased from 76% at normocapnia to 22% at extreme hypercapnia (Pco2 of 87 mm Hg), thus showing an inverse correlation with arterial Pco2 (r=.97). The heterogeneity of intravascular dye concentrations observed in the present experiments indicates heterogeneous velocities of plasma perfusion in different brain capillaries during normocapnia and a more homogeneous distribution pattern during hypercapnic hyperemia.
The dynamics of capillary perfusion in the brain are largely unknown. Recently, attention has focused on the question of whether the brain capillaries are perfused completely or only partially. Our group has measured perfusion of all brain capillaries with plasma at any time point,1 2 since an intravenous bolus injection of a fluorescent dye results in all brain capillaries filling with dye within 3 to 4 seconds.3 In contrast, other authors4 have postulated filling of only a fraction of all brain capillaries lasting for several minutes after intravenous injection of fluorescein isothiocyanate (FITC)–dextran. Our group5 has ascribed these findings to a low sensitivity of detection of perfused capillaries in their experiments. These investigations have been performed after in vivo injection of a fluorescent dye and the detection of capillary filling from cryosections of brain tissue. In other studies,6 7 8 9 the capillary perfusion of the brain surface has been studied more directly with confocal laser microscopy in anesthetized animals. Concerning blood cell flow, discontinuities as well as a lack of flow could be observed, whereas plasma flow was continuous8 9 in all capillaries.
The microcirculation of the brain surface has also been studied using intravital microscopy.10 11 12 These studies have also supported the existence of different red blood cell velocities in single capillaries10 and of variations in plasma and red blood cell flows.11 12 More indirect statements about the capillary perfusion have been based on autoradiographic measurements of local cerebral blood flow combined with measurements of the distribution volume of red blood cells and plasma. These measurements have been performed during variations of arterial Po2 or Pco2,13 14 high-dose pentobarbital anesthesia,15 and subcutaneous injection of nicotine.16 From calculations of mean transit times, it was concluded that changes in flow velocity are the main mechanism by which cerebral blood flow is changed. Capillary recruitment was regarded to be either absent or of minor importance. These findings have invalidated the previous studies of Shockley and LaManna,17 who estimated a moderate increase in microvessel blood volume of the cortex during hypoxic hypercapnia and concluded that the number of perfused capillaries was raised. Meanwhile, Jones et al18 have revised their primary concept of capillary recruitment, concluding that vascular recruitment is not important for the regulation of cerebral blood flow.19
Previous studies investigating the dynamics of the brain microcirculation have been limited by methodological constraints. Autoradiographic studies do not allow direct statements on the capillary perfusion because of the inherent low resolution of this technique with respect to single capillaries. On the other hand, direct recordings of capillary flow in intravital studies have yielded information only on rather few capillaries in each animal. If heterogeneities in the perfusion of single capillaries exist, it is desirable to analyze a large number of capillaries simultaneously. It was our aim in the present study to develop a method that allows the simultaneous analysis of capillary plasma flow distribution in numerous brain vessels. The distribution of capillary flow can be verified from the amount of intravascular marker contained in the different capillaries 3 to 4 seconds after an intravenous bolus injection of the intravascular marker Evans blue. Since the arterial concentration of Evans blue increases continuously during this short period, the amount of Evans blue contained in each capillary at the end of the experiment (3 to 4 seconds after injection) depends on the plasma flow velocity within this capillary. Capillaries perfused by plasma at higher velocities should contain higher concentrations of Evans blue than slowly perfused capillaries. The intravascular Evans blue concentrations measured in numerous adjacent capillary profiles of brain sections should therefore reflect the various plasma flow velocities that exist in the capillaries of the brain structure investigated. Besides the pattern of flow distribution at normal cerebral blood flow, it was of interest to investigate the velocity pattern during an increase in cerebral blood flow. To this end, we induced various degrees of hypercapnia and determined velocity patterns under these conditions.
Materials and Methods
Experiments were performed on 11 conscious and 8 anesthetized (1% to 1.5% halothane, 70% N2O, remainder O2) male Sprague-Dawley rats weighing 280 to 390 g. Analysis of flow velocities was performed in the conscious rats, and the anesthetized rats were used for the measurement of the calibration curve of Evans blue and for the examination of the fading characteristics of Evans blue. The experiments were conducted in accordance with institutional guidelines and the Guide for the Care and Use of Laboratory Animals (National Institutes of Health Publication No. 86-23). Before surgery all animals were anesthetized by the above-mentioned gas mixture. Body temperature was maintained at 37° to 37.6°C using a temperature-controlled heating pad. The left femoral vein was cannulated for application of Evans blue and the left femoral artery for (1) monitoring of arterial blood pressure (Hewlett-Packard), (2) sampling of arterial blood to determine arterial blood gases (AVL 990, AVL GmbH), and (3) determination of the time course of the arterial Evans blue concentration after intravenous bolus injection. All rats used for the analysis of flow velocities were placed in a rat restrainer (Braintree Scientific) after surgery and allowed to recover from anesthesia for 2 hours. Then the rats were allowed to breathe a water vapor–saturated gas mixture containing 0% to 10% CO2, 22% O2, and 68% to 78% N2 for 15 minutes to obtain arterial Pco2 values of 42.9 to 87 mm Hg in different rats. When a constant Pco2 was reached for at least 10 to 15 minutes, 1 mL/kg 2% Evans blue dissolved in 0.9% saline was injected as a bolus (injection time, approximately 0.2 second) into the femoral vein. Timed arterial blood samples (four to seven droplets per second) were taken simultaneously from the free-flowing arterial catheter and dropped on a board covered with aluminum foil that was moved below the end of the catheter. This procedure resulted in separate placing of the droplets on the foil. The rats were decapitated during the phase of increasing arterial concentration of Evans blue, ie, 3 to 4 seconds after the bolus injection of the dye when the first clearly colored droplet became visible.
The brains were rapidly dissected out, frozen in 2-methylbutane chilled to −60°C, and embedded (M-1 embedding matrix, Lipshaw). The brains were cut immediately into 5-μm coronal sections in a cryomicrotome (2800 Frigocut E, Leica) at −20°C. Each section was transferred to a slide that was covered just before with a thin film of a saturated sucrose solution (2 g sucrose/mL H2O) and kept at room temperature. Immediately (less than half a second) after the frozen section was taken up, the slide was dipped into cold acetone (−20°C) for 20 seconds. The slides were then transferred into acetone at room temperature. After 20 seconds they were dried on a heated plate at 35° to 40°C. Then the fixed sections were observed with an incident light fluorescence microscope (Axioplan, Zeiss) equipped with a high-pressure mercury lamp (Zeiss HBO 100) and a microscope photometer (Zeiss MPM). The emitted fluorescence light passed through an aperture into the photomultiplier of the microscope photometer, and its signals were quantified by a microscope system processor (Zeiss MSP 21). Before a series of measurements was performed, the system was adjusted to a standardized sensitivity using a fluorescent standard (Zeiss FL-Standard 47 42 56). The measurements were performed on five brain structures (cerebral white, corpus callosum, inferior colliculus, caudate nucleus, and frontal cortex). In each structure, 40 neighboring capillaries were chosen.
The Evans blue concentration in each capillary was determined by measurement of the fluorescence per cross-sectional area of the dye. The capillaries containing Evans blue were observed and measured with a 546-nm primary filter, a 580-nm dichroic interference mirror, and a 590-nm secondary filter. For fluorescence quantification, a single capillary profile was positioned under 50% excitation light intensity into the middle of an aperture of the photometer with a depiction diameter of 10 μm. The capillaries were selected based on a nearly round shape and an area of less than 45 μm2, which corresponds to a maximal diameter of 7.6 μm. It took maximally 10 seconds to adjust the capillary profile into the right position. The fluorescence intensity was then measured for 0.2 second at 100% excitation light intensity. After the fluorescence intensity had been determined in a capillary profile, the fluorescent cross-sectional area of this profile was measured with a CCD camera (Kappa CF6, Aqua-TV) and an image analyzer (Signum). In such a setting, the cross-sectional area of a capillary profile with a high luminescence (definition: fluorescence intensity per cross-sectional area) of the fluorescent marker would appear larger than the area of the same profile when containing a lower amount of fluorescent marker. Therefore, gray filters were used to attenuate individually the luminescence of different capillary profiles to yield similar values of luminescence. Thirteen different filter combinations were used to attenuate the fluorescence intensities down to 6%, if necessary, in attenuation steps of 5% to 10%. For finding the correct filter combination, it was useful that most capillaries were not perfectly circular but rather angular at some part. When the capillary profiles were observed in the fluorescent microscope, their shape was compared with that displayed on the video screen of the image processor. Gray filters were then added until the two images were the same. Then the cross-sectional area of that capillary profile was measured.
To verify the time course of the arterial concentration of Evans blue, we cut the aluminum foil with the sampled blood droplets into strips with one droplet per strip. The first droplet was weighed on the foil. Then the blood droplet was washed away from the foil with distilled water and the foil was weighed again, the difference representing the weight of the droplet. Pilot experiments have shown that the weight of all droplets of one experimental series varied by less than ±2%. Since the weight of the droplets was defined by this procedure, all other droplets were diluted with 3 mL saline solution and centrifuged. The Evans blue concentration in each droplet was then calculated from the photometric absorption (Zeiss Photometer PMQ II) measured in the supernatant.
To test the fading characteristics of Evans blue in the capillaries during adjustment of the capillary section to the measuring aperture and during measurement of fluorescence intensity, we injected FITC-dextran (molecular weight, 71 000 kD; 0.5 mL; 10%) and Evans blue (0.5 mL; 4%) simultaneously into a femoral vein of one anesthetized Sprague-Dawley rat. After a circulation time of 10 minutes, the rat was decapitated and the brain removed and processed as described above (freezing, embedding, cutting, fixation). Fifty capillary sections were adjusted to the microscopic measuring aperture under FITC excitation to avoid Evans blue fading. The fluorescence intensity of Evans blue was then measured in each capillary section for 1 minute in 5-second intervals under 50% continuous excitation. This procedure was repeated for an additional 50 capillaries at 100% continuous excitation.
To obtain a calibration curve for the intracapillary concentration of Evans blue, we injected the dye in different quantities (0.15 mL, 2%; 0.25 mL, 4%; 0.5 mL, 4%; 1 mL, 4%; 1.75 mL, 4%; 2 mL, 4%; and 1.75 mL, 8%) into the catheterized femoral vein of seven anesthetized rats. The dye was allowed to circulate for 10 minutes to obtain a homogeneous plasma concentration. After an arterial blood sample was taken for the analysis of Evans blue plasma concentration, the rats were decapitated. The brains were dissected out, frozen, and treated as described above (embedding, cutting, fixation). The fluorescence intensity per cross-sectional area was measured in 250 capillary sections at each concentration, and the mean fluorescence intensity per cross-sectional area was related to the respective arterial dye concentration of that rat.
The fading characteristics of Evans blue during 1 minute of excitation were tested by comparing the measured fluorescence intensities at the start and end of the excitation period using the Wilcoxon test for differences of paired values.
The calibration curve taken for the measurement of intravascular Evans blue concentration (Fig 1⇓) was calculated using linear regression analysis. Linear regression analysis was also applied to determine the relation between arterial plasma concentration and measured capillary cross-sectional area (Fig 2⇓).
The slope of the arterial input functions for each animal of the physiological studies was calculated using the last two blood samples that were taken immediately before decapitation. The slopes were correlated with the respective Pco2 value, and the correlation coefficient obtained was tested for its significance compared with zero.
Potential differences between the coefficients of variation of intravascular dye concentration in the five different brain structures investigated were tested using the Meyer-Bahlburg20 test. Forty capillaries were taken for each brain structure in each rat.
For the analysis of the correlation between the coefficient of variation (SD/mean · 100) of intravascular dye concentration and arterial Pco2 (Fig 5⇓), an exponential regression was calculated using the least-squares method for the equation y=a · bx.
In all tests the level of significance was set at a value of P<.05.
Assessment of Method in Anesthetized Rats
To test the fading characteristics of Evans blue, we measured the fluorescence intensity in 50 capillary profiles during 1 minute of continuous excitation at 50% and in 50 additional profiles at 100% intensity of the excitation light. The mean fluorescence intensity decreased significantly by 8.3% at 50% and 16.7% at 100% excitation light intensity.
Calibration Curve for Evans Blue
To calibrate the intracapillary fluorescence intensity, we circulated Evans blue for 10 minutes before decapitation. This assured an equal concentration in the brain capillaries in which the fluorescence intensity was measured and in the peripheral plasma (femoral artery) where the concentration was measured with a photometer. Since the fluorescence intensity measured in a capillary section depends on the amount of dye contained in this capillary section, the plasma volume contained in the capillary section must also be known to yield the capillary concentration. In the brain sections, the plasma volume of each capillary section is defined by its cross-sectional area because all sections had the same thickness of 5 μm. Therefore, the cross-sectional area of each capillary section was determined besides its fluorescence intensity in the calibration studies as well as in the perfusion pattern studies.
Fig 1⇑ shows the calibration curve determined for Evans blue after correction for the cross-sectional area. It is evident that the capillary fluorescence per cross-sectional area is directly proportional to the photometrically determined arterial dye concentration (r=.99). To estimate a potential error that could arise from differences in the intravascular luminescence of the dye, we correlated the measured cross-sectional areas with the arterial dye concentration. Fig 2⇑ shows that the measured cross-sectional areas did not depend on arterial dye concentrations. The identity of measured cross-sectional areas at different intravascular luminescence shows that the attenuation procedure used to correct for different luminescence was effective. Finally, the error inherent in the measurement of the intravascular Evans blue concentrations was calculated. The average coefficient of variation (SD/mean · 100) of intracapillary dye concentration measured in a total of 1750 capillary sections of seven rats after 10 minutes of circulation was found to be 17.4%.
Physiological Studies in Conscious Rats
The Table⇓ shows arterial pH, Po2, and mean arterial blood pressure determined at the different arterial Pco2 values chosen for the studies. Because of hyperventilation at an increased arterial Pco2, arterial Po2 was moderately elevated during hypercapnia. Mean arterial blood pressure remained unchanged with increasing arterial Pco2.
Perfusion Patterns of Brain Capillaries
We investigated the perfusion patterns of capillary plasma flow in brains of 11 conscious rats that had been decapitated during the phase of increasing arterial Evans blue concentration. This is verified by Fig 3⇓, which shows the time course of the arterial Evans blue concentration of each rat. Statistical analysis of the curves showed that the slope of increasing arterial Evans blue concentration did not change significantly with increasing Pco2.
We measured the intracapillary dye concentrations at these different Pco2 values in 200 brain capillaries of each rat. During normocapnia, a wide distribution range of intracapillary dye concentrations was found (Fig 4⇓), indicating a high degree of heterogeneity of perfusion velocities in different capillaries during normocapnia. Perfusion velocities appeared to be randomly distributed in the brain, because the observed extreme variations in dye concentrations could be found in neighboring capillaries. With increasing degrees of hypercapnia, the variation of the intravascular dye concentration was gradually reduced (Fig 4⇓), indicating a decrease in the heterogeneity of capillary plasma perfusion. At an arterial Pco2 of 87 mm Hg, the width of the distribution range was diminished to less than half of the value obtained during normocapnia.
Each of the curves shown in Fig 4⇑ refers to the sum of the intravascular concentrations measured in five brain structures of each rat. To investigate whether differences in the perfusion pattern exist among these brain structures, we calculated the coefficient of variation of intracapillary dye concentration for each brain structure in each rat. Since the coefficients of variation measured for the five brain structures of each rat were not significantly different in any of the rats investigated, the coefficients of variation were pooled for each rat as shown in Fig 5⇓. An exponential inverse correlation exists between the heterogeneity of intracapillary dye concentration and arterial Pco2 (r=.97).
In the present study, we present a method that allows one for the first time to assess the pattern of capillary plasma perfusion simultaneously in all brain areas of interest. The results show a highly heterogeneous distribution of capillary plasma flow in normocapnic rats. The heterogeneity of plasma flow is gradually reduced with increasing hypercapnia, a condition in which cerebral blood flow has been shown to be increased in comparable experiments.1
Discussion of Method
The capillary circulation in the brain has been investigated in previous studies using intravascular markers to detect capillary plasma flow. However, these experiments have been limited by the methodological constraint that they have only allowed the differentiation between perfused and nonperfused capillaries.1 2 4 5 19 The present method is an extension of the previous methods because an effort is made to gain information about the extent of plasma perfusion of single capillaries.
Evans blue was chosen as an intravascular marker because of the following reasons: (1) Evans blue does not diffuse out of the capillaries in vivo under normal conditions21 22 and is not taken up by red blood cells23 ; (2) the intracapillary Evans blue concentration can be quantified; in the calibration studies in anesthetized rats, the fluorescence intensities per cross-sectional area were tightly correlated with the Evans blue plasma concentrations determined photometrically from plasma samples; and (3) Evans blue fading was negligible in the present experimental setup. Fading could result from two different procedures. First, the capillary profile had to be placed into the right position. This procedure took approximately 10 seconds and was performed at an excitation light intensity of 50%. At this intensity, a fading of 8.3% was measured during 1 minute corresponding to a fading of 1.4% during the 10 seconds of placement. Second, the fluorescence intensities were then measured at an excitation light intensity of 100% within 0.2 second. During 1 minute of maximal excitation, fading of approximately 17% was measured in the capillaries, which corresponds to a fading of 0.05% during the measuring period. This low degree of total fading (1.45%) could be achieved without treatment of the brain sections with chemicals that reduce fading of fluorescent dyes.24
A prerequisite for quantification of intracapillary Evans blue concentration is a tissue preparation that precludes diffusion or condensation of the dye. Diffusion of the dye out of the capillary lumen would impede the detection of cross-sectional area, whereas condensation of the dye within the capillary lumen could result in a partial absorption of the emitted fluorescence by the condensed dye, resulting in erroneously low fluorescence intensities. In the present study, a sufficiently homogeneous distribution of the dye inside the capillary lumen without any outward diffusion was achieved by placing the cryosections on slides that had previously been coated with a concentrated sucrose solution and by dehydrating the sections thereafter in acetone.
Determination of Heterogeneity
The intracapillary Evans blue concentration varied from capillary to capillary. It could be argued that the differences in capillary Evans blue concentrations were due to the cutting of a randomly arranged capillary network,10 25 in which some capillaries are cut at the arterial end, others at the venous end, and others in between. Because of the experimental design, the Evans blue concentration along each capillary decreases from the arterial to the venous end, since the decapitation was always performed during an increasing arterial dye concentration. Therefore, the measured intracapillary Evans blue concentration depends on the location of the dye in the capillary. Since in the present study dye concentrations were measured in numerous capillary sections, it appears likely that altogether the capillaries were cut at random locations. Therefore, some degree of heterogeneity of intravascular dye concentration found in the present study in each rat can be ascribed to variations in the location of the intravascular Evans blue concentration along the capillaries. However, the differences in heterogeneity of intravascular dye concentrations observed at various arterial Pco2 values cannot be explained by such variations of the intravascular location of the dye. Another cause of the observed heterogeneity of the intracapillary Evans blue concentration might be the existence of erythrocytes inside the capillary lumen. Erythrocytes do not take up Evans blue.23 Therefore, their existence in the capillary lumen could attenuate the Evans blue fluorescence. The measurement of intravascular Evans blue concentration is not disturbed by the existence of erythrocytes as long as an erythrocyte is not covered by plasma in a capillary section, because the fluorescence intensity is determined exclusively in the remaining capillary lumen that is filled with fluorescent dye. The result is a reduction in the cross-sectional area caused by the lack of fluorescence at the location of the erythrocyte. On the other hand, the capillary dye concentration may be underestimated if an erythrocyte is partly covered by Evans blue. Such an error can be estimated from the experiments in anesthetized rats in which identical Evans blue concentrations were achieved in all capillaries after 10 minutes of circulation. In these experiments, the coefficient of variation of intravascular Evans blue concentrations was 17.4%. This quantifies the combined error that arises from erythrocytes, the inaccurate determination of cross-sectional area, and further inaccuracies such as fading of the fluorescent dye. Given this error, it appears likely that the coefficient of variation measured during normocapnia and the decrease in the coefficient of variation observed during hypercapnia cannot be explained by methodological errors.
In principle, changes in the heterogeneity of intracapillary Evans blue concentrations could be caused by changes in the arterial input function. This could happen if the input function would change systematically with increasing hypercapnia. As shown in Fig 3⇑, a systematic change of the slope of the input function could not be observed.
Comparison With Other Methods
The present method appears to be supplementary to other methods that have been used to investigate capillary blood flow in the brain. Capillary blood flow has been analyzed in vivo using video imaging,10 confocal laser microscopy,6 7 8 9 and the photoelectric recording of carbon black dilution curves.26 These methods allow the observation of the dynamics of capillary perfusion in the superficial layers of the cerebral cortex of anesthetized animals after the skull is opened. In contrast, the present method makes it possible to analyze any brain structure that might be of interest regardless of its location. In addition, Evans blue injection can be performed in conscious animals, and the skull remains intact during the experiment. A potential problem of open-skull methods is the induction of alterations in the microcirculation9 that could be caused by surgery, the combination of a light source and intravascular fluorescent dyes,6 9 10 and the insertion of a light source into the brain tissue.26 The present method circumvents these problems because the dye is applied to a conscious animal and the brain is not exposed to fluorescent light in vivo. However, no discrepancies have been found between our data and those of Villringer et al9 and Hudetz et al.10
Discussion of Results
The present study shows a considerable heterogeneity of Evans blue concentration in cerebral capillaries 3 to 4 seconds after intravenous bolus injection of Evans blue that is diminished when blood flow velocity is increased during hypercapnia. The heterogeneity of Evans blue concentration in the present experimental setup can be best explained by a heterogeneity of plasma flow velocities in the different brain capillaries. The reduced heterogeneity of Evans blue concentration with increased blood flow indicates a less heterogeneous velocity profile. This conclusion is based on the notion that a rapidly perfused capillary will contain more Evans blue than a slowly perfused capillary as long as the arterial concentration is increasing.
Two other causes of changes in the heterogeneity of dye concentration during hypercapnia cannot be excluded. One possibility concerns the profile of intravascular dye concentrations. With a lack of capillary recruitment, an increasing cerebral blood flow should result in an increased flow velocity in the capillaries. With such an increased flow velocity, the concentration difference between the arterial and venous ends of the capillaries should decrease because the dye passes more quickly through the microvessels. Thus, regardless of where along the capillary the concentration is measured, it will be closer to that at the beginning of the capillary, which would mean a decrease in the heterogeneity of intravascular dye concentration. Another cause of the observed decrease in the heterogeneity of intravascular dye concentration during hypercapnia could be a decrease in the number of “plasmatic” capillaries. Several authors7 27 28 have suggested a perfusion of 10% to 15% of all brain capillaries by plasma but not red blood cells under normal conditions. When blood flow increases, these capillaries begin to carry red blood cells. This change would make all capillaries more similar with respect to erythrocyte and plasma flow, which would result in a decreased heterogeneity of plasma flow. The high concentrations of Evans blue measured in the normocapnic experiments (Pco2, 42.9 to 46.2 mm Hg) are in accordance with such an effect. It appears possible that these high concentrations originate from capillaries that were perfused only with plasma. The viscosity of fluid within plasmatic capillaries is probably much less than that in hematic (plasma plus red blood cell) capillaries. Dye would therefore pass more quickly through plasmatic than hematic capillaries. With vasodilation during hypercapnia, red blood cells would pass through all capillaries, which would result in a rise in viscosity in the newly hematic capillaries and a drop in velocity in these capillaries. This would explain the decrease of the highest concentrations of Evans blue in the hypercapnic experiments. Each or both effects, an increase in the number of hematic capillaries and a flattening of the arteriovenous concentration profile of Evans blue, might contribute to the observed decrease in the heterogeneity of intravascular dye concentration.
The conclusion drawn from the present study of a heterogeneity of capillary plasma flow in the brain is in accordance with the findings and conclusions of other studies. Results obtained from patients with various cerebral disorders using the double-indicator method were consistent with the hypothesis of a heterogeneity of capillary flow at normal cerebral blood flow29 30 and an increase of heterogeneity during low flow conditions.30 Photoelectric recordings of the appearance of carbon black at the brain surface of cats after intracarotid injection26 have also been interpreted to indicate flow heterogeneity in the brain during normocapnia and a decrease of heterogeneity during hypercapnia. Flow heterogeneities have been observed directly for erythrocytes in superficial cortical capillaries using confocal laser microscopy,6 7 8 9 video imaging,10 and microtransillumination.29
Altogether, the heterogeneity of Evans blue concentration found in brain capillary sections in the present study indicates a heterogeneity of capillary plasma flow, although direct proof is missing that would totally exclude other possibilities. The data also indicate a decrease in the heterogeneity of plasma flow during hypercapnic hyperemia.
This study was supported by a grant from the Deutsche Forschungsgemeinschaft.
- Received June 2, 1994.
- Accepted September 30, 1994.
- © 1995 American Heart Association, Inc.
Göbel U, Theilen H, Kuschinsky W. Congruence of total and perfused capillary network in rat brains. Circ Res.. 1990;66:271-281.
Theilen H, Schröck H, Kuschinsky W. Capillary perfusion during incomplete forebrain ischemia and reperfusion in rat brain. Am J Physiol.. 1993;265:H642-H648.
Weiss HR, Buchweitz E, Murtha TJ, Auletta M. Quantitative regional determination of morphometric indices of the total and perfused capillary network in rat brain. Circ Res.. 1982;51:494-503.
Villringer A, Dirnagl U, Gebhardt R, Einhäupl KM. An in vivo approach to assess the capillary recruitment hypothesis in the brain microcirculation using confocal laser scanning microscopy. J Cereb Blood Flow Metab. 1991;11(suppl 2):S441. Abstract.
Villringer A, Them A, Lindauer U, Dirnagl U. Capillary perfusion of the rat brain cortex: an in vivo confocal microscopy study. Circ Res.. 1994;75:55-62.
Rovainen CM, Woolsey TA, Blocher NC, Wang D-B, Robinson OF. Blood flow in single surface arterioles and venules on the mouse somatosensory cortex measured with videomicroscopy, fluorescent dextranes, nonoccluding fluorescent beads, and computer-assisted image analysis. J Cereb Blood Flow Metab.. 1993;13:359-371.
Bereczki D, Wei L, Otsuka T, Hans F-J, Acuff V, Patlak C, Fenstermacher J. Hypercapnia slightly raises blood volume and sizably elevates flow velocity in brain microvessels. Am J Physiol.. 1993;264:H1360-H1369.
Hans F-J, Wei L, Bereczki D, Acuff V, Demaro J, Chen J-L, Otsuka T, Patlak C, Fenstermacher J. Nicotine increases microvascular blood flow and flow velocity in three groups of brain areas. Am J Physiol.. 1993;265:H2142-H2150.
Jones SC, Bose B, Furlan AJ, Friel HT, Easley KA, Meredith MP, Little JR. CO2 reactivity and heterogeneity of cerebral blood flow in ischemic, border zone, and normal cortex. Am J Physiol.. 1989;257:H473-H482.
Williams JL, Shea M, Jones SC. Evidence that heterogeneity of cerebral blood flow does not involve vascular recruitment. Am J Physiol.. 1993;264:H1740-H1743.
Meyer-Bahlburg HFL. A nonparametric test for relative spread in unpaired samples. Metrica.. 1970;15:23-29.
Rawson RA. The binding of T-1824 and structurally related diazo dyes by the plasma proteins. Am J Physiol.. 1943;138:708-717.
Gregersen MI, Schiro H. The behavior of the dye T-1824 with respect to its absorption by red blood cells and its fate in blood undergoing coagulation. Am J Physiol. 1938;121:284-292.
Valnes K, Brandtzaeg P. Retardation of immunofluorescence fading during microscopy. J Histochem Cytochem.. 1985;33:755-761.
Tomita M, Gotoh F, Amano T, Tanahashi N, Kobari M, Shinohara T, Mihara B. Transfer function through regional cerebral cortex evaluated by a photoelectric method. Am J Physiol.. 1983;245:H385-H398.
Tajima A, Nakata H, Lin S-Z, Acuff V, Fenstermacher J. Differences and similarities in albumin and red blood cell flows through cerebral microvessels. Am J Physiol.. 1992;262:H1515-H1524.
Hertz MM, Paulson OB. Heterogeneity of cerebral capillary flow in man and its consequences for estimation of blood-brain barrier permeability. J Clin Invest.. 1980;65:1145-1151.