Glutamate receptor antagonists inhibit calpain-mediated cytoskeletal proteolysis in focal cerebral ischemia

Brain Research 810 Ž1998. 181–199

Research report

Glutamate receptor antagonists inhibit calpain-mediated cytoskeletal proteolysis in focal cerebral ischemia Stephen L. Minger a,b,c , James W. Geddes b,d , Mary L. Holtz f,g , Susan D. Craddock Sidney W. Whiteheart e , Robert G. Siman h , L. Creed Pettigrew a,b,c,f,g,)

a,b

,

a

The Stroke Program of the UniÕersity of Kentucky Chandler Medical Center, USA The Sanders-Brown Center on Aging, UniÕersity of Kentucky Chandler Medical Center, USA c Department of Neurology, UniÕersity of Kentucky Chandler Medical Center, Lexington, KY, USA Departments of Anatomy and Neurobiology, UniÕersity of Kentucky Chandler Medical Center, Lexington, KY, USA e Department of Biochemistry, UniÕersity of Kentucky Chandler Medical Center, Lexington, KY, USA f The Graduate Center for Toxicology, UniÕersity of Kentucky Lexington, KY, USA g Department of Veterans Affairs Medical Center, Lexington, KY, USA h Cephalon, West Chester, PA, USA b

d

Accepted 1 September 1998

Abstract Excitatory amino acids may promote microtubular proteolysis observed in ischemic neuronal degeneration by calcium-mediated activation of calpain, a neutral protease. We tested this hypothesis in an animal model of focal cerebral ischemia without reperfusion. Spontaneously hypertensive rats were treated with 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo-ŽF.quinoxaline ŽNBQX., a competitive antagonist of the neuronal receptor for a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ŽAMPA., or cis-4-wphosphono-methylx-2piperidine carboxylic acid ŽCGS 19755., a competitive antagonist of the N-methyl-D-aspartate ŽNMDA. receptor. After treatment, all animals were subjected to permanent occlusion of the middle cerebral artery for 6 or 24 h. Infarct volumes measured in animals pretreated with CGS 19755 after 24 h of ischemia were significantly smaller than those quantified in ischemic controls. Rats pretreated with NBQX showed partial amelioration of cytoskeletal injury with preserved immunolabeling of microtubule-associated protein 2 ŽMAP 2. at 6 and 24 h and reduced accumulation of calpain-cleaved spectrin byproducts only at 6 h. Prevention of cytoskeletal damage was more effective after pretreatment with CGS 19755, as shown by retention of MAP 2 immunolabeling and significant restriction of calpain activity at both 6 and 24 h. Preserved immunolabeling of tau protein was observed at 6 and 24 h only in animals pretreated with CGS 19755. Western analysis performed on ischemic cortex taken from controls or rats pretreated with either NBQX or CGS 19755 suggested that loss of tau protein immunoreactivity was caused by dephosphorylation, rather than proteolysis. These results demonstrate a crucial link between excitotoxic neurotransmission, microtubular proteolysis, and neuronal degeneration in focal cerebral ischemia. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Cerebral ischemia; Microtubule-associated protein; Calpain; Neuronal degeneration

1. Introduction Cytoskeletal degradation, represented by the loss of microtubule-associated proteins ŽMAPs. shown by immunochemical methods, is a sensitive indicator of neuronal injury in ischemic brain. Disruption of microtubular struc) Corresponding author. The Stroke Program of the Sanders-Brown Center on Aging, 101 Sanders-Brown Building, University of Kentucky College of Medicine, 800 South Limestone Street, Lexington, KY 405360 2 3 0 , U S A . F ax : q 1 -6 0 6 -2 5 7 -8 9 9 0 ; E -m ail ad d ress: [email protected]

tures in the apical dendrites of CA1 neurons may begin as soon as 5 min after unilateral common carotid artery ŽCCA. occlusion in gerbils w63x and is accompanied by the simultaneous loss of dendrite-specific MAP 2 immunoreactivity w31x. In global forebrain ischemia, MAP 2 immunostaining begins to disappear from subiculum-CA1 after no more than 1 h of post-ischemic reperfusion in gerbils subjected to bilateral CCA occlusion w64x. Matesic and Lin w40x confirmed that these changes did not simply reflect intracellular redistribution of the MAP 2 antigen by demonstrating that MAP 2 immunoreactivity was no longer detectable in western blots of gerbil hippocampal tissue

0006-8993r98r$ - see front matter q 1998 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 8 . 0 0 9 2 1 - 4

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obtained one day after 5 min of bilateral ischemia. In our study of cytoskeletal changes in rat brain subjected to global ischemia induced by the four-vessel occlusion Ž4VO. technique w21x, we found that loss of MAP 2 immunostaining in the CA1 region was not observed consistently until 72 h afterward. Our results mirrored those of Tomioka et al. w61x, who described similar variability in MAP 2 immunolabeling of CA1 neurons in 4VO animals. Miyazawa et al. w43x have suggested that fluctuations in the pattern of MAP 2 immunoreactivity may be temperaturedependent, a recurrent theme underlying many observations made in rodent models of global cerebral ischemia. A growing body of evidence suggests that cytoskeletal changes observed in ischemic neurons may result from microtubular proteolysis caused by calpain, a nonlysosomal, calcium-activated neutral protease Žsee Bartus w1x for review.. Calpain is distributed ubiquitously in all mammalian cells as procalpain, an inactive oligomer composed of a 75 to 80 kDa catalytic subunit and a 30 kDa regulatory fraction w41x. The two isozymic forms of calpain in cerebral tissue differ in the composition of the catalytic subunit and in the cell of origin. Calpain I Žm-calpain. is localized to neurons and may be activated in the presence of micromolar concentrations of calcium under in vitro conditions w41x. Calpain II Žm-calpain. is present in glial cells and in axonal tracts w36x but must be exposed to millimolar concentrations of calcium to be activated in vitro. The intracellular concentration of calcium in ischemic neurons will reach the micromolar range required for activation of calpain I, due to the release of this ion from stores within cellular organelles or enhancement of calcium conductance from the extracellular space w32x. Calcium-mediated activation of calpain begins with the translocation of procalpain to a target substance in the cell membrane or cytoskeleton, followed by autolytic transformation of the precursor to the active calpain moiety Žsee Croall and DeMartino w13x for review.. Once activated, calpain may cause proteolysis of neurofilaments, MAPs, and other cytoskeletal proteins before returning as a soluble, inactive form to the cytoplasm. Roberts-Lewis et al. w49x developed a family of antibodies that recognize a proteolytic fragment of a-spectrin produced by activated calpain. One of these antibodies, Ab-37, immunolabeled CA1 pyramidal neurons after 5 min of bilateral common carotid arterial occlusion ŽBCCAO. in the gerbil. The immunoreactivity persisted from 24 to 48 h and correlated with the well-recognized vulnerability of CA1 neurons to cellular death associated with reperfusion after global ischemia w30x. Immunostaining was also observed in the neocortex, striatum, and thalamus as early as 30 min after reversal of BCCAO but had begun to fade after 24 h. In our recent work w47x, we used Ab-37 as a functional assay of calpain activity in an animal model of focal brain infarction. The most sensitive indicator of ischemic neuronal change was the coiled, disfigured appearance of apical dendrites observed after no more than

15 min of ischemia. Double-labeled immunofluorescence studies showed that perikaryal accumulation of MAP 2 was observed in ischemic neurons after no more than 30 min of focal ischemia but was preceded by Ab-37 immunoreactivity in the neuronal cytoplasm at 15 min. Subsequently, maximal immunolabeling with Ab-37 was shown to accompany loss of MAP 2 immunoreactivity within ischemic cortex after 6 h of arterial occlusion. Having demonstrated that calpain activation precedes and accompanies microtubular breakdown during the early stages of ischemic neuronal degeneration, we focused the current work upon the role of excitotoxic neurotransmission in calpain-mediated cytoskeletal proteolysis. RobertsLewis et al. w49x found that pretreatment with MK-801, a noncompetitive antagonist of the N-methyl-D-aspartate ŽNMDA. receptor, completely eliminated Ab-37 immunoreactivity outside the hippocampus and caused partial loss of immunostaining in CA1 after transient forebrain ischemia in the gerbil. These findings suggest that NMDA receptor activation is partly responsible for calpain-induced proteolysis of a-spectrin in ischemic brain, probably by increasing the intraneuronal content of calcium. Similar conclusions were drawn by Seubert et al. w52x in an earlier report showing that MK-801 prevented spectrin proteolysis in the same animal model. Siman et al. w56x provided additional evidence for the putative interaction between excitatory neurotransmission and calpain activity by demonstrating that a glutamate receptor antagonist prevented spectrin breakdown and CA1 cell death observed after intracerebroventricular injection of NMDA. Del Cerro et al. w17x confirmed that blockade of NMDA receptors by MK-801 will inhibit calpain-induced spectrin proteolysis in hippocampal cultures. Although excitotoxic mechanisms and calpain activation must figure prominently in ischemic cytoskeletal damage w47x, no previous studies have examined the capacity of glutamate receptor antagonists to reverse these effects in focal brain infarction. In the present investigation, we used an animal model of permanent focal cerebral ischemia to test the hypothesis that calpainmediated cytoskeletal proteolysis may be modulated by excitotoxic neurotransmission.

2. Materials and methods 2.1. Production of 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 ŽSHRs; Harlan Sprague–Dawley, Indianapolis, IN. of 250 to 320 g b.wt. were used in all experiments Žtotal n s 92.. Permanent focal cerebral ischemia was induced by occlusion of the unilateral CCA and middle cerebral artery ŽMCA. as described by Brint et al. w10x and modified in our laboratory w20,47,58x. All rats were fasted overnight prior to

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surgical preparation. For isolation of the MCA and CCA, each animal was anesthetized with an intraperitoneal injection of 68 mgrkg ketamine and 10 mgrkg rompun. Rectal and temporalis muscle temperatures were maintained at 36 to 378C by external warming. One femoral artery and the corresponding vein were cannulated for arterial and venous access. Arterial blood samples taken 10 min before and 15 min after the occlusion procedure were used to determine pH, PaO 2 , PaCO 2 , and mean arterial blood pressure ŽMABP.. Hematocrit and glucose were assayed only on the pre-ischemic samples. The left CCA was isolated through an anterior incision in the neck. The ipsilateral MCA was identified through a second incision made between the lateral canthus of the left eye and the corresponding external auditory canal to bare the underlying skull. Under a Zeiss operating microscope, a 2-mm burrhole was drilled 2 to 3 mm rostral to the fusion of the zygomatic arch and the squamosal bone to expose the MCA. The dura was opened with a sharp needle and an alloy wire Ž0.1-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. Animals included in a shamischemia control group underwent nonocclusive manipulation of the left CCA and craniotomy without penetration of the dura. Several randomly selected animals received pretreatment with glutamate receptor antagonists prior to induction of focal ischemia. One group was given intraperitoneal 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo-ŽF.quinoxaline ŽNBQX; Toronto Research Chemicals, North York, Ontario, Canada., a competitive antagonist of the neuronal receptor for a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ŽAMPA., at a dose of 30 mgrkg 30 min prior to CCA-MCA occlusion. The second group was given 10 mgrkg of cis-4-wphosphono-methylx-2-piperidine carboxylic acid ŽCGS 19755; gift of Dr. Al Kotake, CibaGeigy, Summit, NJ., a competitive antagonist of the Nmethyl-D-aspartate ŽNMDA. receptor, by bolus injection through the femoral vein cannula 5 min before ischemia. Ischemic control animals received drug-free vehicle by the intraperitoneal or intravenous route, depending upon assignment to match with an ischemic rat treated with NBQX or CGS 19755. 2.2. Measurement of infarct Õolume Twenty-four hours after induction of permanent focal ischemia, five animals were randomly chosen from the ischemic group and five from each of the drug-treated groups for quantitation of infarct volume, using the triphenyltetrazolium chloride ŽTTC. staining procedure of Bederson et al. w4x. Each rat was anesthetized with an

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intraperitoneal injection of 500 mgrkg of chloral hydrate before undergoing transcardiac perfusion with heparinized saline. The brain was removed and chilled at y208C for 15 min before being cut into 1-mm coronal sections with a rat brain matrix. The resulting coronal sections were placed in 2% TTC and incubated for 20 min at 378C. Sections were then immersed in 10% formalin and stored in the dark at 48C. The infarct area in each section was determined by computer-assisted videodensitometry, using a system consisting of a Power Macintosh 7100r80 computer ŽApple Computer, Cupertino, CA. equipped with an LG-3 frame grabber card ŽScion, Frederick, MD., an Hitachi CCD camera mounted with a 55-mm Nikon MicroNikkor lens on a photocopy stand, and NIH Image Analysis software ŽScion Image, v. 1.59.. The system was calibrated with a Kodak Optical Density Standard ŽEastman Kodak, Rochester, NY.. An optical density threshold was taken from healthy gray matter in the unaffected right cortex and used to create a black-and-white image that was superimposed over the videophotograph, making the edges of the infarct clearly visible and easily outlined. The software was then used to compute the total area of infarcted cortex, along with the area of the unaffected cortex in the contralateral hemisphere. The method of Swanson et al. w60x was used to correct all measurements of infarct volume for tissue expansion caused by cerebral edema. The total volume of the infarction was determined by multiplying the calculated infarct area in each coronal section by the number of slices Ž n s 7. and the thickness of each slice Ž1 mm uniformly.. Resulting volumes were expressed as percentage of the total area of the affected left hemisphere. 2.3. Primary antibodies For the detection and localization of neuronal cytoskeletal proteins, immunocytochemical procedures were performed with AP20 ŽBoehringer-Mannheim, Indianapolis, IN; 1:10,000 dilution., a monoclonal antibody recognizing all isoforms of MAP 2 w8x, and Tau-1 ŽBoehringerMannheim; 1:10,000 dilution., a monoclonal antibody specific for a phosphorylation-dependent, nonphosphorylated axonal epitope of tau w7x. The same antibodies were used for western analysis at dilutions of 1:7500 for AP20 and 1:10,000 for Tau-1. To determine the total pool of tau protein, 8C11 ŽAthena Neurosciences, South San Francisco, CA., a monoclonal antibody that recognizes a phosphorylation-independent epitope of tau was used in the western studies Ž1:10,000 dilution.. Whereas Tau-1 recognizes only nonphosphorylated tau, 8C11 immunolabels all isoforms of tau, independent of their phosphorylation state w62x. To determine the temporal and spatial activation of calpain, two polyclonal antisera recognizing a proteolytic fragment of a-spectrin cleaved by activated calpain w49x were employed: Ab-37 at 1:10,000 dilution in immuno-

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cytochemistry and Ab-38 at 1:2000 in western analysis Žboth antibodies were gifts of Dr. Robert Siman of Cephalon, West Chester, PA.. Optimal dilutions of all primary antisera used in the immunocytochemical studies have been verified in recent publications from our laboratory w19–21,47,50x. Optimal dilutions for western analysis were obtained by extensive control experiments to verify linearity of protein signal Ždata not shown.. 2.4. Immunocytochemistry After either 6 or 24 h of CCA-MCA occlusion, ischemic controls and animals pretreated with either NBQX or CGS 19755 prior to the onset of permanent focal ischemia were randomly selected for immunocytochemical analysis Ž n s 5–6 for each time point and group.. The first time point was chosen to show the effect of NMDA or AMPA receptor blockade on MAP 2 and spectrin proteolysis occurring concomitantly at 6 h, as demonstrated in our previous work w47x. The 24-h sampling time was selected to correlate with quantitative measurements of infarct volume performed in parallel groups of animals. In addition to nonoperated and sham-ischemic controls, groups of shamischemic animals given either NBQX or CGS 19755 Ž n s 3 for each group. were analyzed 24 h after drug administration. Each animal was anesthetized with an intraperitoneal injection of 500 mgrkg chloral hydrate before being perfused transcardially with 50 ml of heparinized normal saline, followed by 200 ml cold 4% paraformaldehyde in phosphate buffered saline ŽPBS, pH 7.4.. Brains were removed and postfixed overnight in 4% paraformaldehyde at 48C, cryoprotected in 30% sucrose for approximately three days, and then frozen on powdered dry ice and stored at y708C. A sliding microtome ŽMicrom HM440E, Carl Zeiss, Thornwood, NY. was used to obtain 30-mm coronal sections throughout the infarct zone of each animal. Sections adjacent to those used for immunocytochemical analysis were stained with cresyl violet to visualize cell somata and to estimate neuronal survival. The resulting coronal brain sections were processed for immunocytochemical analysis using ‘free-floating’ techniques. Briefly, sections were washed with three changes of TRIS buffered saline ŽTBS; 50 mmolrl, pH 7.5. and then sequentially incubated in 3.0% hydrogen peroxide ŽH 2 0 2 ., 0.1% Triton X-100, and either 1.5% horse serum or goat serum, depending on the animal source of the secondary antibody Žall in TBS.. Sections were incubated overnight with primary antibody at room temperature, washed, and then incubated with either rat serum preadsorbed biotinylated horse anti-mouse Žmonoclonal antibodies. or unadsorbed goat anti-rabbit Žpolyclonal antisera. secondary antibodies ŽVector Laboratories, Burlingame, CA. for 1 h at room temperature. Sections were washed with TBS with 0.1% Triton X-100, incubated for 1 h in TBS with 0.1% Triton X-100 containing an avidin–biotin complex ŽVectastain ABCrElite Kit, Vector Laboratories.,

and developed with diaminobenzidine ŽVectastain Peroxidase Substrate Kit, Vector Laboratories.. Differences in the intensity of immunostaining between normal and infarcted tissue were quantified by computerassisted image analysis, using the system described above for quantitation of infarct volumes. The infarcted region in each tissue section was outlined and the mean optical density measured in comparison to the equivalent area in the opposite hemisphere. For each animal, two tissue sections were processed for each antibody and the optical density differences averaged to yield a final result for each pair of sections per primary antibody. 2.5. Western analysis After either 6 or 24 h of ischemia, five animals were randomly selected for western analysis from the untreated ischemic animals and each of the glutamate receptor antagonist groups. Five nonoperated control animals were also chosen. All animals were anesthetized with chloral hydrate and perfused transcardially with 30 ml of 15% India ink Žvrv. in normal saline. Brains were removed, chilled with a fluorinated hydrocarbon spray ŽCryokwik, International Equipment, Needham Heights, MA., and cut into 2-mm coronal sections. Our experience with this method w47x has shown that normally perfused tissue is stained with ink, but unperfused cortex remains pale and unstained. 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 undamaged contralateral hemisphere. The unperfused sample was not contaminated by stained tissue taken from the penumbral region surrounding the ischemic cortex. All samples were packaged individually and snap-frozen in liquid nitrogen. Each sample was individually homogenized Ž20% wrv. in TBS containing leupeptin Ž10 mmolrl., EDTA Ž1 mmolrl., pepstatin A Ž1 mmolrl., and AEBSF Ž250 mmolrl.. Protein levels were determined by the micro-BCA method ŽPierce Chemical, Rockford, IL. and equivalent concentrations of the samples were separated by SDS polyacrylamide gel electrophoresis ŽSDS-PAGE.. Standard 8.0% polyacrylamide gels were used for electrophoresis of MAP 2 and calpain-cleaved spectrin, and 12% gels for separation of tau proteins. The electrophoretic gels prepared for MAP 2 and tau were loaded with 5 mg of proteinrlane; gels prepared to quantify calpain-cleaved spectrin byproducts were loaded with 20 mg of proteinrlane. A standard homogenate of normal rat cortex of the appropriate protein concentration was applied to each gel to ensure reproducibility and to allow comparison between blots. Following electrophoresis, proteins were electrophoretically transferred onto 0.45-mm pore-size nitrocellulose membrane ŽSchleicher and Schuell, Keene, NH. and immunoreacted with AP-20, Tau 1, 8C11, or Ab-38. The efficiency of protein transfer was determined by staining the nitrocellulose membrane with Ponceau S. Nitrocellulose membranes

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were blocked in 5% dried nonfat milk in TBS for 45 min and then incubated with the optimal concentration of primary antibody in 5% milk overnight at 48C. After three 20-min washes with TBS, the membranes were exposed to the appropriate horseradish peroxidase ŽHRP.-conjugated secondary antibodies diluted in 5% milk for 45 min and washed an additional three times in TBS. The membrane was then incubated in SuperSignal Chemiluminescence HRP Substrate ŽPierce Chemical. for 3 to 5 min, exposed to Kodak T-Mat blue X-ray film for an appropriate period of time Ž1–60 s., and developed. Formal quantification of immunoreactive proteins was accomplished using a red laser densitometer ŽLKB Ultrascan XL, Pharmacia, Piscataway, NJ.. The relative quantities of immunoreactive proteins in the ischemic cortex were expressed as percentages of those in matching tissue obtained from the unaffected contralateral hemisphere. 2.6. Statistical analysis The physiologic data obtained from all animals were expressed as means" S.D. and were analyzed by two-way Žexperimental group condition and sampling time. analysis of variance ŽANOVA. with repeated measures. Volumetric determinations of infarct volumes in TTC-stained sections were compared using unpaired t-tests. The mean corrected optical densities of ischemic animals were compared to ischemic animals pretreated with either NBQX or CGS 19755, using unpaired t-tests. Densitometric measurements of western blots performed to determine the concentration of MAP 2, tau, and calpain-cleaved spectrin protein in the untreated ischemic and drug-treated ischemic cortex were also compared using unpaired t-tests. Data obtained from ischemic control animals given drug-free vehicle by the intraperitoneal or intravenous route before being prepared for the assay of interest were pooled for statistical comparison.

3. Results

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showed no significant group by time interaction on two-way ANOVA, suggesting that the ischemic control and drug treatment groups reacted uniformly to changes in physiologic status caused by ischemia. The lowest mean arterial blood pH measured before CCA-MCA occlusion was 7.33 " 0.10 in ischemic controls Ž n s 20., compared to the lowest value of 7.36 " 0.06 in NBQX-treated animals Ž n s 15. sampled after the onset of ischemia. The corresponding levels of mean arterial blood pCO 2 were 51 " 8 mmHg in ischemic controls sampled before ischemia and 42 " 7 mmHg in NBQX-treated animals studied after its onset. The mild hypercapnea exhibited in the ischemic control group during pre-ischemic sampling is seen typically in lightly-anesthetized rodents not maintained on mechanical ventilation. Mild hyperventilation during ischemia causes reversal of the hypercapnea, as shown in the NBQX-treated animals sampled after the onset of arterial occlusion. Oxygenation remained uniformly adequate in all groups at both sampling times, as shown by the lowest pre-ischemic mean arterial blood pO 2 of 75 " 14 mmHg in CGS 19755-treated animals Ž n s 20. and the lowest mean of 73 " 10 mmHg in the same treatment group sampled after the onset of ischemia Ž n s 20.. The lowest MABP ranged from 96 " 24 mmHg sampled before ischemia in NBQX-treated animals Ž n s 20. to 98 " 18 mmHg in the same group Ž n s 15. studied after the onset of arterial occlusion. 3.2. Infarct Õolume Twenty-four hours after permanent CCA-MCA occlusion, ischemic control animals and a parallel series of rats pretreated with either NBQX or CGS 19755 prior to focal ischemia Ž n s 5 in each group. were euthanized for infarct volume determinations. The administration of 10 mgrkg CGS 19755 5 min prior to CCA-MCA occlusion reduced mean infarct volume to 41 " 21 mm3, compared to 92 " 21 mm3 in ischemic controls Ž p s 0.002.. Pretreatment with 30 mgrkg NBQX given 30 min before ischemia reduced mean infarct volume to 76 " 30 mm3 Ž p s n.s. compared to ischemic controls or CGS 19755-pretreated animals..

3.1. Physiology 3.3. Immunocytochemical analysis Physiologic data were computed from arterial blood samples taken 10 min before and 15 min after the onset of ischemia. The results confirmed that all animals were uniformly stable prior to CCA-MCA occlusion. There were no significant between-group differences in whole blood glucose and hematocrit, which were analyzed only in pre-ischemic samples. The mean glucose level ranged from 4.18 " 0.44 mmolrl in CGS 19755-treated animals Ž n s 20. to 4.58 " 0.48 mmolrl in ischemic controls Ž n s 20.. Mean hematocrit ranged from 52.4 " 1.4% in ischemic controls Ž n s 19. to 53.1 " 2.9% in NBQX-treated rats Ž n s 20.. Arterial blood pH, pCO 2 , and pO 2 sampled before and after the initiation of CCA-MCA occlusion

3.3.1. MAP 2 Coronal brain sections obtained from ischemic controls or animals pretreated with NBQX or CGS 19755 before CCA-MCA occlusion were processed for immunocytochemistry with the AP-20 antibody ŽFig. 1.. In the ischemic control animals, significant reductions in the density of MAP 2 immunoreactivity were already apparent 6 h after the onset of permanent focal ischemia. Most ischemic animals displayed an almost complete loss of MAP 2 immunoreactivity within the infarct; scattered immunoreactive processes were observed to extend from the pial surface to approximately lamina II in four of the five rats.

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Fig. 1. Representative photomicrographs Ž2 = . showing MAP 2 immunoreactivity ŽAP-20. in the infarcted cerebral cortex of ischemic controls ŽIC. and spontaneously hypertensive rats pretreated with either NBQX or CGS 19755 before 6 ŽA. or 24 ŽB. h of permanent focal ischemia. The zone of ischemic tissue showing loss of MAP 2 immunoreactivity appeared smaller in rats pretreated with CGS 19755 before undergoing 6 h of occlusion of the common carotid and middle cerebral arteries. Scale bar s 500 mm.

The remaining animal had a less complete infarct in which islands of residual MAP 2 processes were observed. This pattern of MAP 2 immunoreactivity remained unchanged in samples examined 24 h after ischemia; four of the five rats displayed an almost complete loss of MAP 2 immunoreactivity and one had residual MAP 2 immunoreactive islands within the infarct. Both of the glutamate receptor antagonists were effective in attenuating the loss of MAP 2 immunoreactivity 6 h after permanent focal ischemia, although CGS 19755 was clearly superior to NBQX in preserving cytoskeletal struc-

ture within the infarct. The most significant therapeutic effect of NBQX was in the enhancement of MAP 2 immunoreactivity in processes extending from the pial surface to lamina II. In comparison, at this early postischemic time point, CGS 19755 appeared to almost fully protect MAP 2 immunoreactivity in a transition zone corresponding to the ischemic penumbra, although most of the immunoreactivity was lost in the core Ž3r5 animals.. Surprisingly, three of five animals pretreated with NBQX and examined after 24 h of CCA-MCA occlusion showed increased MAP 2 immunoreactivity within the

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transition zone. This effect was not observed in the parallel group of rats pretreated with the same drug and euthanized for immunocytochemistry after 6 h of ischemia. Preservation of MAP 2 immunoreactivity within the transition zone was also observed in animals pretreated with CGS 19755 and rendered ischemic for 24 h Ž3r5 animals.. Quantification of the intensity of MAP 2 immunoreactivity using computer-assisted image analysis confirmed microscopic comparisons of the immunoreactive specimens from each of the groups. Both NBQX and CGS 19755 significantly attenuated the loss of MAP 2 immuno-

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reactivity when specimens were examined at 6 and 24 h after the onset of permanent focal occlusion ŽFig. 4, NBQX: 6 h p s 0.03, 24 h p s 0.05; CGS 19755: 6 h p s 0.015, 24 h p s 0.02.. 3.3.2. Calpain-cleaÕed spectrin (Ab-37) After 6 h of ischemia, intense and prominent Ab-37-immunoreactive fibrillary processes were observed in the infarct in all ischemic control animals Žsee Fig. 2A, left., while no immunoreactivity was observed in the contralateral hemisphere. When examined after 24 h of ischemia,

Fig. 2. Representative photomicrographs Ž2 = . showing immunoreactive products of calpain-mediated spectrin breakdown ŽAb-37. in the infarcted cerebral cortex of ischemic controls ŽIC. and spontaneously hypertensive rats pretreated with either NBQX or CGS 19755 before 6 ŽA. or 24 ŽB. h of permanent focal ischemia. The zone of ischemic tissue showing calpain-mediated spectrin proteolysis appeared smaller in rats pretreated with CGS 19755 before undergoing 6 h of occlusion of the common carotid and middle cerebral arteries. Scale bar s 500 mm.

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the intensity of immunoreactivity was essentially unchanged ŽFig. 2B, left., although the pattern was quite different. The immunoreactive processes observed at 6 h were no longer visible and had been replaced by immunolabeling of dense particulate and occasional aberrant cellular structures ŽFig. 5; second column from left.. The intensity of the immunoreactivity and the density of the particulate matter within the infarct made it difficult to determine to what extent the Ab-37 immunoreactivity was associated with intact cells. Most of the immunoreactive material appeared to be fragmented particulate debris, most

likely the remnants of immunoreactive cells undergoing degeneration. In the NBQX-treated animals, the pattern of immunoreactivity at 6 h was identical to that observed in the ischemic controls in that the immunoreactivity was associated with fibrillary structures within the infarct rather than with discrete cellular profiles ŽFig. 2A, middle.. In animals pretreated with CGS 19755 prior to permanent focal ischemia, the pattern of immunoreactivity after 6 h of ischemia was much more variable. Two of the five animals displayed primarily the fibrillary pattern of immunoreactiv-

Fig. 3. Representative photomicrographs Ž2 = . of tau immunoreactivity ŽTau 1. in the infarcted cerebral cortex of ischemic controls ŽIC. and spontaneously hypertensive rats pretreated with either NBQX or CGS 19755 after 6 ŽA. or 24 ŽB. h of permanent focal ischemia. Tau immunoreactivity was diminished within ischemic tissue in all animals sampled after 24 h of occlusion of the common carotid and middle cerebral arteries, regardless of treatment status. Scale bar s 500 mm.

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ity observed with the ischemic controls and the NBQXtreated animals. The other three, however, had significant numbers of immunoreactive cell bodies within the intact core and the adjoining transition zone. These cells had the morphological characteristics of neurons. As can be readily appreciated pictorially in Fig. 2A and quantitated in Fig. 4, the intensity of Ab-37 immunoreactivity after 6 h of ischemia was significantly lower in both groups treated with a glutamate receptor antagonist than in the ischemic controls ŽNBQX: p s 0.02; CGS 19755: p s 0.04.. After 24 h of ischemia, the intensity of Ab-37 immunoreactivity in the NBQX-treated animals was greater than that observed in animals examined after 6 h of ischemia, as can be seen by comparing both the normalized optical density measurements for Ab-37 in Fig. 4 and the representative photomicrographs in the middle panels of Fig. 2A and B. Reflecting the gross increase in Ab-37 immunoreactivity, microscopic examination of NBQX-treated animals revealed prominent, intense, and widespread Ab-37immunoreactive neurons throughout the infarct in each NBQX-treated animal ŽFig. 2B, middle, and especially Fig. 5A and B, third column from left., in striking contrast to

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the immunolabeled particulate debris observed in the ischemic control animals at an identical time point Žcompare the second to the third column from the left in Fig. 5.. Normalized optical density measurements of Ab-37 immunoreactivity in rats pretreated with CGS 19755 and examined after 24 h of ischemia revealed highly significant amelioration in the intensity of Ab-37 immunoreactivity in comparison to ischemic control animals Žsee Fig. 2B, right, Fig. 4; p s 0.008.. As was shown in the NBQXtreated animals, Ab-37 immunoreactivity was observed to be associated with neuronal profiles within the infarct after 6 h of ischemia Žsee Fig. 5A and B, right panels.. Although the normalized optical density measurements did not reflect a significant amelioration of Ab-37 immunoreactivity by NBQX pretreatment ŽFig. 4., it is readily apparent that this compound encouraged the preservation of neuronal structures within the infarct as long as 24 h after the onset of ischemia. Animals pretreated with the NMDA receptor antagonist showed a similar effect on isolated neurons within the same time frame but only CGS 19755 promoted significant attenuation of calpain activity throughout the infarct at 24 h.

Fig. 4. Histogram showing the intensity of protein immunolabeling in ischemic tissue, normalized against unaffected cortex in the same animal Ž n s 5 or 6 for each group and time point.. For MAP 2 immunoreactivity ŽAP-20., the mean optical density Žo.d.. of ischemic tissue in NBQX-pretreated spontaneously hypertensive rats was less than that in ischemic controls ŽIC. at both 6 and 24 h Ž) p F 0.05.. This effect was even more pronounced in CGS 19755-pretreated rats Ž)) p F 0.02 when compared to IC at 6 and 24 h.. For immunolabeling of calpain-mediated spectrin proteolysis ŽAb-37., the mean o.d. of ischemic tissue in NBQX-pretreated rats fell below that of IC animals at 6 h Ž)) p F 0.02.. In CGS 19755-pretreated rats, the mean o.d. of the Ab-37-immunolabeled area was less than that in IC animals at both 6 Ž) p F 0.05. and 24 Ž‡ p F 0.01. h. For tau immunoreactivity ŽTau 1., only the mean o.d. of ischemic tissue in CGS 19755-pretreated animals was significantly less than that of IC rats at both 6 and 24 h Ž) p F 0.05..

190 S.L. Minger et al.r Brain Research 810 (1998) 181–199 Fig. 5. Representative photomicrographs ŽA: 10=, B: 40=. showing immunoreactive products of calpain-mediated spectrin breakdown ŽAb-37. in the cerebral cortex of control animals that were not rendered ischemic ŽC., ischemic controls ŽIC., or spontaneously hypertensive rats pretreated with either NBQX or CGS 19755 before undergoing permanent focal ischemia. Tissue sections demonstrating ischemic changes were obtained after 24 h of arterial occlusion. All images taken of ischemic brain focused on the infarct core. Although Ab-37 immunolabeling was confined to dysmorphic cellular structures and particulate matter in IC rats, it could be used to identify surviving neurons in both NBQX and CGS 19755-pretreated animals. Scale bar s100 mm ŽA. or 50 mm ŽB..

S.L. Minger et al.r Brain Research 810 (1998) 181–199 Fig. 6. Representative photomicrographs Ž40=. of tau immunoreactivity ŽTau 1. in the cerebral cortex of control animals that were not rendered ischemic ŽC., ischemic controls ŽIC., or spontaneously hypertensive rats pretreated with either NBQX or CGS 19755 before undergoing permanent focal ischemia. Tissue sections demonstrating ischemic changes were obtained after 6 ŽA. or 24 ŽB. h of arterial occlusion. All images taken of ischemic brain focused on the infarct core. Sections taken from CGS 19755-pretreated animals showed the largest number of tau-immunoreactive cells. Scale bar s 50 mm.

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192 S.L. Minger et al.r Brain Research 810 (1998) 181–199 Fig. 7. Representative photomicrographs ŽA: 10=, B: 40=. of Nissl staining in the cerebral cortex of control animals that were not rendered ischemic ŽC., ischemic controls ŽIC., or spontaneously hypertensive rats pretreated with NBQX or CGS 19755 before undergoing permanent focal ischemia. Tissue sections demonstrating ischemic changes were obtained after 24 h of arterial occlusion. All images taken of ischemic brain focused on the infarct core. Sections taken from rats in the drug treatment groups contained greater numbers of recognizable neurons than those sampled from IC animals. Scale bar s100 mm ŽA. or 50 mm ŽB..

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3.3.3. Tau In animals subjected to 6 h of permanent focal ischemia and processed for Tau 1 immunocytochemistry, greater immunoreactivity was observed in ischemic cortex than in contralateral intact cortex ŽFig. 3A.. The immunoreactivity was predominantly fibrillary in appearance, although immunoreactive cellular profiles were also observed in most animals Ž3r5., most prominently throughout the transition zone between ischemic and healthy tissue Žsee Fig. 6A, second column from left.. In animals pretreated with NBQX and analyzed after 6 h of ischemia, greater Tau 1 immunoreactivity was also observed in the infarcted cortex than in the intact cortex ŽFig. 3A.; optical density measurements revealed that pretreatment with NBQX was ineffective in preventing the increase in Tau 1 immunoreactivity at this time point ŽFig. 4.. More importantly, somatic neuronal immunoreactivity was observed prominently in the more superficial layers ŽI–III. in the core of the infarct Žsee Fig. 6A, third column from left.. In animals pretreated with CGS 19755, there was a significant attenuation of the increase in Tau 1 immunoreactivity in the infarcted cortex as compared to the contralateral undamaged cortex Žsee Fig. 3A, Fig. 4A; p s 0.03.. Similar to the rats pretreated with NBQX, animals given CGS 19755 prior to the onset

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of ischemia displayed an increased number of Tau 1 immunoreactive cells Žsee Fig. 6A, fourth column from left.. Many of these cells had the morphological characteristics of neurons. Their distribution was larger and more widespread than that observed in NBQX-treated animals, immunoreactive neurons often being found throughout the infarct as well as in the deeper cortical layers ŽIV–V.. After 24 h of ischemia, there was a significant reduction in Tau 1 immunoreactivity in the infarcted cortex of ischemic control animals in comparison to the intact hemisphere Žsee Fig. 3B., with a corresponding loss of Tau 1 fibrillary immunoreactivity. Residual Tau 1 immunoreactivity in these animals consisted primarily of round, amorphous, immunoreactive cells and particulate matter, distributed throughout the infarction ŽFig. 6B, second column from left.. NBQX-treated animals displayed a similar decrease in overall Tau 1 immunoreactivity in the infarction ŽFig. 3B, Fig. 4.. Many Tau 1 immunopositive cells also were observed scattered throughout the infarction, including a large subpopulation in which the neuronal morphology was preserved Žsee Fig. 6B, third column from left.. Samples from animals pretreated with CGS 19755 analyzed after 24 h of permanent focal ischemia, however, demonstrated a significant amelioration of the

Fig. 8. Representative western analysis of protein samples obtained from normal or infarcted cortex of spontaneously hypertensive rats after 6 Žlanes 1–4. or 24 Žlanes 5–8. h of permanent focal ischemia, immunolabeled for MAP 2 ŽA. or calpain-cleaved spectrin byproducts ŽB.. Lanes 1 and 5 contain samples taken from unaffected cortex. Samples of ischemic cortex are shown in lanes 2 and 6 Žischemic controls., 3 and 7 ŽCGS 19755., and 4 and 8 ŽNBQX.. There is almost complete loss of MAP 2 immunoreactivity in ischemic tissue, regardless of sampling time or treatment condition. Calpain-mediated spectrin proteolysis appears more robust after 24 h of ischemia, as would be expected in a mature infarct. Quantitative densitometric analysis of protein taken from the different groups of ischemic rats Ž n s 5 animalsrgroup. showed no protective treatment effect Žsee text for details..

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decrease in Tau 1 immunoreactivity in comparison to both control ischemic and NBQX-treated animals ŽFig. 3B, Fig. 4; p s 0.03.. As in samples from animals pretreated with NBQX, large numbers of immunoreactive cells, some of which were morphologically reminiscent of neurons, were scattered throughout the infarct Žsee Fig. 6B, fourth column from the left.. In samples analyzed after 24 h of ischemia from both ischemic control and ischemic animals pretreated with either NBQX or CGS 19755, the distribution of Tau 1 immunoreactive neurons within the infarct was very similar to that observed after 6 h of ischemia and to that observed at 24 h in adjacent sections processed for calpain-cleaved spectrin immunoreactivity Žcompare Fig. 5B with Fig. 6B.. 3.3.4. Cresyl Õiolet Cresyl violet staining was performed on sections adjacent to those used for immunocytochemical procedures to correlate neuronal survival with the pattern of immunoreactivity observed with each set of antibody labeling. Within 6 h of CCA-MCA occlusion, a significant reduction in the density of Nissl-positive cells was observed in the infarcted cortex in comparison to that observed in animals treated with either excitatory amino acid receptor antagonist Ždata not shown.. The cyto- and neuroprotective

effects of both NBQX and CGS 19755 are readily apparent in sections prepared after 24 h of ischemia from animals treated with both these substances ŽFig. 7.. These data support the immunocytochemical observations demonstrating the significant preservation of cells, including neurons, within the infarcted cerebral cortex in animals pretreated with excitatory amino acid receptor antagonists prior to the onset of permanent focal ischemia. 3.4. Western analysis A representative immunoblot of cortical samples processed for MAP 2 is shown in Fig. 8A. As expected, a protein of approximately 220 kDa is seen in samples of unaffected cortex. In comparison, near-complete loss of MAP 2 protein has occurred in the cortex of ischemic control animals as well as those in both drug-treatment groups within 6 h of CCA-MCA occlusion. Similarly, no preservation of MAP 2 protein is observed in any of the ischemic animals after 24 h. Densitometric analysis of brain samples from a series of 5 animalsrtreatment group and time point found no preservation of MAP 2 immunoreactivity by pretreatment with either excitatory amino acid receptor antagonist Ždata not shown., suggesting that neither NBQX nor CGS 19755 is effective in preventing proteolysis of MAP 2 protein within the ischemic core.

Fig. 9. Representative western analysis of non-phosphorylated and phosphorylated tau protein in cytosolic fractions prepared from cortical tissue obtained from spontaneously hypertensive rats after 6 Žlanes 1–4. or 24 Žlanes 5–8. h of permanent focal ischemia. In Panel A, Tau 1 monoclonal antibody has been used to identify phosphorylation-dependent, non-phosphorylated tau protein in cortical homogenates. Lanes 1 and 5 contain samples taken from unaffected cortex. Samples of ischemic cortex are shown in lanes 2 and 6 Žischemic controls., 3 and 7 ŽCGS 19755., and 4 and 8 ŽNBQX.. The arrow in Panel A identifies a densely immunoreactive band associated with a 52-kDa fragment of tau protein observed only after 6 h of ischemia. In Panel B, the same set of samples has been immunolabeled with the 8C11 monoclonal antibody to show all isoforms of tau protein, independent of phosphorylation state. The degenerative fragment is present in ischemic tissue, as shown in Panel A, but only in samples taken after 6 h of occlusion of the common carotid and middle cerebral arteries.

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The samples depicted in Fig. 8A were also immunolabeled with Ab-38 to identify calpain-cleaved spectrin, as shown in Fig. 8B. In the unaffected cortex, the lack of calpain-cleaved spectrin byproduct is consistent with an observation made by Roberts-Lewis et al. w49x that calpain activation is not detectable under normal physiologic conditions. After 6 h of permanent focal ischemia, a calpaincleaved spectrin fragment of approximately 150 kDa is observed in the sample obtained from the untreated ischemic cortex. The same protein is noted in ischemic cortex taken from animals pretreated with either NBQX or CGS 19755. This 150-kDa band becomes more intense in all three groups after 24 h of CCA-MCA occlusion, suggesting that calpain-mediated spectrin proteolysis becomes amplified as the infarct matures. Comparison of densitometric analysis of individual samples from ischemic controls and animals pretreated with NBQX or CGS 19755 Ž n s 5 in each group. found that neither drug produced significant inhibition of calpain activity Ždata not shown.. Finally, cortical samples examined for alterations in tau protein are shown in Fig. 9A. Significant changes in the intensity of individual Tau 1-immunolabeled proteins are readily apparent in samples from the untreated ischemic cortex after 6 h of CCA-MCA occlusion in comparison with those obtained from the unaffected hemisphere. Prominent loss of higher molecular weight Tau 1-immunoreactive isoforms and the emergence of lower weight forms are also observed in the sample taken from the ischemic cortex after 6 h of arterial occlusion. Of particular interest is a 52-kDa fragment that appears in the ischemic cortex of the control rat and persists in samples taken from animals treated with either NBQX or CGS 19755. Neither drug has any effect on the pattern of Tau 1-immunoreactive bands found in ischemic cortex at 6 h. In cortical samples harvested after 24 h of CCA-MCA occlusion, limited preservation of tau protein immunoreactivity is noted only in an ischemic animal pretreated with CGS 19755. Densitometric analysis of samples obtained from the complete set of animals in each of the treatment groups, however, revealed no significant protection of Tau 1-immunoreactive proteins within the core of the infarct with either excitatory amino acid receptor antagonist in comparison to the untreated ischemic control animals. To further explore the nature of the alterations in molecular weight of individual tau proteins, the samples presented in Fig. 9A were immunolabeled with 8C11, a monoclonal antibody specific for a phosphorylation-insensitive epitope found on all tau isoforms w62x, and are displayed again in Fig. 9B. The patterns of immunoreactive bands seen in unaffected cortical samples at 6 and 24 h and in all ischemic animals at 6 h are similar in Fig. 9A and B. The same 52-kDa band representing a degenerative fragment of nonphosphorylated tau produced by 6 h of ischemia in Fig. 9A appears under the same conditions in Fig. 9B. This observation suggests that the downward

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shifts in molecular weight of tau protein fragments in ischemic brain are caused by dephosphorylation that is not reversed by the use of NBQX or CGS 19755. As with Tau 1 in Fig. 9A, no 8C11-immunoreactive tau proteins are observed after 24 h of CCA-MCA occlusion.

4. Discussion Our results demonstrate that using glutamate receptor antagonists to inhibit excitotoxic neurotransmission reduces the loss of neuronal cytoskeletal proteins that occurs within the ischemic core after permanent focal cerebral ischemia. Pre-ischemic administration of these compounds also effectively inhibits the activation of calpain, which is temporally and spatially associated with the cytoskeletal disruption and proteolysis that take place in response to ischemic injury. These studies are the first to demonstrate a relationship between excitotoxicity, calpain activation, and destruction of neuronal cytoskeletal proteins in models of focal brain ischemia. A sizable body of experimental evidence suggests that ischemic neuronal injury is mediated by excitotoxic neurotransmission. Several intracerebral microdialysis studies have demonstrated rapid and significant increases in extracellular glutamate and aspartate concentrations in animal models of both global w5,6x and focal w12,25x cerebral ischemia. These increases in extracellular glutamate translate into significant elevations in intraneuronal calcium caused by opening of ligand-sensitive channels, such as those with affinity for NMDA, and secondary activation of voltage-sensitive calcium channels. Potential downstream targets of this cascade include calcium-dependent neutral cysteine proteases, the calpains Žsee Sorimachi et al. w59x for review.. Of these, calpain I is thought to be the major proteolytically active form under both physiological and pathological conditions because it is activated when intracellular calcium rises to micromolar levels. Studies in several models of neuronal injury that associate increases in intracellular calcium and excessive excitotoxic neurotransmission also show calpain activation. These models include administration of excitotoxic agents both in vitro w17,38,51x and in vivo w55,56x and both transient global w49,52x and focal cerebral ischemia w2,66x. Several other studies in animal models of stroke have shown that various inhibitors of calpain significantly reduce ischemic damage w26,34,39,48x. Despite the wealth of data suggesting that calpain is activated primarily by excitotoxic-mediated increases in intracellular calcium, evidence directly linking these events is currently lacking. To date, only one study from Roberts-Lewis et al. w49x has shown that a glutamate receptor antagonist significantly reduced calpain-mediated neuronal damage. Activation of calpain during ischemia probably causes proteolytic degradation of cytoskeletal proteins in neurons. Such degradation is a characteristic neuropathological fea-

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ture of both global and focal models of ischemia w21,31,52x. The preferred proteolytic substrates of activated calpain are the cytoskeletal proteins spectrin, MAP 2, and tau w28,29x. Recently published studies in this laboratory w47x have further demonstrated that the onset of cytoskeletal abnormalities and calpain activation in focal cerebral ischemia coincide in time and space. In the experiments reported here, pretreatment with CGS 19755 had reduced infarct volume by approximately 60% at 24 h after permanent CCA-MCA occlusion. These observations agree with a number of previous reports demonstrating the efficacy of CGS 19755 in models of focal cerebral ischemia in rats w57x and global ischemia in gerbils w9x. Pretreatment with NBQX, however, had no effect on infarct volume. This finding confirms previous reports demonstrating lack of therapeutic efficacy using similar ischemic and pharmacologic methodology w16,22x. Reports that NBQX effectively prevented neuronal loss in global ischemia at the dosage used in our studies have been sporadic w54x. Other investigators have demonstrated that NBQX is neuroprotective but only at dosages at least double those used in our experiments w22,35x or when given as a continuous infusion w33,44x. Pretreatment with CGS 19755 was more effective than that with NBQX in protecting cytoskeletal proteins and reducing calpain activation in the infarcted cortex. Although both CGS 19755 and NBQX significantly inhibited the activation of calpain at 6 h after the onset of ischemic injury, quantitative analysis of computer-assisted optical density measurements showed that only CGS 19755 remained effective at 24 h. We suspect that this differential treatment effect may originate from functional differences in the NMDA and AMPA receptors and their capacity for enhancement of calcium conductance. Binding of ligand to the NMDA receptor will cause the associated ion channel to open and calcium conductance to increase. Most AMPA receptors are not permeable to calcium upon ligand binding, although a small proportion have the appropriate subunit composition to augment calcium permeability. Therefore, inhibition of a calcium-activated enzyme like calpain should be more effectively achieved by blocking the NMDA receptor. This assumption is supported by the observation that CGS 19755 was more beneficial than NBQX in reducing Ab-37 immunoreactivity in ischemic tissue. The same receptor-mediated differential effect may also be responsible for the failure of NBQX to reduce infarct volume. Our findings are in accordance with a previous study demonstrating that NMDA receptor antagonism significantly reduced Ab-37 immunoreactivity and promoted neuronal survival in a gerbil model of transient forebrain ischemia w49x, but similar investigations examining the efficacy of AMPA-specific receptor antagonists have not been reported. In comparing our results to those of Roberts-Lewis and colleagues w49x, it is important to note that the two studies examined different animal models of ischemic neuronal

degeneration and that both investigations may have shown the neuroprotective benefit of hypothermia. Buchan and Pulsinelli w11x determined that MK-801 may confer neuroprotective effects by inducing hypothermia. In reviewing the findings of Roberts-Lewis et al. w49x, it is not possible to determine whether MK-801 inhibited calpain-mediated cytoskeletal disruption by hypothermia or by successful antagonism of the NMDA receptor. MK-801 has also been shown to have neurotoxic and cytotoxic effects w18,53x. For these reasons, we chose CGS 19755 for our study because it is less likely than MK-801 to induce hypothermia and has no known cytotoxic effects. As an additional experimental control against hypothermia, we kept brain temperature within a range that is not associated with neuroprotective benefit. Neither CGS 19755 nor NBQX was found to induce cytopathic responses in any brain region examined in our animals, including the cingulate and retrosplenial cortices that are affected by MK-801. The rapid and selective loss of MAP 2 immunoreactivity we observed is a characteristic neuropathological feature after focal cerebral w15,47x and global ischemic injury w31,40x. In models of permanent focal cerebral ischemia, including those used in this laboratory, significant alterations in the characteristic morphology of MAP 2-immunoreactive processes have been observed within 30 min of CCA-MCA occlusion w47x, with profound loss of MAP 2 immunoreactivity within 1 h of the onset of ischemia w15,47x. The rapid destruction of MAP 2 or other cytoskeletal structures confined to dendrites may be due to a greater concentration of calpain in this neuronal compartment. In the rodent brain, calpain I immunoreactivity has been observed in cortical pyramidal neurons within cell bodies and throughout the dendritic arborization w45x, whereas axonal structures have been found to be only lightly calpain-immunoreactive w24,45x. More detailed ultrastructural analysis has revealed a dense accumulation of calpain I in neuronal dendritic processes w46x, and biochemical analysis of subcellular fractions obtained from rat brain demonstrated that significant calpain I activity was localized to the fraction containing small cellular processes, which includes dendritic postsynaptic densities w3x. The close apposition of MAPs and calpain within the somatodendritic compartment may underlie the rapid response to brain injury and subsequent degradation of these proteins. The ability of both NBQX and CGS 19755 pretreatment to significantly attenuate reductions in MAP 2-immunoreactivity within the infarcted cortex suggests that dendritic damage and MAP 2 disruption and proteolysis are mediated through both AMPA- and NMDA-type excitatory amino acid receptors. In addition, these observations support the hypothesis that preferential dendritic damage occurs early in the ischemic injury cascade. If damage at this site can be inhibited before alterations take place in cytoskeletal structure, protection against dendritic injury may be sustained even as ischemia spreads and the penumbra expands.

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Only pretreatment with CGS 19755 significantly protected against alterations in tau protein at both 6 and 24 h after CCA-MCA occlusion. Pre-ischemic administration of CGS 19755 significantly attenuated the increase in Tau-1 immunoreactivity observed in untreated ischemic animals at 6 h. At 24 h, Tau-1 immunoreactivity in the ischemic cortex was not significantly different from that in the nonischemic cortex. The greater immunoreactivity observed in the ischemic cortex at 6 h suggests an increased accumulation of tau or reflects the unmasking of Tau-1 epitopes in response to injury. The immunocytochemical and western blot data presented here support previous observations from our laboratory that tau proteins in the ischemic cortex undergo dephosphorylation before proteolysis w47x. All of these results are consistent with in vitro immunocytochemical and biochemical studies demonstrating that exposure of cortical neurons to glutamate results in tau dephosphorylation and increased Tau-1 immunoreactivity prior to overt neuronal degeneration w14x and with intracortical glutamate infusion studies demonstrating increased Tau-1 immunoreactivity as a result of excitotoxic-mediated dephosphorylation w27x. Reductions in Tau1 immunoreactivity found in animals pretreated with CGS 19755 at 6 h after the onset of ischemia suggest that the dephosphorylation of tau may be affected by inhibition of excitotoxic neurotransmission. The phosphorylation state of tau may be crucial in determining its susceptibility to degradation by calpain w28,37x. In vitro studies have found preparations of enriched paired helical filament tau proteins ŽPHF-tau. from Alzheimer’s disease brain, which are composed of abnormally hyperphosphorylated tau, to be significantly resistant to calpain proteolysis w23x. Although PHFs are consistently resistant to calpain proteolysis w42,65x, tau isolated from fetal brain, which is more highly phosphorylated than normal adult tau, did not differ from adult tau in rate and extent of calpain proteolysis w42,65x. The western analysis presented in our study using antibodies specific for both phosphate sensitive ŽTau 1. and insensitive Ž8C11. epitopes demonstrate that the reductions in molecular weight observed prior to overt proteolysis, which most likely represent tau dephosphorylation, appear to be a requisite step prior to in situ degradation. Although both Nissl staining and computer-assisted morphometric analysis of the infarcted cortex indicate that antagonism of glutamate receptors has prominent neuroprotective effects, western analysis of cortical brain samples revealed that these compounds failed to significantly inhibit the degradation of MAP 2 or tau proteins or the calpain-specific proteolysis of spectrin. Brain tissue processed for western analysis, because it was taken from the area that was not stained by India ink, was necessarily restricted to the core of the infarct where blood flow was completely absent. In contrast, morphometric analysis of brain sections prepared for immunocytochemistry included the entire infarcted cortex and no distinction was attempted

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between penumbra and core. The differences in the results of these studies suggest that glutamate receptor blockade is primarily efficacious in the penumbra of the infarct, but may have limited effect on neuronal destruction within the ischemic core. These observations highlight the dynamic character of the ischemic penumbra and suggest that glutamate receptor antagonism and perhaps coadministration of calpain inhibitors may offer a reasonable therapeutic approach to salvage tissue in which blood flow has been compromised or transiently lost. In summary, these studies clearly demonstrate that blocking excitatory amino acid neurotransmission by competing for the binding site at the glutamate receptor reduces the pathophysiology of focal cerebral ischemia. Our findings suggest that inhibition of the NMDA receptor is more efficacious than inhibition of the AMPA receptor. These results support previous work demonstrating the efficacy of NMDA receptor antagonists in reducing ischemic neuronal damage and show that one potential mechanism for the neuroprotective effects of glutamate receptor antagonism is the inhibition of calpain-mediated cytoskeletal disruption. Protection of cytoskeletal proteins and reduction in calpain activity after glutamate receptor antagonism in focal cerebral ischemia provides a cellular and biochemical model for examining neuroprotective interventions targeting preservation of microtubular structure.

Acknowledgements This work was supported by grants to S.L.M. from the Kentucky Affiliate of the American Heart Association and the American Health Assistance FoundationrNational Heart Foundation, NIHrNINDS R01 NS33773 ŽL.C.P.., and Alzheimer’s Disease Research Center grant P50 AG05144 ŽJ.W.G... We thank Sherry C. Williams for editorial assistance.

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