Brain Research, 526 (1990) 177-179 Elsevier
177
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Suppressive effect of E-64c on ischemic degradation of cerebral proteins following occlusion of the middle cerebral artery in rats Takashi Inuzuka 1, Akira Tamura 2, Shuzo Sato ~, Takaaki Kirino 2, Itaru Toyoshima 3 and Tadashi Miyatake ~ 1Department of Neurology, Brain Research Institute, Niigata University, Niigata (Japan); 2Departmentof Neurosurgery, Teikyo University, Tokyo (Japan); and 3Departmentof the 1st Internal Medicine, Akita University, Akita (Japan) (Accepted 22 May 1990)
Key words: Cerebral ischemia; Microtubule-associated protein 2; Myelin-associated glycoprotein; Calpain; E-64c
Microtubule-associated protein 2 (MAP2) levels in the left cerebral hemisphere decreased significantly 3 days after occlusion of the left middle cerebral artery in rats to 29 + 16.3% of control levels. Since MAP2 is one of the substrates of calpain, E-64c, a synthetic calpain inhibitor, was administered at a dose of 400 mg/kg twice a day for 3 days, with the first dose being given before the production of ischemia. This depletion was significantly inhibited in vivo by E-64c (P < 0.05) to increase MAP2 levels to 55 + 25.7% of control levels. E-64c had no significant effect on the ischemia-induced depletion of myelin-associated glycoprotein. Sham-operated rats were used as controls. Our results suggest that calpain is partially involved in the degradation of MAP2, and that the use of calpain inhibitors can be a useful clinical approach to cerebral ischemia.
We have previously reported that focal persistent ischemia for 3 days after occlusion of the left middle cerebral artery (MCA) in rats induced a significant decrease in the concentration of both microtubuleassociated protein 2 (MAP2) and myelin-associated glycoprotein ( M A G ) 5. Since these cytoskeletal proteins are substrates of calpain, the findings suggested the involvement of calpain in postischemic changes in cerebral tissue. Accordingly, we examined the in vivo effect of E-64c, a synthetic calpain inhibitor 4, on the degradation of MAP2 and M A G in the left cerebral hemispheres of Sprague-Dawley rats 3 days after occlusion of the left M C A when MAP2 and M A G were significantly reduced in our previous study 5. Fifteen male Sprague-Dawley rats (weight: 340-440 g) were anesthetized by inhalation of 2% halothane in 30% oxygen and 70% nitrogen. The proximal portion of the left M C A was permanently occluded using a microsurgical technique which was modified from our original method 16. A microbipolar electrocautery unit (KeiseiIka, Tokyo) was used to cauterize and sever the exposed M C A just medial to the olfactory tract. In sham-operated animals, the surgical procedure was carried out in a similar manner, except for the electrocautery and severing of the exposed MCA. After closure of the incision, animals were returned to their cages and permitted free access to food and water. Rats were decapitated 3 days
after surgery and their brains were quickly removed. The left cerebral hemispheres were immediately frozen and stored at -70 °C until use. In the treatment group (n = 5), E-64c (400 mg/kg) was injected intraperitoneally 30 min before M C A occlusion, and an additional 5 doses were given at 12-h intervals. Another 5 rats with MCA occlusion and 5 sham-operated rats were given injections of saline. E-64c was dissolved in a small amount of saturated bicarbonate solution and then was diluted with 0.9% NaCI saline. The pH was adjusted to 7.4 with 0.1 N HCI. The injection volume of E-64c or saline was approx. 2 ml per dose. Homogenate of each cerebral hemisphere was prepared in 10 mM Tris-HCl (pH 7.5), 1 mM PMSF, 1 mM EGTA, 1 mM E-64c and 1 mM camostat mesilate (FOY305) and appropriate aliquots were taken for the assay described below. Electrophoresis was performed on polyacrylamide slab gels according to the method of Laemmli and Favre 7 after delipidation of freeze-dried aliquots of the homogenate with 3:2 (vol/vol) diethylether:ethanol. The running gels used were 5% polyacrylamide for MAP2 and 11% for M A G , with 10/~g of total protein being applied for M A G and 50 ~g for MAP2, so as to obtain a linear response to a decrease in the amount of each of these proteins in the total protein. To assure the validity of the quantitative analysis of each protein,
Correspondence: T. Inuzuka, Department of Neurology, Brain Research Institute, Niigata University, Niigata 951, Japan. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
178 preliminary immunoblot experiments were carried out over a wide range of protein concentrations to determine the linear range of this method (Fig. 1). Protein concentrations of the homogenates were determined by the method of Lowry et al. s. Preparation of rabbit polyclonal antibody directed against rat M A G of the central nervous system was performed according to the method of Quarles et al. 9. A mouse monoclonal antibody to bovine MAP2 was prepared by the standard hybridoma technique 15. Biotinylated anti-mouse IgG, anti-rabbit IgG, and avidinbiotin complex standard kits were purchased from Vector Lab. Inc. (CA, U.S.A.). The anti-MAG and anti-MAP2 antibodies were diluted 1:200 and 1:400, respectively, in 3% bovine serum albumin. Blotting of proteins was carried out essentially according to the method of Towbin et al. 13. The blots were immunostained with antisera by an avidin-biotin complex peroxidase procedure using 4-chloro-l-naphthol as chromogen. The blots were then scanned with a spectrodensitometer (CS-910, Shimadzu, Japan) operating at 560 nm for semiquantitation of protein levels. Changes in the levels of each protein following MCA occlusion with or without E-64c treatment were calculated from the area of the protein in the sample as determined by scanning and expressed as a percentage of the area in the sham-operated control rats. Statistical comparisons were made using Student's t-test. The MAP2 and M A G contents in the left cerebral hemispheres were respectively depleted to 29 + 16.3%, and 58 + 14.7% (mean + S.E.M.) of the sham-operated control levels in the untreated rats with MCA occlusion. E-64c significantly increased (P < 0.05) the MAP2 level to 55 + 25.7% of the sham-operated control level (Fig. 2A). Immunoblotting showed that MAP2 degradation
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Fig. 2. The percentage of intact protein remaining in the left hemisphere. (Protein area determined by scanning for MCAoccluded rats treated with E-64c or saline/corresponding area for the sham-operated rats treated with saline x 100.) Each group consisted of 5 rats. Bars represent the + S.E.M. S, sham-operated rats with saline. O, MCA-occluded rats treated with saline. E, MCAoccluded rats treated with E-64c. A: MAP2, B: MAG.
products were decreased in the E-64c-treated rats (Fig. 3A). E-64c had no significant effect on the depletion of M A G (Figs. 2B, 3B). Activation of calpain by an increase of intraceUular calcium is considered to be a factor involved in the occurrence of the breakdown of cytoskeletal proteins following ischemia 12. We have previously reported that focal persistent ischemia for 3 days after M C A occlusion in rats induced decreases in MAP2 and M A G concentrations and suggested the involvement of calpain in these changes 5. Calcium accumulation in the ischemic region after MCA occlusion has also been reported .1, and calcium channel blockers attenuated the degradation of MAP2 and calspectin in the gerbil brain after 5 min of bilateral carotid artery occlusion 12. Our results demonstrated that E-64c significantly suppressed the in vivo degradation of MAP2 and suggested that calpain is at least partially involved in the degradation of this protein. A tracer experiment has revealed that labeled E-64c shows little uptake by the intact brain 13. The increase of
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177
BRES 24258
Suppressive effect of E-64c on ischemic degradation of cerebral proteins following occlusion of the middle cerebral artery in rats Takashi Inuzuka 1, Akira Tamura 2, Shuzo Sato ~, Takaaki Kirino 2, Itaru Toyoshima 3 and Tadashi Miyatake ~ 1Department of Neurology, Brain Research Institute, Niigata University, Niigata (Japan); 2Departmentof Neurosurgery, Teikyo University, Tokyo (Japan); and 3Departmentof the 1st Internal Medicine, Akita University, Akita (Japan) (Accepted 22 May 1990)
Key words: Cerebral ischemia; Microtubule-associated protein 2; Myelin-associated glycoprotein; Calpain; E-64c
Microtubule-associated protein 2 (MAP2) levels in the left cerebral hemisphere decreased significantly 3 days after occlusion of the left middle cerebral artery in rats to 29 + 16.3% of control levels. Since MAP2 is one of the substrates of calpain, E-64c, a synthetic calpain inhibitor, was administered at a dose of 400 mg/kg twice a day for 3 days, with the first dose being given before the production of ischemia. This depletion was significantly inhibited in vivo by E-64c (P < 0.05) to increase MAP2 levels to 55 + 25.7% of control levels. E-64c had no significant effect on the ischemia-induced depletion of myelin-associated glycoprotein. Sham-operated rats were used as controls. Our results suggest that calpain is partially involved in the degradation of MAP2, and that the use of calpain inhibitors can be a useful clinical approach to cerebral ischemia.
We have previously reported that focal persistent ischemia for 3 days after occlusion of the left middle cerebral artery (MCA) in rats induced a significant decrease in the concentration of both microtubuleassociated protein 2 (MAP2) and myelin-associated glycoprotein ( M A G ) 5. Since these cytoskeletal proteins are substrates of calpain, the findings suggested the involvement of calpain in postischemic changes in cerebral tissue. Accordingly, we examined the in vivo effect of E-64c, a synthetic calpain inhibitor 4, on the degradation of MAP2 and M A G in the left cerebral hemispheres of Sprague-Dawley rats 3 days after occlusion of the left M C A when MAP2 and M A G were significantly reduced in our previous study 5. Fifteen male Sprague-Dawley rats (weight: 340-440 g) were anesthetized by inhalation of 2% halothane in 30% oxygen and 70% nitrogen. The proximal portion of the left M C A was permanently occluded using a microsurgical technique which was modified from our original method 16. A microbipolar electrocautery unit (KeiseiIka, Tokyo) was used to cauterize and sever the exposed M C A just medial to the olfactory tract. In sham-operated animals, the surgical procedure was carried out in a similar manner, except for the electrocautery and severing of the exposed MCA. After closure of the incision, animals were returned to their cages and permitted free access to food and water. Rats were decapitated 3 days
after surgery and their brains were quickly removed. The left cerebral hemispheres were immediately frozen and stored at -70 °C until use. In the treatment group (n = 5), E-64c (400 mg/kg) was injected intraperitoneally 30 min before M C A occlusion, and an additional 5 doses were given at 12-h intervals. Another 5 rats with MCA occlusion and 5 sham-operated rats were given injections of saline. E-64c was dissolved in a small amount of saturated bicarbonate solution and then was diluted with 0.9% NaCI saline. The pH was adjusted to 7.4 with 0.1 N HCI. The injection volume of E-64c or saline was approx. 2 ml per dose. Homogenate of each cerebral hemisphere was prepared in 10 mM Tris-HCl (pH 7.5), 1 mM PMSF, 1 mM EGTA, 1 mM E-64c and 1 mM camostat mesilate (FOY305) and appropriate aliquots were taken for the assay described below. Electrophoresis was performed on polyacrylamide slab gels according to the method of Laemmli and Favre 7 after delipidation of freeze-dried aliquots of the homogenate with 3:2 (vol/vol) diethylether:ethanol. The running gels used were 5% polyacrylamide for MAP2 and 11% for M A G , with 10/~g of total protein being applied for M A G and 50 ~g for MAP2, so as to obtain a linear response to a decrease in the amount of each of these proteins in the total protein. To assure the validity of the quantitative analysis of each protein,
Correspondence: T. Inuzuka, Department of Neurology, Brain Research Institute, Niigata University, Niigata 951, Japan. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
178 preliminary immunoblot experiments were carried out over a wide range of protein concentrations to determine the linear range of this method (Fig. 1). Protein concentrations of the homogenates were determined by the method of Lowry et al. s. Preparation of rabbit polyclonal antibody directed against rat M A G of the central nervous system was performed according to the method of Quarles et al. 9. A mouse monoclonal antibody to bovine MAP2 was prepared by the standard hybridoma technique 15. Biotinylated anti-mouse IgG, anti-rabbit IgG, and avidinbiotin complex standard kits were purchased from Vector Lab. Inc. (CA, U.S.A.). The anti-MAG and anti-MAP2 antibodies were diluted 1:200 and 1:400, respectively, in 3% bovine serum albumin. Blotting of proteins was carried out essentially according to the method of Towbin et al. 13. The blots were immunostained with antisera by an avidin-biotin complex peroxidase procedure using 4-chloro-l-naphthol as chromogen. The blots were then scanned with a spectrodensitometer (CS-910, Shimadzu, Japan) operating at 560 nm for semiquantitation of protein levels. Changes in the levels of each protein following MCA occlusion with or without E-64c treatment were calculated from the area of the protein in the sample as determined by scanning and expressed as a percentage of the area in the sham-operated control rats. Statistical comparisons were made using Student's t-test. The MAP2 and M A G contents in the left cerebral hemispheres were respectively depleted to 29 + 16.3%, and 58 + 14.7% (mean + S.E.M.) of the sham-operated control levels in the untreated rats with MCA occlusion. E-64c significantly increased (P < 0.05) the MAP2 level to 55 + 25.7% of the sham-operated control level (Fig. 2A). Immunoblotting showed that MAP2 degradation
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Fig. 2. The percentage of intact protein remaining in the left hemisphere. (Protein area determined by scanning for MCAoccluded rats treated with E-64c or saline/corresponding area for the sham-operated rats treated with saline x 100.) Each group consisted of 5 rats. Bars represent the + S.E.M. S, sham-operated rats with saline. O, MCA-occluded rats treated with saline. E, MCAoccluded rats treated with E-64c. A: MAP2, B: MAG.
products were decreased in the E-64c-treated rats (Fig. 3A). E-64c had no significant effect on the depletion of M A G (Figs. 2B, 3B). Activation of calpain by an increase of intraceUular calcium is considered to be a factor involved in the occurrence of the breakdown of cytoskeletal proteins following ischemia 12. We have previously reported that focal persistent ischemia for 3 days after M C A occlusion in rats induced decreases in MAP2 and M A G concentrations and suggested the involvement of calpain in these changes 5. Calcium accumulation in the ischemic region after MCA occlusion has also been reported .1, and calcium channel blockers attenuated the degradation of MAP2 and calspectin in the gerbil brain after 5 min of bilateral carotid artery occlusion 12. Our results demonstrated that E-64c significantly suppressed the in vivo degradation of MAP2 and suggested that calpain is at least partially involved in the degradation of this protein. A tracer experiment has revealed that labeled E-64c shows little uptake by the intact brain 13. The increase of
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