| Partâ… Studies of Electro-acupuncture in Different Time Windows on Rats with Cerebral InfarctionObjective:To study the best time window and its possible mechanism of electro-ac upuncture therapy to rats with cerebral infarction.Method:One hundred SD rats are randomly divided into four groups:normal group (n=15), sham-operated group (n=60), model group (n=60) and electro-acupuncture tre ated group (n=60), each group is then divided into five sub-groups according to diff erent time of postoperative treatment:6 h,12 h,24 h,48 h and 72 h. The model group and electro-acupuncture treated group are sutured to cerebral infarction model established by left middle cerebral artery occlusion (MCAO/R), perfusion starts 90mi n later after cerebral ischemia. Each sub-group of electro-acupuncture treated group i s electro-acupunctured according to their individual time of postoperative treatment, while all the other groups including sub-groups are treated at the same time withsha m electro-acupuncture. Treatment lasts for 14d. After treatment each sub-group isscal ed by Modified Neurological Severity Scores (NSS). The Morris water navigation ta sk is applied to examine samples'spatial learning and memory, while infarctionsize and changes in local metabolism are monitored by functional NMRI. In infarcted ar ea, the number of apoptotic cells is measured by TUNEL method, the expression of Caspase-3 and Nestin is assayed by immunohistochemical treatment, and real-time fl uorescent quantitative PCR is used to measure the expression of Bcl-2 mRNAand B DNF mRNA.Results:Comparison of outcomes between the same time window and different grou ps shows that:The NSS score, infarction size, number of apoptotic cells, the expres sion of Caspase-3 and escape latency in electro-acupuncture treated group are all co nsiderably lower than in model group (P<0.05), while the expressions level of NA A/Cr, Bcl-2 mRNA, BDNF mRNA and positive Nestin cell number are all significa ntly higher than in model group (P<0.05). Comparison of outcomes between the sa me group and different time windows shows that:There is significant difference bet ween sub-groups within 24h and sub-groups outside 24h in NSS scores (P<0.05). T here is also significant difference (P<0.05) in the data above, between different sub-groups divided according to their individual time of postoperative treatment (exceptio ns including Naa/Cr ratio between sub-group 48h and 72h, expression level of Casp ase-3 between sub-group 12h and 24h, positive Nestin cells between sub-group 12h and 24h). In sub-group 12h after postoperative treatment, escape latency, infarction s ize and apoptotic cell number are relatively lower, the expression level of Caspase-3 is relatively lower in sub-group 24h after postoperative treatment, while Naa/Cr rati o, positive Nestin cell number, the expression level of BDNFmRNA and Bcl-2mRN A are relatively higher in sub-group 12h after postoperative treatment.Conclusion:Electro-acupuncture is able to improve the recovery of neurological fun ction in rats with infarction, and there is a time window effect during the therapy. The best time window is presumably to be 12h-24h. The mechanism of electro-acup uncture therapy is likely to be improving local metabolism by shrinking the infarctio n size, and improving the recovery of neurological function by inhibiting the apopto sis of neurons and activating endogenous neural stem cells.Part II Studies of Transcranial Magnetic Stimulation in Different Time Windows on Rats with Cerebral Infarction Objective:To study the best time window and its possible mechanism of transcrani al magnetic stimulation therapy to rats with cerebral infarctionMethod:One hundred SD rats are randomly divided into four groups:normal group (n=15), sham-operated group (n=60), model group (n=60) and transcranial magnetic stimulation treated group (n=60), each group is then divided into five sub-groups ac cording to different time of postoperative treatment:6 h,12 h,24 h,48 h and 72 h. The model group and transcranial magnetic stimulation treated group are sutured to cerebral infarction model established by left middle cerebral artery occlusion (MC AO/R), perfusion starts 90min later after cerebral ischemia. Each sub-group of transc ranial magnetic stimulation treated group is treated with transcranial magnetic stimul ation according to their individual time of postoperative treatment, while all the othe r groups including sub-groups are treated at the same time with sham transcranial m agnetic stimulation. Treatment lasts for 14d. After treatment each sub-group is scaled by Modified Neurological Severity Scores (NSS). The Morris water navigation task i s applied to examine samples'spatial learning and memory, while infarction size an d changes in local metabolism are monitored by functional NMRI. In infarcted area, the number of apoptotic cells is measured by TUNEL method, the expression of Ca spase-3 and Nestin is assayed by immunohistochemical treatment, and real-time fluor escent quantitative PCR is used to measure the expression of Bcl-2 mRNA and BD NF mRNA.Results:Comparison of outcomes between the same time window and different grou ps shows that:The NSS score, infarction size, number of apoptotic cells, the expres sion of Caspase-3 and escape latency in transcranial magnetic stimulation treated gro up are all considerably lower than in model group (P<0.05), while the expressions level of NAA/Cr, Bcl-2 mRNA, BDNF mRNA and positive Nestin cell number are all significantly higher than in model group (P<0.05). Comparison of outcomes bet ween the same group and different time windows shows that:There is significant di fference (P<0.05) in the data above, between different sub-groups divided according to their individual time of postoperative treatment (exceptions including NSS score b etween sub-group 6h and 72h, between sub-group 12h,24h and 48h, infarction size, Naa/Cr ratio and expression level of Caspase-3 between sub-group 12h and 24h). In sub-group 24h after postoperative treatment, NSS score, escape latency, infarction siz e, apoptotic cell number and expression level of Caspase-3 are relatively lower, whil e Naa/Cr ratio and expression level of BDNFmRNA is relatively higher. Positive Ne stin cell number and the expression level of Bcl-2mRNA are relatively higher in su b-group 12h after postoperative treatment.Conclusion:Transcranial magnetic stimulation is able to improve the recovery of ne urological function in rats with infarction, and there is a time window effect duringt he therapy. The best time window is presumably to be 12h-24h. The mechanism of transcranial magnetic stimulation therapy is likely to be activating endogenous neural stem cells, forming new synapses, improving local metabolism and inhibiting the ap optosis of neurons in order to shrink the infarction size and improve the recovery o f neurological function.Partâ…¢Studies of electro-acupuncture combined with transcranial magnetic stimulation in Different Time Windows on Rats with Cerebral InfarctionObjective:To study the best time window and its possible mechanism of electro-ac upuncture combined with transcranial magnetic stimulation therapy to rats with cereb ral infarctionMethod:One hundred SD rats are randomly divided into four groups:normal group (n=15), sham-operated group (n=60), model group (n=60) and electro-acupuncture co mbined with transcranial magnetic stimulation treated group (n=60), each group is th en divided into five sub-groups according to different time of postoperative treatmen t:6 h,12 h,24 h,48 h and 72 h. The model group and electro-acupuncture com bined with transcranial magnetic stimulation treated group are sutured to cerebral inf arction model established by left middle cerebral artery occlusion (MCAO/R), perfus ion starts 90min later after cerebral ischemia. Each sub-group of electro-acupuncture combined with transcranial magnetic stimulation treated group is treated with electro-acupuncture combined with transcranial magnetic stimulation according to their indivi dual time of postoperative treatment, while all the other groups including sub-groups are treated at the same time with sham electro-acupuncture combined with transcrani al magnetic stimulation. Treatment lasts for 14d. After treatment each sub-group is s caled by Modified Neurological Severity Scores (NSS). The Morris water navigation task is applied to examine samples'spatial learning and memory, while infarction si ze and changes in local metabolism are monitored by functional NMRI. In infarcted area, the number of apoptotic cells is measured by TUNEL method, the expression of Caspase-3 and Nestin is assayed by immunohistochemical treatment, and real-time fluorescent quantitative PCR is used to measure the expression of Bcl-2 mRNA and BDNF mRNA.Results:Comparison of outcomes between the same time window and different grou ps shows that:The NSS score, infarction size, number of apoptotic cells, the expres sion of Caspase-3 and escape latency in electro-acupuncture combined with transcran ial magnetic stimulation treated group are all considerably lower than in model grou p (P<0.05), while the expressions level of NAA/Cr, Bcl-2 mRNA, BDNF mRNA a nd positive Nestin cell number are all significantly higher than in model group (P< 0.05). Comparison of outcomes between the same group and different time windows shows that:There is significant difference (P<0.05) in the data above, between diffe rent sub-groups divided according to their individual time of postoperative treatment (exceptions including NSS score between sub-group 6h,12h and 72h, between sub-g roup 24h and 48h, infarction size and Naa/Cr ratio between sub-group 24h and 48h). In sub-group 24h after postoperative treatment, NSS score, escape latency, infarctions ize, apoptotic cell number and expression level of Caspase-3 are relatively lower, w hile positive Nestin cell number, Naa/Cr ratio and expression level of Bcl-2mRNA i s relatively higher. The expression level of BDNFmRNA is relatively higher in sub- group 48h after postoperative treatment.Conclusion:Electro-acupuncture combined with transcranial magnetic stimulation is able to improve the recovery of neurological function in rats with infarction, and th ere is a time window effect during the therapy, other time would not result in best curing effect. The best time window is presumably to be 24h-48h. The mechanism of electro-acupuncture combined transcranial magnetic stimulation therapy is likely to be activating endogenous neural stem cells and forming new synapses to recover da maged nerve functions, reducing neuron apoptosis caused by cerebral ischemia and s hrinking the infarction size.Part IV Studies of Passive Movement Therapy in Different Time Windows on Rats with Cerebral InfarctionObjective:To study the best time window and its possible mechanism of passive mo vement therapy to rats with cerebral infarctionMethod:One hundred SD rats are randomly divided into four groups:normal group (n=15), sham-operated group (n=60), model group (n=60) and passive movement trea ted group (n=60), eachgroup is then divided into five sub-groups according to differ ent time of postoperative treatment:6 h,12 h,24 h,48 h and 72 h. The model gr oup and passive movement treated group are sutured to cerebral infarction model est ablished by left middle cerebral artery occlusion (MCAO/R), perfusion starts 90min later after cerebral ischemia. Each sub-group of passive movement treated group is t reated with passive movement according to their individual time of postoperative tre atment, while all the other groups including sub-groups are treated at the same time with sham passive movement. Treatment lasts for 14d. After treatment each sub-grou p is scaled by Modified Neurological Severity Scores (NSS). The Morris water navi gation task is applied to examine samples'spatial learning and memory, while infarc tion size and changes in local metabolism are monitored by functional NMRI. In in farcted area, the number of apoptotic cells is measured by TUNEL method, the expr ession of Caspase-3 and Nestin is assayed by immunohistochemical treatment, and real-time fluorescent quantitative PCR is used to measure the expressi on of Bcl-2mRNA and BDNF mRNA.Results:Comparison of outcomes between the same time window and different grou ps shows that:The NSS score, infarction size, number of apoptotic cells, the expres sion of Caspase-3 and escape latency in passive movement treated group are all con siderably lower than in model group (P<0.05), while the expressions level of NAA /Cr, Bcl-2 mRNA, BDNF mRNA and positive Nestin cell number are all significantl y higher than in model group (P<0.05). Comparison of outcomes between the same group and different time windows shows that:There is significant difference (P<0.05) in the data above, between different sub-groups divided according to their individual time of postoperative treatment (exceptions including NSS score between sub-group 6h,12h and 72h, between sub-group 24h and 48h, infarction size and Naa/Cr ratio between sub-group 24h and 48h). In sub-group 24h after postoperative treatment, NS S score, escape latency, infarction size, apoptotic cell number and expression level o f Caspase-3 are relatively lower, while positive Nestin cell number, Naa/Cr ratio and expression level of Bcl-2mRNA and BDNFmRNA is relatively higher.Conclusion:Passive movement is able to improve the recovery of neurological func tion in rats with infarction, and there is a time window effect during the therapy, e arly start would not result in best curing effect. The best time window is presumabl y to be 24h. The mechanism of passive movement therapy is likely to be reducing neuron apoptosis around ischemic penumbra, shrinking the infarction size, and activa ting endogenous neural stem cells around infarction cortex to help neurotrophic facto r expression, in order to recover damaged neurons and improve their resurrection.
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