Differential role of mGlu1 and mGlu5 receptors in rat hippocampal slice models of ischemic tolerance

Activation of glutamate receptors has been proposed as a key factor in the induction of ischemic tolerance. We used organotypic rat hippocampal slices exposed to 30 min oxygen-glucose deprivation (OGD) to evaluate postischemic pyramidal cell death in the CA1 subregion. In this model, 10 min exposure to OGD 24 h before the exposure to toxic OGD was not lethal and reduced the subsequent OGD neurotoxicity by approximately 53% (ischemic preconditioning). Similarly, a 30 min exposure to the group I mGlu receptor agonist DHPG (10 microM) significantly reduced OGD neurotoxicity 24 h later (pharmacological preconditioning). Ischemic tolerance did not develop when either the selective mGlu1 antagonists LY367385 and 3-MATIDA or the AMPA/KA antagonist CNQX were present in the incubation medium during exposure to sublethal OGD. Neither the NMDA antagonist MK801 nor the mGlu5 antagonist MPEP affected the preconditioning process. On the other hand, pharmacological preconditioning was prevented not only by LY367385 or CNQX, but also by MPEP. In preconditioned slices, the toxic responses to AMPA or NMDA were reduced. The neurotoxicty of 100 microM DHPG in slices simultaneously exposed to a mild (20 min) OGD was differentially altered in the two preconditioning paradigms. After ischemic preconditioning, DHPG neurotoxicity was reduced in a manner that was sensitive to LY367385 but not to MPEP, whereas after pharmacological preconditioning it was enhanced in a manner that was sensitive to MPEP but not to LY367385. Our results show that mGlu1 and mGlu5 receptors are differentially involved in the induction and expression of ischemic tolerance following two diverse preconditioning stimuli.     

Poly(ADP-ribose) polymerase during reperfusion after transient forebrain ischemia

The activation of poly(ADP-ribose) polymerase (PARP) in the reperfused brain after ischemia has been assumed but never has been directly presented. Our studies indicate a different dynamic of PARP activity alteration in hippocampus during reperfusion after 3 and 10 min of transient forebrain ischemia in gerbils. The phasic stimulation of PARP activity was observed during reperfusion 15 min, 120 min, and 4 d after 3 min of ischemia with subsequent lowering of its activity close to control value on the seventh day of reperfusion. After 10 min of ischemic insult, PARP activity significantly increased from the third to the seventh day of reperfusion. The protein level of PARP was not significantly changed during reperfusion after 3 and 10 min of ischemia, with one exception: On the third day after 10 min of ischemia, PARP protein level was 28% lower compared to control; however, no enhancement of 85-kDa protein immunoreactivity was observed. These data indicate the lack of PARP cleavage in hippocampus of gerbils subjected to ischemia-reperfusion injury. The inhibitor of PARP, 3-aminobenzamide (3-AB) in a dose of 30 mg/kg b.w. (body weight) injected intravenously directly after 3 min of ischemia protects >60% of neuronal cells against death in the CA1 layer of hippocampus but has no effect after 10 min of ischemic episode. 3-AB decreased forebrain edema significantly after 3 and 10 min of ischemia. Our data indicate that PARP inhibitor(s) might offer a potent therapeutic strategy for short global ischemia. The combination of PARP inhibitor with potent antioxidant might enhance its ameliorating effect.     

Involvement of independent mechanism upon poly(ADP-ribose) polymerase (PARP) activation in methylmercury cytotoxicity in rat cerebellar granule cell culture

Poly(ADP-ribose) polymerase (PARP) activation plays a role in repairing injured DNA, while its overactivation is involved in various diseases, including neuronal degradation. In the present study, we investigated the use of a PARP inhibitor, 3,4-dihydro-5-[4-(1-piperidinyl)butoxy]-1(2H)-isoquinolinone (DPQ), whether methylmercury-induced cell death in the primary culture of cerebellar granule cells involved PARP activation. DPQ decreased the methylmercury-induced cell death in a dose-dependent manner. Unexpectedly, this protective effect was DPQ specific; none of the other PARP inhibitors--1,5-dihydroxyisoquinoline, 3-aminobenzamide, or PJ34--affected neuronal cell death. Methylmercury-induced cell death involves the decrease of glutathione (GSH) and production of reactive oxygen species. Therefore, to understand the mechanism by which DPQ inhibits cytotoxicity, we first studied the effect of DPQ on buthionine sulfoximine- or diethyl maleate-induced death of primary cultured cells and human neuroblastoma IMR-32 cells, both of which are mediated by GSH depletion. DPQ inhibited the cell death of both cultured cells, but it did not restore the decrease of cellular GSH by buthionine sulfoximine to the control level. Second, we evaluated the antioxidant activity of PARP inhibitors by methods with ABTS (2-2'-azinobis(3-ethylbenzothiazoline 6-sulfonate) or DPPH (1,1-diphenyl-2-picrylhydrazyl) used as a radical because antioxidants also efficiently suppress methylmercury-induced cell death. The antioxidant activity of DPQ was the lowest among the tested PARP inhibitors. Taken together, our results indicate that DPQ effectively protects cells against methylmercury- and GSH depletion-induced death. Furthermore, they suggest that DPQ exerts its protective effect through a mechanism other than PARP inhibition and direct antioxidation, and that PARP activation is not involved in methylmercury-induced neuronal cell death.     

Expression of poly(ADP-ribose) polymerase and distribution of poly(ADP-ribosyl)ation in glioblastoma and in a glioma multicellular tumour spheroid model

Development of necrosis is a characteristic feature of glioblastoma but its pathogenesis remains poorly understood. The process of poly(ADP-ribosyl)ation in response to DNA damage is mediated by poly(ADP-ribose) polymerase (PARP) and results in NAD+ depletion. The consequent ATP and energy depletion may result in cell necrosis. Therefore PARP activation is a potential candidate for a regulatory role in the pathogenesis of necrosis in glioblastoma. This study investigated whether there might be a relationship between both PARP expression and poly(ADP-ribosyl)ation, and necrosis in glioblastoma. The pattern of expression of PARP and of poly(ADP-ribose) groups in an archival series of glioblastoma was examined using immunohistochemistry. These parameters were also studied in multicellular tumour spheroids, derived from human glioma cell lines in which central necrosis develops with increasing spheroid diameter. Poly(ADP-ribose) groups were expressed in peri-necrotic tumour cells in glioblastoma. In the spheroid model poly(ADP-ribosyl)ation was seen centrally in pre-necrotic and necrotic cells with increasing spheroid diameter. PARP was widely expressed in viable tumour cells in the glioblastoma sections. In the spheroids, PARP expression, which was initially diffuse, became confined to the outer proliferative zone with increasing diameter. The pattern of expression of poly(ADP-ribose) groups in the spheroids and in glioblastoma raises the possibility that poly(ADP-ribosyl)ation may play a role in the development of necrosis in glioma. The high basal PARP expression in both glioblastoma and the spheroids suggests that this enzyme may have additional roles in glioma cell biology.     

Sex differences in the response to activation of the poly (ADP-ribose) polymerase pathway after experimental stroke

It is increasingly recognized that histological and functional outcomes after stroke are shaped by biologic sex. Emerging data suggests that ischemic cell death pathways are sexually dimorphic (Hurn, P., Vannucci, S., Hagberg, H. (2005) Adult or perinatal brain injury: does sex matter?. Stroke 36, 193-195 ; Lang, J.T., McCullough, L.D. (2008) Pathways to ischemic neuronal cell death: are sex differences relevant?. J. Transl. Med. 6). Reducing neuronal nitric oxide (NO) or poly-ADP-ribose polymerase (PARP1) activation protects only the male brain (Hagberg, H., et al. PARP-1 gene disruption in mice preferentially protects males from perinatal brain injury. J. Neurochem. 90, 1068-1075 (2004)), and paradoxically enhances ischemic injury in females (McCullough, L.D., et al. Ischemic nitric oxide and poly (ADP-ribose) polymerase-1 in cerebral ischemia: male toxicity, female protection. J. Cereb. Blood Flow Metab. 25, 502-512 (2005)). In this study, we examined downstream mediators of NO/PARP activation to investigate possible mediators of ischemic sexual dimorphism. Nuclear translocation of Apoptosis Inducing Factor (AIF) was equivalent in wild type males and females after stroke and was unaffected by estrogen exposure. Deletion of PARP1 led to a dramatic reduction in stroke-induced poly (ADP-ribose) polymerase (PAR) formation and AIF translocation in both sexes, yet ischemic damage was reduced only in males. Subsequent examination of AIF-deficient Harlequin mice demonstrated that male Harlequin mice had less PAR formation, reduced AIF translocation and less ischemic damage than male wild type mice. In contrast, female Harlequin mice had no neuroprotective effect of gene deletion despite robust reductions in PAR formation and AIF translocation. Although equivalent activation of this cell death pathway occurs in both sexes after ischemia, detrimental effects are only present in males. AIF translocation and PAR formation do not mediate ischemic injury in the female brain, therefore agents designed to reduce PARP1 activation are unlikely to benefit females.     

Effects of deltamethrin on nitric oxide synthase and poly(ADP-ribose) polymerase in rat brain

The effects of deltamethrin on the activities of nitric oxide synthase (NOS) and poly(ADP-ribose) polymerase (PARP) and the protein expression of neuronal NOS (nNOS) and PARP in rat brain were investigated in the present study. The activity of NOS was significantly increased in cortex and hippocampus at 5 h after deltamethrin treatment, and maintained at an increased level at 24 h. The activity of PARP was also elevated at the same time points in the same brain regions of treated rats. By immunohistochemical analysis, it was demonstrated that the nNOS-immunoreactive cells were markedly increased at 24 h after treatment in the cortex and hippocampus, whereas few nNOS-immunoreactive cells were observed in the same brain regions of control and treated rats at 5 h after treatment. The immunoreactivity for PARP was also increased in the same brain regions, showing the similar time course of the induction of nNOS by deltamethrin. These results indicate that deltamethrin increases the activities of NOS and PARP and initiates the protein expression of nNOS and PARP, suggesting that NOS and PARP might play important roles in neurotoxicity of deltamethrin.     

Deleterious poly(ADP-ribose)polymerase-1 pathway activation in traumatic brain injury in rat

Traumatic brain injury produces nitric oxide and reactive oxygen species. Peroxynitrite, resulting from the combination of nitric oxide and superoxide anions, triggers DNA strand breaks, leading to the activation of poly(ADP-ribose)polymerase-1. As excessive activation of this enzyme induces cell death, we examined the production of nitrosative stress, the activation of poly(ADP-ribose)polymerase-1, and the role of this enzyme in the outcomes of traumatic brain injury produced by fluid percussion in rats. Immunohistochemistry showed that 3-nitrotyrosine, an indicator of nitrosative stress, and poly(ADP-ribose), a marker of poly(ADP-ribose)polymerase-1 activation, were present as early as 30 min post-injury, and that persisted for 72 h. The poly(ADP-ribose)polymerase inhibitor, 3-aminobenzamide, at 10 and 30 mg/kg, significantly improved the neurological deficit, with a 60% reduction in the brain lesion volume and inhibition of poly(ADP-ribose)polymerase-1 activation. Thus, poly(ADP-ribose)polymerase-1 is involved in the neurological consequences of traumatic brain injury and may be a promising therapeutic target in clinical treatment of acute brain trauma.     

Neuronal accumulation of poly(ADP-ribose) after brain ischaemia

Animal and in vitro studies suggest that overactivation of poly(ADP-ribose) polymerase (PARP) in response to oxidative DNA damage makes a substantial contribution to cell death after brain ischaemia. We have recently shown that global brain ischaemia due to cardiac arrest in man induces a rapid increase in the amount of neuronal and glial PARP that can be detected by immunohistochemistry. In the present study we sought evidence of a corresponding increase in the amount of poly(ADP-ribose) within the brain, as this would confirm PARP activation and imply resulting consumption of NAD⁺ . We also studied the distribution of poly(ADP-ribose) accumulation in relation to morphological evidence of ischaemic damage, and used double immunolabelling to investigate the types of cell that were affected. We found that global brain ischaemia did cause accumulation of poly(ADP-ribose), particularly during the first 2 days after cardiac arrest. The distribution of cells with accumulation of poly(ADP-ribose) corresponded in general to regions of ischaemic damage or immediately adjacent neocortex. Double immunolabelling for poly(ADP-ribose) and MAP2 showed many of the cells with poly(ADP-ribose) accumulation to be neurons. Our findings are in keeping with experimental evidence of a role for PARP in post-ischaemic necrosis and of the potential for reducing ischaemic brain damage by the use of PARP inhibitors.     

The role of NADPH oxidase and neuronal nitric oxide synthase in zinc-induced poly(ADP-ribose) polymerase activation and cell death in cortical culture

In the present study, we examined the role and the mechanism of poly(ADP-ribose) polymerase (PARP) and poly(ADP-ribose) glycohydrolase (PARG) activation in zinc-induced cell death in cortical culture. After brief exposure to 400 microM zinc, cortical cells exhibited DNA fragmentation, increased poly(ADP-ribosyl)ation, and decreased levels of nicotinamide adenine dinucleotide (NAD) and ATP and subsequently underwent cell death. Inhibitors of PARP/PARG attenuated both zinc-induced NAD/ATP depletion and cell death, thereby implicating the PARP/PARG cascade in these processes. The zinc-inducible enzymes NADPH oxidase and neuronal nitric oxide synthase (nNOS) contributed to PARP activation as their inhibitors attenuated zinc-induced poly(ADP-ribosyl)ation. Levels of nitric oxide and nitrites increased following zinc exposure, consistent with NOS activation. In addition, Western blots and RT-PCR analysis revealed that protein and mRNA levels of nNOS specifically increased following zinc exposure in a manner similar to that of NADPH oxidase. The present study demonstrates that induction of NADPH oxidase and nNOS actively contributes to PARP/PARG-mediated NAD/ATP depletion and cell death induced by zinc in cortical culture.     

Effects of an inhibitor of poly(ADP-ribose) polymerase, desmethylselegiline, trientine, and lipoic acid in transgenic ALS mice

The development of transgenic mouse models of amyotrophic lateral sclerosis (ALS) allows the testing of neuroprotective agents. We evaluated the effects of five agents in transgenic mice with the G93A Cu,Zn superoxide dismutase mutation. A novel inhibitor of poly(ADP-ribose) polymerase showed no effects on survival. Desmethylselegiline and CGP3466 are agents that exert antiapoptotic effects in vitro by preventing nuclear translocation of glyceraldehyde-3-phosphate dehydrogenase. They had no significant effects on survival in the G93A mice. Trientine, a copper chelator, produced a modest significant increase in survival. Similarly administration of lipoic acid in the diet produced a significant improvement in survival. These results therefore provide evidence for potential therapeutic effects of copper chelators and lipoic acid in the treatment of ALS.     

Effects of transient global ischemia and kainate on poly(ADP-ribose) polymerase (PARP) gene expression and proteolytic cleavage in gerbil and rat brains

Poly (ADP-ribose) polymerase (PARP) is involved in various cellular functions, including DNA repair, the cell cycle and cell death. While PARP activation could play a critical role in repairing ischemic brain damage, PARP inactivation caused by caspase 3-cleavage may also be important for apoptotic execution. In this study we investigated the effects of transient global ischemia and kainic acid (KA) neurotoxicity, in gerbil and rat brains, respectively, on PARP gene expression and protein cleavage. PARP mRNA increased in the dentate gyrus of gerbil brains 4 h after 10 min of global ischemia, which returned to basal levels 8 h after ischemia. KA injection (10 mg/kg) also induced a marked elevation in PARP mRNA level selectively in the dentate gyrus of rat brains 1 h following the injection, which returned to basal levels 4 h after the injection. These observations provide the first evidence of altered PARP gene expression in brains subjected to ischemic and excitotoxic insults. Using both monoclonal and polyclonal antibodies to PARP cleavage products, little evidence of significant PARP cleavage was found in gerbil brains within the first 3 days after 10 min of global ischemia. In addition, there was little evidence of significant PARP cleavage in rat brains within 2 days after kainate (KA) injection. Though these findings show that caspase induced PARP cleavage is not substantially activated by global ischemia and excitotoxicity in whole brain, the PARP mRNA induction could suggest a role for PARP in repairing DNA following brain injury.     

Enhanced poly(ADP-ribose) polymerase-1 activation contributes to recombinant tissue plasminogen activator-induced aggravation of ischemic brain injury in vivo

Recombinant tissue plasminogen activator (rt-PA) treatment improves functional outcome after acute ischemic stroke, inducing reperfusion by its thrombolytic activity. Conversely, there is evidence that rt-PA can mediate neuronal damage after ischemic brain injury in vivo. In addition to other mechanisms, enhancement of N-methyl-D-aspartate (NMDA) receptor signalling has been proposed to underlie rt-PA-mediated neurotoxicity. However, the role of poly(ADP-ribose) polymerase-1 (PARP-1) activation, which mediates postischemic excitotoxic cell death, in rt-PA-mediated aggravation of ischemic brain injury has not been established and was therefore addressed in this study. After permanent focal cerebral ischemia, intravenous rt-PA application significantly increased early postischemic PARP-1 activation within ischemic hemispheres and infarct volumes compared with control mice without affecting cerebral blood flow. Rt-PA induced increase in PARP-1 activation, and infarct volumes could be blocked by the PARP inhibitor 3-aminobenzamide. Moreover, the rt-PA-induced increase in PARP-1 activation was also prevented by the NMDA antagonist MK-801. In summary, we demonstrate that rt-PA treatment enhances postischemic PARP-1 activation, which contributes to rt-PA induced aggravation of ischemic brain injury in vivo. Furthermore, we provide evidence that NMDA receptor activation is required for rt-PA-mediated effects on postischemic PARP-1 activation.     

Sustained activation of ERK signaling in astrocytes is critical for neuronal injury‐induced monocyte chemoattractant protein‐1 production in rat corticostriatal slice cultures

We previously demonstrated that N‐methyl‐d ‐aspartate (NMDA) treatment (50 μm , 3 h) induced astrocytic production of monocyte chemoattractant protein‐1 (MCP‐1, CCL2), a CC chemokine implicated in ischemic and excitotoxic brain injury, in rat corticostriatal slice cultures. In this study, we investigated the signaling mechanisms for NMDA‐induced MCP‐1 production in slice cultures. The results showed a close correlation between NMDA‐induced neuronal injury and MCP‐1 production, and an abrogation of NMDA‐induced MCP‐1 production in NMDA‐pretreated slices where neuronal cells had been eliminated. These results collectively indicate that NMDA‐induced neuronal injury led to astrocytic MCP‐1 production. NMDA‐induced MCP‐1 production was significantly inhibited by U0126, an inhibitor of extracellular signal‐regulated kinase (ERK). Immunostaining for phosphorylated ERK revealed that transient neuronal ERK activation was initially induced and subsided within 30 min, followed by sustained ERK activation in astrocytes. Treatment with U0126 during only the early phase (U0126 was washed out at 15 or 30 min after NMDA administration) suppressed early activation of ERK in neuronal cells, but not later activation of ERK in astrocytes. In this case, MCP‐1 production was not suppressed, suggesting that activation of neuronal ERK is not necessary for MCP‐1 production. In contrast, delayed application of U0126 at 3 h after the beginning of NMDA treatment inhibited MCP‐1 production to the same degree as that observed when U0126 was applied from 3 h before NMDA administration. These findings suggest that sustained activation of the ERK signaling pathway in astrocytes plays a key role in neuronal injury‐induced MCP‐1 production.     

Post-treatment with an inhibitor of poly(ADP-ribose) polymerase attenuates cerebral damage in focal ischemia

Poly(ADP-ribose) polymerase (PARP) is thought to play a physio-logical role in maintaining genomic integrity and in the repair of DNA strand breaks. However, the activation of PARP by free radical-damaged DNA plays a pivotal role in mediating ischemia-reperfusion injury. The excessive activation of PARP causes a rapid depletion of intracellular energy leading to cell death. The present study examined the effect of post-ischemic pharmacological inhibition of PARP in a rat focal cerebral ischemia model. In Long-Evans rats, focal cerebral ischemia was produced by cauterization of the right distal middle cerebral artery (MCA) with bilateral temporary common carotid artery (CCA) occlusion for 90 min. A PARP inhibitor, 3, 4-dihydro-5-[4-(1-piperidinyl)butoxy]-1(2H)-isoquinolinone (DPQ; IC50=1 microM/l) was injected i.p. 30 min after the onset of MCA occlusion (control: 10, 20, 40 and 80 mg/kg; n=7 each). Twenty-four hours later, the total infarct volume was measured. Regional blood flow in the right parietal cortex decreased to approximately 20% of the baseline following MCA occlusion in all groups. PARP inhibition lead to a significant decrease in damaged volume in all treated groups with the largest reduction in the 40 mg/kg group (111.5+/-24. 8 mm3, mean+/-SD, p<0.01), compared to the control group (193.5+/-28. 6 mm3). We also found there was a significant increase of poly(ADP-ribose) immunoreactivity in the ischemic region, as compared to the contralateral side, with DPQ treatment diminishing poly(ADP-ribose) production. These findings indicate that DPQ exerts its neuroprotective effects in vivo by PARP inhibition and that PARP inhibitors may be effective for treating ischemic stroke, even when the treatment is initiated after the onset of ischemia.     

Poly(ADP–ribose) polymerase is found in both the nucleus and cytoplasm of human CNS neurons

There is evidence that inhibitors of poly(ADP–ribose) polymerase (PARP) may be therapeutically useful in neurodegenerative diseases. Using immunocytochemistry, we have investigated the distribution of PARP in the human CNS. Some neuronal groups showed cytoplasmic staining in addition to the expected staining of nuclei. Considerable variation between different neuronal groups was noted: motor neurons in the spinal cord showed greatest cytoplasmic staining, whereas staining was virtually absent in other neurons, notably in the hippocampus. These results indicate that PARP can be associated with sub-cellular components other than the nucleus, and may indicate additional roles for this enzyme.     

Long-term neuroprotective effect of inhibiting poly(ADP-ribose) polymerase in rats with middle cerebral artery occlusion using a behavioral assessment

Poly(ADP-ribose) polymerase (PARP) can initiate an energy-consuming and inefficient repair cycle following cerebral ischemia/reperfusion by transferring ADP ribose units to nuclear proteins eventually leading to cellular dysfunction and neuronal death. 3-Aminobenzamide (3-AB) is a selective inhibitor of PARP that can significantly reduce brain damage after focal ischemia in rats and displays a low toxicity in vivo. The goals of this study were to determine if inhibiting PARP with 3-AB has a long-term neuroprotective effect and if functional outcome improves in rats following focal ischemia and treatment with 3-AB. Focal ischemia was induced by a 2-h occlusion of the middle cerebral artery (MCA), using an intraluminal filament. Motor functions were evaluated from 5 to 28 days after reperfusion in four groups of rats: stroke without treatment; stroke treated with 3-AB at doses of 15 mg/kg, stroke treated with 3-AB at doses of 55 mg/kg; and the non-ischemic control rats. Functional behaviors were tested by a series of motor function tasks (foot placing, parallel bar crossing, rope and ladder climbing), as well as a neurological examination. Infarct volume of stroke brain in the same rat was determined by Nissl staining 28 days after surgery. Comparison of the untreated stroke group (n=11) and the treated stroke groups indicates that impairment of motor function was significantly (P<0.001) reduced by administration of 3-AB at doses of 15 mg/kg (n=9) or 55 mg/kg (n=10). Neurological outcome was also improved significantly (P<0.001). Infarct volume was significantly (P<0.01) reduced in both treated groups. Long-term neuroprotection following ischemia/reperfusion injury to the brain can be obtained by administration of a PARP inhibitor. The motor tests employed in this study can be used as sensitive, objective and reproducible measurements of functional impairment in rats following an ischemic stroke.     

亚低温对大鼠局灶性脑缺血再灌注后多聚腺苷二磷酸核糖聚合酶表达的影响

目的研究亚低温对大鼠局灶性脑缺血再灌注后多聚腺苷二磷酸核糖聚合酶(PARP-1)不同时空表达的影响,进一步探讨亚低温脑保护作用的分子机制。方法线栓法建立大鼠大脑中动脉阻塞再灌注模型,分假手术组、假手术 亚低温组、模型组及模型 亚低温组。应用Western blotting和免疫组化技术分别检测再灌注后不同时相缺血侧皮层PARP-1蛋白的表达与裂解。结果模型组PARP-1蛋白表达量随再灌注时间的延长逐渐增加,至再灌注24h达高峰,然后逐渐减少,再灌注72h时仍高于假手术组的水平;模型组PARP-1蛋白出现裂解,随再灌注时间的延长,裂解逐渐增强。每一相同再灌注时间点,模型 亚低温组PARP-1蛋白表达量和裂解片段含量均低于模型组。结论PARP-1的过度表达和裂解是局灶性脑缺血再灌注神经元死亡的重要分子机制。亚低温可通过抑制PARP-1的过度表达及减少PARP-1的裂解而发挥脑保护作用。     

Pattern of labeling of poly (A)-associated RNA in the CA3 region of rat hippocampus during training

Newly synthesized poly (A)-associated mRNA was analysed in the hippocampal CA₃ pyramidal nerve cells in rats subjected to training to reverse handedness. The nerve cells of trained animals showed a smaller percentage of poly (A)-associated mRNA than did the active controls. However, a specific fraction of this poly (A) hippocampal RANA with a S_E value of around 25 was reproducibly stimulated. This qualitative nerve cell mRNA change is discussed in relation to similar changes in the protein pattern as a function of training.     

Ischaemic pre-conditioning in organotypic hippocampal slice cultures is inversely correlated to the induction of the 72 kDa heat shock protein (HSP72)

In vivo, preconditioning with a sublethal insult can confer resistance to normally lethal episodes of cerebral ischaemia. This phenomenon has been linked with the induction of the 72 kDa heat shock protein (HSP72), but this has not been clearly demonstrated in vitro. We have used organotypic hippocampal slice cultures to investigate whether tolerance to lethal ischaemia is dependent on HSP72. Cultures were maintained in vitro for 14 days, and neuronal damage assessed using propidium iodide fluorescence. Prolonged neuronal HSP72 upregulation occurred following exposure to 30 min ischaemia, 45 min hypoxia and 1 microM kainate, but not 1 microM NMDA or 20 min ischaemia, all sublethal insults. Preconditioning with ischaemia, kainate or hypoxia 24 h prior to lethal ischaemia (45 min) was not protective, and when the delay was increased to 48 h, damage in the CA3 pyramidal cell region was significantly increased compared to cultures exposed to 45 min ischaemia alone. Preconditioning with 20 min ischaemia had no effect on the severity of ischaemic damage. Preconditioning with 1 microM NMDA significantly reduced neuronal damage produced by either 45 or 60 min ischaemia when the delay between insults was 48 h. NMDA pre-treatment also prevented neurotoxicity produced by glutamate (5-10 mM) but not NMDA (10-30 microM). These data suggest that in vitro, the increased expression of HSP72 following some sublethal insults should be considered as a marker of cell stress prejudicial to the survival of neurones subsequently exposed to ischaemia, while tolerance can be produced through mechanisms independent of HSP72 induction.