Tetsuya Nagayama

Microarray analysis of hippocampal gene expression in global cerebral ischemia

The brains response to ischemia, which helps determine clinical outcome after stroke, is regulated partly by competing genetic programs that respectively promote cell survival and delayed cell death. Many genes involved in this response have been identified individually or systematically, providing insights into the molecular basis of ischemic injury and potential targets for therapy. The development of microarray systems for gene expression profiling permits screening of large numbers of genes for possible involvement in biological or pathological processes. Therefore, we used an oligodeoxynucleotide‐based microarray consisting of 374 human genes, most implicated previously in apoptosis or related events, to detect alterations in gene expression in the hippocampus of rats subjected to 15 minutes of global cerebral ischemia followed by up to 72 hours of reperfusion. We found 1.7‐fold or greater increases in the expression of 57 genes and 1.7‐fold or greater decreases in the expression of 34 genes at 4, 24, or 72 hours after ischemia. The number of induced genes increased from 4 to 72 hours, whereas the number of repressed genes decreased. The induced genes included genes involved in protein synthesis, genes mutated in hereditary human diseases, proapoptotic genes, antiapoptotic genes, injury‐response genes, receptors, ion channels, and enzymes. We detected transcriptional induction of several genes implicated previously in cerebral ischemia, including ALG2, APP, CASP3, CLU, ERCC3, GADD34, GADD153, IGFBP2, TIAR, VEGF, and VIM, as well as other genes not so implicated. We also found coinduction of several groups of related genes that might represent functional modules within the ischemic neuronal transcriptome, including VEGF and its receptor, NRP1; the IGF1 receptor and the IGF1‐binding protein IGFBP2; Rb, the Rb‐binding protein E2F1, and the E2F‐related transcription factor, TFDP1; the CACNB3 and CACNB4 β‐subunits of the voltage‐gated calcium channel; and caspase‐3 and its substrates, ACINUS, FEM1, and GSN. To test the hypothesis that genes identified through this approach might have roles in the pathophysiology of cerebral ischemia, we measured expression of the products of two induced genes not heretofore implicated in cerebral ischemia—GRB2, an adapter protein involved in growth‐factor signaling pathways, and SMN1, which participates in RNA processing and is deleted in most cases of spinal muscular atrophy. Western analysis showed enhanced expression of both proteins in hippocampus at 24 to 72 hours after ischemia, and SMN1 was localized by immunohistochemistry to hippocampal neurons. These results suggest that microarray analysis of gene expression may be useful for elucidating novel molecular mediators of cell death and survival in the ischemic brain.

Induction of vascular endothelial growth factor and hypoxia-inducible factor-1α by global ischemia in rat brain

Vascular endothelial growth factor is an angiogenic and neurotrophic peptide whose expression is transcriptionally induced in hypoxic tissues through the action of hypoxia-inducible factor-1alpha. To determine if this signaling pathway is activated in the ischemic brain, and might therefore participate in adaptive processes such as angiogenesis and neuroprotection, we examined the expression of vascular endothelial growth factor and hypoxia-inducible factor-1alpha in cerebral cortex and hippocampus following transient global cerebral ischemia in the rat. Northern analysis showed ischemia-inducible expression of multiple vascular endothelial growth factor messenger ribonucleic acid splice variants between 4 and 24h. Western analysis and immunocytochemistry demonstrated the concerted induction of vascular endothelial growth factor and hypoxia-inducible factor-1alpha in the same, apparently neuronal, cells in vulnerable regions of cortex and hippocampus after 15min of ischemia, which persisted for as long as 4 to 72h of reperfusion. These findings demonstrate that hypoxia-sensitive vascular endothelial growth factor signaling can be induced in neurons in global cerebral ischemia in vivo, and are consistent with the hypothesis that ischemic insults trigger hypoxia-sensing and adaptive downstream molecular responses in central neurons.

Induction of oxidative DNA damage in the peri-infarct region after permanent focal cerebral ischemia.

Abstract: To address the role of oxidative DNA damage in focal cerebral ischemia lacking reperfusion, we investigated DNA base and strand damage in a rat model of permanent middle cerebral artery occlusion (MCAO). Contents of 8‐hydroxyl‐2′‐deoxyguanosine (8‐OHdG) and apurinic/apyrimidinic abasic sites (AP sites), hallmarks of oxidative DNA damage, were quantitatively measured in nuclear DNA extracts from brains obtained 4‐72 h after MCAO. DNA single‐ and double‐strand breaks were detected on coronal brain sections using in situ DNA polymerase I‐mediated biotin‐dATP nick‐translation (PANT) and terminal deoxynucleotidyl transferase‐mediated dUTP nick end‐labeling (TUNEL), respectively. Levels of 8‐OHdG and AP sites were markedly elevated 16‐72 h following MCAO in the frontal cortex, representing the peri‐infarct region, but levels did not significantly change within the ischemic core regions of the caudateputamen and parietal cortex. PANT‐ and TUNEL‐positive cells began to be detectable 4‐8 h following MCAO in the caudate‐putamen and parietal cortex and reached maximal levels at 72 h. PANT‐ and TUNEL‐positive cells were also detected 16‐72 h after MCAO in the lateral frontal cortex within the infarct border, where many cells also showed colocalization of DNA single‐strand breaks and DNA fragmentation. In contrast, levels of PANT‐positive cells alone were transiently increased (16 h after MCAO) in the medial frontal cortex, an area distant from the infarct zone. These data suggest that within peri‐infarct brain regions, oxidative injury to nuclear DNA in the form of base and strand damage may be a significant and contributory cause of secondary expansion of brain damage following permanent focal ischemia.

bcl-2 Antisense Treatment Prevents Induction of Tolerance to Focal Ischemia in the Rat Brain

In the rat, 60 minutes of transient ischemia to the middle cerebral artery results in infarction of the caudate putamen. Ischemic preconditioning with 20 minutes of transient focal ischemia produced tolerance (attenuated infarction volume) to 60 minutes of subsequent focal ischemia administered three days, five days, or seven days later. Western blots from tolerant caudate putamen demonstrated increased bcl-2 expression, maximum at 3 days and persisting through 7 days. Immunocytochemical examination found that bcl-2 was expressed in cells with both neuronal and nonneuronal morphology in striatum after preconditioning ischemia. bcl-2 antisense oligodeoxynucleotides (ODNs), bcl-2 sense ODNs, or artificial cerebrospinal fluid (CSF, vehicle) was infused into the lateral ventricle for the 72 hours between the 20-minute ischemic preconditioning and the 60-minute period of ischemia. Antisense ODN treatment reduced expression of bcl-2 in the striatum and blocked the induction of tolerance by preconditioning ischemia. Sense and CSF treatments had no effect on either bcl-2 expression or tolerance. In this model of induced tolerance to focal ischemia, bcl-2 appears to be a major determinant.

Activation of Poly(ADP‐Ribose) Polymerase in the Rat Hippocampus May Contribute to Cellular Recovery Following Sublethal Transient Global Ischemia

Abstract: We have investigated the role of poly(ADP‐ribose) polymerase (PARP) activation in rat brain in a model of sublethal transient global ischemia. Adult male rats were subjected to 15 min of ischemia with brain temperature reduced to 34°C, followed by 1, 2, 4, 8, 16, 24, and 72 h of reperfusion. PARP mRNA expression was examined in the hippocampus using quantitative RT‐PCR, northern blot analysis, and in situ hybridization. Protein expression was assessed using western blot analysis. PARP enzymatic activity was investigated by measuring nuclear [3H]NAD incorporation. The presence of poly(ADP‐ribose) polymers was assessed immunocytochemically. Although PARP mRNA and protein expressions were not altered after ischemia, enzymatic activity was increased 4.37‐fold at 1 h (p < 0.05 vs. sham) and 1.73‐fold (p < 0.05 vs. sham) at 24 h of reperfusion. Immunostaining demonstrated the presence of poly‐(ADP‐ribose) polymers in CA1 neurons. Cellular NAD+ levels were not significantly altered at any time point. Furthermore, systemic administration of 3‐aminobenzamide (30 mg/kg), a PARP inhibitor, prevented the increase in PARP activity at 1 and 24 h of reperfusion, significantly decreased the number of surviving neurons in the hippocampal CA1 region 72 h after ischemia (p < 0.01 vs. sham), and increased DNA single‐strand breaks assessed as DNA polymerase I‐mediated biotin‐dATP nick‐translation (PANT)‐positive cells (p < 0.01 vs. sham). Furthermore, using an in vitro DNA repair assay, 3‐aminobenzamide (30 mg/kg) was shown to block DNA base excision repair activity. These data suggest that the activation of PARP, without subsequent NAD+ depletion, following mild transient ischemia may be neuroprotective in the brain.

Cloning and characterization of rat caspase-9: implications for a role in mediating caspase-3 activation and hippocampal cell death after transient cerebral ischemia.

Delayed hippocampal neurodegeneration after transient global ischemia is mediated, at least in part, through the activation of terminal caspases, particularly caspase-3, and the subsequent proteolytic degradation of critical cellular proteins. Caspase-3 may be activated by the membrane receptor-initiated caspase-8–dependent extrinsic pathway and the mitochondria-initiated caspase-9–dependent intrinsic pathway; however, the precise role of these deduced apoptosis-signaling pathways in activating caspase-3 in ischemic neurons remains elusive. The authors cloned the caspase-9 gene from the rat brain and investigated its potential role in mediating ischemic neuronal death in a rat model of transient global ischemia. Caspase-9 gene expression and protease activity were extremely low in the adult brain, whereas they were developmentally upregulated in newborn rats, especially at postnatal 12 weeks, a finding consistent with the theory of an essential role for caspase-9 in neuronal apoptosis during brain development. After 15-minute transient global ischemia, caspase-9 was overexpressed and proteolytically activated in the hippocampal CA1 neurons at 8 to 72 hours of reperfusion. The temporal profile of caspase-9 activation coincided with that of cytochrome c release and caspase-3 activation, but preceded CA1 neuronal death. Immunoprecipitation experiments revealed that there was enhanced formation of Apaf-1/caspase-9 complex in the hippocampus 8 and 24 hours after ischemia. Furthermore, intracerebral ventricular infusion of the relatively specific caspase-9 inhibitor N-benzyloxycarbonyl-Leu-Glu-His-Asp-fluoro-methylketone before ischemia attenuated caspase-3–like activity and significantly enhanced neuronal survival in the CA1 sector. In contrast, inhibition of caspase-8 activity had no significant effect on caspase-3 activation or neuronal survival. These results suggest that the caspase-9–dependent intrinsic pathway may be the primary mechanism responsible for the activation of caspase-3 in ischemic hippocampal neurons.

Fas (CD95) May Mediate Delayed Cell Death in Hippocampal CA1 Sector after Global Cerebral Ischemia

Cell death–regulatory genes like caspases and bcl-2 family genes are involved in delayed cell death in the CA1 sector of hippocampus after global cerebral ischemia, but little is known about the mechanisms that trigger their expression. The authors found that expression of Fas and Fas-ligand messenger ribonucleic acid and protein was induced in vulnerable CA1 neurons at 24 and 72 hours after global ischemia. Fas-associating protein with a novel death domain (FADD) also was upregulated and immunoprecipitated and co-localized with Fas. Caspase-10 was activated and interacted with FADD protein to an increasing extent as the duration of ischemia increased. Moreover, caspase-10 co-localized with both FADD and caspase-3. These findings suggest that Fas-mediated death signaling may play an important role in signaling hippocampal neuronal death in CA1 after global cerebral ischemia.

Inducible Repair of Oxidative DNA Lesions in the Rat Brain After Transient Focal Ischemia and Reperfusion

To determine the role of oxidative DNA damage and repair in brain injury after focal ischemia and reperfusion, the authors investigated DNA base damage and DNA base excision repair (BER) capacity, the predominant repair mechanism for oxidative DNA lesions, in the rat model of temporary middle cerebral artery occlusion. Contents of 8-hydroxyl-2′-deoxyguanosine (8-oxodG) and apurinic/apyrimidinic abasic site (AP site), hallmarks of oxidative DNA damage, were quantitatively measured in nuclear DNA extracts from brains 0.25 to 72 hours after 1 hour of middle cerebral artery occlusion. In parallel to the detection of DNA lesions, the capacity for 8-oxodG- or AP site-dependent DNA repair synthesis was measured in nuclear protein extracts using specific in vitro DNA repair assays. After postischemic reperfusion, the levels of 8-oxodG and AP sites were markedly increased in ischemic tissues. In frontal/parietal cortex, regions that survived ischemia, 8-oxodG and AP sites were efficiently repaired during reperfusion. However, in the caudate, a region that was destined to infarct, the DNA lesions were poorly repaired. In consistent with the patterns of endogenous lesion repair, a markedly induced and long-lasting (at least 72 hours) BER activity was detected in the cortex but not in the caudate after ischemia. The induced BER activity in ischemic cortex was attributed to the upregulation of gene expression and activation of selective BER enzymes, particularly DNA polymerase-β and OGG1. These results strongly suggest that inducible DNA BER constitutes an important endogenous mechanism that protects brain against ischemia-induced oxidative neuronal injury.

Bcl-w expression is increased in brain regions affected by focal cerebral ischemia in the rat

Proteins of the bcl-2 family are important regulators of apoptosis in many tissues of the embryo and adult and may play a role in cell death following stroke. The recently isolated bcl-w gene encodes a pro-survival member of the bcl-2 family, which is widely expressed. However, it is not known whether bcl-w plays a role in determining cell survival after cerebral ischemia. Using Western blot analysis and immunocytochemistry, regional bcl-w protein expression was studied in rat brain 2, 6, 24 and 72 h following 20 min temporary middle cerebral artery occlusion (MCAO). Focal cerebral ischemia increased bcl-w protein expression within the caudate putamen and parietal cortex, as well as causing milder increases within frontal cortex. Immunocytochemically bcl-w was expressed within neurons (frontal and parietal cortex) and glia (caudate putamen) 24 h after MCAO. These data suggest that bcl-w could play a role in determining cell survival after cerebral ischemia.

Two caspase-2 transcripts are expressed in rat hippocampus after global cerebral ischemia

Caspase family genes play a critical role in the initiation and execution of programmed cell death. Programmed cell death is an important contributor to neuronal loss following cerebral ischemia. We have performed a series of experiments to investigate the role of a specific caspase, caspase‐2, in the development of delayed neuronal death following transient global ischemia in the rat. A rat ischemic brain cDNA library was screened, and two splice‐variants of caspase‐2 mRNA were identified, caspase‐2S and caspase‐2L, which were highly homologous with the sequences of human and mouse caspase‐2S and caspase‐2L genes, respectively. RT‐PCR demonstrated an increase in expression of both caspase‐2S and caspase‐2L mRNA at 8, 24 and 72 h of reperfusion after global ischemia. The ratio of the two PCR fragments did not change significantly throughout the time course of reperfusion. Western blot with monoclonal antibody specific to the pro‐apoptotic caspase‐2L splice variant revealed an increase in procaspase‐2 (51 kDa) protein from 4 to 72 h following ischemia compared with sham‐operated controls. Furthermore, an approximately 30‐kDa cleavage product appeared at 8 h and increased with increasing duration of reperfusion. Thus, caspase‐2L is both translated and activated following transient global ischemia. Finally, intraventricular administration of the caspase‐2‐like inhibitor (VDVAD‐FMK) 30 min before induction of ischemia decreased the number of CA1 neurons staining positively for DNA damage (Klenow‐labeling assay) and increased the number of healthy‐appearing CA1 neurons (cresyl violet) compared with vehicle‐treated controls. Taken together, the data suggest that caspase‐2 induction and activation are important mediators of delayed neuronal death following transient global ischemia.