Kensuke Murakami

Cytosolic Redistribution of Cytochrome C after Transient Focal Cerebral Ischemia in Rats

Recent in vitro cell-free studies have shown that cytochrome c release from mitochondria is a critical step in the apoptotic process. The present study examined the expression of cytochrome c protein after transient focal cerebral ischemia in rats, in which apoptosis was assumed to contribute to the expansion of the ischemic lesion. In situ labeling of DNA breaks in frozen sections after 90 minutes of middle cerebral artery (MCA) occlusion showed a significant number of striatal and cortical neurons, which were maximized at 24 hours after ischemia, exhibiting chromatin condensation, nuclear segmentation, and apoptotic bodies. Cytosolic localization of cytochrome c was detected immunohistochemically in the ischemic area as early as 4 hours after 90 minutes of MCA occlusion. Western blot analysis of the cytosolic fraction revealed a strong single 15-kDa band, characteristic of cytochrome c, only in the samples from the ischemic hemisphere. Western blot analysis of the mitochondrial fraction showed a significant amount of mitochondrial cytochrome c in nonischemic brain, which was decreased in ischemic brain 24 hours after ischemia. These results provide the first evidence that cytochrome c is being released from mitochondria to the cytosol after transient focal ischemia. Although further evaluation is necessary to elucidate its correlation with DNA fragmentation, our results suggest the possibility that cytochrome c release may play a role in DNA-damaged neuronal cell death after transient focal cerebral ischemia in rats.

Overexpression of CuZn-Superoxide Dismutase Reduces Hippocampal Injury After Global Ischemia in Transgenic Mice

BACKGROUND AND PURPOSE The role of copper, zinc-superoxide dismutase (CuZn-SOD) in hippocampal injury after transient global ischemia was studied using transgenic (Tg) mice and wild-type littermates. METHODS Global ischemia was induced by bilateral common carotid artery occlusion. The hemisphere with the hypoplastic posterior communicating artery was determined and then the hippocampus in this hemisphere was evaluated qualitatively using a score of 0 to 4 and quantitatively using an image analyzer. RESULTS Hippocampal injury was reduced in Tg mice after both 5 and 10 minutes of ischemia. In the 5-minute ischemia group, the mean score of the injury was significantly lower in Tg than nontransgenic (nTg) mice at 3 days. In the 10-minute group, the hippocampal injury was reduced more in Tg than nTg mice at 1 day. Quantitative evaluation by an image analyzer confirmed the qualitative data. Neurons with fragmented DNA were also studied in the hippocampal injury. In the 5-minute group, despite the reduction of the injury in Tg mice, their neurons with fragmented DNA were relatively increased at 1 day. In the 10-minute group, this ratio was almost the same in both nTg and Tg mice. CONCLUSIONS CuZn-SOD plays a protective role in the pathogenesis of selective hippocampal injury after brief ischemia, whether the insult is relatively mild or intense. Furthermore, CuZn-SOD may reduce both necrotic and DNA fragmented neuronal death after global ischemia.

Exacerbation of Delayed Cell Injury After Transient Global Ischemia in Mutant Mice With CuZn Superoxide Dismutase Deficiency

BACKGROUND AND PURPOSE We have demonstrated that copper-zinc superoxide dismutase (CuZn-SOD), a cytosolic isoenzyme of SODs, has a protective role in the pathogenesis of superoxide radical-mediated brain injury. Using mice bearing a disruption of the CuZn-SOD gene (Sod1), the present study was designed to clarify the role of superoxide anion in the pathogenesis of selective vulnerability after transient global ischemia. METHODS Sod1 knockout homozygous mutant mice (Sod1 -/-) with a complete absence of endogenous CuZn-SOD activity, heterozygous mutant mice (Sod1 +/-) with a 50% decrease in the activity, and littermate wild-type mice (male, 35 to 45 g) were subjected to global ischemia. Since the plasticity of the posterior communicating artery (PcomA) has been reported to influence the outcome of hippocampal injury, we assessed the relation between the plasticity of PcomAs and the decrease of regional cerebral blood flow in global ischemia. RESULTS The fluorescence intensity of hydroethidine oxidation, a measurement of ethidium fluorescence for superoxide radicals, was increased in mutant mice 1 day after both 5 and 10 minutes of global ischemia, compared with wild-type mice. Hippocampal injury in the PcomA hypoplastic brains showed significant exacerbation in mutant mice compared with wild-type littermates 3 days after 5 minutes of global ischemia, although a marked difference was not observed at 1 day. CONCLUSIONS These data suggest that superoxide radicals play an important role in the pathogenesis of delayed injury in the vulnerable hippocampal CA1 subregion after transient global ischemia.

Effects of Nitric Oxide Synthase Inhibition on Brain Infarction in SOD-1-Transgenic Mice following Transient Focal Cerebral Ischemia

To investigate the role of superoxide in the toxicity of nitric oxide (NO), we examined the effect of nitric oxide synthase (NOS) inhibition on brain infarction in transgenic mice overexpressing CuZn-superoxide dismutase (SOD-1). Male SOD-transgenic mice and nontransgenic littermates (30–35 g) were subjected to 60 min of middle cerebral artery occlusion followed by 24 h of reperfusion. Either N G -nitro-l-arginine methyl ester (l-NAME; 3 mg/kg), a mixed neuronal and endothelial NOS inhibitor, or 7-nitroindazole (7-NI; 25 mg/kg), a selective neuronal NOS inhibitor, was administered intraperitoneally 5 min after the onset of ischemia. At 24 h of reperfusion, the mice were decapitated and the infarct volume was evaluated in each group. In the nontransgenic mice, l-NAME significantly increased the infarct volume as compared with the vehicle, while 7-NI significantly decreased it. In the SOD-transgenic mice, l-NAME-treated animals showed a significantly larger infarct volume than vehicle-treated ones, whereas there were no significant differences between 7-NI- and vehicle-treated mice. Our findings suggest that selective inhibition of neuronal NOS ameliorates ischemic brain injury and that both neuronal and endothelial NOS inhibition may result in the deterioration of ischemic injury due to vasoconstriction of the brain. Since l-NAME increased infarct volume even in SOD-transgenic mice, the protective effect of SOD could result from the vasodilation by increased endothelial NO as well as the reduction of neuronal injury due to less production of peroxynitrite compared to wild-type mice. Moreover, the neurotoxic role of NO might not be dependent on NO itself, but the reaction with superoxide to form peroxynitrite, because of no additive effects of SOD and a neuronal NOS inhibitor.

The development of a new mouse model of global ischemia : focus on the relationships between ischemia duration, anesthesia, cerebral vasculature, and neuronal injury following global ischemia in mice

A new model for mouse global ischemia is presented, and the relationship of ischemia duration, cerebral vasculature, and ischemic neuronal injury has been determined. CD-1 mice anesthetized by chloral hydrate were subjected to global ischemia by bilateral common carotid artery occlusion under controlled ventilation for 3, 5, and 10 min. After evaluating the patency of the posterior communicating artery (PcomA) as hypoplastic or normoplastic, neuronal injury was independently determined in the striatum, cortex, and hippocampus in each hemisphere. Ischemic injury was strongly correlated with not only ischemia duration, but also with the patency of the PcomAs. Furthermore, neuronal injury developed in a delayed fashion after 3-min ischemia, while it was maximized at 24 h after 10-min ischemia. Physiological studies showed the induction of slight hypotension as compared with inhalation anesthesia, and improvement of blood gas data relative to spontaneous respiration. These data demonstrate the usefulness of this method to induce selective vulnerability and delayed neuronal cell death in mice, and to provide a useful model to study the detailed mechanism of global ischemia using transgenic or knockout mutant mice.

Apoptosis in the striatum of rats following intraperitoneal injection of 3-nitropropionic acid

The present study investigated the mechanism of cellular degeneration within the striatum following administration of the mitochondrial toxin, 3-nitropropionic (3-NP) acid. Internucleosomal fragmentation typical of apoptosis was present in the DNA of cells from the striatum of 3-NP-treated rats. DNA fragmentation was also evident in this region by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling. The data suggest that striatal cells die by apoptosis following administration of 3-NP.

Reperfusion following focal cerebral ischemia alters distribution of neuronal cells with DNA fragmentation in mice

To clarify the role of reperfusion in DNA damage following focal cerebral ischemia, we determined the distribution of cells with DNA fragmentation following permanent or transient focal ischemia. Less DNA-damaged cells were observed in the permanent than in the transient group in the ischemic core, but there was no difference in the boundary zone. We conclude that reperfusion following transient ischemia may exacerbate neuronal death following DNA damage.

Decreased expression of bcl-2 and bcl-x mRNA coincides with apoptosis following intracerebral administration of 3-nitropropionic acid

The mitochondrial toxin, 3-nitropropionic acid (3-NP), is an irreversible inhibitor of succinate dehydrogenase that induces apoptosis in vitro and in vivo. We injected 3-NP into the striatum of rats to examine the potential role of Bcl-2 or Bcl-x, proteins that can inhibit apoptosis, in brain injury due to 3-NP. Electrophoretic examination of striatal tissue indicated that 3-NP induced internucleosomal fragmentation typical of apoptosis. There was also histologic evidence of apoptosis based on staining by the terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling (TUNEL) method. Apoptosis was first observed 6 h after injection, was maximal at 1 day, and was still observed on day 7. Expression of bcl-2, bcl-x, and c-jun mRNA expression was evaluated 1, 3, 6, and 12 h and 1, 3, 5, and 7 days after injection using in situ hybridization. Both bcl-2 and bcl-x mRNA expression in the striatum decreased starting at 6 h and continued to 5 days after injection. This was in contrast to an apparent increase in c-jun expression. The similarity in the time course of apoptosis to that of suppression of bcl-2 and bcl-x mRNA suggests that changes in expression of these genes may contribute to apoptosis following 3-NP injection.

Expression of hsp 70 mRNA is induced in the brain of transgenic mice overexpressing human CuZn-superoxide dismutase following transient global cerebral ischemia

We examined hsp70 mRNA expression in the brains of transgenic (Tg) mice overexpressing CuZn-superoxide dismutase and in wild-type (Wt) littermates after 3 min of bilateral common carotid artery occlusion. Significant induction of hsp70 mRNA occurred in the hippocampus, the cortex, and other brain regions of the Tg mice 4 h after ischemia compared to the Wt mice. However, there was no histological damage in the brains of Tg and Wt mice as assessed by both Cresyl violet staining and by TUNEL staining for DNA fragmentation. These data suggest that high levels of CuZn-superoxide dismutase activity increase hsp70 mRNA expression and that intense hsp70 mRNA expression does not predict neuronal damage, even in vulnerable hippocampal CA1 neurons.

Acute ischemic complications after therapeutic parent artery occlusion with revascularization for complex internal carotid artery aneurysms

BACKGROUND Parent artery occlusion (PAO) is an alternative surgical strategy for complex internal carotid artery (ICA) aneurysms, which are unclippable because of their anatomical structures, including a broad neck, fragile dome, critical branch, and cavernous sinus location. Despite revascularization, ischemic complications occur after the PAO because of several factors, such as hypoperfusion, embolism, and perforator impairment. METHODS Acute ischemic complications and their mechanisms were analyzed in a consecutive series of 32 patients presenting with complex ICA aneurysms treated by PAO. RESULTS Fourteen ruptured and 18 unruptured aneurysms were located in the cavernous sinus (n = 16) and paraclinoid portion (n = 16). Preoperative balloon test occlusion was performed to investigate ischemic tolerance and to select the bypass method in 24 cases. An extracranial-intracranial bypass was constructed using the superficial temporal artery in 17, and a saphenous vein graft was done in 12 cases. Although postoperative hypoperfusion was prevented by revascularization based on the balloon test occlusion, acute ischemic complications due to perforator occlusion and thromboembolism were observed in 10 cases after the surgery. Five cases presented with impairment of perforating branches adjacent to the occluded parent artery, resulting in prolonged neurologic deterioration. CONCLUSION The distal location of ICA aneurysms is a risk factor for the perforator impairment, when treated by PAO, and PAO by clip placement is preferred to endovascular coiling to prevent of perforator impairment.