Structural alterations and changes in cytoskeletal proteins and proteoglycans after focal cortical ischemia

Pergamon

PII:

Neuroscience Vol. 82, No. 2, pp. 397–420, 1997 Copyright ? 1997 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306–4522/98 $19.00+0.00 S0306-4522(97)00289-3

STRUCTURAL ALTERATIONS AND CHANGES IN CYTOSKELETAL PROTEINS AND PROTEOGLYCANS AFTER FOCAL CORTICAL ISCHEMIA H.-J. BIDMON,*¶ V. JANCSIK,‡ A. SCHLEICHER,* G. HAGEMANN,† O. W. WITTE,† P. WOODHAMS§ and K. ZILLES* *Departments of Neuroanatomy and †Neurology, Heinrich-Heine University, Moorenstrasse 5, D-40225 Du¨sseldorf, Germany ‡Department of Anatomy and Histology, University of Veterinary Science, Istvan utca 2, H-1078 Budapest, Hungary §National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, U.K. Abstract––In order to study structural alterations which occur after a defined unilateral cortical infarct, the hindlimb region of the rat cortex was photochemically lesioned. The infarcts caused edema restricted to the perilesional cortex which affected allocortical and isocortical areas differently. Postlesional changes in cytoskeletal marker proteins such as microtubule-associated protein 2, non-phosphorylated (SMI32) and phosphorylated (SMI35, SMI31 and 200,000 mol. wt) neurofilaments and 146,000 mol. wt glycoprotein Py as well as changes in proteoglycans visualized with Wisteria floribunda lectin binding (WFA) were studied at various time points and related to glial scar formation. The results obtained by the combination of these markers revealed six distinct regions in which transient, epitope-specific changes occurred: the core, demarcation zone, rim, perilesional cortex, ipsilateral thalamus and contralateral homotopic cortical area. Within the core immunoreactivity for microtubule-associated protein 2 and SMI32 decreased and the cellular components showed structural disintegration 4 h post lesion, but partial recovery of somatodendritic staining was seen after 24 h. Microtubule-associated protein 2 and SMI32 persisted up to days 7 and 5 respectively in the core, whereas the number of glial fibrillary acidic protein- and WFA-positive cells decreased between days 7 and 14. The demarcation zone showed a dramatic loss of immunoreactivity for all epitopes 4 h post lesion which was not followed by a phase of recovery. In the inner region of the demarcation zone there was an invasion and accumulation of non-neuronal WFA-positive cells which formed a tight capsule around the core. Neuronal immunoreactivities for microtubule-associated protein 2, SMI31 and Py as well as astrocytic glial fibrillary acidic protein increased strongly within an approximately 0.4–1.0 mm-wide rim region directly bordering the demarcation zone. Py immunoreactivity increased significantly in the perilesional cortex, whereas glial fibrillary acidic protein-positive astrocytes became transiently more numerous in the entire lesioned hemisphere including strongly enhanced immunoreactivity in the thalamus by days 5–7 post lesion. Glial fibrillary acidic protein immunoreactivity increased in the corpus callosum and the homotopic cortical area of the unlesioned hemisphere by days 5–7. In this homotopic area additional changes in SMI31 immunoreactivity occurred. Our results showed that a cortical infarct is not only a locally restricted lesion, but leads to a variety of cytoskeletal and other structural changes in widely-distributed functionally-related areas of the brain. ? 1997 IBRO. Published by Elsevier Science Ltd. Key words: brain, edema, microtubule-associated protein 2, neurofilaments, fibrillary acidic protein, diaschisis.

Within the forebrain, the hippocampus and subcortical regions such as the striatum, as well as the ¶To whom correspondence should be addressed. Abbreviations: BSA, bovine serum albumin; CaBP, calcium-binding proteins; DAB, 3,3*-diaminobenzidine tetrachloride; Fr1/2, frontal motor cortex 1 and 2; GFAP, glial fibrillary acidic protein; LI–VI, cortical layers I–VI; MAP2, microtubule-associated protein 2; MCAO, middle cerebral artery occlusion; MK-801, dizocilpine maleate; NGS, normal goat serum; NMDA, N-methyl-aspartate; NO, nitric oxide; NOS, NO synthase; Par1–2, parietal cortex area 1 or 2; PBS, phosphate-buffered saline; Py, pyramidal 146,000 mol. wt glycoprotein; RSA, agranular retrosplenial cortex; RSG, granular retrosple-

neocortex itself, are most vulnerable to ischemic injury.54,75 Most studies addressing the issue of postlesional cortical changes use middle cerebral artery occlusion (MCAO) as an experimental model to induce cerebral infarction. New insights have been gained into the processes which play important

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nial cortex; SD, spreading depression; SMI31, highly phosphorylated neurofilament 160–200,000 mol. wt; SMI32, non-phosphorylated neurofilament 160–200,000 mol. wt; SMI35, medium-phosphorylated neurofilament 160–200,000 mol. wt; TB, Tris buffer; TBS, Tris-buffered saline; WFA, Wisteria floribunda agglutinin.

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roles during postischemic neuronal degeneration and functional recovery. However, these MCAO lesions are relatively large, and show great individual variation.67 While MCAO is the model of choice to study larger lesions, which include subcortical and cortical ischemia as well as serious damage to the choroid plexus,30 we were interested in the changes occurring after small, clearlycircumscribed cortical lesions. The photothrombosis model induces small, topographically-defined lesions within the cerebral cortex.26,31,41,89 These focal lesions cause functional and structural changes throughout the brain during the initial phase, as well as during postlesional degeneration and regeneration.54,69,75 We have monitored the effects of small photothrombotic lesions by studying a number of markers such as neurofilaments and microtubule-associated protein 2 (MAP2) which are not only important structural components of the normal neuronal cytoskeleton but also indicators of pathological changes.3,34,51,52,86,90 MAP2 is a somatodendritic marker58 which rapidly disappears after ischemic insults1,46,72,95 and after death.77 Changes in the amount of neurofilaments or their degradation products are indicative of several neurodegenerative diseases.1,8,33,35,39,46,95 Nonphosphorylated neurofilament 160–200,000 mol. wt (SMI32) is a somatodendritic marker for pyramidal cells which stains intra- and interhemispheric projection neurons in primates14,15,37,62 and shows an area-specific cortical staining pattern in rats.5,6 Rapid post mortem changes have been reported in both nonphosphorylated and phosphorylated neurofilaments.28 In humans, however, ischemic insults lead to a transient change in the degree of phosphorylation of the usually nonphosphorylated neurofilament 160–200,000 mol. wt in a homotopic area contralateral to a lesion.34 The degree of neurofilament phosphorylation is also indicative of the amount of axonal neurofilament transport.90 Pyramidal 146,000 mol. wt glycoprotein (Py) is a marker expressed during the development of large pyramidal neurons such as those in hippocampal field CA3, which is less vulnerable to ischemic insults than other hippocampal regions.93 Wisteria floribunda agglutinin (WFA) identifies N-acetylgalactosamine, a component of the proteoglycans which characterize perineuronal nets, and which appear to be involved in glia–neuron interactions.12,17 Furthermore, proteoglycans have been identified as potent inhibitors of axonal growth59,60 and may influence glial scar formation and regeneration following axon damage. In order to investigate the specific postlesional changes which are associated with small, defined and topographically localized photothrombotic cortical lesions, we have used antibodies against the dendritic and axonal markers described above, as well as markers for glia and extracellular matrix.

EXPERIMENTAL PROCEDURES

Induction of photothrombosis Topographically defined lesions were produced in male Wistar rats (280–320 g body weight, HAN:WIST multiplication stock, TVA, Du¨sseldorf ) according to Watson et al.89 and Domann et al.26 In brief, rats were anaesthetized with halothane (1.5% during operation and 0.8% during lesioning). After the animals had been placed in a stereotactic frame, focal lesions were produced in the cortical hindlimb area.66,99 The skin of the skull was incised and an optic fibre bundle (aperture 1.5 mm), mounted onto a cold light source (Schott, Mainz, FRG) was stereotactically placed 4 mm posterior to bregma and 4 mm lateral to the midline. The light was turned on for 20 min. During the first 2 min Rose Bengal (Aldrich, 1.3 mg/100 g body weight) was injected i.v. After illumination, catheter and light source were removed and incisions sutured. The treatment produced cylindrical lesions (J 2.82&0.75 mm).76 Histological examination revealed that the lesions spanned all cortical layers (LI–VI) but did not involve the underlying white matter. Control animals were treated similarly to the ones described above but either the light was not turned on or Rose Bengal was not injected. Histochemical investigation Lesioned and control rats were studied at several intervals: 4 h, one, two, three, five, seven or 14 days and for certain epitopes also 21, 30 and 60 days after lesioning. At each interval five to seven individuals were either decapitated and the dissected brains immersed in Bodians fixative, or they were perfused with physiological saline containing 2.2 mM CaCl2 followed by Zamboni’s fixative (pH 7.2). Brains fixed in Bodian’s fixative for 48 h were dehydrated in ethanol followed by methylbenzoate, and embedded in paraffin. Sections (10 µm) were mounted on siliconized slides and stored until staining. Perfusion-fixed brains were postfixed in the same fixative overnight and either cut directly on a Vibratome (50–60 µm) or submerged in 30% sucrose in 0.1 M phosphate-buffered saline (PBS) for 48 h, and frozen on dry ice. These brains were cut on a cryotome into 50 or 70 µm-thick serial sections shortly before use. Both types of fixation were necessary. Complete serial sections through the brain and the infarct region were obtained from the paraffin-embedded brains, but the centre of the lesion was often randomly lost in sections during processing of Vibratome or cryotome sections. Some stainings such as WFA binding, were only successful on free-floating sections. For immunohistochemical identification of nonphosphorylated neurofilament, phosphorylated neurofilament, MAP2, basic myelin protein and glial fibrillary acidic protein (GFAP), deparaffinized 10 µm sections were rehydrated and endogenous peroxidase was blocked for 30 min in absolute methanol containing 0.33% H2O2. Sections were rinsed three times 5 min each in PBS (0.1 M, pH 7.4) and then incubated in 2% normal goat serum (NGS) and 10% heat inactivated fetal calf serum diluted with Tris-buffered saline (TBS) (pH 7.4). These sections were incubated overnight at 4)C with first antibody diluted in TBS containing 1% NGS. Primary antibodies for deparaffinized sections were used at the following final dilutions: MAP2 polyclonal rabbit,40 1:500; SMI31, SMI32, SMI35 monoclonal (Sternberger Monoclonals) 1:500; anti-basic myelin protein, monoclonal (Boehringer, Mannheim) 1:500; neurofilament 200,000 mol. wt, monoclonal (Boehringer), 1:25; GFAP, monoclonal (Boehringer) 1:25. After incubation in primary antibody, sections were rinsed 3#5 min in TBS and incubated for 3 h in TBS containing 1% NGS and either second peroxidase-coupled anti-mouse serum (diluted 1:50 for monoclonals) or second

Cytoskeletal changes after cortical ischemia peroxidase-coupled anti-rabbit serum (diluted 1:200 for polyclonals; Sigma, Deisenhofen). Sections were rinsed two times in TBS and twice in Tris buffer (TB) (0.05 M, pH 7.6) for 5 min each followed by the visualization of antibody binding by incubation in TB containing 0.75 mg/ml 3,3*diaminobenzidine (DAB; Sigma) and 0.003% H2O2. After 8 min, a brown precipitate had formed and sections were rinsed in TB, dehydrated in ethanol and coverslipped with DePeX (Hoechst). Free-floating sections were rinsed four times 15 min in PBS followed by the blocking of endogenous peroxidase in PBS containing 3% H2O2 for 30 min. After four further rinses of 15 min each, nonspecific binding was blocked as described above followed by a 48 h incubation at 4)C in TBS containing 1% NGS and the primary antibody at the following dilutions: MAP2, 1:1500; SMI31, SMI32, SMI35, 1:1000; GFAP, 1:40; Py monoclonal 1:100 (from Dr P. Woodhams). After four rinses in TBS, 15 min each, sections were incubated in the same second antibodies as described above for 14 h at 4)C. Sections were rinsed twice in TBS and twice in TB for 15 min each and antibody binding was visualized as described above. Afterwards sections were rinsed in TB, submerged in chrome-alum–gelatine, mounted on slides, air-dried and coverslipped with DePex. For lectin binding, free-floating sections were rinsed as described. Endogenous peroxidase was blocked with PBS containing 1% H2O2 and after the four rinses, non-specific binding was blocked with 2% bovine serum albumin (BSA) dissolved in TBS for 30 min. Sections were incubated for 48 h at 4)C in biotinylated WFA (1 µg/ml; Sigma) diluted in TBS+1% BSA followed by four rinses in TBS. Sections were then incubated for 90 min in avidin–biotin complex (EliteKit, Vectastain) diluted 1:300 with TBS, rinsed, stained with DAB and further processed as described. For histological examination and photographic documentation we used a Zeiss Axiomat and Kodak T64 film. RESULTS

General pathological changes The animals recovered from scalp surgery and illumination without complications. No gross signs of functional deficits could be observed. After photothrombosis a lesion (diameter 2 mm) developed progressively through all six cortical layers as shown in Fig. 1. Four hours post lesion, the core showed fewer Nissl-stained cells than perilesional areas (Fig. 1a). Cell loss was complete in all animals by day 14 (Fig. 1b). A generally widespread, longlasting edema developed within the lesioned hemisphere, resulting in increased cortical thickness compared to the unlesioned side. This edema was most pronounced in perilesional areas. It was quantified by measuring cortical thickness at the three positions indicated by arrowheads in Fig. 1a and b: the retrosplenial cortices (agranular retrosplenial cortex [RSA]/granular retrosplenial cortex [RSG]) the frontal motor cortex (Fr1) and the parietal area 1 (Par1). The latter represents most of the primary somatosensory barrel cortex. Differences in cortical thickness between the right (lesioned) and left (unlesioned) hemispheres were calculated and plotted for comparison to those calculated from control animals (Fig. 2). In all three areas a similar time schedule of progression and regression of right–left differences in cortical swelling was registered.

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Significant values were measurable at 4 h, reached a maximum between 24 h and three days post lesion. The values declined thereafter but were still measurable up to 30 days (720 h) post lesion. The values then became negative for the frontal and parietal perilesional cortices, due to the shrinkage of the core and a partial closure of the scar by the perilesional cortex (Fig. 1c,d). In more than 50% of the animals the shrinkage of the core caused the normally closely attached ependyma to become separated from the ipsilateral corpus callosum and hippocampus (Figs 1c,d, 6d, 7a,c). This ependymal dissociation was most pronounced by day 60 post lesion. At 60 days two reactions in response to the scar had taken place: (i) the contour of the scar and its surroundings created a shallow cone and the corpus callosum was distinctly separated from the hippocampus and drawn towards the scar (Fig. 1c); (ii) the cone-shaped depression was much deeper but there was only a small gap between corpus callosum and hippocampus (Fig. 1d). Both kinds of reactions led to a slight reduction in the thickness of the perilesional cortex post edema. The edema was more pronounced in the Fr2/1 and Par1 regions than in retrosplenial areas (Fig. 2). This was not due to the fact that the frontal and parietal areas are directly adjacent to the lesion, since in some rats in which the lesion was slightly larger and bordered directly on RSA the swelling was still less severe than in Par1 (not shown). These latter animals were excluded from further evaluations. As seen in Fig. 1a,b a narrow unstained zone encapsulated the core. This unstained zone was surrounded by a wider rim with increased immunoreactivity for certain markers. The perilesional cortex surrounding the rim showed more moderate changes in immunoreactivity for Py and GFAP (Figs 3, 4, 5, 9). We therefore defined four pathologicallyaltered cortical areas (Fig. 3); (i) the centre of the lesion is defined as the core; (ii) the unstained zone surrounding it is the demarcation zone; (iii) the area adjacent to the demarcation zone with increased immunoreactivity is the rim; and (iv) the surrounding cortex with slightly altered immunoreactivity for certain antigens is defined as perilesional cortex. Alterations of cortical markers Microtubule-associated protein 2. To study changes in the distribution of MAP2, we used mainly paraffin sections because changes occurred within the core of the lesion. Similar changes were seen in Vibratome sections in which the core had not been lost. Photothrombosis resulted in a rapid loss of MAP2 immunoreactivity within the core and even more so in the demarcation zone. Many MAP2-positive neurons were still present in the core at 4 h (Fig. 4a) but immunoreactivity was reduced to small granulelike drops within the dendrites, and the somata showed only weak uniform staining (Fig. 4b,c).

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Fig. 1. Ag–Nissl- (a–c) or neurofilament 200,000 mol. wt- (d) stained frontal sections of rats 4 h (a), 14 days (336 h; b) and 60 days (c–d) post lesion. After 4 h there are fewer stained cell somata in the lesion and edema has begun. At 14 days the core is mainly disintegrated and partially infiltrated by the overlaying connective tissue. After 60 days the ventricles are extended by ependymal separation (ES) between the corpus callosum (CC) and hippocampus (c, the white triangle marks one remaining ependymal adhesion). In (d) ES is less pronounced, but cortical thickness is reduced, mainly at the pial surface (arrowtips). Retrosplenial cortex (RSA), 1; frontal cortex (Fr1) 2 and 1, 2; hindlimb area, 3; parietal cortex 1 (Par1); 4. Arrowheads mark the distances measured to evaluate cortical thickness (see Fig. 2) and arrows mark the rim region of the lesion. Demarcation zone, d. Scale bars=1 mm.

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Fig. 2. Differences in cortical thickness of rats (n=6) expressed as areas of the right lesioned hemisphere minus the left non-lesioned hemisphere. Positive values show that perilesional areas are thickened by edema for up to 30 days. Note that edema is less pronounced in RSA. Solid&dashed lines=value of control animals. For abbreviations see Fig. 1.

Pyramidal neurons in layers II/III stained more intensely than those in the deeper layers V and VI at all times (Figs 4a, e–g, 5a,d, Table 1). From 4 h onward, a complete loss of the MAP2 epitope defined the narrow demarcation zone. However, in the adjacent 400 µm-wide rim, MAP2 immunoreactivity was increased, especially in LII–V. It appeared in darkly stained somata, apical dendrites and their branches (Fig. 4a,c). One day post lesion the initial, progressive disintegration of MAP2-positive structures and the loss of MAP2 epitope was arrested or even reversed in the core. The MAP2-positive neurons with uniformly distributed immunoreactivity in somata and main apical dendrites were clearly visible in the upper layers (Fig. 4e–g). In the deeper layers the loss of immunoreactivity progressed further, whereas the

almost complete loss of MAP2 within the demarcation zone as well as the increased immunoreactivity in the rim remained unchanged (Fig. 4d–e). At 24 h the core showed a general and sometimes irregular widening of blood vessels and some cyst-like spaces in the deeper layers (Fig. 4e). These spaces grew in size to become quite large by day 3. A ‘‘secondary’’ decline in the number of MAP2-positive pyramidal neurons began between days 1 and 3 (Fig. 5a–b) and continued up to day 5 (Fig. 5d). The increased MAP2 staining in the rim became limited to an approximately 80–100 µm wide perilesional area by day 5. By day 3 only the upper pyramidal neurons in the core still expressed MAP2 which was seen in the main apical dendrites and in the perisomal parts of the basal dendrites (Fig. 5b). The distribution pattern and staining intensity for MAP2

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Fig. 3. Drawing showing the lesioned dorsomedial cortex. The defined regions which develop are indicated. Corpus callosum, cc; Core: centre of the lesion which disintegrates and is lost after several days. Demarcation zone: zone which looses immunoreactivity for MAP2 and all studied neurofilaments during the first day. Perilesional cortex: clearly surviving but affected cortex which shows altered immunoreactivity for certain epitopes. Rim: part of the perilesional cortex which lies directly adjacent to the demarcation zone and which shows strongest alterations in immunoreactivity for certain epitopes, such as MAP2, SMI31, Py, and GFAP.

in the homotopic area to the core in the contralateral hemisphere appeared relatively normal (Fig. 5c), whereas the few remaining MAP2-positive neurons within the core show clear signs of degeneration. It is striking that the somatodendritic staining at three and five days exhibited a better structural integrity than at 4 h. However, by seven days, MAP2 staining had completely disappeared within the core, whereas an increased number of MAP2-stained somata and dendrites still persisted within the rim. The core and the demarcation zone were now filled with cyst-like enlarged remnants of blood vessels and an amorphous mass of degenerated tissue, which showed non-specific, light staining (Fig. 6a–b, Table 1). At 14 days this condition was more or less unchanged (Fig. 6c). Above the white matter underlying the lesioned cortex a non-immunoreactive tissue ‘‘cushion’’ was seen which gradually replaced the amorphous degenerated tissue remnants between itself and the overlaying meninges. This tissue cushion became slightly thicker from day 14 to day 60 (Fig. 6d) and contained GFAP-positive astrocytes (not shown) and other cells which participate in the formation of the glial scar as described by Schroeter et al.76 Non-phosphorylated neurofilament H (SMI32). Immunoreactivity for SMI32, a well-established somatodendritic marker of pyramidal neurons, showed similar changes to those described for MAP2. The

loss of SMI32 epitope was, however, more rapid than that observed for MAP2. Four hours post lesion, SMI32-immunostained dendrites showed degeneration which made them appear like strings of beads, mainly in the lower cortical layers within the core (Fig. 7a–b). The intensity of immunoreactivity was reduced throughout the core and demarcation zone. Some somata remained uniformly stained (Fig. 7b). One day post lesion, SMI32 immunoreactivity was markedly more reduced in layers I and II within the core, although it was more enhanced in somata and dendrites in deeper layers (Fig. 7c) than after 4 h. At three days (not shown) and five days (Fig. 7d), only dot-like remnants of SMI32-immunoreactive material could be found in layer VI of the core and slightly more of it towards LVIc. As with MAP2, the demarcation zone showed no immunoreactivity (Fig. 7c–e). No increase in SMI32 immunoreactivity occurred in the perilesional cortex. This was also seen in the frontal motor cortices Fr2/1 which showed only weak SMI32 immunoreactivity in layer VI compared to the parietal and retrosplenial areas (Fig. 7c–e, Table 1). Seven days post lesion the core and the demarcation zone showed only weak non-specific staining for SMI32, which was associated with the remnants of degenerated tissue (Fig. 7e). Figure 7e also shows the normal staining pattern for SMI32 in the homotopic area to the lesion within the contralateral hemisphere. It becomes evident that under normal conditions almost no SMI32 immunoreactivity occurs in Fr1 and 2. Phosphorylated neurofilaments (SMI31, SMI35, 200,000 mol. wt). As seen with MAP2 and SMI32, immunoreactivity for the medium-phosphorylated SMI35 epitope (Fig. 8a–b), fully phosphorylated SMI31 (Fig. 8c–e, Table 1), and neurofilament 200,000 mol. wt (Fig. 8f ) epitopes rapidly decreased within the core and almost completely in the demarcation zone. No specific immunoreactivity was found for neurofilament 200,000 mol. wt after day one (e.g., Fig. 8f ). Levels of immunoreactivity in the perilesional cortex were not elevated above those for control animals. In contrast, whilst SMI35 immunoreactivity disappeared almost completely within the demarcation zone, some diffuse staining remained in the upper parts of the core and was seen as large particles (degenerating axon fragments) in the deeper part of the lesion (Fig. 8a). Increased SMI35 immunoreactivity was still apparent in a 200 µm-wide rim after 14 days and remained up to 30 days. Many

Fig. 4. (a–g) Changes in MAP2 immunoreactivity within the core and perilesional cortex 4 h (a–c) and 24 h (d–g). Four hours post lesion somatodendritic MAP2 immunoreactivity is reduced in the core (a,b) and even more so in the demarcation zone, whereas an increase of staining is seen in the rim (a,c). Within the core immunoreactivity in the dendrites is reduced to a string-of-beads pattern (b, arrows). At 24 h the pattern in the demarcation zone and rim is unchanged but in the upper layers of the core dendrites of immunoreactive pyramidal neurons show normal, uniform staining (e,f and g show enlargements of layers III and V pyramidal neurons, respectively). Demarcation zone, d; rim, r. Scale bars=250 µm (a); 50 µm (b,c,e); 10 µm (d,f,g,).

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Fig. 5. (a–d) Immunoreactivity for MAP2 is still present in some neurons of layer III, and a few deeper pyramidal neurons in the core after three days (a,b), but the amount of staining is reduced and confined to the somata and the main dendrite. Compare with staining of the homotopic area of the intact contralateral hemisphere (c). A further reduction in immunoreactive neurons is seen in the core after five days and no immunoreactivity is present in the demarcation zone (d). Scale bars=100 µm (a); 50 µm (b–d).

specifically SMI35-stained small and large globular structures appeared between seven and 14 days in the glial scar ‘‘cushion’’ between the underlying white

matter and the remnants of the submeningeal tissue as well as to a lesser degree in the demarcation zone (Fig. 8a–b).

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Fig. 6. (a–d) MAP2 immunoreactivity is completely lost in the core after seven days (a) and the lesion is filled with diffusely stained, vacuolized tissue remnants trapped between arachnoid and glial scar (a,b). With progressing time the core becomes filled with connective tissue as seen at 30 (c) and 60 days (d), whereas staining intensity is still slightly increases in the rim. Demarcation zone, arrowheads. Scale bar=100 µm.

Similar to SMI35, SMI31 immunoreactivity in fibres and some somata increased and remained raised in the rim (Fig. 8c–d). Whereas this antibody diffusely stained degenerated tissue remnants more intensely than SMI35, globular structures within the glial scar cushion were smaller and less intensely stained than for SMI35. Since in humans strong homotopic contralateral staining has been reported for SMI35 and SMI31 immunoreactivity,34 we also studied the homotopic area. In two out of seven animals we found a few pyramidal neurons with intensely SMI31-positive somata and main apical dendrites in layers V and VI (Fig. 8e). Somatodendritic SMI31 staining was absent in unlesioned

controls as well as in the contralateral hemispheres of other five animals, except for occasional somata in the piriform cortex. Py immunoreactivity. Immunoreactivity for the 146,000 mol. wt glycoprotein Py also decreased rapidly within the core and demarcation zone. Four hours post lesioning similar results as for the SMI32 epitope were observed: a few stronglyimmunoreactive somata were present mainly in layer V of the core (not shown). One day post lesion no specific immunostaining was found within the core and the demarcation zone, and changes in the Py immunoreactivity occurred mainly within the

MAP2 SMI32 SMI31 SMI35 Py NF200,000 WFA GFAP MAP2 SMI32 SMI31 SMI35 Py NF200,000 WFA GFAP MAP2 SMI32 SMI31 SMI35 Py NF200,000 WFA GFAP MAP2 SMI32 SMI31 SMI35 Py NF200,000 WFA GFAP MAP2 SMI32 SMI31 SMI35 Py NF200,000 WFA GFAP

4h

5 days

3 days

2 days

1 day

Marker

Time

mol. wt

mol. wt

mol. wt

mol. wt

mol. wt

"4 "3 "3 "2 "3 "2

"4 "4 "4 "4 "4 "4 "4

"4 "4 "4 "4 "4 "4 "3**

"3 "3 "3 "2 "3 "2

"2 "2 "3 "3 "4 "4 "1

"2 "2 "4 "4 "4 "4 "1 +1 "2 "3 "4 "4 "4 "4 "1 +2 "3 "4 "4 "4 "4 "4 "1 +3

"4 "4 "4 "4 "4 "4 "2**

"4 "4 "4 "4 "4 "4 "3**

inner

core Fr/HL

"4 "4 "4 "4 "4 "4 "4 +2 "4 "4 "4 "4 "4 "4 "4 +2 "4 "4 "4 "4 "4 "4 "4 +2

"4 "4 "4 "4 "4 "4 "4

"3 "3 "3 "2 "3 "2

outer

demarcation zone

+3 +2

+4

+2

+2

+2

+2

Par1

+1

+2

+1

+1

+1

+1

RSA/G

perilesional cortex

+2 +2 +4

(+*) +4 +4

+1 +1 +3

(+*) +3 +4

+1 +1 +3

+1 +2

+1

+2

rim

Ipsilateral hemisphere

+2

+1

+1

+1

hc

+2

+2

+1

th

Table 1. Gross changes in neuronal and glial markers after cortical photothrombosis

+1

+1

+1

RSA/G

+2

+1 ("*)

("*)

h.a. Fr/HL

+1

+1

lateral cortex

Contralateral hemisphere

+1

+1

hc

+1

+1

th

406 H.-J. Bidmon et al.

MAP2 SMI32 SMI31 SMI35 Py NF200,000 WFA GFAP MAP2 SMI32 SMI31 SMI35 Py NF200,000 WFA GFAP MAP2 SMI32 SMI31 SMI35 Py NF200,000 WFA GFAP MAP2 SMI32 SMI31 SMI35 Py NF200,000 WFA GFAP

7 days

mol. wt

mol. wt

mol. wt

mol. wt

"4 "4 "4 "3* "4 "4 "4

"4 "4 "4 "3* "4 "4 "1 +3 "4 "4 "3 "3* "4 "4 "3 ? "4 "4 "3* "3* "4 "4 "4

core Fr/HL

"4 "4 "3 "3* "4 "4 "2** ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ?

"4 "4 "4 "3 "4 "4 "1**

"4 "4 "4 "3 "4 "4 "4 +2 "4 "4 "3 "3* "4 "4 "4 ? ? ? ? ? ? ? "4 ? ? ? ? ? ? ? ? ?

outer

demarcation zone inner

+2

+1

+2 +2

+2 +1 +1

+3 +2

+2 +2 +2

+4 +4

+2 +2 +4

+4

rim

+1

+1

+2

+3

+1

+1

+1

+2

RSA/G

perilesional cortex Par1

+1

+2

+1

hc

+1

+2

+1

th

+1

RSA/G

(+*)

+3

("*) ("*) (+*)

h.a. Fr/HL

+1

lateral cortex

Contralateral hemisphere

+1

hc

+1

th

No entry, no significant change; (+) increase; (") decrease; 1, slight; 2, strong; 3, stronger; 4, highest; ?, region could be no longer identified; (+*) increase seen in some animals; ("*) decrease seen in some animals; *axon terminals; **microglia or leucocytes; h.a., homotopic area; hc, hippocampus; th, thalamus; Fr, frontal motor cortex 1; HL, hindlimb area; Par1, parietal cortex 1; RSA/G, retrosplenial cortex, agranular/granular.

60 days

30 days

14 days

Marker

Time

Ipsilateral hemisphere

Table 1. Continued

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Fig. 7. (a–e) SMI32 immunoreactivity in the somata and dendrites of pyramidal neurons decreased 4 h after photothrombosis in the core (a). Some neurons in deeper layers V and VI show darkly stained somata with dispersed staining of dendrites (b, enlargement marked in a). After 24 h the intensity of staining recovered within the core but not in the demarcation zone (c). At five days immunoreactive remnants of neurons were present in deeper layers only (d). A complete loss of specific staining was seen in the core after seven days compared to the contralateral side. Under normal conditions the frontal cortical motor areas Fr2/1 (arrows) contain only weak SMI32 staining in a few neurons of layer VI as seen in the unlesioned hemisphere (e). Large filled arrowheads indicate the demarcation zone, d. Hippocampal subfields, CA1, CA2, CA3; corpus callosum, CC. Scale bars=10 µm (b), 100 µm (a), 250 µm (c–d), 500 µm (e).

perilesional cortex. After two days a clear increase in immunoreactivity was evident in the rim as seen in a coronal section through the whole brain (Fig. 9a–b, Table 1). This increase in staining intensity was

mostly due to the formation of a dense dendritic fibre plexus in LIII and V of the rim (Fig. 9b). Within the rim this fibre plexus occurred to a lesser extent also in LVI but filled LIV entirely, forming a bridge of small

Fig. 8. (a–f ) SMI35 immunoreactivity after 14 days is confined to some tissue remnants in the upper part of the core and to larger particles (arrowheads) in the deeper part of the lesion (a,detail in b). SMI31 immunoreactivity is similar to that of SMI35 in the deeper part, the core and demarcation zone after 14 days (c). Within the rim a few single stained somata are present between the axons (d enlargement of c, star marks same position in c and d). A few isolated immunoreactive somata are also present in the deeper layers of the homotopic area of the contralateral hemisphere (e). Specific neurofilament 200,000 mol. wt immunoreactivity is lost within the core and demarcation zone after 14 days (f ). Demarcation zone, d; triangles, immunoreactive neurons. Scale bars=100 µm.

Fig. 9. (a–e) Py immunoreactivity shows an area- and lamina-specific staining pattern of somata and dendrites of larger pyramidal neurons after two (a,b) and seven days (c,d). Py immunoreactivity was increased in the rim, as seen in (b) where the section is cut not through the centre of the lesion but tangentially through the occipital part of the rim (detail at arrows is shown in a). Increase in Py immunoreactivity was clearly visible in the entire perilesional cortex but not so in non-lesioned hemisphere at seven days (d; detail at arrows is shown in c). (e) Control section showed similar Py staining in both hemispheres (e; compare also with Fig. 7e). Arrowheads mark the border of increased Py staining in the lateral perilesional cortex. Scale bars=100 µm (a,c); 1 mm (b,d,e).

Cytoskeletal changes after cortical ischemia Table 2. Changes in intensity of Py immunoreactivity among cortical areas Differences in optical density&S.D.(right–left hemisphere) Days post lesion 2 7 0 (control)

RSG/RSA

Par1+2

0.027&0.008 0.026&0.019 0.006&0.004

0.023&0.021 0.043&0.0095 0.005&0.006

n=5.

dendritic or axonal fibres between LIII and V. The main dendrites of LIII pyramidal neurons clustered together and formed darkly-stained bundles within the rim. There was also a slight increase in Py immunoreactivity within the perilesional, retrosplenial and parietal cortex compared to the contralateral side as determined by the measurement of optical densities within these areas (Table 2). The immunoreactivity in the perilesional cortex decreased slightly towards the more laterocaudal area Par2. At seven days all sections showed a clear increase in Py immunoreactivity in LII–III and V–VI of the perilesional cortex compared with the contralateral side (Fig. 9c,d; Table 2). In both controls and the intact contralateral cortex of lesioned animals there was only weak Py immunoreactivity present in LVIa, seen as a small light band between LV and VIb (Fig. 9b,d,e). At day 7, LVIa showed similar immunoreactivity as is seen in LV and VIb of the perilesional Par1 and to a lesser degree in the parietal cortex 2 (Fig. 9d). At 30 days post lesion no differences in Py immunoreactivity were seen compared to controls except for some strongly stained bundles of main dendrites in the rim toward the demarcation zone and core which were still present after 60 days in a few animals. Wisteria floribunda agglutinin. Proteoglycans are important components of the extracellular matrix.9 WFA binding to the N-acetylgalactosamine component of proteoglycans produced an area- and lamina-specific staining pattern throughout the cortex (Fig. 10b, d). No consistent changes in normal staining were observed after lesioning, but a few animals showed increased staining in LII/III and V/VI in the rim and perilesional parietal cortex. WFA binding could only be carried out on Vibratome or cryosections, usually resulting in the loss of the core precluding its consistent evaluation. In sections in which the core remained intact, WFA binding declined slowly from 4 h to day 14, with a few weakly-stained cell surfaces remaining in about 20% of the animals by day 14. As with neurofilament staining, WFA binding decreased markedly in the demarcation zone, which appeared as a narrow unstained band between the core and the WFA-positive perilesional cortex

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(Fig. 10a–e, Table 1). However, beginning 24 h after lesioning the rim and demarcation zone were progressively invaded by increasing numbers of ovoid cells exhibiting strong WFA binding (Fig. 10a,b), so that by seven days they formed a tightly packed small band between the inner part of the demarcation zone, forming a capsule around the core (Fig. 10d,e). A gradual decrease in their number was observed by 14 days, but cells were still present after 30 days. Glial fibrillary acidic protein. GFAPimmunoreactive cells showed a similar time schedule of loss within the core as described for WFA binding. However, immunoreactivity in the remaining cells was enhanced and the immunoreactive remnants formed a darkly-stained cluster within the core. An 800–1000 µm-wide rim of increased GFAP immunoreactivity surrounded the core and the partly immunonegative demarcation zone. The latter contained numerous cyst-like vacuoles. Within the rim, GFAP immunoreactivity increased continuously up to day 7 forming an astrocytic (glial) scar around the demarcation zone and core (Fig. 11a,b, Table 1). In addition, a continuous increase in the number of GFAP-immunoreactive cells occurred throughout the entire lesioned hemisphere (Fig. 11b). This general ipsilateral increase was less pronounced or even absent in retrosplenial areas despite the fact that they were closer to the lesion than the piriform cortex. Five and seven days post lesion a slight increase in the number of GFAP-immunoreactive astrocytes was also seen in a cortical area homotopic to the lesion within the non-lesioned hemisphere (Fig. 11b,c). At 14 days, GFAP immunoreactivity had decreased within the lesioned hemisphere but remained raised within the rim up to 60 days (Table 1). DISCUSSION

General observations Focal cortical photothrombotic lesions in our animals resulted in transient epitope-selective immunohistochemical changes of neuronal and glial components. We identified six pathologically altered areas. Four areas were directly lesion-associated: the core, the demarcation zone, the rim and the perilesional cortex, whilst two others were distant, but functionally connected to the lesion: the ipsilateral thalamus and the homotopic cortical area of the unlesioned hemisphere. Identification of four of these six areas was marker-dependent, and only the core and the demarcation zone were clearly seen with all markers. The rim was characterized by enhanced immunoreactivity for MAP2, SMI31, SMI35, Py and GFAP but not by SMI32 and WFA binding. The perilesional cortex and the ipsilateral thalamus were characterized by increased Py and GFAP, respectively, whereas the contralateral homotopic area showed a weak, inconsistent increase in SMI31 and a more pronounced consistent increase in GFAP.

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Fig. 10. (a–e) WFA binding to N-acetylgalactosamines in the rat brain at three (a–c) and seven days (d–e). WFA shows a lamina- and area-specific staining pattern throughout the cortex which is not specifically changed in the lesioned hemisphere when compared to the intact one after three and seven days (b,d). In the demarcation zone of the lesion an increasing number of round, strongly WFA-positive leucocyte-like cells (white arrowheads) accumulate and form a small capsule by day 7 (a=enlargement of b and e=enlargement of d). Between this capsule and the normal cortical WFA-binding, a small clearly unstained outer demarcation zone (od) became obvious. Note the presence of WFA-positive cells within blood vessels (e). Within the core (c) WFA-binding disintegrated. Fat arrows in (b) and (d) mark enlarged regions seen in (a) and (e). Blood vessel, b; demarcation zone, small arrows; rim, r; triangles normal WFA-positive cerebral cells. Scale bars=50 µm (c,e); 100 µm (a); 1 mm (b,d).

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Fig. 11. (a–c) GFAP immunoreactivity shows the distribution pattern of activated astrocytes after seven days (a, detail of b showing the lateral rim). The glial scar is tightly closed around the core and the number of reactive astrocytes is strongly increased towards the demarcation zone and core. With exception of the retrosplenial areas, the entire lesioned hemisphere shows an increased number of reactive astrocytes (b,c). A slight increase in reactive astrocytes is also found in an area homotopic to the lesion (arrowheads) as well as in the ipsilateral laterodorsal nuclei of the thalamus (arrows, LTV, LTD in c and small arrows in b) which is clearly visible after contrast enhancement in a digitized image of b (c). Scale bars=25 µm (a); 1 mm (b).

Normally the ependyme between the corpus callosum and hippocampus are well attached by adhesions,44 but in most of the lesioned animals these adhesions were lost between days 14 to 60. This loss was probably due to tension resulting from shrinkage of the core. The functional consequences of these changes have still to be evaluated. Core, demarcation zone and rim Delayed neuronal and glial degeneration in the core seems to depend more on perilesional changes than on the initial thrombosis. The intra-dendritic distribution pattern for MAP2-positive neurons is partially restored within the core during the first day. These are signs of neuronal survival. This partial

recovery was unexpected, especially since the MAP2 protein is one of the proteins most vulnerable to experimental ischemia in adults as well as during ontogeny1,8,35,57,71,72 and is rapidly lost within about 8 h after death.77 The recovery of dendritic MAP2 staining is also indicative of restored oxygen supply to the core, since oxygen and protein phosphorylation are essential for MAP2 maintenance. The relatively long persistence of cells showing WFA binding is also indicative of restored oxygen supply. The observed time schedule of neuronal and glial cell loss in the core parallels that of the inner demarcation zone and rim in which WFA-positive cells and astrocytes form a capsule or glial scar separating the core from the surrounding cortex. This astrocytic scar was also found by Schroeter et al.76 who used the same

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experimental setup. The time schedule for its appearance also fits well to that seen for the restoration of blood–brain barrier in other lesion models.79 In our study we observed a similar time schedule for the formation of the astrocytic scar. The onset of the secondary decrease following the initial restorative phase of MAP2 and SMI32 somatodendritic staining within the core also corresponds well with the onset and progression of gliosis in the rim. Gliosis in the rim is evidently reciprocally coupled with the accumulation of WFA-positive cells in the demarcation zone and cellular disintegration of the core. Since neuronal nutrition and the maintenance of the oxygen supply are highly dependent on functioning astrocytes,56 the increase in reactive astrocytes within the rim as well as within the entire lesioned hemisphere may lead to a nutritional imbalance which affects injured neurons most severely. Additional processes described for the penumbra and perilesional cortex may also contribute to multifactorially-mediated processes of degeneration and regeneration.54,63 One of these processes could be hyperexcitation which has been observed in most lesion models.13,35,50,55,74,83,98,100 It leads to increased energy consumption in the perilesional tissue and to increased nitric oxide (NO) synthesis via the N-methyl--aspartate (NMDA) receptormediated induction of neuronal NO synthase (NOS).18,23,61,65,67,94,96 This post lesional increase in NO production, by neuronal, endothelial NOS and inducible NOS,18,21,47 consumes large amounts of oxygen48 and may contribute to increasing oxidative stress around the core. Furthermore, increased NO production results in an increase of MAP2 in vivo43 which may explain the observed rise in MAP2 within the rim, a region in which the number of astrocytes which express inducible NOS is strongly increased after lesioning.4 These observations suggest that a pharmacological management of perilesional glial scar formation may support functional recovery of the core in small lesions. Perilesional cortex As seen in other experimental models,16,49,54 GFAP immunoreactivity is not restricted to the rim and perilesional cortex, usually called the penumbra. The amount of GFAP-immunoreactive structures increased within the entire ipsilateral hemisphere. The increase was most pronounced in regions connected to the lesioned cortical area such as the thalamus and even the corpus callosum, through which the glial reaction spread into the white matter of the contralateral hemisphere as well as into the contralateral homotopic cortical area. Whereas the latter findings may be due to retrograde reactions following a lesion-induced deafferentation, ipsilateral gliosis may be caused by spreading depression (SD) since inhibition of SD by the NMDA antagonist dizocilpine maleate (MK 801) blocks GFAP expres-

sion,76 and MK 801 also reduces injury-induced edema in most models.60,97 The time schedule found by Grome et al.30 for the progression and regression of edema after cortical photothrombosis was very similar to ours. However, we observed regional differences in the extend of edema. Despite the fact that the retrosplenial areas lie close to the lesion, perilesional edema as well as all other markers were reduced in the retrosplenial cortex. This suggests that the retrosplenial cortices may be less affected by cerebral edema, possibly due to differences in the blood supply between retrosplenial and medial frontal cortical areas compared with more lateral frontal and parietal cortices. The latter are supplied by branches of the medial cerebral artery, whereas the retrosplenial areas are supplied by the azygos pericallosal artery (a branch of the anterior cerebral artery) and branches of the posterior cerebral artery.78 Demarcation zone, rim and perilesional cortex vs penumbra Another general observation was the very early appearance of a narrow demarcation zone surrounding the core, which rapidly lost immunoreactivity for all epitopes, with the exception of cells expressing N-acetylgalactosamine in its inner zone. This inner zone showed the most severe signs of necrosis, with many cyst-like vacuoles appearing after the onset of glial scar formation. A similar immunonegative rim zone is also seen after MCAO23,67 but most authors include it in the core region. However, in our animals this zone did not exhibit recovery of somatodendritic MAP2 and SMI32 and it lost its normal WFA binding pattern shortly after lesioning (in contrast to the core, where WFA persisted for more than seven days). This selective vulnerability of the demarcation zone was unexpected since the photolesion was not produced by a spread light but by a dot-like beam: a beam might have been expected to spare the area surrounding its centre. Further ultrastructural studies may provide information on the alterations taking place within this zone. We suggest that the demarcation zone is caused during the early phase (<4–24 h post lesion) by the surviving cells within the perilesional cortex. These cells (as well as the more distant neurons which send afferents to the lesioned area) might retract their immunoreactive processes from the core, causing a zone free of immunoreactivity for cytoskeletal and glial markers. The term rim is not always clearly defined and is often restricted to the penumbral region which borders the lesion. The term penumbra is defined as a zone of reduced blood flow but with intact energy metabolism as reviewed by Nedergaard63 and Hossmann38 and/or as a region with altered protein synthesis.19 In regard to the first definition, the penumbra, for example in the MCAO model, may extend from the border of the core up to 3 mm into

Cytoskeletal changes after cortical ischemia

the perilesional tissue.54 We observed epitope-specific differences in the size of the rim and/or perilesional cortex. In addition, the medial perilesional cortex bordering the retrosplenial cortex, in which changes in immunoreactivity occurred, was always narrower than the affected lateral perilesional cortex. Therefore, the definition of a penumbra and its extent as a region with altered protein synthesis may depend on the location of the lesion and the protein studied, since the perilesional area in which MAP2 had increased was much smaller than that for Py or GFAP, and SMI32 immunoreactivity showed no perilesional enhancement. We therefore, prefer the term perilesional cortex rather than penumbra. In order to clearly separate the non-immunoreactive zone around the core from the hyper- or hypoimmunoreactive perilesional cortex and its hyperreactive rim, we would suggest the neutral term ‘‘demarcation zone’’ (Fig. 3). Microtubule-associated protein 2 This study showed three different reactions within the post lesional distribution pattern of the exclusively somatodendritic marker MAP2: (i) immunoreactivity disintegrated during the first 4 h within the core and shows a consistent restoration after 24 h, followed by a continuous decrease; (ii) a complete and permanent loss of immunoreactivity within 24 h in the demarcation zone; and (iii) an increased staining intensity within dendrites of the rim. MAP2 has been identified as one of the markers most vulnerable to ischemic insults.46,52,71,72 Most investigations of the lesion-induced MAP2 decline have been undertaken in the hippocampus of various species.1,75 In contrast to its condition in the hippocampus, MAP2 was more stable under certain experimental ischemic conditions in the cortex87 suggesting regional differences in MAP2 vulnerability. In rat cortical and hippocampal neurons loss of MAP2 is associated with and precedes neuronal death.8 Our results on the loss of MAP2 within the core are closely comparable to those of Dawson and Hallenbeck23 and Araki et al.2 after MCAO lesion. The first group reported a more rapid decrease in MAP2 within the lesion but mentioned that staining was variable within the core one day after the insult, probably due to differences in the severity of the lesion. Araki et al.2 used a mild MCAO (10 min) which resulted in a significant reduction in glycine binding sites 5 h later, whereas most of the decline in cortical MAP2 took place between the second and seventh day, a time range which parallels our findings. Compared to lesions produced with the MCAO model, our photothrombosis model produced much smaller lesions without the accompanying damage to subcortical structures. This may explain the differences observed in the amount and the time schedule of MAP2 decrease. However, during the first 24 h the lesioned tissue is not separated from the surrounding

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perilesional cortex by cysts and several neurotrophins which have beneficial effects on neuronal and cytoskeletal recovery33,36 are enhanced after ischemia, making it likely that a short-term recovery of the somatodendritic MAP2 pattern in the core reflects the action of neurotrophins up to the time before the demarcation zone and glial scar have fully developed. The increase of MAP2 immunoreactivity seen within the rim region of the perilesional cortex was not due to a general increase in background staining. It was confined mainly to dendrites as noted in paraffin as well as in Vibratome sections. However, in comparison to the results obtained for Py-immunopositive fibres, MAP2 immunoreactivity was restricted to the apical dendrites. Alterations in the metabolism of neurotransmitters and hyperexcitability of penumbral neurons have been suggested to be the major post-ischemic mechanisms leading to delayed neurotoxicity13,24,64,75,98,100 as well as to changes in MAP-associated proteins.68 Hyperexcitation is caused by an increase in glutamate and glutamate receptors, especially of the NMDA type, by a loss of inhibition due to the reduction of receptors for GABA55,83,91,96,98,100 and glycine2 and by a reduction of glutamate uptake from the synaptic cleft by glial cells.16,42,45,49,50 Hyperexcitation results in increased MAP2 expression.43 Neurofilaments Generally, the breakdown of immunoreactivity for non-phosphorylated and phosphorylated neurofilaments was much more rapid than that for MAP2 within the core. Changes in the pattern of neurofilament immunoreactivity after 4 h were somewhat similar to those described by Posmantur et al.71 for dendritic markers during the first 3 h after cortical injury as well as to those described by Geddes et al.28 for early post mortem changes. There were also differences between the lamina-specific reduction of SMI32 and that of MAP2 within the core. SMI32 immunoreactivity persisted longer in the deeper layers whereas MAP2 remained more pronounced in the upper layers. This may be due to the fact that SMI32 is more abundant in larger pyramidal cells than in the smaller ones of LII/III. The partial restoration and prolonged persistence of intralesional SMI32 immunoreactivity clearly distinguishes it from post mortem SMI32 immunoreactivity. The latter shows a rapid and continuous decrease which is obviously related to post mortem time.28 Blood vessels penetrate from the surface into the cortex, becoming thinner from upper to lower layers. It has been suggested that deeper vessels become closed by coagulated blood earlier than larger vessels in upper cortical layers. However, the partial recovery seen for SMI32 after 24 h below LIV indicates that diminished oxygen supply through blood vessels or other sources is not solely responsible for the early changes in the core.

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Neurofilament degeneration within the core may be linked to factors similar to those discussed above for MAP2, such as glial scar formation and widening and infiltration of the demarcation zone by WFA-positive cells. Interestingly enough, only a slight increase in SMI31 immunoreactivity occurred within the rim, whereas immunoreactive particles within the rim and the glial scar were observed for SMI31, SMI35 and to some extent also for neurofilament 200,000 mol. wt. These particles may represent remnants of retracted axons since these antibodies are axon-specific.69,73 In humans it has been found that SMI32 immunoreactivity is shifted to SMI35 and SMI31 immunoreactivity in homotopic cortical areas in the contralateral hemisphere.34 Somewhat similar findings were noted in transplanted neuronal grafts which also showed a slight decrease in nonphosphorylated neurofilaments and an increase in phosphorylated epitopes. These results were suggested to be related to the degree of neuronal injury.73 In our rats this contralateral, homotopic increase in neurofilaments was not very pronounced and consistently observed. However, a slight, homotopic increase in the number of GFAP-positive astrocytes was consistently present. Both observations show that retrograde transhemispheric neuro- and gliochemical changes are induced by the injury. The results are in agreement with observations indicative of a slight contralateral reduction in SMI32 and MAP2 immunoreactivity in comparison to controls.6 Such transhemispheric contralateral changes are certainly not restricted to GFAP and neurofilaments but have been observed for amyloid precursor protein,69 synaptophysin, GAP4382 and certain receptors for neurotransmitters (Zilles, unpublished observations) as well as in electrophysiological studies.13 146,000 mol. wt glycoprotein Py Py immunoreactivity was observed in frozen and Vibratome sections as well as in paraffin sections. However, in paraffin sections staining intensity was much weaker than in frozen or Vibratome sections. In paraffin sections similar changes within the core were found as described for SMI32. However, the rapid loss (within 24 h) of Py in the core was different from that seen for MAP2 and SMI32 and was not due to the weak staining observed after paraffin embedding, since Py was also lost in frozen sections in which the core was retained. This loss of Py is similar to that seen for the somato-axonal phosphorylated neurofilament epitopes and may reflect its intermediate position since Py is normally present in dendrites, somata and axons.93 The most prominent post-lesional change was the appearance of dense Py-positive fibre plexuses in the rim. Their appearance may be due to an increase in immunoreactivity within the basal dendrites as well as in axons terminating in the rim. The second increase of Py in the

ipsilateral lateroventral cortex was restricted to regions, which also contained Py staining under normal conditions. These perilesional changes contrasted sharply to the lack of change for SMI32. The functional significance of increasing amounts of Py is not yet clear. Py is most intensely expressed in hippocampal pyramids and hilar interneurons in fields CA3 and CA4, respectively, which are less vulnerable to ischemic insults than CA1 and CA2 pyramids.93 These findings suggest that Py may be part of the mechanisms involved in protecting neurons from ischemia. Proteoglycans Proteoglycans are involved in the formation of the so-called perineuronal nets and are parts of the extracellular matrix composed of polysaccharides linked to proteins of plasmalemmata. They can be detected by various methods, one of which is WFA binding which specifically recognizes N-acetylgalactosamines beta 1 (GalNAc beta 1–3 Gal) containing polysaccharides.17,20 WFA is present throughout the rodent cortex in an area- and lamina-specific pattern and is a component of the glia–neuron interface.12 Several proteoglycans have been identified in the CNS which inhibit or promote post-lesional axonal outgrowth53,59,60,81,88 and are involved in neurodegenerative diseases.29,80 In rat lesion models it has been found that the post-lesional expression of proteoglycans as well as that of GAP43, synaptophysin and fibroblast growth factor depends on the technique used to produce the lesions.84 The latter authors found clear differences in the expression patterns of these markers and other cell adhesion molecules between lesions produced by aspiration of cortical tissue or by thermocoagulation of pial blood vessels. We noticed only slight changes in staining intensity of WFA during the first days. These early changes may be due to edema and changes in calcium binding and GABA turnover, since WFA-positive interneurons also contain calcium-binding protein (CaBP) and/or GABA.10,11,32 Both CaBP, which acts as a calcium buffer, and GABA (an inhibitory neurotransmitter) are involved in lesion-induced hyperexcitation. Our study shows that WFA-positive proteoglycans do not contribute to the astrocyte–neuron interface within the rim, because WFA staining and GFAP immunoreactivity are not associated and exhibit different distribution patterns. One of the most striking changes occurred within the demarcation zone, which had no laminar-specific WFA binding. The inner part of the demarcation zone became invaded by strongly WFA-positive cells which separated the WFA-positive core from the outer part of the demarcation zone and the adjacent rim. The time at which these WFA-positive cells formed a capsule around the core coincides with the

Cytoskeletal changes after cortical ischemia

appearance of cyst-like vacuoles and with the second decrease in SMI32 and MAP2 in the core. It is likely that these WFA-positive cells represent lymphocytes and/or macrophages similar to cells seen in peripheral organs, where most thymocytes and splenocytes are WFA-positive.70 Macrophages involved in host–parasite interactions also bind WFA.25 This suggests that the WFA-positive cells in the demarcation zone are leucocytes involved in the digestion of cell debris. Several of these cells were seen in contact with injured blood vessels and their shape and appearance was very similar to endothelial cells which are rich in proteoglycans7,27,85,92 and known to invade cortical lesions.47 CONCLUSION

Defined photothrombosis of the cortex induces different reactions in tissue components of the lesion and perilesional cortex. Differences in cytoskeletal and glial alterations can be distinguished in zones

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surrounding the focus of the lesion. Some of the markers used to detect alterations were also affected in areas distant from the lesion, including even the contralateral hemisphere. Our model of defined cortical photothrombosis leads to transient and longlasting neurochemical and gliochemical alterations, which are in part similar to those seen in some other lesion models. In contrast to models utilizing larger lesions, defined cortical photothrombosis is a reliable model for studying small, restricted lesions. These occur frequently in humans22 where they are largely unnoticed, but where they have the potential to become initiators for slowly progressing neurodegenerative conditions. Acknowledgements—The authors thank Dr K. Rascher, Department of Morphological Endocrinology, for helpful discussions and corrections, K. Schubert for technical assistance and H. Hoffmann and A. Opfermann-Ru¨ngler for photographic and artwork. The study was supported by the Deutsche Forschungsgemeinschaft (SFB 194-A6 and B2).

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