Acupuncture Treatment Rules For Spinal Cord Injury And Experimental Study On Electroacupuncture Combined With Conductive Hydrogel In The Treatment Of Spinal Cord Transection In Rat

Background:Spinal Cord Injury(SCI)is a devastating disease of the central nervous system(CNS)that causes irreversible motor and sensory dysfunction,as well as loss of bladder,bowel and sexual functions.The morbidity and mortality rates are high,which seriously affects patient’s life and health conditions.Till now,there is still no definite and effective treatment regimen.The pathological mechanism of SCI is complicated,and the changes of astrocytes may be an important part.Due to changes in the local microenvironment after SCI,astrocytes are transformed into reactive ones with drastic morphological changes,and get involved in the development of glial scars together with other cells in the spinal cord.In previous view,the formation of glial scars in the late stages of SCI is one of the main reasons for hindering mammalian axonal regeneration and functional recovery.However,in a follow-up study,mice in which genes related to glial scarring were knocked out not only affected the reconstruction of the blood-spine barrier and the blood-brain barrier after SCI,but also developed angiogenic edema,enlarged inflammation,and aggravated tissue injuries.A new understanding of the role of glial scar after SCI emerges.In the early stage of injury,glial scar can effectively inhibit the spread of inflammatory response and the occurrence of secondary injury,while in the later stage,glial scar can hinder the recovery of spinal cord tissues and functions from physical and chemical aspects.In 2012,the bidirectional effects of glial scarring were further explained by cytotoxic A1 and neuroprotective A2 astrocytes.Many clinical studies have confirmed that electroacupuncture(EA)has good therapeutic effect on SCI-induced complications such as motor dysfunction,sensory dysfunction,and urinary retention.Relevant animal experiments also proved its certain protective effect on the CNS by promoting nerve repair.But at present,it seems that for complete SCI,effects of in vitro therapies such as acupuncture and EA are limited due to lack of spared neural pathway connections.Tissue regeneration is another key factor in treating spinal cord dysfunction.tissue engineering techniques have been widely used in regenerative medicine to replace,restore or enhance tissue function.Meanwhile,to enhance the therapeutic effect of EA,we used oriented conductive biologic scaffold materials in this experiment.Oriented conductive bioprotein hydrogel(OCBH)can provide a platform for regenerative medicine therapy,while serving as a bridge to temporarily connect the upper and lower ends of SCIs.EA,an electrical stimulation,is combined with conductive bioscaffolds that deliver electrical signals to nerve cells to well promote nerve regeneration.In this experiment,we attempted to explore the effect and potential mechanism of neurorestoration after SCI in model rats treated by combination of EA and OCBH.The OCBH fiber scaffold designed in regenerative medicine was used to assist the regeneration of neural pathways to promote the therapeutic effect of EA on the nervous system.Objective:①To explore the rules of limbs movement after acupuncture for spinal cord injury and the rules of acupoint selection for sensory dysfunction by using data mining technology;② To observe the effect of electroacupuncture(EA)combined with oriented conductive bioprotein hydrogel(OCBH)on the recovery of nerve function in rats with complete spinal cord injury(SCI),and to explore its effect and mechanism on the formation and changes of glial scars at the material-spinal cord interface.Methods:① Search on CNKI,Wanfang,VIP.com.and sort out the clinical control study literature about spinal cord injury and sensory dysfunction which chose acupuncture as the main cure method since the establishment of the databases and perform descriptive analysis,association analysis,and cluster analysis.② A total of 72 female Sprague-Dawley rats were randomly divided into sham operation group,model group,material group,and material+EA group,with 18 rats in each group.Spinal cord transection was used to construct the rat model of complete SCI.OCBH was implanted in the spinal cord defect of the rats in the material group and the material+EA group.In the material+EA group,EA at Jiaji acupoints on the 3rd day after operation was performed until sample collection.On day 1,day 3,week 1,week 2,week 4,week 8,and week 12 after operation,5 rats in each group were selected for Basso,Beattie and Bresnahan(BBB)scoring.The tail-flick test was performed at week 2,week 4,and week 12 after operation.Motor evoked potential detection was conducted at week 12 after operation.At week 2,week 4,and week 12 after operation,frozen sections were prepared after cardiac perfusion and fixation.Hematoxylin-eosin staining was used to observe the morphology of the injured spinal cord tissues,and immunofluorescence staining was used to detect the expression of glial fibrillary acidic protein(GFAP)and complement C3.Meanwhile,the fresh spinal cords around the injury were collected for Western blotting to detect the expressions of GFAP protein,nuclear factor-κB(NF-κB),tumor necrosis factor(TNF)-α,interleukin(IL)-1α,and(glial-derived neurotrophic factor)GDNF.Results:①Bring acupuncture into 120 studies and 133 prescriptions,using 115 Shu Points in total with the frequency of 1395 times;The main intervention method is electro-acupuncture therapy;The most frequently used acupoints are Jiaji acupoints in the adjacent segment,Dumai acupoints in the adjacent segment,and 15 other acupoints in total;The most relevant acupoints combinations are Zusanli-Waiguan-Sanyinj iao,Quchi-Hegu-Yanglingquan,Quchi-Hegu-Waiguan;The most effective acupoints combinations are Quchi-Waiguan-Hegu,Yanglingquan-Zhu Sanli,Taixi-Shousanli and Mingmen-Dazhui.②In the experimental behavior test,the BBB scores at week 2,week 4,week 8,and week 12 after operation,and tail-flick test at week 4 and week 8,and MEP at week 12 in the material group and the material+EA group were significantly higher than those in the model group(all P<0.05).The BBB score at week 8 and week 12,the tail-flick test at week 12,and MEP at week 12 in the material+EA group were better than those in the material group(all P<0.05).H&E staining showed that the primary injury area spreading to the head and tail after operation,unclear boundary of the injury,and a large area of syringomyelia in the model group,which improved slightly at week 12.The improvement was more obvious in the material group and the material+EA group characterized by obvious injury boundary and no spreading of the injury area.Compared with the material group at week 12 after operation,the tissue boundary in the material+EA group was slightly blurred,but the material and cells were distributed more evenly,and the area of the spinal cord-material interface gap was much smaller.In immunofluorescence staining,no obvious activated astrocytes were found in the sham operation group at each time point.There was almost no GFAP protein expression in the spinal cord injury area or the tail of the injured area in the model group,and GFAP protein expression was obvious at the head of the injured area at week 12.In the material group and the material+ EA group,GFAP expression in lateral distribution was obvious at both ends of the spinal cord injury area at week 4,indicating gradual development of glial scars.The density of GFAP expression in the material+EA group was higher than that in the material group,while at week 12,decreased significantly compared with that in the material group.After spinal cord injury,the expression of complement C3 in the model group,material group,and material+EA group was significant,suggesting that astrocytes were activated and transformed into A1 type,but the expression of complement C3 protein in the latter two groups was significantly lower than that of the model group,even at week 12.Besides,the expression of complement C3 protein in the material+EA group was significantly lower than that of the model group.Western blotting showed that the expression of GFAP protein in material group and material+EA group was higher than that in model group at each time point after SCI(all P<0.05).The expression of GFAP protein in the material+EA group was higher than that in the material group at week 2 and week 4 after operation(P<0.01 and P=0.011,respectively),but was significantly lower than that in the material group at week 12(P<0.01).At week 2 and week 4,and week 12 after operation,the levels of IL-1α,TNF-α,and NF-κB(p65)in the material+EA group were significantly lower than those in the model group(All P<0.05).At week 12 after operation,the expression of GDNF protein in the material+EA group was significantly higher than that in the model group and the material group(both P<0.01).Conclusion:①On the acupuncture treatment of limb dysfunction after spinal cord injury,the main idea is to "Restore Yang and replenish energy and blood",achieving the treatment through the cooperation of the proximal Jiaji and Du meridian points and the distal yang meridian points of the limbs.②EA combined with OCBH can facilitate the formation of glial scars at the spinal cord-material interface in the early stage of SCI,prevent the further spread of inflammatory response,and change the morphology of glial scars at the spinal cord-material interface in its late stage.It can increase the expression of some neurotrophic factors,benefit for the regeneration and growth of axons,and thereby promote the recovery of motor functions after SCI.The mechanism may be related to the bidirectional regulation of EA,which inhibits the activation of astrocytes to A1 type in the early stage of SCI,and promotes the activation of astrocytes to A2 type in the late stage.