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Localization of Fas antigen mRNA induced in postischemic murine forebrain by in situ hybridization
MOLECULAR BRAIN RESEARCH
ELSEVIER
Molecular Brain Research 34 (1995) 166-172
Short communication
Localization of Fas antigen mRNA induced in postischemic murine forebrain by in situ hybridization Tomohiro Matsuyama a,* Ryuji Hata a, Yoshihiro Yamamoto b, Masafumi Tagaya a Hiroshi Akita a, Hisakazu Uno a, Akio Wanaka c, Jun-ichi Furuyama b, Minoru Sugita a a Fifth Department of Internal Medicine, Hyogo College of Medicine, 1-1 Mukogawacho, Nishinomiya 663, Japan b Department of Genetics, Hyogo College of Medicine, 1-1 Mukogawacho, Nishinomiya 663, Japan c Department of Anatomy, Osaka University Medical School, 2-2 Yamadaoka, Suita 565, Japan Accepted 28 June 1995
Abstract
The expression of mRNA for the Fas antigen, a membrane-associated protein mediating apoptosis, was localized by in situ hybridization histochemistry in murine brains following 30 min of global cerebral ischemia. Six hours following the ischemia, many labeled cells were detected anew throughout the brain. The hybridization was seen in the small neural cells and in the cells along the walls of the ventricles and vessels, and became undetectable 24 h following the ischemia. These results suggest that the Fas antigen is expressed in the neuron, glia and periventricular cells of the post-ischemic brain. Keywords: Fas antigen; Apoptosis; Cerebral ischemia; BALB/c mouse; In situ hybridization histochemistry
Apoptosis is a morphologically distinct form of cell death that is involved in many physiologic and pathologic processes [7,8,15], and it requires the activation of a 'cell death' gene program [24,33]. Recent studies have shown that apoptosis may account, at least in part, for ischemic injury of the brain [13,14,25]. Fas antigen is an apoptosis-associated polypeptide [10,31]. It belongs to a family of cell surface proteins which includes nerve growth factor receptor, tumor necrosis factor receptor and B-cell antigen CD40, all of which mediate cell death. Fas expression has been detected in Band T-lymphoid cell lines and in various organs including the thymus and liver. It is suggested that the Fas/Fas ligand pathway is one of the major mechanisms for Tcell-mediated cytotoxicity [11,21]. We previously reported using Northern blot analysis that Fas antigen mRNA was expressed in brains following transient global cerebral ischemia [18]. Fas expression was induced transiently, peaked at 6 h, and became undetectable 24 h following restoration of cerebral circulation. However, it has not been confirmed whether Fas antigen is expressed in the neural cells or in the circulating peripheral
* Corresponding author. Fax: (81) (798) 45-6597. 0169-328X/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved
SSDI 0 1 6 9 - 3 2 8 X ( 9 5 ) 0 0 1 6 2 - X
blood cells. In this study, we examined the localization of Fas antigen mRNA in post-ischemic brains using in situ hybridization histochemistry. Male B A L B / c mice (25-30 g) (Charles River, Yokohama, Japan) were anesthetized lightly by ether inhalation and cerebral ischemia was produced by the occlusion of both c o m m o n carotid arteries with microaneurysm clips for 30 min. Their rectal temperature was monitored and was maintained at 37.0-37.2°C during the ischemic insult. The neurological status of each animal was recorded during a 30-min period of occlusion. For prompt neurological assessment, disturbance of consciousness as well as some neurological deficits such as piloerection, ptosis, eye fixed open, jumping, running, rolling and a splayed out hind limb were observed. Animals were divided into two groups depending on the consciousness status: (1) animals showing coma (disappearing of righting reflex) and (2) animals without coma. In the preliminary study, 53 of 100 animals which underwent the surgery showed loss of righting reflex during the ischemic insult. Twenty-seven animals out of 53 comatose mice spontaneously died during the ischemic insult. Twenty-four animals out of the remaining 26 mice showed a clear neuronal degeneration in their hippocampus 4 days after the ischemic insult, but the other two had not. In this study,
T. Matsuyama et aL /Molecular Brain Research 34 (1995) 166-172
eight out of 36 operated mice survived the carotid occlusion and showed coma during the ischemic insult. These animals were decapitated 6 h or 24 h following clip removal (four animals each). The carotid arteries of the control animals ( n = 4) were exposed through the neck, but clips were not used. The brain was removed quickly, frozen in powdered dry ice, and sectioned on a cryostat. 16 /xm thick sections were thaw-mounted on T E S T A (3-
167
aminopropyl triethoxysilane)-treated slides. The slides were kept at - 8 0 ° C until use. R N A probe was prepared for in situ hybridization of Fas antigen mRNA. Mouse Fas antigen c D N A was obtained from total R N A (2 /xg) of a B A L B / c mouse thymus using the reverse transcriptase polymerase chain reaction (RT-PCR) [30,31]. The amplified D N A fragment (about 1100 bp) was gel-purified, digested with E c o R I and
Fig. 1. Bright-field (A, B and C) and dark-field (D, E and F) photomicrographs show Fas antigen hybridization in the dorsal hippocampus from a sham-operated mouse (A, D) and from mice 6 h (B, E) and 24 h (C, F) after 30 min of bilateral carotid occlusion. Almost no hybridization was observed in the control hippocampus (D). Strong hybridization was detected in numerous cells throughout the hippocampus, 6 h after the ischemia (E). Note that almost no hybridization signal exists in the CA1 pyramidal layer. The signal was reduced at 24 h (F), but a few labeled cells were still detected in the dentate gyrus (DG). * indicates the lateral ventricle. Scale bar = 300 /zm.
168
I". Matsuyama et al. / Molecular Brain Research 34 (1995) 166-172
S a l I and subcloned into the pBluescript K S ( + ) vector
(Stratagene, La Jolla, CA). The resultant plasmid named pBMF [18] was linearized by digesting with E c o R I (antisense) or S a l I (sense). In vitro transcription was performed using the appropriate RNA polymerase (T3 RNA polymerase for the antisense strand; T7 RNA polymerase for the sense strand) and [c~-35S]UTP. In situ hybridization procedures were based on those of Wilkinson et al. with some modifications [29,32]. The sections were fixed in 4% paraformaldehyde in 0.1 M sodium phosphate buffer (NaPB) for 20 min, and were treated with 1 0 / z g / m l of proteinase K in 50 mM Tris-HCl, 5 mM EDTA (pH 8.0) for 5 min at room temperature. They were post-fixed in the same fixative as described above, acetylated with acetic anhydride in 0.1 M triethanolamine. The slides then were dehydrated in serial alcohol solutions, defatted in chloroform, and air-dried. Hybridization was performed overnight at 55°C with the 35S-labeled probes (1-5 X 105 dpm per slide) in hybridization buffer, which consisted of 50% deionized formamide, 0.3 M NaCI, 20 mM Tris-HCl, 5 mM EDTA, 10 mM NaPB, 10% dextran sulfate, 1 X Denhardt's solution (0.2% polyvinylpyrrolidone, 0.2% Ficoll, 0.2% bovine serum albumin), 0.2% Sarcosyl, 500 p,g/ml yeast tRNA, and 200 /zg/ml of salmon sperm DNA (pH 8.0). The subsequent stringency washes were performed using 5 x SSC (1 X SSC is 0.15 M NaC1, 0.015 M sodium citrate) at 55°C for 20 min, and 2 x SSC, 0.1 M DTT, 50% deionized formamide at 65°C for 30 min. Following rinsing with ribonuclease (RNase) buffer (0.5 M NaC1, 10 mM Tris-HC1, 5 mM EDTA, pH 8.0) four times for 10 min each at 37°C, the sections were treated with 1 /.~g/ml of RNase A in RNase buffer for 30 min at 37°C. Following an additional wash in RNase buffer, the slides were incubated at 65°C in 50% formamide, 2 x SSC for 30 min, and rinsed with 2 X SSC and 0.1 x SSC for 15 min each at room temperature. They then were dehydrated in a series of graded alcohol solutions and dried. The slides were dipped in Kodak NTB-2 emulsion, developed following exposure for 3 weeks at 4°C and counterstained with cresyl violet. Histologic examination revealed that the brains of comatose animals exhibited few changes in their forebrain including the caudate putamen, cerebral cortex and hippocampus, at 6 h (Fig. 1B) and at 24 h (Fig. 1C) following the ischemic insult. In situ hybridization revealed that little Fas mRNA hybridization was observed in the sham-operated mice brain (Fig. 1D). At 6 h following the ischemia, significant hybridization in numerous cells was detected throughout the brain including the cerebral cortex (Fig. 3A), caudate putamen (Fig. 3B), thalamus and the hippocampus (Fig. 1E). The periventricular cells including the ependymal cells and the choroid plexus had a strong hybridization signal for Fas antigen mRNA (Figs. 2 and 3). In the parenchyma, the hybridized cells resembled glial cells, because of their rather small size and location. They were
located in the white matter region such as the corpus callosum, fimbria and optic nerve tract (Fig. 2B). The intensity of the hybridization signals was reduced considerably throughout the brain at 24 h (Fig. 1F). At 6 h, the caudate putamen exhibited a number of cells which had a positive hybridization signal (Fig. 2B). They were distributed rather evenly, and were predominantly present outside the fiber bundles of the caudate putamen. The hybridization was also seen in association with the vascular structures. In the hippocampus, the Fas hybridization signal was more predominant in the white matter regions such as the stratum radiatum, lacunosum moleculare and fftratum oriens (Fig. 1E). Little hybridization was seen in the pyramidal neurons of the CA regions or in the granular cells of the dentate gyrus, although a few small cells scattered among these neurons had a strong hybridization signal. In the cerebral cortex, the hybridizing cells were distributed in all cortical layers (Fig. 3A), but the large pyramidal neurons in layer V exhibited no significant hybridization for the Fas antigen. This study demonstrated that Fas mRNA expression was induced transiently in some neural cells, periventricular cells and perivascular cells of the brain following ischemic insult. This is in good agreement with the findings from our previous study which employed Northern blot analysis [18]. The cells which were positive for Fas antigen expression in the brain parenchyma are likely to be glial cells, because they are distributed in the white matter region of the brain. However, it has not yet been proven that Fas antigen mRNA is expressed in small neuronal cells such as interneurons. The Fas antigen is an apoptosis-associated cell surface molecule, which seems to be a receptor protein mediating a cell death signal intercellularly [10]. The Fas ligand, in contrast, induces apoptosis in Fas antigen-expressing target cells [27] and is considered to be an effector molecule in the Ca 2+-independent cytotoxicity of the cytotoxic T lymphocyte [23]. Thus, the Fas antigen expressed in the thymus has been suggested to be involved in the clonal deletion of T cells in the thymus [1,30], and the Fas/Fas ligand system may contribute to T cell-mediated cytotoxicity [11]. However, the intracellular signal transduction via the Fas antigen which leads to cell death is not clear [21], although recent reports have proposed the sphingomyelin pathway as a candidate of Fas antigen-mediated signal transduction [6,9]. The Fas antigen-expressing lymphocytes do not always undergo cell death [19]. Alderson et al. have suggested that the Fas protein may also play an important role in the proliferation of normal T-lymphocytes [2]. The functional significance of the Fas antigen expressed in neural cells should be examined in the future. In situ hybridization showed that during the entire period of recirculation, Fas antigen mRNA was not expressed in the pyramidal neurons in the hippocampus which are vulnerable to ischemia [3,12]. Therefore, it is not likely that the delayed neuronal death of the CA1 pyrami-
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dal neurons involves Fas-mediated cell death. In the central nervous system (CNS), neurons are not the only neural cell type that undergoes apoptosis. Some glial cells also die during the development of the CNS [4,26]. Recent studies suggest that a subpopulation of astrocytes and oligodendrocytes undergoes apoptosis when they die [20]. Another study has also shown that in situ DNA fragmentation is seen in astrocytes as well as in neurons after ischemic insult [13]. Our result suggests, but does not prove, that the Fas antigen-expressing glial cells as well as neurons may undergo apoptosis following an ischemic insult. The effects of an ischemic insult on the ependymal cells and the choroid plexus are unclear, although they likely have some response to oxidative stress, which can be a mediator of apoptosis [5]. The superoxide dismutases as well as the heat shock stress proteins have been reported to be induced in the periventricular cells following ischemia in gerbils [16,17,22,28]. Further study is needed to provide evidence for the role of the induced Fas antigen in the periventricular cells following ischemia. In conclusion, we first demonstrated that Fas antigen mRNA expression is induced in some neural cells following cerebral ischemia, suggesting that apoptotic cell death is mediated by the Fas antigen in the post-ischemic brain. These results also suggest that novel therapeutic approaches directed at blocking the action of the Fas antigen may be useful for preserving functional neural tissue following an ischemic insult.
Acknowledgements This work was supported in part by Grants-in-Aid for Scientific Research by the Ministry of Education, Science and Culture, Japan.
References [1] Adachi, M., Watanabe-Fukunaga, R. and Nagata, S., Aberrant transcription caused by the insertion of an early transposable element in an intron of the Fas antigen gene of lpr mouse, Proc. Natl. Acad. Sci. USA, 90 (1993) 1756-1760. [2] Alderson, M.R., Armitage, R.J., Maraskovsky, E., Tough, T.W., Roux, E., Schooley, K., Ramsdell, F. and Lynch, D.H., Fas transduces activation signals in normal human T lymphocytes, J. Exp. Med., 178 (1993) 2231-2235. [3] Barone, F.C., Knudsen, D.J., Nelson, A.H., Feuerstein, G.Z. and Willette, R.N., Mouse strain differences in susceptibility to cerebral ischemia are related to cerebral vascular anatomy, J. Cereb. Blood Flow Metab., 13 (1993) 683-692. [4] Barres, B.A., Hart, I.K., Coles, H.S.R., Burne, J.F., Voyvodic, J.T., Richadson, W.D. and Raft, M.C., Cell death and control of cell survival in the oligodendroglial lineage, Cell, 70 (1992) 31-46. [5] Buttke, T.M. and Sandstrom, P.A., Oxidative stress as a mediator of apoptosis, Immunol. Today, 15 (1994) 7-10. [6] Cleveland, J.L. and Ihle, J.N., Contenders in FasL/TNF death signaling, Cell, 81 (1995) 479-482.
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[7] Cohen, J.J., Apoptosis, lmmunol. Today, 14 (1993) 126-130. [8] Fesus, L., Davies, P.J.A. and Piacentini, M., Apoptosis: molecular mechanisms in programmed cell death, Eur. J. Cell Biol., 56 (1991) 170-177. [9] Heller, R.A. and Krfnke, M., Tumor necrosis factor receptor-mediated signaling pathways, J. Cell Biol., 126 (1994) 5-9. [10] ltoh, N., Yonehara, S., Ishii, A., Yonehara, M., Mizushima, S.-I., Sameshima, M., Hase, A., Seto, Y. and Nagata, S., The polypeptide encoded by the cDNA for human cell surface antigen Fas can mediate apoptosis, Cell, 66 (1991) 233-243. [11] Kagi, D., Vignaux, F., Lederman, B., Burki, K., Depraetere, V., Nagata, S., Hengartner, H. and Golstein, P., Fas and perforin pathways as major mechanisms of T cell-mediated cytotoxicity, Science, 265 (1994) 528-530. [12] Kirino, T. and Sano, K., Fine structure of delayed neuronal death following ischemia in the gerbil hippocampus, Acta Neuropathol., 62 (1984) 209-218. [13] Li, Y., Chopp, M., Jiang, N. and Zaloga, C., In situ detection of DNA fragmentation after focal cerebral ischemia in mice, Mol. Brain Res., 28 (1995) 164-168. [14] Linnik, M.D., Zobrist, R.H. and Hatfield, M.D., Evidence supporting a role for programmed cell death in focal cerebral ischemia in rats, Stroke, 24 (1993) 2002-2009. [15] Lockshin, R.A. and Zakeri, Z., Programmed cell death and apoptosis. In L.D. Tomei and F.O. Cope (Eds.), Apoptosis: The Molecular Basis of Cell Death, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1991, pp. 47-60. [16] Matsuyama, T., Michishita, H., Nakamura, H., Tsuchiyama, M., Shimizu, S., Watanabe, K. and Sugita, M., Induction of copper-zinc superoxide dismutase in gerbil hippocampus after ischemia, ./. Cereb. Blood Flow Metab., 13 (1993) 135-144. [17] Matsuyama, T., Shimizu, S., Nakamura, H., Michishita, H., Tagaya, M. and Sugita, M., Effect of recombinant superoxide dismutase on manganese superoxide dismutase gene expression in gerbil hippocampus after ischemia, Stroke, 25 (1994) 1417-1424. [18] Matsuyama, T., Hata, R., Tagaya, M., Yamamoto, Y., Nakajima, T., Furuyama, J., Wanaka, A. and Sugita, M., Fas antigen mRNA induction in postischemic murine brain, Brain Res., 657 (1994) 342-346. [19] Miyawaki, T., Uehara, T., Nibu, R., Tsuji, T., Yachie, A., Yonehara, S. and Taniguchi, N., Differential expression of apoptosis-related Fas antigen on lymphocyte subpopulations in human peripheral blood, J. lmmunol., 149 (1992) 3753-3758. [20] Mizuguchi, M., Ikeda, K., Asada, M., Mizutani, S. and Kamoshita, S., Expression of Bcl-2 protein in murine neural cells in culture, Brain Res., 649 (1994) 197-202. [21] Nagata, S. and Golstein, P., The Fas death factor, Science, 267 (1995) 1449-1456. [22] Nowak, T.S. Jr., Localization of 70kDa stress protein mRNA induction in gerbil brain after ischemia, J. Cereb. Blood Flow Metab., 11 (1991) 432-439. [23] Rouvier, E., Luciani, M.-F. and Goldstein, P., Fas involvement in Ca2+-independent T cell-mediated cytotoxicity, J. Exp. Med., 177 (1993) 195-200. [24] Schwartz, L.M. and Osborne, B.A., Programmed cell death, apoptosis and killer genes, Immunol. Today, 14 (1993) 582-590. [25] Shigeno, T., Yamasaki, Y., Kato, G., Kusaka, K., Mima, T., Takakura, K., Graham, D.I. and Furukawa, S., Reduction of delayed neuronal death by inhibition of protein synthesis, Neurosci. Lett., 120 (1990) 117-119. [26] Sturrock, R.R., A quantitative lifespan study of changes in cell number, cell division and cell death in various regions of the mouse forebrain, Neuropathol. Appl. Neurobiol., 5 (1979) 433-456. [27] Suda, T., Takahashi, T., Golstein, P. and Nagata, S., Molecular cloning and expression of Fas ligand, a novel member of the tumor necrosis factor family, Cell, 75 (1993) 1169-1178. [28] Vass, K., Welch, W.J. and Nowak, T.S. Jr., Localization of 70kDa
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stress protein induction in gerbil brain after ischemia, Acta Neuropathol., 77 (1988) 128-135. [29] Wanaka, A., Johnson, E.M. Jr. and Milbrandt, J., Localization of FGF receptor mRNA in the adult rat central nervous system by in situ hybridization, Neuron, 5 (1990) 267-281. [30] Watanabe-Fukunaga, R., Brannan, C.I., Copeland, N.G., Jenkins, N.A. and Nagata, S., Lymphoproliferation disorder in mice explained by defects in Fas antigen that mediates apoptosis, Nature, 356 (1992) 314-317. [31] Watanabe-Fukunaga, R., Brannan, C.I., ltoh, N., Yonehara, S.,
Copeland, N.G., Jenkins, N.A. and Nagata, S., The cDNA structure, expression and chromosomal assignment of the mouse Fas antigen, J. Immunol., 148 (1992) 1274-1279. [32] Wilkinson, D.G., Peters, C., Dickson, C. and MacMahon, A.P., Expression of the FGF-related proto-oncogene int-2 during gastrulation and neurulation in the mouse, EMBO J., 7 (1988) 691-695. [33] Wyllie, A.H., Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation, Nature, 284 (1980) 555-556.
ELSEVIER
Molecular Brain Research 34 (1995) 166-172
Short communication
Localization of Fas antigen mRNA induced in postischemic murine forebrain by in situ hybridization Tomohiro Matsuyama a,* Ryuji Hata a, Yoshihiro Yamamoto b, Masafumi Tagaya a Hiroshi Akita a, Hisakazu Uno a, Akio Wanaka c, Jun-ichi Furuyama b, Minoru Sugita a a Fifth Department of Internal Medicine, Hyogo College of Medicine, 1-1 Mukogawacho, Nishinomiya 663, Japan b Department of Genetics, Hyogo College of Medicine, 1-1 Mukogawacho, Nishinomiya 663, Japan c Department of Anatomy, Osaka University Medical School, 2-2 Yamadaoka, Suita 565, Japan Accepted 28 June 1995
Abstract
The expression of mRNA for the Fas antigen, a membrane-associated protein mediating apoptosis, was localized by in situ hybridization histochemistry in murine brains following 30 min of global cerebral ischemia. Six hours following the ischemia, many labeled cells were detected anew throughout the brain. The hybridization was seen in the small neural cells and in the cells along the walls of the ventricles and vessels, and became undetectable 24 h following the ischemia. These results suggest that the Fas antigen is expressed in the neuron, glia and periventricular cells of the post-ischemic brain. Keywords: Fas antigen; Apoptosis; Cerebral ischemia; BALB/c mouse; In situ hybridization histochemistry
Apoptosis is a morphologically distinct form of cell death that is involved in many physiologic and pathologic processes [7,8,15], and it requires the activation of a 'cell death' gene program [24,33]. Recent studies have shown that apoptosis may account, at least in part, for ischemic injury of the brain [13,14,25]. Fas antigen is an apoptosis-associated polypeptide [10,31]. It belongs to a family of cell surface proteins which includes nerve growth factor receptor, tumor necrosis factor receptor and B-cell antigen CD40, all of which mediate cell death. Fas expression has been detected in Band T-lymphoid cell lines and in various organs including the thymus and liver. It is suggested that the Fas/Fas ligand pathway is one of the major mechanisms for Tcell-mediated cytotoxicity [11,21]. We previously reported using Northern blot analysis that Fas antigen mRNA was expressed in brains following transient global cerebral ischemia [18]. Fas expression was induced transiently, peaked at 6 h, and became undetectable 24 h following restoration of cerebral circulation. However, it has not been confirmed whether Fas antigen is expressed in the neural cells or in the circulating peripheral
* Corresponding author. Fax: (81) (798) 45-6597. 0169-328X/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved
SSDI 0 1 6 9 - 3 2 8 X ( 9 5 ) 0 0 1 6 2 - X
blood cells. In this study, we examined the localization of Fas antigen mRNA in post-ischemic brains using in situ hybridization histochemistry. Male B A L B / c mice (25-30 g) (Charles River, Yokohama, Japan) were anesthetized lightly by ether inhalation and cerebral ischemia was produced by the occlusion of both c o m m o n carotid arteries with microaneurysm clips for 30 min. Their rectal temperature was monitored and was maintained at 37.0-37.2°C during the ischemic insult. The neurological status of each animal was recorded during a 30-min period of occlusion. For prompt neurological assessment, disturbance of consciousness as well as some neurological deficits such as piloerection, ptosis, eye fixed open, jumping, running, rolling and a splayed out hind limb were observed. Animals were divided into two groups depending on the consciousness status: (1) animals showing coma (disappearing of righting reflex) and (2) animals without coma. In the preliminary study, 53 of 100 animals which underwent the surgery showed loss of righting reflex during the ischemic insult. Twenty-seven animals out of 53 comatose mice spontaneously died during the ischemic insult. Twenty-four animals out of the remaining 26 mice showed a clear neuronal degeneration in their hippocampus 4 days after the ischemic insult, but the other two had not. In this study,
T. Matsuyama et aL /Molecular Brain Research 34 (1995) 166-172
eight out of 36 operated mice survived the carotid occlusion and showed coma during the ischemic insult. These animals were decapitated 6 h or 24 h following clip removal (four animals each). The carotid arteries of the control animals ( n = 4) were exposed through the neck, but clips were not used. The brain was removed quickly, frozen in powdered dry ice, and sectioned on a cryostat. 16 /xm thick sections were thaw-mounted on T E S T A (3-
167
aminopropyl triethoxysilane)-treated slides. The slides were kept at - 8 0 ° C until use. R N A probe was prepared for in situ hybridization of Fas antigen mRNA. Mouse Fas antigen c D N A was obtained from total R N A (2 /xg) of a B A L B / c mouse thymus using the reverse transcriptase polymerase chain reaction (RT-PCR) [30,31]. The amplified D N A fragment (about 1100 bp) was gel-purified, digested with E c o R I and
Fig. 1. Bright-field (A, B and C) and dark-field (D, E and F) photomicrographs show Fas antigen hybridization in the dorsal hippocampus from a sham-operated mouse (A, D) and from mice 6 h (B, E) and 24 h (C, F) after 30 min of bilateral carotid occlusion. Almost no hybridization was observed in the control hippocampus (D). Strong hybridization was detected in numerous cells throughout the hippocampus, 6 h after the ischemia (E). Note that almost no hybridization signal exists in the CA1 pyramidal layer. The signal was reduced at 24 h (F), but a few labeled cells were still detected in the dentate gyrus (DG). * indicates the lateral ventricle. Scale bar = 300 /zm.
168
I". Matsuyama et al. / Molecular Brain Research 34 (1995) 166-172
S a l I and subcloned into the pBluescript K S ( + ) vector
(Stratagene, La Jolla, CA). The resultant plasmid named pBMF [18] was linearized by digesting with E c o R I (antisense) or S a l I (sense). In vitro transcription was performed using the appropriate RNA polymerase (T3 RNA polymerase for the antisense strand; T7 RNA polymerase for the sense strand) and [c~-35S]UTP. In situ hybridization procedures were based on those of Wilkinson et al. with some modifications [29,32]. The sections were fixed in 4% paraformaldehyde in 0.1 M sodium phosphate buffer (NaPB) for 20 min, and were treated with 1 0 / z g / m l of proteinase K in 50 mM Tris-HCl, 5 mM EDTA (pH 8.0) for 5 min at room temperature. They were post-fixed in the same fixative as described above, acetylated with acetic anhydride in 0.1 M triethanolamine. The slides then were dehydrated in serial alcohol solutions, defatted in chloroform, and air-dried. Hybridization was performed overnight at 55°C with the 35S-labeled probes (1-5 X 105 dpm per slide) in hybridization buffer, which consisted of 50% deionized formamide, 0.3 M NaCI, 20 mM Tris-HCl, 5 mM EDTA, 10 mM NaPB, 10% dextran sulfate, 1 X Denhardt's solution (0.2% polyvinylpyrrolidone, 0.2% Ficoll, 0.2% bovine serum albumin), 0.2% Sarcosyl, 500 p,g/ml yeast tRNA, and 200 /zg/ml of salmon sperm DNA (pH 8.0). The subsequent stringency washes were performed using 5 x SSC (1 X SSC is 0.15 M NaC1, 0.015 M sodium citrate) at 55°C for 20 min, and 2 x SSC, 0.1 M DTT, 50% deionized formamide at 65°C for 30 min. Following rinsing with ribonuclease (RNase) buffer (0.5 M NaC1, 10 mM Tris-HC1, 5 mM EDTA, pH 8.0) four times for 10 min each at 37°C, the sections were treated with 1 /.~g/ml of RNase A in RNase buffer for 30 min at 37°C. Following an additional wash in RNase buffer, the slides were incubated at 65°C in 50% formamide, 2 x SSC for 30 min, and rinsed with 2 X SSC and 0.1 x SSC for 15 min each at room temperature. They then were dehydrated in a series of graded alcohol solutions and dried. The slides were dipped in Kodak NTB-2 emulsion, developed following exposure for 3 weeks at 4°C and counterstained with cresyl violet. Histologic examination revealed that the brains of comatose animals exhibited few changes in their forebrain including the caudate putamen, cerebral cortex and hippocampus, at 6 h (Fig. 1B) and at 24 h (Fig. 1C) following the ischemic insult. In situ hybridization revealed that little Fas mRNA hybridization was observed in the sham-operated mice brain (Fig. 1D). At 6 h following the ischemia, significant hybridization in numerous cells was detected throughout the brain including the cerebral cortex (Fig. 3A), caudate putamen (Fig. 3B), thalamus and the hippocampus (Fig. 1E). The periventricular cells including the ependymal cells and the choroid plexus had a strong hybridization signal for Fas antigen mRNA (Figs. 2 and 3). In the parenchyma, the hybridized cells resembled glial cells, because of their rather small size and location. They were
located in the white matter region such as the corpus callosum, fimbria and optic nerve tract (Fig. 2B). The intensity of the hybridization signals was reduced considerably throughout the brain at 24 h (Fig. 1F). At 6 h, the caudate putamen exhibited a number of cells which had a positive hybridization signal (Fig. 2B). They were distributed rather evenly, and were predominantly present outside the fiber bundles of the caudate putamen. The hybridization was also seen in association with the vascular structures. In the hippocampus, the Fas hybridization signal was more predominant in the white matter regions such as the stratum radiatum, lacunosum moleculare and fftratum oriens (Fig. 1E). Little hybridization was seen in the pyramidal neurons of the CA regions or in the granular cells of the dentate gyrus, although a few small cells scattered among these neurons had a strong hybridization signal. In the cerebral cortex, the hybridizing cells were distributed in all cortical layers (Fig. 3A), but the large pyramidal neurons in layer V exhibited no significant hybridization for the Fas antigen. This study demonstrated that Fas mRNA expression was induced transiently in some neural cells, periventricular cells and perivascular cells of the brain following ischemic insult. This is in good agreement with the findings from our previous study which employed Northern blot analysis [18]. The cells which were positive for Fas antigen expression in the brain parenchyma are likely to be glial cells, because they are distributed in the white matter region of the brain. However, it has not yet been proven that Fas antigen mRNA is expressed in small neuronal cells such as interneurons. The Fas antigen is an apoptosis-associated cell surface molecule, which seems to be a receptor protein mediating a cell death signal intercellularly [10]. The Fas ligand, in contrast, induces apoptosis in Fas antigen-expressing target cells [27] and is considered to be an effector molecule in the Ca 2+-independent cytotoxicity of the cytotoxic T lymphocyte [23]. Thus, the Fas antigen expressed in the thymus has been suggested to be involved in the clonal deletion of T cells in the thymus [1,30], and the Fas/Fas ligand system may contribute to T cell-mediated cytotoxicity [11]. However, the intracellular signal transduction via the Fas antigen which leads to cell death is not clear [21], although recent reports have proposed the sphingomyelin pathway as a candidate of Fas antigen-mediated signal transduction [6,9]. The Fas antigen-expressing lymphocytes do not always undergo cell death [19]. Alderson et al. have suggested that the Fas protein may also play an important role in the proliferation of normal T-lymphocytes [2]. The functional significance of the Fas antigen expressed in neural cells should be examined in the future. In situ hybridization showed that during the entire period of recirculation, Fas antigen mRNA was not expressed in the pyramidal neurons in the hippocampus which are vulnerable to ischemia [3,12]. Therefore, it is not likely that the delayed neuronal death of the CA1 pyrami-
T. Matsuyama et al. / Molecular Brain Research 34 (1995) 166-172
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T. Matsuyama et al. / Molecular Brain Research 34 (1995) 166-172
dal neurons involves Fas-mediated cell death. In the central nervous system (CNS), neurons are not the only neural cell type that undergoes apoptosis. Some glial cells also die during the development of the CNS [4,26]. Recent studies suggest that a subpopulation of astrocytes and oligodendrocytes undergoes apoptosis when they die [20]. Another study has also shown that in situ DNA fragmentation is seen in astrocytes as well as in neurons after ischemic insult [13]. Our result suggests, but does not prove, that the Fas antigen-expressing glial cells as well as neurons may undergo apoptosis following an ischemic insult. The effects of an ischemic insult on the ependymal cells and the choroid plexus are unclear, although they likely have some response to oxidative stress, which can be a mediator of apoptosis [5]. The superoxide dismutases as well as the heat shock stress proteins have been reported to be induced in the periventricular cells following ischemia in gerbils [16,17,22,28]. Further study is needed to provide evidence for the role of the induced Fas antigen in the periventricular cells following ischemia. In conclusion, we first demonstrated that Fas antigen mRNA expression is induced in some neural cells following cerebral ischemia, suggesting that apoptotic cell death is mediated by the Fas antigen in the post-ischemic brain. These results also suggest that novel therapeutic approaches directed at blocking the action of the Fas antigen may be useful for preserving functional neural tissue following an ischemic insult.
Acknowledgements This work was supported in part by Grants-in-Aid for Scientific Research by the Ministry of Education, Science and Culture, Japan.
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