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(Stroke. 1995;26:2120-2126.)
© 1995 American Heart Association, Inc.

Articles

Microvascular Basal Lamina Antigens Disappear During Cerebral Ischemia and Reperfusion

Presented in part at the 20th International Joint Conference on Stroke of the American Heart Association, Charleston, SC, February 9-11, 1995.

Gerhard F. Hamann, MD; Yasushi Okada, MD; Robert Fitridge, MD Gregory J. del Zoppo, MD

From the Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, Calif (G.F.H., Y.O., R.F., G.J. del Z.); the Department of Neurology, University of the Saarland, Homburg/Saar, Germany (G.F.H.); the Cerebrovascular Disease Clinic, National Kyushu Medical Center Hospital, Fukuoka, Japan (Y.O.); and the Department of Vascular Surgery, Queen Elizabeth Hospital, Woodville, South Australia, Australia (R.F.).

Correspondence to Gregory J. del Zoppo, MD, Department of Molecular and Experimental Medicine, SBR5, The Scripps Research Institute, 10666 N Torrey Pines Rd, La Jolla, CA 92037.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Background and Purpose Changes in vascular permeability are well-known and important consequences of cerebral ischemia. The development of edema and of petechial hemorrhage is connected to altered vascular integrity. A major part in microvascular integrity is played by the basal lamina.

Methods The fates of the basal lamina components laminin, fibronectin, and type IV collagen during middle cerebral artery occlusion (2 hours, n=3) and occlusion (3 hours) with reperfusion (1 hour, n=3; 4 hours, n=3; and 24 hours, n=4) were evaluated in the nonhuman primate. Specific monoclonal antibodies against these components were used. The number and size distribution of the microvessels in each specimen were determined by video-imaging microscopy, and the relative fluorescence intensity of laminin was semiquantified by laser confocal microscopy. Basal lamina antigen presentations were compared by double-stain immunofluorescence histochemistry.

Results The number of microvascular structures defined by the presence of each basal lamina antigen decreased significantly up to 24 hours of reperfusion (P<.0001). The ratio of laminin-containing vessels between the ischemic and nonischemic territories decreased significantly from control (0.98±0.04) to 2 hours of ischemia (0.83±0.09) and 1 hour (0.79±0.08), 4 hours (0.77±0.06), and 24 hours of reperfusion (0.55±0.07). The ratio of fibronectin (cellular) and of collagen (IV)-containing vessels decreased from 0.98±0.04 to 0.75±0.1 and from 1.02±0.03 to 0.57±0.1, respectively. Mean laminin fluorescence intensity decreased from 76.1±6.0 U (controls) to 52.0±14.6 U (24 hours of reperfusion; P<.001).

Conclusions The significant parallel losses of three basal lamina components, both in number and intensity, contribute to loss of microvascular integrity. These phenomena may be important for understanding cell extravasation and the hemorrhagic consequences of acute stroke.

Key Words: basal lamina • cerebral ischemia • collagen • fibronectin • laminin • microscopy, confocal

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The increase in permeability of the cerebral vessels to transvascular solute transport during and after an ischemic event is an experimentally^{1 2} and clinically^{3 4} important change. Hemorrhagic transformation of the ischemic area also requires major alterations in vascular permeability and vessel wall integrity.⁵ It is unclear whether these events may be augmented in number and severity by the therapeutic use of plasminogen activators.^{6 7 8} The early development of vasogenic edema and petechial hemorrhage during focal cerebral ischemia is consistent with the concept of at least two different levels of local microvascular-structural changes.

Vascular integrity is provided by the cerebral microvascular intima, which contains two anatomic and functional barriers to solute transport and the transmigration of circulating blood cells.^{9 10} The blood-brain barrier, the primary barrier, requires the integrity of tight inter–endothelial cell junctions of capillary and postcapillary venule endothelium for retention of fluid within the plasma space.¹¹

A potential second barrier to the transvascular passage of blood cells is the BL, on which endothelial cells rest.¹² The BL is a fabric containing type IV collagen, laminin, fibronectin, entactin, thrombospondin, various proteoglycans, and heparan sulfates.^{12 13 14 15 16 17} It is a specialized part of the ECM, which connects the endothelial cell compartment to the subjacent cell layers (astrocytes in the glia limitans) and the smooth muscle of the media.¹³ The BL matrix is constructed from collagen (IV) chains and a second polymer network derived from laminin. Entactin connects both complexes, and fibronectin connects the BL with the surrounding tissue and the ECM.¹² Fibronectin, a 450-kD glycoprotein that derives from plasma or cellular sources, is especially important for cell adhesion and the interaction with blood cells or blood components.^{16 18 19 20}

Little is known about the effect of focal cerebral ischemia and reperfusion on the integrity of BL or its individual components. The hypothesis tested here states that MCAO and subsequent reperfusion result in alteration in and eventual loss of the major structural antigens of BL (and ECM). To test this hypothesis, a careful quantitative immunohistochemical assessment of the presence of laminin, cellular fibronectin, and collagen (IV) was made of microvessels of less than 100 µm in the lenticulostriatal territory of primates undergoing 2- or 3-hour MCAO and subsequent reperfusion for various times.

	Materials and Methods

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The procedures used throughout this study were approved by the institutional animal research committee and were performed in accordance with standards published by the National Research Council (The Guide for the Care and Use of Laboratory Animals), the National Institutes of Health Policy on Human Care and Use of Laboratory Animals, and the US Department of Agriculture Animal Welfare Act. In accordance with these standards, every effort was made to ensure that the subjects were free of pain and/or discomfort. The principal investigator, a veterinarian, and the primate handling staff were present for all procedures.

Thirteen adolescent male baboons were used for the MCAO and MCAO/R studies, and three separate primates served as unoperated control animals. Material from these subjects has been used for related studies.^{2 21} Preparation of the awake baboon MCAO/R stroke model has been described in detail in previous studies of this group.^{2 21 22 23} Central to this preparation is the surgical placement of an inflatable balloon catheter assembly, with the balloon around the MCA at the takeoff point of the lenticulostriate arteries. After the surgical implantation procedure, a 7-day interval for observation and recovery was allowed in each animal. All subjects were neurologically normal and clinically free of infections or apparent inflammation.

Three animals underwent MCAO for 2 hours, while the remaining 10 subjects underwent 3-hour MCAO followed by 1 hour (n=3), 4 hours (n=3), or 24 hours of reperfusion (n=4). The experimental plan previously described was followed here exactly.² In short, compression of one MCA was achieved by inflation of the implanted 100-µL extrinsic balloon in the awake animal. Each experiment was terminated by left ventricular transcardiac perfusion with isosmotic perfusion fluid containing heparin (2000 IU/L), sodium nitroprusside (6.7 µmol/L), and bovine serum albumin (50 g/L) with subjects under thiopental sodium anesthesia.² Tissue blocks (1 cmx1 cmx0.2 to 0.5 cm) from symmetrically located sites in both basal ganglia were embedded in Tissue-Tek OCT compound (Miles Inc), frozen in 2-methylbutane/dry ice, and stored at -80°C in preparation for sectioning and the following immunohistological experiments.

Well-characterized murine anti-human MoAb were used, including laminin (IgG; clone LAM 89),^{24 25} cellular fibronectin (IgM; clone FN-3E2: against the 240-kD band of cellular fibronectin but lacking cross-reactivity to plasma fibronectin),²⁶ and type IV collagen (IgG; clone CO6-94: against the ₁ and/or ₂ chain of type IV collagen, which lacks cross-reactivity to collagen types I, II, III, V, VI, and VII)^{27 28} (Sigma Chemical Co). Immunohistochemical procedures were performed as previously described.² Consecutive 10-µm cryostat sections from matched regions of the MCAO/R (right) or normal nonischemic (left) basal ganglia were used. Sections were fixed with acetone for 10 minutes at -20°C and immersed in 100 mmol/L glycine in PBS (100 mmol/L Na₂HPO₄ and 140 mmol/L NaCl, adjusted at pH 7.4) for 10 minutes. After sections were rinsed in PBS wash solution, a 30-minute incubation with Blotto followed to reduce nonspecific binding.^{2 29} Each section was incubated with 100 µL of the primary antibody solution over 2 hours at 37°C. The working concentration of the primary antibody for laminin was 1:800, for collagen type IV 1:400, and for cellular fibronectin 1:50. After the sections were washed with PBS, biotinylated horse serum against mouse IgG (for laminin and collagen type IV) or IgM (cellular fibronectin) was incubated over 30 minutes at 37°C (Vector Laboratories). Chromogen 3-amino-9-ethylcarbazole (AEC KIT, Biomeda Corp) was used for the development of the peroxidase signal. All sections were counterstained with Mayer's hematoxylin (Biomeda Corp) for 45 seconds, blued in saturated sodium bicarbonate, and mounted in crystal mount. Negative and positive controls were routinely performed in each staining experiment as described previously.²

The absolute number and minimum transverse diameters of microvessels (<100 µm) visually identified by laminin, cellular fibronectin, or collagen (IV) antigen from the basal ganglia of control and from ischemia or ischemia/reperfusion specimens were determined using a computerized video-imaging system.^{2 23} A checkerboard array of 250 image fields (18.3 mm² total) in each block was scanned in a meandering pattern. Off-line analyses of the quantity and microvascular diameter distributions were performed using resident statistical programs. Using double-stain techniques with FITC anti-IgM (against fibronectin-IgM) or TRITC anti-IgG (against laminin-IgG or collagen-IgG), the vascular distribution of each antigen was independently derived. The counting of the double-stained structures was performed manually over 100 contiguous complete microscopic fields (magnification x400) in each block in each animal. The number of vessels not stained for one or the other epitope was recorded separately.

Fluorescence intensity measurements of microvessel-associated FITC anti-murine IgG against the murine anti-laminin MoAb were performed with laser confocal microscopy (LSM Invert 410, Karl Zeiss). After standardization of a stable sample of FITC beads (MultiSpeck, Molecular Probe Inc), all measurements were performed with the same pinhole size, brightness and contrast, zoom, and laser time (4 seconds). Each vessel was scanned in the z plane (10 scans per 1 µm), and a summed stereoscopic image was calculated. Also, from the background area in the neighborhood of each measured microvessel, a summed z-scan stereoscopic image was obtained. The intensity of the vessel was normalized to the background. The normalized intensity is expressed as mean±SD of each microvessel using a relative scale from 0 to 255 (highest intensity) arbitrary units (U). Ten microvessels each of 4.0 to 7.5 µm in diameter and 7.5 to 30 µm in diameter were measured in each specimen.

Neuronal damage was classified according to Eke et al.³⁰ For the comparison of neuronal to vessel change, a simultaneous manual counting of counterstained neuron silhouettes and peroxidase-stained microvascular structures in 100 complete microscopic fields at magnification of x400 was performed.

All values are expressed as mean±SD. Data for vessel numbers and laminin distribution were analyzed using the Kruskal-Wallis nonparametric ANOVA test. The Mann-Whitney U test was used for the comparison of the numbers and distributions of fibronectin (cellular) and collagen (IV)-stained vessels and for the laminin intensity values. The test for the equality of slopes in linear regression was applied to the relative differences in neuron number and microvessel number during MCAO/R. Significance was set at P<.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
As reported elsewhere, within 5 to 10 minutes after MCAO, each subject developed hemiparesis and/or variable degrees of visual-field or seventh cranial nerve impairment. The recorded neurological scores dropped from 100 to 41.1±14.7 after 1 hour of MCAO. Baseline blood cell counts were not affected by MCAO/R compared with those in control animals.

Laminin, collagen (IV), and cellular fibronectin antigens were readily detected in the microvasculature of the nonischemic cortical gray matter and basal ganglia in the MCA territory of all subjects. The number of microvascular structures in the ischemic lenticulostriatal territory defined by the presence of each BL component decreased significantly during MCAO/R (laminin, P<.0001; collagen [IV] and cellular fibronectin, P<.001) (Figs 1 and 2, bottom; Table 1). An evaluation of neuron silhouettes in identical image fields, to assess neuronal damage and the contribution of edema, demonstrated a reduction in the absolute numbers of neurons between 2-hour MCAO and 3-hour MCAO with 24-hour reperfusion. The reduction was most prominent by 3 hours of MCAO and 1 hour of reperfusion (Fig 2). The overall decrease in intact BL structures as demonstrated for laminin was significantly greater than that of intact neurons (P<.003). Absolute vessel counts based on intact BL structures and relative vessel numbers normalized for the reduction in neuron silhouette numbers were analyzed. Both data sets provided results of similar significance (Fig 2, bottom).

View larger version (0K): [in this window] [in a new window]   Figure 1. Photomicrographs show lenticulostriatal microvessels stained by the anti-laminin MoAb LAM 89 in the basal ganglia after 3 hours of MCAO and 24 hours of reperfusion (B) compared with no MCAO (A). In addition to the obvious reduction in the number of vessels per field, there is substantial destruction of microvessels in the ischemic region (original magnification x200; magnification bar=100 µm).

View larger version (32K): [in this window] [in a new window]   Figure 2. Top, Graph shows comparison of reduction in ratio of neuron silhouettes () and of microvascular structures defined by LAM 89 () in the ischemic with the matched nonischemic basal ganglia after MCAO (see text). The significance of the difference of each group compared with the control group is P<.0001 (Kruskal-Wallis analysis). Bottom, Bar graph shows reduction of microvessels stained for laminin (open bars), type IV collagen (hatched bars), and fibronectin (black bars) after 3 hours of MCAO and 24 hours of reperfusion compared with control subjects. *P<.0001 and **P<.001 relative to respective control. I/R indicates ischemia/reperfusion.

View this table: [in this window] [in a new window]   Table 1. Fate of Laminin After Ischemia/Reperfusion

The decrease of the laminin-containing microvascular structures was significant at each time point after MCAO and overall (P<.0001) (Fig 2, Table 1). The difference between the number of laminin structures in nonischemic basal ganglia of the controls and within the ischemic basal ganglia at 24-hour MCAO/R was especially remarkable. In addition to the obvious reduction in vessel number by 24 hours of reperfusion, there was the impression of severely injured vessels with disrupted walls and less intense stain associated with alterations in neuron silhouettes (Fig 1). According to the classification system of Eke et al,³⁰ the observed reduction in laminin structures occurred exclusively in the area of significant neuronal damage (data not shown).

Regarding the apparent alterations in peroxidase stain intensity (Fig 1), a separate semiquantitative study of fluorescence intensity of FITC IgG anti-LAM 89 was performed (Table 2, Fig 3). The mean fluorescence intensity in 240 different microvessels in the nonischemic BL of control subjects (n=3) was 76.1±5.5 U, which decreased to 52.0±4.6 in subjects undergoing 24-hour reperfusion (n=4) measured in 320 different microvessels (P<.001). Although the decrease in mean intensity in the 1-hour MCAO/R cohort was also significant (P<.01), the major intensity loss appeared after longer reperfusion.

View this table: [in this window] [in a new window]   Table 2. Relative Intensities for FITC Anti-Laminin by Laser Confocal Microscopy

View larger version (0K): [in this window] [in a new window]   Figure 3. Photomicrograph shows typical microvessel stained with the anti-laminin MoAb LAM 89 after 3 hours of MCAO and 24 hours of reperfusion compared with nonischemic vessel (left). Laser confocal microscopic image was summed from 1 µm serially acquired and used for the intensity measurement. Green is higher and blue is lower intensity. The control microvessel is 30 µm in diameter; the 24-hour MCAO/R vessel is 25 µm, maximum diameter (original magnification x400).

The MoAb CO6-94 against collagen (IV) detected nearly identical numbers of structures compared with LAM 89 (against laminin) in the ischemic and contralateral nonischemic zones at 24 hours of reperfusion (Table 3). As noted in Table 4, mean paired microvascular numbers after 24-hour reperfusion decreased from 1293±50 and 1272±69 in the control cohorts to 854±125 microvascular structures in the ischemic zone (P<.0001).

View this table: [in this window] [in a new window]   Table 3. Reduction in Laminin and Fibronectin Structures Relative to Type IV Collagen

View this table: [in this window] [in a new window]   Table 4. Fate of Type IV Collagen and Cellular Fibronectin After Ischemia/Reperfusion

Because of the ubiquitous presence of fibronectin in plasma, fibronectin could be readily identified among the extravasated proteins in the ischemic basal ganglia in all specimens after 3-hour MCAO (data not shown). Therefore, only fibronectin of cellular origin was examined. Fewer microvascular structures were identified with the MoAb FN-3E2 against cellular fibronectin in both control basal ganglia and the nonischemic basal ganglia than with laminin, as corroborated by a separate fluorescence colocalization study (Table 3; eg, ratio of fibronectin to laminin=0.75). However, the absolute numbers of vessels expressing cellular fibronectin in the ischemic zone was similar to that detected with LAM 89 against laminin. In the control group, the mean vessel numbers in the two nonischemic basal ganglia were 935±81 and 947±63, whereas the ischemic basal ganglia at 24-hour reperfusion displayed only 715±111 (P<.001) (Table 4 and Fig 2, bottom). Compared with the observed reduction in laminin and collagen (IV) antigen, the relative number of microvascular structures displaying fibronectin antigen of cellular origin increased by 24 hours of reperfusion (Table 3). This relative increase in cellular fibronectin is consistent with stimulation of fibronectin generation from cellular sources.

By 24 hours, reduction in the BL antigens was seen in all microvascular size classes. The relative distribution of microvascular structures as defined by their minimum diameter was not significantly different among those undergoing different periods of ischemia and reperfusion and the control tissues for each of the BL antigens. The reduction in microvascular number by 24 hours did not affect any diameter class predominantly (data not shown).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
This study demonstrates that significant coincident disappearance of antigens of three of the essential components of microvascular BL and ECM, laminin, collagen (IV), and cellular fibronectin occurs during experimental focal cerebral ischemia and reperfusion. As seen for laminin antigen, a monotonic decrease in the BL localization of these epitopes occurs during reperfusion. Similar reductions in microvascular silhouettes with duration of ischemia (and reperfusion) defined by antigens of the BL components type IV collagen and cellular fibronectin were observed in the basal ganglia. By 24 hours of reperfusion, significant interruptions of the microvascular structure with rudiments of former intact microvessels in the ischemic zone supported the gradual, but sustained, dissolution of the BL and digestion of the microvascular substructure. The coincident loss of BL/ECM antigens suggests degradation of the structural components of the BL. However, it cannot be excluded that a reduction of presentation or covering of the antigen under these conditions might have occurred. Direct measurement of matrix proteins may also be possible by Western blotting, which could confirm the pathophysiological cascade of BL degradation. However, the loss of vascular architectural information and the limitations imposed in obtaining valuable corpus striatal tissue for immunoblotting supported the immunohistochemical approach here.

Dissolution of the BL is supported most clearly by the findings of the laminin intensity measurements. Application of laser scan confocal microscopy to the problem of fluorescence intensity quantification is novel. Measurement of laminin-related intensity was highly reproducible (coefficient of variation=0.04). Serial 1-µm images revealed that the vessels, which were almost intact in their visible structure, had a consistent, significant reduced concentration of laminin antigen. Additionally, normal-appearing microvessels could be imaged with an intermediate decreased FITC signal compared with the disrupted vessels of 24-hour MCAO/R. In a number of microvessels, BL antigen dropout was focal. For laminin antigen during focal cerebral ischemia, the postischemic state represents a continuum from intact BL (controls) to partially digested forms (decreased laminin intensity) to complete loss of the substructure by 24 hours (laminin, collagen [IV], cellular fibronectin).

The duration of ischemia and reperfusion would seem to be an important correlate to loss in the vessel BL integrity. Whether a shorter period of MCAO might modulate the degree of laminin, cellular fibronectin, or collagen (IV) loss is not yet known. Certainly, the reduction in BL integrity and epitope expression was time dependent. This finding may be correlated to the clinical observation of higher numbers of hemorrhagic complications after long-term ischemia/reperfusion in contrast to early ischemia.⁶ A similar suggestion was offered by J.S. Meyer,³¹ who described necrosis and phagocytosis of selected cerebral microvessel walls after experimental brain infarction. Perivascular hemorrhage was noted at blood flow restoration, attributed to the degree of circulation and the duration of ischemia.³¹

Several mechanisms may be responsible for the dissolution of the microvascular substructures. Two different noncellular systems of the degradation of the ECM components and the BL are of practical importance: the plasmin system and metalloproteinases of endothelial origin.^{32 33} Whereas laminin and fibronectin are substrates of plasmin, collagen is not directly degraded by plasmin but is exposed to other proteinases.³⁴ For instance, plasmin plays a key role in activation of gelatinase, an enzyme that cleaves collagen as a specific substrate.³⁵ Thus, the interaction of plasmin and gelatinase may effect degradation of the ECM and BL. This may have been responsible for the increase in vascular permeability and extravasation of serum components observed in a rabbit mesenteric model after administration of the plasminogen activator rTPA.³⁶ The same effect was observed in an in vitro model using confluent endothelial cell layers.³⁷ Endothelial cell–derived neutral metalloproteinases degrade collagen types I through V, fibronectin, laminin, and proteoglycans.^{38 39}

With activation and adherence to postcapillary endothelium,^{40 41 42} polymorphonuclear leukocytes transmigrate into the perivascular parenchyma. Garcia et al⁴³ have demonstrated that granulocyte invasion is maximal by 24 hours after MCAO (rodent) but is earlier when reperfusion is superimposed. This time course accords with that of the BL antigen alterations. Both gelatinase and elastase are secreted on granulocyte activation.

Increased vascular permeability after polymorphonuclear leukocyte adhesion has been reported during experimental inflammation.^{44 45} During leukocyte activation and endothelial cell adhesion, primary granule release allows secretion of myeloperoxidase, elastase,^{46 47} collagenase,⁴⁸ and other enzymes. These may contribute to degradation of fibronectin and ECM.⁴⁹ In response, cellular production of fibronectin is stimulated by polymorphonuclear leukocytes.⁵⁰ In addition to fibronectin, laminin has chemotactic properties for leukocytes and is a stimulus for BL attachment and penetration.⁵¹ Fibronectin acts as a chemotactic stimulus for monocytes and stimulates cluster formation of lymphocytes.^{52 53} Interestingly, fibronectin fragments enhance the phagocytic activity of macrophages, which may be important in the digestion of damaged cellular and vascular structures.

The reaction of the vasculature to degrading stimuli of the BL is of great interest. Because laminin may contribute to the resistance of endothelium to mechanical stress, it is possible that the observed reduced laminin content after ischemia/reperfusion may be associated with reduced mechanical resistance.⁵⁴ Also, endothelial cell injury leads to an increase in the production of the components of the ECM.⁵⁵ This reaction can be explained as a repair mechanism to sustain the vascular integrity.⁵⁵ In the hemolytic-uremic syndrome, for instance, there is a close relationship between endothelial cell leakage and fibronectin degradation caused by activated neutrophils.⁵⁶ Experimentally, there is a close relationship between granulocyte-mediated endothelial damage and fibronectin degradation.⁵⁷ Endotoxin-induced endothelial cell injury leads to a reactive increase in fibronectin production to provide reendothelialization.⁵⁸

During experimental focal cerebral ischemia, the alterations in fibronectin, type IV collagen, and laminin antigen, which reflect important changes in microvascular BL, may involve the plasmin system and leukocyte activation. If so, conversion of plasminogen to plasmin locally, even by therapeutic activation, and the microvascular endothelial cell adhesion of leukocytes with granule release and subsequent stimulation of proteolytic activity by plasmin, metalloproteinases, elastase, and various other proteases may contribute to BL dissolution. The resultant degradation of ECM and BL components increases microvascular permeability and allows the extravasation of fluid (edema) and fibrin² and erythrocytes as hemorrhage.

The results of this study connect the early findings of postischemic microvascular damage and perivascular hemorrhage by Meyer³¹ to specific molecular components of the microvessel walls and their fate. While the early permeability increase during cerebral ischemia/reperfusion with extravasation of plasma components is probably due to endothelial dysfunction,² loss of BL matrix integrity offers an explanation for erythrocyte leakage and the development of hemorrhagic complications in stroke.⁶

	Selected Abbreviations and Acronyms

 

BL = basal lamina ECM = extracellular matrix MCAO = middle cerebral artery occlusion MCAO/R = middle cerebral artery occlusion/reperfusion MoAb = monoclonal antibody

	Acknowledgments

 
This work was performed with the support of grant NS 26945 of the National Institute of Neurological Disorders and Stroke (Dr del Zoppo) and grant Ha 2078/2-1 from the DFG (Dr Hamann). We wish to thank Pearl Akamine for her technical and laboratory expertise and Zaverio Ruggeri, MD, and Rolf Habermann, PhD, for their assistance with the laser confocal microscopy studies. Dr Hamann is the 1995 recipient of the Robert G. Siebert Young Investigator Award of the American Heart Association.

	Footnotes

 
This is publication No. MEM-9207 from The Scripps Research Institute, La Jolla, Calif.

Received March 3, 1995; revision received June 16, 1995; accepted June 20, 1995.

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