Regional variation in brain capillary density and vascular response to ischemia

Brain Research 910 (2001) 81–93 www.elsevier.com / locate / bres

Research report

Regional variation in brain capillary density and vascular response to ischemia Marco Cavaglia a,b , Stephen M. Dombrowski a , Judith Drazba c , Amit Vasanji c , b a,d , Paula M. Bokesch , Damir Janigro * a Cerebrovascular Research Center, Department of Neurological Surgery, Cleveland Clinic Foundation, Cleveland, OH 44195, USA Cerebrovascular Research Center, Department of Cardiothoracic Anesthesia, Cleveland Clinic Foundation, Cleveland, OH 44195, USA c Cerebrovascular Research Center, Department of Confocal Microscopy Core, Cleveland Clinic Foundation, Cleveland, OH 44195, USA d Cerebrovascular Research Center, Department of Cell Biology, Cleveland Clinic Foundation, Cleveland, OH 44195, USA b

Accepted 24 May 2001

Abstract Differences in brain neuroarchitecture have been extensively studied and recent results demonstrated that regional differences in the physiological properties of glial cells are equally common. Relatively little is known on the topographic differences in vascular supply, distribution and density of brain capillaries in different CNS regions. We developed a simple method consisting of intravascular injection of fluorescent dyes coupled to immunocytochemical techniques that allows for simultaneous observation of glia–neuronal–vascular interactions in immersion-fixed brain specimens from small rodents. This technique permits quantitative evaluation of regional differences in glial / neuronal distribution and the study of their relationship to vascular densities. Variations of this technique also allow the detection of abnormal microvasculature (i.e. ‘leaky’ vessels), a useful feature for studies of blood–brain barrier function in health and disease. By use of quantitative confocal microscopy, the three-dimensional geometry of cortical and hippocampal structures revealed remarkable differences in vascularization between cortical gray / white matter junction, and hippocampal formation (CA1 and CA3 regions). Significant differences were also observed within the same investigative region: CA1 was characterized by low capillary density compared to neighboring CA3. Following an ischemic insult, CA1 vessels had more extensive blood–brain barrier leakage than CA3 vessels. We conclude that in addition to neuronal and glial heterogeneity, cortical structures are also endowed with region-specific vascular patterns characterized by distinct pathophysiological responses.  2001 Elsevier Science B.V. All rights reserved. Theme: Other systems of the CNS Topic: Brain metabolism and blood flow Keywords: Cerebrovascular disorder; Blood–brain barrier; CA1; Selective vulnerability; Vasculogenesis; Cerebral blood flow; Hippocampus

1. Introduction Unlike other organs, the brain regulates its own blood supply primarily by translating parenchymal signals into arteriolar diameter changes. Brief interruption of vascular supply to the CNS results in altered neuronal function and, if the ischemic interval is of sufficient duration, to neuronal cell death [29,49]. The vascular supply to different brain regions is not homogeneous, and large differences in *Corresponding author. Cerebrovascular Research, Cleveland Clinic Foundation / NB20, 9500 Euclid Avenue, Cleveland, OH 44195, USA. Tel.: 11-216-445-0561; fax: 11-216-445-1466. E-mail address: [email protected] (D. Janigro).

capillary density exist between gray and white matter. The higher capillary density in gray matter appears to be related to the higher metabolic demand of principal cells and synapses compared to axons [15]. It is not clear whether vascular density gradients also exist within cortical regions. Since certain regions are more sensitive to a variety of insults including ischemia [29,49], it is possible that in addition to intrinsic neuronal factors, differences in cerebrovascular supply or regulation may play a role in this selective vulnerability. In addition to providing blood supply to the brain, the cerebral vasculature is also engaged in the protection of neurons from systemic influences by highly specialized endothelial cells (ECs) forming the blood–brain barrier

0006-8993 / 01 / $ – see front matter  2001 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 01 )02637-3

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(BBB). BBB properties are not innate in ECs, and differentiation of cerebral microvessels is induced by yet unknown astrocytic ‘factors’ [19,26]. In spite of the fact that regional differences in astrocytic morphology and function exist [11,16,45], and that one would expect regional variation in how they influence neighboring ECs, it is generally assumed that the BBB is a homogeneous structure (for review see [17]). The anatomical arrangement of brain arterial supply can be studied by a variety of techniques, including horseradish peroxidase [50], ink, [57] and BaSO 4 angiography [46]. While these approaches have contributed significantly to our knowledge of vasculogenesis, this information was usually limited to larger, or superficial vessels. Direct observation of BBB function is achieved by a variety of methods, based on monitoring extravasation of exogenously injected molecules [3,59] [5,8,18]. Since a number of cerebrovascular diseases affect both intravascular perfusion and BBB function, it is desirable to simultaneously visualize the entire vascular function and anatomy of the brain. One limitation of these methods is their inability to simultaneously monitor vascular anatomy and blood–brain barrier function. A recently developed microangiographic technique based on the injection of fluorescent probes enables confocal visualization of the entire brain micro- and macrovasculature [60,61]. Simultaneous observation of functional changes in BBB permeability after ischemia or hypoxia is also possible if appropriate microangiographic molecules are employed. To this end, fluorescent albumin was used, since albumin does not extravasate abluminally if the BBB is intact [22]. The experiments described herein were designed to address the following questions: (1) are capillary densities homogeneously distributed across gray matter in the rodent brain? (2) do vascularization patterns parallel any known anatomical differences in parenchymal cells? and (3) are cerebral blood vessels equally sensitive to changes induced by an ischemic insult that causes selective neuronal death?

ventricle was cannulated with a 24-gauge polyethylene catheter (Insyte, Vialon), and perfusion (1 ml / min) was performed with a fluorescent dye solution (see Solution composition). The right atrium was incised to prevent elevation in systemic blood pressure. Immediately following perfusion, animals were sacrificed, decapitated and the brains removed. The lag time between decapitation and immersion fixation never exceeded 2 min. In contrast to cardiac injection, intravascular dye application did not require thoracic exposure, and animals were not intubated. After skin incisions, bilateral carotid arteries or jugular veins were exposed with the aid of a dissecting microscope and cannulated with 24-gauge polyurethane catheters. Fluorescent dye solution was perfused through the carotid arteries or jugular veins at a rate of 1 ml / min (10 ml / kg). In order to avoid systemic blood pressure elevation, the same amount of blood was withdrawn from the femoral vein (see Fig. 1). Brains were immersion fixed for 24–48 h in 10% formalin solution, then cryoprotected in a 30% sucrose solution. Brains were mounted on a freezing microtome and cut in the coronal plane at 50 or 100 mm. Sections were collected and floated in 0.1 M phosphate buffer solution and mounted on gelatin-subbed, glass slides. Sections were dried overnight and coverslipped using

2. Materials and methods

2.1. Surgical procedures Adult Sprague–Dawley rats (200–270 g) were anesthetized with an intraperitoneal injection of 40 mg / kg sodium pentobarbital (Abbott Laboratories). Animals were positioned supine on a surgical towel to expose the ventral surface, and the thoracic skin removed. To prevent blood clot formation, the femoral vein was exposed and heparin (100 I.U. / kg) administered intravenously with a 26-gauge needle. Intracardiac injections were performed under deep anesthetic conditions with controlled ventilation. After surgical exposure of the rib cage, the midline thoracic cutis was incised and the sternum opened. The apex of the left

Fig. 1. Comparison of different injection techniques used to visualize cortical vascular structures. The schematic drawings in the left panel illustrate the site of dye injection. Note that the results obtained were essentially identical, and that intravascularly stained cerebral vessels were easily detected by conventional confocal microscopy. The arrows indicate penetrating pial vessels, while the arrowheads show examples of dyefilled capillaries. The dashed arrows point towards the pial surface (bar 35 mm).

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Vectashield mounting medium with Dapi (Vector, Burlingame, CA, USA). In two experiments, the vasodilator sodium nitroprusside (1.5 mg / kg) was injected intravenously to decrease the total vascular resistance. This procedure was not used for the ischemia–reperfusion experiments since the presence of vasodilators may interfere with the hyperperfusion / no reflow phenomena [51]. No significant effect of this vasodilator was observed.

2.2. Solution composition The fluorescent albumin solution was prepared by reconstituting 500 mg of bovine desiccate albumin– fluorescein isothiocyanate (FITC, Sigma A 9771, Mw 69 kD, 12 mol / mol albumin) in 50 ml of phosphate buffered saline (0.1 M PBS) lacking magnesium and calcium ions. The FITC solution was stirred at room temperature in the dark for 5 min prior to injection.

2.3. Morphology and computer reconstruction The sections were analyzed with a Leica TCS-SP spectrophotometric laser scanning confocal microscope (Heidelberg, Germany). Microscopic data were acquired with a 53 objective (0.1 numerical aperture). Green (FITC–albumin) fluorochromes, on the sections were excited by a laser beam at 488 mm and emission was detected with a photomultiplier tube (PMT) through a 522-mm filter. Laser intensity was set at 45% of laser power and black levels were zero for all data acquisition. PMT gain ranged from 920 to 980. Because the size of fluorescent spots in a two dimensional image depends on the laser power, pinhole, zoom, focus, gain, and duration of sampling time were fixed within the same section during the acquisition of data. For both group of rats (control and ischemic), sections of interest were scanned in 204832048 pixel format in the x–y direction using an 8-frame-scan average. The dimension of the final image was 4 mm 2 . Slightly overlapping images were collected sequentially across the horizontal axis and then down the vertical axis of each brain slice. Anatomical reference points (e.g. ventricle, pial surface) were used to determine overlap. All acquired images were imported into the image analysis system Adobe PHOTOSHOP (version 5.5, Adobe System, San Jose, CA, USA) for image analysis. To create a single brain section, images collected by methods described above were aligned by overlapping common anatomical reference points (see Fig. 3). Analysis of vessel density was performed by isolating regions of interest (ROI) within each montage using a marquee tool and applying filters from the Image Processing (IP) TOOLKIT (v.3.0) (Reindeer games, Ashville, NC, USA). For each ROI a median filter (2–3 pixel radius) was applied to reduce background noise and thresholding was performed

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to produce a binary ROI (vessels were given a pixel intensity of 255 and background pixel intensity was set at 0). Since each brain slice may have been uneven in total thickness, thresholding was based on maximizing surface vessels (brightest pixels) removing the contribution of vessels buried deeper in the slice. A skeletonizing filter was applied to each binary ROI, reducing each vessel to a single, one pixel width black line (pixel intensity of 0) on a white background (pixel intensity of 255). Each line or vessel had end points with a pixel intensity of 200. Finally, the regions of interest were thresholded to 200 so that only the end points of vessels were visible. These end points were counted by an IP filter and divided by two (two end points5one line or vessel) to determine the number of vessels in a given ROI. Vessel density was determined by dividing the number of vessels in a ROI by the area of the ROI. The values for density of vessels are presented as the vessel(s) per mm 2 (mean6S.E.M.), although the surface of different regions of interest (e.g. CA1 and CA3 regions) were ,1 mm 2 . By a different toolkit of the same IMAGE program system, analysis of brightness was performed in order to quantify BBB leakage as determined by visualization of albumin extravasation (Fig. 2). For each ROI, the pixel intensity was measured using a gray scale of 0 to 255, where 0 corresponds to black and 255 to white following conversion to gray scale. We arbitrarily developed a mean pixel intensity range scale consisting of 5 different intervals in increments of 50. A grid was constructed to represent the intensity scales where 0–50 represents no leakage; 51–100 represents mild leakage; moderate leakage is represented by 101–150; severe leakage by 151–200; and complete leakage, by values .200.

2.4. Immunocytochemistry To identify astrocytes and neurons, the sections were stained with rabbit anti-cow glial fibrillary acidic protein antibody (GFAP, Dako, USA) and with mouse anti-neuronal nuclei monoclonal antibody (NeuN, Chemicon, USA), respectively. Free floating sections, of 50 mm thickness, were incubated in a solution of 3% normal goat serum (NGS), 0.1% Triton-X (TX), and 1.0% bovine serum albumin (BSA) in 0.1 M Tris-buffered saline (TBS; pH 7.4) for 1 h to block nonspecific staining. Sections were then transferred to the primary antisera containing 1:100 dilution of antibody against GFAP and 1:500 for NeuN in 0.1 M TBS with 3% NGS, 0.1% TX, and 1.0% BSA, and incubated for 24 h at temperature of 48C. Sections were then rinsed five times in 0.1 M PBS and incubated for 3 h at room temperature in the dark with secondary antibodies for chicken anti-rabbit (GFAP) and goat anti-mouse (NeuN), 1:50 IgG, conjugated to either rhodamine (TRITC) or fluorescein (FITC); (Rockland, Gilbertsville, PA, USA). Finally, sections were rinsed in 0.1 M PBS and mounted on glass slides, using Vectashield

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Fig. 2. Techniques used to quantify vessel density and blood–brain barrier leakage. (A) Coronal sections (A1) of cortical regions used to demonstrate the thresholding technique used for quantitative determination of vascular densities. See Methods for complete description. Briefly, analysis of vessel density was performed by isolating areas or regions of interest. For each ROI, thresholding was performed to produce a binary ROI; vessels were given a pixel intensity of 255 and background pixel intensity was set to 0. Thresholding (A2) was based on maximizing surface vessels (brightest pixels) thus removing the contribution of vessels buried deeper in the slice. A skeletonizing filter was applied to each binary ROI, reducing each vessel to a single, one pixel width black line (A3; pixel intensity of 0) on a white background (pixel intensity of 255). The inset shows a color-coded example demonstrating how end and start points (shown in red) were obtained. Finally, the regions of interest were thresholded to 200 so that only the end points of vessels were visible (A4). The numbers in bold indicate the final vessel density, in this case expressed as vessels / field. (B) Examples of different levels of blood–brain barrier leakage and definition of mean intensity range scale. See text for details.

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(Vector, Burlingame, CA, USA) mounting medium with DAPI.

2.5. Ischemia–reperfusion Reversible forebrain ischemia was achieved as described elsewhere [47]. In brief, animals were anesthetized as described above, the femoral artery was exposed and cannulated with a pressure monitor. The same outlet was used to withdraw appropriate amounts of blood to induce controlled hypotension (systolic pressure of 40 mmHg maintained for 8 min). The blood pressure drop was concomitant with the occlusion of previously exposed carotid arteries for an identical duration. The latter was achieved by vascular clamp devices. The reperfusion period lasted 45 min; after reperfusion, intravascular dye injection was performed, and the animal sacrificed by decapitation after 5 min.

2.6. Statistics Data were analyzed by ANOVA followed by nonparametric analysis (Mann–Whitney) in Figs. 4 and 6. All values are presented as means6S.E.M. Statistical significance was set at p,0.05.

3. Results The experiments described herein were performed on a total of fifteen Sprague–Dawley rats: nine control animals and six animals subjected to forebrain ischemia. Three different injection methods were used to fill with fluorescent albumin the rat cerebral vasculature (Fig. 1). Dyes were injected either in the arterial blood stream (by bilateral carotid injection, Fig. 1A), in the venous outflow (jugular injection; Fig. 1B), or directly into the left ventricle (1C). Since the three methods essentially yielded the same morphological results, we have chosen the arterial injection method owing to the following considerations: (1) a smaller amount of fluorescent dye could be used, thus minimizing hemodilution and cost; (2) the surgical procedure for intracarotid injections is less traumatic and assures 100% survival and (3) reproducibility following intra-arterial injection was greater compared to intravenous or intracardiac injection procedures.The results obtained by the three different methods were comparable in cortical sections (Fig. 1). Filling of the intravascular compartment as well as filling of both large, penetrating pial vessels, and capillaries was achieved successfully regardless of the technique used. Large penetrating vessels (.50 mm) were found perpendicular to the pial surface, while smaller vessels (,20 mm) occupied all boundaries of the parenchymal space.

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3.1. Vascular anatomy of whole brain mounts Bilateral carotid injection reliably and symmetrically filled the intravascular compartments of the encephalon. Whole brain sections were observed at both low magnification and at increasing higher resolution. An example of computer reconstruction of a coronal section clearly showing the posterior aspect of the parietal cortex (25.7 mm from bregma [30]) is shown in Fig. 3. Cellular nuclei were reliably stained blue for Dapi under UV fluorescence (Fig. 3A, B and D) or red using NeuN immunocytochemistry (Fig. 3C). A consistent pattern of distribution was observed in the vascularization of elongated penetrating pial arteries; these vessels run perpendicular to the cortical layers and often bifurcate before entering the adjacent white matter (Figs. 3 and 4A). Shorter, smaller caliber arteries were confined mostly to the cortex, forming a compact network with the longitudinal vessels in the intermediate cellular layer of the gray matter. Under microscopic visualization, vascular densities were highest in neocortex and by comparison substantially less at the gray matter–white matter transition, for example the external capsula and alveus (ec and alv; Fig. 3B). Blood vessels situated in white matter oriented preferentially parallel to axonal fibers and gave rise to relatively scarce capillaries (Fig. 3B). Vascular pattern changes are clearly seen at the boundary between extracortical structures. For example, substantia nigra pars reticulata and pars compacta could be differentiated based on the distribution of their vasculature (Fig. 3C). Differences in vascular network could also be used to recognize midline nuclei such as the superior mamillari and medial mamillari nuclei (Fig. 3D).

3.2. Glia–vascular interactions The relationship between glial and vascular elements was investigated in sections obtained from animals perfused through the carotids with fluorescent albumin and subsequently processed for GFAP immunocytochemistry. Processes from astrocytic endfeet were found to engulf penetrating pial vessels (Fig. 4). Fenestrations in the glial envelope were occasionally present, as was an empty space interposed between glial cells and the extravascular compartment corresponding to Virchow-Robin’s space (Fig. 4A). At higher magnification, the ensheathment of individual capillaries by astrocytic endfeet was also evident (Fig. 4B). It was also possible to determine the relationship between neuronal nuclei, glial cells, blood vessels, using stains for NeuN (neurons), GFAP (astrocytes) immunocytochemistry (Fig. 4C).

3.3. Regional differences in vascular density Regions with the highest synaptic activity and metabolic demand were generally endowed with higher levels of vascularization. This was particularly evident when com-

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Fig. 3. Heterogeneity of vascular patterns in a 100-mm hemisection of rat brain. This image was obtained by acquiring a high definition series of sections that were then mounted together to faithfully reproduce the original structures. The quality of this high definition image cannot be fully appreciated at low power magnification, and selected regions are thus shown in more detail (bar51 mm). Note that the image itself contains sufficient detail to clearly display capillary structures. The expanded detail pictures also show DAPI staining for nuclear profiles, allowing the visualization of principal cell layers and the vasculature. (A1) Frontal cortex: note the perpendicular orientation of pial vessels to the pyramidal cell layer (bar5500 mm). (A2) More detailed visualization of a region with pial vessels intercalated by a dense capillary network (bar 100 mm). (B) Transition between gray and white matter (ec, external capsula; alv, alveus; CA1, pyramidal cell layer). Note the dramatic difference in vascular densities (bar 100 mm). (C) Vascularization of the substantia nigra pars compacta (SNc) and reticulata (SNr) (bar 200 mm). In this image, nuclear DAPI stain is indicated in red. (D) Supramammillari nuclei (SMn) and medial mamillari nuclei display high levels of vascularization, comparable to cortical gray matter.

paring gray matter to white matter. While the differences between gray and white matter were apparent after simple examination, using confocal microscopy, more subtle differences were revealed quantitatively by the technical approach described here (see Methods), aimed at calculating the density of capillaries per surface area. Comparisons were made between neighboring hippocampal regions (Figs. 5A–C), parietal, frontal and entorhinal cortices (Fig. 3), and white matter (corpus callosum, Fig. 3). The results of this quantitative analysis are shown in Fig. 5D. Hippocampal CA1 exhibited fewer capillaries than the neighboring CA3 region. Marked differences in the quantitative distribution of blood vessels were also observed among these three cortical regions. The differences between CA1, entorhinal cortex and hilus where not statistically significant ( p.0.05).

3.4. Functional evaluation of blood–brain barrier function by microangiography The permeability of the BBB is controlled by a number of cellular and diffusible factors, but ultimately depends on intact tight junctions [1,7,20]. Pharmacological studies have clearly demonstrated that large molecules traverse the intact endothelium to a negligible extent; this is in particularly true for large polypeptides and proteins. Thus, under normal conditions plasma proteins are sequestered and extravasation into the brain interstitial space occurs only when the BBB is breached. This constitutes the physiological basis for the use of fluorescent labeled albumin for the visualization of the intravascular compartment of various central nervous system structures, pending that an intact BBB is present.

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Fig. 4. Glia–vascular interactions can be studied in confocal sections where fluorescing-field vessels can be counter stained for GFAP immunocytochemistry. (A) Typical appearance of the cortex after visualization of the intravascular compartments (bar 200 mm). A magnified pial vessel is shown in the inset to demonstrate the relationship between GFAP positive envelope and the vessel itself. The two arrows point at clefts frequently observed in the astrocytic cuffing of vessels. At higher magnification (B) glial apposition on capillaries can be observed (bar 50 mm). The white dots present in the intraluminal compartment are artifacts due to the presence of residual blood cells within the lumen (As, indicates a GFAP positive astrocyte). Neuronal glial vascular interactions can also be investigated as demonstrated in (C) where neuronal cells (bright green), astrocytes (red) and blood vessels can be visualized in the same section. Gc represents the granular cell layer, hi, the hippocampal hilus.

Under the experimental conditions used for control experiments, extravasation of the fluorescent dye was never observed, demonstrating that this tracer itself may be used as an indicator of BBB intactness. To test this hypothesis, six rats underwent forebrain ischemia, an insult known to affect BBB integrity [52,54]. Fig. 6 shows the results of these experiments. Under preischemic conditions, normal hippocampal vascular morphology was observed. Following 8 min of ischemia and 45 min of reperfusion, cerebral blood flow was reinstated and the fluorescent dye injected through the carotid arteries. Following this procedure, the brains were processed as described in the Methods section. Scattered regions of impaired blood–brain barrier function were detected as regions with abnormal extravasation of the fluorescent dye (see Fig. 3). In particular, all CA1 regions

analyzed (n56), showed a mean pixel intensity range corresponding to a mean value of 3.12 on the arbitrary leakage scale shown in Fig. 2. In the CA3 regions the analysis showed no leakage in four cases and two cases of mild leakage corresponding to a mean value of 2.05 arbitrary units. Taken together, these results demonstrate that differences in vascular density exist between various cortical regions, and that region-specific paradigms of pathological responsiveness to noxious stimuli are also present.

4. Discussion The significance of the present study is twofold. On the one hand, we report the modification of an existing

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Fig. 5. Heterogeneous vascular densities in the rat hippocampus. Confocal microscopic images of the hippocampus were obtained from six animals. Capillary density was quantified as described in the methods section. Note that at low power (A) regional differences are already evident. Large vessels penetrating into the hippocampus are seen between the stratum radiatum and the molecular layer of the dentate gyrus (Gc). Note that the neocortex in the upper right corner has a higher capillary density than any hippocampal region. At high magnification (B, C) differences between CA3 and CA1 become more evident, as noted for (A). The capillary density in the neocortex is, however, comparably much higher. The results obtained by quantitative analysis of vascular densities are summarized in (D) where the mean6S.E.M. capillary densities are shown. Note that the CA3 region together with parietal and frontal cortex had the highest vascular density, while hippocampal CA1 had a density comparable to white matter (corpus callosum). Bars 100 mm. Asterisks denote significant differences ( p,0.05; t-test). The differences between CA1, entorhinal cortex and hilus where not statistically significant. Taken individually, these regions were however significantly different from white matter (corpus callosum).

microangiographic technique that allows morphological and functional evaluation of vascular patterns and BBB intactness in the CNS. By use of this technique, we were able to dissect out regional vascular differences in the CNS. In particular, two hippocampal subfields characterized by divergent sensitivity to ischemia displayed

profound differences in vascular density and region-specific changes following ischemia / reperfusion.

4.1. Methodological considerations Techniques aimed at visualization of vascular structures

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Fig. 6. Vascular changes following forebrain ischemia and reperfusion. Marked areas of vascular engorgement were observed in the neocortex, in hippocampal CA3 as well as in other cortical areas (* in A; bar 250 mm). By comparison, the CA1 region was frequently characterized by apparent no reflow phenomena (white circles in A1). Additional functional information was obtained from these sections by determining the amount of FITC–albumin leakage from the intravascular compartment to the brain parenchyma (A2 and B). Note numerous leaky vessels indicated by arrows in A2 and arrowheads in B. Also note the area of marked extravasation in the granular cell layer (dotted line in B, bar 50 mm), and at higher magnification (inset) spotted leakage from capillaries in the CA3 region indicated by the arrowhead. (C) Summary of the ischemia–reperfusion experiments to determine BBB intactness. Note that leakage was more common in CA1 then in CA3 ( p,0.01, n56).

in intact tissue are not novel, and have not historically been limited to investigations of the cerebrovasculature. These techniques are generally based on the intravascular application of ‘contrast’ agents that do not significantly extravasate during the procedure itself. The chemical nature of these agents and the method of microscopic examination determines the resolution of these techniques. Radioopaque agents give a sharp image of large vessels, but fail to visualize capillaries [33]. Other limitations of in vivo

angiography relate to the toxicity of the media used, the potential ischemic response to the injection procedure, etc. Modern diagnostic techniques have demonstrated the feasibility of brain angiography in humans, and important information on the anatomy of human brain vasculature has recently emerged [56]. Since rodents and guinea pigs have historically been the most commonly used experimental animals for the study of neurovascular insults, it is surprising that only a few studies have focused on

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detailed investigation of the anatomy and anatomical variation of their cerebrovasculature [32,33,35– 38,57,60,61]. These studies revealed a number of vascular patterns that are also present in the brain of primates, including individual variability in vascular supply to the brain even within the same inbred population [6]. The use of fluorescent tags to visualize the brain vasculature was introduced many years ago [41], but the use of this intravital technique was, however, limited to the observation of functional changes in pial vessels. An important feature of FITC–albumin microangiography is intrinsic to the nature of the tracer itself. Albumin is a non-toxic component of serum and addition of FITC does not appear to add significant acute toxicity to the molecule. Short-term survival following injection was achieved in 100% of the experiments, and no significant signs of brain damage (such as BBB leakage or neuronal cell death) were observed in control animals. Furthermore, albumin-based microangiography has the advantage of allowing simultaneous assessment of vascular architecture and permeability to serum proteins. Thus, while previous reports have described the three-dimensional network of the cerebrovasculature by use of fluorescent molecules, no information on functional changes in BBB function was reported [60]. Increased protein extravasation is a hallmark of brain injury, and has often been used to assess BBB integrity in animal models of neurological disease [8,9,19,53]. On this extent we used albumin instead of dextran–FITC because it has been shown by many investigators that high-molecular-weight dextrans leak out from the vascular system over time [43]. Our experimental technique was not limited to observation of superficial vessels, but rather allowed visualization of the entire cerebral vasculature. Furthermore, by combining the intravascular angiographic approach to quantitative confocal microscopy, we were able to obtain the reconstructions of vessels in discrete areas of the brain. An additional advantage of this approach was the fact that tissue samples could be processed for immunocytochemistry, revealing interactions between cerebral blood vessels and parenchymal elements.

4.2. Heterogeneity of vascular density The coupling of brain cell function to the vascular system is the basis for a number of functional neuroimaging methods relevant for human studies [56]. These methods map specific brain activation through a vascular response, such as an increase in CBF or a change in blood oxygenation. The mechanisms by which CNS neurons sense and regulate CBF have been so far elusive, as a result of owing to our scant knowledge of glia–neuronal– vascular interactions. Proximity of sites of neurotransmitter release and neuronal electrical activity to the cerebrovasculature facilitates coupling of neuronal activity and cerebral blood flow. This communication depends on

neuronal signals released synaptically (e.g. nitric oxide [14]), or directly linked to electric current flow through excitable membranes (e.g. potassium ions lost during action potential repolarization [31,44]). As a consequence, it is believed that the microvascular density in the CNS is associated with the rate of metabolic (and electrical) activity of different regions [39,56]. Our results in part support this notion, since differences in vasculature were found between synaptically active regions (e.g. neocortex) and fiber tracts (corpus callosum). We did, however, find significant differences between regions where no obvious metabolic differences are believed to exist, such as hippocampal CA1 and CA3. This finding adds to the numerous reports showing intrinsic differences between these neighboring hippocampal regions, differences ranging from vulnerability to ischemia [29], epileptic seizures [2] and anoxia / spreading depression [12,23] to differential expression of GABA responses [24,25], size of extracellular space [42], and properties of glial cells [11]. The fundamental principles of morphometric analysis have been successfully applied to determine parameters of interest concerning the capillary network of the brain [21,43,58]. By using confocal microscopy, Chopp et al. obtained a three-dimensional reconstruction of selected brain regions. This study, however, did not focus on hippocampal vascularization, and differences between hippocampal sectors were not determined [61].

4.3. Regional vascular responses to ischemia– reperfusion In addition to regional variations in vascular densities, we also found differences in vascular responsiveness to ischemia–reperfusion. Our results demonstrate that the CA1 region of the hippocampus undergoes postischemic BBB changes that may participate in the region’s vulnerability in vivo. Since selective CA1 neuronal damage can be easily reproduced in vitro where the influences of CBF are irrelevant (e.g. [4], our finding does not suggest a novel mechanism to explain the exquisite sensitivity to anoxia / hypoglycemia in CA1. The differential response of the CA1 region, with its higher propensity to BBB leakage and no reflow phenomena may be rather an etiologic factor in other pathological conditions, such as vascular dementia [51]. Interestingly, de la Torre and co-workers using a chronic model of reduced cerebrovascular perfusion reported the largest changes in metabolic activity in the CA1 subfield of the hippocampus [13,51]. This area demonstrated the largest decrease in cytochrome oxidase activity possibly linked to delayed neuronal cell death. These results indirectly support our hypothesis linking region-specific pathological vascular changes to neuronal damage and possibly cell death.

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4.4. Functional significance and relationship to parenchymal cell populations Our results are in agreement with previous reports demonstrating different densities of capillaries in the human hippocampus with a greater vascularization in CA3 vs. CA1 [40]. It was concluded that CA3 receives two major vascular afferents (dorsal arteries), while CA1 is vascularized by only one arterial branch (ventral artery) and that this may be the underlying anatomical source of the capillary density difference. Given that such a difference exists in both rodent and human hippocampi, what may be the ontogenetic reason for the observed vascular differences between CA1 and CA3 regions? Previous results from our laboratory have described regional differences in functional gap junction coupling between CA1 and CA3 astrocytes [11]. Differences in astrocytic ion channel expression were also found, suggesting that the differential vulnerability of these two regions to acute brain injury (such as head trauma) may be, in part, due to selective ablation of specific mechanisms of glial physiology [10]. It was also shown that functional coupling between CA3 hippocampal astroglia was much less prominent than among their CA1 counterpart. These findings, together with the results presented herein, suggest that perhaps the link between regional variations in capillary density in CA1 vs. CA3 may be related to region-

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specific variability of glial physiology. Thus, the more vascularized CA3 may require fewer functional intercellular conduits since the average distance between neurons and vessels is less (Fig. 7). According to this hypothesis, the large number of functional gap junctions in CA1 may conversely be a consequence of a lower capillary density. These intrinsic differences between seemingly identical regions may play a role in the selective vulnerability of CA1 to a variety of insults in vivo. This hypothesis is based on the fact that the mean distance between neurons and capillaries plays an important role in mechanisms of catabolic and metabolic transfer. In fact, energy substrates are carried from the lumen to neurons by transcellular astrocytic pathways [34,48], while clearance of [K 1 ] out involves a reverse process [27,28,55]. It is thus possible that the deficits associated with the existence of a gradient in vascular density may be overcome by increased availability of the transcellular, gap junction-mediated pathways. In conclusion, we demonstrate the feasibility of combined immunocytochemical–confocal microscopy–microangiographic methods as a tool to investigate regional interactions between cerebrovasculature and parenchymal cells. Application of these techniques to control and postischemic animals revealed differential distribution of blood vessels in cortical and white matter regions, as well as differences in vascular responses to ischemia–reperfusion.

Acknowledgements This work was supported by a AHA-9951512V to PMB and NIH-2RO1-06-HL51614 and NIH-RO1 NS38195 to DJ. The helpful suggestions of Dr. Ben Albensi are also gratefully acknowledged.

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Fig. 7. Hypothetic link between heterogeneous capillary density and glial cell physiology between CA1 and CA3 hippocampal subfields. The upper portion of the figure depicts the quantitative relationship between CA3 neurons, vasculature and glia-to-glia coupling. The data in support of this model were derived from the experiments shown herein and from D’Ambrosio et al. [11]. The CA3 region is characterized by a comparably large density of vascular profiles, and thus requires less functional coupling between astrocytes. In contrast, CA1 vasculature is comparably sparse, and well developed intracellular networks are established by neighboring glial cells. Full description in text.

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