- Email: [email protected]
Induction of SPI-3 mRNA, encoding a serine protease inhibitor, in gerbil hippocampus after transient forebrain ischemia
•
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MOLECULAR BRAIN RESEARCH
..
11 ELSEVIER
Molecular Brain Research 35 (1996) 314-318
Short communication
Induction of SPI-3 mRNA, encoding a serine protease inhibitor, in gerbil hippocampus after transient forebrain ischemia M. Tsuda ,,d,,, K. Kitagawa b, K. Imaizumi ~.d, A. Wanaka ~, M. Tohyama ~, T. Takagi
~'
Department o[ Molecular Neurobtology "Tanabe ", Osaka Umtersitv Medical School, 2-2 Yamadaoka, Suita, Osaka 565, Japan h First Department of Internal Medicine, Osaka Unicersitv Medical School, Osaka, Japan • Department of Anatomy and Neuroscience, O~aka Unit'ersi~.' Medical School, Osaka, Japan J Lead Generation Research Laboratory, Tanahe Seivaku Co. Ltd.. Osaka, Japan Accepted 16 August 1995
Abstract
We cloned genes the expression of which is induced in the Mongolian gerbil (Meriones unguiculatus) hippocampus after transient forebrain ischemia by a differential display technique. Among these genes, a rat serine protease inhibitor SPI-3 homologue was isolated. Present analyses suggested that the expression of gerbil SPI-3 mRNA was closely associated with delayed neuronal death and may block activities of proteases leaking from degenerating neurons or may support neuronal survival. Keyvvords: lschemia; Differential display: SPI-3: Astrocytc
Transient arrest of cerebral blood circulation leads to neuronal cell death in the hippocampal CA1 region [9]. Neuronal cell death, however, does not occur rapidly after ischemic insult, and neurons survive for several days, a phenomenon known as delayed neuronal death (DND) [8], The molecular mechanism of DND has attracted considerable attention. In the brain, expression of some genes, such as c-fos protooncogene [6] and those encoding heat shock proteins (HSPs) [14], and brain-derived neurotrophic factor (BDNF) [12], is induced during DND. These genes arc thought to regulate further gene expressions or to support neuronal survival. However, the molecular mechanism of DND is still not understood. To investigate DND after ischemia, we searched for new ischemia-related genes with the differential display technique [11], which is an effective method to identify and isolate genes which are differentially expressed in various cells or under altered conditions. Adult male Mongolian gerbils weighing 60-8(I g were used in the present study. The surgical procedure was described previously [7]. Briefly, each gerbil was lightly anesthetized by ether inhalation and both common carotid
• Corresponding author. Fax: {~1) (6) 879-3648. 0169-328X/96/$15.(~1 ~ 1996 Elsevier Science B.V, All rights reserved
SSDI 0 1 6 9 - 3 2 8 X ( 9 5 ) 0 0 2 1 1-1
arteries were exposed. Bilateral cerebral ischemia was produced by occlusion of both common carotid arteries with miniture aneurysm clips. Body temperature was maintained at 37°C using warming blankets and a heating lamp throughout the surgical procedure. Animals were divided randomly into a sham-operated group and a 5 min ischemic group. Twenty-four hours after clip removal, the animals were again anesthetized by ether inhalation, decapitated and their hippocampi were promptly removed for RNA purification. Total RNA was isolated by the acid guanidine thiocyanate-phenol-chloroform method [2] from the hippocampi in each group, and differential display was carried out as described previously [5]. Briefly, cDNAs were synthesized from the total RNA with reverse transcriptase after DNase treatment and PCR was performed with the synthesized cDNA as template. Four cDNA fragments which were amplified only from the cDNA derived from ischemic hippocampi were observed. These fragments were recovered, subcloned into the pGEM-T vector (Promega) and sequenced. The sequences were analyzed for homology with the Software Development Co. data bank (EMBL and GenBank DNA databases) using its program. The sequence of clone P017 (916 bp), showed a high degree of homology to the rat SPI
315
M. Tsuda et al. / Molecular Brain Research 35 (1996) 314-318
gene family, which consists of three serine protease inhibitors (SPI-1, 2, and 3) reported to be expressed in rat liver [15,16]. Clone P017 had a region encoding a C-terminal 132 amino acid sequence including the reactive center. SPI-1, 2 and 3 have a high degree of homology with each other, but there is no homology in the reactive center. The amino acid sequence homologies between P017 and SPI-1, P017 and SPI-2, P017 and SPI-3 were 62%, 60%, 73%, respectively. The amino acid sequence at the reactive center of P017 was different from those of SPI-1 and SPI-2 (Fig. 1B), whereas it was very similar to that of SPI-3. In addition, the P1 residue, according to the nomenclature defined by Schechter and Berger [18], thought to be crucial in determining the anti-protease specificity [20] was methionine in both P017 and SPI-3 (Fig. 1A). Both SPI-1 and SPI-2 m R N A s are approximately 1.8 kb, and that of SPI-3 is about 2.2 kb. From Northern blot analysis, the length of clone P017 m R N A was about 2.2 kb (Fig. 2). Taking all of these findings into consideration, we concluded that clone P017 is gerbil SPI-3. To investigate the expression pattern of gerbil SPI-3, we prepared total R N A from the hippocampus of the animals at 1 h, 6 h, 24 h, 3 days, 7 days, and 1 month after bilateral common carotid occulusion. 15 /zg of total R N A was fractioned by electrophoresis. A c D N A probe synthesized from a fragment of SPI-3 (clone P017) was shown to hybridized with a single m R N A species of about 2.2 kb (Fig. 2). In sham-operated animals, expression of SPI-3 was low. A significant increase in level of expression of SPI-3 was observed from 6 h after ischemia, and attained a maximum level at 24 h. Subsequently, a gradual reduction in level of SPI-3 m R N A was seen, and it was hardly detectable after 1 month. In situ hybridization using c R N A probe was carried out to analyze the distribution of cells expressing SPI-3 m R N A after ischemia. Digoxigenin-labeled c R N A was generated by in vitro transcription using the c D N A subcloned into
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Fig. 1. Alignmentof rat SPI family and P017 amino acid sequences. A: Alignment of rat SPI-3 and P017 amino acid sequences. The amino acid sequence of rat SPI-3 shown refers to that reported by Pages et al. [16], and that of clone P017 was deduced from the cDNA sequence. Numbering of rat SPI-3 amino acid residues is indicated on both sides above the sequences given in the one-letter code. Asterisks indicate identical residues conserved between rat SPI-3 and clone P017. The anti-protease reactive center region is boxed and the P1 residues in the reactive sites are circled. B: Alignment of the anti-protease reactive center region of SPI-I. SPI-2 and P017.
the p G E M - T vector as a template in the presence of digoxigenin-labeled UTP. Thaw-mounted frozen sections 15 /zm thick were postfixed for 20 min in 4% formal-
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Fig. 2. Pattern of gerbil SPI-3 mRNA expression after ischemic insult. A: Each lane was loaded with 15 p.g of total RNA purified from the hippocampus at specified times after ischemic insult. Ethidium bromide-stained ribosomal RNAs (28S and 18S) indicate that the amounts of total RNA loaded in each lane were equivalent. Lane 1, sham-operated; 2, 6 h; 3, 24 h; 4, 3 days; 5, 7 days; 6, 1 month. B: Membrane-transferredRNAs were hybridized with gerbil SPI-3 (clone P017) probe. The arrow indicates SPI-3 signal.
316
M. l~vuda et al. / Moh'cular Brain Rexearch 35 t 1996) 3 1 4 - 3 1 8
Fig, 3. In situ hybridization of gerbil brain sections 3 days after transient ischcmia with gerbil SPI-3 cRNA probe. SPI-3 signals were seen only in the inner side of CA1 region, especially in stratum oriens and stratum radiatum. In stratum pyramidale there were just weak signals. The section 24 h after transient ischemia was almost the same as that of 3 days. a, alveus: o, stratum oriens; p, stratum pyramidale; r, stratum radiatum. Bar = 250 /zm.
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Fig. 4. In situ hybridization with SPI-3 cRNA probe and immunohistochemistry with anti-GFAP antibody. A: In situ hybridization of CA1 region with SPI-3 cRNA probe. B: Immunohistochemistry of the section adjacent to that in A with anti-GFAP antibody. Cells containing SPI-3 mRNA and GFAP are shown by arrows in the consecutive sections. The stars denote the same capillaries in the serial sections. C: In situ hybridization of CAI region with SPI-3 cRNA probe. D: Immunohistochemistry of section C reacted with anti-GFAP antibody and FITC-labeled anti-mouse lgG antibody. Bar = 30 /,tin. Arrows indicate cells containing both SPI-3 mRNA signals and GFAP immunoreactivity in their cell bodies. Arrowheads indicate cells showing SPI-3 mRNA signals in their cell bodies and GFAP immunoreactivities in their processes.
M. Tsuda et al. / Molecular Brain Research 35 (1996) 314-318
dehyde, followed by two 5 min washes in 0.1 M phosphate buffer (pH 7.2). All subsequent procedures were performed as described previously [5]. In sham-operated gerbils, SPI-3 mRNA signals were not detected. In gerbil brains obtained 24 h and 3 days after 5 min ischemia, SPI-3 mRNA was detected in the alveus, stratum oriens and stratum radiatum. In the stratum pyramidale, weak signals were observed (Fig. 3). Twenty-four hours to 3 days after the operation, SPI-3 mRNA signals reached their peak in accordance with Northern blot analysis, showing a subsequent reduction. Cells expressing SPI-3 seemed to be glial cells on the basis of their distribution pattern and size. To confirm this possibility, immunohistochemistry and in situ hybridization were carried out on pairs of alternate adjacent serial sections. One section in each pair was reacted with antiGFAP monoclonal antibody (Chemicon) followed by the Vectstain ABC Kit (Vector Lab), and the other was hybridized with SPI-3 cRNA probe. SPI-3 mRNA signals and GFAP immunoreactivity were colocalized in some cells (Fig. 4A,B). Moreover, the sections which reacted with the SPI-3 probe were also then stained for GFAP by fluorescent immunohistochemistry. SPI-3 signals overlapped widely with positive immunofluorescence GFAP (Fig. 4C,D). From these results, cells expressing SPI-3 mRNA were confirmed to be astrocytes. In this investigation, four clones induced in the gerbil hippocampus after ischemia were isolated. We analyzed clone P017, which was thought to be gerbil SPI-3 judging from DNA and amino acid sequence analyses and the length of its transcript. Gerbil SPI-3 was induced in the hippocampal CA1 region after ischemia. Recent reports suggested that possible interleukin-6 (IL-6)-responsive elements were included in the promoter region of rat SPI-3 gene [17] and that IL-6 was responsible for the induction of SPI-3 in rat hepatocytes [10]. It is well known that IL-6 is immediately induced during brain injury [21] and has a protective effect on neurons [3]. Therefore, it is possible that the expression of SPI-3 from astrocytes is mediated by IL-6 against ischemic damage. SPI-3 was induced in hippocampal CA1 6 h after ischemia, when neurons have not yet degenerated morphologically. Activation of astrocytes or microglia, which are thought to be markers of irreversible ischemic damage, are not induced only in CA1 but also in CA3 where DND does not occur. As SPI-3 was induced only in CA1, SPI-3 may be a useful sensitive marker of irreversible ischemic damage. The function of SPI-3 in the brain is not yet understood. However it is presumably involved in (1) blocking the protease activity leaking from degenerative neurons, (2) supporting neurons and playing a role in structural rearrangement and (3) leading to neuronal death. Other protease inhibitors such as glia-derived nexin (GDN) [4,13] or amyloid precursor protein (APP) 751/770 [1] are known to be induced after ischemia. They have
317
neurite promoting activity, and GDN is thought to support neuronal cells and to play a role in structural rearrangement of the central nervous system because GDN is expressed widely in CA1 more than 90 days after transient ischemia. On the other hand, the expression of SPI-3 disappeared within a month. SPI-3 may not support neurons because its expression pattern was different from that of GDN. To investigate this possibility, more detailed studies are needed. Also, from the present results, we are not able to discuss whether SPI-3 leads to neuronal death or not. In the present study, (i) the expression of SPI-3 was localized to the degenerative region in CA1, (ii) the time course of the expression seemed to coincide with DND, (iii) many SPI-3-positive cells were seen in the stratum oriens and the stratum radiatum, areas containing many dendrites. DND begins with degeneration of dendrites 3 h after ischemia [19]. Therefore, it is possible that SPI-3 inhibits a protease activity leaking from dying neurons to maintain the environment around those areas where DND occurs.
References [I] Abe, K., Tanzi, R.E. and Kogure, K., Selective induction of Kunitztype protease inhibitor domain-containing amyloid precursor protein mRNA after persistent focal ischemia in rat cerebral cortex, Neurosci. Lett., 125 (1991) 172-174. [2] Chomczynski, P. and Sacchi, N., Single-step method of RNA i~lation by acid guanidinium thiocyanate-phenol-chloroform extraction, Anal. Biochem., 162 (1987) 156-159. [3] Hama, T., Kushima, Y. Miyamoto, M., Kubota, M., Takei, N. and Hatanaka, H., Inteleukine-6 improves the survival of mesencephalic and septal cholinergic neurons from postnatal, two-week-old rats in culture, Neuroscience, 40 (1991) 445-452. [4] Hoffman, M.C., Nitsch, C., Scotti, A.L., Reinhard, E. and Monard, D., The prolonged presence of glia-derived nexin, an endogenous protease inhibitor in the hippocampus after ischemia-induced delayed neuronal death, Neuroscience, 49 (1992) 397-408. [5] imaizumi, K., Tsuda, M., Wanaka, A., Tohyama, M. and Takagi, T., Differential expression of sgk mRNA, a member of the Ser/Thr protein kinase gene family, in rat brain after CNS injury, Mol. Brain Res., 26 (1994) 189-196. [6] Jorgensen, M.B., Deckert, J., Wright, D.C. and Gehlert, D.R., Delayed c-los protooncogene expression in the rat hippocampus induced by transient global ischemia: an in situ hybridization study, Brain Res., 484 (1989) 393-398. [7] Kitagawa, K., Matsumoto, M., Tagaya, M., Hata, R., Ueda, H., Niinobe, M., Handa, N., Fukunaga, R., Kimura, K., Mikoshiba, K. and Kamada, T., 'l~hemic tolerance' phenomenon found in the brain, Brain Res., 528 (1990) 21-24. [8] Kirino, T., Delayed neuronal death in the gerbil hippocampus following ischemia, Brain Res., 239 (1982) 57-69. [9] Kirino, T. and Sano, K., Selective vulnerability in the gerbil hippocampus following transient ischemia, Acta. Neuropathol. (Berl.), 62 (1984) 201-208. [10] Kordula, T., Buguo, M., Lason, W., Prezewlocki, R. and Koj, A., Rat contrapsins are the type II acute phase proteins: regulation by interleukine-6 on the mRNA level, Biochem. Biophys. Bes. Commun., 201 (1994) 222-227. [11] Liang, P. and Pardee, A.B., Differential display of eukaryotic m e s -
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[12]
[13]
[14] [15]
[16]
M. lXuda et al. / Molecular Brain Re,search 35 (1996) 314-318
senger RNA by means of the polymerasc chain reaction, Science. 257 (1992) 967-971. Lindvall, O., Ernfors, P., Bengzon, J., Kokaia, Z., Smith, M.t,.. Siesjo, B.K. and Persson, It., Differntial regulation of mRNAs for nerve growth factor, brain-derived neurotrophic factor, and neurotrophin 3 in the adult rat brain following cerebral ischemia and hypoglycemic coma, Proc. Natl. Acad. Sci. USA, 89 (1992) 648-652. Nitsh, C., Scotti, A.I,., Monard, D., Helm, C. and Sontag, K.H., The gila-derived protea,~ nexinl persists for over I year in rat brain areas selectively lesioned by transient global ischemia. Eur..I. Neurosci., 5 (1993) 292-297. Nowark Jr., T.S., Synthesis of a stress protein toIlowing transient ischemia in the gerbil, .1. Neurochem., 45 (1985) 1635-1641. Ohkubo, K., Ogata, S., Misumi, Y., "l'akami, N. and Ikchara, Y., Molecular cloning and characterization of rat contrapsin-likc pr~tease inhibitor and related proteins. J. Biochem. 1(19 (1991) 24325(I. Pages, G., Rouayrenc, J.F.. IJc Cam, G., Mariller, M. ;~nd Ix: Cam, A., Molecular charactarization of three rat liver scrinc proteasc
inhibitors affected by inflammation and hyIx)physectomy, Eur..I. Biochem., 191) (199(I) 385-391.
[17] Rossi, V., Rouayrenc, J.F.. Paquereau, L., Vilarem, M.J. and l,c (?am, A., Analysis of proteins binding to the proximal promoter region of two rat serine proteasc inhibitor genes. Nucleic Acids Re.~., 2{1 (1992) 1061 - 1068. [18] Schcchter, I. and Berger. A., On the size of the active site in proteases, Biochem. Biophys. Res. Commun., 27 (1967) 157-162. [l~,q Shigeno, T., Mira, T., Takamura, K., Graham, D.I., Kato, G., Hashimoto, Y. and Furukawa, S., Amelioration of delayed neuronal death in the hippocampus by nerve growth factor, J. Neuro.sci.. 11 (1991) 2914-2919. 12Ill Travis, J. and Salvescn, S., Human plasma proteinasc inhibitors, Annu. Rel. Biochem., 52 (1983)655-709. [21] Woodrt×ffe, M.N., Sarna, G.S.. Wadhwa, M., Hayes, G.M.. Lx)ughlin, A.J., Tinker, A. and Curzner, M.L., Detection of interlcukine-I and interleukine-6 in adult rat brain following machanical injury, by in vitro microdialysis: evidence of a role for microglia in cytokinc production, J. Neuroimmunol., 33 ( 1991 ) 227-236.
,.
L..
y-j !
MOLECULAR BRAIN RESEARCH
..
11 ELSEVIER
Molecular Brain Research 35 (1996) 314-318
Short communication
Induction of SPI-3 mRNA, encoding a serine protease inhibitor, in gerbil hippocampus after transient forebrain ischemia M. Tsuda ,,d,,, K. Kitagawa b, K. Imaizumi ~.d, A. Wanaka ~, M. Tohyama ~, T. Takagi
~'
Department o[ Molecular Neurobtology "Tanabe ", Osaka Umtersitv Medical School, 2-2 Yamadaoka, Suita, Osaka 565, Japan h First Department of Internal Medicine, Osaka Unicersitv Medical School, Osaka, Japan • Department of Anatomy and Neuroscience, O~aka Unit'ersi~.' Medical School, Osaka, Japan J Lead Generation Research Laboratory, Tanahe Seivaku Co. Ltd.. Osaka, Japan Accepted 16 August 1995
Abstract
We cloned genes the expression of which is induced in the Mongolian gerbil (Meriones unguiculatus) hippocampus after transient forebrain ischemia by a differential display technique. Among these genes, a rat serine protease inhibitor SPI-3 homologue was isolated. Present analyses suggested that the expression of gerbil SPI-3 mRNA was closely associated with delayed neuronal death and may block activities of proteases leaking from degenerating neurons or may support neuronal survival. Keyvvords: lschemia; Differential display: SPI-3: Astrocytc
Transient arrest of cerebral blood circulation leads to neuronal cell death in the hippocampal CA1 region [9]. Neuronal cell death, however, does not occur rapidly after ischemic insult, and neurons survive for several days, a phenomenon known as delayed neuronal death (DND) [8], The molecular mechanism of DND has attracted considerable attention. In the brain, expression of some genes, such as c-fos protooncogene [6] and those encoding heat shock proteins (HSPs) [14], and brain-derived neurotrophic factor (BDNF) [12], is induced during DND. These genes arc thought to regulate further gene expressions or to support neuronal survival. However, the molecular mechanism of DND is still not understood. To investigate DND after ischemia, we searched for new ischemia-related genes with the differential display technique [11], which is an effective method to identify and isolate genes which are differentially expressed in various cells or under altered conditions. Adult male Mongolian gerbils weighing 60-8(I g were used in the present study. The surgical procedure was described previously [7]. Briefly, each gerbil was lightly anesthetized by ether inhalation and both common carotid
• Corresponding author. Fax: {~1) (6) 879-3648. 0169-328X/96/$15.(~1 ~ 1996 Elsevier Science B.V, All rights reserved
SSDI 0 1 6 9 - 3 2 8 X ( 9 5 ) 0 0 2 1 1-1
arteries were exposed. Bilateral cerebral ischemia was produced by occlusion of both common carotid arteries with miniture aneurysm clips. Body temperature was maintained at 37°C using warming blankets and a heating lamp throughout the surgical procedure. Animals were divided randomly into a sham-operated group and a 5 min ischemic group. Twenty-four hours after clip removal, the animals were again anesthetized by ether inhalation, decapitated and their hippocampi were promptly removed for RNA purification. Total RNA was isolated by the acid guanidine thiocyanate-phenol-chloroform method [2] from the hippocampi in each group, and differential display was carried out as described previously [5]. Briefly, cDNAs were synthesized from the total RNA with reverse transcriptase after DNase treatment and PCR was performed with the synthesized cDNA as template. Four cDNA fragments which were amplified only from the cDNA derived from ischemic hippocampi were observed. These fragments were recovered, subcloned into the pGEM-T vector (Promega) and sequenced. The sequences were analyzed for homology with the Software Development Co. data bank (EMBL and GenBank DNA databases) using its program. The sequence of clone P017 (916 bp), showed a high degree of homology to the rat SPI
315
M. Tsuda et al. / Molecular Brain Research 35 (1996) 314-318
gene family, which consists of three serine protease inhibitors (SPI-1, 2, and 3) reported to be expressed in rat liver [15,16]. Clone P017 had a region encoding a C-terminal 132 amino acid sequence including the reactive center. SPI-1, 2 and 3 have a high degree of homology with each other, but there is no homology in the reactive center. The amino acid sequence homologies between P017 and SPI-1, P017 and SPI-2, P017 and SPI-3 were 62%, 60%, 73%, respectively. The amino acid sequence at the reactive center of P017 was different from those of SPI-1 and SPI-2 (Fig. 1B), whereas it was very similar to that of SPI-3. In addition, the P1 residue, according to the nomenclature defined by Schechter and Berger [18], thought to be crucial in determining the anti-protease specificity [20] was methionine in both P017 and SPI-3 (Fig. 1A). Both SPI-1 and SPI-2 m R N A s are approximately 1.8 kb, and that of SPI-3 is about 2.2 kb. From Northern blot analysis, the length of clone P017 m R N A was about 2.2 kb (Fig. 2). Taking all of these findings into consideration, we concluded that clone P017 is gerbil SPI-3. To investigate the expression pattern of gerbil SPI-3, we prepared total R N A from the hippocampus of the animals at 1 h, 6 h, 24 h, 3 days, 7 days, and 1 month after bilateral common carotid occulusion. 15 /zg of total R N A was fractioned by electrophoresis. A c D N A probe synthesized from a fragment of SPI-3 (clone P017) was shown to hybridized with a single m R N A species of about 2.2 kb (Fig. 2). In sham-operated animals, expression of SPI-3 was low. A significant increase in level of expression of SPI-3 was observed from 6 h after ischemia, and attained a maximum level at 24 h. Subsequently, a gradual reduction in level of SPI-3 m R N A was seen, and it was hardly detectable after 1 month. In situ hybridization using c R N A probe was carried out to analyze the distribution of cells expressing SPI-3 m R N A after ischemia. Digoxigenin-labeled c R N A was generated by in vitro transcription using the c D N A subcloned into
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Fig. 1. Alignmentof rat SPI family and P017 amino acid sequences. A: Alignment of rat SPI-3 and P017 amino acid sequences. The amino acid sequence of rat SPI-3 shown refers to that reported by Pages et al. [16], and that of clone P017 was deduced from the cDNA sequence. Numbering of rat SPI-3 amino acid residues is indicated on both sides above the sequences given in the one-letter code. Asterisks indicate identical residues conserved between rat SPI-3 and clone P017. The anti-protease reactive center region is boxed and the P1 residues in the reactive sites are circled. B: Alignment of the anti-protease reactive center region of SPI-I. SPI-2 and P017.
the p G E M - T vector as a template in the presence of digoxigenin-labeled UTP. Thaw-mounted frozen sections 15 /zm thick were postfixed for 20 min in 4% formal-
B 1
2
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4
5
6
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2
3
4
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Fig. 2. Pattern of gerbil SPI-3 mRNA expression after ischemic insult. A: Each lane was loaded with 15 p.g of total RNA purified from the hippocampus at specified times after ischemic insult. Ethidium bromide-stained ribosomal RNAs (28S and 18S) indicate that the amounts of total RNA loaded in each lane were equivalent. Lane 1, sham-operated; 2, 6 h; 3, 24 h; 4, 3 days; 5, 7 days; 6, 1 month. B: Membrane-transferredRNAs were hybridized with gerbil SPI-3 (clone P017) probe. The arrow indicates SPI-3 signal.
316
M. l~vuda et al. / Moh'cular Brain Rexearch 35 t 1996) 3 1 4 - 3 1 8
Fig, 3. In situ hybridization of gerbil brain sections 3 days after transient ischcmia with gerbil SPI-3 cRNA probe. SPI-3 signals were seen only in the inner side of CA1 region, especially in stratum oriens and stratum radiatum. In stratum pyramidale there were just weak signals. The section 24 h after transient ischemia was almost the same as that of 3 days. a, alveus: o, stratum oriens; p, stratum pyramidale; r, stratum radiatum. Bar = 250 /zm.
f ¢".i.-"
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Fig. 4. In situ hybridization with SPI-3 cRNA probe and immunohistochemistry with anti-GFAP antibody. A: In situ hybridization of CA1 region with SPI-3 cRNA probe. B: Immunohistochemistry of the section adjacent to that in A with anti-GFAP antibody. Cells containing SPI-3 mRNA and GFAP are shown by arrows in the consecutive sections. The stars denote the same capillaries in the serial sections. C: In situ hybridization of CAI region with SPI-3 cRNA probe. D: Immunohistochemistry of section C reacted with anti-GFAP antibody and FITC-labeled anti-mouse lgG antibody. Bar = 30 /,tin. Arrows indicate cells containing both SPI-3 mRNA signals and GFAP immunoreactivity in their cell bodies. Arrowheads indicate cells showing SPI-3 mRNA signals in their cell bodies and GFAP immunoreactivities in their processes.
M. Tsuda et al. / Molecular Brain Research 35 (1996) 314-318
dehyde, followed by two 5 min washes in 0.1 M phosphate buffer (pH 7.2). All subsequent procedures were performed as described previously [5]. In sham-operated gerbils, SPI-3 mRNA signals were not detected. In gerbil brains obtained 24 h and 3 days after 5 min ischemia, SPI-3 mRNA was detected in the alveus, stratum oriens and stratum radiatum. In the stratum pyramidale, weak signals were observed (Fig. 3). Twenty-four hours to 3 days after the operation, SPI-3 mRNA signals reached their peak in accordance with Northern blot analysis, showing a subsequent reduction. Cells expressing SPI-3 seemed to be glial cells on the basis of their distribution pattern and size. To confirm this possibility, immunohistochemistry and in situ hybridization were carried out on pairs of alternate adjacent serial sections. One section in each pair was reacted with antiGFAP monoclonal antibody (Chemicon) followed by the Vectstain ABC Kit (Vector Lab), and the other was hybridized with SPI-3 cRNA probe. SPI-3 mRNA signals and GFAP immunoreactivity were colocalized in some cells (Fig. 4A,B). Moreover, the sections which reacted with the SPI-3 probe were also then stained for GFAP by fluorescent immunohistochemistry. SPI-3 signals overlapped widely with positive immunofluorescence GFAP (Fig. 4C,D). From these results, cells expressing SPI-3 mRNA were confirmed to be astrocytes. In this investigation, four clones induced in the gerbil hippocampus after ischemia were isolated. We analyzed clone P017, which was thought to be gerbil SPI-3 judging from DNA and amino acid sequence analyses and the length of its transcript. Gerbil SPI-3 was induced in the hippocampal CA1 region after ischemia. Recent reports suggested that possible interleukin-6 (IL-6)-responsive elements were included in the promoter region of rat SPI-3 gene [17] and that IL-6 was responsible for the induction of SPI-3 in rat hepatocytes [10]. It is well known that IL-6 is immediately induced during brain injury [21] and has a protective effect on neurons [3]. Therefore, it is possible that the expression of SPI-3 from astrocytes is mediated by IL-6 against ischemic damage. SPI-3 was induced in hippocampal CA1 6 h after ischemia, when neurons have not yet degenerated morphologically. Activation of astrocytes or microglia, which are thought to be markers of irreversible ischemic damage, are not induced only in CA1 but also in CA3 where DND does not occur. As SPI-3 was induced only in CA1, SPI-3 may be a useful sensitive marker of irreversible ischemic damage. The function of SPI-3 in the brain is not yet understood. However it is presumably involved in (1) blocking the protease activity leaking from degenerative neurons, (2) supporting neurons and playing a role in structural rearrangement and (3) leading to neuronal death. Other protease inhibitors such as glia-derived nexin (GDN) [4,13] or amyloid precursor protein (APP) 751/770 [1] are known to be induced after ischemia. They have
317
neurite promoting activity, and GDN is thought to support neuronal cells and to play a role in structural rearrangement of the central nervous system because GDN is expressed widely in CA1 more than 90 days after transient ischemia. On the other hand, the expression of SPI-3 disappeared within a month. SPI-3 may not support neurons because its expression pattern was different from that of GDN. To investigate this possibility, more detailed studies are needed. Also, from the present results, we are not able to discuss whether SPI-3 leads to neuronal death or not. In the present study, (i) the expression of SPI-3 was localized to the degenerative region in CA1, (ii) the time course of the expression seemed to coincide with DND, (iii) many SPI-3-positive cells were seen in the stratum oriens and the stratum radiatum, areas containing many dendrites. DND begins with degeneration of dendrites 3 h after ischemia [19]. Therefore, it is possible that SPI-3 inhibits a protease activity leaking from dying neurons to maintain the environment around those areas where DND occurs.
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