| Cerebral ischemic stroke, well known is a life-theatening event in which part of the brain does not receive enough oxygen, usually due to a blood clot lodged in a cerebral artery. Cerebral ischemic stroke is a leading cause of death and disability in adults in the world. Among the cerebral ischemic stroke patients who survive approximately half of them are expected to have lasting disabilities as a direct result of the event, requiring assistance in daily living. Cerebral ischemic stroke not only impaired patients health, but also brought burden to the patients'family and society. Therefore, it is an urgent task to further definite the pathophysiological mechanism of cerebral ischemic stroke and to develop effective drugs of preventing and treating cerebral ischemic stroke.By now, there is lack of the novel therapeutic efficacy yet on cerebral ischemic stroke. On the contrary, therapeusis of Traditional Chinese Medicine have special treatment preponderance to improve the neurological function recovery after cerebral ischemic stroke. Catalpol is a active component from Chinese Traditional and Herbal Drugs which has outstanding clinical and experimental effect. Catalpol on protecting cerebral ischemia injury was studied from different levels with the methods of behavioral performance, biochemical assay, immunohistochemistry, enzyme-linked immunosorbent assay, molecular biology, etc. So that it can provide the experiment proof for studying Catalpol pharmacodynamic mechanism of curing cerebral ischemic stroke. Here, in the study we were interested in the effect of Catalpol on the recovery of neurological function after cerebral ischemia injury, using middle cerebral artery occlusion model during a relatively long term of 14 days. We investigated the effect of Catalpol on the pathogenic change and neuron apoptosis in the early reparative period post ischemia in brain.So that we might provide experimental evidence for Catalpol clinical application and explore the rationality of optimal therapeutic window with Chinese Medicine intervention on cerebral ischemic stroke. 1 Effect of Catalpol on the Recovery of Neurological Function after Permanent Cerebral Ischemia in rats and its Primary Mechanisms1.1 Effect of Catalpol on the Recovery of Neurological Function and Injury of Neuron after Permanent Cerebral Ischemia in RatsObjective To investigate the effect of Catalpol on the recovery of neurological function and injury of neuron after permanent cerebral ischemia in rats.Methods Permanent cerebral ischemia was induced by permanent middle cerebral artery occlusion (pMCAO) in rats which were departed into 7 groups: sham, model, CTP 15 mg/kg, CTP 30 mg/kg, CTP 60 mg/kg, NBP 70 mg/kg and BC 7 mg/kg group.Ischemic medication animals received intragastric administration except BC i.p. once a day 3-14 day post-ischemia. Neurological function was evaluated with neurologic deficit, beam-walking, adhesivetape-exposing and bar-grasping performance at 3,7,10 and 14 day post-ischemia. Animals were killed at 14 days post-ischemia.Brains were made into ice brain sections, pathomorph-ology by histologic sections stained with HE and Nissl.Neuron ultrastructure was investigated by transmission electron microscopy.Results Model rats showed severe neurological disfuction compared with sham group. Rats receiving CTP at a dose of 30 mg/kg and 60 mg/kg had a more rapid recovery of neurologic deficit, beam-walking, adhesivetape-exposion and bar-grasping performance than model rats, and the impovement became significant at 14 days after ischemia (p<0.05, p<0.01). Model rats also showed severe injury of neuron compared with sham group. Neuron pathomorphology and ultrastructure were abnormal change in model rats, but there were obvious recovery for the abnormal neuron pathomorphology and ultrastructure in CTP 30 mg/kg and 60mg/kg groups (p<0.05, p<0.01).Conclusin The results demonstrate that CTP can improve the recovery of neurological function follwing focal permanent cerebral ischemia in rats. The ability of CTP might be related to the attenuating of cerebral pathomorphology and ultrastructure damage.1.2 Effect of Catalpol on the Cell Apoptosis in Rats with Permanent Cerebral Ischemia InjuryObjective To study the effects of Catalpol on the cell apoptosis in rats with pMCAO injury. Methods Permanent cerebral ischemia was induced by pMCAO in rats which were departed into 7 groups:sham, model, CTP 15 mg/kg, CTP 30 mg/kg, CTP 60 mg/kg, NBP 70 mg/kg and BC 7 mg/kg group. Ischemic medication animals received intragastric administration except BC i. p. once a day 3-14 day post ischemia. Animals were killed at 14 days post ischemia. Brains were removed for the detection of apoptotic neurons by the method of TUNEL and the cerebral expressions of Bcl-2 and Bax proteins were analyzed with immunohistochemical technique.Results In cerebral cortex area surrounding the ischemic core the apoptosis rates of neuronal cells in CTP groups were significantly lower than model group (p<0.05, p<0.01). The expression of Bcl-2 protein in cerebral cortex area surrounding the ischemic core enhanced quite obviously, the expression of Bcl-2 protein in model group was obviously lower than that in CTP groups (p<0.05, p<0.01). However, expression of Bax in cerebral cortex area surrounding the ischemic core enhanced quite obviously. The expression of Bax in model group were obviously better than that in CTP groups(p<0.05, p<0.01).Conclusin Catalpol intervention treatment can reduce neuronal apoptosis in the ischemic penumbra, through up-regulating Bcl-2 expression and reduced Bax expression, and thus play a role in nerve cell protection.1.3 Effect of Catalpol on Expression of NGF, BDNF, GDNF, VEGF and bFGF Proteins in Rats with Permanent Cerebral Ischemia InjuryObjective To study the effects of Catalpol on expression of NGF, BDNF, GDNF, VEGF and bFGF proteins in rats with pMCAO injury.Methods Permanent cerebral ischemia was induced by pMCAO in rats which were departed into 7 groups:sham, model, CTP 15 mg/kg, CTP 30 mg/kg, CTP 60 mg/kg, NBP 70 mg/kg and BC 7 mg/kg group. Ischemic medication animals received intragastric administration except BC i. p. once a day 3-14 day post ischemia. Animals were killed at 14 days post ischemia. The cerebral expressions of NGF, BDNF, GDNF, VEGF and bFGF proteins were analyzed with immunohistochemical technique.Result Compared with the sham group, NGF, BDNF, VEGF and bFGF proteins expression in model group were reduced (p<0.05, p<0.01), but there was no significantly reduced in GDNF protein expression. Compared with the model group, CTP 30 mg/kg group was significantly increased in NGF, BDNF, VEGF and bFGF proteins expression and reduced in GDNF protein expression (p<0.05, p<0.01).Conclusin CTP might play a role in the treatment of ischemic stroke through increasing the expression of NGF, BDNF, VEGF and bFGF proteins.1.4 Effect of Catalpol on Brain Energy Metabolism in Rats with Permanent Cerebral Ischemia InjuryObjective To study the effects of Catalpol on brain energy metabolism in rats with pMCAO.Methods Permanent cerebral ischemia was induced by pMCAO in rats which were departed into 7 groups:sham, model, CTP 15 mg/kg, CTP 30 mg/kg, CTP 60 mg/kg, NBP 70 mg/kg and BC 7 mg/kg group. Ischemic medication animals received intragastric administration except BC i.p. once a day 3-14 day post ischemia. Animals were killed at 14 days post ischemia, and ischemic cortex Lac and Pyru content, Na+,K+-ATPase and Ca2+,Mg2+-ATPase activity were measured by the method of colorimetry.Result Compared with the sham group, ischemic cortex Lac and Pyru content in model group were increased (p<0.01), but ischemic cortex Na+,K+-ATPase and Ca2+,Mg2+-ATPase activity were reduced (p<0.01). Compared with the model group, CTP groups were significantly increased Pyru, Na+, K+-ATPase and Ca2+, Mg2+-ATPase content (p<0.01) and reduced Lac content (p<0.01).Conclusin CTP might play a role in the treatment of ischemic stroke through increasing the brain energy.2 Effect of Catalpol on the Recovery of Neurological Function in Rats with Focal Cerebral Ischemia-Reperfusion Injury2.1 Effect of Catalpol on the Recovery of Neurological Function and Brain Energy Metabolism in Rats with Focal Cerebral Ischemia-Reperfusion InjuryObjective To investigate the effect of Catalpol on the recovery of neurological function and brain energy metabolism in rats with focal cerebral ischemia-reperfusion injury.Methods Focal cerebral ischemia-reperfusion model was established in rats by reversible right middle cerebral artery occlusion with filament. Right cerebral ischemia was for 2 h and then with 14 days reperfusion. Animals were departed into 6 groups:sham, model, CTP 15 mg/kg, CTP 30 mg/kg, CTP 60 mg/kg and NBP 70 mg/kg group.Ischemia-reperfusion medication animals received intragastric administration once a day 3-14 day post ischemia-reperfusion. Neurological function was evaluated with neurologic deficit, beam-walking, adhesivetape-exposing and bar-grasping performance at 3,7,10 and 14 day post ischemia-reperfusion. Animals were killed at 14 days post ischemia, and ischemic cortex Lac and Pyru content, Na+,K+-ATPase and Ca2+, Mg2+-ATPase activity were measured by the method of colorimetry.Results Model rats showed severe neurological disfuction compared with sham group. Rats receiving CTP at a dose of 30 mg/kg and 60 mg/kg had a more rapid recovery of neurologic deficit, beam-walking, adhesivetape-exposion and bar-grasping performance than model rats, and the impovement became significant at 14 days after ischemia-reperfusion (p<0.05, p<0.01). Compared with the sham group, ischemic cortex Lac and Pyru content in model group were increased significantly(p<0.01), but ischemic cortex Na+,K+-ATPase and Ca2+, Mg2+-ATPase activity were reduced significantly (p<0.01). Compared with the model group, CTP groups were significantly increased Pyru, Na+,K+-ATPase and Ca2+, Mg2+-ATPase and reduced Lac content (p<0.05, p<0.01).Conclusin The results demonstrate that CTP can improve the recovery of neurological function follwing focal permanent cerebral ischemia in rats. The ability of CTP might be related to increase the brain energy metabolism.2.2 Effect of Catalpol on Oxidative stress and Inflammation Reaction in Rats with Focal Cerebral Ischemia-Reperfusion InjuryObjective To investigate the effect of Catalpol on oxidative stress and inflammation reaction in rats with focal cerebral ischemia-reperfusion injury.Methods Focal cerebral ischemia-reperfusion model was established in rats by reversible right middle cerebral artery occlusion with filament.Right cerebral ischemia was for 2 h and then with 14 days reperfusion. Animals were departed into 6 groups:sham, model, CTP 15 mg/kg, CTP 30 mg/kg, CTP 60 mg/kg and NBP 70 mg/kg group. Ischemia-reperfusion medication animals received intragastric administration once a day 3-14 day post ischemia-reperfusion. Animals were killed at 14 days post-ischemia, and ischemic cortex SOD and MDA content were measured by the method of colorimetry, and IL-6, IL-10 and NF-kBp65 activity were assayed by the method of ELISA. Results Compared with the sham group, ischemic cortex MDA and IL-10 content in model group were increased (p<0.01), but ischemic cortex SOD content was reduced(p<0.05). Compared with the model group, CTP groups were significantly increased SOD content and reduced MDA and IL-10 content (p<0.05, p<0.01).Conclusin The results demonstrate that CTP can improve the recovery of neurological function follwing focal permanent cerebral ischemia in rats.The ability of CTP might be related to inhibit oxidative stress and inflammation reaction.3 Effect of Catalpol on the Signaling Pathway of Neuroprotection and neurorestoration after Permanent Cerebral Ischemia in Rats3.1 Effect of Catalpol on the Regulating Pathway of NGF, TrKA, BDNF, TrKB and mRNA in Neuron after Permanent Cerebral Ischemia in RatsObjective To investigate the effect of Catalpol on the expression of NGF, TrKA, BDNF, TrKB and mRNA in neuron after permanent cerebral ischemia in rats.Methods Permanent cerebral ischemia was induced by pMCAO in rats which were departed into 7 groups:sham, model, CTP 15 mg/kg, CTP 30 mg/kg, CTP 60 mg/kg, NBP 70 mg/kg and BC 7 mg/kg group.Ischemic medication animals received intragastric administration except BC i. p. once a day 3-14 day post-ischemia. Animals were killed at 14 days post-ischemia. Western Blot methods were used to evaluate the expression of NGF, TrKA, BDNF and TrKB proteins, and Realtime PCR methods were used to evaluate the expression of NGFmRNA, TrKAmRNA, BDNFmRNA and TrKBmRNA of ischemic cortex.Results Compared with the sham group, the expression of NGF, TrKA, BDNF, TrKB, NGFmRNA, TrKAmRNA, BDNFmRNA and TrKBmRNA of ischemic cortex in model group were reduced(p<0.05, p<0.01). Compared with the model group, CTP groups were significantly increased the expression of NGF, TrKA, BDNF, TrKB, NGFmRNA, TrKAmRNA, BDNFmRNA and TrKBmRNA of ischemic cortex (p<0.05, p<0.01).Conclusin The results demonstrate that CTP can improve the neurological function follwing focal permanent cerebral ischemia in rats. Which might related to the upregulating of the expression of NGF, TrKA, BDNF, TrKB and mRNA of ischemic cortex.3.2 Effect of Catalpol on the PI3K/Akt Signaling Pathway after Permanent Cerebral Ischemia in Rats Objective To investigate the effect of Catalpol on the expression of PI3K, Akt and mRNA in the PI3K/Akt signaling pathway after permanent cerebral ischemia in rats.Methods Permanent cerebral ischemia was induced by pMCAO in rats which were departed into 7 groups:sham, model, CTP 15 mg/kg, CTP 30 mg/kg, CTP 60 mg/kg, NBP 70 mg/kg and BC 7 mg/kg group. Ischemic medication animals received intragastric administration except BC i.p. once a day 3-14 day post ischemia. Animals were killed at 14 days post ischemia. Western Blot methods were used to evaluate the expression of PI3K and Akt proteins, and Realtime PCR methods were used to evaluate the expression of PI3KmRNA and AktmRNA of ischemic cortex.Results Compared with the sham group, the expression of PI3K, Akt and AktmRNA of ischemic cortex in model group were reduced (p<0.05, p<0.01), but the expression of PI3KmRNA was incresed (p<0.01). Compared with the model group, CTP groups were significantly increased the expression of PI3K, Akt, PI3KmRNA and AktmRNA of ischemic cortex (p<0.05, p<0.01).Conclusin The results demonstrate that CTP can stimulate the PI3K/Akt signaling pathway after permanent cerebral ischemia in rats by upregulating the expression of NGF, TrKA, BDNF, TrKB and mRNA. Which might related to decrease neuronal apoptosis, relieve the brain injury and promote neurological function recovery follwing focal permanent cerebral ischemia in rats.The Innovation of the Research1.At the early recovery stage of cerebral ischemia in rats, model rats have significant neurological disfuction, and brain pathophysiological changes are small compare with the subacute stage and the recovery stage of cerebral ischemia in rats. So, to study the effects of Traditional Chinese Medicine on the recovery of neurological function after cerebral ischemia 3-14 day in rats will be better on the cerebral ischemic stroke patients.2. To investigate the neuroprotective and neurorestorative effects of Catalpol on rats with cerebral ischemia injury by received intragastric administration once a day 3-14 day post ischemia. The experiments included neuron apoptosis, neurotrophic factors, energy metabolism failure, oxidative stress, inflammation reaction, etc. So that it can provide the experiment proof for studying Catalpol pharmacodynamic mechanism of curing cerebral ischemic stroke.3. The researcher observed the behavioral performance of neurological deficit and detect level of NGF, TrKA, BDNF, TrKB, PI3K and Akt in neuroprotection and neurorestoration-regulated classic PI3K/Akt signal pathway in brain to explore the effect and mechanism of Catalpol on the recovery of neurological function after cerebral ischemia in rats.
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