Alterations of oxidative stress markers and apoptosis markers in the striatum after transient focal cerebral ischemia in rats

Cumulative evidence demonstrates that apoptosis caused by oxidative stress plays a key role in neuronal cell death after transient focal cerebral ischemia. In this study, we investigated exactly the immunohistochemical alterations of neuronal nuclei (NeuN), Cu/Zn-SOD (superoxide dismutase), Mn-SOD, 4-hydroxy-2-nonenal (HNE), and single strand DNA (ssDNA) in the striatum from 3 h up to 15 days after transient focal cerebral ischemia in rats under the same conditions. A conspicuous decrease of NeuN immunoreactive neurons was observed in the ipsilateral striatum from 3 h up to 15 days after focal ischemia. For Cu/Zn-SOD, Mn-SOD and HNE immunostainings, the alteration of Cu/Zn-SOD and HNE immunoreactivity was more pronounced than that of Mn-SOD immunoreactivity in the shrunken or atrophic neurons of ipsilateral striatum 3 h after focal ischemia. Thereafter, a significant increase of HNE immunoreactivity was observed in the shrunken or atrophic neurons of ipsilateral striatum up to 15 days after focal ischemia. In contrast, a significant decrease of Cu/Zn-SOD immunoreactivity was found in the ipsilateral striatum from 3 up to 15 days after focal ischemia. On the other hand, a significant increase of Mn-SOD immunereactivity was observed in the ipsilateral striatum from 1 up to 7 days after focal ischemia. In addition, our Western blot analysis also showed a significant increase of Cu/Zn-SOD and Mn-SOD in the ipsilateral striatum 1 day after focal ischemia, as compared to sham-operated group. In contrast, a significant increase in the number of ssDNA immunoreactive apoptotic neurons was observed in the ipsilateral striatum from 3 h to 3 days after focal cerebral ischemia. The present results also suggest that increased reactive oxygen species (ROS) production during reperfusion may contribute to the induction of the alteration of lipid peroxidation and could thereby lead to apoptosis in neurons of the ipsilateral striatum after transient focal ischemia, because of an insufficient expression of Cu/Zn-SOD and Mn-SOD. Furthermore, our findings demonstrate that the lipid peroxidation against mitochondrial membrane may contribute to apoptosis of striatal neurons after transient focal ischemia. Thus our findings demonstrate that the protection of lipid peroxidation against mitochondrial membrane may offer a novel therapeutic strategy for brain stroke in humans.     

Time dependent changes of striatal interneurons after focal cerebral ischemia in rats

Summary. The cellular damage over time and the alterations of neuronal subtypes was characterized in the striatum after 90-min middle cerebral artery occlusion and reperfusion in rats. We investigated the immunohistochemical alterations of choline acetyltransferase (ChAT)-positive (cholinergic-positive), γ-aminobutyric acid (GABA)ergic parvalbumin (PV)-positive, GABAergic nNOS (neuronal nitric oxide synthase)-positive interneurons, neuronal nuclei (NeuN)-positive spiny projection neurons, glial fibrillary acidic protein (GFAP)-positive strocytes and microglial response factor-1 (MRF-1)-positive microglia in the striatum after focal cerebral ischemia in rats. In the present study, transient focal cerebral ischemia in rats caused severe damage against interneurons as well as spiny projection neurons in the striatum. In contrast, a significant increase in the number of GFAP-immunopositive astrocytes was observed in the ipsilateral striatum 15 days after focal cerebral ischemia. Furthermore, a significant increase of MRF-1 immunoreactivity was observed in microglia of the ipsilateral striatum 7 days and 15 days after focal cerebral ischemia. Among three types of cholinergic interneurons, GABAergic PV-positive interneurons and GABAergic nNOS-positive interneurons, the severe damage of cholinergic and GABAergic PV-positive interneurons was more pronounced than that of GABAergic nNOS-positive interneurons after transient focal cerebral ischemia in rats. Furthermore, the present results suggest that GABAergic nNOS-positive interneurons in the striatum after focal cerebral ischemia undergo cellular death in a delayed manner. Correspondence: Tsutomu Araki, Department of Neurobiology and Therapeutics, Graduate School and Faculty of Pharmaceutical Sciences, The University of Tokushima, 1-78 Sho-machi, Tokushima 770-8505, Japan     

Immunohistochemical detection of oxidative DNA damage induced by ischemia–reperfusion insults in gerbil hippocampus in vivo

There is much evidence to suggest that ischemic injury occurs during the reperfusion phase of ischemia–reperfusion insults, and that the injury may be due to reactive-oxygen-species (ROS)-mediated oxidative events, including lipid peroxidation and DNA damage. However, oxidative DNA damage has until now not been examined in situ. In the present study, we report for the first time observation of cell type- and region-specific oxidative DNA damages in 5 min transient ischemic model by immunohistochemical methods, using monoclonal antibody against 8-hydroxy-2′-deoxyguanosine (8-OHdG), an oxidative DNA product. The cell types containing 8-OHdG immunoreactivity were neurons, glia and endothelial cells in the hippocampus. The 8-OHdG immunoreactivity was present in the nucleus but not the cytoplasm of these cells. The level of 8-OHdG in CA1 increased significantly (P<0.05) at the end of 30 min after ischemia, but there was no increase within CA2 and CA3 areas. The 8-OHdG levels in the hippocampus increased significantly (about fourfold) after 3 h of reperfusion and remained significantly (P<0.01) elevated for at least 12 h. At 4 days after ischemia, 8-OHdG levels in the CA2 and CA3 areas decreased to levels of the sham without neuronal loss, while disappearance of 8-OHdG immunoreactivity in the CA1 coincided with neuronal death in this area. These findings strongly suggest that ischemia-induced DNA damage evolves temporally and spatially, and that oxidative DNA damage may be involved in delayed neuronal death in the CA1 region.     

阿魏酸钠对大鼠脑缺血—再灌流后氧化性DNA损伤的保护作用

为研究脑缺血 再灌流后氧化性DNA损伤及阿魏酸钠的保护作用 ,采用线栓法制成大鼠大脑中动脉阻塞及再通模型。大脑中动脉再通时静脉注射阿魏酸钠 (15mg/kg) ,用免疫组织化学方法检测脑缺血 2h ,再灌流 4 8h后缺血再灌流组、阿魏酸钠组及对照组脑组织再灌流后氧化性DNA损伤产物 8 羟基脱氧鸟苷(8 OHdG)的表达。结果发现对照组脑区仅见少数散在微弱的 8 OHdG阳性表达 ;缺血再灌流组大脑中动脉阻塞侧额顶叶上部皮质和内侧尾壳核脑区有大量的 8 OHdG阳性表达 ,较对照组显著增加 (P <0 .0 1) ;阿魏酸钠组 8 OHdG阳性表达在脑区分布与缺血再灌流组相似 ,但较缺血再灌流组显著减少 (P <0 .0 1)。上述结果表明脑缺血—再灌流所致的氧化性DNA损伤主要存在于缺血半暗带 ,阿魏酸钠对氧化性DNA损伤具有保护作用。     

Cell cycle protein expression in proliferating microglia and astrocytes following transient global cerebral ischemia in the rat

Cerebral ischemia induces microglial and astroglial activation, which may play a crucial role in the development of ischemic neuronal damage. In this study, we examined the role of cell cycle proteins in glial proliferation in the hippocampus following 10min of global cerebral ischemia in the rat. Proliferating cells were identified with immunostaining for proliferating cell nuclear antigen (PCNA), and glial cells were visualized with immunostaining for microglial response factor-1 (microglia/macrophages) and glial fibrillary acidic protein (astrocytes). Expression of cyclin D1 and cyclin-dependent kinase-4 was also examined with double label immunohistochemistry. Proliferating cells in the CA1 region after ischemia consisted of microglia and much fewer astrocytes. Microglial activation and proliferation (7.6-fold increase in number after 7 days) were preceded by an increase in PCNA-positive microglia; 83% of microglia were PCNA-positive after 2 days. Astrocytes increased by 1.8-fold after 7 days, and only 6% of astrocytes became PCNA-positive by day 7. Cyclin D1 and cyclin-dependent kinase-4 immunoreactivity appeared in these glial cells in parallel with the expression of PCNA. The findings suggest that the accumulation of brain macrophages elicited by transient cerebral ischemia is caused predominantly by activation and proliferation of resident microglia through the upregulation of cell cycle proteins.     

Peripheral benzodiazepine receptor ligand PK11195 reduces microglial activation and neuronal death in quinolinic acid-injected rat striatum

The effects of the peripheral benzodiazepine receptor (PBR) ligand, PK11195, were investigated in the rat striatum following the administration of quinolinic acid (QUIN). Intrastriatal QUIN injection caused an increase of PBR expression in the lesioned striatum as demonstrated by immunohistochemical analysis. Double immunofluorescent staining indicated PBR was primarily expressed in ED1-immunoreactive microglia but not in GFAP-immunoreactive astrocytes or NeuN-immunoreactive neurons. PK11195 treatment significantly reduced the level of microglial activation and the expression of pro-inflammatory cytokines and iNOS in QUIN-injected striatum. Oxidative-mediated striatal QUIN damage, characterized by increased expression of markers for lipid peroxidation (4-HNE) and oxidative DNA damage (8-OHdG), was significantly diminished by PK11195 administration. Furthermore, intrastriatal injection of PK11195 with QUIN significantly reduced striatal lesions induced by the excitatory amino acid and diminished QUIN-mediated caspase-3 activation in striatal neurons. These results suggest that inflammatory responses from activated microglia are damaging to striatal neurons and pharmacological targeting of PBR in microglia may be an effective strategy in protecting neurons in neurological disorders such as Huntington's disease.     

Upregulation of neuronal nitric oxide synthase and mRNA, and selective sparing of nitric oxide synthase-containing neurons after local cerebral ischemia in rat

Nitric oxide synthase-containing neurons are presumed to be resistant to neurodegeneration and neurotoxicity, however this resistance has not been demonstrated after focal cerebral ischemia. We therefore measured the temporal profile of neuronal nitric oxide synthase (NOS-I) mRNA and immunoreactivity and NADPH-diaphorase reactivity over a one week period after permanent middle cerebral artery (MCA) occlusion in 48 male Wistar rats and compared these data to ischemic cell damage as evaluated on hematoxylin and eosin (H & E) stained sections by light microscopy. NOS-I mRNA increased as early as 15 min after MCA occlusion in the ipsilateral striatum and maximal expression of NOS-I was found in the ipsilateral cortex and striatum 1 h after MCA occlusion. The numbers of NOS-I-containing neurons in the ipsilateral cortex and striatum were significantly greater (P < 0.05) than NOS-I-containing neurons in the contralateral hemisphere at 2–48 h after the onset of ischemia. The number of NOS-I-containing neurons peaked at 4 h after MCA occlusion. Neurons exhibited shrinkage or were swollen at 1 to 4 h after MCA occlusion. At 24–48 h after ischemia, neurons in the ischemia lesion appeared to be eosinophilic or ghost like on H & E stained sections. However, some of these neurons retained morphological integrity on the NOS-I immunohistochemical sections. At 168 h after ischemia, all neurons within the lesion appeared necrotic on H & E stained sections; however, scatterred neurons expressed NOS-I and NADPH-diaphorase. The rapid upregulation of NOS-I and mRNA in the ischemic lesion suggests that NOS-I is involved in focal cerebral ischemic injury; the expression of NOS-I by neurons that retain their morphological structure in the area of the infarct suggests that NOS-I-containing neurons are more resistant to the ischemic insult. Our data also indicate a close association of NOS-I immunoreactivity and NADPH-diaphorase reactivity in ischemic brain.     

Focal cerebral ischemia enhances glial expression of ecto-5′-nucleotidase

The effect of ischemia on the reactive expression of ecto-5′-nucleotidase in rat brain was studied 6 h and 1, 2 and 7 days after permanent middle cerebral artery occlusion (MCAO). The distribution of 5′-nucleotidase in the infarcted brain was compared to markers for astrocytes (glial fibrillary acidic protein (GFAP)) and microglia (complement receptor type 3, antibody OX42) using histological staining or immunohistochemistry. 5′-Nucleotidase could be associated with reactive astrocytes by immunohistochemistry and with reactive microglia by enzyme histochemistry. In the untreated control 5′-nucleotidase was associated with astrocytes only in the hippocampus and the submeningeal space. After ischemia the enzyme was expressed on reactive astrocytes in the tissue surrounding the volume of infarction. Individual reactive astrocytes were observed 6 h after MCAO and the astrocytic expression became continuously enhanced during the following days. An enzyme histochemical analysis of 5′-nucleotidase activity revealed a postischemic increase in reaction product around the infarcted tissue. Seven days after MCAO a discrete band (0.2–0.4 mm) of reaction product characterized the rim of the infarcted area. This band of activity of 5′-nucleotidase colocalized with a band of immunoreactivity for OX42, indicative of an intense accumulation of 5′-nucleotidase expressing microglia. Our results suggest that ischemia following permanent MCAO results in an upregulation of the capacity for the hydrolysis of nucleotides within the tissue adjacent to the infarcted volume. Nucleotides released from the damaged cells can be hydrolyzed and the adenosine eventually formed may exert neuroprotective functions limiting the extent of damage.     

Folic acid deficiency increases delayed neuronal death, DNA damage, platelet endothelial cell adhesion molecule-1 immunoreactivity, and gliosis in the hippocampus after transient cerebral ischemia

Folic acid deficiency increases stroke risk. In the present study, we examined whether folic acid deficiency enhances neuronal damage and gliosis via oxidative stress in the gerbil hippocampus after transient forebrain ischemia. Animals were exposed to a folic acid-deficient diet (FAD) for 3 months and then subjected to occlusion of both common carotid arteries for 5 min. Exposure to an FAD increased plasma homocysteine levels by five- to eightfold compared with those of animals fed with a control diet (CD). In CD-treated animals, most neurons were dead in the hippocampal CA1 region 4 days after ischemia/reperfusion, whereas, in FAD-treated animals, this occurred 3 days after ischemia/reperfusion. Immunostaining for 8-hydroxy-2'-deoxyguanosine (8-OHdG) was performed to examine DNA damage in CA1 neurons in both groups after ischemia, and it was found that 8-OHdG immunoreactivity in both FAD and CD groups peaked at 12 hr after reperfusion, although the immunoreactivity in the FAD group was much greater than that in the CD group. Platelet endothelial cell adhesion molecule-1 (PECAM-1; a final mediator of neutrophil transendothelial migration) immunoreactivity in both groups increased with time after ischemia/reperfusion: Its immunoreactivity in the FAD group was much higher than that in the CD group 3 days after ischemia/reperfusion. In addition, reactive gliosis in the ischemic CA1 region increased with time after ischemia in both groups, but astrocytosis and microgliosis in the FAD group were more severe than in the CD group at all times after ischemia. Our results suggest that folic acid deficiency enhances neuronal damage induced by ischemia.     

Monocyte chemoattractant protein-1 plays a critical role in neuroblast migration after focal cerebral ischemia.

Transient focal ischemia is known to induce proliferation of neural progenitors in adult rodent brain. We presently report that doublecortin positive neuroblasts formed in the subventricular zone (SVZ) and the posterior peri-ventricle region migrate towards the cortical and striatal penumbra after transient middle cerebral artery occlusion (MCAO) in adult rodents. Cultured neural progenitor cells grafted into the non-infarcted area of the ipsilateral cortex migrated preferentially towards the infarct. As chemokines are known to induce cell migration, we investigated if monocyte chemoattractant protein-1 (MCP-1) has a role in post-ischemic neuroblast migration. Transient MCAO induced an increased expression of MCP-1 mRNA in the ipsilateral cortex and striatum. Immunostaining showed that the expression of MCP-1 was localized in the activated microglia and astrocytes present in the ischemic areas between days 1 and 3 of reperfusion. Furthermore, infusion of MCP-1 into the normal striatum induced neuroblast migration to the infusion site. The migrating neuroblasts expressed the MCP-1 receptor CCR2. In knockout mice that lacked either MCP-1 or its receptor CCR2, there was a significant decrease in the number of migrating neuroblasts from the ipsilateral SVZ to the ischemic striatum. These results show that MCP-1 is one of the factors that attract the migration of newly formed neuroblasts from neurogenic regions to the damaged regions of brain after focal ischemia.     

Increased 8-OHdG levels in the urine, serum, and substantia nigra of hemiparkinsonian rats

8-Hydroxy-2'-deoxyguanosine (8-OHdG), the predominant marker of oxidative DNA damage, may be a good biomarker for monitoring the progression of Parkinson's disease (PD). Unfortunately, there are no basic laboratory data examining 8-OHdG levels in animal models of PD. In this study, we demonstrate that rats lesioned with 6-hydroxydopamine (6-OHDA) in the medial forebrain bundle display significantly elevated 8-OHdG levels in urine, serum, and substantia nigra, but not cerebrospinal fluid and striatum, compared to sham controls. These increments in 8-OHdG levels were detected at 2 days, but not at 7 days after the lesion suggesting that oxidative stress is restricted to the acute phase of 6-OHDA neurotoxicity. The present results support 8-OHdG as a biomarker that may aid both in the diagnosis and in the documentation of progression in PD.     

Activation of the JAK/STAT pathway following transient focal cerebral ischemia: signaling through Jak1 and Stat3 in astrocytes

JAK/STAT is one of the pathways bearing signals from the cell membrane to the nucleus in response to extracellular growth factors and cytokines. In the present study, we examined the cellular distribution of Jak1 and Stat3, and activation of the JAK/STAT pathway following transient focal cerebral ischemia in the rat. Jak1 was mainly seen in white matter astrocytes and in certain neurons. Notably, large pyramidal neurons of cortical layer V showed the highest neuronal Jak1 expression within cerebral cortex and, in addition, expressed Stat3 indicating that the JAK/STAT pathway is involved in signaling in the corticofugal projection system. Shortly following ischemia, Jak1 immunoreactive astrocytes located in the ipsilateral neighbouring white matter and ischemic cortex and striatum showed nuclear translocation of Stat3. These features were maintained in large reactive astrocytes that surrounded the infarct from 3 to 7 days. At these later times, the abundant reactive microglia/macrophages were strongly immunoreactive to Stat3 and, to a lesser extent, Jak1. Two main protein complexes showing DNA binding activity at the sis-inducible element site were found under basal conditions, followed by changes in this pattern following ischemia concomitant with neuronal cell loss and activation of glia. This study showed basal cerebral activity of JAK/STAT signaling pathway, involving Jak1 and Stat3 proteins, and selective activation following ischemia. It is suggested that the kinase activity of Jak1 mediates nuclear translocation of Stat3 in astrocytes, and that this signaling pathway is involved in the astroglial response to focal cerebral ischemia.     

Oxidative damage and breakage of DNA in rat brain after transient MCA occlusion.

As thrombolytic therapy for treatment of ischemic stroke was propagated, much attention has been paid to reperfusion brain injury. Oxidative stress is one of the most important factors that exacerbate tissue damage by reperfusion. Thus, we investigated the extent of oxidative damage in rat brain after transient middle cerebral artery (MCA) occlusion by immunohistochemical analysis for 8-hydroxy-2'-deoxyguanosine (8-OHdG), which is one of the best markers of oxidative damage. Furthermore, in order to investigate its role in neuronal cell death, we performed terminal deoxynucleotidyl transferase-mediated dUTP-biotin in situ nick end labeling (TUNEL) study, and compared the results with that of 8-OHdG immunohistochemistry. There was no immunoreactive 8-OHdG in sham-operated brain, but it became present in neurons of MCA territory at 3 h of reperfusion after 90-min ischemia. At 48 h after reperfusion, cerebral tissue of MCA territory was severely destroyed, and many cells in that area revealed TUNEL positivity. Some neurons in MCA territory showed mild immunoreactivity for 8-OHdG at that time, but it was strongest in neurons in the outer area of MCA territory. Those cells did not show TUNEL positivity, suggesting that 8-OHdG production is not necessarily followed by early cell death. Here, it was demonstrated that oxidative DNA damage occurs in more extended area than that where cell death is recognized. Although this damage does not cause early cell death, this might result in more prolonged cell dysfunction and eventual neuronal loss. Anti-oxidant therapy might be required for treatment of stroke in the future.     

Early expressions of hypoxia-inducible factor 1alpha and vascular endothelial growth factor increase the neuronal plasticity of activated endogenous neural stem cells after focal cerebral ischemia

Endogenous neural stem cells become "activated" after neuronal injury, but the activation sequence and fate of endogenous neural stem cells in focal cerebral ischemia model are little known. We evaluated the relationships between neural stem cells and hypoxia-inducible factor-1α and vascular endothelial growth factor expression in a photothromobotic rat stroke model using immunohistochemistry and western blot analysis. We also evaluated the chronological changes of neural stem cells by 5-bromo-2′-deoxyuridine(BrdU) incorporation. Hypoxia-inducible factor-1α expression was initially increased from 1 hour after ischemic injury, followed by vascular endothelial growth factor expression. Hypoxia-inducible factor-1α immunoreactivity was detected in the ipsilateral cortical neurons of the infarct core and peri-infarct area. Vascular endothelial growth factor immunoreactivity was detected in bilateral cortex, but ipsilateral cortex staining intensity and numbers were greater than the contralateral cortex. Vascular endothelial growth factor immunoreactive cells were easily found along the peri-infarct area 12 hours after focal cerebral ischemia. The expression of nestin increased throughout the microvasculature in the ischemic core and the peri-infarct area in all experimental rats after 24 hours of ischemic injury. Nestin immunoreactivity increased in the subventricular zone during 12 hours to 3 days, and prominently increased in the ipsilateral cortex between 3–7 days. Nestin-labeled cells showed dual differentiation with microvessels near the infarct core and reactive astrocytes in the peri-infarct area. BrdU-labeled cells were increased gradually from day 1 in the ipsilateral subventricular zone and cortex, and numerous BrdU-labeled cells were observed in the peri-infarct area and non-lesioned cortex at 3 days. BrdU-labeled cells rather than neurons, were mainly co-labeled with nestin and GFAP. Early expressions of hypoxia-inducible factor-1α and vascular endothelial growth factor after ischemia made up the microenvironment to increase the neuronal plasticity of activated endogenous neural stem cells. Moreover, neural precursor cells after large-scale cortical injury could be recruited from the cortex nearby infarct core and subventricular zone.     

Transient ischemia-induced change of CCR7 immunoreactivity in neurons and its new expression in astrocytes in the gerbil hippocampus

Chemokines and their receptors are important players in organism homeostasis, development and immune response to inflammatory stimuli. In the present study, we examined effects of ischemia-reperfusion injury on the immunoreactivity and protein levels of chemokine C–C motif receptor 7 (CCR7) in the gerbil hippocampus (CA1–3 regions) after 5 min of transient global cerebral ischemia. CCR7 immunoreactivity was dramatically changed in the pyramidal neurons of the CA1, not CA2/3, region after ischemia-reperfusion. The immunoreactivity was increased after ischemia-reperfusion, and it was barely found from 5 days post-ischemia. In addition, CCR7 immunoreactivity was newly expressed in astrocytes, not microglia, in the ischemic CA1 region from 5 days post-ischemia. However, we did not observe this finding in the ischemic CA2/3 region. Furthermore, CCR7 protein levels in the ischemic CA1 region were changed like the change pattern of its immunoreactivity. These results indicate that both CCR7 immunoreactivity and protein levels are distinctively altered only in the CA1 region after transient cerebral ischemia and that the changes in CCR7 expression may be related to the ischemia-induced delayed neuronal death.     

Zonisamide reduces the increase in 8-hydroxy-2'-deoxyguanosine levels formed during iron-induced epileptogenesis in the brains of rats

PURPOSE: To examine the change of 8-hydroxy-2'deoxyguanosine (8-OHdG) levels, which are used as a marker for oxidative DNA damage, in iron-induced epileptogenic foci of the rat cerebrum. METHOD: Male Wistar rats were given a cortical injection of ferric chloride, and their 8-OHdG levels were determined over time. Additional animals were pretreated with the antiepileptic drug zonisamide (ZNS) before the ferric chloride injection, and their 8-OHdG levels were compared with the nonpretreated rats. RESULTS: Fifteen minutes after ferric chloride solution injection, the level of 8-OHdG increased, reaching a maximum 30 minutes after injection. Sixty minutes after injection, the levels coincided with those of controls. ZNS, in concentrations of 50 and 100 mg/kg body weight, prevented the increase of 8-OHdG levels within the cerebrum 30 minutes after iron solution injection. CONCLUSIONS: These results indicate that the formation of iron-induced epileptogenic foci in rats is related to DNA-damage-induced reactive oxygen species and that the inhibition of 8-OHdG formation by ZNS after iron injection may be due to the drug's antioxidant activity. The data suggest that free radical species known to be formed during iron salts-induced focal epileptogenesis cause damage to isocortical DNA. Furthermore, ZNS appears to inhibit the focal injuring response to DNA that occurs following iron salts-induced acute epileptogenesis.     

Heme oxygenase-1 (HSP-32) and heme oxygenase-2 induction in neurons and glial cells of cerebral regions and its relation to iron accumulation after focal cortical photothrombosis

Cerebral ischemic injury results in the liberation of heme from degenerating heme-containing proteins. The neurotoxic heme is usually detoxified by the constitutive heme oxygenase-2 (HO-2) and its inducible isoform HO-1(heat shock protein 32) resulting in the formation of biliverdin which becomes reduced to bilirubin, carbon monoxide (CO), and iron. Biliverdin and bilirubin have antioxidative properties whereas CO is discussed as a signaling molecule. Iron if it remains free could catalyze Haber--Weiss and Fenton reactions causing the formation of highly toxic radicals. We have studied the alterations of cerebral HO-2 and HO-1 in relation to iron accumulations after defined cortical photothrombosis within the hindlimb area of the rat. HO-2 immunohistochemistry showed that the number of HO-2-positive neurons in most perilesional regions remained constant. However, much stronger systemic immunoreactivity for HO-2 was observed between days 1 and 7 postlesion. For HO-1 a systemic increase of immunoreactivity occurred also between days 1 and 7. In addition HO-1-positive astrocytes and microglia appeared as early as 4 h postlesion and increased up to day 3 followed by a sharp decline toward day 14 within the injured hemisphere. HO-1-positive astrocytes and microglia occurred in ipsilateral cortex, corpus callosum, hippocampus, striatum, and thalamic nuclei. Additionally an increase of HO-1 in myelin-associated globulin-positive oligodendrocytes was found in ipsilateral and contralateral cortex. Next to the lesion iron accumulation occurred after day 3 and increased strongly toward day 14 at times when HO-1 and -2 had decreased, suggesting that HO activity does not directly contribute to postlesional iron deposition.     

Magnetic resonance imaging shows delayed ischemic striatal neurodegeneration

Brief focal ischemia leading to temporary neurological deficits induces delayed hyperintensity on T1-weighted magnetic resonance imaging (MRI) in the striatum of humans and rats. The T1 hyperintensity may stem from biochemical alterations including manganese (Mn) accumulation after ischemia. To clarify the significance of this MRI modification, we investigated the changes in the dorsolateral striatum of rats from 4 hours through 16 weeks after a 15-minute period of middle cerebral artery occlusion (MCAO), for MRI changes, Mn concentration, neuronal number, reactivities of astrocytes and microglia/macrophages, mitochondrial Mn-superoxide dismutase (Mn-SOD), glutamine synthetase (GS), and amyloid precursor protein. The cognitive and behavioral studies were performed in patients and rats and compared with striatal T1 hyperintensity to show whether alteration in brain function correlated with MRI and histological changes. The T1-weighted MRI signal intensity of the dorsolateral striatum increased from 5 days to 4 weeks after 15-minute MCAO, and subsequently decreased until 16 weeks. The Mn concentration of the dorsolateral striatum increased after ischemia in concert with induction of Mn-SOD and GS in reactive astrocytes. The neuronal survival ratio in the dorsolateral striatum decreased significantly from 4 hours through 16 weeks, accompanied by extracellular amyloid precursor protein accumulation and chronic glial/inflammatory responses. The patients and rats with neuroradiological striatal degeneration had late-onset cognitive and/or behavioral declines after brief focal ischemia. This study suggests that (1) the hyperintensity on T1-weighted MRI after mild ischemia may involve tissue Mn accumulation accompanied by Mn-SOD and GS induction in reactive astrocytes, (2) the MRI changes correspond to striatal neurodegeneration with a chronic inflammatory response and signs of oxidative stress, and (3) the subjects with these MRI changes are at risk for showing a late impairment of brain function even though the transient ischemia is followed by total neurological recovery.     

Postischemic changes in the immunophilin FKBP12 in the rat brain

An immunosuppressant tacrolimus (FK506) protects against neuronal damage following cerebral ischemia. On the other hand, the major physiological role of the immunophilin FK506-binding protein-12 (FKBP12) is a modulation of intracellular calcium flux. Since an increase in intracellular calcium concentration is a major mediator of ischemic neuronal death, we investigated the changes in FKBP12 following cerebral ischemia in the rat. We induced focal cerebral ischemia by intraluminal occlusion of the middle cerebral artery for 1 h, and global cerebral ischemia for 10 min by bilateral carotid artery occlusion combined with hypotension. The animals were killed at 4 h to 7 days after reperfusion. Immunohistochemistry was performed on paraffin sections using a monoclonal antibody raised against recombinant FKBP12. Immunoreactivity to FKBP12 in control brains was most pronounced in the CA1 subfield of the hippocampus and the striatum, the localization being primarily neuronal. Following focal ischemia, FKBP12 immunoreactivity decreased rapidly in the ischemic core by 4 h, but increased in surviving neurons in penumbra areas (4 h-7 days). Within an area of infarction, invading leukocytes and macrophages exhibited immunoreactivity to FKBP12 (3-7 days). Following global ischemia, FKBP12 immunoreactivity in CA1 neurons decreased after 1 day, and then it was lost between 2 and 7 days, although many CA1 neurons showed a transient increase in FKBP12 at 2 days. No FKBP12 immunoreactivity was observed in reactive glial cells. Thus, FKBP12 declined in dying neurons, whereas FKBP12 was upregulated in less severely injured neurons. The findings suggest that (1) FKBP12 plays an important role in the process of neuronal survival and death following cerebral ischemia, and (2) FKBP12 is involved in inflammatory reactions that occur within an area of infarction.