The Study Of Histological And Pathological Changes Of Skeletal Muscle Injury Induced By Local Soft Tissue Lysis With Common Needles

BackgroundAcupuncture is an important treatment method under the guidance of Chinese medicine meridian theory,and it has been considered as a "green" treatment method that is safe,effective and has minimal toxic and side effects.For a long time,a variety of needles,to needle as the representative,been widely used in clinical rehabilitation treatment of chronic diseases of soft tissue injury,and achieved good clinical efficacy.In recent decades,various news needles,researched and developed on the basis of needle,were constantly emerging.And those needles were widely used in the treatm-ent of myofascial pain syndrome and chronic soft tissue injury in clinical.However,the related basic research on the new needles dose not go hand in hand.Basic research related to new needles is very weak,and the specific therapeutic mechanism of those needles is not yet clear.At present,based on documented the Huang Di Nei Jing,always a partial soft tissue release adhesions and clear the meridians to illustrate its mechanism of action in introducing its treatment mechanism.The local soft tissue releasing therapy with new needles is an invasive therapeutic method,at present,there is no relevant research to explore and observe whether local tissue damage caused by local releasing therapy with those new needles and its pathological changes after intervened.It is of great importance to clarify the histomorphological changes and pathological regression of local tissue after local releasing therapy with those new needles.So 6 types of needles,the Xiao-zhen needle,Ren-needle,Pi-needle,Yin-zhi needle,Song-jin needle and Chang-yuan needle(in Chinese),were choosed and ani-mal experiments was carried out so that provided histological and pathological basis for the clinical use of new needles.Research Objectives1.At a general level,to observe the effects of local releasing therapy with the new needles on local muscle with eyes.2.At the organizational level,to observe the effects of local releasing therapy with new needles on local muscle tissue morphology under the light microscope,and observe the effects of local releasing therapy with new needles on ultrastructural changes under the transmission electron microscope(TEM).3.At the organizational level,to observe the pathological changes and prognosis at differernt times after local releasing therapy with the new needles.Research Methods1.Laboratory animal grouping and operation.A total of 108 male Sprague-Dawley rats were randomly divided into six groups for the Song Jin-needle group,YinZhi-needle group,Chang Yuan-needle group,XiaoZhen-needle group,Ren-needle group and Pi-needle group(in Chinese)and 18 per group.All rats were kept under constant temperature and humidity conditions,free drinking water and diet.After one week feeding adaptation,rats were anestheti-zed by intraperitoneal injection of 3%sodium pentobarbital(50mg/kg mass).The rats were supine position fixed to the console,shaving hair and disinfected.According to the operation method of the local soft tissue lysis,the analog surgery were carried out in the middle part of gastrocnemius muscle of rats with the SongJin-needle,YinZhi-needle,ChangYuan-needle,XiaoZhen-needle,Ren-needle and Pi-needle(piercing depth of 0.5～0.8cm,operating 2 to 3 times).The operation requires the operator to loosen the intensity,scope and time as far as possible to maintain the same,After surgery with a sterile cotton ball press the surgery area 2～3 min,and sterile band-aid bandage.In addition,one rats were reserved in the SongJin-needle group,YinZhi-needle group,XiaoZhen-needle group and Pi-needle group to observe the local loose solution.2.Gross observationFour rats from the SongJin-needle group,YinZhi-needle group,XiaoZhen-needle group and Pi-needle group were anesthetized,opened the chest cavity and exposed the heart,and stabbed the perfusion needle from the left ventricle anterior wall into the left ventricle,and perfusion with normal saline.At the same time,cut open the right ear,so that normal saline outflow from the right heart,when the liver turned white and stopped perfusion with normal saline.With 4%paraformaldehyde fixative perfusion fixation,and after the hind limb hardens,the analog surgery were carried out in the middle part of gastrocnemius muscle of rats with the SongJin-needle,YinZhi-needle,XiaoZhen-needle and Pi-needle.The local damage was observed by the naked eye after the operation,and photographed with digital camera.The rest of the rats were anesthed by 3%sodium pentobarbital intraperitoneal injection on the 3rd day,7 days and 15 days after the intervention.Aseptic conditions open the area of skin,the local morphological changes of each time point were observed with the anatomical microscope and the naked eye,and took photos with digital camera.3.Observation of histological and pathological changesRats in each group were anesthetized and sacrificed at 3rd,7th,14th,21th and 30th days respectively after the intervention.To dissect and isolate the gastrocnemius muscle,and to quickly cut the 1～2cm of the intervention site at low temperature,via the saline rinse,put in 4%paraformaldehyde fixative and fixed over 24 hours at 4℃.Gradient alcohol dehydration,xylene transparent,embedded in paraffin and 4μm serial sliced.Hematoxylineosin staining(HE staining),Mallory trichrome staining and Ⅰ/Ⅲcollagen immunohistochemistry were performed respectively.The tissue morphology and pathological changes of the local muscle tissue after loosening were observed under light microscope.The images(TIFF format)were collected with cellsens-entry1.15 software to collect images(TIFF format),using Image Pro-Plus 6.0 analysis software to analyze and process all the pictures and calculate the relative area of the far fiber and the I/III type collagen integral absorbance value(intensive optical Density,IOD).Inflammatory cell counts,fibroblast counts and microvessel density(MVD)count analysis were also performed.4.Transmission electron microscope observationAfter each group of anatomical separation of gastrocnemius muscle in low temperature condition,rinse 2-3 in PBS buffer quickly.The rinsed tissue was cut into 2 mm×2 mm strips under low temperature conditions and quickly put into a petri dish containing 2.5%glutaraldehyde fixing solution for 1 to 2 minutes.The tissue pieces were cut into 1 mm3 and put into a 2.5%glutaraldehyde fixing solution for 4 hours at 4℃after the tissues were fixed slightly.Ultra-thin sections used uranyl acetate-lead citrate double staining,Ultrastructural changes of local muscle tissue were observed under transmission electron microscope.Results1.The gross observation results:The gross observation with eyes show that local organizations were more likely to hemorrhage with edge-needles than blunt-needles acupuncture,and oozed last.However,The local organizations had basically no bleeding or only a small amount of bleeding,and oozed less.It may be related to the sharpness of the needle.Then the rats were perfused with saline and paraformaldehyde and carried out the simulation of lysis.A hole-like area in varying degrees could be seen in the local tissues after the simulation of lysis.Opening the acupuncture part of the skin,it is showed that local tissues had different levels of extracellular secretions accumulation,and Local collag-en fibers and skin adhesion could be seen under the dissecting microscope on the 3th day after acupuncture.On the 7th day,more secretions in the local wound area could be seen by means of eyes,and Higher than the local tissue plane.On the 15th day,local area secrets were absorbed basically with eyes,only a small amount of fibers and local skin adhesion could be seen under the dissecting microscope.2.The Observation results of tissue morphology and pathological changesOn the 3th day after the intervention,the muscle fibers dissolution,necrosis and a large number of inflammatory cells infiltration could be seen in the operation area under light microscope.Activated satellite cells were observed by electron micr-oscopy.It was showed that reduction of inflammatory cells,fibroblasts activation and proliferation,and granulation tissue hyperplasia on the7th day.On the 14th day,local accumulation of collagen with substantial formation of granulation tissue.On the 21th day,the collagen fibers gradually decreased and granulation tissue began organi-zing.Light microscopy revealed local tissue scarring but not yet stable,and the electron microscopy showed interstitial collagen deposition on the 30th day.Compar-ed with the relative area of collagen fibers,at the different time,and there were signi-ficant differences between the different types of needles and diameter(P<0.01).there is an interaction between the needle type and the diameter(P<0.01).3.The results of immunohistochemical stainingImmunohistochemical staining showed that there were type I collagen fibers at different times in the operation area after the intervention.The changes of MVD,fibr-oblast count and IOD values show the trend of first increased and then decreased gradually after after two types of needle intervention.On the 7th day,these indicators peaked and then dropped gradually.At the same time,Compared with MVD,there were significant differences between the different diameters of same types of needles(P<0.01),Compared with fibroblast count,there were significant differences betwe-en the different diameters of same types of needles(P<0.01),Compared with IOD value,there were significant differences between the different diameters of same types of needles(P<0.01);Compared with MVD,there were significant differences between the different times of same needles(P<0.01),Compared with fibroblast count,there were significant differences between the different times of same needles(P<0.01),Compared with IOD value,there were significant differences between the different times of same needles(P<0.01).4.TEM observationsNormal skeletal muscle fibers are arranged neatly,z-lines are clear,the mitocho-ndria are evenly distributed and the rules are arranged.After the intervention of the ChangYuan-needle and Ren-needle 3h,some of the muscle fibers were disturbed,broken,dissolved,Z-line blurred,mitochondria swelling and cavitation,mitochondria crest shortened or disappeared under the electron microscope.The first 3d,both groups were visible activated muscle satellite cells,cytoplasm one pole or two poles to form cytoplasm and extension.There are many myofilaments around the nucleus,suggesting that damaged myofibrils are repaired;On the 30th day,myofibrillar lytic lesions and lysosomes were observed.There were fibroblasts in the interstitium and massive proliferation of collagen fibers.Conclusions1.The gross observation showed that the simulation of lysis,carried out with these new needles,had some damage for the local muscle tissue;The edged-needles were more easily damage the small blood vessels and nervous tissue than blunt-needles.2.The simulation of lysis,carried out with these new needles,had a certain trau-ma for the local muscle tissue,local tissue is not yet fully restored and produce scarr-ing after it was injured.Statistical analysis showed that Both the needle type and dia-meter were the decisive factors of the trauma,namely,larger diameter by its damag-ing effect was more obvious in the same type of needle under the conditions,and the round blunt needle with a greater damage than with blade type in the needle with the same diameter conditions.3.Pathological changes observation showed that operation area muscle fiber frac-ture,dissolution and necrosis happened,and local abnormal inflammatory response,fibroblast activation and proliferation could be observed under the light microscope.Collagen and others hypersecretion,eventually leading to irreversible damage to local tissue.