Neuroscience Letters, 115 (1990) 19-23
19
Elsevier Scientific Publishers Ireland Ltd. NSL 06984
Immunocytochemical localization of manganese superoxide dismutase (Mn-SOD) in the hippocampus of the rat Fumiharu Akai I, Mitsuyo Maeda 2, Keiichiro Suzuki 3, Shinobu Inagaki 4, Hiroshi Takagi 4 and Naoyuki Taniguchi 3 Departments of lNeurosurgery and 2Pathology, Kinki University School of Medicine, Osaka-Savama (Japan), 3Department of Biochemistry, Osaka University Medical School, Osaka (Japan) and 4First Department of Anatomy, Osaka City University Medical School, Osaka (Japan)
(Received 8 March 1990; Accepted 11 March 1990) Key words." Manganese superoxide dismutase; Hippocampus; Immunocytochemistry;Selective vulnera-
bility The immunocytochemicaldistribution of manganese superoxide dismutase (Mn-SOD) was determined in the rat hippocampus. The enzymewas localized in the mitochondria. CA I pyramidal cells were weakly immunostained, whereas CA3 pyramidal cells were strongly reactive. These differences in the intensity of the Mn-SOD immunostaining reactions may relate to variations in the sensitivityof subfields of the hippocampus to ischemia.
Superoxide dismutases (SODs) catalyze the conversion of the superoxide radicals to hydrogen peroxide and molecular oxygen. These enzymes may protect cells against reactive free radicals produced under different physiological conditions, such as reoxygenation following ischemia [16]. This is supported by animal experiments which show that ischemic neuronal damage is significantly prevented by the pre-administration of exogenous SODs [9, 11]. The hippocampus is one of the most sensitive areas to ischemia. Following transient cerebral ischemia, delayed neuronal death occurs most prominently in the CA1 area as compared with other subfields [8]. The reason for such selective vulnerability of specific regions has not been elucidated [10, 12, 14]. Therefore, we have studied the immunocytochemical localization of manganese (Mn)-SOD, in order to provide a morphological basis for understanding the possible role of this enzyme in the hippocampus. A total of 12 adult male, 150g Wistar rats were used in this study. Ten animals were anesthetized and perfused with 200 ml of Zamboni's fixative [17] for light microCorrespondence: H. Takagi, First Department of Anatomy, Osaka City University Medical School, 1-4-54 Asahimachi, Abenoku, Osaka 545, Japan.
0304-3940/90/$ 03.50 © 1990 ElsevierScientific Publishers Ireland Ltd.
20
Fig. 1. Light micrographs of the rat hippocampus stained for Mn-SOD. A gradient of the reaction intensity a m o n g the subfields is seen (A). Arrows indicate both ends of CA1 where pyramidal cell bodies are almost devoid of immunoreactivity in contrast to possible interneurons with strong reactivity (arrowheads). Higher magnification shows pyramidal cells (B) in CA3 staining strongly as contrasted with weak staining CA l cells (C). Scales: A = 50/~m; B, C = 20 ~m.
21
scopic studies. Two animals were perfused with 200 ml of picric acid (0.2 %)-glutaraldehyde (0.05 %)-paraformaldehyde (4 %) fixative [15] for electron microscopic studies. A portion of the brain containing the hippocampus was immersed in these fixatives for 4 h at 4°C, followed by immersion in 0.1 M phosphate buffer (pH 7.4) containing 30% sucrose. Frontal sections were cut at a thickness of 40/am with a Microslicer (Dosaka EM). These were placed in a small vial and incubated overnight at 4°C with an 1:100 dilution of the primary antiserum. They were then stained according to the avidin-biotin-peroxidase (ABC) method [4] (Vectastain ABC kit). The primary antiserum employed was raised in New Zealand white rabbits to Mn-SOD which was purified from normal rat liver as described previously [5]. The enzyme used had a specific activity of 3200 U/mg protein and gave a single band on SDS polyacrylamide gel electrophoresis. The specificity of the antiserum was established by an enzyme immunoassay technique [5]. The immunostained sections used for light microscopy were mounted, dehydrated and coverslipped. Sections for electron microscopy were postfixed with 1% OsO4 for 1 h and embedded in epoxy resin. Ultrathin sections were stained with 1% uranyl acetate and lead citrate. Electron micrographs were taken at 100 kV on a Hitachi H-12A. To test for the specificity of the immunocytochemical procedure, control experiments were performed, i.e., omission of the primary antiserum, replacement of the primary antiserum with normal rabbit serum, and use of the primary antiserum following its absorption, with purified Mn-SOD (20 -6 M). No tissue sections prepared by the latter methods showed immunostaining. A marked topographical difference in the intensity of the immunoreactions was apparent. Most of the pyramidal cells in CA1 were weakly stained, whereas those in CA3 were stained strongly (Fig. 1). CA4 pyramidal cells showed a lower reactivity than those of CA3, but more than for CA1 cells. Within the CA3 area there was a gradient in the intensity of reaction, the CA3a and CA3b areas being more strongly stained than CA3c whose reaction was still more pronounced than CA4. Judging from the perikaryal size and shape, some non-pyramidal cells in all of the subfields appeared to have strong immunoreactivity in their somata and processes (Fig. 1C). Such cells were also found in other layers of the hippocampus (Fig. 1B,C). These immunoreactive cells showed numerous granular or rod-shape precipitates (Fig. 1B,C). Nuclei were always devoid of such reactivity. The electron microscopy results confirmed the localization of the Mn-SOD as being in mitochondria associated with the inner membrane and matrix in both reactive pyramidal and non-pyramidal cells (Fig. 2). Little information is available on the morphological localization of Mn-SOD in the brain [3]. The present study has demonstrated a characteristic localization of this enzyme in pyramidal cell layers of the rat hippocampus. The activity of antioxidative enzymes, i.e., Mn-SOD and Cu/Zn-SOD, may relate to the defense capability of Fig. 2. Electron micrographs of Mn-SOD-immunoreactive non-pyramidal cell in the stratum oriens of CA I. Immunoprecipit.-ates are seen in most of the mitochondria (A). Higher magnification shows they are associated with the inner membrane and matrix (B). G, Golgi apparatus; N, nucleus. Scales: A =0.5/~m; B = 0.2/am.
22 b r a i n tissues, for example, h i p p o c a m p u s a n d cerebral cortex, against reactive oxygen free radicals formed as the result o f ischemia a n d s u b s e q u e n t r e o x y g e n a t i o n [1]. Therefore, the differences in the intensity o f the M n - S O D i m m u n o s t a i n i n g reactions m a y relate to v a r i a t i o n s in the sensitivity of subfields of the h i p p o c a m p u s to ischemia. O u r study revealed that some cells in C A 1 exhibited a strong i m m u n o r e a c t i v i t y for M n - S O D . These cells m a y c o r r e s p o n d to ischemia-resistant i n t e r n e u r o n s [6]. N e u r o nal d a m a g e in C A I appears to be limited to p y r a m i d a l cells, whereas G A B A e r g i c i n t e r n e u r o n s are relatively resistant [13]. It will be interesting to determine which category of n e u r o n s these i m m u n o s t a i n e d , n o n - p y r a m i d a l cells belong to. The present study also d e m o n s t r a t e d that M n - S O D is present in the m i t o c h o n d r i a o f n e u r o nal cells. This specific localization o f M n - S O D has been also reported in h u m a n hepatocytes [7], whereas C u / Z n - S O D is m a i n l y located in the cytoplasmic matrix a n d nucleus, b u t does n o t appear to be associated with the m i t o c h o n d r i a [2]. The a u t h o r s wish to t h a n k Prof. H.F. Deutsch for his kind review o f the text. 1 Chan, P.H., Chu, L. and Fishman, R.A., Reduction of activities of superoxide dismutase but not of glutathione peroxidase in rat brain regions following decapitation ischemia, Brain Res., 439 (1988) 388-390. 2 Chang, L.-Y., Slot, J.W., Geuze, H.J. and Crapo, J.D., Molecular immunocytochemistryof the CuZn superoxide dismutase in rat hepatocytes, J. Cell Biol., 107 (1989) 2169-2179. 3 Dobashi, K., Asayama, K., Kato, K., Kobayashi, M. and Kawaoi, A., Immunohistochemicallocalization of copper-zinc and manganese superoxide dismutases in rat tissues, Acta Histochem. Cytochem., 22 (1989) 351 365. 4 Hsu, S., Raine, L. and Fanger, H., Use of avidin-biotin-peroxidasecomplex (ABC) in immunoperoxidase technique: a comparison between ABC and unlabelled antibody (PAP) procedures, J. Histochem. Cytochem., 29 (1981) 577-580. 5 Iizuka, S., Taniguchi, N. and Makita, A., Enzyme-likedimmunosorbent assay for human manganesecontaining superoxide dismutase and its content in lung cancer, J. Natl. Cancer Inst., 72 (1984) 10431049. 6 Johansen, F.F., Jorgensen, M.B. and Diemer, N.H., Resistance of hippocampal CA-I interneurons to 20 min of transient cerebral ischemia in the rat, Acta Neuropathol., 61 (1983) 135-140. 7 Kawaguchi, T., Noji, S., Uda, T., Nakashima, Y., Takeyasu, A., Kawai, Y., Takagi, H., Tohyama, M. and Taniguchi, N., A monoclonal antibody against COOH-terminal peptide of human liver manganese superoxide dismutase, J. Biol. Chem., 264 (1989) 5762-5767. 8 Kirino, T. and Sano, K., Selective vulnerability in the gerbil hippocampus following ischemia, Acta Neuropathol., 62 (1984) 201-208. 9 Liu, T.H., Beckman, J.S., Freeman, B.A., Hogan, E.L. and Hsu, C.Y., Polyethylen glycol-conjugated superoxide dismutase and catalase reduce ischemic brain injury, Am. J. Physiol., 256 (1989) H589H593. 10 Matsumoto, M., Hatakeyama, T., Akai, F., Brengman, J.M. and Yanagihara, T., Prediction of stroke before and after unilateral occlusion of the common carotid artery in the gerbil, Stroke, 19 (1988) 49(~ 497. 11 Pigott, J.P., Donovan, D.L., Fink, J.A. and Sharp, W.V., Experimental pharmacologic cerebroprotection, J. Vasc. Surg., 7 (1988) 625~530. 12 Rothman, S., Synaptic release of excitatory amino acid neurotransmitter mediates anoxic neuronal death, J. Neurosci., 4 (1984) 1884-1891. 13 Schlander, M., Hoyer, S. and Frotscher, M., Glutamate decarboxylase-immunoreactiveneurons in the aging rat hippocampus are more resistant to ischemia than CAI pyramidal cells, Neurosci. Lett., 91 (1988) 241 246.
23 14 Simon, R.P., Swan, J.H. and Gritiiths, T., Blockade of N-methyl-D-aspartate receptors may protect against ischemic damage in the brain, Science, 226 (1984) 850-852. 15 Somogyi, P. and Takagi, H., A note on the use of picric acid-paraformaldehyde-glutaraldehyde fixative for correlated light and electron microscopic immunocytochemistry, Neuroscience, 7 (1982) 17791783. 16 Yoshida, S., Inoh, S., Asano, T., Sano, K., Kubota, M., Shimazaki, H. and Ueta, N., Effect of transient ischemia on free fatty acids and phospholipids in the gerbil brain: lipid peroxidation as possible cause of postischemic injury, J. Neurosurg., 53 (1980) 323-331. 17 Zamboni, L. and De Martino, C., Buffered picric acid formaldehyde: a new fixative for electron-microscopy, J. Cell Biol., 35 (1967) 148/A.
19
Elsevier Scientific Publishers Ireland Ltd. NSL 06984
Immunocytochemical localization of manganese superoxide dismutase (Mn-SOD) in the hippocampus of the rat Fumiharu Akai I, Mitsuyo Maeda 2, Keiichiro Suzuki 3, Shinobu Inagaki 4, Hiroshi Takagi 4 and Naoyuki Taniguchi 3 Departments of lNeurosurgery and 2Pathology, Kinki University School of Medicine, Osaka-Savama (Japan), 3Department of Biochemistry, Osaka University Medical School, Osaka (Japan) and 4First Department of Anatomy, Osaka City University Medical School, Osaka (Japan)
(Received 8 March 1990; Accepted 11 March 1990) Key words." Manganese superoxide dismutase; Hippocampus; Immunocytochemistry;Selective vulnera-
bility The immunocytochemicaldistribution of manganese superoxide dismutase (Mn-SOD) was determined in the rat hippocampus. The enzymewas localized in the mitochondria. CA I pyramidal cells were weakly immunostained, whereas CA3 pyramidal cells were strongly reactive. These differences in the intensity of the Mn-SOD immunostaining reactions may relate to variations in the sensitivityof subfields of the hippocampus to ischemia.
Superoxide dismutases (SODs) catalyze the conversion of the superoxide radicals to hydrogen peroxide and molecular oxygen. These enzymes may protect cells against reactive free radicals produced under different physiological conditions, such as reoxygenation following ischemia [16]. This is supported by animal experiments which show that ischemic neuronal damage is significantly prevented by the pre-administration of exogenous SODs [9, 11]. The hippocampus is one of the most sensitive areas to ischemia. Following transient cerebral ischemia, delayed neuronal death occurs most prominently in the CA1 area as compared with other subfields [8]. The reason for such selective vulnerability of specific regions has not been elucidated [10, 12, 14]. Therefore, we have studied the immunocytochemical localization of manganese (Mn)-SOD, in order to provide a morphological basis for understanding the possible role of this enzyme in the hippocampus. A total of 12 adult male, 150g Wistar rats were used in this study. Ten animals were anesthetized and perfused with 200 ml of Zamboni's fixative [17] for light microCorrespondence: H. Takagi, First Department of Anatomy, Osaka City University Medical School, 1-4-54 Asahimachi, Abenoku, Osaka 545, Japan.
0304-3940/90/$ 03.50 © 1990 ElsevierScientific Publishers Ireland Ltd.
20
Fig. 1. Light micrographs of the rat hippocampus stained for Mn-SOD. A gradient of the reaction intensity a m o n g the subfields is seen (A). Arrows indicate both ends of CA1 where pyramidal cell bodies are almost devoid of immunoreactivity in contrast to possible interneurons with strong reactivity (arrowheads). Higher magnification shows pyramidal cells (B) in CA3 staining strongly as contrasted with weak staining CA l cells (C). Scales: A = 50/~m; B, C = 20 ~m.
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scopic studies. Two animals were perfused with 200 ml of picric acid (0.2 %)-glutaraldehyde (0.05 %)-paraformaldehyde (4 %) fixative [15] for electron microscopic studies. A portion of the brain containing the hippocampus was immersed in these fixatives for 4 h at 4°C, followed by immersion in 0.1 M phosphate buffer (pH 7.4) containing 30% sucrose. Frontal sections were cut at a thickness of 40/am with a Microslicer (Dosaka EM). These were placed in a small vial and incubated overnight at 4°C with an 1:100 dilution of the primary antiserum. They were then stained according to the avidin-biotin-peroxidase (ABC) method [4] (Vectastain ABC kit). The primary antiserum employed was raised in New Zealand white rabbits to Mn-SOD which was purified from normal rat liver as described previously [5]. The enzyme used had a specific activity of 3200 U/mg protein and gave a single band on SDS polyacrylamide gel electrophoresis. The specificity of the antiserum was established by an enzyme immunoassay technique [5]. The immunostained sections used for light microscopy were mounted, dehydrated and coverslipped. Sections for electron microscopy were postfixed with 1% OsO4 for 1 h and embedded in epoxy resin. Ultrathin sections were stained with 1% uranyl acetate and lead citrate. Electron micrographs were taken at 100 kV on a Hitachi H-12A. To test for the specificity of the immunocytochemical procedure, control experiments were performed, i.e., omission of the primary antiserum, replacement of the primary antiserum with normal rabbit serum, and use of the primary antiserum following its absorption, with purified Mn-SOD (20 -6 M). No tissue sections prepared by the latter methods showed immunostaining. A marked topographical difference in the intensity of the immunoreactions was apparent. Most of the pyramidal cells in CA1 were weakly stained, whereas those in CA3 were stained strongly (Fig. 1). CA4 pyramidal cells showed a lower reactivity than those of CA3, but more than for CA1 cells. Within the CA3 area there was a gradient in the intensity of reaction, the CA3a and CA3b areas being more strongly stained than CA3c whose reaction was still more pronounced than CA4. Judging from the perikaryal size and shape, some non-pyramidal cells in all of the subfields appeared to have strong immunoreactivity in their somata and processes (Fig. 1C). Such cells were also found in other layers of the hippocampus (Fig. 1B,C). These immunoreactive cells showed numerous granular or rod-shape precipitates (Fig. 1B,C). Nuclei were always devoid of such reactivity. The electron microscopy results confirmed the localization of the Mn-SOD as being in mitochondria associated with the inner membrane and matrix in both reactive pyramidal and non-pyramidal cells (Fig. 2). Little information is available on the morphological localization of Mn-SOD in the brain [3]. The present study has demonstrated a characteristic localization of this enzyme in pyramidal cell layers of the rat hippocampus. The activity of antioxidative enzymes, i.e., Mn-SOD and Cu/Zn-SOD, may relate to the defense capability of Fig. 2. Electron micrographs of Mn-SOD-immunoreactive non-pyramidal cell in the stratum oriens of CA I. Immunoprecipit.-ates are seen in most of the mitochondria (A). Higher magnification shows they are associated with the inner membrane and matrix (B). G, Golgi apparatus; N, nucleus. Scales: A =0.5/~m; B = 0.2/am.
22 b r a i n tissues, for example, h i p p o c a m p u s a n d cerebral cortex, against reactive oxygen free radicals formed as the result o f ischemia a n d s u b s e q u e n t r e o x y g e n a t i o n [1]. Therefore, the differences in the intensity o f the M n - S O D i m m u n o s t a i n i n g reactions m a y relate to v a r i a t i o n s in the sensitivity of subfields of the h i p p o c a m p u s to ischemia. O u r study revealed that some cells in C A 1 exhibited a strong i m m u n o r e a c t i v i t y for M n - S O D . These cells m a y c o r r e s p o n d to ischemia-resistant i n t e r n e u r o n s [6]. N e u r o nal d a m a g e in C A I appears to be limited to p y r a m i d a l cells, whereas G A B A e r g i c i n t e r n e u r o n s are relatively resistant [13]. It will be interesting to determine which category of n e u r o n s these i m m u n o s t a i n e d , n o n - p y r a m i d a l cells belong to. The present study also d e m o n s t r a t e d that M n - S O D is present in the m i t o c h o n d r i a o f n e u r o nal cells. This specific localization o f M n - S O D has been also reported in h u m a n hepatocytes [7], whereas C u / Z n - S O D is m a i n l y located in the cytoplasmic matrix a n d nucleus, b u t does n o t appear to be associated with the m i t o c h o n d r i a [2]. The a u t h o r s wish to t h a n k Prof. H.F. Deutsch for his kind review o f the text. 1 Chan, P.H., Chu, L. and Fishman, R.A., Reduction of activities of superoxide dismutase but not of glutathione peroxidase in rat brain regions following decapitation ischemia, Brain Res., 439 (1988) 388-390. 2 Chang, L.-Y., Slot, J.W., Geuze, H.J. and Crapo, J.D., Molecular immunocytochemistryof the CuZn superoxide dismutase in rat hepatocytes, J. Cell Biol., 107 (1989) 2169-2179. 3 Dobashi, K., Asayama, K., Kato, K., Kobayashi, M. and Kawaoi, A., Immunohistochemicallocalization of copper-zinc and manganese superoxide dismutases in rat tissues, Acta Histochem. Cytochem., 22 (1989) 351 365. 4 Hsu, S., Raine, L. and Fanger, H., Use of avidin-biotin-peroxidasecomplex (ABC) in immunoperoxidase technique: a comparison between ABC and unlabelled antibody (PAP) procedures, J. Histochem. Cytochem., 29 (1981) 577-580. 5 Iizuka, S., Taniguchi, N. and Makita, A., Enzyme-likedimmunosorbent assay for human manganesecontaining superoxide dismutase and its content in lung cancer, J. Natl. Cancer Inst., 72 (1984) 10431049. 6 Johansen, F.F., Jorgensen, M.B. and Diemer, N.H., Resistance of hippocampal CA-I interneurons to 20 min of transient cerebral ischemia in the rat, Acta Neuropathol., 61 (1983) 135-140. 7 Kawaguchi, T., Noji, S., Uda, T., Nakashima, Y., Takeyasu, A., Kawai, Y., Takagi, H., Tohyama, M. and Taniguchi, N., A monoclonal antibody against COOH-terminal peptide of human liver manganese superoxide dismutase, J. Biol. Chem., 264 (1989) 5762-5767. 8 Kirino, T. and Sano, K., Selective vulnerability in the gerbil hippocampus following ischemia, Acta Neuropathol., 62 (1984) 201-208. 9 Liu, T.H., Beckman, J.S., Freeman, B.A., Hogan, E.L. and Hsu, C.Y., Polyethylen glycol-conjugated superoxide dismutase and catalase reduce ischemic brain injury, Am. J. Physiol., 256 (1989) H589H593. 10 Matsumoto, M., Hatakeyama, T., Akai, F., Brengman, J.M. and Yanagihara, T., Prediction of stroke before and after unilateral occlusion of the common carotid artery in the gerbil, Stroke, 19 (1988) 49(~ 497. 11 Pigott, J.P., Donovan, D.L., Fink, J.A. and Sharp, W.V., Experimental pharmacologic cerebroprotection, J. Vasc. Surg., 7 (1988) 625~530. 12 Rothman, S., Synaptic release of excitatory amino acid neurotransmitter mediates anoxic neuronal death, J. Neurosci., 4 (1984) 1884-1891. 13 Schlander, M., Hoyer, S. and Frotscher, M., Glutamate decarboxylase-immunoreactiveneurons in the aging rat hippocampus are more resistant to ischemia than CAI pyramidal cells, Neurosci. Lett., 91 (1988) 241 246.
23 14 Simon, R.P., Swan, J.H. and Gritiiths, T., Blockade of N-methyl-D-aspartate receptors may protect against ischemic damage in the brain, Science, 226 (1984) 850-852. 15 Somogyi, P. and Takagi, H., A note on the use of picric acid-paraformaldehyde-glutaraldehyde fixative for correlated light and electron microscopic immunocytochemistry, Neuroscience, 7 (1982) 17791783. 16 Yoshida, S., Inoh, S., Asano, T., Sano, K., Kubota, M., Shimazaki, H. and Ueta, N., Effect of transient ischemia on free fatty acids and phospholipids in the gerbil brain: lipid peroxidation as possible cause of postischemic injury, J. Neurosurg., 53 (1980) 323-331. 17 Zamboni, L. and De Martino, C., Buffered picric acid formaldehyde: a new fixative for electron-microscopy, J. Cell Biol., 35 (1967) 148/A.
