Kazuhiko Nozaki

Mitogen-activated protein kinases and cerebral ischemia.

Mitogen-activated protein kinases (MAPKs) have crucial roles in signal transduction from the cell surface to the nucleus and regulate cell death and survival. Recent papers support the hypothesis that neuronal apoptosis and cerebral ischemia induce the robust activation of MAPK cascades. Although extracellular signal-regulated kinases pathways promote cell survival and proliferation, and c-Jun N-terminal protein kinases/p38 pathways induce apoptosis in general, the roles of MAPK cascades in neuronal death and survival seem to be complicated and altered by the type of cells and the magnitude and timing of insults. Some specific inhibitors of MAPK cascades provide important information in clarifying the roles of each molecule in neuronal death and survival, but the results are still controversial. Further studies are necessary to elucidate the activated signal transduction upstream and downstream of the cascades in cerebral ischemia, and to define the crosstalk between the cascades and other signaling pathways, before MAPK cascades can be candidate molecules in the treatment of cerebral ischemia.

Primate embryonic stem cell-derived neuronal progenitors transplanted into ischemic brain

Transplantation of stem cells has the possibility of restoring neural functions after stroke damage. Therefore, we transplanted neuronal progenitors generated from monkey embryonic stem (ES) cells into the ischemic mouse brain to test this possibility. Monkey ES cells were caused to differentiate into neuronal progenitors by the stromal cell-derived inducing activity method. Focal cerebral ischemia was induced by occluding the middle cerebral artery by the intraluminal filament technique. The donor cells were transplanted into the ischemic lateral striatum at 24 h after the start of reperfusion. The cells transplanted into the ischemic brain became located widely around the ischemic area, and, moreover, the transplanted cells differentiated into various types of neurons and glial cells. Furthermore, at 28 days after the transplantation, over 10 times more cells in the graft were labeled with Fluorogold (FG) by stereotactic focal injection of FG into the anterior thalamus and substantia nigra on the grafted side when compared with the number at 14 days. From these results we confirmed the survival and differentiation of, as well as network formation by, monkey ES-cell-derived neuronal progenitors transplanted into the ischemic mouse brain.

Enhanced Poly(ADP-ribosyl)ation after Focal Ischemia in Rat Brain

Nitric oxide from neuronal cells plays detrimental roles in glutamate neurotoxicity and in focal brain ischemia. Nitric oxide directly damages DNA, and breaks in the DNA strands activate poly(ADP-ribose) polymerase (PARP), which brings poly(ADP-ribosyl)ation of the nuclear proteins. The excessive activation of PARP is thought to cause depletion of ATP and the energy failure resulting in cell death. To clarify the involvement of poly(ADP-ribosyl)ation in ischemic insult, we examined poly(ADP ribosyl)ation by immunohistochemical methods and the protective effect of 3-aminobenzamide, which is a PARP inhibitor, on focal brain ischemia using an intraluminal permanent middle cerebral artery occlusion model in rats. Poly(ADP ribosyl)ation was widely and markedly detected 2 hours after the ischemic insult in the cerebral cortex and striatum in which infarction developed 24 hours later. The enhanced immunoreactivity of poly(ADP-ribose) gradually decreased, and 16 hours later, no immunoreactivity was detected. Intraventricular administration of 3-aminobenzamide (1 to 30 mg/kg) 30 minutes before the ischemic insult decreased infarction volume in a dose-dependent manner along with the immunohistochemical reduction of poly(ADP-ribosyl)ation. Pretreatment with 7-nitroindazole (25 mg/kg, intraperitoneally), a selective neuronal nitric oxide synthetase inhibitor, partially reduced poly(ADP-ribosyl)ation. These data suggest the involvement of poly(ADP-ribosyl)ation in the development of cerebral infarction.

Intravenous Administration of Thioredoxin Decreases Brain Damage Following Transient Focal Cerebral Ischemia in Mice

Thioredoxin (TRX) is induced by a variety of oxidative stimuli and shows cytoprotective roles against oxidative stress. To clarify the possibility of clinical application, we examined the effects of intravenously administered TRX in a model of transient focal cerebral ischemia in this study. Mature male C57BL/6j mice received either continuous intravenous infusion of recombinant human TRX (rhTRX) over a range of 1-10 mg/kg, bovine serum albumin, or vehicle alone for 2 h after 90-min transient middle cerebral artery occlusion (MCAO). Twenty-four hours after the transient MCAO, the animals were evaluated neurologically and the infarct volumes were assessed. Infarct volume, neurological deficit, and protein carbonyl contents, a marker of protein oxidation, in the brain were significantly ameliorated in rhTRX-treated mice at the dose of 3 and 10 mg/kg versus these parameters in control animals. Moreover, activation of p38 mitogen-activated protein kinase, whose pathway is involved in ischemic neuronal death, was suppressed in the rhTRX-treated mice. Further, rhTRX was detected in the ischemic hemisphere by western blot analysis, suggesting that rhTRX was able to permeate the blood-brain barrier in the ischemic hemisphere. These data indicate that exogenous TRX exerts distinct cytoprotective effects on cerebral ischemia/reperfusion injury in mice by means of its redox-regulating activity.

Redox Control of Neuronal Damage during Brain Ischemia after Middle Cerebral Artery Occlusion in the Rat: Immunohistochemical and Hybridization Studies of Thioredoxin

Thioredoxin (TRX) is a small, multifunctional protein with a redox-active site and multiple biological functions that include reducing activity for reactive oxygen intermediates. We assayed TRX and TRX mRNA by immunohistochemical methods and hybridization experiments in the rat brain after middle cerebral artery (MCA) occlusion. During ischemia, the immunoreactivity for TRX decreased; it disappeared after MCA occlusion in the ischemic regions. It rapidly decreased and nearly disappeared at 4 and 16 hours after MCA occlusion in the lateral striatum and frontoparietal cortex, respectively. On the other hand, in the perifocal ischemic region, the penumbra, TRX immunoreactivity began to increase 4 hours after MCA occlusion and continued to increase until 24 hours after occlusion. In hybridization experiments, TRX mRNA decreased and nearly disappeared 4 hours after MCA occlusion in the lateral striatum. In the frontoparietal cortex, it decreased until 24 hours after MCA occlusion. In the perifocal ischemic region, TRX mRNA began to increase 4 hours after MCA occlusion and continued to increase until 24 hours. Northern blot analysis showed that total TRX mRNA in the operated hemispheres was induced from 8 hours and increased until 24 hours after the surgical procedures. We previously reported that recombinant TRX promotes the in vitro survival of primary cultured neurons. We now suggest that TRX in the penumbra has neuroprotective functions and that decreased levels of TRX in the ischemic core modify neuronal damage during focal brain ischemia.

3-Nitropropionic acid induces ischemic tolerance in gerbil hippocampus in vivo

Systemic administration of high dose of 3-nitropropionic acid (3-NP), an irreversible inhibitor of succinate dehydrogenase, causes neurodegeneration within the striatum in vivo. However, it has been reported that pretreatment with low dose of 3-NP may increase tolerance to subsequent hypoxia in hippocampal slices. The present study investigated whether ischemic tolerance can be induced in gerbil hippocampus in vivo by low dose of 3-NP. After pretreatment with 3-NP (single i.p. injection of 3 mg/kg, body weight 2-4 days prior to forebrain ischemia), the number of surviving neurons in CA1 region of hippocampus against succeeding forebrain ischemia was significantly higher than in the ischemic control group. Our results show that chemical preconditioning with low dose of 3-NP induces ischemic tolerance in gerbil hippocampus in vivo.

Hypoxia-ischemia induces thioredoxin expression and nitrotyrosine formation in new-born rat brain

Abstract Thioredoxin (TRX) is a 13 kDa protein with antioxidant effect and redox regulating functions. Peroxynitrite is a strong oxidizing and nitrating agent which can react with all classes of biomolecules. In the present study, we focused on the association between TRX and nitrotyrosine, which served as a marker of peroxynitrite formation, in the neonatal hypoxia-ischemia (HI) rat brain. At 4-16 h after HI, the immunoreactivity for TRX was diminished in the injured region in the cortex and striatum, whereas nitrotyrosine immunoreactivity was enhanced. In contrast, around the injured region, TRX immunoreactivity was enhanced in survival neurons at 4-24 h after HI, while the immunoreactivity for nitrotyrosine was mostly not detected. Northern blot analysis showed increased TRX mRNA induction in the cerebral hemisphere ipsilateral to the carotid ligation from 4-24 h after HI but not in the contralateral hypoxic hemisphere. These findings suggest that production of peroxynitrite is involved in HI brain injury, and that induced TRX plays a neuroprotective role against oxidative stress resulting from HI.

Excitotoxic hippocampal injury is attenuated in thioredoxin transgenic mice.

Thioredoxin is a small, multifunctional protein with a redox-active disulfide/dithiol in the active site. Thioredoxin plays several important biologic roles both in intracellular and extracellular compartments with its redox-regulating and reactive oxygen intermediates scavenging activities. We assayed the seizure response and the excitotoxic hippocampal injury in thioredoxin transgenic and wild-type C57BL/6 mice. Seizure score after kainic acid treatment was significantly lower in thioredoxin transgenic mice. Seven days after kainic acid administration, the damage in the hippocampal CA1 and CA3 regions was significantly attenuated in thioredoxin transgenic mice. Thioredoxin and redox regulation play an important role in excitotoxic brain damage.

3-Nitropropionic acid induces poly(ADP-ribosyl)ation and apoptosis related gene expression in the striatum in vivo

Impaired energy metabolism plays an important role in neuronal cell death after brain ischemia, and apoptosis has been implicated in cell death induced by metabolic impairment. In the present study, metabolic impairment was induced by 3-nitropropionic acid (3-NP), an irreversible inhibitor of succinate dehydrogenase. In order to clarify the involvement of poly(ADP-ribosyl)ation and apoptotic pathway in 3-NP induced cell death, we examined poly(ADP-ribosyl)ation and the apoptosis related gene protein expression after systemic administration of 3-NP by immunohistochemistry. Poly(ADP-ribosyl)ation was evidently detected in the striatal lesion but not in any other region. Immunoreactive ratio of Bcl-2 to Bax significantly increased both in the striatum and cortex. The data suggest that striatal cell death involves poly(ADP-ribosyl)ation and also apoptotic pathway in part following administration of 3-NP.

Expression and distribution of redox regulatory protein, thioredoxin after metabolic impairment by 3-nitropropionic acid in rat brain

Thioredoxin (TRX) is a small, multifunctional protein with a redox-active site and multiple biological functions that include reducing activity for reactive oxygen intermediates. We assayed TRX by immunohistochemical methods in the rat brain after intraperitoneal injection of 3-nitropropionic acid (3-NP), an irreversible inhibitor of succinate dehydrogenase. Systemic administration of 3-NP produced lateral striatal, hippocampal CA1 and CA3 lesions in the present study. The immunoreactivity for TRX was enhanced in hippocampal CA3, dentate gyrus and lateral striatum, but not detected in hippocampal CA1 subfield after 3-NP intoxication. The data suggest that TRX may play an important role in the pathogenesis of 3-NP neurotoxicity.