| Part Ⅰ Effects of Wei’s Triple Nine Needling Therapy on Visual Electrophysiology and Histomorphology in Rats with Optic Nerve InjuryObjective:Wei’s triple nine needling therapy is a traditional acupuncture therapy that has been developed based on Yanjing Wei’s ophthalmic acupuncture prescription.This study aims to investigate the effects of Wei’s triple nine needling therapy on optic nerve protection in rats with traumatic optic neuropathy(TON).Specifically,this study aims to evaluate the effects of the acupuncture therapy on in vivo visual electrophysiology and retinal ganglion cell(RGC)histomorphology and survival rate after optic nerve injury in rats,as well as to evaluate the TON model and acupuncture effects.Methods:The study was conducted on 36 Wistar rats,randomly divided into four groups(n=9),and 27 of them were surgically modeled in the right eye.Among them,18 rats were randomly divided into the Wei’s triple nine needling therapy group and the model group,9 rats each,after making the rat TON model using the transverse quantitative retraction method,and the other 9 rats were the sham-operated group,with only the optic nerve exposed and no retraction treatment.Another 9 were divided into normal control group.On the next day of modelling,the Wei’s triple nine needling therapy group was treated with the acupuncture therapy for 20 minutes once a day for 14 days,while the model group,sham-operated group,and normal control group received no intervention.On day 14,the following indexes were observed:(1)flash visual evoked potential(FVEP)to detect the visual electrophysiology of each group of rats;(2)HE-stained retinal sections to observe the histopathological changes of each group of rats under a light microscope;(3)transmission electron microscopy to observe the ultrastructure of RGCs in each group of rats;(4)Brn3a immunofluorescence staining of frozen sections of retinas to mark the survival of RGCs.Results:(1)FVEP:Compared with the normal control group,the N2 wave crest latency,P2 wave crest latency,and N2-P2 wave amplitude differences in the sham-operated group were not statistically significant(P>0.05).However,compared with the model group,the N2 wave crest latency and P2 wave crest latency were prolonged(P<0.05),and the N2-P2 wave amplitude was reduced(P<0.05).Compared with the model group,the P2 wave latency was shortened(P<0.05),and the N2-P2 wave amplitude was increased(P<0.05)in the Wei’s triple nine needling therapy group,while its N2 wave latency was not statistically different(P>0.05),although it also tended to be reduced compared with the model group.(2)HE staining:The retinal tissue structure of the model group was obviously abnormal,with reduced numbers of RGCs,disordered arrangement,crinkled and dense staining of the nucleus,swollen or crinkled cytosol of RGCs,and enlarged cell gaps.The retinal tissue structure and RGC damage in the Wei’s triple nine needling therapy group were improved,with more neatly arranged RGCs and locally missing nuclei,although some RGCs still had abnormal morphology and uneven staining.(3)Transmission electron microscopy:The ultrastructure of RGCs in the model group rats was severely damaged,with apoptotic cells,blurred ultrastructure,disappearance of nuclear membrane,reduced number of organelles in the cytoplasm,obvious swelling of mitochondria,and loss of organelle structures with vacuoles.The degree of lesions in the ultrastructure of RGCs in the Wei’s triple nine needling therapy group was improved to some extent,with less severe apoptosis of RGCs,mild crinkling of some cells,fewer cytoplasm showing vacuolar degeneration,and less swollen mitochondria.However,compared with the normal control group,the ultrastructure of RGCs was still blurred and the organelle structure was partially disappeared.(4)Brn3a staining:The positive rate of Brn3a in the sham-operated group showed no statistical difference compared to the normal control group.The Brn3a positive rate was significantly lower in the model group compared to the normal control group and the sham-operated group.However,the Brn3a positive rate was significantly higher in the Wei’s triple nine needling therapy group compared to the model group.Conclusion:(1)The transverse quantitative traction method successfully established a TON rat model.(1)In this experiment,the TON rat model was successfully established by the transverse quantitative traction method.After modeling for 14 days,the visual electrophysiological activity of rats decreased,RGC survival decreased,and tissue histology and ultrastructure were severely damaged.(2)Wei’s triple nine needling therapy has been found to exert a protective effect on the optic nerve in rats with TON.Specifically,this effect is manifested by the therapy’s ability to partially restore the electrophysiological function of optic nerve,ameliorate histopathological changes in RGCs,and increase the survival rate of these cells.Part Ⅱ Wei’s Triple Nine Needling Therapy Modulates the Mitochondrial Apoptotic Pathway in Retinal Ganglion Cells of Optic Nerve Injury RatsObjective:To explore the effects of Wei’s triple nine needling therapy on the mitochondrial apoptotic pathway of RGCs in a transverse quantitative traction model of optic nerve injury in rats,and further clarify whether the protective effect of Wei’s triple nine needling therapy on the optic nerve of rats with traumatic optic neuropathy(TON)is achieved by upregulating the anti-apoptotic genes Bcl-xl/Bcl-2,downregulating the pro-apoptotic genes Bax/Bak,increasing the mitochondrial membrane potential(MMP),inhibiting the activation of apoptotic execution protein Caspase-3,and blocking RGCs apoptosis.Methods:The study was conducted on 144 Wistar rats,randomly divided into four groups(n=36),and 108 of them were surgically modeled in the right eye.Among them,72 rats were randomly divided into the Wei’s triple nine needling therapy group and the model group,36 rats each,after making the rat TON model using the transverse quantitative retraction method,and the other 36 rats were the sham-operated group,with only the optic nerve exposed and no retraction treatment.Another 36 were divided into normal control group.On the next day of modelling,the Wei’s triple nine needling therapy group was treated with the acupuncture therapy for 20 minutes once a day for 14 days,while the model group,sham-operated group,and normal control group received no intervention.On day 14,the following indexes were observed:(1)Terminal deoxynucleotidyl transferase(TdT)mediated dUTP-biotin nick end labelling(TUNEL)staining to detect apoptosis of RGCs in the retina;(2)Western Blot to detect Bclxl/Bcl-2,Bax/Bak,and Caspase-3 protein levels in each group of rats;(3)immunohistochemistry to detect Bcl-xl/Bcl-2,Bax/Bak,and Caspase-3 expression in RGCs;(4)real-time quantitative PCR(RT-PCR)to detect Bcl-xl/Bcl-2,Bax/Bak,and Caspase-3 transcript levels in the retina.(5)detection of MMP in RGCs by JC-1.Results:(1)TUNEL staining:There was no significant difference in apoptosis rate between the sham-operated group and the normal control group(P>0.05).Compared with them,the apoptosis rate increased in the model group(P<0.05).In comparison with the model group,Wei’s triple nine needling therapy group showed a decrease in cell apoptosis rate(P<0.05).(2)Western blot:Compared with the normal control group,there was no significant difference in the expression levels of Bcl-2,Bcl-xl,Bax,Bak,and Caspase-3 in the sham-operated group(P>0.05).In comparison with them,the expression levels of Bax,Bak,and Caspase-3 in the model group tended to increase,but only Bax showed a statistically significant difference(P<0.05);the expression levels of Bcl-2 and Bcl-xl decreased(P<0.05).Compared with the model group,the expression levels of Bax,Bak,and Caspase-3 in Wei’s triple nine needling therapy group tended to decrease,but only Bax showed a statistically significant difference(P<0.05);the expression levels of Bcl-2 and Bcl-xl tended to increase,but only Bcl-xl had statistical significance(P<0.05).(3)Immunohistochemistry:There was no significant difference in the expression of the aforementioned five proteins between the normal control group and the sham-operated group(P>0.05).Compared with them,the expression levels of Bax,Bak,and Caspase-3 in the model group increased(P<0.05);the expression levels of Bcl2 and Bcl-xl decreased(P<0.05).Compared with the model group,the expression levels of Bax,Bak,and Caspase-3 in Wei’s triple nine needling therapy group decreased,while the expression levels of Bcl-2 and Bcl-xl increased(P<0.05).(4)RT-PCR:Compared with the normal control group,the transcription levels of Bax,Bak,and Caspase-3 increased,and the transcription levels of Bcl-2 and Bcl-xl decreased in the model group.Compared with the model group,the transcription levels of Bax,Bak,and Caspase-3 in Wei’s triple nine needling therapy group decreased,while the transcription levels of Bcl-2 and Bcl-xl increased.(5)MMP:There was no significant difference in the MMP between the normal control group and the sham-operated group(P>0.05).The MMP in the model group decreased compared with the normal control group(P<0.05).Compared with the model group,the MMP in Wei’s triple nine needling therapy group increased(P<0.05).Conclusion:(1)After transverse quantitative retraction injury of the optic nerve,the apoptosis rate of RGCs in rats increased,MMP decreased,and the expression of pro-apoptotic proteins Bax,Bak,and Caspase-3 was up-regulated,while the expression of anti-apoptotic proteins Bcl-2 and Bcl-xl was down-regulated.This indicates that the activation of the RGCs apoptotic mitochondrial pathway is the pathological basis of TON.(2)Wei’s triple nine needling therapy can reduce the level of RGCs apoptosis in TON rats.This therapy works by regulating the mitochondrial apoptotic pathway,increasing MMP,promoting the expression of anti-apoptotic proteins Bcl-2 and Bcl-xl,and reducing the expression of pro-apoptotic proteins Bax,Bak,and Caspase-3,thereby exerting a therapeutic effect.Part Ⅲ Modulation of Wei’s Triple Nine Needling Therapy on BDNF/TrkB Signaling Pathway in RGCs of Rats with Optic Nerve InjuryObjective:To investigate the effect of Wei’s triple nine needling therapy on the regulation of BDNF/TrkB signaling pathway in RGCs of transverse quantitative pull model rats,and to further clarify whether the protective effect of Wei’s triple nine needling therapy on the optic nerve of TON rats is achieved by regulating the expression of BDNF-TrKB signaling pathway,interfering with the mitochondrial apoptosis pathway,and promoting the growth and repair of RGCs.Methods:The study was conducted on 40 Wistar rats,randomly divided into four groups(n=10),and 30 of them were surgically modeled in the right eye.Among them,20 rats were randomly divided into the Wei’s triple nine needling therapy group and the model group,10 rats each,after making the rat TON model using the transverse quantitative retraction method,and the other 10 rats were the sham-operated group,with only the optic nerve exposed and no retraction treatment.Another 10 were divided into normal control group.On the next day of modelling,the Wei’s triple nine needling therapy group was treated with the acupuncture therapy for 20 minutes once a day for 14 days,while the model group,sham-operated group,and normal control group received no intervention.On day 14,the following indexes were observed:(1)Brn3a immunofluorescence staining of retinal pavement to detect the survival of RGCs;(2)Western Blot to detect the levels of BDNF and TrkB protein in the retina of each group of rats;(3)immunohistochemistry to detect the expression of BDNF and TrkB in RGCs.Results:(1)RGCs Density:There was no significant difference in RGCs density between the sham-operated group and the normal control group.However,the model group and Wei’s triple nine needling therapy group showed a significant decrease in RGCs number.The degree of RGCs reduction in Wei’s triple nine needling therapy group was improved compared to the model group.(2)Western Blot:Compared with the normal control group,there was no statistical difference in the expression levels of BDNF and TrkB in the sham-operated group(P>0.05).However,the expression levels of BDNF and TrkB in the model group were significantly reduced(P<0.05).Compared with the model group,Wei’s triple nine needling therapy group showed a tendency of increase in the expression levels of BDNF and TrkB.However,only the difference in BDNF was statistically significant(P<0.05).(3)Immunohistochemistry:Compared with the normal control group,there was no statistical difference in the expression levels of BDNF and TrkB in the sham-operated group(P>0.05).However,the expression levels of BDNF and TrkB in the model group were significantly reduced(P<0.05).Compared with the model group,Wei’s triple nine needling therapy group showed an increase in the expression levels of BDNF and TrkB(P<0.05).Conclusion:(1)Transverse quantitative retraction of the optic nerve leads to a decrease in the number of RGCs in rats and inhibits the BDNF/TrkB signaling pathway in RGCs,and the decrease in the protein level of this pathway may aggravate the apoptosis of RGCs.(2)Wei’s triple nine needling therapy can promote the expression levels of BDNF and its receptor TrkB protein up-regulated in RGCs of transverse quantitative retraction TON model rats,interfere with the mitochondrial apoptosis pathway and promote the growth and repair of RGCs,which has positive significance for the treatment of diseases related to optic nerve injury. |
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