| [Background]Ischemic stroke(cerebral infarction),called "Zhong feng" in traditional Chinese medicine,of which the core is the neurological functional deficit caused by ischemia.Acupuncture and moxibustion are widely used for the definite clinical effectiveness,with its mechanisms being constantly enriching and deepening.Along with the continuous development of neuroscience,more and more basic researches of stroke focus on the holistic concept of neurovascular unit,studying on the changes of structure,function and interaction among its cellular components,in order to explain the relevant mechanisms and provide reference for the prevention,intervention and sequelae treatment.Taking it as a starting point,this study intended to explore the relevant mechanism of acupuncture in the experimental stroke model.[Purpose]After clarifying the behavioral and morphological changes of multiple cerebral infarction model rats at different time points,the appropriate time window of treatment was selected.Then the electroacupuncture(EA)treatment was applied to the model animals,and comprehensively methods and techniques,such as behavioral,morphological and molecular biology methods were used to evaluate the therapeutic effect of EA,to explore the underlying mechanism of EA alleviating the injuries of neurovascular unit,providing the theoretical basis for clinical practice.[Method]This study was divided into two parts:Experiment Ⅰ:Changes of neurovascular unit in rats with multiple cerebral infarction at different time pointsIn experiment Ⅰ,thirty-seven SD rats were randomly divided into normal group(n=7),6 h group(n=7),1 d group(n=9),7 d group(n=7),and 14 d group(n=7).The model was prepared by the injection of 0.6 mL 45-53 μm green fluorescent microspheres solution(about 1000 microspheres)to the clipped right common carotid artery.The behavioral changes were evaluated by Zea-Longa score and gait analysis,and the distribution of infarction was identified by 2,3,5-triphenyltetrazolium chloride(TTC)staining,the morphological alteration was observed by immunofluorescence staining:the blood vessels in the infarcted area were labeled with phalloidin(Pha)and platelet-endothelial cell adhesion molecule-1(CD31),astrocytes were labeled with glial fibrillary acidic protein(GFAP),microglia were labeled with ionized calcium binding adapter molecule 1(Ibal),neurons were labeled with Nissl and neuronal nuclear antigen(NeuN),and CX3CL1,cell apoptosis marker caspase-3 were also used to clarify the pathological process of the model and provided evidence for selecting appropriate intervention time-window.Experiment Ⅱ:Effect of electroacupuncture on neurovascular unit in multiple cerebral infarction model animalsIn experiment Ⅱ,rats with the same conditions were randomly divided into normal group(n=10),model group(n=10)and EA group(n=10).The modeling method was same as above.The EA intervention was at ST36(Zusanli)and GB34(Yanglingquan)on both side(parameters:2/15 Hz,1 mA,15 min;once a day for 5 days).Apart from the above methods,pericytes labeled with platelet-derived growth factor receptors(PDGFR),smooth muscle cells labeled with a-Smooth muscle actin(α-SMA),TUNEL,and nuclear factor-κB(NF-κB),transforming growth factor-β(TGF-β)were also stained in this part.Besides,interleukin-1β(IL-1β),IL-6、IL-10、CX3CL1、NF-κB and vascular endothelial growth factor(VEGF)in ischemic hemisphere were detected by ELISA,and PDGFR in the ischemic hemisphere were detected by Western blot.The above examinations were combined to confirmed effectiveness of EA on rats with multiple cerebral infarction.[Results]Experiment Ⅰ:Changes of neurovascular unit in rats with multiple cerebral infarction at different time points1 Changes of neurological deficit score and gait analysis parameters in model rats at different time pointsThe Zea-Longa score significantly increased at each time point after modeling(P<0.05).The gait parameters were calculated and analyzed with the ratio calculated by(right forelimb/left forelimb)*100%and(right hind limb/left hind limb)*100%.The Swing ratio of the forelimb significantly increased at 6 h and 1 d after modeling(P<0.05),and then returned to normal,while the one in the hind limbs had no significant difference compared with the normal group.The Mean Intensity ratio of the forelimb significantly increased at 6 hours and 7 days after modeling(P<0.05),while the hind limbs showed significant difference only at 6 h after modeling(P<0.05),then returned to normal.The Max Intensity At ratio of the forelimb significantly increased at 6 h and 1 d after modeling(P<0.05),and the ratio of the hindlimb was significantly increased at 6 h after modeling(P<0.05),then returned to normal.The Max Contact Max Intensity ratio of the forelimb significantly increased at 6 h and 7 d after modeling(P<0.05),and the ratio of the hind limb significant increased at 6 h after modeling(P<0.05),then returned to normal.2 Distribution of fluorescent microspheres and infarcted areas in the brain of model ratsThe distribution of fluorescent microspheres and infarcted areas was consistent.The distribution rate of fluorescent microspheres(the number of microspheres observed in this brain area/the total number of microspheres observed)in the ipsilateral cerebral cortex was significantly higher than that in other brain areas(such as the hippocampus,striatum,thalamus,and inner capsule)(P<0.05).3 Changes of blood vessels in the infarcted area of model rats at different time pointsIn normal group,the Pha-labeled arteries were continuous and complete,with the low expression of CD31-labeled micro vessels.At each time point after modeling,the downstream vessels of the blocked artery gradually degenerated or even necrosed,and the peripheral micro vessel density increased significantly(P<0.05).4 Changes of activation of astrocytes and microglia in the infarcted area of model rats at different time pointsIn normal group,GFAP-labeled astrocytes stretched to the blood vessel wall with processes in cerebral cortex,Ibal-labeled microglia distributed evenly and quiescently,and Nissl-labeled neurons were healthy.At each time point after modeling,astrocytes and microglia activated and gathered in the downstream area of the microspheres embedded in the blood vessels,and the astrocyte processes became longer and thicker,with the increasing density.The microglia turned to amoeboid from ramified,and gradually gathered in the infarcted area,resulting in the significant increase of its density(P<0.05),and Nissl-labeled neurons decreased.Under normal condition,there was a little co-labeled microglia of Ibal and CD68,at 6 h and 1 d after modeling,the co-labeled microglia in the infarcted area distributed along the blood vessels,and at 7 d and 14 d,the co-labeled microglia gradually increased and accumulated massively.5 Changes of neurons,microglia and CX3CL1 in the infarcted area of model rats at different time pointsUnder normal conditions,Nissl-labeled neurons expressed CX3CL1,remaining the Iba1-labeled microglia rested through the contact and interaction.At each time point after modeling,the neurons in the ischemic core-area degenerated and necrosed rapidly,and those in the penumbra secreted CXC3CL1 in a large amount,which recruited microglia to its surroundings.The density of CX3CL1(+)staining in the infarcted area increased significantly in 1 d,7 d and 14 d after the modeling(P<0.05),while the microglial number within the 100 μm-radius of CX3CL1(+)neurons also increased significantly at each time point after modeling(P<0.05).6 Neuron injury and necrosis in the infarcted area of model rats at different time pointsUnder normal conditions,neurons labeled with NeuN had complete appearance with the low expression of caspase-3,a marker of apoptosis.After modeling,with the expression of caspase-3 increased,the neurons gradually shrunk,damaged,and even necrosed.The density of neurons gradually decreased after modeling,and compared with normal group,there was significant difference on the group of 1 d,7 d and 14 d after modeling(P<0.05),and the expression of caspase-3 significantly increased at 6 h after modeling(P<0.05).Experiment Ⅱ:Effect of electroacupuncture on neurovascular unit in multiple cerebral infarction model animals7 Effect of electroacupuncture on neurological deficit score and gait analysis parameters improvement of model ratsThere was no neurological deficit in the normal group,and the Zea-Longa score in the model group increased significantly versus the normal(P<0.05).Although there was a downward trend in the EA group versus model group,there was still significant difference compared with the normal level(P<0.05).The "Stand" of the four limbs of normal group was less than that in the model group(P<0.05),while EA could significantly reduce it(P<0.05)and return to the normal level.The "Swing" of the right forelimb and left hind limb in the model group was significantly increased than that of the normal group and the EA group(P<0.05);the Swing of the left forelimb in the model group and the EA group significantly increased(P<0.05),but compared with the former,the latter had a downward trend.The Swing of the right hind limb in the model group was significantly higher than that in the normal group(P<0.05),the EA group had a downward trend and returned to the normal level.The Max Contact Max Intensity of all limbs in the normal group were more than both the model and EA group.For the right forelimb,the model group significantly decreased versus the normal group(P<0.05),and the EA group had an upward trend,returned to the normal level.For the left forelimb,both the model group and the EA group decreased versus the normal group(P<0.05),and the EA group had an increasing trend.For the right hind limb,the model group significantly decreased versus the normal and EA group(P<0.05),and the EA group had returned to the normal level.For the left hind limb,although there was no significant statistical difference among the three groups,the model group has a lower trend than the normal and EA group,and the EA group had an improved trend.The Mean Intensity of the right forelimb and right hind limb in the model group was significantly decreased than that of the normal and EA group(P<0.05),and the EA group returned to the normal level.For the left forelimb,the model group was significantly decreased than the normal group(P<0.05),and the EA group had an increasing trend to normal.For the left hind limb,both the model and the EA group decreased versus the normal level(P<0.05),although there was still a gap to the normal level,the EA group significantly increased versus the model group(P<0.05).8 Electroacupuncture alleviated the neuronal injuries in the infarcted area of model animalsIn the cerebral cortex of normal group,Nissl-labeled neurons had complete appearance,rarely co-labeled with TUNEL,represented with the low Pearson’s Rr.In the model group,the neurons in infarcted area were atrophic and degenerated,the area of affected neurons became larger,with TUNEL co-labeling,represented with the higher Pearson’s Rr(P<0.05).In the EA group,the degree of neuronal atrophy and degeneration was lower than that in the model group,the area of neurons involved was more limited,and the Pearson’s Rr of TUNEL significant decreased versus the model group(P<0.05),but there was still a certain difference from the normal level(P<0.05).The results of neuron density(the ratio of the area of Nissl positive area to the total area)showed that compared with the normal group,the neurons in the model group were severely degenerated and lost(P<0.05),and compared with the model group,the EA group had a tendency to alleviate the degeneration and necrosis of neurons to the normal level.9 Electroacupuncture regulated the excessive activation of astrocytes in the infarcted area and decreased NF-κB p65 in ischemic hemisphere of model animalsUnder normal circumstances,the density of astrocytes was low,and significantly increased in the model and EA group(P<0.05),though there was still a significant difference between the normal and EA group(P<0.05),the latter significantly reduced the aggregation of astrocytes versus the model group(P<0.05).The total number of branches and end-point voxels of astrocytes in the normal group were relatively small,showing a resting state.The total number of branches in the model and EA group was significantly increased(P<0.05),showing the active state.The average branch length of astrocytes in the normal group was smaller,which was significantly increased in the model group(P<0.05).Compared with the model group,it significantly decreased in the EA group(P<0.05)and returned to the normal level.The quiescent astrocytes in the normal group hardly express NF-κB p65,with the low Pearson’s Rr.In the infarcted area,the downstream of the blood vessel embedded by the microspheres in the model group,a large number of astrocytes gathered,their processes became more,longer,thicker,with the expression of NF-κB p65 increased significantly(P<0.05);and in contrast,the activation and aggregation of astrocytes in the EA group decreased,with less NF-κB p65 in Pearson’s Rr(P<0.05),although there was still a certain gap to the normal level(P<0.05).The results of ELISA showed that there was low NF-κB p65 expression in the cerebral hemisphere of the normal group,which significantly increased in ischemic hemisphere of model group(P<0.05),and EA could effectively reduce its expression(P<0.05)and reached to the normal level.10 Electroacupuncture regulated the excessive activation of microglia in the infarcted area and decreased the inflammatory reaction in ischemic hemisphere of model animalsUnder normal circumstances,the density of microglia was low,and the model and EA group significantly increased(P<0.05).Although there was still significant difference between the normal and the EA group(P<0.05),EA significantly decreased the aggregation of microglia(P<0.05).The total number of microglia branches in the normal group was small,showing the resting state,while significantly increased in the model and EA group(P<0.05),showing an active state.Compared with the model group,the total number of microglial branches in the EA group was significantly decreased(P<0.05),although there was still a significant difference to the normal group(P<0.05).The total number of end-point voxels in the normal group was low,while significantly increased in the model group(P<0.05).Compared with the model group,the number of total numbers of end-point voxels in the EA group showed a decreasing trend and returned to the normal level.The average branch length of microglia in the normal group was small,which was significantly increased in the model group(P<0.05),and compared with the model group,it significantly decreased in the EA group(P<0.05)and returned to the normal level.In the normal group,Ibal-labeled microglia were evenly distributed and in a resting state,with less co-labeling with CD68 and TGF-β respectively.The co-labeling of Ibal and CD68 in the ischemic core area of the model group increased significantly(P<0.05),and the co-labeling of Ibal and TGF-β was at an increasing trend.Compared with the model group,the EA group has a tendency to reduce the co-labeling of CD68,but significantly increased the co-labeling of TGF-β(P<0.05).ELISA results showed that IL-1β in cerebral hemisphere of normal group was low,which in ischemic hemisphere of model group significantly increased(P<0.05),and EA significantly decreased(P<0.05)and returned to the normal level.The expression of IL-6 was low in the normal group,significantly increased in the model group(P<0.05),and there was a decreasing trend in the EA group and returned to the normal level.The expression of IL-10 was higher in the normal group,and significantly decreased in the model group(P<0.05),but significantly increased in the EA group(P<0.05)and returned to the normal level.11 Electroacupuncture reduced the expression of VEGF in ischemic hemisphere of model ratsThe results of ELISA’ showed that the expression of VEGF in the cerebral hemisphere of the normal group was low,which of the model group significantly increased(P<0.05),while EA significantly reduced it(P<0.05),and returned to the normal level.12 Electroacupuncture reduced microvascular hyperplasia and protected pericytes in infarcted area of model ratsIn the normal group,the density of CD31-labeled micro vessels was low,and the PDGFR-labeled pericytes attached to the vascular wall with abundant expression,αSMA-labeled smooth muscle was twining with little expression in the penetrating vessels of the cortex,and was co-labeled with CD31.In the model group,the expression of CD31 in the infarcted area were strong,the density of micro vessels significantly increased(P<0.05),the pericytes decreased(P<0.05),and the vascular structure ofα-SMA(+)and CD31(+)decreased.Although the micro vessels density in the EA group was significantly higher than that of the normal group(P<0.05),compared with the model group,there was a downward trend,the expression of pericytes increased(P<0.05),which returned to the normal level,and there could be seen that the blood vessels co-labeled by α-SMA and CD31.13 Electroacupuncture reduced the level of CX3CL1 in the ischemic hemisphere and the expression of CX3CL1 in neurons in the infarcted area of model animalsThe neurons in the normal group generally expressed CX3CL1,and the quiescent microglia were evenly distributed with little contact with the neurons expressing CX3CL1.The expression of CX3CL1 in the ischemic hemisphere of the model group significantly decreased(P<0.05),while the expression of CX3CL1 in the neurons in the infarcted area showed a downward trend,and the microglia migrated and gathered in the ischemic core area,with the contact with the neurons expressing CX3CL1 increasing.The Pearson’s Rr of CX3 CL 1 and neurons in ischemic hemisphere of the EA group significantly decreased compared with the normal and the model group(P<0.05)in the infarcted area,that is,the neuronal expression of CX3CL1 significantly decreased than that of the normal group.[Conclusion]1.Fluorescent microspheres and infarcted areas were mainly distributed in the ipsilateral cerebral cortex.The infarcted area mostly located at the downstream of the blood vessels lodged by fluorescent microspheres,the lodged blood vessel itself degenerated and necrosed,and the density of micro vessels around increased.2.At 6 h and 1 d after modeling,the asymmetry of gait emerged,suggesting the motor dysfunction of the model animals in the acute stage,with the neuronal dysfunction existing.As time passed by,the gait asymmetry returned to normal at the 7 d and 14 d after modeling,while there was still neuronal dysfunction.3.Under normal conditions,microglia and astrocytes were in resting state,once the cerebral infarction occurred,caspase-3 caused by ischemia and hypoxia stimulation rapidly led to irreversible neuronal damage or even apoptosis in the hyperacute phase.CX3CL1 in the ischemic core area and penumbra increased,along with the contact of microglia with it also raised due to its chemotaxis.At 6 h after modeling,they activated and aggregated to the infarcted area,with the processes of astrocytes being thicker and longer,the branches of microglia increasing until it became amoebalike,which lasted until 14 d after modeling.In this process,7 d after modeling was the key time node.4.Electroacupuncture had a certain therapeutic effect on multiple cerebral infarction model rats,which could be reflected in:EA could improve the neuronal function of model rats,improve the time and pressure parameters of gait,reduce the neuronal injuries and limit the affected area,decrease the expression of NF-κB p65,VEGF,CX3CL1 and the inflammatory reaction in ischemic hemisphere,inhibit the overactivation of astrocytes and microglia,inhibit the over-proliferation of micro vessels,and increase the expression PDGFR to some extent.To sum up,as the dividing point between the acute and chronic phases,7 d after modeling was also the key time point for the injury and repairment of the neurovascular unit.Therefore,this study applied the EA treatment in the acute phase to model rats and evaluated the behavioral,morphological and molecular biological alterations at 7 d after modeling.The results confirmed that effectiveness of EA,of which the mechanism may include the regulation of CX3CL1,NF-κB p65,VEGF and inflammatory reaction and the protection effect on the structure and function of neurons,astrocytes,microglia,endothelial cells and pericytes. |
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