Involvement of oxidative stress and caspase-3 in cortical infarction after photothrombotic ischemia in mice.

Apoptosis-related cell death is linked to oxidative stress and caspases in experimental cerebral ischemia. However, the role of oxidative stress in caspase activation and subsequent apoptotic cell death after cerebral ischemia is unknown. The authors evaluated the role of oxidative stress in ischemic cerebral infarction after photothrombosis and the relation between oxidative stress and caspase-related cell death 6 and 24 hours after ischemia with and without U-74389G, a potent free radical scavenger (10 mg/kg, 30 minutes before and after ischemia induction). Reactive oxygen species, detected by hydroethidine oxidation, and cytosolic cytochrome c were detected in early ischemic lesions. Western blot analysis showed the cleaved form and the increased level of the proform of caspase-3 in the ischemic lesion 24 hours after ischemia. Decreased caspase-3 immunoreactivity was detected in the antioxidant-treated group after ischemia. Decreased DNA fragmentation and laddering were detected and the lesion was smaller in the treated group after ischemia compared with the untreated group. Oxidative stress and cytochrome c release occur in the ischemic lesion after photothrombotic ischemia. The free radical scavenger attenuated caspase-3 up-regulation, DNA fragmentation, and the final lesion. The authors concluded that oxidative stress may mediate caspase-related apoptotic cell death and subsequent cortical infarction after photothrombotic ischemia.     

Both caspase-dependent and caspase-independent pathways may be involved in hippocampal CA1 neuronal death because of loss of cytochrome c From mitochondria in a rat forebrain ischemia model.

In a rat forebrain ischemia model, the authors examined whether loss of cytochrome c from mitochondria correlates with ischemic hippocampal CA1 neuronal death and how cytochrome c release may shape neuronal death. Forebrain ischemia was induced by bilateral common carotid artery occlusion with simultaneous hypotension for 10 minutes. After reperfusion, an early rapid depletion of mitochondrial cytochrome c and a late phase of diffuse redistribution of cytochrome c occurred in the hippocampal CA1 region, but not in the dentate gyrus and CA3 regions. Intracerebroventricular administration of Z-DEVD-FMK, a relatively selective caspase-3 inhibitor, provided limited but significant protection against ischemic neuronal damage on day 7 after reperfusion. Treatment with 3 minutes of ischemia (ischemic preconditioning) 48 hours before the 10-minute ischemia attenuated both the early and late phases of cytochrome c redistribution. In another subset of animals treated with cycloheximide, a general protein synthesis inhibitor, the late phase of cytochrome c redistribution was inhibited, whereas most hippocampal CA1 neurons never regained mitochondrial cytochrome c. Examination of neuronal survival revealed that ischemic preconditioning prevents, whereas cycloheximide only delays, ischemic hippocampal CA1 neuronal death. DNA fragmentation detected by terminal deoxytransferase-mediated dUTP-nick end labeling (TUNEL) in situ was largely attenuated by ischemic preconditioning and moderately reduced by cycloheximide. These results indicate that the loss of cytochrome c from mitochondria correlates with hippocampal CA1 neuronal death after transient cerebral ischemia in relation to both caspase-dependent and -independent pathways. The amount of mitochondrial cytochrome c regained may determine whether ischemic hippocampal CA1 neurons survive or succumb to late-phase death.     

Hypoxia induces caspase-9 and caspase-3 activation without neuronal death in gerbil brains

To investigate the in vivo apoptotic machinery in oxygen deprived brain, we examined the expression of caspase-9 and caspase-3 in the hippocampus of Mongolian gerbils subjected to either transient hypoxia (4% O2 for 6 min) or forebrain ischemia (10 min bilateral carotid artery occlusion) followed by 8 h to 7 days of reoxygenation or blood recirculation. Apoptotic death was characterized by isolating hippocampal genomic DNA and analysing DNA fragmentation as well as histological studies including TUNEL assay and toluidine blue staining of brain sections. The results showed that both hypoxic and ischemic gerbil brains exhibited an increase in caspase-9 and caspase-3 gene expression. However, no cell damage was detectable following hypoxia, while marked DNA fragmentation and extensive cell death was observed following ischemia. Moreover, although hypoxia did not lead to cell death, both hypoxia and ischemia were associated with cleavage of procaspase-9 and procaspase-3 and increases in their activities as well as cleavage of poly(ADP-ribose) polymerase-1 (PARP-1), a major caspase-3 substrate. These results indicate that, in vivo, even late apoptotic events such as caspase activation and PARP-1 cleavage in hypoxic brains do not necessarily induce an irreversible commitment to apoptotic neuronal death.     

Caspase-3 activation and DNA fragmentation in primary hippocampal neurons following glutamate excitotoxicity

Excitotoxic glutamate CNS stimulation can result in neuronal cell death. Contributing mechanisms and markers of cell death are the activation of caspase-3 and DNA fragmentation. It remains to be resolved to which extent both cellular reactions overlap and/or indicate different processes of neurodegeneration. In this study, mixed neuronal cultures from newborn mice pubs (0-24 h) were stimulated with glutamate, and the co-localization of active caspase-3 and DNA fragmentation was investigated by immunocytochemistry and the TUNEL nick-end labelling. In untreated cultures, 8% scattered neurons (marked by MAP-2) displayed activated caspase-3 at different morphological stages of degeneration. TUNEL staining was detected in 5% of cell nuclei including GFAP-positive astrocytes. However, co-localization of active caspase-3 with TUNEL was less than 2%. After glutamate stimulation (125 microM), the majority of neurons was dying between 12 and 24 h. The absolute number of active caspase-3 neurons increased only moderately but in relation of surviving neurons after 24 h from 8 to 36% (125 microM), to 53% (250 microM) or to 32% (500 microM). TUNEL staining also increased after 24 h following glutamate treatment to 37% but the co-localization with active caspase-3 remained at the basal low level of 2%. In our system, glutamate-mediated excitotoxicity effects the DNA fragmentation and caspase-3 activation. Co-localization of both parameters, however, is very poor. Active caspase-3 in the absence of TUNEL indicates a dynamic degenerative process, whereas TUNEL marks the end stage of severe irreversible cell damage regardless to the origin of the cell.     

Neuroprotective effect of diazoxide on brain injury induced by cerebral ischemia/reperfusion during deep hypothermia

OBJECT: The purpose of this study was to determine the effects of diazoxide on apoptosis and the relative mechanisms in a model of brain injury induced by cerebral ischemia/reperfusion (I/R) during deep hypothermia. METHODS: Three-week-old Sprague-Dawley male rats were randomly and equitably divided into sham-operated group, placebo-treated group and diazoxide-treated group respectively. Specific examination of the regional cerebral blood flow (rCBF) was measured in the three groups continuously during the operation by laser Doppler flowmetry. Terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) was showed DNA fragmentation. The mRNA expressions of cytochrome c and full-length caspase-3 were determined by RT-PCR, while the protein expressions of cytochrome c and cleaved caspase-3 were determined by immunohistochemistry at 1 h, 6 h, 24 h, 72 h and 7 days after I/R, respectively. Cytosolic release of cytochrome c at 24 h after I/R was also confirmed by Western blot. RESULTS: rCBF was significantly decreased in both of placebo-treated and diazoxide-treated group just after ischemia in the time interval 0-5 min, and had no obvious changes in all the time intervals during the operation. Diazoxide preconditioning significantly decreased the percentage of TUNEL-positive staining cells. The mRNA expressions of cytochrome c and full-length caspase-3 in diazoxide-treated group were significantly decreased. In addition, diazoxide provided a significant reduction in the protein expressions of cytochrome c and cleaved caspase-3. CONCLUSION: These results suggested that the neuroprotective effects of diazoxide against cerebral I/R injury during deep hypothermia correlated with the reduction of DNA fragmentation, prevention of mitochondrial cytochrome c release and inhibition of caspase-3 activation.     

Automated analysis of global ischemia-induced CA1 neuronal death using terminal UTP nick end labeling (TUNEL)

In the brain, DNA fragmentation is associated with apoptotic cell death following ischemic/excitotoxic damage. Fragmented DNA can be detected in situ by labeling the 3'OH termini of the internucleosomal generated fragments with deoxynucleotides, through a process known as terminal deoxynucleotidyl transferase (TdT) dUTP nick end labeling, or TUNEL. TUNEL is frequently being used to assess neuronal death following cerebral ischemia in a number of animal models. However, conventional techniques for TUNEL can be time consuming, and are often subjective and thus can lead to inconsistencies among investigators. Moreover, the lack of tools for its quantification and standardization limits the use of this technique in assessing the magnitude of cell death. In the present report, we describe an improved higher throughput technique for TUNEL staining at room temperature on a BioGenex automated stainer, and its subsequent quantitative analysis using NORTHERN ECLIPSE, an imaging analysis program. Its implementation allows us to effectively quantify TUNEL positive cells in the CA1 region of the hippocampus following global forebrain ischemia in rats. We conclude that this general histological technique can be applied to the study of cell death in numerous other experimental models.     

Evidence of phosphorylation of Akt and neuronal survival after transient focal cerebral ischemia in mice.

The serine-threonine kinase, Akt, prevents apoptosis by phosphorylation at serine-473 in several cell systems. After phosphorylation, activated Akt inactivates other apoptogenic factors, such as Bad or caspase-9, thereby inhibiting cell death. The present study examined phosphorylation of Akt at serine-473 and DNA fragmentation after transient focal cerebral ischemia in mice subjected to 60 minutes of focal cerebral ischemia by intraluminal blockade of the middle cerebral artery. Phospho-Akt was analyzed by immunohistochemistry and Western blot analysis. The DNA fragmentation was evaluated by terminal deoxynucleotidyl transferase-mediated uridine 5-triphosphate-biotin nick end-labeling (TUNEL). Immunohistochemistry showed the expression of phospho-Akt was markedly increased in the middle cerebral artery territory cortex at 4 hours of reperfusion compared with the control, whereas it was decreased by 24 hours. Western blot analysis showed a significant increase of phospho-Akt 4 hours after focal cerebral ischemia in the cortex, whereas phospho-Akt was decreased in the ischemic core. Double staining with phospho-Akt and TUNEL showed different cellular distributions of phospho-Akt and TUNEL-positive staining. Phosphorylation of Akt was prevented after focal cerebral ischemia by LY294002, a phosphatidylinositol 3-kinase inhibitor, which facilitated subsequent DNA fragmentation. These results suggest that phosphorylation of Akt may be involved in determining cell survival or cell death after transient focal cerebral ischemia.     

Hypoxia-induced apoptotic cell death is prevented by oestradiol via oestrogen receptors in the developing central nervous system

The neuroprotective effects of oestrogens have been demonstrated against a variety of insults, including excitotoxicity, oxidative stress and cerebral ischemia under certain conditions. However, the molecular mechanisms underlying oestrogen neuroprotection are still unclear. We aimed to determine whether 17beta-oestradiol (E(2)) administration post-hypoxia (p-hx) was neuroprotective and whether these actions were mediated through oestrogen receptors (ER). For this purpose, 12-embyonic day-old chickens were subjected to acute hypoxia [8% (O(2)), 60 min], followed by different reoxygenation periods. To test the neuroprotective effect of E(2) and its mechanism, embryos were injected 30 min after the end of hypoxia with E(2) alone or with ICI 182 780, a competitive antagonist of ER. Cytochrome c (cyt c) release, an indicator of mitochondrial apoptotic pathway, was measured by western blot in optic lobe cytosolic extracts. DNA fragmentation by TUNEL fluorescence and caspase-3 fragmentation by immunofluorescence were detected on optic lobe sections. Acute hypoxia produces a significant increase in cyt c release from mitochondria at 4 h p-hx, followed by an increase in TUNEL positive cells 2 h later (6 h p-hx). Administration of E(2) (0.5 mg/egg) produced a significant decrease in cytosolic cyt c levels at 4 h p-hx, in caspase-3 activation and in TUNEL positive cells at 6 h p-hx compared to vehicle treated embryos. In the E(2)-ICI 182 780 treated embryos, cyt c release, caspase-3 fragmentation and TUNEL positive cells were similar to the hypoxic embryos, thus suggesting the requirement of an E(2)-ER interaction for E(2) mediated neuroprotective effects. In conclusion, E(2) prevents hypoxia-induced cyt c release and posterior cell death and these effects are mediated by oestrogen receptors.     

EGP-314 is expressed differentially in three brain zones at an early time in an experimentally induced ischemia rat model

Gene expression in frontal, occipital, and hippocampal regions of rat brains at 15 min of ischemic injury was studied in a rat model by producing focal cerebral ischemia through middle cerebral artery (MCA) occlusion without reperfusion. Catalase, epithelial glycoprotein (EGP-314), cytochrome C oxidase-subunit 1, ribosomal L31 protein, and ceruloplasmin were found to be differentially expressed. Specific primers were designed to study this newly reported brain EGP-314, a cellular adhesion molecule involved in cell-cell and cell-extracellular matrix interactions and related with cytoskeletal organization, differentiation, and proliferation. In the frontal and occipital lobes, EGP-314 expression was low in control and ischemic conditions and increased in sham injured conditions, whereas in the hippocampal region its expression was induced only by ischemia. In situ hybridization and immunohistochemistry revealed that EGP-314 mRNA and the protein were present in the ischemic hippocampus pyramidal neurons. DNA fragmentation was demonstrated by TUNEL and LM-PCR analysis in hippocampus region. TUNEL positive pyramidal neurons were observed at 15 min of ischemia. DNA ladder was found at 12 and 15 min of ischemia.     

Activation of caspase-9 and -3 during H2O2-induced apoptosis of PC12 cells independent of ceramide formation

The treatment of PC12 cells with H2O2 (100-500 microM) resulted in typical apoptotic changes including fragmentation and condensation of nuclei, and DNA fragmentation observed as DNA ladder. H2O2-induced apoptosis was associated with activation of caspase-3 as assessed by cleavage of specific fluorogenic substrate peptide and processing of procaspase-3 and poly(ADP-ribose) polymerase. However, formation of ceramide, which often locates upstream of caspase-3, was not observed. The inhibitory peptide relatively specific for caspase-3, z-DEVD-FMK and non-selective caspase inhibitor z-VAD-FMK inhibited activation of caspase-3 and apoptotic cell death. However, the relatively specific inhibitors, Ac-YVKD for caspase-1 and Ac-IETD for caspase-8/6, did not affect the occurrence of apoptotic cell death. As an upstream activation of caspase-3, induction of cytochrome c release followed by processing of procaspase-9 was observed by Western blotting, although the formation of intracellular ceramide was not observed. On the other hand, in PC12 cells overexpressing Bcl-2, the number of apoptotic cells was markedly decreased and activation of both caspases-9 and -3 was prevented. These results suggest that cytochrome c and caspase-9 initiate the activation of executor caspase-3 in H2O2-treated PC12 cells, and that Bcl-2 inhibits H2O2-induced release of cytochrome c from mitochondria and then proteolytic processing of procaspase-9.     

Augmentation of endoplasmic reticulum stress in cerebral ischemia/reperfusion injury associated with comorbid type 2 diabetes

Abstract

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Objective: Diabetes is one of the major risk factors for ischemic stroke and is reported to aggravate the ischemic brain damage in different experimental models as well as clinical situations. However, the mechanisms underlying the exacerbated ischemia/reperfusion (I/R) brain injury associated with comorbid diabetes are still not clear. This study investigated the role of endoplasmic reticulum (ER) stress in pathophysiology of aggravated I/R brain injury associated with diabetes.

Methods: Focal cerebral ischemia was induced by middle cerebral artery occlusion for 2 hours followed by 22 hours of reperfusion in high-fat diet-fed and low-dose streptozotocin-treated type 2 diabetic rats. Immunohistochemistry and western blotting analysis were performed to detect the changes in expression of various ER stress and apoptotic markers such as 78 kDa glucose-regulated protein (GRP78), CCAAT/enhancer binding protein homologous protein or growth arrest DNA damage-inducible gene 153 (CHOP/GADD153), and caspase-12. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay was performed to detect the extent of DNA fragmentation and cell death.

Results: The diabetic rats subjected to I/R manifested significantly larger brain infarct volume and severe deterioration in neurological deficits than their normal, non-diabetic counterparts. There was a marked upregulation of GRP78 observed in brains of diabetic rats after 22 hours of reperfusion. Furthermore, augmentation of CHOP/GADD153 expression and activation of caspase-12 (ER stress-induced apoptotic factors) were observed in parallel with enhanced TUNEL-positive cells or DNA fragmentation in diabetic rats compared to normal rats following cerebral I/R.

Discussion: Taken together, the current experimental findings demonstrate that diabetes exacerbates brain I/R injury which may be mediated through enhanced ER stress and cell death involving CHOP/GADD153 and caspase-12 activation.     

Inhibition of caspase-3-like activity reduces glutamate induced cell death in adult rat retina

Retinal cell death induced by over-stimulation of glutamate receptors is related to the programmed cell death or apoptosis. However, little is known about the intracellular events that lead to this cell death process in the retina. In this study, we asked if caspase-3 family cysteine proteases regulate cell death in an explant culture of adult rat retina after exposure to excessive glutamate. Cells with DNA fragmentation were first detected in the ganglion cell layer 3 h after a brief exposure to 20 mM glutamate; whilst those in the inner nuclear layer were first observed 6 h after the glutamate lesion. Caspase-3-like activity, as indicated by immunostaining of the fractin antibody that recognizes actin fragments generated by caspase-3 family proteases, was seen 40 min after glutamate treatment. Staining was first detected in the ganglion cell layer and then in the inner nuclear layer, preceding the appearance of cells with DNA fragmentation in these layers. Colocalization study showed that all cells with DNA breaks were fractin positive, indicating that caspase-3 family activity was involved in the glutamate-induced cell death in the adult rat retina. Furthermore, DEVD-CHO, a tetrapeptide inhibitor for caspase-3 family members, reduced dramatically the fractin staining and significantly alleviated glutamate-induced cell death and DNA fragmentation in the ganglion cell layer and inner nuclear layer. Inhibitor for caspase-1-like activity, YVAD-CHO, neither reduced the fractin staining nor showed comparable neuroprotective effects to the retina. We conclude that glutamate-induced apoptotic cell death in adult rat retina is mediated by a specific activation of cysteine proteases related to the caspase-3 family, and an intervention to the caspase-3 proteases provides effective protection to retinal neurons against glutamate excitotoxicity.     

大鼠局灶性脑缺血再灌流后Caspase-3激活与神经元凋亡的关系

目的探讨Caspase-3与局灶性脑缺血后神经元凋亡的关系。方法局灶性脑缺血再灌流大鼠模型,按再灌流时间不同随机分为5组。用半定量RT-PCR观察了脑缺血后不同时程Caspase-3mRNA表达及四肽荧光底物法检测Caspase-3蛋白酶活性;用TUNEL方法检测不同时程细胞凋亡。结果脑缺血2h再灌流2h组Caspase-3mRNA水干即已升高(P＜0.05)),蛋白酶活性轻度升高,再灌流6h后两者均明显升高(P＜0.05);脑缺血再灌流后不同时程细胞凋亡具有与Caspase-3相似的变化趋势。结论提示脑缺血后细胞凋亡的发生与Caspase-3的激活有关。     

Necrosis, apoptosis and hybrid death in the cortex and thalamus after barrel cortex ischemia in rats

Focal ischemia in the cerebral cortex results in acute and delayed cell death in the ischemic cortex and non-ischemic thalamus. We examined the hypothesis that neurons in ischemic and non-ischemic regions died from different mechanisms; specifically, we tested whether a mixed form of cell death containing both necrotic and apoptotic changes could be identified in individual cells.Focal barrel cortex ischemia in rats was induced by occlusion of small branches of the middle cerebral artery (MCA) corresponding to the barrel cortex, local blood flow was measured by quantitative autoradiography. Cell death was visualized by 2,3,5-triphenyltetrazolium chloride (TTC) staining, hematoxylin-eosin (H&E) staining, the terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), and caspase-3 staining 1 to 10 days after the ischemia. Electron microscopy was used for ultrastructural examination. Cell death occurred in the ipsilateral cortex 24 h after ischemia, followed by selective neuronal death in the ventrobasal (VB) thalamus 3 days later. TUNEL positive neurons were found in these two regions, but with striking morphological differences, designated as type I and type II TUNEL positive cells. The type I TUNEL positive cells in the ischemic cortex underwent necrotic changes. The type II TUNEL positive cells in the thalamus and the cortex penumbra region represented a hybrid death, featured by concurrent apoptotic and necrotic alterations in individual cells, including marked caspase-3 activation, nuclear condensation/fragmentation, but with swollen cytoplasm, damaged organelles and deteriorated membranes. Cell death in the thalamus and the cortex penumbra were attenuated by delayed administration of the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp fluoromethylketone (Z-VAD-FMK). Our data suggest that TUNEL staining should be evaluated with morphological changes, the hybrid death but not typical apoptosis occurs in the penumbra region and non-ischemic thalamus after cerebral ischemia.     

Inhibition of apoptosis by hyperbaric oxygen in a rat focal cerebral ischemic model.

The hypothesis was tested that hyperbaric oxygen therapy (HBO) reduced brain infarction by preventing apoptotic death in ischemic cortex in a rat model of focal cerebral ischemia. Male Sprague-Dawley rats were subjected to middle cerebral artery occlusion/reperfusion (MCAO/R) and subsequently were exposed to HBO (2.5 atmospheres absolute) for 2 h, at 6 h after reperfusion. Rats were killed and brain samples were collected at 24, 48, 72 h, and 7 days after reperfusion. Neurologic deficits, infarction area, and apoptotic changes were evaluated by clinical scores, 2,3,7-triphenyltetrazolium chloride staining, caspase-3 expression, DNA fragmentation assay, and terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate-biotin nick end labeling (TUNEL)-hematoxylin and eosin (H&E) costaining. In MCAO/R without HBO treatment animals, DNA fragmentation was observed in injured cortex at 24, 48, and 72 h but not in samples at 7 days after reperfusion. Double labeling of brain slides with NeuN and caspase-3 demonstrated neurons in the injured cortex labeled with caspase-3. TUNEL+H&E costaining revealed morphologic apoptotic changes at 24, 48, and 72 h after reperfusion. Hyperbaric oxygen therapy abolished DNA fragmentation and reduced the number of TUNEL-positive cells. Hyperbaric oxygen therapy reduced infarct area and improved neurologic scores at 7 days after reperfusion. One of the molecular mechanisms of HBO-induced brain protection is to prevent apoptosis, and this effect of HBO might preserve more brain tissues and promote neurologic functional recovery.     

Neuroprotective effect of tacrolimus (FK506) on ischemic brain damage following permanent focal cerebral ischemia in the rat

We investigated the neuroprotective effect of tacrolimus (FK506) on the ischemic cell death with respect to cytochrome c translocation and DNA fragmentation, which are pivotal events in the necrotic and apoptotic signaling pathway, using permanent focal cerebral ischemia in rats. Immunohistochemically, cytochrome c was observed in the cytoplasm as early as 1 h after middle cerebral artery (MCA) occlusion in the infarcted hemisphere. Cytosolic release of cytochrome c after MCA occlusion was also confirmed by Western blot analysis and enzyme immunoassay. Terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling (TUNEL) showed DNA fragmentation evolving in the ipsilateral cortex and the caudate putamen after 3 and 6 h, respectively, following MCA occlusion. Tacrolimus (1 mg/kg, i.v.), administered immediately after MCA occlusion, significantly attenuated the release of cytochrome c in the ischemic region, the number of TUNEL-positive cells in the ischemic penumbra zone, and the size of cortical ischemic lesions. This study demonstrated that tacrolimus ameliorated the accumulation of cytochrome c in the cytosol and the increase of TUNEL-positive cells induced by cerebral ischemia, indicating that the neuroprotective action of tacrolimus on ischemic brain injury caused by permanent focal cerebral ischemia could partially be attributed to the attenuation of the activation of the apoptotic execution machinery.     

NMDA and Kainate Induce Internucleosomal DNA Cleavage Associated With Both Apoptotic and Necrotic Cell Death in the Neonatal Rat Brain

Injection of N-methyl-D-aspartate (NMDA) or kainate in the striatum of 7-day-old rats induced massive cell loss in the ipsilateral striatum, hippocampus and inner cortical layers. In order to examine whether apoptosis contributes to cell death in this model of excitotoxic injury we examined the progression of internucleosomal DNA fragmentation and changes in cellular ultrastructure. Agarose gel electrophoresis of DNA extracted from the ipsilateral striatum, cerebral cortex and hippocampus clearly showed breakdown of DNA into oligonucleosome-sized fragments, indicative of apoptosis, 12 h post-NMDA injection. In addition, an increase between 12 and 24 h was observed as well as a continuous presence 5 days later. Kainate induced a similar time course of oligonucleosomal DNA fragmentation, but the intensity of the ethidium bromide stained bands was less compared with that observed for NMDA. DNA fragmentation was not detected in animals intrastriatally injected with Tris-HCl or in animals treated with MK-801 [(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohept-5,10-imine hydrogen maleate, 1 mg/kg] 30 min after NMDA injection. MK-801 had no effect on DNA fragmentation induced by kainate. In addition to agarose gel electrophoresis, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labelling (TUNEL) was used for detection of DNA fragmentation in sections. A gradual increase in the density of both apoptotic and non-apoptotic TUNEL nuclei was found in the anterior cingulate (ACC) and retrosplenial (RSC) areas of the cortex, the striatum, and the CA1 area and dentate gyrus of the hippocampus over the first 24 h post-NMDA or kainate injection. In the contralateral hemisphere hardly any TUNEL nuclei were present and their density was comparable with that in animals injected with vehicle only. In the ipsilateral mammillary nucleus (MN), which showed no signs of acute cell swelling after intrastriatal injection with NMDA, internucleosomal DNA fragmentation was found 24 and 48 h after intrastriatal NMDA injection. Here, the density of TUNEL cells with apoptotic morphology was high at 12 and 24 h post-NMDA injection but returned to control levels by 5 days. Electron microscopy showed cells with a clearly apoptotic morphology in the ACC and RSC and in the MN 24 h after NMDA injection. In the CA1 area of the hippocampus a necrotic, rather than an apoptotic, ultrastructure prevailed, indicating that the TUNEL method stained both apoptotic and necrotic cells. Based on biochemical and morphological criteria this study provides strong evidence that both apoptosis and necrosis are involved in NMDA- or kainate-induced excitotoxic cell death in the neonatal rat brain.     

Fas receptor and neuronal cell death after spinal cord ischemia.

Cell death from spinal cord injury is mediated in part by apoptotic mechanisms involving downstream caspases (e.g., caspase-3). Upstream mechanisms may involve other caspases such as procaspase-8, a 55 kDa apical caspase, which we found constitutively expressed within spinal cord neurons along with Fas. As early as 1.5 hr after transient ischemia, activated caspase-8 (p18) and caspase-8 mRNA appeared within neurons in intermediate gray matter and in medial ventral horn. We also detected evidence for an increase in death receptor complex by co-immunoprecipitation using Fas and anti-procaspase-8 after ischemia. At early time points, Fas and p18 were co-expressed within individual neurons, as were activated caspase-8 and caspase-3. Moreover, we detected p18 in cells before procaspase-3 cleavage product (p20), suggesting sequential activation. The appearance of cytosolic cytochrome c and gelsolin cleavage after ischemia was consistent with mitochondrial release and caspase-3 activation, respectively. Numerous terminal deoxynucleotidyl transferase-mediated DNA nick end-labeling-positive neurons contained p18 or p20 (65 and 80%, respectively), thereby supporting the idea that cells undergoing cell death contain both processed caspases. Our data are consistent with the idea that transient spinal cord ischemia induces the formation of a death-inducing signaling complex, which may participate in caspase-8 activation and sequential caspase-3 cleavage. Death receptors as well as downstream caspases may be useful therapeutic targets for limiting the death of cells in spinal cord.     

GDNF protects against aluminum-induced apoptosis in rabbits by upregulating Bcl-2 and Bcl-XL and inhibiting mitochondrial Bax translocation

Direct (intracisternal) injection of aluminum complexes into rabbit brain results in a number of similarities with the neuropathological and biochemical changes observed in Alzheimer's disease and provides the opportunity to assess early events in neurodegeneration. This mode of administration induces cytochrome c release from mitochondria, a decrease in Bcl-2 in both mitochondria and endoplasmic reticulum, Bax translocation into mitochondria, activation of caspase-3, and DNA fragmentation. Coadministration of glial cell neuronal-derived factor (GDNF) inhibits these Bcl-2 and Bax changes, upregulates Bcl-XL, and abolishes the caspase-3 activity. Furthermore, treatment with GDNF dramatically inhibits apoptosis, as assessed by the TUNEL technique for detecting DNA damage. Treatment with GDNF may represent a therapeutic strategy to reverse the neuronal death associated with Alzheimer's disease and may exert its effect on apoptosis-regulatory proteins.     

Temporal profile of cytochrome c and caspase-3 immunoreactivities and TUNEL staining after permanent middle cerebral artery occlusion in rats

Although apoptotic pathways play important roles in ischemic neuronal injury, exact mechanism of apoptotic enzyme cascade has not been fully studied. Immunohistochemical stainings for cytochrome c and caspase-3, and histochemical staining for a terminal deoxynucleotidyl-transferase (TdT)-mediated dUTP-biotin nick end-labeling method (TUNEL) were examined in a rat model of permanent middle cerebral artery (MCA) occlusion. Cytochrome c was strongly induced in neurons of the ischemic penumbra from 3 h after MCA occlusion, and caspase-3 began to be induced in the same area from 3 h with a peak at 8 h. Neuronal cells in MCA area became TUNEL positive at delayed time, reaching a peak at 24 h. Thus, the peak of induction of cytochrome c preceded that of caspase-3, and these two peaks were also precedence of the peak of DNA-fragmentation. Western blot analysis showed cytosolic expression of cytochrome c from mitochondria. This study demonstrated 1. Rapid release of cytochrome c from mitochondria to the cytosol, mainly in neurons of the cortex at 3 h after ischemia. 2. Subsequent peaks of caspase-3 and TUNEL in this order. These temporal profiles suggest a serial cascadic activation of apoptotic pathways in neuronal death after permanent MCA occlusion of rats.