| Tissue plasminogen activator (t-PA), is presently the only approved treatment for stroke, and works by dissolving blood clots in the brain. But t-PA must be administered within three hours of symptom onset, increases the risk of brain hemorrhage, and only patients in whom cerebral hemorrhage has been excluded are eligible for treatment. So there is an urgent medical need for new treatment strategies for stroke. In china,Traditional Chinese Medicine is the primary treatment strategy for stroke,it may effectively reduce the behavioral deficits and is of benefit to neuro-function recovery for stroke patients in clinical practice. Radix Rehmanniae is the primary drug to treat stroke,the main active component of it is Catalpol which has exact neuroprotective effect. At the same time, a large number of neuroprotective drugs failed in clinical trials, due to the most important reason that little attention was paid on the neuro-functional recovery (the end point index) during design and research. Therefore, it is essential to research in the therapeutic actions and mechanisms of Catalpol in stroke based on neuroplastisity.OBJECTIVE 1. To observe the effects of Catalpol on the neuronal survival activity and axon outgrowth of the primary cortex in culture;2. To observe the effects of Catalpol on behavioral outcome after permanent occlusion of the distal right middle cerebral artery (pMCAO);3. To observe whether Catalpol could go through the BBB;4. To observe whether Catalpol could enhance axon sprouting or dendrite growth;5. To observe whether Catalpol could effectively improve axon sprouting or enhance synaptic reorganization;6. To investigate the mechanism of Catalpol in post-ischemic stroke based on promoting and inhibition factor.METHODSExperiment I--In vitroAt the cellular level,primary cerebral cortical neurons were cultured, and we observed the effects of Catalpol on survival activity and axon growth of primary cortical neurons in culture, using MTT and micrometer respectively.Experimentâ…¡--In vivo1. To investigate the effects of Catalpol on pMCAO rats, we divided the rats into five groups which included the sham-operation group, the model group, the group treatment with different dose of Catalpol, the control group treatment with Citicoline and the control group treatment with saline. Catalpol was injected with a time-to-treatment of 6h or 24h after pMCAO intraperitoneally, and injected once a day for 7 days with the dose of 1,5.0,10 mg·kg-1. 2. The effects of Catalpol on behavioral outcome was evaluated by a battery of behavioral testing, including Bederson scores, muscle strength scores, Beam walking scores and affected forelimb skilled reaching test, at the different time point of 1, 4, 7, 14, 21d after pMCAO.3. To evaluate the possibility of Catalpol to go through the BBB by HPLC assay.4. To observe the effects on dendrite branch and spine density induced by Catalpol with Golgi-Cox staining.5. To observe the effects on neuronal axon sprouting and synaptic reorganization of Catalpol, We investigated the expression of GAP-43 and P38 protein in pMCAO rats brain for each group using immunofluorescence method and Western blot analysis.6. Ultrastructural changes of nerve cells and synapse were investigated by electron microscopy.7. To observe the effects on long distance neurite sprouitng of Catalpol, We investigated the corticospinal tract (CST) sprouting in cervical enlargement of spinal cord and observed the axon sprouting with cortex surrounding the ischemic lesion no matter it got in touch with the sensorimotor cortex of uninjured side or not, using biotinlated dextran amine labeling tracing and immunofluorescence double labelling method.8. To investigate the mechanism of catalpol on neurorepairment, We investigated the expression of Nogo A,NgR,BDNF and TrkB protein in pMCAO rats brain for each group using immunofluorescence method and Western blot analysis.RESULTSâ… . Results in vitroPrimary cortical neurons was cultured successfully and identified by NF-200 antibody. Its purity was more than 95%.Catalpol didn't affect survival activity of primary cortical neurons by MTT assay. Compared with blank group and Citicoline group, the neurons survival activity in each dose group has no remarkably difference(p>0.05). At the dose of 15mg·ml-1, a statistically significant dose-dependent increase in the axon growth of primary cortical neurons was observed, with the peak occurring at 2.5mg·ml-1 of catalpol, there was significant difference(p<0.05). But cortical neurons axon growth began to decline at dose of 5mg·ml-1, and there was no difference(p>0.05).â…¡. Results in vivo1. Catalpol improved neurobehavioral outcomeTo explore the effects of catalpol on the functional recovery of nerves(NFR) in pMCAO rats, Bederson score, balance function, muscle strength and percent success on forelimb skilled reaching were assessed at 1 d, 4 d, 7 d, 15 d and 21 d after the operation. The percent of success on forelimb skilled reaching in catalpol group was 48.7%±5.4% and 47.3%±4.8% at dose of 5 and 10 mg·kg-1 respectively, it was increased significantly than that of control group and model group (p<0.05). It showed that catalpol can contribute to functional recovery of nerves after pMCAO.2. Catalpol went through the BBBThe ability of Catalpol to go through the blood-brain barrier (BBB) was investigated, Intravenous (i.v.) Catalpol was rapidly distributed into brain. After 40min, Catalpol could be detected by HPLC in CSF.3. Catalpol promoted neuronal axon sprouting and dendrite growth and its possible mechanisms in pMCAO rats brain.(1)Catalpol upregulated GAP-43 protein expression GAP-43 is the marker of axon growth. There was no difference between the group of model or normal saline and sham operation group, although GAP-43 positive cell population in model and normal saline group were higher than that in sham operation group. Compared with model, normal saline and sham operation group, GAP-43 positive cell population in Citicoline and Catalpol group increased obviously (p<0.05) ,among the Catalpol group, Catalpol at the dose of 5 mg·kg-1 remarkably upregulated GAP-43 expression, and there was significant difference (p<0.05). Western blot analysis showed the results similarly. The results suggested that catalpol can promote neuronal axon sprouting.。(2)Catalpol promoted neuronal dendrite growthDendrite branches and spine density of the pyramidal cells in the survival cerebral cortex surrounding the ischemic lesion were examined using Golgi-Cox procedure.The mean of dendrite branching points in the model group (24.67±4.04) was significantly lower than that in sham operation group(14.67±2.08)(p< 0.05). The mean of dendrite branching points in the Catalpol middle dose group was 23.67±3.51, which was significantly higher than that in model group and Citicoline group(p< 0.05). The changes were also observed in the spine density. It demonstrated that Catalpol can increase dendrite arborization and spine density.(3)Catalpol increased the fibers correlation between the lesioned sensorimotor cortex and the unlesioned sensorimotor cortex.Using tracing and immunofluorescence double technique to show the fibers correlation between the unlesioned sensorimotor cortex and the lesioned sensorimotor cortex. The cells and fibers labeled with BDA were shown in green, the cells and axons labeled with GAP-43 were shown in red, BDA and GAP-43 colocalization was shown in yellow. The Pearson's correlation coefficient, the most quantitative estimate of colocalization, showed the correlation between the axon sprouting surrounding the lesioned sensorimotor cortex and the fibers originating in the unlesioned sensorimotor cortex. Our work has shown that green fluorescence intensity was no difference for each group(p>0.05, it means that catalpol didn't increase the number of crossing fibers from the unlesioned sensorimotor cortex to the lesioned sensorimotor cortex. Red fluorescence intensity and the Pearson's correlation coefficient in Catalpol group increased remarkably, compared with model and citicoline group(p<0.05). The results indicatd that Catalpol increased the fibers correlation between the unlesioned sensorimotor cortex and the lesioned sensorimotor cortex.(4)Catalpol increased the number of midline crossing fibers in the spinal cordTracing was used to show that CST (corticospinal tract) in cervical enlargement centralize at dorsal funiculus of spinal cord. Under normal circumstances, there are few midline crossing fibers. Midline crossing fibers in the model group were increased more than in the sham operation group(p<0.05), it showed the role of midline crossing fibers originating in the unlesioned sensorimotor cortex during the behavioral recovery after cerebral ischemia; compared with the model and the citicoline group, midline crossing fibers treatment with Catalpol were increased obviously(p<0.05). It demonstrated that Catalpol promoted the long distance neurite sprouting after cerebral ischemia.(5)Catalpol promoted CST (corticospinal tract) axonal sprouting in the spinal cord Using tracing and immunofluorescence double technique to show the CST axonal sprouting originating in the unlesioned sensorimotor cortex in the cervical enlargement of the spinal cord, CST labeled with BDA was shown in green, sprouting axonal fibers were shown in red, BDA and GAP-43 colocalization was shown in yellow. The more red fluorescence intensity was , the higher expression of GAP-43 was; the more the Pearson's correlation coefficient was, the more new axonal was formed. Our work has shown that red fluorescence intensity and the Pearson's correlation coefficient in model group were higher than that in sham operation group (p<0.05); Catalpol group increased remarkably, compared with model and citicoline group(p<0.05). The results indicated that cerebral ischemia damage stimulated per se uninjured side CST axonal sprouting, and Catalpol promoted and sustained CST axonal sprouting in the cervical enlargement of the spinal cord.(6)Catalpol downregulated the Nogo-A protein expressionImmunofluorescence results demonstreated that Nogo-A positive cell was red labled with Cy3. The number of Nogo-A positive cell increased obviously, there was significant difference between the model group and the sham operation group(p>0.05); but Catalpol at dose of 5,10 mg·kg-1 reduced the number of Nogo-A positive cell remarkably, compared with the model group, the normal saline group and the Citicoline group, there was significant difference(P<0.05). The same results were obtained by western blot analysis. It showed that Catalpol can downregulate the Nogo-A protein expression in the unlesioned sensorimotor cortex after cerebral ischemia(7)Catalpol downregulated the NgR protein expression Immunofluorescence results demonstreated that NgR positive cell was green labled with FITC. There were no differences among the model group, the normal saline group and the sham operation group(p>0.05), it showed that the NgR protein expression approached the normal level at 15 days after stroke. But Catalpol at dose of 5, 10 mg·kg1 reduced the number of NgR positive cell remarkably, compared with the model group, the normal saline group and the Citicoline group, there was significant difference(p<0.05). The similar results were obtained by western blot analysis. It showed that Catalpol can downregulate the NgR protein expression in the unlesioned sensorimotor cortex after cerebral ischemia4. Synaptic reorganization and possible mechanisms induced by Catalpol(1)Catalpol upregulated the protein expression of synaptophysin Synaptophysin(P38), a marker of synapse, which was labeled with FITC and shown in punctiform or granules with green. There were no differences among the model group, the normal saline group and the sham operation group(p>0.05); but Catalpol at dose of 5 mg·kg-1 increased the IOD of synaptophysin(P38)positive expression obviously(P<0.05), compared with the citicoline group. It demonstrated that Catalpol can promotes the synaptophysin(P38)expression after cerebral ischemia.(2)Catalpol increased synapse numbers in sensorimotor cortex neuropilTo observe the synapse changes in the neuropil of the sensorimotor cortex, Electron microscope was employed. Compared with that in uninjured side, synapse numbers in sensorimotor cortex neuropil reduced for each group, excluding the sham operation group. There was no difference in uninjured side between the model group and the sham operation group(p>0.05),but in injured side, synapse numbers were less than that in the sham operation group obviously(p<0.05), it showed that there existed synapse loss after cerebral ischemia. Compared with the model group and the normal saline group, Catalpol at dose of 5 mg·kg-1 increased synapse numbers in the neuropil of the sensorimotor cortex obviously(p<0.05), but there was no difference between the catalpol and Citicoline group(p>0.05). It demonstrated that Catalpol can promote the synaptic reorganization after cerebral ischemia.(3)Catalpol promoted axon sprouting to form synapseUsing immunofluorescence double technique with LCSM to show the axon sprouting formed synapse. P38 labeled with FITC was shown in green. GAP-43, new axonal sprouting fiber marker, labeled with Cy3 was shown in red, P38 and GAP-43 colocalization was shown in yellow. The more the Pearson's correlation coefficient was, the more new presynaptic terminus was formed. Our work has shown that the Pearson's correlation coefficient in the model group has great than in the sham operation group (p<0.05), it meant cerebral ischemia damage stimulated per se axonal sprouting and formed new synapse connection. The Pearson's correlation coefficient in the Catalpol group increased remarkably, compared with the model group and the citicoline group(p<0.05). It showed that Catalpol can enhence the new axon sprouting to form new synapse connection in effect.(4)Catalpol upregulated the BDNF protein expressionImmunofluorescence results demonstreated that BDNF positive cell was red labled with Cy3. BDNF positive cell in the model group was increased, compared with the sham operation group, there was significant difference(p<0.05); compared with the model group, Catalpol at each dose group remarkably increased BDNF positive cell, there was significant difference(p<0.05); compared with the Citicoline group, Catalpol at dose of 5 and 10 mg·kg-1 increased BDNF positive cell obviously(p<0.05). The similar results were obtained by Western blot analysis. It showed that Catalpol upregulated the BDNF protein expression in brain tissue after cerebral ischemia.(5)Catalpol upregulated the TrkB protein expressionImmunofluorescence results demonstreated that TrkB positive cell was green labled with FITC. TrkB positive cell population in the model group was reduced, compared with the sham operation group, but there was no difference(P>0.05); compared with the normal saline group, and the Citicoline group, catalpol at the dose of 5 mg·kg-1 remarkably upregulated TrkB positive cell population, there was significant difference(P<0.05). The similar results were obtained by western blot analysis. It showed that Catalpol upregulated the TrkB protein expression in brain tissue after cerebral ischemia.CONCLUSIONCatalpol can downregulate the Nogo-A and NgR protein expression in brain tissue after cerebral ischemia, and enhance the neuronal axon sprouting and dendrite growth. Catalpol can upregulate BDNF and TrkB protein expression, and enhance the synaptic reorganization. Catalpol can improve behavioral outcome and neural plasticity in rats with cerebral ischemia obviously. The results suggest that Catalpol has great potential as a candidate drug for ischemic stoke by promoting neural repair.
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