大鼠脑缺血再灌流后神经细胞凋亡与caspase-3和caspase-9基因表达

目的：探讨大鼠局灶性脑缺血再灌流后神经细胞凋亡及其与caspase-3和caspase-9基因表达的关系。方法：应用原位末端标记和原位杂交技术分别观察细胞凋亡与caspase-3mRNA和caspase-9mRNA表达。结果：脑缺血再灌流后，凋亡神经细胞主要分布于缺血半影区，随着时间的延长凋亡细胞数逐渐增加，至24h达高峰。在缺血半影区，再灌流后神经细胞caspase-3mRNA和caspase-9mRNA表达逐渐增强，到24h阳性细胞数目最多，COD值最高，而缺血中心区两基因均弱表达。结论：脑缺血再灌流后神经细胞凋亡是一个动态的渐进过程。caspase-3和caspase-9基因表达在介导细胞凋亡过程中起重要作用。     

Tacrolimus (FK506) attenuates biphasic cytochrome c release and Bad phosphorylation following transient cerebral ischemia in mice

Tacrolimus (FK506) has a neuroprotective action on cerebral infarction produced by cerebral ischemia, however, detailed mechanisms underlying this action have not been fully elucidated. We examined temporal profiles of survival-and death-related signals, Bad phosphorylation, release of cytochrome c (cyt.c), activation of caspase 3 and DNA fragmentation in the brain during and after middle cerebral artery occlusion (MCAo) in mice, and then examined the effect of tacrolimus on these signals. C57BL/6J mice were subjected to transient MCAo by intraluminal suture insertion for 60 min. Tacrolimus (1 mg/kg, i.p.) was administered immediately after MCAo. There were biphasic increases in the release of cyt.c in the ischemic core and penumbra; with the first increase toward the end of the occlusion period and the second increase 3-12 h after reperfusion. Tacrolimus significantly inhibited the increase of cytosolic cyt.c during ischemia and reperfusion. Phosphorylated Bad, Ser-136 (P-Bad(136)) and Ser-155 (P-Bad(155)) were detected 30 min after MCAo and after reperfusion in the ischemic cortex, respectively. Tacrolimus increased P-Bad(136) during ischemia and prolonged P-Bad(155) expression after reperfusion. Tacrolimus also decreased caspase-3 and terminal deoxynucleotidyl transferase-mediated DNA nick-end labeling-positive cells, and reduced the size of infarct 24 h after reperfusion. Our study provided the first evidence that the neuroprotective action of tacrolimus involved inhibition of biphasic cyt.c release from mitochondria, possibly via up-regulation of Bad phosphorylation at different sites after focal cerebral ischemia and reperfusion.     

Immunohistochemical and biochemical assessment of caspase-3 activation and DNA fragmentation following transient focal ischemia in the rat

In the present study, we evaluated the time-course of caspase-3 activation, and the evolution of cell death following focal cerebral ischemia produced by transient middle cerebral artery occlusion in rats. Ischemia-induced active caspase-3 immunoreactivity in the striatum but not the cortex at 3 and 6 h time points post-reperfusion. Furthermore, using a novel approach to visualize enzymatic activity, deltaC-APP, a C-terminal cleavage product of APP generated by caspase-3, was found to immunolocalize to the same areas as active caspase-3. Double-labeling studies demonstrated co-localization of these two proteins at the cellular level. Further double-labeling experiments revealed that active caspase-3 was confined to neuronal cells which were still viable and thus immunoreactive for NeuN. DNA fragmentation, assessed histologically by terminal dUTP nick-end labeling (TUNEL), was observed in a small number of cells in the striatum as early as 3 h, but only began to appear in the cortex by 6 h. DNA fragmentation was progressive, and by 24 h post-reperfusion, large portions of both the striatum and cortex showed TUNEL positive cells. However, double-labeling of active caspase-3 with TUNEL showed only minimal co-localization at all time-points. Thus, caspase-3 activation is an event that appears to occur prior to DNA fragmentation. As a confirmation of the histological TUNEL data, 24 h ischemia also induced the generation of nucleosome fragments, evidenced by cell death enzyme-linked immunosorbent assay. Using a novel ischemia-induced substrate cleavage biochemical approach, spectrin P120 fragment, a caspase-specific cleavage product of alpha II spectrin, a cytoskeletal protein, was shown to be elevated by western blotting. Brain concentrations of both nucleosomes and spectrin P120 correlate with the degree of injury previously assessed by triphenyltetrazolium chloride staining and infarct volume calculation. Together, our findings suggest a possible association between caspase-3 activation and ischemic cell death following middle cerebral artery occlusion brain injury.     

局灶性脑缺血神经细胞DNA损伤与修复机制探讨

目的:观察脑缺血后半胱氨酸蛋白酶3(caspase-3)和脱嘌呤/脱嘧啶核酸内切酶(APE/Ref-1)的表达, 探讨脑缺血损伤与修复机制。方法: 采用大鼠大脑中动脉阻塞脑缺血模型, 应用免疫组化染色观察caspase-3和APE/Ref-1的表达, 应用TUNEL染色观察神经细胞DNA损伤, 采用免疫双标染色观察APE/Ref-1与DNA损伤的关系。结果:Caspase-3的活性单位P₂₀蛋白主要在半暗带表达, 表达高峰时间早于DNA损伤最高峰的时间。随着缺血时间延长, 缺血周边区APE/Ref-1免疫阳性细胞数量逐渐减少。结论:脑缺血后半胱氨酸蛋白酶级联反应启动, 促使DNA破坏, 同时, DNA损伤的修复分子表达水平下降, DNA修复失败, 进一步加速了细胞凋亡。     

Activation of caspase-12 by endoplasmic reticulum stress induced by transient middle cerebral artery occlusion in mice

We sought to clarify the involvement of caspase-12, a representative molecule related to endoplasmic reticulum (ER) stress-induced cell-death signaling pathways, in neuronal death resulting from ischemia/reperfusion in mice. Transient focal cerebral ischemia (1 h) was produced by intraluminal occlusion of the middle cerebral artery (MCA). We assessed the expression patterns of caspase-12, Bip/GRP78, an ER-resident molecular chaperone whose expression serves as a good marker of ER stress, and caspase-7 by Western blotting and/or immunohistochemistry. Double-fluorescent staining of caspase-12 immunohistochemistry and the terminal deoxynucleotidyl transferase-mediated DNA nick-end labeling (TUNEL) method was performed to clarify the involvement of caspase-12 in cell death. We confirmed that ER stress was induced during reperfusion in our model, as witnessed by up-regulated Bip/GRP78 expression in the MCA territory. Western blot analysis revealed that caspase-12 activation occurred at 5-23 h of reperfusion, and immunoreactivity for caspase-12 was enhanced mainly in striatal neurons on the ischemic side at the same time points. We found the co-localization of caspase-12 immunoreactivity and DNA fragmentation detectable by the TUNEL method. We did not detect the presence of caspase-7 in the ER fraction at the period of caspase-12 cleavage. Our results imply that cerebral ischemia/reperfusion induces ER stress and that caspase-12 activation concurred with ER stress. Caspase-12 seems to be involved in neuronal death induced by ischemia/reperfusion. Caspase-7 is not likely to contribute to the cleavage of caspase-12 in our experimental model.     

Possible mechanisms of Cyclosporin A ameliorated the ischemic microenvironment and inhibited mitochondria stress in tree shrews’ hippocampus

Objective: The ischemic brain damage is always accompanied by the significant accumulation of glutamate and calcium ions (Ca²⁺). Our objectives were to observe the effects of glutamate and Ca²⁺ overloading in tree shrew's hippocampal microenvironment on mitochondrial stress resulting in cytochrome C release and caspase apoptotic gene activation, and to explore the possible mechanism of Cyclosporin A (CsA) inhibiting mitochondrial stress. Methods: The thrombotic focal cerebral ischemia was induced by photochemical reaction in tree shrews. The extracellular contents of amino acidic neurotransmitters and Ca²⁺ were determined, respectively, with high performance liquid chromatography (HPLC) and atomic absorption spectrophotometry at 4, 24 and 72 h after cerebral ischemia. The glutamate–calcium chloride solutions were microperfused into hippocampus by a kind of single-pumped push–pull perfusion (SPPP) system under three-dimensional orientation instrument in tree shrews. At 24 h, the expression of cytochrome C was observed in perfused lateral hippocampus by immunochemistry. Also, the hippocampus was removed, then mitochondria and cytoplasmic fragment were divided by low temperature centrifugation and the distribution of cytochrome C was assessed through Western blot. Real time fluorescence polymerase chain reaction was used to evaluate the relative amounts of caspase-3 and caspase-9 mRNA. In the treated group, CsA (40 mg/kg) was intravenously injected at 6 h after the microperfuse or cerebral ischemia. The glutamate–calcium solutions were perfused into the hippocampus and inspected the above-mentioned items at 24 h. Data were compared between the two groups (ischemia group vs. sham group, or ischemia group vs. CsA group). Results: Thrombotic cerebral ischemia led to significant increase in extracellular glutamate and Ca²⁺ level of hippocampus (P < 0.01). The cerebral ischemia group and the microperfusion group, which cytochrome C immunoreactivity increased and Western blot analysis demonstrated that the cytochrome C content in the mitochondria of hippocampal cells decreased (P < 0.01), but the cytochrome C in the cytosol increased (P < 0.01). When CsA was intravenously injected at 6 h after the microperfusion or cerebral ischemia, the cytochrome C expression weakened and its release was diminished to a lesser extent. By real time PCR, in relation to the control group, the caspase-3 and caspase-9 mRNA was higher in the glutamate–calcium chloride solution perfused group. CsA treatment cut down the contents of caspase-3 mRNA and caspase-9 mRNA (P < 0.01). Conclusions: It is a primary factor that glutamate and Ca²⁺ accumulate in hippocampal microenvironment, which results in proapoptotic protein cytochrome C release from mitochondria into cytoplasm and caspase cascade activation, and finally mitochondria stress and neuronal secondary injury appear. The neuroprotection of CsA is in relation to inhibiting glutamate receptor overactivation and reducing the Ca²⁺ influx, which can decrease cytochrome C release and caspase mRNA transition.     

Immunohistochemical expression of Bcl-2, Bax and cytochrome c following focal cerebral ischemia and effect of hypothermia in rat

Recent studies suggest that mild hypothermia significantly alleviate damage following cerebral ischemia though the precise mechanism is poorly defined. In the present study, middle cerebral artery occlusion (MCAo) was induced in Sprague-Dawley (SD) rats for 1 h followed by varying periods of reperfusion. Cerebral infarcts identified by hematoxylin & eosin (H&E) staining revealed extensive lesion in normothermic (NT) 37 degrees C and small lesion in hypothermic (HT) 33 degrees C group of rats. Immunohistochemical analysis revealed Bcl-2 was induced in many neurons of HT group, while Bax and cytochrome c was induced in few neurons. In situ detection of DNA fragmentation using 3'-OH end labeling method (terminal dUTP nick-end labelling (TUNEL)) indicated, higher number of TUNEL-positive cells in NT group, but significantly decreased in HT group. The expression pattern revealed many neurons at the penumbra region could survive in HT group whereas, many neurons are committed to die in NT group. Our results suggest that hypothermia is selectively interfering at more than one place and providing protection.     

大鼠局灶性脑梗死后细胞凋亡及热休克蛋白70表达的变化

目的 探讨大鼠局灶性脑梗死后热休克蛋白70(HSP70)及细胞凋亡的变化。方法采用光化学法诱导制作大鼠局灶性脑梗死模型，冰冻切片行HSP70免疫组化染色，并用TdT-介导duTP-生物缺口末端标记(TUNEL)法检测凋亡细胞。结果免疫组化显示对照组和假手术组动物无HSP70免疫反应；局灶性脑梗死组12h即有：HSP70的表达，48h时HSP70表达至高峰，阳性反应局限于半暗带。TUNEL结果显示，对照组和假手术动物无凋亡细胞；局灶性脑梗死组6h时在半暗区凋亡细胞出现，3d达高峰。结论局灶性脑梗死可诱导HSP70的表达、神经细胞凋亡，两者的分布一致，但HSP70的表达高峰明显早于细胞凋亡。     

高血糖和脑缺血对树鼩皮层不同区域VEGF表达的影响

目的：研究高血糖及局灶性脑缺血条件下,树鼩皮层不同区域VEGF表达的变化,探讨脑缺血、高血糖与VEGF之间的相互关系。方法：用链脲佐菌素复制树鼩高血糖模型,并建立光化学诱导皮层局灶性脑缺血,观察缺血4 h、24 h及72 h的病理形态学改变并计数海马神经元密度,用免疫组化法测定上述时间树鼩缺血中心区、半暗带、对侧皮层VEGF表达的动态变化。结果：形态学观察显示,光化学反应后4 h照射区皮层可见梗塞灶;24 h病损达高峰;72 h伴随胶质细胞增生等修复性反应。相应时点高血糖加缺血组的损伤大于缺血组,以缺血后24 h(P<0.01)和72 h(P<0.05)尤为显著。免疫组化染色表明,缺血后4 h皮层缺血半暗区可见VEGF表达增加, 24 h达高峰,72 h减弱;单纯高血糖也使VEGF表达上调;高血糖加缺血组VEGF表达强于单纯高血糖组(P<0.05),但高血糖加缺血组与缺血组的同期值比较,无显著差异。结论：(1)在低等灵长类动物树鼩体内注射链脲佐菌素,并结合血栓性局部脑缺血方法学的应用能成功复制出实验性高血糖及脑缺血模型;(2)实验证明高血糖对局灶性脑缺血有恶化加重作用;(3)脑缺血及高血糖均可分别作为独立因素诱导VEGF的表达;但缺血与高血糖相加对VEGF表达未显示出叠加效应。     

脑缺血再灌注后神经元和内皮细胞凋亡与Bc1-2和Bax表达的时相关系

目的　探讨大鼠局灶性脑缺血再灌注后受损伤的神经细胞和血管内皮细胞凋亡 ,以及Bcl 2和Bax蛋白表达与再灌注时间的关系。　方法　应用原位末端标记 (TUNEL)技术和免疫组织化学方法 ,分别观察脑缺血再灌注 2h、6h、12h、2 4h、2d、3d、7d、14d和 2 1d等不同时间点神经细胞和血管内皮细胞凋亡数及Bcl 2和Bax蛋白的表达。　结果　 1.脑缺血周围区 ,再灌注 2h神经细胞和内皮细胞凋亡开始明显增多 ,12～ 2 4h达高峰 ,之后逐渐减少 ,7～ 14d降至假手术组水平 ;血管内皮细胞凋亡迟于神经元凋亡约 12h。 2 .Bcl 2蛋白表达于缺血再灌注 2h开始逐渐增强 ,12～ 2 4h达高峰 ,之后逐渐下降 ,至 7～ 14d接近假手术组水平 ;3.Bax蛋白表达于缺血再灌注 6h开始逐步增高 ,2 4～ 4 8h达高峰 ,之后逐渐下降 ,至 14d与假手术组已无显著性差异。 4 .Bcl 2表达与细胞凋亡的时相变化基本一致 ,Bax表达时相迟于细胞凋亡。　结论　细胞凋亡是脑缺血再灌注损伤细胞死亡的形式之一 ,血管内皮细胞凋亡迟于神经细胞凋亡 ,Bcl 2和Bax参与细胞凋亡的调节。     

Rapid apoptosis induced by Shiga toxin in HeLa cells

Apoptosis was induced rapidly in HeLa cells after exposure to bacterial Shiga toxin (Stx1 and Stx2; 10 ng/ml). Approximately 60% of HeLa cells became apoptotic within 4 h as detected by DNA fragmentation, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay, and electron microscopy. Stx1-induced apoptosis required enzymatic activity of the Stx1A subunit, and apoptosis was not induced by the Stx2B subunit alone or by the anti-globotriaosylceramide antibody. This activity was also inhibited by brefeldin A, indicating the need for toxin processing through the Golgi apparatus. The intracellular pathway leading to apoptosis was further defined. Exposure of HeLa cells to Stx1 activated caspases 3, 6, 8, and 9, as measured both by an enzymatic assay with synthetic substrates and by detection of proteolytically activated forms of these caspases by Western immunoblotting. Preincubation of HeLa cells with substrate inhibitors of caspases 3, 6, and 8 protected the cells against Stx1-dependent apoptosis. These results led to a more detailed examination of the mitochondrial pathway of apoptosis. Apoptosis induced by Stx1 was accompanied by damage to mitochondrial membranes, measured as a reduced mitochondrial membrane potential, and increased release of cytochrome c from mitochondria at 3 to 4 h. Bid, an endogenous protein known to permeabilize mitochondrial membranes, was activated in a Stx1-dependent manner. Caspase-8 is known to activate Bid, and a specific inhibitor of caspase-8 prevented the mitochondrial damage. Although these data suggested that caspase-8-mediated cleavage of Bid with release of cytochrome c from mitochondria and activation of caspase-9 were responsible for the apoptosis, preincubation of HeLa cells with a specific inhibitor of caspase-9 did not protect against apoptosis. These results were explained by the discovery of a simultaneous Stx1-dependent increase in endogenous XIAP, a direct inhibitor of caspase-9. We conclude that the primary pathway of Stx1-induced apoptosis and DNA fragmentation in HeLa cells is unique and includes caspases 8, 6, and 3 but is independent of events in the mitochondrial pathway.     

beta1 integrin antagonism on adherent, differentiated human neuroblastoma cells triggers an apoptotic signaling pathway

Integrin receptors mediate several functions including prevention of matrix detachment-induced apoptosis (anoikis) of several adherent cell types. We report here that antagonists of beta1 integrins trigger an apoptotic signaling pathway in adherent differentiated LAN-5 human neuroblastoma cells, a cell line which represents a model system for the study of human neurons. The pathway is characterized by cytochrome c release into the cytoplasm, and activation of caspase-9 and caspase-3, 4-6h after treatment; cleavage products of caspase-8 and caspase-2 were not detectable in the cells. Coordinate inactivation of cell survival pathways, including cleavage of focal adhesion kinase, decreased expression of protein kinase B, and reduced phosphorylation of the pro-apoptotic protein, Bad, also characterized the signaling pathway. These events occurred in adherent cells; DNA fragmentation and detachment followed as late events 18-24h after addition of beta1 integrin antagonists. zDEVD-fmk, an irreversible inhibitor of caspase-3-like enzymes, and cytochalasin D, an actin depolymerizing agent, blocked caspase-3 cleavage and delayed cell death. In contrast to these results, undifferentiated, adherent and dividing LAN-5 cells did not die in response to beta1 integrin antagonists.These studies identify a distinct apoptotic pathway which is triggered by antagonists of beta1 integrins on differentiated adherent neuronal cells.     

Expression of redox factor-1, p53-activated gene 608 and caspase-3 messenger RNAs following repeated unilateral common carotid artery occlusion in gerbils--relationship to delayed cell injury and secondary failure of energy state

The temporospatial expression pattern of the nuclear DNA repair enzyme redox factor-1 (ref-1), the p53-activated gene (pag) 608 and the effector caspase-3 was examined by in situ hybridization histochemistry in gerbils subjected to two 10-min episodes of unilateral common carotid artery occlusion, separated by 5h. Gene responses were correlated with the metabolic state, as revealed by regional adenosine 5'-triphosphate bioluminescent imaging, and with the degree of histological damage, as assessed by haematoxylin-eosin staining and terminal deoxynucleotidyl transferase-mediated-dUTP nick end labeling (TUNEL), in order to evaluate the role of these genes in the maturation of injury. Focal infarcts developed in the dorsolateral cerebral cortex at the bregma level and the nucleus caudate-putamen within four days after repeated unilateral ischemia, as indicated by a secondary adenosine 5'-triphosphate loss after initial adenosine 5'-triphosphate recovery and by histomorphological signs of pannecrosis. The more caudal cortex at hippocampal levels and the hippocampus (CA1>CA3 area), however, exhibited selective neuronal injury without adenosine 5'-triphosphate depletion. TUNEL+ cells appeared starting 5h after repeated unilateral ischemia. TUNEL+ cells reached maximum levels in the caudate-putamen at 12-24h, but much later in the cortex and hippocampus at two days after ischemia. Remarkably few TUNEL+ cells were noticed in the thalamus, where adenosine 5'-triphosphate state did not recover after reperfusion. Following repeated unilateral ischemia, a transient elevation of ref-1 mRNA was detected after 5h in the cerebral cortex and hippocampal CA1 area. Ref-1 mRNA levels decreased within 12-24h, before the onset of tissue damage. Subsequently, pag608 and caspase-3 mRNA levels increased, closely in parallel with the appearance of DNA fragmented cells, but slightly prior to the deterioration of adenosine 5'-triphosphate state. In the caudate-putamen, pag608 and caspase-3 mRNAs reached maximum levels already 12-24h after repeated common carotid artery occlusion, when DNA fragmentation was most prominent, and declined thereafter. In the cortex and hippocampal CA1-3 areas, where DNA damage appeared more slowly, pag608 and caspase-3 mRNAs were induced starting 24h after ischemia, and remained elevated even after two to four days. The levels of pag608 and caspase-3 mRNAs were similar at rostral and caudal levels of the cortex, as well as in the hippocampal CA1 and CA3 area, although the degree of injury differed considerably between these structures. Notably, pag608 and caspase-3 mRNAs were not elevated in the thalamus after repeated unilateral ischemia. The present report shows a close temporal association between the induction of ref-1, pag608 and caspase-3 mRNAs, the manifestation of cell injury and the secondary adenosine 5'-triphosphate depletion in infarcting brain areas, suggesting (i) that de novo responses of these genes may be involved in the maturation of cell injury and (ii) that apoptotic programs and the secondary deterioration of cerebral energy state may interfere with each other after ischemia.     

Cytochrome c-dependent activation of caspase-3 by tumor necrosis factor requires induction of the mitochondrial permeability transition

The killing of L929 mouse fibroblasts by tumor necrosis factor-alpha (TNF-alpha) in the presence of 0.5 microg/ml actinomycin D (Act D) is prevented by inhibition of the mitochondrial permeability transition (MPT) with cyclosporin A (CyA) in combination with the phospholipase A(2) inhibitor aristolochic acid (ArA). The MPT is accompanied by the release of cytochrome c from the mitochondria, caspase-8 and caspase-3 activation in the cytosol, cleavage of the nuclear enzyme poly(ADP-ribose)polymerase (PARP), and DNA fragmentation, all of which were inhibited by CyA plus ArA. The caspase-3 inhibitor z-Asp-Glu-Val-aspartic acid fluoromethyl-ketone (Z-DEVD-FMK) did not prevent the loss of viability or the redistribution of cytochrome c, but it did prevent caspase-3 activation, PARP cleavage, and DNA fragmentation. Inhibition of the MPT reduced the activation of caspase-8 to the level occurring with TNF-alpha alone (no ActD). The caspase-8 inhibitor z-Ile-Glu(OMe)-Thr-Asp(OMe) fluoromethylketone (Z-IETD-FMK) did not prevent the cell killing and decreased only slightly the translocation of Bid to the mitochondria. These data indicate that induction of the MTP by TNF-alpha causes a release of cytochrome c, caspase-3 activation with PARP cleavage and DNA fragmentation. The loss of viability is dependent on the MPT but independent of the activation of caspase-3. The activation of caspase-8 is not dependent on the MPT. There is no evidence linking this enzyme to the loss of viability. Thus, the killing of L929 fibroblasts by TNF-alpha can occur in the absence of either caspase-3 or caspase-8 activity. Alternatively, cell death can be prevented despite an activation of caspase-8.     

Time-course of c-Jun N-terminal kinase activation after cerebral ischemia and effect of D-JNKI1 on c-Jun and caspase-3 activation

The c-Jun N-terminal kinase (JNK) signaling pathway plays a critical role in ischemic brain injury. The d-retro-inverso form of c-Jun N-terminal kinase-inhibitor (D-JNKI1), a cell-permeable inhibitor of JNK, powerfully reduces neuronal death induced by permanent and transient ischemia, even when administered 6 h after the ischemic insult, offering a clinically relevant window. We investigated the JNK molecular cascade activation in rat cerebral ischemia and the effects of D-JNKI1 on this cascade. c-Jun activation starts after 3 h after ischemia and peaks at 6 h in the ischemic core and in the penumbra at 1 h and at 6 h respectively. The 6 h c-Jun activation peak correlates well with that of P-JNK. We also examined the activation of the two direct JNK activators, MAP kinase kinase 4 (MKK4) and MAP kinase kinase 7 (MKK7). MKK4 showed the same time course as JNK in both core and penumbra, reaching peak activation at 6 h. MKK7 did not show any significant increase of phosphorylation in either core or penumbra. D-JNKI1 markedly prevented the increase of P-c-Jun in both core and penumbra and powerfully inhibited caspase-3 activation in the core. These results confirm that targeting the JNK cascade using the TAT cell-penetrating peptide offers a promising therapeutic approach for ischemia, raising hopes for human neuroprotection, and elucidates the molecular pathways leading to and following JNK activation.     

Photodynamic therapy induced Fas-mediated apoptosis in human carcinoma cells

Photodynamic therapy (PDT) is a clinical approach that utilizes light-activated drugs for the treatment of a variety of pathologic conditions. Human poorly (CNE2) and moderately differentiated (TW0-1) human nasopharyngeal carcinoma (NPC) cells undergo rapid apoptosis when treated with PDT sensitized with Hypocrellin A (HA) and Hypocrellin B (HB). It has been shown that these compounds have a strong photodynamic effect on tumors and viruses. The initiating events of PDT sensitized HA and HB-induced apoptosis are poorly defined. In the current study, we sought to determine whether Fas/FasL upregulation and involvement of mitochondrial events are an early event in HA and HB-treated PDT induced apoptosis. Loss of mitochondrial transmembrane potential, release of cytochrome c, involvement of caspases-8 and -3 and the status caspase-3 specific substrate PARP, were evaluated in PDT treated tumor cells. Photoactivation of HA and HB enhanced both CD95/CD95L expression and induced CD95-signaling dependent cell death in all tumor cell lines studied. CD95/ CD95L expression appeared within 2 h following light activation and appeared to be a primary event in PDT induced apoptosis. Furthermore, these results indicate that release of mitochondrial cytochrome c into the cytoplasm is a secondary event following the activation of initiator caspase-8 preceding caspase-3 activation, cleavage of PARP and DNA fragmentation. Cytochrome c appeared in the cytosol within 2-3 h post PDT. Cleavage of PARP was observed at 3-4 h following PDT and caspase-3 specific inhibitor DEVD-CHO and broad-spectrum caspases inhibitor z-VAD-fmk blocked caspase-3 activation and PARP cleavage suggesting that caspase-3 plays an important role in HA and HB-induced apoptosis.     

局灶性脑梗死后半暗带区皮层血流量与神经细胞凋亡的实验研究

目的：研究大鼠局灶性脑梗死后半暗带区皮层血流量及神经细胞凋亡。 方法： 光化学法诱导大鼠局灶性脑梗死模型，应用激光多普勒微循环测量仪检测局灶性脑梗死后各时间点坏死区和半暗带皮层血流量；TdT-介导dUTP-生物缺口末端标记（TUNEL）法检测凋亡细胞。 结果： 脑梗死后坏死区皮层血流量呈波浪样改变，3 h降至最低，为正常血流量的9.36%±1.72%，6h后回升，12 h达高峰，为正常31.18%±1.44%，24-48 h血流量再度下降，与3 h相比无显著差异（P＞0.05）。半暗带皮层血流量6 h降至最低，为正常皮层血流量的30.88%±7.11%，9-48 h上升，至48 h血流恢复至正常91.90%±4.56%，与其它组差异显著 (P<0.05)。TUNEL结果显示，局灶性脑梗死组 6 h 开始出现凋亡细胞，主要集中于半暗带区，呈半球状向四周放射样扩展，随梗死时间的延长凋亡细胞逐渐增多，梗死48 h后凋亡细胞达峰值。 结论： 局灶性脑梗死后半暗带区皮层血流量与神经细胞凋亡具有明显的关系，其血流量的恢复引起的再灌注损伤最终导致了迟发性神经细胞凋亡。     

Relationship between metabolic dysfunctions, gene responses and delayed cell death after mild focal cerebral ischemia in mice.

The evolution of brain injury was examined in mice subjected to focal cerebral ischemia as induced by 30 min of intraluminar thread occlusion of the middle cerebral artery, followed by 3 h to 3 days of reperfusion. Metabolic dysfunctions were studied by 3H-leucine autoradiography for the measurement of cerebral protein synthesis and by regional ATP bioluminescent imaging. Metabolic changes were compared with responses of the genes c-fos, c-jun, heat-shock protein gene (hsp)72, p53-activated gene (pag)608 and caspase-3, which were investigated by in situ hybridization histochemistry and immunocytochemistry, and correlated with the degree of DNA fragmentation, as assessed by the terminal TdT-mediated dUTP-biotin nick end labeling method. Intraluminar thread occlusion led to a reproducible reduction of cerebral laser Doppler flow to 20-30% of control. Thread withdrawal was followed by a short-lasting post-ischemic hyperperfusion to approximately 120%. In non-ischemic control animals, fractional protein synthesis values of 0.81+/-0.26 and 0.94+/-0.23 were obtained. Thread occlusion resulted in a suppression of protein synthesis throughout the territory of the middle cerebral artery after 3 h of reperfusion (0.04+/-0.08 in caudate-putamen and 0.14+/-0.19 in somatosensory cortex, P<0.05). Protein synthesis partly recovered in the cortex after 24 h and 3 days (0.71+/-0.40 and 0.63+/-0.26, respectively), but remained suppressed in the caudate-putamen (0.14+/-0.22 and 0.28+/-0.28). Regional ATP levels did not show any major disturbances at the reperfusion times examined. Thread occlusion resulted in a transient increase of c-fos mRNA levels in ischemic and non-ischemic parts of the cortex and caudate-putamen at 3 h after ischemia, which suggests that spreading depressions were elicited in the tissue. At the same time, c-jun and hsp72 mRNAs were elevated only in ischemic brain areas showing inhibition of protein synthesis. C-fos and c-jun responses completely disappeared within 24 h of reperfusion. Hsp72 mRNA levels remained elevated in the cortex after 24 h, but decreased to basal values in the caudate-putamen. Twenty-four hours after reperfusion, pag608 and caspase-3 mRNA levels increased in the caudate-putamen, where protein synthesis rates were still reduced, and remained elevated even after 3 days. However, pag608 and caspase-3 mRNA levels did not increase in the cortex, where protein synthesis recovered. After 24 h and 3 days, functionally active p20 fragment of caspase-3 was detected in the caudate-putamen, closely associated with the appearance of DNA fragmented cells. Neither activated caspase-3 nor DNA fragmentation were noticed in the cortex.In summary, the suppression of protein synthesis is reversible in the ischemia-resistant cortex following 30 min of thread occlusion in mice, but persists in the vulnerable caudate-putamen. In the caudate-putamen, apoptotic programs are induced, closely in parallel with the manifestation of delayed cell death. Thus, the recovery of protein synthesis may be a major factor influencing tissue survival after transient focal ischemia.     

葡萄糖转运体3在脑缺血/再灌注后半影区表达的变化及意义

目的:研究大鼠局灶性脑缺血不同缺血时间和不同再灌注时间的脑梗塞体积比、皮质半影区葡萄糖转运体3(GLUT3)转录水平和蛋白水平的表达。方法:用线栓法复制大鼠局灶性脑缺血模型,用Kontron IBAS2.5全自动图像分析系统检测脑梗塞体积比;剥取缺血半影区皮质组织,采用反转录-聚合酶链反应(RT-PCR)测定GLUT3 mRNA水平的变化;用免疫组织化学方法半定量测定GLUT3蛋白水平的变化。结果:脑缺血1 h后再灌注组的脑梗塞体积明显小于缺血3 h再灌注组梗塞体积。GLUT3自3 h即开始升高,24 h到达高峰,1周时仍高于假手术对照组;缺血3 h再灌注组在3 h有一下降点,然后升高,24 h到高峰,1周时接近正常水平。GLUT3蛋白水平的表达与mRNA相符合。结论:GLUT3在缺血半影区的表达上调,可能是机体对缺血／再灌注的保护性反应。