New Patterns of Intracortical Projections after Focal Cortical Stroke

Neurobiology of Disease 8, 910 –922 (2001) doi:10.1006/nbdi.2001.0425, available online at http://www.idealibrary.com on

New Patterns of Intracortical Projections after Focal Cortical Stroke S. Thomas Carmichael,* ,1 Ling Wei,* ,† Carl M. Rovainen, † and Thomas A. Woolsey* ,† *Department of Neurology and Neurological Surgery and †Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110 Received March 1, 2001; revised May 29, 2001; accepted for publication June 5, 2001

Cortical strokes alter functional maps but associated changes in connections have not been documented. The neuroanatomical tracer biotinylated dextran amine (BDA) was injected into cortex bordering infarcts 3 weeks after focal strokes in rat whisker barrel (somatosensory) cortex. The mirror locus in the opposite hemisphere was injected as a control. After 1 week of survival, brains were processed for cytochrome oxidase (CO)-, Nissl-, and BDA-labeled neurons. Cortex bordering the infarct (peri-infarct cortex) had abnormal CO and Nissl structure. BDA-labeled neurons were plotted and projections were analyzed quantitatively. Animals with small strokes had intracortical projections, arising from peri-infarct cortex, not seen in normal hemispheres: the overall orientation was statistically significantly different from and rotated 157° relative to the controls. Compared to the controls, significantly fewer cells were labeled in the thalamus. Thus, after focal cortical stroke, the peri-infarct cortex is structurally abnormal, loses thalamic connections, and develops new horizontal cortical connections by axonal sprouting. © 2001 Academic Press Key Words: neuronal plasticity; functional reorganization; cerebral ischemia; focal; repair; axonal sprouting.

INTRODUCTION

dritic remodeling (Jones & Schallert, 1992) and induce stem cells to differentiate into projection neurons that establish long-distance cortical connections (Magavi et al., 2000). This demonstrates the capacity of the adult brain to establish new neuronal circuits but these results are from cortical injury models other than stroke. Stroke has been shown to produce changes in indirect markers of new neuronal connections, such as GAP-43 and synaptophysin (Stroemer et al., 1995), but no studies have directly demonstrated the formation of new patterns of neuronal connections. The present study was undertaken to determine the extent and distribution of axonal sprouting after focal cortical stroke. Small strokes were targeted to a portion of the somatosensory cortex (SI) containing the cortical representation of the facial whiskers, known as the barrel field (see Figs. 1 and 2 for details; Wei et al., 1995, 1998). This cortical region contains a regular array of cortical modules, the barrels, that have a 1:1 correspondence with the facial whiskers (Woolsey & Van der Loos, 1970; Chapin & Lin, 1990). The barrel

Stroke is a principal cause of disability in the United States. There is considerable understanding of the mechanisms of acute cell death but little is known of the basis of brain reorganization and repair after stroke. Recent studies have shown that the adult brain is capable of a striking degree of remodeling after focal cortical lesions. An understanding of the mechanisms of brain reorganization after stroke may allow for the development of novel therapies targeted toward neurorehabilitation of the disabled stroke patient. Cortical lesions produce physiological plasticity of cortical sensory and motor maps, including shifted limb representations to new loci around a lesion site (Nudo & Milliken, 1996; Nudo et al., 1996; Traversa et al., 1997). Focal cortical lesions provoke distant den1 To whom correspondence should be addressed at Department of Neurology, UCLA School of Medicine, 710 Westwood Plaza, Los Angeles, CA 90095. E-mail: [email protected].

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field has been studied extensively and its connections and structure are well defined and stereotypic (see, for example, Jones & Diamond, 1995). The patterns of cortical connections found after focal ischemia can thus be easily compared with those in control studies and in the literature. We utilized quantitative methods to statistically compare the distribution of projections within cortex subjected to ischemia with those of control cortex. This evaluation obviates some of limitations of the traditional case by case analysis of cortical projections in neuroanatomical studies.

METHODS Experimental protocol. Animal housing, maintenance, and preparation met or exceeded federal and institutional animal care guidelines. Focal strokes in the distribution of the large whisker barrels (Fig. 1) in adult rats were induced by ligating selected distal branches of the middle cerebral artery under direct vision (Wei et al., 1995, 1998). Because this model is related to a variable local arterial pattern, stroke size and location are variable (see Discussion). Three weeks after arterial occlusion, the cortex was reexposed, the infarct was identified, and a small injection of an axonal tracer was placed into the barrel field near the stroke. An identical injection was targeted by stereotactic coordinates to the mirror site in the other hemisphere. One week later the animals were sacrificed, and the brains were fixed and processed for visualization of the axonal tracer, cytochrome oxidase (CO) histochemistry, and Nissl staining. Cortical structure, CO staining patterns, and location of the neurons projecting to the injection site were then compared between the peri-infarct cortex on the side of the stroke (experimental) and the opposite (control) hemisphere. No differences were found in the connections or structure between the control hemisphere opposite the stroke and reports of similar studies of normal rats (Hoefflinger et al., 1995; Miller et al., 2001b). Experimental stroke. Focal strokes in the barrel field were made in adult Wistar rats of both sexes (n ⫽ 20; 250 –270 g; Wei et al., 1995, 1998). The rats were anesthetized (87 mg/kg ketamine and 13.5 mg/kg xylazine ip), placed on a servo controlled heating pad with rectal temperature probe and fixed in a specially designed head holder. Core temperature was maintained between 36 and 37°C. The parietal cortex was carefully exposed and the location of the barrel cortex was estimated from the pattern of the surface vessels under direct vision (Cox et al., 1993). In some cases the

911 whisker cortex was functionally identified by an intrinsic optical signal with whisker stimulation (Dowling et al., 1996). Typically, three to five branches of the middle cerebral artery supply the posteromedial barrel field (PMBSF) (Wei et al., 1995) and these were ligated with 10-0 suture through the intact dura. Video images of the brain surface blood vessel patterns before and after placing the ligatures were recorded and saved for future reference. The published method for this model (Wei et al., 1995) was modified by temporally clamping both common carotid arteries for 1 h to consistently produce infarcts. The wounds were closed with 6-0 sutures and the animals were returned to their cages. Axonal tracer injection. Twenty-one days later, the animals were anesthetized again, the opposite parietal cortex was exposed, and the hemisphere with the stroke was reexposed. The infarct was identified under a stereomicroscope in reference to the saved video images and the stereotaxic coordinates of its margins were determined relative to the bregma. Based on previous results (Wei et al., 1995) barrel D3 was determined to routinely be in the cortex that borders, but is spared by, the infarct and this barrel was targeted for injection (stereotaxic coordinates from bregma of ⫺2.2 mm anterior and 5.25 mm lateral, Chapin & Lin, 1990). Because of variability in the location of the infarct, if the region of D3 was found to be within the infarct, the location of the injection was moved just anterior and/or lateral to the infarct margin to label cortex bordering the infarct. A 10% solution of the axonal tracer biotinylated dextran amine (BDA, Molecular Probes, Eugene, OR) was injected through a glass micropipette (internal diameter, 20 ␮m) by pressure into peri-infarct cortex. Small injections were made at several depths between 200 and1200 ␮m deep to the cortical surface along a single radial penetration. The final volume of all injections was between 50 and 80 nL. Similar injections were then made at the mirror stereotactic locus in the opposite hemisphere. The scalp was closed and the animal was returned to its cage. Tissue processing. One week after BDA injection (4 weeks after the focal stroke), the animals were deeply anesthetized and perfused through the heart with phosphate-buffered saline and then 4% paraformaldehyde followed by 4% paraformaldehyde in 10% sucrose. The brains were removed and the cortex was dissected free from the thalamus and flattened between two glass slides (Welker, 1976). After overnight postfixation in 4% paraformaldehyde and 30% sucrose, the brains were frozen and sectioned serially on Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.

912 a sliding microtome. The flattened cortex was cut tangentially at 50 ␮m and the thalamus was cut coronally at 75 ␮m. Sections were immediately rinsed and processed for CO histochemistry (Wong-Riley, 1979). BDA was visualized in the same sections (Veenman et al., 1992), with a Vectastain Elite kit (Vector Labs, Burlingame, CA) and DAB as the chromogen; the DAB reaction product was enhanced with CoCl 2 and nickel ammonium sulfate. Every section through the thalamus and cortex was stained and used for quantitative analysis. The sections were mounted on subbed slides, dried, cleared in xylenes, and coverslipped. After the analysis of CO staining and plotting the distribution of retrogradely labeled cells, coverslips were removed; the sections were counterstained with a Nissl stain (cresyl violet) and coverslipped again. Analysis. Seven animals had a small stroke in the PMBSF and a BDA tracer injection into peri-lesion cortex. Two animals in this group also had a tracer injection centered in a barrel in the contralateral hemisphere. In five animals the stroke destroyed the entire barrel field in the experimental hemispheres, but the contralateral, control cortices had tracer injections centered on a barrel which were used in the control set. Thus a total of seven experimental hemispheres with partial barrel field strokes and BDA tracer injections and seven control hemispheres with PMBSF BDA injections were analyzed. In one additional animal, the stroke destroyed only a portion of the PMBSF, but the BDA tracer injection was placed outside of somatosensory cortex (Fig. 1). The infarcts were circumscribed by an organized glial border. The distributions of retrogradely labeled cells and the patterns of CO and Nissl staining were compared between control and experimental hemispheres. The pattern of cortical connections between animals was compared first on a case by case basis. Next, the location of all cells within the tangential plane of the cortex between animals was compared statistically. Each section was plotted using a microscope digitizing system (Minnesota Datametrics, Inc., St. Paul, MN) to map the location of the infarct and labeled cell bodies in relation to the tracer injection site and the barrels. Plots were constructed for control and experimental hemispheres. In this way, each labeled neuron was converted to a point with x and y coordinates in the tangential plane of the cortex, with the tracer injection as the center for reference. From the data direct and statistical comparisons were made between patterns of cortical connections in the barrel fields of control and ischemic hemispheres. Cell positions were plotted as polar histograms around the injection site. In these plots the number Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.

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of cells is represented by the length of a vector in each of twelve 30° arcs. Polar plots were made for each cortical layer of each experimental and control hemisphere. To directly compare the tangential distribution of cells in the infarcted versus control hemispheres, mean vectors were computed for supragranular layers, layer IV, and infragranular layers. The polar plots from different layers were aligned to each other using section outlines and penetrating vessels as fiducial marks and were summed together to produce a mean orientation vector for each hemisphere. For comparisons between different animals, the plots were aligned using the barrel pattern as a template (Woolsey & Van der Loos, 1970; Cox et al., 1993). Mean vector orientations for the distribution of cells in all hemispheres with an infarction and all control hemispheres were computed. Confidence intervals for these mean vectors were determined by circular statistics, with the total number of observations (the sample size or “n”) corresponding to the number of labeled cells in a cortical layer or hemisphere (Batschelet, 1981; Miller et al., 2001). The statistical significance of the length and direction of the vectors for each cortical layer, for each hemisphere, and for all hemispheres with strokes compared with these same vectors from the control hemispheres was tested (Batschelet, 1981). Cells labeled retrogradely in the thalamus after cortical BDA injections were counted with the same digitized counting system. The labeled cells in the entire ventroposterior nucleus of the thalamus (VPM; Jones & Diamond, 1995) that project to the barrel field were counted for each case. The mean numbers of retrogradely labeled cells in control and ischemic hemispheres were evaluated with a nonpaired T test (Statview 5.0.1, SAS Institute, Cary, NC).

RESULTS Location of Focal Strokes within Somatosensory Cortex The structure and connections of the largest barrels, termed the PMBSF (Fig. 1), were analyzed in this study. The PMBSF contains five rows of barrels in layer IV which are the cortical representation of the somatosensory input from five rows of larger facial whiskers (Fig. 1; Woolsey & Van der Loos, 1970; Welker, 1976; Chapin & Lin, 1990). Each barrel stains densely for CO (Land & Simons, 1985a) and consists of

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FIG. 1. CO-stained tangential sections through layer IV of rat somatosensory cortex after small BDA injections. (a) CO staining defines the somatic representation (hindlimb, trunk, lower lip, etc.) in SI. The posteromedial barrel subfield (PMBSF) is the cortical representation of the large facial whiskers within somatosensory cortex. Other CO patches relate to the lips and paws. The BDA injection is centered in barrel C4 and extends into barrel D3 (see legend to Fig. 2 for nomenclature.) (b) A BDA injection into the contralateral hemisphere of the same animal. The infarct was in the barrel field. CO staining is absent immediately adjacent to the infarct. The BDA injection is located at the edge of the infarct in the position of the C6 barrel as reconstructed from the context of remaining CO patches. The BDA injection is approximately one-third the diameter of the dense label from local projections (see Fig. 4b). This and all subsequent figures are oriented as if from the right hemisphere.

a group of neurons which coincides with terminal arbors of afferents from the VPM of the thalamus (Chapin & Lin, 1990). In the seven animals with strokes, the strokes uniformly involved the posterior and lateral aspects of the PMBSF, infarcting mainly rows A and B while sparing rows E and D and lateral potions of row C (Figs. 1 and 2). In some cases the infarct extended into visual cortex. In one case there was a second small infarct in the mouth area of the

somatosensory cortex anterior and medial to the larger stroke (case e, Fig. 2). Topical Pathology of Infarcted Somatosensory Cortex Twenty-eight days after the strokes three structurally different cortical zones were related to infarcts in the barrel field. The necrotic core was devoid of neuCopyright © 2001 by Academic Press All rights of reproduction in any form reserved.

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rons; a dense rim of nonneuronal cells bordered this region. Adjacent cortex, not part of the infarct core, had altered metabolic and cytological features. This region extended 2 to 4 mm away from the infarct border (Figs. 1– 4). CO staining intensity in the barrels within this zone was markedly diminished or completely absent, lacked the “texture” of normal barrels, and showed less contrast with the septa that separate the barrels (Figs. 3 and 4). Nissl staining revealed reduced barrel definition within the same region. This region is hereafter referred to as “peri-infarct” cortex (Fig. 3c). The third zone, surrounding peri-infarct cortex, had normal barrel CO patterns and normal barrel cytoarchitecture (Figs. 2 and 3). Peri-infarct cortex did not border the infarct in a simple radial fashion, as there were regions of cortex with normal metabolic and cytological barrel structure directly abutting the infarct (Fig. 2). The distribution of structurally abnormal peri-infarct cortex may reflect patterns of collateral flow at the time the stroke is produced (Wei et al., 1998; see Discussion). Intracortical Connections of the Peri-infarct Cortex Distributions of cells retrogradely labeled after BDA injections into peri-infarct cortex were compared to similar injections in the opposite, control, barrel cortex (Figs. 2 and 4). With the volume injected, BDA injections produced a small and dense zone of extracellular tracer approximately 500 to 700 ␮m in diameter that was the actual area of uptake; this is defined as the injection site (Fig. 4b). Adjacent areas were densely labeled by filled axons, cell bodies, and dendrites, but were not sites of active neuronal uptake and transport (Miller et al., 2001a). In the ischemic hemispheres five of seven BDA injections were located in the periinfarct cortex. (Figs. 2 and 4). Tracer injections into barrel columns in control hemispheres labeled the neurons in neighboring barrel columns along the barrel row or in adjacent barrel columns in neighboring rows (Figs. 5a and 5c). Most of these projections arose from cells in barrel columns that were lateral and anterior to the injected barrel. The pattern of the labeled cells in the plane of the FIG. 2. Location of tracer injections in the barrel cortex and structural changes in the barrel cortex in all cases used in this study. The left column illustrates the location of tracer injections (black ovals) within the barrel cortex in seven control, noninfarcted hemispheres. Barrel nomenclature is indicated in the second control case. In the right column, barrels destroyed by the infarct are dashed and barrels adjacent to the infarct with abnormal CO or Nissl structure are

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light gray. Numbers or letters above each case identify the specific brains for reference in the text and other figures. Focal infarcts in the barrel cortex alter the structure of cortex within 2– 4 mm of the edge of the infarct, the “peri-infarct” cortex.

Anatomical Plasticity after Stroke

FIG. 3. Metabolic and cellular abnormalities in peri-infarct cortex. Tangential sections through barrel somatosensory cortex stained for CO (a, b) and Nissl (c). (a) CO staining reveals a decrement and then loss of barrel staining in increasing proximity to the infarct (arrows) (same animal as in Fig. 1). Sections in (b) and (c) are adjacent from the same brain (case c, Fig. 2). Barrels have diminished CO staining and less distinct septa near the infarct (arrow). In the Nissl stain, the barrels near the infarct have a more homogenous cellular structure (arrow) and are less sharply separated. Asterisks in (b) and (c) denote the same barrel in both panels. Bar in (c) applies also to (a) and (b).

cortex resulted in mean vectors for all cortical layers that were directed along the barrel row in the anterior and lateral directions (Fig. 5c). This pattern of connec-

915 tions was indistinguishable from that previously reported (Koraleck et al., 1990; Bernardo et at., 1990; Hoefflinger et al., 1995). Tracer injections into barrels in the peri-infarct cortex labeled neurons in the anterior and lateral barrel field, as in controls, but also in posterior and medial portions of the PMBSF (Fig. 5b and 5e). This projection pattern from posterior and medial barrels was not seen in control hemispheres, nor has it been described in published connections of the barrel cortex (Koraleck et al., 1990; Bernardo et at., 1990; Hoefflinger et al., 1995). The cells that give rise to this new projection pattern are located in cortex adjacent to the infarct (Fig. 5d). The cells in the infarcted hemisphere were well labeled, with BDA filling soma, dendrites, and axons (see inset Fig. 4d), just as in control animals. Retrogradely labeled cells were found right up to the border of the infarct, indicating that neurons in this region were viable and capable of transporting the tracer (Fig. 4d). Neurons in the motor cortex and the second somatosensory area were also labeled retrogradely following BDA injections into the peri-infarct cortex. The distribution of these labeled cells was not distinguishable from that seen on the control side. The robustness of this new poststroke cortical projection pattern was tested statistically from digitized plots of cell location and number. From polar plots of labeled cells within the tangential plane of the cortex, the mean vectors of labeled cells were significantly different between the experimental (Fig. 5f) and control hemispheres (Fig. 5e). The mean vector for the distribution of labeled cells in all the infarcted hemispheres was 73.3 ⫾ 25°, compared with a mean vector for all the control hemispheres of 276 ⫾ 15°. The difference of 157.3° between the mean orientation of labeled cells in control vs infarcted barrel fields is significant (alpha ⬍ 0.01; Fig. 5). Two injections in the experimental group fell outside of the PMBSF, but were still within the barrel field (cases f and g, Fig. 2). The orientation of the vectors from these two cases was not different from the other injections placed within the posteroemedial barrel field (Fig. 5f). When the data from these two cases are excluded from the overall analysis, the mean vector for the experimental group becomes 93 ⫾ 31°. This mean vector for ischemic hemispheres with cases f and g excluded is significantly different from the distribution of cells in control cases (177°; alpha ⬍ 0.01). The difference in vector angle between infarcted and noninfarcted somatosensory cortex indicates an increase in cells projecting from the posterior and medial portions of the barrel field, adjacent to the Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.

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FIG. 4. Neuroanatomical tracer injections into peri-infarct cortex with retrogradely labeled cells. Sections are taken from four different cases showing BDA injections into barrels within the peri-infarct cortex. (a) BDA injection into small barrels adjacent to row A (case g in Fig. 2). CO staining is diminished in barrels adjacent to the infarct (barrels E1, E2, D2, etc.). In this and subsequent figures, the white line demarcates the edge of the injection. (b) BDA injection centered on barrel D3 (case b in Fig. 2). Local connections are heavily labeled in barrels E1–E4. Inset in (b) is an enlarged view of region within the white square after Nissl counterstain. The border of the BDA injection can be defined as the area with the dense extracellular label, as indicated by the white arrow. (c) BDA injection into barrel E4 (case a in Fig. 2). Heavily labeled connections are seen in anterior and lateral portions of rows D and E. (d) BDA injection into peri-infarct cortex (case d in Fig. 2). BDA fills axons, somata, and proximal dendrites. The arrows point to several labeled neurons at the edge of the infarct. Inset in (d) is an enlarged view of region bounded by dashed line in (d) and shows good labeling of neuronal processes.

stroke in peri-infarct cortex (Fig. 5). In addition to the summed vector for all cortical layers, the mean vectors for each cortical layer between the infarcted and control hemispheres were also significantly different (alpha ⬍ 0.01). In each case the position of the mean vector indicated that a different population of cells was projecting from the posterior and medial portion of the barrel field after focal ischemia, in the periinfarct cortex, compared to the controls (compare Figs. 5e and 5f). Thalamic Connections The VPM, the nucleus projecting to the barrels in layer IV from the thalamus, showed classical retrograde degeneration with gliosis and neuronal atrophy Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.

after focal cortical strokes (e.g., Woolsey, 1978). There was an overall reduction in the size of the nucleus and an increase in nonneuronal cells throughout the nucleus. The cortical strokes produced a substantial loss in the projection from the thalamus to the barrel cortex (Fig. 6). There were fewer BDA-labeled somata in VPM in ischemic hemispheres. The mean number of retrogradely labeled cells in the VPM in ischemic hemispheres was 75.3 ⫾ 22.6 and the mean number of labeled cells in the VPM of control hemispheres was 250.9 ⫾ 85.6. This difference is significant (P ⬍ 0.0002). Barrel field strokes also produced a loss in the projection from the cortex to the thalamus, with fewer labeled axons in VPM in ischemic vs control hemispheres (Fig. 6). In cases in which BDA injections were placed within similar barrels in control and ischemic

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hemispheres of the same animal, very few labeled cells and axons were present at the same anatomical level within the thalamus (Fig. 6). This represents a striking loss of reciprocal thalamic connections with the cortex near focal infarcts.

DISCUSSION The principal findings of this study are that periinfarct cortex adjacent to a focal cortical stroke: (1) is structurally abnormal; (2) loses thalamic connections; and (3) forms new patterns of horizontal cortical connections. The formation of new connections after stroke is statistically significant. The predominant orientation of the new projection pattern after stroke is shifted nearly 160° compared with the orientation of normal horizontal projections in the barrel field. This axonal sprouting arises from neurons within the periinfarct cortex. Methodological Issues The extent of the infarcts varied between animals. The cortical strokes were produced by tying off small arterial branches of the middle cerebral artery that supply the barrel field. The advantage of this model is that ischemic tissue destruction occurs in a verifiable portion of anatomically and functionally defined cortex; however, stroke location and size depend on a

FIG. 5. Summary of intracortical projections in control and infarcted hemispheres. (a) Dots indicate BDA-labeled cell bodies. The injection site is encircled with a white line (case 2 in Fig. 2). On the right, the numbers of cells and their location in relation to the injection site were used to compute equivalent vectors in 30° seg-

ments using the coordinate system shown in the inset. The lengths of the lines indicate the number of cells in each 30° bin. The coordinate system is shown in the inset. The gray line is the orientation of the mean vector for all of the cells for this brain. (b) Retrogradely labeled cells and polar histogram from an injection into D5 (case b in fig. 2). Same conventions as in (a). (c) Retrogradely labeled cells and polar histogram from case 1 in fig. 2. (d) Retrogradely labeled cells and polar histogram from case a in Fig. 2. (e) Mean vectors for seven control, noninfarcted hemispheres. The length of each vector represents the weighted average of the overall location of labeled cells in each brain. For example, if all of the cells in one brain were located in the same 30° segment than the vector length would be 1.0; if the cells were equally distributed in all 30° segments the mean vector would have a length of 0. The numbers next to each vector designate the brain from which the data was derived, as listed in Fig. 2. The gray line is the orientation of mean vector for the distribution of a cells in the control hemispheres. (f) Mean vectors for seven infarcted hemispheres. The letters next to each vector designate the brain from which the data was derived, as shown in Fig. 2.Other conventions as in (e). Note that the orientation of the mean vector for the position of labeled cells in the infarcted hemispheres is 157.3° counterclockwise to that of the control hemispheres.

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FIG. 6. Changes in projections between the cortex and thalamus after cortical stroke. Two cases to show robust anterograde and retrograde BDA labeling in the ventroposterior medial nucleus (VPM) of the thalamus from barrel cortex in the control hemisphere (left) that is lacking after an infarct in the opposite hemisphere (right). (a) and (b) are from the same animal (hemispheres from cases a and 4 in Fig. 2) with a BDA injection into the equivalent regions of the barrel cortex. (a) Field from the control hemisphere (the sections in the insets were subsequently stained for Nissl) and (b) from the hemisphere with the infarct. (c and d) From a section from a different animal (hemispheres from cases g and 2 in Fig. 2). The white squares in the insets show the areas that are enlarged in the full figures.

local arterial distribution that is unique for each hemisphere. This is in contrast to the stroke pattern after experimental occlusion of the middle cerebral artery (Chen et al., 1986; McAuley, 1995). But unlike the large strokes produced by middle cerebral artery occlusion, the extent of each stroke in the present model was precisely located in somatosensory cortex and this allowed strokes that involved a large region of the somatosensory cortex to be discarded. Moreover, the remaining barrels outside of the infarct define an essentially invariant context to determine the detailed structural and functional changes after stroke and to align data from different animals for analysis of intracortical projections. The loss of CO activity and the increase in nonneuronal cells within the peri-infarct cortex suggest that the axonal sprouting occurs within a region that was partially ischemic, but survived (see Wei et al., 2001). Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.

The variability in infarct location may have affected these results. It is possible that changes in the pattern of cortical connections did not reflect new patterns of cortical connections but simply of altered cortical connections as a result of tissue loss. However, instead of a loss of connections as this would predict, there were substantially more horizontally connecting neurons in this peri-infarct region than in the controls. This indicates that, rather than causing differential loss of connections related to tissue loss, ischemia has induced new connections to form by axonal sprouting. Because BDA was also injected into the contralateral barrel field, it is possible that labeled neurons around the stroke were labeled from the opposite barrel field via the corpus callosum. There were no detectable alterations in the pattern of corticocortical connections in the control hemispheres, whether or not there were tracer injections in the contralateral barrel field. The

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organization of intracortical connections in these control hemispheres was indistinguishable from patterns found by us and others after unilateral injections of axonal tracers in normal rats and mice (Koraleck et al., 1990; Bernardo et at., 1990; Hoefflinger et al., 1995). While the BDA injections involved septa between barrels which receive callosal connections, these callosal connections are especially sparse in the PMBSF (Olavarria et al., 1984; Koraleck et al., 1990; AroniadouAnderjaska & Keller, 1996; Hyama & Ogama, 1997), which is the cortex labeled with BDA in this study. Peri-infarct Cortex In this study, peri-infarct cortex was defined as that region bordering the infarct with abnormal metabolic and cytological features. This may encompass a portion of the ischemic penumbra. However, the penumbra has been defined functionally (Astrup et al., 1981) and subsequently extended to blood flow studies (Furlan et al., 1996; Obrenovitch, 1995). Since these techniques were not used here, the structurally abnormal peri-infarct cortex cannot be directly related to the penumbra. However, the size and location of the periinfarct cortex may reflect a degree of initial ischemia and reperfusion by collateral flow. Collateral flow has been directly visualized in this stroke model using in vivo, intraarterial fluorescein as a blood flow marker. Blood flow in this stroke model demonstrates initially reduced perfusion and delayed collateral flow within a portion of the region identified as the peri-infarct cortex in the present study (Wei et al., 1998). In addition to axonal sprouting in this region, angiogenesis, a second process of structural tissue remodeling, occurs within this region of partial damage and partial collateral flow and reperfusion (Wei et al., 2001). Compared with the mechanisms of cell death in the infarct core, reperfusion induces a unique set of destructive processes, particularly free radical damage (Lipton, 1999). The overlap of zones of reperfusion, partial damage, and axonal sprouting after stroke suggests that processes involved in reperfusion injury may be important in the causal mechanisms of axonal sprouting. Dense CO staining in a barrel is related to the concentration and activity of CO in mitochondria, principally in dendrites in the barrel cortex that receive thalamic afferents (Wong-Riley, 1989). Thalamic lesions reduce CO activity in the barrels (Fuji et al., 1993). We show that the thalamic projection was substantially lost in the peri-infarct cortex, likely by direct ischemic damage either to the thalamocortical projec-

919 tions in the cortex or to immediately subjacent white matter. Obviously cortical neurons that are targets of the thalamic neurons are lost also. However, reduced whisker use after stroke could contribute to the observed metabolic changes in peri-infarct cortex, as disuse lowers CO activity in the barrel field (Wong-Riley & Welt, 1980; Land & Simmons, 1985b). There were no detected changes in whisker use by these animals, but quantitation of behavior is necessary to rule this out (Carvell & Simons, 1990; Bermejo et al., 1998). Altogether, the data in this study show that focal cortical stroke has persistent metabolic and cellular effects on neighboring cortical tissue and these may play a role in the axonal sprouting occurring within this region. Comparison with Previous Findings of Cortical Plasticity This is the first direct demonstration of axonal sprouting in the peri-infarct cortex following focal stroke. Anatomical plasticity in corticocortical connections has been well documented after peripheral deafferentation and after other types of central lesions. For instance, small retinal lesions induce axonal sprouting in horizontal cortical connections into the appropriate area of the primary visual cortex (Darian-Smith & Gilbert, 1994). Physiological reorganization on the scale of centimeters has been described in the somatosensory cortex of primates months to years after peripheral deafferentation (Pons et al., 1991) to which axonal sprouting by cortical neurons is correlated (Florence et al., 1998; Florence & Kaas, 2000). Many of these studies have evaluated axonal sprouting months to years after deafferentation. The present study documents substantial sprouting within 3 weeks in the adult and without changes in peripheral input. Interestingly, this sprouting occurs into peri-infarct cortex, a region largely cut off from its thalamic projections. The timing of axonal sprouting after focal stroke is similar to that reported for changes in sprouting associated proteins in peri-infarct cortex and for anatomical changes in cortex contralateral to other types of cortical lesions. Substantial increases in the levels of the synaptic protein, synaptophysin, and the growth cone-associated protein, GAP-43, occur in the periinfarct cortex (Stroemer et al., 1995; Li et al., 1998). GAP-43 levels increase from 3 to 14 days after the infarct, followed by an increase in synaptophysin which begins at 14 days (Stroemer et al., 1995). This suggests a progression from growth cone formation to synapse formation in peri-infarct cortex within the first 2 weeks after experimental stroke (Stroemer et al., Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.

920 1995). Dendritic arbors on layer V pyramidal neurons contralateral to electrolytic cortical lesions grow more complex, peaking at 18 days after the lesion (Jones & Schallert, 1992). Striatal projection neurons in cortex contralateral to a cortical thermocoagulatory lesion also sprout within 1 month (Napieralski et al., 1996). These data and those from the present study suggest that a potent stimulus for morphological changes occurs between the first week and could extend to 1 month after focal cortical lesions. Mechanisms of Axonal Sprouting in Peri-infarct Cortex New connections after focal stroke may be triggered by local excitability changes. Evidence from the hippocampus supports a mechanism for hyperexcitability-induced axonal sprouting. Ablations of entorhinal cortex or lesions to the contralateral dentate gyrus produce robust sprouting in several projections to the dentate gyrus (Ramirez, 1997). Electrical stimulation of the entorhinal projection to the dentate gyrus, the perforant path, also produces sprouting in the mossy fiber system emanating from the dentate gyrus (Cavazos et al., 1991). Axonal sprouting in this system is correlated with altered expression of neurotrophins (such as NGF, BDNF, and NT-3) and other growth factors with seizures (Gall et al., 1997; Hughes et al., 1998). The importance of hyperexcitablity as a stimulus for neurotrophin expression is underscored by the finding that only one evoked afterdischarge can elicit robust changes in growth factor expression in the hippocampus (Gall et al., 1997). Hyperexcitability has been observed in peri-infarct cortex. Following focal ischemia, inhibition evoked by afferent stimulation is reduced, the number of GABAergic binding sites falls, epileptiform postsynaptic potentials develop and facilitated long-term potentiation appears (Luhmann et al., 1995; NeumannHaefelin et al., 1995; Schiene et al., 1996; Hagemann et al., 1998). While axotomy alone produces some hyperexcitability in cortical pyramidal neurons (Salin et al., 1995), there is a substantial increase in cortical excitation following reperfusion after cortical ischemia (Luhmann & Heineman, 1992; Luhmann et al., 1995). Reperfusion through collateral flow has been documented within the region of the peri-infarct cortex in the present stroke model (Wei et al., 2001), and correlative evidence suggests reperfusion around focal infarcts in humans. It is therefore possible that the hyperexcitability that derives from reperfusion in the peri-infarct cortex, in conjunction with axotomy, proCopyright © 2001 by Academic Press All rights of reproduction in any form reserved.

Carmichael et al.

duces a stimulus for local axonal sprouting. Studies are now in progress to test the hypothesis that, as in the hippocampus, peri-infarct hyperexcitability correlates with axonal sprouting.

ACKNOWLEDGMENTS This study was supported by NIH Grants NS 17763, NS 28781, and NS37372; the McDonnell Center for Studies of Higher Brain Function; and an award from the Spastic Paralysis Foundation of the Illinois-Eastern Iowa District of the Kiwanis International. The authors thank Dr. Joseph L. Price for the use of the Minnesota Datametrics system, Dr. Joseph P. Erinjeri and Dr. Brad Miller for technical assistance; and Kathryn Diekmann for help in manuscript preparation.

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