Activation of cell death pathway after a brief period of global ischemia in diabetic and non-diabetic animals

Mitochondria play a critical role in the pathogenesis of cerebral ischemia. Acute hyperglycemia has been shown to activate the mitochondria-initiated cell death pathway after an intermediate period of ischemia. The objective of the present study was to determine if diabetic hyperglycemia induced by streptozotocin activates the cell death pathway after a brief period of global ischemia. Five minutes of global ischemia was induced in nondiabetic and diabetic rats. Brain samples were collected after 30 min, 6 h, 1, 3, and 7 days of recirculation as well as from sham-operated controls. Histopathological examination in the hippocampal CA1, CA3, hilus, and dentate gyrus regions, as well as in the cortical and thalamic areas, showed that neuronal death in diabetic animals increased compared to nondiabetic ischemic controls. Neuronal damage maturation occurred after 7 days of recovery in nondiabetic rats, while it was shortened to 3 days of recovery in diabetic animals. Western blot analyses revealed that release of cytochrome c markedly increased after 1 and 3 days of reperfusion in diabetic rats. Caspase-3 activation was evident in the nuclear fraction of the cortex of diabetic rats after 3 days recovery and it was preceded by activation of caspase-9, but not activation of caspase-8. Electron microscopy demonstrated that chromatin condensation and mitochondrial swelling were features of the diabetes-mediated ischemic neuronal damage. However, no apoptotic bodies were observed in any sections examined. These results suggest that a brief period of global ischemia in diabetic animals activates a neuronal cell death pathway involving cytochrome c release, caspase-9 activation, and caspase-3 cleavage, all of which are most likely initiated by early mitochondria damage.     

Caspase-activated DNase/DNA fragmentation factor 40 mediates apoptotic DNA fragmentation in transient cerebral ischemia and in neuronal cultures

Nuclear changes, including internucleosomal DNA fragmentation, are characteristic features of neuronal apoptosis resulting from transient cerebral ischemia and related brain insults for which the molecular mechanism has not been elucidated. Recent studies suggest that a caspase-3-mediated mechanism may be involved in the process of nuclear degradation in ischemic neurons. In this study, we cloned from rat brain a homolog cDNA encoding caspase-activated deoxyribonuclease (CAD)/DNA fragmentation factor 40 (DFF40), a 40 kDa nuclear enzyme that is activated by caspase-3 and promotes apoptotic DNA degradation. Subsequently, we investigated the role of CAD/DFF40 in the induction of internucleosomal DNA fragmentation in the hippocampus in a rat model of transient global ischemia and in primary neuronal cultures under ischemia-like conditions. At 8-72 hr after ischemia, CAD/DFF40 mRNA and protein were induced in the degenerating hippocampal CA1 neurons. CAD/DFF40 formed a heterodimeric complex in the nucleus with its natural inhibitor CAD (ICAD) and was activated after ischemia in a delayed manner (>24 hr) by caspase-3, which translocated into the nucleus and cleaved ICAD. Furthermore, an induced CAD/DFF40 activity was detected in nuclear extracts in both in vivo and in vitro models, and the DNA degradation activity of CAD/DFF40 was inhibited by purified ICAD protein. These results strongly suggest that CAD/DFF40 is the endogenous endonuclease that mediates caspase-3-dependent internucleosomal DNA degradation and related nuclear alterations in ischemic neurons.     

Distinct spatial and temporal activation of caspase pathways in neurons and glial cells after excitotoxic damage to the immature rat brain

Although cleaved caspase-3 is known to be involved in apoptotic cell death mechanisms in neurons, it can also be involved in a nonapoptotic role in astrocytes after postnatal excitotoxic injury. Here we evaluate participation of upstream pathways activating caspase-3 in neurons and glial cells, by studying the intrinsic pathway via caspase-9, the extrinsic pathway via caspase-8, and activation of the p53-dependent pathway. N-methyl-D-aspartate (NMDA) was injected intracortically in 9-day-old postnatal rats, which were sacrificed at several survival times between 4 hr postlesion (pl) and 7 days pl. We analyzed temporal and spatial expression of caspase-8, caspase-9, and p53 and correlation with neuronal and glial markers and caspase-3 activation. Caspase-9 was significantly activated at 10 hpl, strongly correlating with caspase-3. It was present mainly in damaged cortical and hippocampal neurons but was also seen in astrocytes and oligodendrocytes in layer VI and corpus callosum (cc). Caspase-8 showed a diminished correlation with caspase-3. It was present in cortical neurons at 10-72 hpl, showing layer specificity, and also in astroglial and microglial nuclei, mainly in layer VI and cc. p53 Expression increased at 10-72 hpl but did not correlate with caspase-3. p53 Was seen in neurons of the degenerating cortex and in some astrocytes and microglial cells of layer VI and cc. In conclusion, after neonatal excitotoxicity, mainly the mitochondrial intrinsic pathway mediates neuronal caspase-3 and cell death. In astrocytes, caspase-3 is not widely correlated with caspase-8, caspase-9, or p53, except in layer VI-cc astrocytes, where activation of upstream cascades occurs.     

The neuroprotective action of SP600125, a new inhibitor of JNK, on transient brain ischemia/reperfusion-induced neuronal death in rat hippocampal CA1 via nuclear and non-nuclear pathways

Increasing evidence suggests that c-Jun N-terminal kinase (JNK) is an important kinase mediating neuronal apoptosis in brain ischemia. To further study the roles of JNK activation in hippocampal CA1 neurons in a rat model of transient global ischemia, we assessed the effect of JNK inhibition by SP600125 on the degree of brain injury. Our results demonstrated that SP600125 significantly increased the number of surviving cells in hippocampal CA1 subfield and decreased the activation of p-JNK1/2 and p-JNK3 at 30 min and 3 days after brain ischemia. Moreover, SP600125 significantly diminished the increased levels of phosphorylated-c-Jun (Ser63/73) and phosphorylated-Bcl-2 (Ser87) at 3 h after brain ischemia. These results indicate that SP600125, a new inhibitor of JNK, protected transient brain ischemia/reperfusion-induced neuronal death in rat hippocampal CA1 region at least via suppressing the activation of nuclear substrate (c-Jun) and inactivating non-nuclear substrate (Bcl-2) induced by ischemic insult. Thus, inhibiting JNK activity by SP600125 may represent a new and effective strategy to treat ischemic stoke.     

Inhibition of caspase-9 activation and apoptosis is involved in ischemic preconditioning-induced neuroprotection in rat brain

OBJECTIVES: Cerebral ischemic pre-conditioning (IPC) is capable of protecting hippocampal neurons from ischemia/reperfusion (I/R) injury. In the current study, we investigated the role of activated caspase-9 in the protective process induced by IPC and related it to cytochrome c release and apoptosis. METHODS: I/R injury was induced by a four-vessel occlusion model in Wistar rats which were randomly divided into ischemia/reperfusion group (I/R), ischemic pre-conditioning + I/R group (IPC + I/R) and control group. Histologic changes in the pyramidal layer of the hippocampal CA1 region were determined by hematoxylin and eosin (H&E) staining. The relative proportion of apoptotic neurons in this area was assessed with TUNEL staining. The redistribution of cytochrome c and activation of caspase-9 were detected in the same area with immunohistochemistry and Western blotting respectively. RESULTS: Compared to the I/R group, IPC increased the number of surviving neurons in the hippocampal CA1 region (p<0.001), markedly reduced the number of apoptotic pyramidal neurons (p<0.001), inhibited the release of cytochrome c from mitochondria to cytoplasm (p<0.001 for positively stained neurons) and decreased the amount of activated caspase-9 (p<0.001). DISCUSSION: These findings confirm that IPC is capable of protecting neurons from injury by apoptosis. The release of cytochrome c to the cytosol demonstrates that the mitochondrial pathway was involved, and the reduction in this release caused by IPC was clearly associated with reduced caspase-9 activation. Together, these results suggest that IPC protects neurons via action on the mitochondrial/caspase-9 pathway of apoptosis.     

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.     

Biphasic cytochrome c release after transient global ischemia and its inhibition by hypothermia.

Hypothermia is effective in preventing ischemic damage. A caspase-dependent apoptotic pathway is involved in ischemic damage, but how hypothermia inhibits this pathway after global cerebral ischemia has not been well explored. It was determined whether hypothermia protects the brain by altering cytochrome c release and caspase activity. Cerebral ischemia was produced by two-vessel occlusion plus hypotension for 10 mins. Body temperature in hypothermic animals was reduced to 33 degrees C before ischemia onset and maintained for 3 h after reperfusion. Western blots of subcellular fractions revealed biphasic cytosolic cytochrome c release, with an initial peak at about 5 h after ischemia, which decreased at 12 to 24 h, and a second, larger peak at 48 h. Caspase-3 and -9 activity increased at 12 and 24 h. A caspase inhibitor, Z-DEVD-FMK, administered 5 and 24 h after ischemia onset, protected hippocampal CA1 neurons from injury and blocked the second cytochrome c peak, suggesting that caspases mediate this second phase. Hypothermia (33 degrees C), which prevented CA1 injury, did not inhibit cytochrome c release at 5 h, but reduced cytochrome c release at 48 h. Caspase-3 and -9 activity was markedly attenuated by hypothermia at 12 and 24 h. Thus, biphasic cytochrome c release occurs after transient global ischemia and mild hypothermia protects against ischemic damage by blocking the second phase of cytochrome c release, possibly by blocking caspase activity.     

Temporal assessment of caspase activation in experimental models of focal and global ischemia

Rodent models of focal and global ischemia were used to examine caspase activation. Several readouts were employed on identical tissue to provide correlative measurement of caspase induction, activation and enzymatic activity. In a rat focal ischemia model, caspase-3 enzymatic activity, as recorded by DEVD-AMC cleavage, peaked in penumbral cortex at 6-12 h following ischemia, correlating with increases in caspase 3-cleaved substrates of PARP and alpha-spectrin and subsequent disappearance of caspase-3 zymogen. Although induction of caspases 8 and 2 proteins was detectable as early as 6 h following ischemia, examination of the same tissues for caspase 8 or 2 enzymatic activities did not show significant modulation up to 12 h after ischemic insult. Caspase 9 induction was evident only after 24 h postischemia and did not correlate with elevated LDHD-AMC cleavage. Following global ischemia in gerbils, levels of caspase-3 enzyme activity peaked at 12 h in hippocampal tissue extracts. Cleaved caspase-3 signal was prominent in NeuN-positive layers in the CA1 region 6-12 h following ischemia. Interestingly, strong caspase-3 immunoreactivity was also detected in the subgranular zone of the dentate gyrus, a known region of ischemia-induced neurogenesis. Caspase-3 activation may be responsible for the loss of these cells, thereby hindering the endogenous recovery process.     

Atypical role of proximal caspase-8 in truncated Tau-induced neurite regression and neuronal cell death

Abnormal Tau protein is known to be closely associated with several neurodegenerative diseases. Previously, we showed that Tau was cleaved by caspase-3 to generate the cleavage product lacking the C-terminus (DeltaTau-1) during neuronal cell death. Here we characterized caspase-8-dependent neurotoxicity of the truncated Tau. Introduction of DeltaTau-1 into primary hippocampal neurons induced loss of neurites in a caspase-dependent manner. Caspase-8 and -6 were proteolytically activated during DeltaTau-1-triggered neuronal cell death, which was suppressed by IETD-fmk, caspase-8 inhibitor. Direct targeting of caspase-8 and its associated FADD with antisense approaches and transient expression of their dominant-negative mutants reduced DeltaTau-1-induced apopotosis. Cells deficient in caspase-8, but not caspase-3, became sensitized to DeltaTau-1-mediated toxicity upon reconstitution with caspase-8. In addition, ectopic expression of mitochondrial antiapoptotic Bcl-2, Bcl-X(L), or inactive caspase-9 short form suppressed DeltaTau-1 toxicity. These results suggest that the truncated Tau protein activates proximal caspase-8 through FADD as a necessary step leading to neuronal cell death and neurite regression, contributing to the progression of abnormal Tau-associated neurodegeneracy.     

In situ immunodetection of neuronal caspase-3 activation in Alzheimer disease.

The mechanism by which cells die in Alzheimer disease (AD) is unknown. Several investigators speculate that much of the cell loss may be due to apoptosis, a highly regulated form of programmed cell death. Caspase-3 is a critical effector of neuronal apoptosis and may be inappropriately activated in AD. To address this possibility, we examined cortical and hippocampal brain sections from AD patients, as well as 2 animal models of AD, for in situ evidence of caspase-3 activation. We report here that senile plaques and neurofibrillary tangles in the AD brain are not associated with caspase-3 activation. Furthermore, amyloid beta (A beta) deposition in the APPsw transgenic mouse model of AD does not result in caspase-3 activation despite the ability of A beta to induce caspase-3 activation and neuronal apoptosis in vitro. AD brain sections do, however, exhibit caspase-3 activation in hippocampal neurons undergoing granulovacuolar degeneration. Our data suggests that caspase-3 does not have a significant role in the widespread neuronal cell death that occurs in AD, but may contribute to the specific loss of hippocampal neurons involved in learning and memory.     

Translocation of apoptosis-inducing factor in vulnerable neurons after transient cerebral ischemia and in neuronal cultures after oxygen-glucose deprivation.

Loss of mitochondrial membrane integrity and the resulting release of apoptogenic factors may play a critical role in mediating hippocampal neurodegeneration after transient global ischemia. In the present study, the authors have cloned and characterized the rat cDNA encoding apoptosis-inducing factor (AIF), an intramitochondrial protein that promotes cell death in a caspase-independent manner upon release into nonmitochondrial compartments. In contrast to the expression patterns of a number of apoptosis-regulatory gene products during brain development, the expression of AIF protein increases gradually with brain maturation and peaks in adulthood. In a rat model of transient global ischemia, AIF was found to translocate from mitochondria to the nucleus in the hippocampal CA1 neurons after ischemia and to manifest a DNA-degrading activity that mimicked the purified AIF protein and was inhibitable by AIF immunodepletion. The temporal profile of AIF translocation after ischemia (24 to 72 hours) coincided with the induction of large-scale DNA fragmentation at the size of 50 kbp, a well-characterized hallmark of AIF-like activity but preceded the formation of internucleosomal DNA fragmentation (72 hours), a DNA degradation associated with the terminal stage of cell death. Further, the nuclear translocation of AIF after ischemia was not blocked by inhibiting caspase-3/-7 activities, but, as shown in neuronal cultures that were challenged with transient oxygen-glucose deprivation, it can be prevented by intracellular delivery of the mitochondria-associated antiapoptotic protein Bcl-xL. The results presented here strongly suggest that mitochondrial release of AIF may be an important factor, in addition to the previously reported cytochrome c and Smac, which could contribute to the selective vulnerability of CA1 neurons to transient global ischemic injury.     

Activation of mitogen-activated protein kinases after transient forebrain ischemia in gerbil hippocampus.

We investigated the expression, activation, and distribution of c-Jun N-terminal kinases (JNKs), p38 mitogen-activated protein kinases (p38s) and extracellular signal-regulated kinases (ERKs) using Western blotting and immunohistochemistry in gerbil hippocampus after transient forebrain ischemia to clarify the role of these kinases in delayed neuronal death (DND) in the CA1 subfield. Immunoblot analysis demonstrated that activities of JNK, p38, and ERK in whole hippocampus were increased after 5 min of global ischemia. We used an immunohistochemical study to elucidate the temporal and spatial expression of these kinases after transient global ischemia. The immunohistochemical study showed that active JNK and p38 immunoreactivities were enhanced at 15 min of reperfusion and then gradually reduced and disappeared in the hippocampal CA1 region. On the other hand, in CA3 neurons, active JNK and p38 immunoreactivities were enhanced at 15 min of reperfusion and peaked at 6 hr of reperfusion and then gradually reduced but was continuously detected 72 hr after ischemia. Active ERK immunoreactivity was observed transiently in CA3 fibers and dentate gyrus. Pretreatment with SB203580, a p38 inhibitor, but not with PD98059, an ERK kinase 1/2 inhibitor, reduced ischemic cell death in the CA1 region after transient global ischemia by inhibiting the activity of p38. These findings indicate that the p38 pathway may play an important role in DND during brain ischemia in gerbil. Components of the pathway are important target molecules for clarifying the mechanism of neuronal death.     

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.     

Protective effects of peroxisome proliferator‐activated receptors γ coactivator‐1α against neuronal cell death in the hippocampal CA1 subfield after transient global ischemia

Peroxisome proliferator‐activated receptors γ coactivator‐1α (PGC‐1α) may regulate the mitochondrial antioxidant defense system under many neuropathological settings. However, the exact role of PGC‐1α in ischemic brain damage is still under debate. Based on an experimental model of transient global ischemia (TGI), this study evaluated the hypothesis that the activation of PGC‐1α signaling pathway protects hippocampal CA1 neurons against delayed neuronal death after TGI. In Sprague‐Dawley rats, significantly increased content of oxidized proteins in the hippocampal CA1 tissue was observed as early as 30 min after TGI, followed by augmentation of PGC‐1α expression at 1 hr. Expression of uncoupling protein 2 (UCP2) and superoxide dismutases 2 (SOD2) in the hippocampal CA1 neurons was upregulated 4–48 hr after TGI. In addition, knock‐down of PGC‐1α expression by pretreatment with a specific antisense oligodeoxynucleotide in the hippocampal CA1 subfield downregulated the expression of UCP2 and SOD2 with resultant exacerbation of oxidative stress and augmentation of delayed neuronal cell death in the hippocampus after TGI. Overall, our results indicate that PGC‐1α is induced by cerebral ischemia leading to upregulation of UCP2 and SOD2, thereby providing a neuroprotective effect against ischemic brain injury in the hippocampus by ameliorating oxidative stress. © 2009 Wiley‐Liss, Inc.     

Intracellular Bax translocation after transient cerebral ischemia: implications for a role of the mitochondrial apoptotic signaling pathway in ischemic neuronal death.

Activation of terminal caspases such as caspase-3 plays an important role in the execution of neuronal cell death after transient cerebral ischemia. Although the precise mechanism by which terminal caspases are activated in ischemic neurons remains elusive, recent studies have postulated that the mitochondrial cell death-signaling pathway may participate in this process. The bcl-2 family member protein Bax is a potent proapoptotic molecule that, on translocation from cytosol to mitochondria, triggers the activation of terminal caspases by increasing mitochondrial membrane permeability and resulting in the release of apoptosis-promoting factors, including cytochrome c. In the present study, the role of intracellular Bax translocation in ischemic brain injury was investigated in a rat model of transient focal ischemia (30 minutes) and reperfusion (1 to 72 hours). Immunochemical studies revealed that transient ischemia induced a rapid translocation of Bax from cytosol to mitochondria in caudate neurons, with a temporal profile and regional distribution coinciding with the mitochondrial release of cytochrome c and caspase-9. Further, in postischemic caudate putamen in vivo and in isolated brain mitochondria in vitro, the authors found enhanced heterodimerization between Bax and the mitochondrial membrane permeabilization-related proteins adenine nucleotide translocator (ANT) and voltage-dependent anion channel. The ANT inhibitor bongkrekic acid prevented Bax and ANT interactions and inhibited Bax-triggered caspase-9 release from isolated brain mitochondria in vitro. Bongkrekic acid also offered significant neuroprotection against ischemia-induced caspase-3 and caspase-9 activation and cell death in the brain. These results strongly suggest that the Bax-mediated mitochondrial apoptotic signaling pathway may play an important role in ischemic neuronal injury.     

Aging is neuroprotective during global ischemia but leads to increased caspase-3 and apoptotic activity in hippocampal neurons

Previously, we found a significantly greater number of surviving CA1 neurons to global ischemia in the aged (24-month-old) F344 rats than in young (4-month-old) rats. The present study tests the hypothesis that aging retards neuronal death in the hippocampal CA1 region following cerebral ischemia. The CA1 "living cell ratio" was significantly greater in aged than in young rats at three days (62+/-8% vs. 30+/-8%) and at eight days (36+/-6% vs. 17+/-5%), but not at 14 days (15+/-12% vs. 18+/-12%) following ischemia. The number of the CA1 cells exhibiting co-localized TdT-mediated X-dUTP nick end labeling reaction and caspase-3 active peptide (C3AP) immunoreactivity was greater in aged than young animals at three and eight days following ischemia (36+/-8/mm vs. 3+/-1/mm and 36+/-14 vs. 0+/-0, p<0.05 respectively). Also, the total number of C3AP-positive cells in the CA1 region in the aged group was significantly greater than in the young group at three and eight days post-ischemia (p<0.05). Aging appears to delay caspase-3-dependent apoptotic cell death induced by global ischemia in the CA1 region of the hippocampus, consistent with an age-induced neuroprotective process.     

亚低温对大鼠短暂全脑缺血后神经元凋亡的影响

目的 探讨亚低温对大鼠脑缺血后神经元凋亡的影响，揭示亚低温的部分神经保护机制。方法 采用“双侧颈总动脉阻断＋全身低血压”方法来建立大鼠短暂性全脑缺血模型。用神经元尼氏体亚甲兰特殊染色法观察大鼠脑缺血后海马CA1区神经元损害情况；原位细胞凋亡检测法（TUNEL染色）及电镜观察脑缺血后CA1区神经元凋亡情况。结果 与假手术组、低温缺血组相比，常温缺血组海马CA1区神经元缺失明显（P＜0．01）。常温及低温缺血组海马CA1区均存在神经元凋亡，但低温缺血组海马CA1区凋亡神经元数明显少于缺血组（P＜0．01）。结论 经“双侧颈总动脉阻断＋全身低血压”方法建立的大鼠短暂全脑缺血模型证实了亚低温的脑保护作用。全脑缺血后的迟发性神经元死亡很可能经由凋亡途径，而亚低温可通过抑制缺血性神经元凋亡而发挥一定的神经保护作用。     

Effects of berberine on hippocampal neuronal damage and matrix metalloproteinase-9 activity following transient global cerebral ischemia

Berberine, an isoquinoline alkaloid with a long history of use in Chinese medicine, has several important pharmacological effects. Several studies have revealed that berberine has neuroprotective and neuropsychiatric effects. However, there are few reports regarding the protective effect of berberine against neuronal damage following transient global cerebral ischemia. In this study, mice were subjected to 20 min of global brain ischemia and sacrificed 72 hr later. Berberine was administered for 7 days prior to ischemia and daily until sacrifice. Mice treated with berberine showed reduced matrix metalloproteinase-9 (MMP-9) activity. Berberine inhibited gelatinase activity directly in in situ zymography and reduced neuronal damage following global ischemia. Laminin expression and NeuN expression were markedly reduced in CA1 and CA2 areas after ischemia, and berberine reduced the laminin degradation and neuronal loss. In the TUNEL assay, damaged neurons were also apparent in the CA1 and CA2 areas, and berberine reduced TUNEL-positive cells. These data demonstrate that berberine, a plant alkaloid, may protect from hippocampal neuronal damage following transient global ischemia by reducing MMP-9 activity.     

Differential changes of bax, caspase-3 and p21 mRNA expression after transient focal brain ischemia in the rat

Recent studies of transient focal ischemia have focused interest on apoptotic mechanisms of neuronal cell death involving constitutive pro-apoptotic proteins. The finding of specific patterns of novel gene expression might indicate the activation of pro-apoptotic genes in previously ischemic areas. Thus, we investigated gene expression for the pro-apoptotic regulators, Bax and caspase-3, after transient focal brain ischemia, together with the p53-regulated cell cycle inhibitor, p21/WAF1/CIP1. Reversible occlusion of the middle cerebral artery for 2 h was carried out in halothane-anesthetized rats using the poly-L-lysine coated filament method. In situ hybridization was performed at 0, 1, 3, 6 h and 1, 3 and 7 d of recirculation and in sham controls. Radioactive antisense probes served for detection of bax, p21 and caspase-3 mRNAs on brain sections, and quantitative film autoradiography was combined with image-averaging techniques. Bax mRNA tended to decline after focal brain ischemia within 1 d. p21 mRNA was upregulated with a perifocal pattern at 3 h and 1 d after ischemia whereas the ischemic regions themselves failed to show significant upregulation. Caspase-3 mRNA was elevated in the resistant dorsomedial cortex at 1 d. A pro-apoptotic pattern of novel gene expression, involving Bax and caspase-3, was not observed after transient focal brain ischemia. Rather, the perifocal expression of p21 and caspase-3 mRNAs observed at 1 d after ischemia points to reactive changes in resistant brain areas.