| Ischemic stroke, which is the most common type of stroke, is one of the main causes of death and disability in adults. Secondary brain injury after cerebral ischemia is a cascade including inflammation, oxidative stress,calcium overload, excitatory toxicity, apoptosis and a series of complex factors. These factors influence each other and form as the second-strike on brain tissue, then exacerbate the disease. Therefore, taking the key link of the cascade as a target and inhibiting the progressive death of cells in the penumbra may protect the brain and have potential value in clinical application.Retigabine(RTG), which has effects of resting the membrane potential and anti-excitability, is currently used as an adjuvant therapy for partial epilepsy in adult,. In addition to being a new anti-epileptic drug, retigabine has potential clinical value in treatment of neural pain, neurodegenerative disease,dystonia and so on. In cultured hippocampal slice with serum deprivation and oxygen deprivation, retigabine exerted an anti-oxidant effect and reduced cell death. We hypothesized that, after cerebral ischemia, cellular energy could be preserved by retigabine via limiting the discharge of neurons, which could reduce the brain damage in the case of ischemia.Ischemic neuronal death includes cell necrosis and apoptosis. After ischemic stroke, cells in the penumbra with relatively mild injury may undergo independent and orderly death, which is regulated by genes.Oxidative stress damage, excessive inflammatory response, large amount of glutamate, calcium overload and so on are likely to trigger cell apoptosis signal to make the cell "suicide". The DNA of apoptotic cell is degraded by apoptotic endonuclease and then apoptotic bodies formed. Studies showed thatone of cysteinyl aspartate specific proteinase(caspase) family, caspase-3, was the main performer of apoptosis, its activation cleaved various protein and led to cell apoptosis. The level of caspase protein in the brain after ischemic stroke was increased, and inhibiting the activition of caspase alleviated the ischemic injury and protected the brain.The process of cell apoptosis involves expression of pro-apoptotic factors and inhibition of anti-apoptotic factors. After ischemic stroke, expression level of Bax which is a kind of pro-apoptotic protein in the brain is increased.Inhibition of Bax or overexpression of Bcl-2 may play a protective role in ischemic brain. Calpain is a general term for a family of calcium-dependent cysteine endopeptidase, it is activated by elevated intracellular calcium and cause apoptosis through a variety of ways. Calpain can also cleave Bcl-2protein to promote cell apoptosis, and inhibiting the activity of calpain can protect the brain.Male, healthy adult CD-1 mice were used as the objects, permanent middle cerebral artery occlusion in mice were established by the suture method. After administrated with retigabine, neurological deficit scores,infarct volumes and brain water contents of mice were evaluated to observe whether retigabine has protective effect on brain of cerebral ischemia. DNA fracture and expression of cleaved caspase-3 protein in the ischemic brain of mice were measured to observe the effect of retigabine on cell apoptosis.Expression of Bax, calpain-1 and Bcl-2 in the ischemic brain of mice were measured to observe the effect of retigabine on pro-apoptotic and antiapoptotic factors, to explore the possible mechanism of retigabine, and to provide theoretical support and experimental basis for the further study and clinical application of retigabine. This study is divided into three parts, each part of the contents are summarized as follows.Part one The neuroprotective effect of retigabine in the mouse model of cerebral ischemiaObjective: To observe the effect of retigabine in the mouse model of focal cerebral ischemia through evaluating neurological deficit scores, infarct volumes and brain water contents.Methods:1 Male, healthy adult CD-1 mice were used as the objects, permanent middle cerebral artery occlusion(MCAO) in mice were established by the suture method. CD-1 mice were randomly divided into five groups: Sham group, MCAO group, RTG5 group, RTG10 group and RTG20 group. Mice in Sham group received the sham operation and intraperitoneal injection of normal saline at 1 hour after the operation. Mice in MCAO group received the operation of middle cerebral artery occlusion and intraperitoneal injection of normal saline at 1 hour after the operation. Mice in RTG5 group received the operation of middle cerebral artery occlusion and intraperitoneal injection of RTG at 5mg/kg at 1 hour after the operation. Mice in RTG10 group received the operation of middle cerebral artery occlusion and intraperitoneal injection of RTG at 10mg/kg at 1 hour after the operation. Mice in RTG15 group received the operation of middle cerebral artery occlusion and intraperitoneal injection of RTG at 15mg/kg at 1 hour after the operation. Neurological defect scores of mice in each group were evaluated at 24 hours after the operation,cerebral infarction volumes of mice in each group were measured by 2,3,5-triphenyltetrazolium chloride(TTC) staining at 24 hours after the operation.Brain water contents were measured by wet-dry method at 24 hours after the operation.2 To further study the neuroprotective time window of RTG in the mouse model of focal cerebral ischemia, we also observed the effect of RTG when administrated at different times after cerebral ischemia. CD-1 mice were randomly divided into five groups: MCAO group, RTG-0h group, RTG-1h group, RTG-2h group and RTG-3h group. Mice in MCAO group received the operation of middle cerebral artery occlusion. Mice in RTG-0h group receivedthe operation of middle cerebral artery occlusion and intraperitoneal injection of RTG at 10mg/kg after the operation immediately. Mice in RTG-1h group received the operation of middle cerebral artery occlusion and intraperitoneal injection of RTG at 10mg/kg at 1 hour after the operation. Mice in RTG-2h group received the operation of middle cerebral artery occlusion and intraperitoneal injection of RTG at 10mg/kg at 2 hours after the operation.Mice in RTG-3h group received the operation of middle cerebral artery occlusion and intraperitoneal injection of RTG at 10mg/kg at 3 hours after the operation. Mice without RTG administration were injected intraperitoneally with saline at each time point. Cerebral infarction volumes of mice in each group were measured by TTC staining at 24 hours after the operation.Results:1 Neurological deficit scores of MCAO group were significantly higher than Sham group which were 0, the difference was statistically significant(P <0.05). Neurological deficit scores of RTG5 group and RTG10 group were decreased significantly compared with MCAO group, the difference was statistically significant(P < 0.05). There was no significant difference in neurological deficit scores of RTG15 group compared with MCAO group, the difference was not statistically significant(P > 0.05).2 After TTC staining, the brain slices of Sham group were red uniformly,the MCAO group showed a large white infarct area on the right cortex and basal ganglia. Compared with MCAO group, the infarct volumes of RTG5 group and RTG10 group were reduced, the difference was statistically significance(P < 0.05). There was no significant difference in the infarct volumes of RTG15 group compared with MCAO group, the difference was not statistically significant(P > 0.05).3 After measurement of brain water contents, compared with MCAO group, the brain water contents of RTG5 group and RTG10 group were reduced, the difference was statistically significance(P < 0.05). There was no significant difference in the brain water contents of RTG15 group compared with MCAO group, the difference was not statistically significant(P > 0.05).The results showed that RTG administrated at 5mg/kg and 10mg/kg conferred neuroprotection, we focused on the effect of RTG at 10mg/kg for the subsequent experiments.4 After TTC staining, the infarct volumes of RTG-0h group, RTG-1h group, RTG-2h group and RTG-3h group were significantly reduced compared with MCAO group, the difference was statistically significant(P <0.05). There was no significant difference in the infarct volumes among RTG-0h group, RTG-1h group, RTG-2h group and RTG-3h group, the difference was not statistically significant(P > 0.05). The results showed that RTG at10mg/kg conferred neuroprotection when administrated at 0-3 hours after cerebral ischemia.Part two The effect of retigabine on apoptosis in the mouse model of cerebral ischemiaObjective: To evaluate the effect of RTG on apoptosis in the mouse model of focal cerebral ischemia by measuring the nuclear DNA fragmentation in the penumbra of ischemic brain and the protein levels of cleaved caspase-3.Methods: Male, healthy adult CD-1 mice were used as the objects,permanent middle cerebral artery occlusion in mice were established by the suture method. CD-1 mice were randomly divided into three groups: Sham group, MCAO group, RTG group. Mice in Sham group received the sham operation and intraperitoneal injection of normal saline at 1 hour after the operation. Mice in MCAO group received the operation of middle cerebral artery occlusion and intraperitoneal injection of normal saline at 1 hour after the operation. Mice in RTG group received the operation of middle cerebral artery occlusion and intraperitoneal injection of RTG at 10mg/kg at 1 hour after the operation. At 24 hours after the operation, TUNEL kit was used to detect the nuclear DNA fragmentation in the penumbra of ischemic brain.Western-blot was used to measure the protein levels of cleaved caspase-3.Results:1 Numbers of TUNEL-positive cells in brain slices of mice in MCAO group were significantly higher compared with Sham group, the difference was statistically significant(P < 0.05). Numbers of TUNEL-positive cells in brain slices of mice in RTG group were significantly lower compared with MCAO group, the difference was statistically significant(P < 0.05).2 After Western-blot analysis, cleaved caspase-3 was expressed in the ischemic brain of mice in each group. The protein levels of cleaved caspase-3in MCAO group were significantly increased compared with Sham group, the difference was statistically significant(P < 0.05). The protein levels of cleaved caspase-3 in RTG group were significantly decreased compared with MCAO group, the difference was statistically significant(P < 0.05).Part three Retigabine down-regulated the expression of calpain-1 and Bax, up regulated Bcl-2 in the mouse model of cerebral ischemiaObjective: To observe the effect of RTG on anti- or pro- apoptotic factors in focal ischemic brain of mice by measuring the expression of calpain-1, Bax, Bcl-2, and to explore its possible mechanism of neuroprotection.Methods: Male, healthy adult CD-1 mice were used as the objects,permanent middle cerebral artery occlusion in mice were established by the suture method. CD-1 mice were randomly divided into three groups: Sham group, MCAO group, RTG group. Mice in Sham group received the sham operation and intraperitoneal injection of normal saline at 1 hour after the operation. Mice in MCAO group received the operation of middle cerebral artery occlusion and intraperitoneal injection of normal saline at 1 hour after the operation. Mice in RTG group received the operation of middle cerebral artery occlusion and intraperitoneal injection of RTG at 10mg/kg at 1 hour after the operation. At 24 hours after the operation, q RT-PCR was used to detect the mRNA levels of calpain-1, Bax and Bcl-2. Western-blot was used to measure the protein levels of calpain-1, Bax and Bcl-2.Results:1 After qRT-PCR analysis, the mRNA levels of calpain-1 and Bax in MCAO group were significantly increased compared with Sham group, the difference was statistically significant(P < 0.05). The mRNA levels of calpain-1 and Bax in RTG group were significantly decreased compared with MCAO group, the difference was statistically significant(P < 0.05). The mRNA levels of Bcl-2 in RTG group were significantly increased compared with MCAO group, the difference was statistically significant(P < 0.05).2 After Western-blot analysis, calpain-1, Bax and Bcl-2 were expressed in the ischemic brain of mice in each group. The protein levels of calpain-1and Bax in MCAO group were significantly increased compared with Sham group, the difference was statistically significant(P < 0.05). The protein levels of calpain-1 and Bax in RTG group were significantly decreased compared with MCAO group, the difference was statistically significant(P < 0.05). The protein levels of Bcl-2 in RTG group were significantly increased compared with MCAO group, the difference was statistically significant(P < 0.05).3 After the immunohistochemistry analysis, the positive cells of calpain-1,Bax and Bcl-2 were expressed in the ischemic brain of mice in each group.The positive cells of calpain-1 and Bax in MCAO group were increased compared with Sham group. The positive cells of calpain-1 and Bax in RTG group were decreased compared with MCAO group. The positive cells of Bcl-2 in RTG group were increased compared with MCAO group.Conclusions:1 The mouse model of focal cerebral ischemia was successfully established by the operation of permanent right middle cerebral artery occlusion, intraperitoneal injection of RTG at 5mg/kg and 10mg/kg at 1 hour after the operation significantly reduced the neurological deficit scores, infarct volumes and brain water contents. When administrated at 10mg/kg at 0-3hours after the operation, RTG protected the brain from focal cerebral ischemia.2 Numbers of TUNEL-positive cells in the penumbra of RTGadministrated mice were decreased, RTG inhibited the nuclear DNAfragmentation in the penumbra. RTG also reduced the protein levels of cleaved caspase-3 in focal cerebral ischemia, it inhibited the activation of caspase-3. RTG protected the ischemic brain by attenuating the apoptosis of the brain tissue.3 RTG down-regulated the mRNA and protein levels of calpain-1 and Bax, and up-regulated the mRNA and protein levels of Bcl-2 in the mouse model of focal cerebral ischemia. RTG attenuated the apoptosis of the ischemic brain tissue, this provided further support for the neuroprotectve effect of RTG in the mouse model of focal cerebral ischemia. |
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