Research About Antagonistic Effect And Mechanism Of Spinal Manipulation On Inflammation Of Juvenile Rat’s Cortex With Cerebral Palsy

ObjectivesObserving the changes presented from spinal manipulation on the infected cytokines TNF-α and IL-10 of the Juvenile Rat’s cortex with cerebral palsy and methylation level of DNA;exploring antagonistic effect and mechanism of spinal manipulation on inflammation of juvenile rat’s cortex with cerebral palsy.MethodsScreening 30 qualified male juvenile rats strictly from 69 new born rats according to the research standards,and dividing randomly into 3 groups: blank group(n=10)、model group(n=10)、experimental group(n=10).Experimental group and model group adopt occlusion of left common carotid and hypoxia making cerebral palsy model after P3(3-day-postnatal),with blank group no disposal.P4 groups verify the correctness of the model by reversal experiment and then conduct a spinal manipulation,once everyday,15min/times,and continuous for 28 days,without the other two groups interfering.These three groups are giving the same living condition and feeding system.They will have weight and exercise balance function checked at the same time,and experiment of balance beam and hang plate of P21 begins.P33 executes all the rats in the experimental group,gets sample in 24 h,tests inflammation response of TNF-α,and IL-10 and changes presented by protein,and methylation level of gene promoter of cortex of rats with the technology of immunohistochemical staining,western Blot and pyrosequencing.Results1.Display of monitoring BMI: Rats of the experimental group are in good mental status,responsive,normal,fast-growing,and no obvious difference compared with the blank group.Rats in the model group are dull-furred,slowly-responded,and in slow weight growth.Rats in three groups are compared in weight with each other P>0.05 before experiments and no statistical differences show.Rats of P13 have statistical differences(P<0.05)and gain weight fast compared with those in model group.The weight blank of rats before execution are in sequence of blank group>experimental group>model group.2.Results of model successfully established: Three group of rats compare with each other of righting reflex,as a result,blank group and experimental group、model group have statistical differences separately(P<0.05),whereas experimental group and model group have no statistical differences of P=0.293>0.05.3.Motion balancing function,1)Results of balance beam experiment: Time passing through balance beam(P21-P23 and rats in three groups): All the three groups have statistical differences(P<0.05)compared to each other,and the transit time are as below: blank group<experimental group<model group;Declines of back-end of the balance beam(P21-P23 and rats in three groups): All the three groups have statistical differences(P<0.05)compared to each other,and the transit time are as below: blank group<experimental group<model group.2)Results of hang plate experiment: All the rats of three groups have statistical differences(P<0.05)compared with each other,with the angel of hang plate,the sequence is blank group>experimental group>model group.4.Inflammation of the cerebral cortex,Results of immunohistochemical staining,1)A comparison of positive cells staining in the infected cytokines TNF-α of the rat’s cortex indicates that cells from model group are densely distributed contrasted to blank group and experimental group.2)A comparison of positive cells staining in the infected cytokines IL-10 of the rat’s cortex indicates that cells from experimental group are densely distributed contrasted to blank group and model group.5.Expression of inflammatory cytokines in the cortex,Results of Western Blot experiment:1)Protein expression level of infected cytokines TNF-α of the rat’s cortex: blank group and model group have statistical differences(P<0.05),blank group and experimental group have no statistical differences(P=0.0645>0.05),experimental group and model group also have statistical differences(P<0.05),and the protein expression level of infected cytokines TNF-α are as following:model group>experimental group>blank group.2)Protein expression level of infected cytokines IL-10 of the rat’s cortex: blank group,experimental group and model group have statistical differences(P<0.05)respectively,experimental group and model group have statistical differences(P<0.05)too,and the protein expression level of infected cytokines IL-10 are as following: experimental group> model group>blank group.6.The level of DNA methylation of inflammatory cytokines in the cortex,Indication of pyrosequencing:1)Methylation level of cytokines TNF-αon Cp G point shows that experimental group,model group,blank group have statistical differences in all other points(P<0.05)except in TNF-αCp G5 point(P>0.05),and compared blank group with experimental group,model group,all of them have statistical differences(P<0.05),while experimental group and model group have no statistical differences in 8 points(P<0.05).The outcome indicates that the DNA Methylation level of inflammatory factors TNF-ɑ from experimental group and model group reduces markedly in Cp G1 、 Cp G2 、 Cp G3 、 Cp G4 、Cp G6、Cp G7,which increases in Cp G8 point.2)DNA Methylation frequency of inflammatory factors IL-10 of experimental group,model group,blank group have no differences in all points of cytokines area(P>0.05);which shows that the Methylation level of cytokines IL-10 in subarea have no differences among three groups.Conclusions1.spinal manipulation is beneficial for improving balance of the rats with cerebral palsy.2.spinal manipulation could adjust the protein expression level of infected cytokines CP,reduce the protein expression level of infected cytokines TNF-α,and enhance protein expression level of the anti-inflammatory cytokine through reconstruction of inflammation of cerebral palsy leading to recovery of brain damage.3.The antagonistic effect of spinal manipulation on infected cytokines of rats in cerebral palsy are associated with the methylation level of adjustion of the infected cytokines TNF-αand IL-10.