Microtubular Proteolysis in Focal Cerebral Ischemia

Focal cerebral ischemia

All experimental methods were approved by the Institutional Animal Care and Use Committee of the University of Kentucky. Male spontaneously hypertensive rats (SHRs; Harlan Sprague-Dawley; Indianapolis, IN, U.S.A.) of 250–350 g body weight were used for each experiment described in this report. CCA-middle cerebral artery (MCA) occlusion was performed by a modification of the technique described by Brint et al. (1988). All rats were fasted overnight prior to surgical preparation. Each animal was anesthetized via intraperitoneal injection with 0.5 mg/g chloral hydrate for isolation of the MCA and CCA. One femoral artery was cannulated for blood sampling and measurement of MABP. Arterial blood was sampled 10 min before CCA–MCA occlusion in each animal prepared for immunocytochemical studies (group A; see below) and used to determine pH, P_ao₂, P_aco₂, hematocrit (Hct), and glucose. Rectal and temporalis muscle temperatures were maintained at 36–37°C by external warming. The left CCA was isolated through an anterior incision in the neck. For identification of the ipsilateral MCA, a second incision was made between the lateral canthus of the left eye and the corresponding external auditory canal to bare the underlying skull. The MCA was exposed through a 2-mm burrhole drilled 2–3 mm rostral to the fusion of the zygomatic arch and the squamosal bone under direct visualization with a Zeiss operating microscope. The dura was opened with a sharp needle, and an alloy wire (0.1 mm diameter) was inserted beneath the MCA just superior to the inferior cortical vein. The MCA was elevated from the cortical surface and cauterized by applying electrical current to the wire. The CCA was tied off with 4–0 silk and both incisions were closed. The animal was then returned to its cage and given free access to water and rat chow.

Thirty-two rats (group A) were killed and prepared for immunocytochemical studies after permanent CCA–MCA occlusion lasting for 15 min or 1, 6, 24, or 72 h (n = 5 or 6 at each time point). Group A also included five sham-ischemic animals subjected to nonocclusive manipulation of the left CCA and craniotomy performed without opening the dura before being killed for immunocytochemical studies 24 h later. An additional 16 animals (group B) underwent permanent CCA–MCA occlusion for 15 min or 1, 6, or 24 h (n = 4 at each time point), before being killed for sampling of normal and ischemic brain tissue used in Western blot analysis.

Primary antibodies

The primary antibodies used for immunocytochemical studies and Western blot analysis included anti-MAP2 (clone AP20, Boehringer–Mannheim Corporation, Indianapolis, IN, U.S.A.), Ab-37 (gift from Dr. Robert Siman of Cephalon, Incorporated, West Chester, PA, U.S.A.), and anti-T protein (τ-1, Boehringer–Mannheim Corporation). The AP20 monoclonal antibody recognizes all isoforms of dendrite-specific MAP2. Ab-37 is a polyclonal antibody that identifies a proteolytic fragment of spectrin produced by activated calpain (Roberts-Lewis et al., 1994). The anti-τ monoclonal antibody recognizes a phosphorylation-sensitive, nonphosphorylated epitope usually confined to axons (Binder et al., 1985). However, enzymatic dephosphorylation of tissue sections taken from adult rat brain will enhance τ-1 immunoreactivity in somatodendritic structures (Papasozomenos and Binder, 1987). Our Western blot studies were designed to include a second anti-τ monoclonal antibody, 8C11 (Athena Neurosciences, South San Francisco, CA, U.S.A.), that identifies a phosphorylation-independent epitope. Whereas τ-1 will recognize only nonphosphorylated protein, 8C11 will immunolabel all isoforms of τ. Optimal dilutions of all primary antibodies were verified in preliminary experiments and in recent publications from our laboratory (Geddes et al., 1994a,b; Schwab et al., 1994). Production, characterization, and specificity have been described for each antibody [AP20 (Binder et al., 1986); Ab-37 (Roberts-Lewis et al., 1994; Geddes et al., 1995); τ-1 (Binder et al., 1985; Papasozomenos and Binder, 1987; Szendrei et al., 1993); 8C11 (Vigo-Pelfrey et al., 1995)]. In the present study, controls for the specificity of the immunostaining included testing of primary antibodies in Western blots containing purified target antigen and omission of the same antibodies from immunocytochemical preparations (Schwab et al., 1994).

Immunocytochemistry and double-labeled immunofluorescence

After the designated period of CCA–MCA occlusion, each rat in group A was reanesthetized with an intraperitoneal injection of 0.5 mg/g chloral hydrate and perfused transcardially with 50 ml of heparinized normal saline, followed by 200 ml cold 4% paraformaldehyde in phosphate buffered saline (pH 7.4). The brain was removed and stored in the same fixative for 24 h, before being cryoprotected in 30% sucrose and frozen in powdered dry ice. The frozen brain was stored at −70°C before being cut into 30-μm coronal sections with a Leitz freezing sledge microtome. Three changes of Tris-buffered saline (TBS; 50 mM, pH 7.5) were used to rinse free-floating tissue sections. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide followed by transfer of the sections into TBS containing 0.1% Triton X-100. All tissue sections designated for immunolabeling of MAP2 or τ protein were then incubated with 1.5% normal horse serum, while those chosen for Ab-37 immunostaining were exposed to 1.5% normal goat serum. Sections prepared for immunolabeling of MAP2 or τ protein were incubated overnight in a solution of primary antibody (AP20 1:10,000; τ-1 1:20,000) maintained at room temperature. Other sections used to demonstrate calpain-mediated spectrin proteolysis were incubated with Ab-37 (1:10,000) under the same conditions. Biotinylated secondary antibodies were used for identification of AP20 and τ-1 (anti-mouse preadsorbed against rat serum, Vector Laboratories, Burlingame, CA, U.S.A.) or Ab-37 (anti-rabbit, Zymed Laboratories, Inc., South San Francisco, CA, U.S.A.). With colorimetric detection by the avidin-biotin technique (Elite ABC kit, Vector Laboratories). Diaminobenzidine was chosen as the peroxidase substrate. Cresyl violet was used to stain corresponding tissue sections.

Differences in the intensity of immunostaining between normal and ischemic tissue were quantified with a computer-assisted image analysis system, consisting of a Macintosh IIci computer (Apple Computer, Cupertino, CA, U.S.A.), a Quick Capture frame grabber card (Data Translations, Marlboro, MA, U.S.A.), an Hitachi CCD camera mounted on an Olympus BH2 microscope, and NIH Image Analysis software, v. 1.55. The system was calibrated against a Kodak Optical Density Standard (Eastman Kodak, Rochester, NY, U.S.A.). Quantification of immunostaining was conducted on brain sections obtained from the group A rats and exposed to the AP20, Ab-37, or τ-1 antibodies. The region of altered immunoreactivity within ischemic tissue was outlined in each coronal section and the optical density was determined in comparison to the equivalent area in the opposite hemisphere. Two tissue sections were examined for each animal and averaged to yield a final result.

After completion of the immunocytochemical studies, selected specimens were prepared for double-labeled immunofluorescence to determine if there was a temporal or spatial correlation between the cytoskeletal changes and the activation of calpain. Tissue sections were blocked in 1.5% horse and 1.5% goat serum before being incubated with both the mouse monoclonal AP20 (1:2,000) and the rabbit polyclonal Ab-37 (1:7,500) overnight at room temperature. The sections were washed three times in TBS and incubated for 1 h with a fluorescein isothiocyanate (FITC)-conjugated horse anti-mouse secondary antibody (Vector). To maximize the specificity of the Ab-37 antibody, the same sections were incubated for 1 h each with a biotinylated goat anti-rabbit secondary antibody and Texas Red avidin D (both Vector products). The sections were mounted on glass slides, cover-slipped with VectaShield to retard quenching of the fluorescent signal, and viewed with the Olympus BH2 microscope equipped for epifluorescence and filtered to differentiate the FITC and Texas Red flurophores.

Western blot analysis

Each animal in group B was reanesthetized with an intraperitoneal injection of 0.5 mg/g chloral hydrate, as described in the preceding paragraphs. It was then perfused transcardially with 30 ml of 15% India ink (v/v) diluted in normal saline. The brain was removed from the skull and chilled with a fluorinated hydrocarbon spray (Cryokwik, International Equipment Corporation, Needham Heights, MA, U.S.A.), before being cut into 2-mm coronal sections. Examination of the brain sections revealed that normally perfused tissue had been stained by the ink, whereas unperfused cortex remained pale. The core of unperfused tissue in the left hemisphere was resected with a scalpel and a comparable volume was removed from the same location in the right hemisphere. The samples were packaged individually and snap-frozen in liquid nitrogen. Each hemispheric sample was homogenized (20% w/v) in Tris-saline containing leupeptin (10 μ/M), EDTA-Na²⁺ (1 μM), pepstatin A (1 μM), and AEBSF (0.25 μM). The homogenates were centrifuged at 100,000 g for 45 min at 4°C. Preliminary studies showed that MAP2 and τ protein immunoreactivity was confined almost exclusively to the supernatant fractions, which were chosen for all quantitative procedures. The protein levels in the supernatants were determined by the micro-BCA method (Pierce Chemical Company, Rockford, IL, U.S.A.) and equivalent aliquots (each containing 20 μg protein) were separated by SDS-polyacrylamide gel electrophoresis. Isolation of MAP2 was conducted with 6.5% gels, but τ protein fractions were separated on preformed 10–20% linear gradient gels (Millipore, Bedford, MA, U.S.A.). A standard supernatant prepared from normal rat cortex was applied to each gel to ensure reproducibility and allow comparison of assays. The proteins were transferred electrophoretically onto polyvinylidene fluoride membranes (Millipore) and immunoreacted with the antibodies of interest (AP20 1:1,000; τ-1 1:2,000; 8C11 1:10,000). Formal quantification of immunolabeled proteins was accomplished by using the peroxidase substrate SG (Vector Laboratories) for colorimetric detection by a red laser densitometer (LKB Ultroscan XL, Pharmacia, Piscataway, NJ, U.S.A.). The relative quantities of proteins in the ischemic cortex were expressed as percentages of those in matching tissue obtained from the unaffected right hemisphere.

Statistical analysis

The physiologic data obtained from group A animals were expressed as means ± SD. Mean whole blood glucose levels, Hct, and arterial blood pH, P_aco₂, and P_ao₂ were compared between subgroups identified by duration of CCA–MCA occlusion, using Kruskal-Wallis one-way analysis of variance (ANOVA) on ranks. The mean optical density measurements made on unaffected and ischemic tissue immunolabeled with the different primary antibodies were tested by the same method, followed by post hoc analysis with paired t tests. Densitometrie readings of Western blots performed to determine the concentration of MAP2 and τ protein in the ischemic and unaffected cortex were compared by the same approach undertaken for the optical densities calculated from tissue sections, except that post hoc analysis was conducted with the Student-Newman-Keuls method for nonparametric multiple comparisons.

Immunocytochemistry of MAP2

Figure 1 shows representative 30-μm coronal sections of brain taken from the right cerebral hemisphere serving as an unaffected control and the left hemisphere demonstrating ischemic tissue sampled at 15 min or 1, 6, 24, or 72 h after initiation of CCA–MCA occlusion. Each section was immunostained with AP20 identifying all isoforms of MAP2, τ-1 recognizing a nonphosphorylated epitope of τ protein, or Ab-37 labeling calpain-cleaved spectrin by-products. The pattern of immunoreactivity observed with the AP20 antibody was identical in unaffected hemispheres and in sham-ischemic controls. The antibody labeled MAP2 fibers in healthy dendrites (Fig. 2A), as described in previous reports (Bernhardt and Matus, 1984; DeCamilli et al., 1984). Immunoreactivity was relatively consistent across cortical regions but was much fainter in the striatum. At higher magnification, a rich plexus of fiber staining was evident in the molecular layer of cerebral cortex. Layers 3 and 4 of the cortex featured prominent immunolabeling of straight apical dendrites and fainter background staining of dendritic branches. In layer 5, neuronal perikarya were only faintly immunostained (Fig. 3A).

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Fig. 1.

Representative 30 μm coronal sections of rat brain taken from the unaffected right hemisphere (C) or the ischemic left hemisphere between 15 min and 72 h after initiation of CCA–MCA occlusion. All photographs were taken at 1× magnification. The loss of MAP2 immunostaining by the AP20 antibody is gradual, beginning in isolated patches of ischemic tissue between 15 min and 1 h and progressing to complete absence of signal within the infarct zone after 24 h. Corresponding inversely to the loss of MAP2 signal is a gradual increase in the intensity of immunostaining with the Ab-37 antibody, representing accumulation of spectrin breakdown products resulting from calpain-mediated proteolysis. Tau-1 immunostaining of nonphosphorylated τ protein becomes intensified within the infarct zone as early as 15 min after CCA–MCA occlusion, but appears diminished in the adjacent transition area representing the ischemic penumbra. This apparent concentration of τ-1 immunostaining persists for 6 h, but decreases gradually between 24 and 72 h. The scale bar represents 2 mm.

Within 15 min of CCA–MCA occlusion, the dendritic processes of some neurons within the infarct had become coiled (Fig. 2B). Fine dendritic processes appeared less intense in ischemic tissue, such that the surviving apical dendrites stood out against an unstained background. After 1 h of CCA–MCA occlusion, MAP2 immunoreactivity appeared patchy within the infarct zone and could not be detected in the dorsolateral aspect of the ipsilateral striatum (see AP20 immunolabeling in Fig. 1). The loss of MAP2 immunolabeling was most prominent in layers 5 and 6 of ischemic cortex, as well as in the outer molecular layer. Toward the boundary of the infarct adjacent to the transition zone representing the ischemic penumbra, MAP2 immunostaining was diminished across all cortical layers. Coiled dendrites were abundant in the regions of decreased MAP2 immunostaining (Fig. 2C). Increased perikaryal MAP2 immunoreactivity was also evident (Fig. 3B), particularly in the transition area. After 6 h of ischemia, MAP2 immunolabeling was markedly diminished in the infarct zone (see AP20 immunolabeling in Fig. 1). While some apical dendritic fragments were visible, neuronal perikarya and the background neuropil were not immunostained. The infarct zone remained devoid of MAP2 immunostaining after 24 and 72 h of ischemia. There was an abrupt transition between the infarct and adjacent normal tissue, with an occasional neuron in the transition zone displaying enhanced perikaryal MAP2 immunoreactivity.

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Fig. 2.

Representative photomicrographs (×40) of alterations in dendritic MAP2 immunoreactivity during focal ischemia. In the unaffected right hemisphere (A) of an animal subjected to 15 min of CCA–MCA occlusion, MAP2 immunoreactivity is prominent in apical dendrites but is also evident in finerdendritic branches. In cortical tissue sectioned from the ischemic left hemisphere of the same rat (B), apical dendrites appear coiled and more intensely immunostained. The diffuse immunoreactivity of the neuropil is less intense than is shown in the unaffected hemisphere. In tissue subjected to 1 h of CCA–MCA occlusion (C), coiled apical dendrites remain visible whereas background immunostaining is largely absent. The scale bar represents 50 um.

Immunocytochemistry of calpain-cleaved spectrin fragments

As shown in Fig. 1, no specific Ab-37 immunoreactivity was observed in normal, nonischemic tissue. Scattered neuronal perikarya were visible in unaffected cortex of ischemic animals in both hemispheres of sham-ischemic rats, but the staining intensity was barely above background. After 15 min of CCA–MCA occlusion, isolated neurons within the infarct zone displayed enhanced perikaryal immunostaining (Fig. 3C). After 1 h of ischemia, neuronal perikarya were labeled more intensely, with immunoreactivity also evident in apical dendrites (Fig. 3D). The staining intensity of background neuropil was increased throughout the infarct zone. Figure 1 shows that intense Ab-37 immunolabeling was observed in a discrete region of the dorsolateral striatum ipsilateral to the cortical infarct in two of five rats, corresponding to the loss of MAP2 immunoreactivity seen in the same region. At 6 h following arterial occlusion, intense Ab-37 immunolabeling was evident throughout the infarct in all animals studied. Neuronal perikarya were no longer stained, but dendrites remained immunopositive. Of the five rats examined at this time point, one showed perikarya immunostaining of isolated cells in the striatum contralateral to the infarction, a second animal displayed perikaryal immunolabeling in cells of the ipsilateral reticular thalamic nucleus, and a third exhibited a large patch of immunostaining in the ipsilateral striatum. After 24 h of ischemia, Ab-37 immunostaining of the infarct remained intense and well demarcated in four of the five animals examined. The remaining animal showed no difference in Ab-37 immunoreactivity of ischemic tissue compared to its unaffected cortex or sham-ischemic controls. After 72 h of CCA–MCA occlusion, Ab-37 immunoreactivity persisted in the infarct zone and was also evident in the ipsilateral external capsule (3/6 rats), the lower ipsilateral cortex (1/6), and the striatum (2/6).

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Fig. 3.

Representative photomicrographs (×40) demonstrating increased perikaryal immunostaining of cytoskeletal proteins during focal ischemia. In the unaffected hemisphere (A), MAP2 immunoreactivity faintly outlines neuronal perikarya but causes intense labeling of the apical dendrites. In the infarct zone of the same animal subjected to 1 h of CCA–MCA occlusion (B), MAP2 immunoreactivity becomes intensified within perikarya of ischemic neurons. C: Scattered neurons within ischemic tissue sampled after 15 min of CCA–MCA occlusion show perikaryal immunostaining with the Ab-37 antibody. In tissue reacted with Ab-37 after 1 h of ischemia (D), spectrin breakdown products resulting from calpain-mediated proteolysis have accumulated in many apical dendrites and remain in isolated neurons. E demonstrates that perikaryal immunostaining with the τ-1 antibody becomes prominent in neurons within the transition zone representing the ischemic penumbra after 6 h of CCA–MCA occlusion. The scale bar represents 50 μm.

Immunocytochemistry of τ protein

Immunostaining with τ-1 in normal cortex was confined primarily to axons, as reported originally by Binder et al. (1985). In the cortex and striatum of sham-ischemic controls and in the unaffected hemisphere of animals subjected to CCA–MCA occlusion, τ-1 immunoreactivity was present in the neuropil whereas neuronal perikarya remained unlabeled. There were regional differences in τ-1 immunostaining of normal cortex. The frontoparietal region displayed prominent τ-1 immunostaining in cortical layers 3 and 4. However, this pattern of immunoreactivity was not seen in primary olfactory cortex, which had a clear zone in layer 2 and prominent labeling of the outer molecular layer. As is demonstrated in Fig. 1, alterations in the normal pattern of τ-1 immunoreactivity were noticeable within 15 min of CCA–MCA occlusion. There was enhancement of τ-1 immunostaining within the infarct zone, while immunolabeling became diminished within the transition area representing the ischemic penumbra. This pattern was also evident after 1 and 6 h of arterial occlusion, when some rats displayed increased τ-1 immunostaining in the dorsolateral striatum ipsilateral to the infarct (Fig. 1). Perikaryal immunostaining of neurons within the transition zone between ischemic, and unaffected tissue became increasingly prominent between 1 and 6 h (Fig. 3E). After 24 h of ischemia, τ-1 immunoreactivity in the neuropil was reduced and had been replaced by enhanced immunostaining in neuronal perikarya (Fig. 1). Increased τ-1 immunoreactivity was also evident in cells, presumably oligodendroglia, found in the corpus callosum. At 72 h, τ-1 immunoreactivity was diminished in the infarct zone and perikaryal immunostaining was no longer observed (Fig. 1).

Cresyl violet

Cresyl violet staining has been used previously to measure the brain infarct area in animal models of arterial occlusion (Benveniste et al, 1991). In this study, we relied upon tissue staining with cresyl violet as a qualitative measure demonstrating the progressive loss of neuronal architecture over time. We sought to determine the correlation between the early changes in the distribution of τ protein, MAP2, or calpain-cleaved spectrin and the alteration of Nissl substance stained by the cresyl violet. Figure 4 shows that only healthy cells with good laminar detail were observed in the unaffected cortex (C), as expected. There was no significant difference in cresyl violet staining of cells in the unaffected cortex and those in the infarct zone after 15 min of CCA–MCA occlusion. After 1 h of ischemia, there was minimal loss of staining but no pyknosis or neuronolysis. After 6 h of arterial occlusion, inspection of ischemic cortex showed shrinkage of neuronal volume with an inflammatory infiltrate in one animal and loss of approximately 30% of cells in another. By 24 h, the infarct zone had developed islands of necrosis with loss of 50–70% of neurons in all animals. Complete loss of cells occurred after 72 h, and the infarct zone appeared uniformly necrotic.

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Fig. 4.

Cresyl violet staining of unaffected cortex and ischemic tissue sampled at 1, 6, or 24 h (upper row: ×4; lower row: ×20). The expected laminar distribution of healthy cells was seen in unaffected cortex (C), shown in the upper row. The higher magnification view of the same sample (C in first panel of lower row) revealed the neuronal density and appearance found customarily in normal cortex. Ischemic tissue sampled after 1 h of CCA–MCA occlusion showed no pyknosis or neuronolysis, but widespread shrinkage of neuronal volume was observed at 6 h. Isolated neurons showed degenerating nuclei in tissue sampled after 6 h (arrows in second panel of lower row). By 24 h, the infarct zone in all animals showed islands of necrosis with loss of 50–70% of neurons. The scale bars represent 0.5 mm in the photographs shown in the upper row and 100 pm in the higher magnification views in the lower row.

Optical density measurements

Figure 5 shows optical densities measured from normal and ischemic cortex obtained from group A animals and processed for immunolabeling of MAP2, calpain-cleaved spectrin, and τ protein. The upper panel in Fig. 5 reveals data acquired from tissue immunolabeled with the AP20 antibody used to identify MAP2. Comparison of group means using the Kruskal-Wallis ANOVA revealed that MAP2 immunoreactivity differed significantly in the infarct zone over varying durations of ischemia (p ≤ 0.002). Post hoc analysis by paired t testing confirmed that ischemic tissue was significantly less immunoreactive than unaffected cortex after 1 (p ≤ 0.05), 6 (p ≤ 0.0001), 24 (p ≤ 0.0001), and 72 (p ≤ 0.02) h of CCA–MCA occlusion.

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Fig. 5.

Histogram of mean optical densities (ODs) ± SD measured in immunocytochemical specimens prepared from animals subjected to varying durations of ischemia (n = 5 or 6 for each sampling time). An overall mean OD measured from unaffected cortex in all animals (n = 32) is shown for interpretive convenience. For AP20 immunolabeling, the mean OD in ischemic tissue was significantly lower than that measured in unaffected cortex taken from the same animals after 1, 6, 24, and 72 h of CCA–MCA occlusion. For Ab-37, mean OD in ischemic cortex differed significantly from unaffected tissue at 6, 24, and 72 h. For the τ-1 antibody, only the relative increase in immunoreactivity measured in ischemic tissue at 6 h was significantly different from unaffected cortex, (a) ≤ 0.05, (b) ≤ 0.02, (c) ≤ 0.02, (d) p ≤ 0.001, and (e) p ≤ 0.0001.

The middle panel of Fig. 5 shows mean optical densities measured from ischemic cortex immunolabeled with Ab-37 to identify calpain-cleaved spectrin. Kruskal–Wallis ANOVA again revealed a highly significant difference between optical density means in groups sampled at varying durations of ischemia (p ≤ 2.0 × 10⁻¹⁰). Post hoc analysis showed that immunoreactivity of calpain-cleaved spectrin rose significantly in ischemic cortex at 6 (p ≤ 0.02), 24 (p ≤ 0.0001), and 72 (p ≤ 0.001) h. The lower panel of Fig. 5 shows mean optical densities measured from unaffected and ischemic cortex immunolabeled with τ-1. Kruskal-Wallis ANOVA revealed a significant difference between optical density means calculated for ischemic tissue in groups sampled at varying times (p ≤ 0.002). Post hoc analysis demonstrated that the increased τ-1 immunoreactivity observed in the infarct zone at 6 h differed significantly from the baseline signal in the unaffected cortex (p ≤ 0.002).

Quantitative Western blot studies

We performed quantitative Western blot analysis to determine if the qualitative differences in immunoreactivity between normal and ischemic cortex were associated with actual changes in the concentration of τ or MAP2 protein. Figure 6 shows a photograph of a representative Western blot incubated with the AP20 antibody recognizing MAP2 in unaffected and ischemic cortex. A single immunoreactive band at 220 kDa was used for quantitation of changes in MAP2 protein concentrations. Sampling of tissue during maturation of the infarct demonstrated a progressive loss of MAP2 as the duration of CCA–MCA occlusion extended to 24 h. This time-dependent loss of MAP2 protein correlated with the disappearance of MAP2 immunoreactivity observed in the infarct zone after 6 h, as shown in Fig. 1.

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Fig. 6.

Representative Western analysis of MAP2 in supernatant prepared by centrifugation of homogenized cortical tissue. Lane 1 shows the MAP2 content in cortical tissue removed from the unaffected hemisphere of an ischemic rat after 15 min of CCA–MCA occlusion. Lane 2 contains MAP2 from ischemic cortical tissue in the same animal. The remaining lanes show MAP2 in ischemic cortex sampled after 1 (lane 3), 6 (lane 4), or 24 (lane 5) h of arterial occlusion. Note the progressive loss of MAP2 protein from ischemic tissue over this time course. A single molecular weight marker of 180 kDa is shown on the left margin. The arrow on the right indicates the 220-kDa band used for quantification of MAP2 by laser densitometry.

Figure 7 contains two photographic images of the same samples displayed in Figure 6, but now immunoreacted with τ-1 (panel A) or 8C11 (panel B). Im-munolabeling with either antibody reveals that a well-demarcated pair of bands exceeding 48.5 kDa was observed in unaffected cortex. This doublet lost immunoreactivity and converted into a single band as the duration of CCA–MCA occlusion extended to 24 h. Correspondingly, a single band of lower molecular weight observed in unaffected cortex became more pronounced as ischemia progressed. This reciprocal phenomenon suggests that dephosphorylation of τ protein occurs as cortical tissue becomes more ischemic, correlating with our observation of a similar process in postmortem brain (Schwab et al, 1994).

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Fig. 7.

Representative Western analysis of τ protein in the same set of samples displayed in Fig. 6. The τ protein shown in A and B was obtained from an unaffected hemisphere (lane 1) or from ischemic cortical tissue sampled after 15 min (lane 2) or 1 (lane 3), 6 (lane 4), or 24 (lane 5) h of CCA–MCA occlusion. Panel A demonstrates the results of immunolabeling with τ-1 antibody to identify a phosphorylation-sensitive, nonphosphorylated epitope. Panel B shows the same set of samples processed for identification of a phosphorylation-independent epitope by the 8C11 antibody. The upper arrow to the right of both panels indicates a well-defined pair of bands in unaffected cortex that loses immunoreactivity and converts into a single band as the duration of CCA–MCA occlusion extends to 24 h. The lower arrow to the right of both panels identifies a single band that becomes more immunoreactive as ischemia is prolonged. Molecular weight markers in kilodaltons are shown on the left margin of both panels.

To better characterize the alterations in τ and MAP2 proteins, laser densitometry was performed on Western blot of unaffected and ischemic cortex sampled between 15 min and 24 h of MCA-CCA occlusion (Fig. 8). For this quantitative analysis, τ protein was identified only by the τ-1 antibody. Comparison of optical density values associated with MAP2 quantified in ischemic cortex showed that there was heterogeneity of group means detected by Kruskal-Wallis ANOVA (p ≤ 0.04). Post hoc testing demonstrated that the mean optical density representing MAP2 antigen in ischemic cortex sampled at 24 h was significantly lower than those obtained at earlier time points (p ≤ 0.05). The optical densities associated with MAP2 antigen in unaffected cortex remained uniform across all sampling times. There were no significant differences in optical densities associated with τ protein in unaffected or ischemic tissue.

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Fig. 8.

Histograms of mean optical densities (ODs) ± SD obtained from quantitative laser densitometry of Western blots similar to those shown in Figs. 6 and 7. Four animals were prepared for each duration of CCA–MCA occlusion. Note the progressive reduction of OD associated with AP20 immunostaining of MAP2 in ischemic tissue subjected to increasing duration of hypoperfusion, (a) ≤ 0.05 between mean OD at 24 h and all preceding time points.

Double-labeled immunofluorescence

To determine the temporal and spatial relationship between cytoskeletal alterations and the activation of calpain in individual neurons affected by ischemia, tissue sections taken from group A animals were incubated simultaneously with AP20 and Ab-37 complexed with fluorescently labeled secondary antibodies. All images used for this double-labeled immunofluorescence technique were taken from within the infarct, avoiding the transition zone between ischemic and unaffected tissue. As is shown in Fig. 9B, perikaryal Ab-37 immunoreactivity was already apparent after 15 min of MCA-CCA occlusion. The same region immunolabeled with AP20 (Fig. 9A) showed no somal accumulation of MAP2. To better delineate when somal accumulation of MAP2 would be seen after calpain activation occurred at 15 min, three rats were subjected to 30 min of arterial occlusion and added to those already prepared for later time points in group A. Representative photomicrographs taken of the ischemic tissue in one of these animals are shown in Fig. 9 (C and D) and demonstrate that somal accumulation of MAP2 and calpain-activated spectrin proteolysis colocalized in the same ischemic neurons. Identical findings were noted in the other two rats in the 30-min group. This colocalization of AP20 and Ab-37 immunolabeling persisted in animals subjected to 60 min of CCA–MCA occlusion (data not shown). However, prolonging cortical ischemia to 6 h produced little demonstrable perikaryal MAP2 immunoreactivity and caused fragmentation of Ab-37 immunofluorescent labeling (data not shown). Surprisingly, somatic MAP2 immunoreactivity reappeared in neurons located within the infarct after 24 h of CCA–MCA occlusion (Fig. 9E). Although there was widespread Ab-37 immunofluorescent labeling within the same area at 24 h (Figure 9F), only rarely did cells coexpress both activities. Photomicrographs of ischemic tissue obtained after 72 h of arterial occlusion were not included in Fig. 9, because the severity of necrotic damage prevented reliable interpretation of the immunofluorescent labeling.

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Fig. 9.

Representative photomicrographs (×40) of neurons within ischemic cortex prepared for double-labeled immunofluorescent studies of MAP2 and calpain-cleaved spectrin immunoreactivity. A, C, E: Tissue sections incubated with AP 20 complexed with a fluorescein isothiocyanate-conjugated secondary antibody to identify MAP2 protein. B, D, and F: Calpain activity in the same tissue sections incubated with Ab-37 complexed with Texas Red avidin D. Panels A and B are focused on ischemic neurons in brain sampled after 15 min of CCA–MCA occlusion. The photomicrograph in panel A demonstrates that there is no somal accumulation of MAP2 at 15 min. In panel B, calpain activity appears to be distributed in cytoplasmic granules. Panels C and D show ischemic neurons in brain sampled after 30 min of CCA–MCA occlusion and reveal that somal MAP2 accumulation and calpain activity are colocalized in the same cells. Panels E and F demonstrate the effects of 24 h of ischemia. There is robust immunofluorescent labeling of somal MAP2 protein in panel E, without true colocalization of calpain activity in panel F (see text for additional details). The scale bar represents 50 μm.