Transferase-mediated dUTP-biotin In Situ Nick End Labeling Research Articles

Recent Progress in Therapeutic Strategies for Ischemic Stroke.

Possible strategies for treating stroke include neuroprotection in the acute phase of cerebral ischemia and stem cell therapy in the chronic phase of cerebral ischemia. Previously, we have studied the temporal and spatial expression patterns of c-fos, hypoxia inducible factor-1α (HIF-1α), heat shock protein 70 (HSP70), and annexin V after 90 min of transient middle cerebral occlusion in rats and concluded that there is a time window for neuroprotection from 12 to 48 h after ischemia. In addition, we have estimated the neuroprotective effect of glial cell line-derived neurotrophic factor (GDNF) by injecting Sendai viral vector containing the GDNF gene into the postischemic brain. This Sendai virus-mediated gene transfer of GDNF showed a significant neuroprotective effect in the ischemic brain. Additionally, we have administered GDNF and hepatocyte growth factor (HGF) protein into the postischemic rat brain and estimated the infarct size and antiapoptotic and antiautophagic effects. GDNF and HGF significantly reduced infarct size, the number of microtubule-associated protein 1 light chain 3 (LC3)-positive cells, and the number of terminal deoxynucleotidyl transferase-mediated dUTP-biotin in situ nick-end labeling (TUNEL)-positive cells, indicating that GDNF and HGF were greatly associated with not only the antiapoptotic effect but also the antiautophagic effects. Finally, we have previously transplanted undifferentiated iPSCs into the ipsilateral striatum and cortex at 24 h after cerebral ischemia. Histological analysis was performed at 14 and 28 days after cell transplantation, and we found that iPSCs could supply a great number of doublecortin-positive neuroblasts but also formed tridermal teratoma in the ischemic brain. Our results suggest that iPSCs have a potential to provide neural cells after ischemic brain injury if tumorigenesis is properly controlled. In the future, we will combine these strategies to develop more effective therapies for the treatment of strokes.

Amlodipine and atorvastatin exert protective and additive effects via antiapoptotic and antiautophagic mechanisms after transient middle cerebral artery occlusion in Zucker metabolic syndrome rats

We examined the neuroprotective effects amlodipine and/or atorvastatin in metabolic syndrome (MetS) Zucker fatty rats against transient (90 min) middle cerebral artery occlusion (MCAO). The rats were pretreated with vehicle, amlodipine, atorvastatin, or amlodipine plus atorvastatin for 28 days, and 24 hr after transient MCAO the infarct size was assessed via hematoxylin and eosin staining, and terminal deoxynucleotidyl transferase-mediated dUTP-biotin in situ nick end labeling (TUNEL) and microtubule-associated protein 1 light chain 3 (LC3) expression were examined by immunohistochemistry to evaluate apoptosis and autophagy, respectively. Compared with the vehicle group, rats treated with amlodipine or atorvastatin alone showed a significant decrease in infarct volume (P < 0.01), which was further decreased in the amlodipine plus atorvastatin group (P < 0.001). Compared with the vehicle group, the numbers of TUNEL- and LC3-positive cells were markedly reduced by amlodipine or atorvastatin alone (P < 0.01) and further decreased by amlodipine plus atorvastatin (P < 0.001). The number of apoptotic TUNEL/autophagic LC3 double-positive cells was also significantly decreased with amlodipine or atorvastatin alone compared with vehicle (P < 0.01) and was further decreased by amlodipine plus atorvastatin (P < 0.001). These data suggest additive neuroprotective effects of combination amlodipine and atorvastatin treatment after acute ischemic stroke in MetS model Zucker rats. These effects are mediated, at least in part, via antiapoptotic and antiautophagic mechanisms. Further studies are now needed to expand these preliminary results to understand fully the mechanisms involved in the protective effects of amlodipine and atorvastatin against ischemic stroke.

Antiapoptotic and antiautophagic effects of glial cell line‐derived neurotrophic factor and hepatocyte growth factor after transient middle cerebral artery occlusion in rats

Glial cell line-derived neurotrophic factor (GDNF) and hepatocyte growth factor (HGF) are strong neurotrophic factors, which function as antiapoptotic factors. However, the neuroprotective effect of GDNF and HGF in ameliorating ischemic brain injury via an antiautophagic effect has not been examined. Therefore, we investigated GDNF and HGF for changes of infarct size and antiapoptotic and antiautophagic effects after transient middle cerebral artery occlusion (tMCAO) in rats. For the estimation of ischemic brain injury, the infarct size was calculated at 24 hr after tMCAO by HE staining. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin in situ nick end labeling (TUNEL) was performed for evaluating the antiapoptotic effect. Western blot analysis of microtubule-associated protein 1 light chain 3 (LC3) and immunofluorescence analysis of LC3 and phosphorylated mTOR/Ser(2448) (p-mTOR) were performed for evaluating the antiautophagic effect. GDNF and HGF significantly reduced infarct size after cerebral ischemia. The amounts of LC3-I plus LC3-II (relative to beta-tubulin) were significantly increased after tMCAO, and GDNF and HGF significantly decreased them. GDNF and HGF significantly increased p-mTOR-positive cells. GDNF and HGF significantly decreased the numbers of TUNEL-, LC3-, and LC3/TUNEL double-positive cells. LC3/TUNEL double-positive cells accounted for about 34.3% of LC3 plus TUNEL-positive cells. This study suggests that the protective effects of GDNF and HGF were greatly associated with not only the antiapoptotic but also the antiautophagic effects; maybe two types of cell death can occur in the same cell at the same time, and GDNF and HGF are capable of ameliorating these two pathways.

Extension of ischemic therapeutic time window by a free radical scavenger, Edaravone, reperfused with tPA in rat brain

3-methyl-1-phenyl-2-pyrazolin-5-one (Edaravone) is a free radical scavenger. We tested the hypothesis that combination treatment of Edaravone and recombinant tissue plasminogen activator (tPA) extends the therapeutic time window. Male Wistar rats were subjected to 1.5-, 3.0- or 4.5-hour middle cerebral artery (MCA) occlusion (MCAO) by a nylon thread. Animals were randomly divided into four groups. The Sham group rats were operated without MCAO and drug injection. In the Vehicle-treated group the same volume of saline was given every 1.5 hours from just after MCAO to just before reperfusion. In the Vehicle + tPA-treated group saline injection was given as above and tPA (5 mg/kg, i.v.) was given once just after reperfusion. Edaravone+tPA-treated group: Edaravone (3 mg/kg, i.v.) was given every 1.5 hours instead of saline and tPA injection as above. Survival rate, infarct size and evidence of apoptosis and hemorrhage were examined in the animals. Combining administration of Edaravone+tPAsignificantly increased survival rate after 3 hours of transient MCAO, and reduced infarct volume after 1.5 hours of transient MCAO compared with the vehicle or vehicle+tPAgroups. In Edaravone+tPA-treated group, the number of terminal deoxynucleotidyl transferase-mediated dUTP-biotin in situ nick end labeling (TUNEL) and 4-hydroxynonenal (4-HNE) positive cells were reduced at 16 hours after 3 hours of transient MCAO, but not in advanced glycation end products (AGEs) and 8-hydroxy-2'-deoxyguanosine (8-OHdG). Hemorrhage rate and the area decreased in the Edaravone+tPA-treated group. The combination therapy of Edaravone+tPA increased survival rate, and reduced the infarct volume and hemorrhage with reduction of lipid peroxidation. Therefore, Edaravone combination is expected to extend the therapeutic time window of tPA in the clinical situation.

Involvement of apoptosis in 3-nitropropionic acid-induced ischemic tolerance to transient focal cerebral ischemia in rats

The involvement of apoptosis in mitochondrial toxin 3-nitropropionic acid (3-NPA)-induced ischemic tolerance to transient focal cerebral ischemia in rats and the mechanism was investigated. 3-NPA at a dose of 20 mg/kg or vehicle control was intraperitoneally into the rats. Three days later, rats were exposed to 2 h of middle cerebral artery occlusion followed by 24 h of reperfusion. Infarct volumes were assessed by 2,3,5-triphenyltetrazolinm chloride (TTC) staining 24 h after reperfusion. Neural cell apoptosis in cerebral ischemic penumbra was detected by terminal deoxynucleotidyl transferase-mediated dUTP-biotin in situ nick end labeling (TUNEL) and flow cytometry methods (FCM). The results showed that as compared to the vehicle-treated group, pretreatment with 3-NPA could reduce the infarct volume by 23.3% and decrease the number of TUNEL-positive neural cells and apoptotic percentage by 47% (P<0.05) and 44.9% (P<0.01), respectively. It was concluded that the development of 3-NPA-induced ischemic tolerance in brain might be related to the decreases in neural cell apoptosis.

Inhibition of caspase-3 activation and apoptosis is involved in 3-nitropropionic acid-induced ischemic tolerance to transient focal cerebral ischemia in rats.

Our aim was to investigate the involvement of caspase-3 activation and apoptotic cell death in mitochondrial toxin 3-nitropropionic acid (3-NPA)-induced ischemic tolerance to transient focal cerebral ischemia in rats. Rats were administrated either vehicle control or 3-NPA ip doses of 20 mg/kg. Three days later, rats were exposed to 2 h of middle cerebral artery occlusion, followed by 24 h of reperfusion. Infarct volumes were assessed by 2,3,5-triphenyltetrazolium chloride (TTC) staining 24 h after reperfusion. We measured neural cell apoptosis in the cerebral ischemic penumbra by terminal deoxynucleotidyl transferase-mediated dUTP-biotin in situ nick end labeling (TUNEL) and flow cytometry (FCM). Cleavage of the fluorogenic substrate zDEVD-afc was used to assay caspase-3 activity. Compared with the vehicle-injected group, pretreatment with 3-NPA reduced the infarct volume by 22.3% and decreased the number of TUNEL-positive neural cells and apoptotic percentages by 47% (p <0.05) and 43.9% (p <0.01), respectively. In terms of caspase-3 activity in ischemic penumbral tissues, the 3-NPA-pretreated group showed 13.9% (p <0.05) less caspase-3 activity than the control group. The development of 3-NPA-induced ischemic tolerance in brain may be related to decreases in caspase-3 activation, which leads to decreased neural cell apoptosis.

Therapeutic time window of Ad-GDNF after transient middle cerebral artery occlusion in rat

Time-dependent influence of adenovirus-mediated glial cell line-derived neurotrophic factor (GDNF) gene (Ad-GDNF) was examined after 90 min of transient middle cerebral artery occlusion (MCAO) in rats. Treatment with Ad-GDNF significantly reduced the infarct volume when immediately administered after reperfusion, but became insignificant when administered at 1 h after the reperfusion as were the cases treated with vehicle and adenoviral vector containing the Escherichia coli lacZ gene (Ad-LacZ)-treated groups. The protective effect of GDNF was related to the significant reduction of the number of terminal deoxynucleotidyl transferase-mediated dUTP-biotin in situ nick end labeling (TUNEL)-positive cells as well as immunohistochemical positive cells for active caspase-3 but not caspase-9. These results showed that exogenous GDNF gene transfer successfully reduced the infarct size in a time-dependant manner by suppressing active caspase-3 but not active caspase-9. However, the therapeutic time window was shorter than the effect of GDNF protein itself previously reported.

Neuroprotection by selective nitric oxide synthase inhibition at 24 hours after perinatal hypoxia-ischemia.

Perinatal hypoxia-ischemia is a major cause of neonatal morbidity and mortality. Until now no established neuroprotective intervention after perinatal hypoxia-ischemia has been available. The delay in cell death after perinatal hypoxia-ischemia creates possibilities for therapeutic intervention after the initial insult. Excessive nitric oxide and reactive oxygen species generated on hypoxia-ischemia and reperfusion play a key role in the neurotoxic cascade. The present study examines the neuroprotective properties of neuronal and inducible but not endothelial nitric oxide synthase inhibition by 2-iminobiotin in a piglet model of perinatal hypoxia-ischemia. Twenty-three newborn piglets were subjected to 60 minutes of hypoxia-ischemia, followed by 24 hours of reperfusion and reoxygenation. Five additional piglets served as sham-operated controls. On reperfusion, piglets were randomly treated with either vehicle (n=12) or 2-iminobiotin (n=11). At 24 hours after hypoxia-ischemia, the cerebral energy state, presence of vasogenic edema, amount of apparently normal neuronal cells, caspase-3 activity, amount of terminal deoxynucleotidyl transferase-mediated dUTP-biotin in situ nick end labeling (TUNEL)-positive cells, and degree of tyrosine nitration were assessed. A 90% improvement in cerebral energy state, 90% reduction in vasogenic edema, and 60% to 80% reduction in apoptosis-related neuronal cell death were demonstrated in 2-iminobiotin-treated piglets at 24 hours after hypoxia- ischemia. A significant reduction in tyrosine nitration in the cerebral cortex was observed in 2-iminobiotin-treated piglets, indicating decreased formation of reactive nitrogen species. Simultaneous and selective inhibition of neuronal and inducible nitric oxide synthase by 2-iminobiotin is a promising strategy for neuroprotection after perinatal hypoxia-ischemia.

Induction of PML immunoreactivity in rat brain neurons after transient middle cerebral artery occlusion

Promyelocytic leukemia (PML) protein is involved in apoptotic death of cultured neuronal cells, but its role in ischemic brain damage remains uncertain. In this study, we investigated change of immunoreactivity for PML protein in rat brain after transient middle cerebral artery occlusion, and compared the results with that of terminal deoxynucleotidyl transferase-mediated dUTP-biotin in situ nick end labeling (TUNEL). Western blotting analysis revealed that PML immunoreactivity was only scant in the sham-control brain, but it increased at 1 h and 1 day after reperfusion, and decreased in density thereafter. Immunohistochemical analysis revealed that nuclei of neurons were most densely stained. TUNEL positive cells appeared at 1 day and peaked at 3 days of reperfusion, indicating that PML protein induction preceded DNA fragmentation in neurons. The present results suggest that PML protein may be one of the key molecules in ischemic neuronal cell death. [Neurol Res 2001; 23: 772-776]

Attenuation of oxidative DNA damage with a novel antioxidant EPC-K1 in rat brain neuronal cells after transient middle cerebral artery occlusion

EPC-K1, L-ascorbic acid 2-[3,4-dihydro-2,5,7,8-tetramethyl-2-(4,8,12-trimethyltridecyl)-2H-1-benzopyran-6-yl-hydrogen phosphate] potassium salt, is a novel antioxidant. In this study, we investigated a reduction of oxidative neuronal cell damage with EPC-K1 by immunohistochemical analysis for 8-hydroxy-2′-deoxyguanosine (8-OHdG) in rat brain with 60 min transient middle cerebral artery occlusion, in association with terminal deoxynucleotidyl transferase-mediated dUTP-biotin in situ nick end labeling (TUNEL) and staining for total and active caspase-3. Treatment with EPC-K1 (20mg kg-1 i.v.) significantly reduced infarct size (p < 0.05) at 24 h of reperfusion. There were no positive cells for 8-OHdG and TUNEL in sham-operated brain, but numerous cells became positive for 8-OHdG, TUNEL and caspase-3 in the brains with ischemia. The number was markedly reduced in the EPC-K1 treated group. These reductions were particularly evident in the border zone of the infarct area, but the degree of reduction was less in caspase-3 staining than in 8-OHdG and TUNEL stainings. These results indicate EPC-K1 attenuates oxidative neuronal cell damage and prevents neuronal cell death. [Neurol Res 2001; 23: 676-680]

Different expression of glycogen synthase kinase-3β between young and old rat brains after transient middle cerebral artery occlusion

Ischemia is a common stress to human brain and is difficult to cure in older individuals. To examine the differences of the response to cerebral ischemia between young and old rat brains, distributions of glycogen synthase kinase-3β (GSK3β) and tau proteins were analyzed after 90 min of transient middle cerebral artery occlusion (MCAO) in young (10-11 weeks) and old (15 months) rats by immunohistochemical analyses. At 4 h of reperfusion, strong cytoplasmic and nuclear immunoreactivity for GSK3β was induced in neurons of lamina I, II, V and VI of the cerebral cortex and dorsal caudate in young brains, while the induction was not observed in lamina I and II of old cerebral cortex. The staining in lamina V and VI and dorsal caudate then gradually decreased until seven days of reperfusion in both animal groups. The staining of tau protein and terminal deoxynucleotidyl transferase-mediated dUTP-biotin in situ nick end labeling (TUNEL) did not show any positive signals in the control brain, but showed positive signals after ischemia with a peak at 24 h and 3 days, respectively. No significant difference was observed in the temporal and spatial patterns of tau and TUNEL stainings between these two groups. These data suggest that GSK3β may have a role in ischemic neuronal cell death, and that the different spatial expression of GSK3β between young and old rat brains may partly explain the vulnerability of older neurons after ischemia. [Neurol Res 2001; 23: 588-592]

Expressions of nitrotyrosine and TUNEL immunoreactivities in cultured rat spinal cord neurons after exposure to glutamate, nitric oxide, or peroxynitrite.

Although excitotoxic and oxidative stress play important roles in spinal neuron death, the exact mechanism is not fully understood. We examined cell damage of primary culture of 11-day-old rat spinal cord by addition of glutamate, nitric oxide (NO) or peroxynitrite (PN) with detection of nitrotyrosine (NT) or terminal deoxynucleotidyl transferase-mediated dUTP-biotin in situ nick end labeling (TUNEL). With addition of glutamate, NOC18 (a slow NO releaser) or PN, immunoreactivity for NT became stronger in the cytoplasm of large motor neurons in the ventral horn at 6 to 48 hr and positive in the axons of the ventral horn at 24 to 48 hr. TUNEL positive nuclei were found in spinal large motor neurons from 24 hr, and the positive cell number greatly increased at 48 hr in contrast to the vehicle. Pretreatment of cultures with alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)/kainate receptor antagonist, NO-suppressing agent, and antioxidant protected the immunoreactivity for NT or TUNEL. The present results suggest that both excitotoxic and oxidative stress play an important role in the upregulation of NT nitration and the apoptotic pathway in cultured rat spinal neurons.

Detection of the exogenous hGDNF in gerbils under the treatment with AxCAhGDNF adenoviral vector

Glial cell line-derived neurotrophic factor (GDNF) is one of the most potent neurotrophic factors and promotes survival in many populations of cells. We examined the neuroprotective effect of an adenoviral vector encoding glial cell line-derived neurotrophic factor (AxCAhGDNF) on the transient global ischemia [Brain Res. 885 (2000) 273–282]. Gerbils received AxCAhGDNF or an adenoviral vector encoding bacterial β-galactosidase gene (AxCALacZ) through administration into the lateral ventricle. Two days later, occlusion of the common carotid arteries for 5 min bilaterally using aneurysm clips produced transient global forebrain ischemia. Animals showed intense immunolabeling for GDNF in ependymal cells on 2, 4 and 7 days after the operation. The exogenous gene transducted by the adenovirus in the same cells was detected by in situ hybridization. The treatment with AxCAhGDNF significantly prevented the loss of hippocampal CA-1 pyramidal neurons 2 to 7 days after the operation, as compared to AxCALacZ treatment. Also terminal deoxynucleotidyl transferase-mediated dUTP-biotin in situ nick end labeling (TUNEL) staining was markedly reduced in the case with AxCAhGDNF treatment at 7 days after the operation. In this paper, we describe in detail the techniques for the detection of the exogenous gene of hGDNF under the treatment with AxCAhGDNF.

Time dependent amelioration against ischemic brain damage by glial cell line-derived neurotrophic factor after transient middle cerebral artery occlusion in rat

Time dependent influence of glial cell line-derived neurotrophic factor (GDNF) was examined after 90 min of transient middle cerebral artery occlusion (MCAO) in rats. Treatment with GDNF significantly reduced the infarct volume stained with 2,3,5-triphenyltetrazolium chloride (TTC) when GDNF was topically applied at 0 and 1 h of reperfusion, but became insignificant at 3 h as compared to vehicle group. The protective effect of GDNF was closely related to the significant reduction of the number of terminal deoxynucleotidyl transferase-mediated dUTP-biotin in situ nick end labeling (TUNEL) positive cells as well as immunofluorescently positive cells for active forms of caspases, especially active caspase-3 but not -9. Thus, the present study showed that topical application of GDNF significantly reduced infarct size in a time-dependent manner, while the therapeutic time window was shorter than other chemical compounds such as an NMDA receptor antagonist (MK-801) and a free radical scavenger (alpha-phenyl-tert-butyl-nitrone, PBN). The effect of GDNF was stronger in suppressing active caspase-3 than active caspase-9.

Expressions of caspase-3, Tunel, and Hsp72 immunoreactivities in cultured spinal cord neurons of rat after exposure to glutamate, nitric oxide, or peroxynitrite.

Although excitotoxic and oxidative stress play important roles in spinal neuron death, the exact mechanisms are not fully understood. We examined cell damage of primary culture of 11 day-old rat spinal cord by addition of glutamate, nitric oxice (NO) or peroxynitrite (PN) with detection of caspase-3, terminal deoxynucleotidyl transferase-mediated dUTP-biotin in situ nick end labeling (TUNEL) or 72 kDa heat shock protein (HSP72). With addition of glutamate, NOC18 (a slow NO releaser) or PN, immunoreactivity for caspase-3 became stronger in the cytoplasm of large motor neurons in the ventral horn at 6 to 24 hr. TUNEL positive nuclei were found in spinal large motor neurons from 24 h and the positive cell proportion greatly increased at 48 h in contrast to the vehicle. On the other hand, the immunoreactivity of HSP72 in the ventral horn was already positive at 0 h, and gradually decreased in the course of time with glutamate, NOC18 or PN than vehicle treatment. In the dorsal horn, the proportion of caspase-3 positive small neurons greatly increased at 6 to 48 h after addition of glutamate. The present results suggest that both excitotoxic and oxidative stress play important roles in the apoptotic pathway in cultured spinal neurons.

Glutamate enhances DNA fragmentation in cultured spinal motor neurons of rat

The role of glutamate in the mechanism of spinal motor neuron death is not fully understood. With addition of glutamate to primary culture of 11-day-old rat spinal cord, terminal deoxynucleotidyl transferasemediated dUTP-biotin in situ nick end labeling (TUNEL) positive nuclei were found in spinal large motor neurons from 24 h, and the number of TUNEL positive large motor neurons greatly increased at 48 h. In contrast, only a small number of large motor neurons became TUNEL positive at 48 h with addition of vehicle to the primary spinal cord culture. The present results show that excessive amount of glutamate enhances DNA fragmentation in developing large motor neuron of cultured spinal cord by involving in apoptotic process of the neurons. [Neurol Res 2001; 23: 79-82]

Rescue of ischemic brain injury by adenoviral gene transfer of glial cell line-derived neurotrophic factor after transient global ischemia in gerbils

Glial cell line-derived neurotrophic factor (GDNF), a member of the transforming growth factor (TGF)–β superfamily, is one of the most potent neurotrophic factors and promotes survival of many populations of cells. We examined neuroprotective effect of an adenoviral vector encoding glial cell line-derived neurotrophic factor (AxCAhGDNF) on the transient global ischemia. Gerbils received administration of AxCAhGDNF or an adenoviral vector encoding bacterial β-galactosidase gene (AxCALacZ) through the lateral ventricle. Two days later, occluding bilateral common carotid arteries for 5 min using aneurysm clips produced the transient global forebrain ischemia. Animals showed intense immunolabeling for GDNF in ependymal cells on 2, 4 and 7 days after the operation. The exogenous gene transducted by adenovirus in the same cells was detected by in situ hybridization. The treatment with AxCAhGDNF significantly prevented the loss of hippocampal CA1 pyramidal neurons 2 to 7 days after the operation, as compared to AxCALacZ treatment. Also terminal deoxynucleotidyl transferase-mediated dUTP-biotin in situ nick end labeling (TUNEL) staining was markedly reduced in the case with AxCAhGDNF treatment at 7 days after the operation. These results indicated that the adenovirus-mediated gene transfer of GDNF might prevent the delayed neuronal death of stroke and other disorders of the cerebral vasculature.

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.

Ameliorative effect of glial cell line-derived neurotrophic factor on brain edema formation after permanent middle cerebral artery occlusion in rats

Glial cell line-derived neurotrophic factor (GDNF) was applied topically on the brain surface immediately after permanent middle cerebral artery (MCA) occlusion in rats. In contrast to the cases treated with vehicle, a formation of brain edema was significantly reduced at one day by the treatment with GDNF. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin in situ nick end labeling (TUNEL) staining was markedly reduced in the cases with GDNF treatment at 12 h after MCA occlusion. However, the induction of immunoreactive 70-kd heat shock protein (HSP70) was slightly ameliorated by the GDNF treatment. The present results suggest that the treatment with GDNF has a significant effect on ameliorating brain edema formation after continuous brain ischemia, and the effect is greatly associated with the reduction of apoptotic changes! but slightly with that of stress response of cells. [Neural Res 1998; 20: 333-336]

Apoptosis of motor neurons with induction of caspases in the spinal cord after ischemia.

Some neuronal subpopulations are especially vulnerable to ischemic injury. In the spinal cord, large motor neurons are vulnerable to ischemia and are selectively lost after transient ischemia. However, the mechanisms of the neuronal loss have been uncertain. We hypothesized that spinal motor neurons might be lost by apoptosis and investigated a possible mechanism of neuronal death by detection of double-strand breaks in genomic DNA and immunohistochemical analysis for caspases, ie, interleukin-1beta converting enzyme (ICE), Nedd-2, and CPP32. We used a rabbit spinal cord ischemia model created with a balloon catheter. The spinal cord was removed at 8 hours, 1, 2, or 7 days after 15 minutes of transient ischemia, and histological changes were studied with hematoxylin-eosin staining. To detect double-strand breaks in DNA, a staining with terminal deoxynucleotidyl transferase-mediated dUTP-biotin in situ nick end labeling (TUNEL) was performed. Furthermore, expression of ICE, Nedd-2, and CPP32 was investigated by Western blotting and immunohistochemical analysis. Motor neurons were selectively lost at 7 days after transient ischemia. TUNEL study demonstrated that no cells were positively labeled until 1 day after ischemia, but nuclei of some motor neurons were positively labeled at 2 days. Western blot analysis revealed no immunoreactivity for ICE and slight immunoreactivities for Nedd-2 and CPP32 in the sham-operated spinal cords. However, immunoreactivity became apparent at 8 hours after transient ischemia, decreased at 1 day, and returned to baseline level at 2 days. Immunohistochemical analysis demonstrated that motor neurons were responsible for induction of those caspases. Double-strand breaks in genomic DNA and induction of three caspases were demonstrated. These results indicate that motor neuron death in the spinal cord after transient ischemia is profoundly associated with activation of apoptotic processes.