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enhancer binding proteins are expressed in the gerbil hippocampus after transient forebrain ischemia
Neuroscience Letters 337 (2003) 106–110 www.elsevier.com/locate/neulet
CCAAT/enhancer binding proteins are expressed in the gerbil hippocampus after transient forebrain ischemia Youji Soga*, Ryuya Yamanaka, Kazuhiko Nishino, Ryuichi Tanaka Department of Neurosurgery, Brain Research Institute, Niigata University, 1-757 Asahimachi Niigata, Japan Received 25 June 2002; received in revised form 31 October 2002; accepted 5 November 2002
Abstract We analyzed CCAAT/enhancer binding protein (C/EBP) family protein levels during reperfusion after a single episode of sublethal forebrain ischemia in the gerbil hippocampus to investigate their expression after ischemia and correlation with neuronal cell death. The common carotid arteries were surgically exposed bilaterally and occluded for 10 min to induce forebrain ischemia in adult Mongolian gerbils. C/EBPa, b, d, 1, z protein immunoreactivity was expressed in the hippocampal layer of the CA1 region at 72 h after ischemia and peaked at 96 h. These results appear to correlate with neuronal degeneration as shown by hematoxylin and eosin staining and DNA fragmentation in the terminal transferase biotinylated-UTP nick end labeled-method. The present results demonstrate that C/EBP family proteins appear in the selectively vulnerable CA1 pyramidal cell layer in gerbils during neuronal degeneration, and may serve as a signal that neurons are progressing to neuronal cell death and DNA fragmentation. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Cerebral ischemia; Transcription factor; CCAAT/enhancer binding protein family; Gerbil; Immunohistochemistry; Neuronal cell death; Hippocampus
Cerebral ischemia is a severe form of metabolic stress that disturbs most biochemical and molecular-biological pathways of cells. It induces short-term and long-lasting alterations of genomic programs, which may be involved in neuronal degeneration, in addition to astrocytic and microglial activation. Selective and delayed neuronal degeneration occurs in the CA1 pyramidal cell layer of the hippocampus following transient forebrain ischemia [10]; however, its mechanism is not well understood. To clarify the detailed mechanism of ischemic neuronal cell injury, we have focused on the stress response and signals that will trigger programmed cell death. The most common and well-studied response to ischemia is an activation of stress-gene expression and a suppression of global protein synthesis. Signal molecules such as c-fos, jun B, c-jun, heat shock proteins [8] and nuclear factor-kappa B [4] have been investigated for their ability to serve as signals associated with neuronal cell death. CAAT/enhancer binding protein (C/EBP) families belong to the basic leucine zipper class of transcription factors. At
* Corresponding author. Tel.: 181-25-227-0653; fax 181-25227-0819. E-mail address: [email protected] (Y. Soga).
present, six members have been characterized by highly conserved basic DNA bindings and leucine zipper dimerization domains. C/EBP families are reported to play important roles in the control of cellular proliferation and differentiation and as essential transducers of intracellular responses to extracellular control signals [5]. We have been studying the roles of the C/EBP family in neuronal function, but at present its distribution and functions are still poorly understood. In the present study, we measured C/EBP family proteins during reperfusion after a single episode of sublethal ischemia in gerbil hippocampus to investigate their expression after ischemia and correlation with neuronal cell death. We also examined the terminal transferase biotinylated-UTP nick end labeled (TUNEL) staining to identify DNA fragmentation, which may be related to apoptosis, as an additional marker for cell death. Adult, mixed gender Mongolian gerbils weighing 60–80 g were used in this study. Animals were anesthetized with 1.75% of halothane mixed with 70% nitrous oxide and 30% oxygen. The common carotid arteries were surgically exposed bilaterally and occluded for 10 min. Since there was a risk of spontaneous hyperthermia during recirculation, we maintained a continuous rectal temperature of
0304-3940/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0 30 4- 39 40 (0 2) 0 131 5- 0
Y. Soga et al. / Neuroscience Letters 337 (2003) 106–110
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Fig. 1. The left column shows hematoxylin and eosin staining, the middle left column shows C/EBPa immunoreactivity, the middle right column shows C/EBPb immunoreactivity and the right column shows C/EBPg immunoreactivity. Each row shows representative findings from each time: sham operated control, 48 h, 72 h, 96 h, 120 h and 7 days after ischemia.
37.5 ^ 0.5 8C for the entire 90 min period of recirculation by careful management of the levels of anesthesia and thermostatically controlled heat lamps. The animals that underwent surgery were allowed to recover, and they were then perfused transcardially with heparinized phosphate-buffered saline (PBS; pH 7.4), followed by fixative containing 4% paraformaledehyde (PFA) postoperatively at 6 h, 12 h, 24 h, 48 h, 72 h, 96 h, 120 h, 7 days, and 14 days respectively. Brains were removed gently and stored in the same fixative overnight at 4 8C. Blocks of neuronal tissue, including the dorsal hippocampus, were dehydrated and embedded in paraffin. Serial sections (7 mm thick) of the dorsal hippo-
campus were cut with a microtome (MICROM HM355, Carl Zeiss, Tokyo, Japan). Dorsal hippocampal sections from each animal were stained with hematoxylin and eosin to estimate the ischemic neuronal damage. For immunostaining, an avidin-biotin-peroxidase system (Vectasin elite ABC kit, Vector Labs, Burlingame, CA) was used. Briefly, after deparaffinization, the sections were incubated overnight at 4 8C with the monoclonal antibody against C/ EBPa (sc-61-G diluted 1:500), C/EBPb (sc-7962 diluted 1:250), C/EBPz (sc-7351 diluted 1:250), polyclonal antibody against C/EBPg (sc-7659 diluted 1:500), C/EBPd(sc151 diluted 1:250) and C/EBP1(sc-158 diluted 1:500). All
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Y. Soga et al. / Neuroscience Letters 337 (2003) 106–110
Fig. 2. The left column shows C/EBPd immunoreactivity, the middle left column shows C/EBP1 immunoreactivity, the middle right column shows C/EBPz immunoreactivity and the right column shows TUNEL staining. Each row shows representative findings from each time: sham operated control, 48 h, 72 h, 96 h, 120 h and 7 days after ischemia.
the antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). The sections were incubated for 30 min with biotinated anti-mouse IgG or anti-goat IgG at room temperature and then stained by the avidin-biotin-peroxidase complex method at room temperature. Between the above incubation steps, the sections were rinsed for 30 min with PBS at room temperature. Finally, the sections were exposed for 10–20 min to 0.01% 3,3-diaminobenzidine (DAB) (Sigma-Aldrich, Tokyo, Japan) and PBS containing 0.01% hydrogen peroxide. To examine the process of DNA fragmentation in the hippocampus, TUNEL-staining was performed using the DeadEnd Colori-
metric Apoptosis Detection System (Promega Corporation, Madison, WI) according to the manufacturer’s instructions. Briefly, sections were treated with 20 mg/ml proteinase K and incubated for 15 min at room temperature, washed in PBS and refixed in 4% PFA. DNA was end-labeled using biotinylated dUTP in TdT buffer at 37 8C for 60 min. After washing, 0.3% hydrogen peroxidase was used to block endogenous peroxidase. Streptavidin horseradish peroxidase (1:500 in PBS) was added for 30 min at room temperature. DNA strand breaks were visualized using hydrogen peroxidase and the stable chromogen DAB. In hematoxylin and eosin stains, severe neuronal damage,
Y. Soga et al. / Neuroscience Letters 337 (2003) 106–110
which was characterized by nuclear shrinkage and eosinophilic chromatin compaction, and neuronal disappearance was seen in the CA1 pyramidal neurons and some neurons in the dentate hilus begging at 72 h after the ischemia (Fig. 1). Neurons in CA3, the dentate gyrus and cerebral cortex were spared. There were no significant C/EBP family immunoreactions detected in the regions of the hippocampus in the sham-operated control groups. The immunoreactivity of C/EBPa, C/EBPb, C/EBPd, C/EBP1, C/EBPz increased 72 h after ischemia in the selectively vulnerable CA1 pyramidal cell layer and peaked at 96 h. However, clear immunoreactivity of C/EBPg was not detected at the same period. The expression of C/EBPb and C/EBPz was also observed at the strata radiatum, strata oriens and dentate hilus at 96 h after the ischemia. The expression of the C/EBP family was not detected in hippocampal regions at 14 days after the ischemia. No clear immunosignals were detected in the CA3 region or cerebral cortex throughout the entire period (Figs. 1 and 2). No TUNEL-positive cells were detected in the sham-operated control brains. Scattered TUNEL-positive neurons were found at 72 h after ischemia in the CA1 neurons. The number of TUNEL-positive cells in which the nuclei were severely impaired peaked at 96 h. The positive staining in the neurons was not localized only in the cell bodies, but also in the dendrites. The CA1 pyramidal neurons that showed positive staining were also detected at 120 h and even 7 days after ischemia. There were no positively stained neurons detected 14 days after ischemia (Fig. 2). The C/EBP family includes six members characterized by highly conserved basic DNA bindings and leucine zipper dimerization domains. C/EBP families homodimerize and heterodimerize each other, which is necessary for binding the same C/EBP consensus DNA sites. They display a high degree of homology in the carboxyl-terminal portion that includes the bZIP domain. In the present study, C/EBPa, b, d, 1 and z proteins were shown to express in the CA1 region of the hippocampus after sublethal forebrain ischemia in gerbils. The C/EBPa, b, d, 1, z( levels increased within 72 h, reached the maximum at 96 h and were extinguished at 14 days, which correlated well with neuronal degeneration shown by hematoxylin and eosin staining. No C/EBP family protein activation was observed in the sham-operated control hippocampus. At present, the precise mechanism of C/EBP family protein expression in the CA1 cell layer after ischemia is not known. However, we developed three hypotheses based on the present findings and those of recent studies. One hypothesis is that the C/EBP correlates with apoptosis. The second is that each C/EBP family responds to the stress induced by ischemia. The third that C/EBP correlates with the cytokine reaction in ischemia. To examine the first hypothesis, we used the TUNEL method to observe simultaneous DNA fragmentation. DNA fragmentation occurred not only in the same region of the hippocampus, but the time of the expression also
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overlapped the activation of immunoreactivity against the C/EBP family protein. The expression of C/EBP b and z in strata radiatum and strata oriens, which we think are due to breakdown of axons and dendrites, were also observed in the TUNEL stain. C/EBP family proteins may have been expressed because there is a correlation between apoptosis signaling and C/EBP. Although the delayed neuronal injury that occurs in CA1 pyramidal neurons after global ischemia demonstrates many of the features associated with apoptosis, the specific genes regulating apoptosis are unknown. Recently, expression of C/EBPb and C/EBPz were reported to be linked to apoptosis [5,19]. The present results support these ideas and, furthermore, may also suggest that other C/ EBP family members play a role in apoptosis. As the basis of the second hypothesis, C/EBP family members have been reported to respond to several stresses, which may explain the present results. C/EBPa, which is expressed in many tissues including the liver, blood, and adipose tissue regulates gene expression and is critical for the establishment and maintenance of energy homeostasis [15]. C/EBPa was recently reported to regulate gene expression and consequently to play role in the activation and/or proliferation of microglia following brain injury[14]. C/ EBPb is highly expressed in liver nuclei and has multiple biological functions, such as inflammation and other acute phase reactions [1], cell differentiation [11], immunoreaction and in the host-defense mechanism [13]. C/EBPd exhibits an expression pattern similar to that of C/EBPb and has been shown to be involved in the regulation of several genes induced during inflammation [9]. C/EBPz, which is known as the growth arrest and DNA-damage inducible gene 153 (gadd153), is markedly induced in response to a variety of cellular stresses including glucose deprivation, exposure to genotoxic agents and other growth-arresting situations [7]. Recently, C/EBPz was reported to be specifically activated under conditions that disturb the functioning of the endoplasmic reticulum, such as transient cerebral ischemia [12]. Several studies have demonstrated heterodimer formation between the C/EBP family members, including interactions of C/EBP1 with C/EBPa, C/EBPb and C/EBPd [16]. For the third hypothesis, reperfusion after cerebral ischemia induces an inflammatory response, and several cytokines are considered to be related to the pathogenesis [6]. C/EBPb may be responsible for the regulation of genes encoding many acute phase proteins and cytokines [2]. In cultured endothelial cells, induction of interleukin 6 (IL-6) was observed through activation of C/EBPb during hypoxic conditions [18]. Lipopolysaccharides, IL-1b and TNF-a induce the expression of the C/EBPb and d genes in mouse primary astrocytes, and C/EBPb and d could be pivotal transcription factors involved in brain inflammation and ischemia [3]. C/EBP1 is also implicated in cytokine signaling such as IL-2, IL-4, IL-12 and TNF-a [17]. We have demonstrated that C/EBP family proteins appear in the selectively vulnerable CA1 pyramidal cell layer in gerbils during neuronal degeneration, and may serve as a
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signal from neurons that are progressing to neuronal death and DNA fragmentation. We have also proposed several hypothesis that may explain these results, but it is unclear whether one of them is valid or not. Further studies are necessary to elucidate the precise mechanism of C/EBP family response to cerebral ischemia, and they are underway. [1] Akira, S., Isshiki, H., Sugita, T., Tanabe, O., Kinoshita, S., Nishio, Y., Nakajima, T., Hirano, T. and Kishimoto, T., A nuclear factor for IL-6 expression (NF-IL6) is a member of a C/EBP family, EMBOJ., 9 (1990) 1897–1906. [2] Akira, S. and Kishimoto, T., IL-6 and NF-IL6 in acute-phase response and viral infection, Immunol. Rev., 127 (1992) 25– 50. [3] Cardinaux, J.R., Allaman, I. and Magistretti, P.J., Proinflammatory cytokines induce the transcription factors C/ EBPbeta and C/EBPdelta in astrocytes, Glia, 29 (2000) 91–97. [4] Clemens, J.A., Stephenson, D.T., Smalstig, E.B., Dixon, E.P. and Little, S.P., Global ischemia activates nuclear factorkappa B in forebrain neurons of rats, Stroke, 28 (1997) 1073–1080. [5] Cortes-Canteli, M., Pignatelli, M., Santos, A. and PerezCastillo, A., CCAAT/enhancer binding protein beta plays a regulatory role in differentiation and apoptosis of neuroblastoma cells, J. Biol. Chem., 227 (2002) 5460–5467. [6] DeGraba, T.J., The role of inflammation after acute stroke: utility of pursuing anti-adhesion molecule therapy, Neurology, 51 (1998) S62–S68. [7] Fawcett, T.W., Eastman, H.B., Martindale, J.L. and Holbrook, N.J., Physical and functional association between GADD153 and CCAAT/enhancer-binding protein beta during cellular stress, J. Biol. Chem., 271 (1996) 14285–14289. [8] Kiessling, M., Stumm, G., Xie, Y., Herdegen, T., Aguzzi, A., Bravo, R. and Gass, P., Differential transcription and translation of immediate early genes in the gerbil hippocampus after transient global ischemia, J. Cereb. Blood Flow Metab., 13 (1993) 914–924. [9] Kinoshita, S., Akira, S. and Kishimoto, T., A member of the C/EBP family, NF-IL6 beta, forms a heterodimer and transcriptionally synergizes with NF-IL6, Proc. Natl. Acad. Sci. USA, 89 (1992) 1473–1476.
[10] Kirino, T., Delayed neuronal death in the gerbil hippocampus following ischemia, Brain Res., 239 (1982) 57–69. [11] Natsuka, S., Akira, S., Nishio, Y., Hashimoto, S., Sugita, T., Isshiki, H. and Kishimoto, T., Macrophage differentiationspecific expression of NF-IL6, a transcription factor for interleukin-6, Blood, 79 (1992) 460–466. [12] Paschen, W., Gissel, C., Linden, T., Althausen, S. and Doutheil, J., Activation of gadd153 expression through transient cerebral ischemia: evidence that ischemia causes endoplasmic reticulum dysfunction, Brain Res. Mol. Brain Res., 60 (1998) 115–122. [13] Tanaka, T., Akira, S., Yoshida, K., Umemoto, M., Yoneda, Y., Shirafuji, N., Fujiwara, H., Suematsu, S., Yoshida, N. and Kishimoto, T., Targeted disruption of the NF-IL6 gene discloses its essential role in bacteria killing and tumor cytotoxicity by macrophages, Cell, 80 (1995) 353–361. [14] Walton, M., Saura, J., Young, D., MacGibbon, G., Hansen, W., Lawlor, P., Sirimanne, E., Gluckman, P. and Dragunow, M., CCAAT-enhancer binding protein alpha is expressed in activated microglial cells after brain injury, Brain Res. Mol. Brain Res., 61 (1998) 11–22. [15] Wang, N.D., Finegold, M.J., Bradley, A., Ou, C.N., Abdelsayed, S.V., Wilde, M.D., Taylor, L.R., Wilson, D.R. and Darlington, G.J., Impaired energy homeostasis in C/EBP alpha knockout mice, Science, 269 (1995) 1108–1112. [16] Williams, S.C., Cantwell, C.A. and Johnson, P.F., A family of C/EBP-related proteins capable of forming covalently linked leucine zipper dimers in vitro, Genes Dev., 5 (1991) 1553– 1567. [17] Yamanaka, R., Barlow, C., Lekstrom-Himes, J., Castilla, L.H., Liu, P.P., Eckhaus, M., Decker, T., Wynshaw-Boris, A. and Xanthopoulos, K.G., Impaired granulopoiesis, myelodysplasia, and early lethality in CCAAT/enhancer binding protein epsilon-deficient mice, Proc. Natl. Acad. Sci. USA, 94 (1997) 13187–13192. [18] Yan, S.F., Tritto, I., Pinsky, D., Liao, H., Huang, J., Fuller, G., Brett, J., May, L. and Stern, D., Induction of interleukin 6 (IL6) by hypoxia in vascular cells: central role of the binding site for nuclear factor-IL-6, J. Biol. Chem., 270 (1995) 11463– 11471. [19] Zinszner, H., Kuroda, M., Wang, X., Batchvarova, N., Lightfoot, R.T., Remotti, H., Stevens, J.L. and Ron, D., CHOP is implicated in programmed cell death in response to impaired function of the endoplasmic reticulum, Genes Dev., 12 (1998) 982–995.
CCAAT/enhancer binding proteins are expressed in the gerbil hippocampus after transient forebrain ischemia Youji Soga*, Ryuya Yamanaka, Kazuhiko Nishino, Ryuichi Tanaka Department of Neurosurgery, Brain Research Institute, Niigata University, 1-757 Asahimachi Niigata, Japan Received 25 June 2002; received in revised form 31 October 2002; accepted 5 November 2002
Abstract We analyzed CCAAT/enhancer binding protein (C/EBP) family protein levels during reperfusion after a single episode of sublethal forebrain ischemia in the gerbil hippocampus to investigate their expression after ischemia and correlation with neuronal cell death. The common carotid arteries were surgically exposed bilaterally and occluded for 10 min to induce forebrain ischemia in adult Mongolian gerbils. C/EBPa, b, d, 1, z protein immunoreactivity was expressed in the hippocampal layer of the CA1 region at 72 h after ischemia and peaked at 96 h. These results appear to correlate with neuronal degeneration as shown by hematoxylin and eosin staining and DNA fragmentation in the terminal transferase biotinylated-UTP nick end labeled-method. The present results demonstrate that C/EBP family proteins appear in the selectively vulnerable CA1 pyramidal cell layer in gerbils during neuronal degeneration, and may serve as a signal that neurons are progressing to neuronal cell death and DNA fragmentation. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Cerebral ischemia; Transcription factor; CCAAT/enhancer binding protein family; Gerbil; Immunohistochemistry; Neuronal cell death; Hippocampus
Cerebral ischemia is a severe form of metabolic stress that disturbs most biochemical and molecular-biological pathways of cells. It induces short-term and long-lasting alterations of genomic programs, which may be involved in neuronal degeneration, in addition to astrocytic and microglial activation. Selective and delayed neuronal degeneration occurs in the CA1 pyramidal cell layer of the hippocampus following transient forebrain ischemia [10]; however, its mechanism is not well understood. To clarify the detailed mechanism of ischemic neuronal cell injury, we have focused on the stress response and signals that will trigger programmed cell death. The most common and well-studied response to ischemia is an activation of stress-gene expression and a suppression of global protein synthesis. Signal molecules such as c-fos, jun B, c-jun, heat shock proteins [8] and nuclear factor-kappa B [4] have been investigated for their ability to serve as signals associated with neuronal cell death. CAAT/enhancer binding protein (C/EBP) families belong to the basic leucine zipper class of transcription factors. At
* Corresponding author. Tel.: 181-25-227-0653; fax 181-25227-0819. E-mail address: [email protected] (Y. Soga).
present, six members have been characterized by highly conserved basic DNA bindings and leucine zipper dimerization domains. C/EBP families are reported to play important roles in the control of cellular proliferation and differentiation and as essential transducers of intracellular responses to extracellular control signals [5]. We have been studying the roles of the C/EBP family in neuronal function, but at present its distribution and functions are still poorly understood. In the present study, we measured C/EBP family proteins during reperfusion after a single episode of sublethal ischemia in gerbil hippocampus to investigate their expression after ischemia and correlation with neuronal cell death. We also examined the terminal transferase biotinylated-UTP nick end labeled (TUNEL) staining to identify DNA fragmentation, which may be related to apoptosis, as an additional marker for cell death. Adult, mixed gender Mongolian gerbils weighing 60–80 g were used in this study. Animals were anesthetized with 1.75% of halothane mixed with 70% nitrous oxide and 30% oxygen. The common carotid arteries were surgically exposed bilaterally and occluded for 10 min. Since there was a risk of spontaneous hyperthermia during recirculation, we maintained a continuous rectal temperature of
0304-3940/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0 30 4- 39 40 (0 2) 0 131 5- 0
Y. Soga et al. / Neuroscience Letters 337 (2003) 106–110
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Fig. 1. The left column shows hematoxylin and eosin staining, the middle left column shows C/EBPa immunoreactivity, the middle right column shows C/EBPb immunoreactivity and the right column shows C/EBPg immunoreactivity. Each row shows representative findings from each time: sham operated control, 48 h, 72 h, 96 h, 120 h and 7 days after ischemia.
37.5 ^ 0.5 8C for the entire 90 min period of recirculation by careful management of the levels of anesthesia and thermostatically controlled heat lamps. The animals that underwent surgery were allowed to recover, and they were then perfused transcardially with heparinized phosphate-buffered saline (PBS; pH 7.4), followed by fixative containing 4% paraformaledehyde (PFA) postoperatively at 6 h, 12 h, 24 h, 48 h, 72 h, 96 h, 120 h, 7 days, and 14 days respectively. Brains were removed gently and stored in the same fixative overnight at 4 8C. Blocks of neuronal tissue, including the dorsal hippocampus, were dehydrated and embedded in paraffin. Serial sections (7 mm thick) of the dorsal hippo-
campus were cut with a microtome (MICROM HM355, Carl Zeiss, Tokyo, Japan). Dorsal hippocampal sections from each animal were stained with hematoxylin and eosin to estimate the ischemic neuronal damage. For immunostaining, an avidin-biotin-peroxidase system (Vectasin elite ABC kit, Vector Labs, Burlingame, CA) was used. Briefly, after deparaffinization, the sections were incubated overnight at 4 8C with the monoclonal antibody against C/ EBPa (sc-61-G diluted 1:500), C/EBPb (sc-7962 diluted 1:250), C/EBPz (sc-7351 diluted 1:250), polyclonal antibody against C/EBPg (sc-7659 diluted 1:500), C/EBPd(sc151 diluted 1:250) and C/EBP1(sc-158 diluted 1:500). All
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Y. Soga et al. / Neuroscience Letters 337 (2003) 106–110
Fig. 2. The left column shows C/EBPd immunoreactivity, the middle left column shows C/EBP1 immunoreactivity, the middle right column shows C/EBPz immunoreactivity and the right column shows TUNEL staining. Each row shows representative findings from each time: sham operated control, 48 h, 72 h, 96 h, 120 h and 7 days after ischemia.
the antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). The sections were incubated for 30 min with biotinated anti-mouse IgG or anti-goat IgG at room temperature and then stained by the avidin-biotin-peroxidase complex method at room temperature. Between the above incubation steps, the sections were rinsed for 30 min with PBS at room temperature. Finally, the sections were exposed for 10–20 min to 0.01% 3,3-diaminobenzidine (DAB) (Sigma-Aldrich, Tokyo, Japan) and PBS containing 0.01% hydrogen peroxide. To examine the process of DNA fragmentation in the hippocampus, TUNEL-staining was performed using the DeadEnd Colori-
metric Apoptosis Detection System (Promega Corporation, Madison, WI) according to the manufacturer’s instructions. Briefly, sections were treated with 20 mg/ml proteinase K and incubated for 15 min at room temperature, washed in PBS and refixed in 4% PFA. DNA was end-labeled using biotinylated dUTP in TdT buffer at 37 8C for 60 min. After washing, 0.3% hydrogen peroxidase was used to block endogenous peroxidase. Streptavidin horseradish peroxidase (1:500 in PBS) was added for 30 min at room temperature. DNA strand breaks were visualized using hydrogen peroxidase and the stable chromogen DAB. In hematoxylin and eosin stains, severe neuronal damage,
Y. Soga et al. / Neuroscience Letters 337 (2003) 106–110
which was characterized by nuclear shrinkage and eosinophilic chromatin compaction, and neuronal disappearance was seen in the CA1 pyramidal neurons and some neurons in the dentate hilus begging at 72 h after the ischemia (Fig. 1). Neurons in CA3, the dentate gyrus and cerebral cortex were spared. There were no significant C/EBP family immunoreactions detected in the regions of the hippocampus in the sham-operated control groups. The immunoreactivity of C/EBPa, C/EBPb, C/EBPd, C/EBP1, C/EBPz increased 72 h after ischemia in the selectively vulnerable CA1 pyramidal cell layer and peaked at 96 h. However, clear immunoreactivity of C/EBPg was not detected at the same period. The expression of C/EBPb and C/EBPz was also observed at the strata radiatum, strata oriens and dentate hilus at 96 h after the ischemia. The expression of the C/EBP family was not detected in hippocampal regions at 14 days after the ischemia. No clear immunosignals were detected in the CA3 region or cerebral cortex throughout the entire period (Figs. 1 and 2). No TUNEL-positive cells were detected in the sham-operated control brains. Scattered TUNEL-positive neurons were found at 72 h after ischemia in the CA1 neurons. The number of TUNEL-positive cells in which the nuclei were severely impaired peaked at 96 h. The positive staining in the neurons was not localized only in the cell bodies, but also in the dendrites. The CA1 pyramidal neurons that showed positive staining were also detected at 120 h and even 7 days after ischemia. There were no positively stained neurons detected 14 days after ischemia (Fig. 2). The C/EBP family includes six members characterized by highly conserved basic DNA bindings and leucine zipper dimerization domains. C/EBP families homodimerize and heterodimerize each other, which is necessary for binding the same C/EBP consensus DNA sites. They display a high degree of homology in the carboxyl-terminal portion that includes the bZIP domain. In the present study, C/EBPa, b, d, 1 and z proteins were shown to express in the CA1 region of the hippocampus after sublethal forebrain ischemia in gerbils. The C/EBPa, b, d, 1, z( levels increased within 72 h, reached the maximum at 96 h and were extinguished at 14 days, which correlated well with neuronal degeneration shown by hematoxylin and eosin staining. No C/EBP family protein activation was observed in the sham-operated control hippocampus. At present, the precise mechanism of C/EBP family protein expression in the CA1 cell layer after ischemia is not known. However, we developed three hypotheses based on the present findings and those of recent studies. One hypothesis is that the C/EBP correlates with apoptosis. The second is that each C/EBP family responds to the stress induced by ischemia. The third that C/EBP correlates with the cytokine reaction in ischemia. To examine the first hypothesis, we used the TUNEL method to observe simultaneous DNA fragmentation. DNA fragmentation occurred not only in the same region of the hippocampus, but the time of the expression also
109
overlapped the activation of immunoreactivity against the C/EBP family protein. The expression of C/EBP b and z in strata radiatum and strata oriens, which we think are due to breakdown of axons and dendrites, were also observed in the TUNEL stain. C/EBP family proteins may have been expressed because there is a correlation between apoptosis signaling and C/EBP. Although the delayed neuronal injury that occurs in CA1 pyramidal neurons after global ischemia demonstrates many of the features associated with apoptosis, the specific genes regulating apoptosis are unknown. Recently, expression of C/EBPb and C/EBPz were reported to be linked to apoptosis [5,19]. The present results support these ideas and, furthermore, may also suggest that other C/ EBP family members play a role in apoptosis. As the basis of the second hypothesis, C/EBP family members have been reported to respond to several stresses, which may explain the present results. C/EBPa, which is expressed in many tissues including the liver, blood, and adipose tissue regulates gene expression and is critical for the establishment and maintenance of energy homeostasis [15]. C/EBPa was recently reported to regulate gene expression and consequently to play role in the activation and/or proliferation of microglia following brain injury[14]. C/ EBPb is highly expressed in liver nuclei and has multiple biological functions, such as inflammation and other acute phase reactions [1], cell differentiation [11], immunoreaction and in the host-defense mechanism [13]. C/EBPd exhibits an expression pattern similar to that of C/EBPb and has been shown to be involved in the regulation of several genes induced during inflammation [9]. C/EBPz, which is known as the growth arrest and DNA-damage inducible gene 153 (gadd153), is markedly induced in response to a variety of cellular stresses including glucose deprivation, exposure to genotoxic agents and other growth-arresting situations [7]. Recently, C/EBPz was reported to be specifically activated under conditions that disturb the functioning of the endoplasmic reticulum, such as transient cerebral ischemia [12]. Several studies have demonstrated heterodimer formation between the C/EBP family members, including interactions of C/EBP1 with C/EBPa, C/EBPb and C/EBPd [16]. For the third hypothesis, reperfusion after cerebral ischemia induces an inflammatory response, and several cytokines are considered to be related to the pathogenesis [6]. C/EBPb may be responsible for the regulation of genes encoding many acute phase proteins and cytokines [2]. In cultured endothelial cells, induction of interleukin 6 (IL-6) was observed through activation of C/EBPb during hypoxic conditions [18]. Lipopolysaccharides, IL-1b and TNF-a induce the expression of the C/EBPb and d genes in mouse primary astrocytes, and C/EBPb and d could be pivotal transcription factors involved in brain inflammation and ischemia [3]. C/EBP1 is also implicated in cytokine signaling such as IL-2, IL-4, IL-12 and TNF-a [17]. We have demonstrated that C/EBP family proteins appear in the selectively vulnerable CA1 pyramidal cell layer in gerbils during neuronal degeneration, and may serve as a
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