| Purpose:To observe if needling at Huantiao acupuncture is effective on nerve repair in rats with sciatic nerve injury,Sprague-Dawley?SPF grade?rats aged 2-3 months were randomly divided into4 groups and sciatic nerve chronic compression injury models were established.By means of sciatic nerve function index?SFI?,motor nerve conduction velocity?MNCV?and electron microscopic ultrastructure,the different changes of nerve function and structure in rats in different state of nerve repair and reconstruction were analyzed caused by the different interference factors.By using transcriptome sequencing method,the differentially expressed genes were screened openly within deep needling Huantiao acupoint group,model group and control group.The differentially expressed genes were enriched and analyzed by GO and KEGG.The signal pathways closely related to the rehabilitation of nerve injury would be obtained,which served as an important basis for laboratory and clinical research of Huantiao acupoint needling method.Experiment 1Material and method:1 Animal groupingForty-eight SD rats of SPF grade?body mass 300±50g?were randomly divided into blank control group?group C?,model group?group M?,superficial needling group?group T1?,deep needling group?group T2?,12 rats in each group.2 Preparation of chronic nerve entrapment modelingAfter 3 days of normal feeding,chronic nerve entrapment model rats were established in the left hind limbs of rats in groupM,group T1 and group T2.The main method:cut the skin of rat near the midpoint of femur in sterile laboratory,isolate muscle tissue bluntly,expose and free sciatic nerve.Sciatic nerve was inserted into medical sterile silica gel tube which was5 mm?inner tube 1.0 mm,outer tube 2.0 mm?,the broken ends of silica gel tube were sutured,wounds were sutured and gentamicin was injected to prevent infection.3 Different interference methods?1?Interference in group C:The interference was normal feeding and there was no interference mentioned in any other groups.?2?Interference in group T1:From the 15th day after the modeling,the interference started at 8:30 a.m.every day.After fixing,the left hind limb of the rats was acupunctured at the"Huantiao"?GB30?acupoint.Needling requirements:Insertion angle,about 90°;Visual Sonics Vevo 2100 imaging system was used for needling guidance,only penetrating subcutaneous muscle tissue?needle tip is not near or touching the sciatic nerve?,needling depth of 5-10 mm;SDZ-II electronic acupuncture therapy instrument was connected,select dense wave,set current 1 mA,frequency 2 Hz.The procedure of aucupuncture was 15minutes every day and the treatment was 14 days totally.?3?Interference in group T2:From the 15th day after the modeling,the interference started at 8:30 a.m.every day.After fixing,the left hind limb of the rats was acupunctured at the"Huantiao"?GB30?acupoint.Needling requirements:Insertion angle,about 90°;Visual Sonics Vevo 2100 imaging system was used for needling guidance.When the needle was near the sciatic nerve and triggered the muscle tissue around the nerve,the exploring was stopped.Needling depth of 8-17 mm;SDZ-II electronic acupuncture therapy instrument was connected,selected dense wave;set current,1mA;set frequency,2Hz.The procedure of aucupuncture was 15 minutes every day and the treatment was 14 days totally.?4?Interference in group M:Except for normal feeding,the interference started at 8:30 a.m.every day.Rats were given fixing stimulus a day for 15 minutes each time for 14 days.4 Indicators detection?1?Sciatic nerve function index?SFI?test:12 rats in each group were selected.Sciatic nerve function index was measured according to Bain J R method.The detection time was the second day of normal feeding,the 14th day after modeling and the 28th day after modeling.?2?Detection of motor nerve conduction velocity?MNCV?:On the 28th day after modeling,8 rats were selected from each group.The motor nerve conduction velocity of sciatic nerve was measured by BL-420 Biological Function Detector?Chengdu Taimeng Electronics Co.,Ltd.?.At least five valid data should be collected with each rat for statistical purposes.5 Morphological observation?1?Observation of Sciatic Nerve State by High Frequency Animal Ultrasound on the 14th Day of Acupuncture InterventionInstrument and equipment:On the 14th day of acupuncture intervention,12 rats in each group.Using Vevo2100 high resolution small animal ultrasound imaging system?Visual Sonics?,high frequency probe S400 was used to detect the sciatic nerve of rats in each group.Sciatic nerve was scanned along the direction of sciatic nerve.The characteristic images,diameters of epineurium,diameters of distal and proximal nerve of silica gel tube in rats of group M,group T1 and group T2 were preserved.The diameters of sciatic nerve were analyzed statistically.?2?TEM observation of sciatic nerve in rats?1?draw materials:On the 15th day of acupuncture intervention,three rats in each group were randomly selected.After execution,the sciatic nerve was exposed rapidly,and a segment of the distal sciatic nerve from the silica gel tube?less than 1 mm3 in volume?was cut of the sciatic nerve.?2?Fixed embedding:osmium tetroxide fixed solution,gradient dehydration,immersion and embedding,ultra-thin slicing machine cutting 1 micron thick cross section?4000x?.To observe the nerve structure.?3?Fixed embedding:The materials were fixed in osmium tetroxide fixing solution,dehydrated gradiently,after soaked and embedded,the materials were then cut 1 micron thick cross section by ultra-thin slicing machine to observe the nerve structure?4000x?.Results:1 The effect of different interference factors on SFI of rats in each group?1?Comparisons before modeling:SFI detection on the 2nd day after feeding showed that SFI was basically the same and there was no significant difference between groups?P>0.05?,which provided the basis for model preparation.?2?Comparisons on the 14th day after modeling:Compared with group C,group M,group T1 and group T2,it showed significant difference?P<0.05?,indicating that chronic nerve compression had a nerve damage effect on rats.Combined with the appearance of the left lower limb,it showed the motor functions had decreased of the left limb with dragging state and the toes and feet were swelling deformity too.Comparisions among rats in group T1,group T2 and group M showed no difference?P>0.05?,indicating that the method of chronic sciatic nerve entrapment model compression was stable and could be used to observe the difference of therapeutic intervention within the groups.?3?Comparisons on the 28th day after modeling:Compared with group C,There were differences in group M,group T1and group T2?P<0.05?,indicating that the effect of chronic nerve compression on the motor function of rats in these three groups were still existing.Combined with the appearance of the left lower limb,it could be found that the motor functions had decreased of the left limb with dragging state and the toes and feet were swelling deformity too.However,the symptoms above were alleviated in group M,group T1 and T2.there was no difference between group M and group T1?P>0.05?,indicating that there was no significant difference in the recovery of sciatic nerve function.Compared with group T2,group M and group T1 showed significant differences?P<0.05?.It can be inferred that deep needling at Huantiao?GB30?acpoint had better motor function recovery for chronic compression injury of sciatic nerve in group T2.?4?SFI comparison before and after treatment.There was no significant difference in group M and group T1 with the comparision before and after treatment?P>0.05?;there was significant difference between group T2 before and after treatment?P<0.05?.The results showed that group T2 promoted the recovery of sciatic nerve injury.2 Effects of different interference factors on MNCV in rats of each group compared with group C,there were differences in group M,group T1 and group T2?P<0.05?,which indicated that chronic nerve compression had an effect on nerve conduction velocity of sciatic nerve in rats,and it was inferred that the compression model might cause myelin sheath damage.There was no difference between group M and group T1?P>0.05?,which indicated that there was no obvious nerve injury repair in superficial needling at Huantiao?GB30?acupoint?P>0.05?.Comparing group group T2,there was a significant difference in group M and group T1?P<0.05?,which indicated that deep needling at Huantiao?GB30?acupoint could increase nerve conduction velocity.It was inferred that the injury of myelin sheath could be repaired or the number of axons could be increased.3 High Frequency Ultrasound Observation of Sciatic NerveIn group C,the demarcation between nerve and surrounding tissues was clear,the nerve traveled naturally,and the epineurium of sciatic nerve was parallel and hyperechoic,Smooth and flat with continuity.The internal echo of nerve is strong.In group M,sciatic nerve of rats with chronic entrapment,the demarcation between sciatic nerve and peripheral tissue?outside silica gel tube?is not clear;the course of sciatic nerve is slightly unnatural;the epineurium of sciatic nerve is continuous;the internal echo of nerve is slightly low.In group T1,sciatic nerve of rats,the demarcation between sciatic nerve and peripheral tissue is not clear;the course of sciatic nerve is slightly unnatural;the epineurium of sciatic nerve is continuous,and the internal echo of nerve is slightly low.In group T2,sciatic nerve of rats,the demarcation between sciatic nerve and peripheral tissue is not clear;the course of sciatic nerve is slightly unnatural;the epineurium of sciatic nerve is continuous,and the internal echo of nerve is slightly low.Diameter measurements showed that the diameter of the sciatic nerve in group C was0.95±0.45mm,the sciatic nerve diameterdiameter of the proximal side in group M was2.28±1.10mm,the diameter of the distal nerve was 3.70±1.01mm,and the degree of edema in the distal nerve was higher than that in the proximal nerve?P<0.05?.In group T1,the diameter of the sciatic nerve in the proximal part of the sciatic nerve was 2.34±0.35mm,the distal nerve diameter was 3.66±0.47mm,and the edema degree of the distal nerve was higher than that of the proximal part?P<0.05?.In group T2,the diameter of the proximal sciatic nerve and the distal sciatic nerve of the silica gel tube of the sciatic nerve was 2.32±0.75 mm and 3.62±0.64 mm respectively,and the degree of edema of the distal nerve was higher than that of the proximal nerve?P<0.05?.4 Ultrastructural observation of sciatic nerve?electron microscopy 10000 x??1?In group C,the sciatic nerve was full in appearance and intact in structure.The thickness of the myelin sheath was uniform and the Schwann cells were distributed outside and wrapped up to form the myelin sheath.The inner ring of the nerve fibers was smooth and the central structure was axon,and microtubules or microfilaments could be seen in normal shape.?2?In group M,sciatic nerve myelin nerve fibers could be seen in the center of the picture.the thickness of myelin sheath was not uniform and fingerprint appearance could be found.Axonal atrophy and vacuolation could be seen and it indicate the formation of Wallerian degeneration.?3?The sciatic nerve of rats in group T1.There are myelinated nerve fibers in the center,Schwann cells,spotted nuclei and matrix in the peripheral area,myelinated nerve fibers could be seen in the upper right and left sides of the figure.The thickness of myelin sheath is thinner.The inner ring of nerve fibers is smooth and the central part is axon.Microtubules or microfilaments or mitochondria can be seen in normal shape.?4?In group T2,Schwann cells before the formation of myelin could be seen around the nerve fibers which was forming myelin sheath in the middle of the picture.The thickness of myelin sheath was not uniform and there were fingerprint appearance,axon atrophy and WD formation on the right side of the picture.The myelinated nerve fibers with uniform but thinner myelin sheath could be seen on the left of the picture.The appearance of the myelinated nerve fibers was full,the thickness of the myelin sheath was uniform but thinner,and Schwann cells could be seen outside;the inner ring of the nerve fibers was smooth,with axons in the center,and microtubules or microfilaments could be seen in normal shape.Experiment 2Material and method:1 Animal groupingTwelve SPF SD rats?body mass 300+50g?were randomly divided into three groups:blank control group?group C?,model group?M?,superficial acupoint group?S?,deep acupoint group?T?,three rats in each group.2 Preparation of chronic nerve entrapment modelingAfter 3 days of normal feeding,chronic nerve entrapment model rats were established in the left hind limbs of rats in groupM,group T and group S.The main method:cut the skin of rat near the midpoint of femur in sterile laboratory,isolate muscle tissue bluntly,expose and free sciatic nerve.Sciatic nerve was inserted into medical sterile silica gel tube which was 5mm?inner tube 1.0 mm,outer tube 2.0 mm?,the broken ends of silica gel tube were sutured,wounds were sutured and gentamicin was injected to prevent infection.3 Different interference methods?1?Interference in group C:The interference was normal feeding and there was no interference mentioned in any other groups.?2?Interference in group M:Except for normal feeding,the interference started at 8:30 a.m.every day.Rats were given fixing stimulus a day for 15 minutes each time for 14 days.?3?Interference in group T:From the 15th day after the modeling,the interference started at8:30 a.m.every day.After fixing,the left hind limb of the rats was acupunctured at the"Huantiao"?GB30?acupoint.Needling requirements:Insertion angle,about 90°;Visual Sonics Vevo 2100 imaging system was used for needling guidance.When the needle was near the sciatic nerve and triggered the muscle tissue around the nerve,the exploring was stopped.Needling depth of 8-17 mm;SDZ-II electronic acupuncture therapy instrument was connected,selected dense wave;set current,1mA;set frequency,2Hz.The procedure of aucupuncture was 15 minutes every day and the treatment was 14 days totally.?Interference in group S:From the 15th day after the modeling,the interference started at8:30 a.m.every day.After fixing,the left hind limb of the rats was acupunctured at the"Huantiao"?GB30?acupoint.Needling requirements:Insertion angle,about 90°;Visual Sonics Vevo 2100 imaging system was used for needling guidance,only penetrating subcutaneous muscle tissue?needle tip is not near or touching the sciatic nerve?,needling depth of 5-10 mm;SDZ-II electronic acupuncture therapy instrument was connected,select dense wave,set current 1 mA,frequency 2 Hz.The procedure of aucupuncture was 15minutes every day and the treatment was 14 days totally.4 Indicators detectionThe gene analysis of dorsal root ganglion of sciatic nerve?1?Draw materials of dorsal root ganglion:On the fifteenth day after acupuncture intervention,three rats in each group were anesthetized with chloral hydrate?10%,I.P.3ml/kg?.The spinal canal was exposed rapidly and L4-5 segments of spinal ganglion were removed under non-polluting conditions?operation time<15 min?.Draw materials were immediately frozen in liquid nitrogen and sent for testing.?2?RNA extraction method:RNA extraction was accomplished by Trizol method:?1?fresh samples 100-200mg were ground into powder rapidly under liquid nitrogen freezing and placed in a 1.5 ml centrifugal tube treated by DEPC,and 1 ml Trizol was added to shake and mix;1 ml Trizol was added to every 5×106-7 cells,shaking and mixing;?2?Nucleic acid protein complex was made by static position at room temperature for 10 minutes till Completely separated.?3?Add 0.2 ml chloroform,swirl for 15 seconds,and place at room temperature for 3 minutes.?4?12000rpm,centrifuge for 15 minutes,then take the colorless water phase from the upper layer,transfer the water phase to the new tube,add isopropanol of equal volume,mix,and rest for 2 hours at-80?.?5?4?12000 rpm,centrifuge for 15minutes,discard the supernatant.?6?The precipitation was washed by adding 75%ethanol1.0ml?newly prepared with DEPC water?,and centrifuged at 4?12000 rpm for 2 minutes.?7?Wash twice with 75%ethanol,discard supernatant,centrifuge instantaneously for 30seconds,absorb residual liquid with gun,dry at room temperature?no water droplets on the wall of centrifugal tube?.?8?The extracted RNA was immediately stored at-80?after quality inspection.?3?Transcriptome sequencing experiments:?1?Separation and fragmentation of RNA;?2?FirstStrandcDNA synthesis;?3?SecondStrandcDNA synthesis;?4?Purification of double-stranded DNA by AgencourtAMPureXP magnetic beads with 1.8X volume;?5?End repair and add addition of A;?6?Connection Adaptor;?7?Sequence screening;?8?Enrichment of PCR library;?9?Purification of PCR products;?10?Quality inspection of library.?4?high-throughput sequencing platform Illumina HiSeq 2000 was used to sequence the DNA samples by Paired-End.The joint sequence and the low quality sequence at both ends of the library were removed from the original sequencing results.The filtered sequence was analyzed by software Tophat2 and Rattus norvegicus genome.HTSeq software was used to analyze the level of gene expression by counting the sequence?reads?located in genome region or gene exon region.The RPKM value of 0.1 was used as the threshold to judge whether the gene was expressed or not,and|logFC|>1,pvalue<0.05,FDR<0.1 was used as the threshold of differential gene expression to screen differentially expressed genes?DGEs?.According to the function annotation information of NCBI database,the GO entries of different genes were obtained by Blast2GO software,and the GO functions of all different genes were classified and counted by WEGO software.Then,the metabolic pathways of different genes were analyzed by Kyoto Encyclopedia of genes and genomes?KEGG?.Results:1 The rats in each group?group C,group M group Sand GroupT?were counted before and after the transcriptome quality control.In this study,each sample's Q20 was more than 97%and Q30 was more than 94%,GC content was close to 50%.It suggesting that higher sequencing quality and depth were in line with the conditions for analysis of gene expression differences between groups.2 Analysis of differential gene expression.Gene expression has temporal and spatial specificity.In this study,we used cufflinks command of cuffdiff software to screen differentially expressed genes.In this study,we found that the number of differential genes?DEG?was 53 in group C and M,75 in group C and T,49 in C and S,63 in group M and T,.44 in M and S;51 in T and S.3 In group S/T,the main signaling pathways were:cell adhesion molecule;allograft rejection;graft-versus-host disease;autoimmune thyroid disease;type I diabetes mellitus;antigen treatment and presentation;viral myocarditis;axonal guidance;receptor-like signaling pathway;antigen processing and presentation;the main differential genes were MHC1,MHC2,CLDN,NTN1,EFNB,ISG15,DHX58,CTL,etc.A4,HLA-DR3,etc.In group M/T,the main signaling pathways were cell adhesion molecule,phage,axon guidance and so on.The main differentially expressed genes were MHC1,MHC2,CLDN,NTN1,EFNB,ISG15,etc.The results of KEGG analysis in group C/M?P<0.05?showed that the main signaling pathways were acetaldehyde and dicarboxylic acid metabolism;glycine,serine and threonine metabolism;glycerol ester metabolism;PPAR signaling pathway;and leukocyte migration through endothelium.The main differentially upregulated genes were PDE6A,PDE6B,PDE6G,and downregulated genes were GLDC,gcvPA,gcvPB,gcvT,DLD,gcvH,LPL,LPL,CLDN,OCLN,ESAM,etc.Conclusion:1 Compared with model group and shallow needling group,the data of SFI and MNCV in deep needling group showed statistical difference,which indicated that deep needling group had a better tendency of neurological rehabilitation,and this effect was related to the structural repair of nerve.2 Ultrasound examination showed that the demarcation line between sciatic nerve and peripheral nerve in model group,superficial needling group and deep needling group was slightly unclear or unclear,and the internal echo of sciatic nerve was decreased,which was related to nerve compression injury.The nerve compression injury caused degeneration or reduction of nerve fibers.Sciatic nerve diameter measurements showed nerve edema at both ends of the silica gel tube in all groups except the control group.There was no difference between the groups,suggesting that compression injury caused sciatic nerve edema,but it could not sensitively reflect the structural changes of sciatic nerve among the groups.After treatment,the internal echo intensity of sciatic nerve in model group,superficial needling group and deep needling group decreased,which indicated that the three groups had different degrees of nerve damage.Compared with model group and superficial needling group,the echo intensity of deep needling group was higher,with statistical difference.The change of echo intensity between groups was positively correlated with nerve regeneration and repair.Trend of regeneration and repair.3 The ultrastructure of the sciatic nerve?distal part of the injured area?in each group showed that the model group,superficial needling group and deep needling group all had the histological characteristics of nerve injury repair:WD degeneration,macrophage phagocytosis of myelin sheath and axon,etc.In the deep needling group,a large number of new axons and Schwann cell matrix surrounded axons and formed myelin sheath,which showed more active features of nerve regeneration and repair.It can be concluded that deep needling at Huantiao Point group has a better tendency of nerve repair,which ensures the recovery of motor nerve conduction and the improvement of nerve function.4 The analysis of gene recombination and sequencing showed that there were a certain number of differentially expressed genes in each group of dorsal root ganglion of sciatica.Compared with model group and deep needling group,the differential gene expression of shallow needling group mainly focused on acetaldehyde and dicarboxylic acid metabolism,glycine,serine and threonine metabolism,glycerol ester metabolism,etc.It was related to inflammation,immune regulation,cell tight junction and other aspects.The main signal pathways were leukocyte endothelial migration,acetaldehyde and dicarboxylic acid metabolism,light transmission.Such signaling pathways mainly affect the process of inflammation and cell damage inhibition.5 The analysis of gene recombination sequencing showed that the differential gene expression in deep needling group was mainly related to nerve injury,apoptosis,development and regeneration,such as WD inhibition,axon guidance,intercellular junction,phagocyte development and formation of Schwann cell migration and development,compared with shallow needling group and model group.The related signaling pathways are cell adhesion molecule signaling pathway,axon guidance signaling pathway and phagosome signaling pathway.These signaling pathways can affect the process of apoptosis,repair and regeneration of sciatic nerve. |
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