| BackgroundStroke,also called cerebral apoplexy,is one of the major diseases that threaten the human life and health seriously.Researches reveal that the annually new cases of stroke in China are increasing every year with a number of about 2.5 million,and the number of deaths from stroke accounted for 22.45%of the total deaths,which affects people’s living standard seriously and causes heavy economic burden to the family as well as the society.Cerebral infarction accounts for about three quarters of the total type in the pathological classification of stroke,and the syndrome of Qi deficiency and blood stasis is the most common one in the syndrome differentiation of Chinese Medicine.Therefore,the Qi-deficiency and Blood-stasis Syndrome of cerebral infarction should be paid more attention in the study of cerebrovascular disease.To establish combination of disease and syndrome animal model is one of the most important means on disease study and new drug development in Chinese Medicine.Multifactor evaluation rat model of Qi-deficiency and Blood-stasis Syndrome of cerebral infarction are commonly used in current researches,and some progresses have been made,indeed.However,the lack of normalization and standardization in modeling methods and evaluation systems,unclear pathologic mechanism due to complicated disturbing factors,and the certain differences compared with natural state of disease makes the existing models difficult to be accepted.Based on previous research,this study aimed to establish a stable and reliable combination rat model of multiple cerebral infarctions with Qi-deficiency and Blood-stasis Syndrome,and to supplement the evaluation system of modeling.Besides,on reference to the target and subject of“973”project,this study investigated the changes of biological indicators associated with qi and blood during the formation of the combination model,and provided valuable material for research and development of new drug,as well as illustrated the material basis of Traditional Chinese Medicine.Part 1 Establishment of combination rat model of multiple cerebral infarctions with Qi-deficiency and Blood-stasis Syndrome1.ObjectiveTo establish a combination rat model of multiple cerebral infarctions with Qi-deficiency and Blood-stasis Syndrome using different modeling factors based on the basic theory of TCM.2.Material and methodsSD male rats were randomly divided into 5 groups:A.the Normal Group(N):normally fed,n=11;B.Sham Group(S):Received incision and threading,without injection of fluorescent microspheres,normally fed,n=11;C.Sleep Deprivation Group(D):16h of sleep deprivation every day for 5 weeks,n=11;D.Multiple Cerebral Infarction Group(MCI):multiple cerebral infarction operation was performed after normally fed for 1 week and followed by normally fed for 4 weeks after operation,n=14;E.Combination of Sleep Deprivation and Multiple Cerebral Infarction group(Combination):multiple cerebral infarction operation was performed after 16h of sleep deprivation every day for 1 week,followed by sleep deprivation for 4 weeks after operation,n=14.At the end of the experiment,the following indicators were collected or detected:general status,body weight,grasping strength of fore limb,duration of loaded swimming to exhaust,spontaneous activity,pulse amplitude,blood viscosity,R,G,B value of tongue image,coagulation function,neurologic deficit score,fluorescent microspheres distribution on the surface of brain,infarct distribution on the brain slice,pathological structure of brain ultrastructure of neuronal cell,MDA content,Na+-k+-ATPase,Ca2+-ATPase and SOD activity.3.Results1.General status:Compared with the normal group and the sham group,the rats in the deprivation group,the MCI group and combination group manifested as lazy and apathetic,especially the in the combination group.2.Qi deficiency indexes:Compared with the sham group,the body weight,grasping strength of fore limb,duration of loaded swimming to exhaust,spontaneous activity and pulse amplitude of rats in the two single-factor model groups and in the combination group were significantly decreased(P<0.05 or P<0.01),moreover,the above indicators in the combination group were significantly decreased compared with the other two single-factor model groups(P<0.05 or P<0.01)3.Blood stasis indexes:①R,G,B value of tongue image:Compared with the sham group,the R,G,B value of tongue image of rats in the three model groups were significantly decreased(P<0.05 or P<0.01);and the R,G,B value of tongue image of rats in the combination group were significantly decreased compared with the other two single-factor model groups(P<0.05 or P<0.01).②blood rheology indexes:Compared with the sham group,the whole blood viscosity under the shear rate of 5s-1 of rats in the three model groups was significantly increased(P<0.05),while there was no significant difference in blood viscosity among the three model groups at each shear rate.③Coagulation function:Compared with the sham group,TT of rats in the deprivation group was significantly decreased(P<0.05),PT、APTT、TT of rats in the MCI model group and combination group were significantly decreased(P<0.05 or P<0.01),PT of rats in the combination group was significantly decreased compared with the other two single-factor model groups(P<0.05).4.Brain infarction Indexes:①neurologic deficit score:The neurologic function of rats in the Normal group,Sham group and Sleep deprivation group are normal,and the neurologic function of rats in the MCI group and combination group was defected,while there was no significant difference in the neurologic deficit score between the two groups 24 hours after operation;the neurologic deficit score of rats in the combination group was significantly decreased compared with MCI group 4 weeks after operation(P<0.01).②Microspheres distribution and cerebral infarction area observation:The fluorescent of microspheres was distributed scattered on the surface of ipsilateral hemisphere with local necrosis in the MCI group and combination group,and there were white-colored infarct areas of different sizes on the TTC stained brain slices in the MCI group and combination group.③ Pathological morphology observation:By HE staining and the electron microscopy observation,the morphology of rats brain in the normal group,sham group and sleep deprivation group have no significant changes,and the morphology of rats brain in the MCI group and combination group have serious pathological changes.5.Biochemical indexes:Compared with the sham group,the MDA content of rats in the three model groups was significantly increased(P<0.05 or P<0.01),the SOD,Na+-K+-ATPase,Ca2+-ATPase activity of rats in the three model groups was significantly decreased(P<0.01);Compared with sleep deprivation group and MCI group,the MDA content of rats in the combination group was significantly increased(P<0.05);the SOD,Na+-K+-ATPase,Ca2+-ATPase activity of rats in the combination group was significantly decreased(P<0.05 or P<0.01).4.Conclusion(1)Simple syndrome modeling,namely prolonged sleep deprivation can make rats appeared qi deficiency and blood stasis syndrome,but it still cannot cause the brain infarction pathological changes.We use the method of multi-factor superposition and simple disease model both can set multiple cerebral infarction combined with qi deficiency and blood stasis model on rats.(2)Compared with simple disease modeling,model setted up by adopting the composite factor is the integration of syndrome animal model of TCM and western medicine pathologic animal model.Part2 Pathophysiological process of the Combination of Sleep Deprivation and Multiple Cerebral Infarction Rat Model with Qi Deficiency and Blood Stasis and the Interventional Effects of YQHX Formula1.ObjectiveTo investigate the pathophysiological process of combination of Sleep Deprivation and Multiple Cerebral Infarction rat model with Qi Deficiency and Blood Stasis by measuring specific indicators at different periods,to study the interventional effects of YQHX Formula,and to illustrate the stability and reliability of this model.2.Material and methodsSD male rats were randomly divided into 4 groups:A.the Normal Group(N):normally fed and administrated by distilled water daily at the dose of 10ml/kg(i.g.)for 4 weeks,n=31;B.Sham Group(S):Received incision and threading,without injection of fluorescent microspheres,normally fed and administrated by distilled water daily at the dose of 10ml/kg(i.g.)for 4 weeks,n=31;C.Combination of Sleep Deprivation and Multiple Cerebral Infarction group(Model):multiple cerebral infarction operation was performed after 16h of sleep deprivation every day for 1 week,followed by sleep deprivation for 4 weeks after operation,and administrated by distilled water daily at the dose of 10ml/kg(i.g.)for 4 weeks,n=30;D.YQHX Formula Group(YQHX):multiple cerebral infarction operation was performed after 16h of sleep deprivation every day for 1 week,followed by sleep deprivation for 4 weeks after operation,and administrated by YQHX Formula(26g crude drug/kg)at the dose of 10ml/kg(i.g.)for 4 weeks,n=30.Each group was then randomly subdivided into 3 sub-groups according to different period of intervention,which is 48h,2w,and 4w after operation,10 rats for each sub-group.The samples were collected and measured at each time point and the indexes were the same as those in the PART 1,besides five new indexes were added in this part of experiment.3.Results1.General status:Rats in the Model group and YQHX group were lazy and apathetic.The status of rats in the Model group became more serious as modeling time goes on;compared with the model group,the status of rats in the YQHX group were improved significantly.2.Qi deficiency indexes:Compared with the sham group,the body weight,grasping strength of fore limb,duration of loaded swimming to exhaust,spontaneous activity and pulse amplitude of rats in the model group were significantly decreased in three time point(P<0.01);The above indexes of rats in the YQHX group were significantly increased compared with the model group(P<0.05 or P<0.01).3.Blood stasis indexes:?R,G,B value of tongue image:Compared with the sham group,the R,G,B value of tongue image of rats in the model group were significantly decreased in three time points(P<0.05 or P<0.01);compared with the model group,there was no significant difference of the R,G,B value of tongue image of rats in the YQHX group 48h after operation;the R value of tongue image of rats in the YQHX group was significantly increased 2 weeks after operation(P<0.01);and the R,G,B value of tongue image of rats in the YQHX group were significantly increased 4 weeks after operation(P<0.05);②Blood rheology indexes:Compared with the sham group,the whole blood viscosity under shear rate of 5s-1,60s-1,120s-1 of rats in the model group were significantly increased(P<0.05 or P<0.01);compared with the model group,the whole blood viscosity under shear rate of 5s-1,60s-1 of rats in the YQHX group were significantly decreased at the second time point(P<0.01);the whole blood viscosity under shear rate of 5 s-1 of rats in the YQHX group was significantly decreased at the third time point(P<0.05);③Coagulation function:Compared with the sham group,PT、APTT、TT of rats in the Model group were significantly decreased at the second and third time point(P<0.05 or P<0.01);compared with the model group,PT、APTT、TT of rats in the YQHX group were significantly increased in the second and third time point(P<0.05 or P<0.01).4.Brain infarction Indexes:①neurologic deficit score:The neurologic function of rats in the normal group and sham group were normal at all the three time points.The neurologic function of rats in the model group and YQHX group was defected at different extent.There was no significant difference in the neurologic function defect grades of rats between the model group and YQHX group at the second and third time point.The neurologic deficit score of rats in the YQHX group was significantly decreased compared with that in the model group at the third time point(P<0.05).②Microspheres distribution and cerebral infarction area observation:The fluorescent of microspheres was distributed scattered on the surface of ipsilateral hemisphere with local necrosis in the model group and YQHX group at three time points and and there were white-colored infarct areas of different sizes on the TTC stained brain slices of rats in the model group and the YQHX group 24 hours after operation.③Pathology observation:The morphology of rats’brain in the normal group,sham group have no significant changes;The morphology of rats brain in the model group have serious pathological changes.Compared with the model group,the morphology of rats’ brain in YQHX group was significantly improved.5.Biochemical indexes:Compared with the sham group,the SOD,Na`-K+-ATPase and Ca2+-ATPase activity of rats in the model group were significantly increased(P<0.05orP<0.01);and the LDH activity of rats in the model group was significantly increased in all the three time points(P<0.01);the MDA、LD、TXB2 content of rats in the model group were significantly increased in all the three time points(P<0.01);the 6-keto-PGF1α、5-HT content of rats in model group were significantly decreased in three time points(P<0.05orP<0.01).Compared with the model group,the 5-HT content of rats in YQHX group was significantly increased 2w after operation(P<0.05),the Na`-K+-ATPase、Ca2+-ATPase activity of rats in the YQHX group was significantly decreased 4w after operation(P<0.05),LDH content of rats in the YQHX group was significantly decreased 4w after operation(P<0.01),the LD、MDA、TXB2 content of rats in the YQHX group was significantly decreased 4w after operation(P<0.05orP<0.01),the 6-keto-PGFia,5-HT content of rats in the YQHX group was significantly increased 4w after operation(P<0.05).4.Conclusion(1)Combination of Sleep Deprivation and Multiple Cerebral Infarction with Qi deficiency and blood stasis syndrome rat model was established and used to evaluate specific indicators at different periods,and preliminarily revealed the process of qi deficiency and blood stasis syndrome and changes of related biochemical indicators of qi and blood.(2)YQHX Formula have positive effect on the model rats,manifested as changes in general characterization,status of qi deficiency and blood stasis,energy metabolic disorders,blood rheology and blood coagulation function and brain tissue pathological,etc.,and verified the stability and reliability of the integrated disease and syndrome rat model that was established.In conclusion,by comparing different multi-factor modeling method,the sleep deprivation combine with cerebral infarction method is applicable to establish multiple cerebral infarction combine with qi-deficiency and blood-stasis rat model,and this model was used to evaluate specific indicators at different periods,and preliminarily revealed the process of qi deficiency and blood stasis syndrome and changes of related biochemical indicators of qi and blood.Then the YiQiHuoXue(YQHX)Formula was used to prove the stability and reliability of the model.The study preliminary revealed the process of qi-deficiency and blood-stasis syndrome and biological basis,providing a valuable research model and means for studying and developing new drugs of Chinese Medicine and clarifying the compound efficacy of Chinese medicine. |
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