Expression Of In Situ Zymography Of Gelatinase After Rats' Ischiadic Nerve Crushing Injury

Objective: To investigate the potential response that the axon of the peripheralnerve after crushing injury and it is very important to comprehend the regenerationand reparation of the injuried nerves.The gelatinases participated all the pathologyand physiology processes of the reparations of the injuried nerves includingdegradation of the extracellular, migration and proliferation of the cells,they aresignificant during the recovery of injuried nerves.This experiment is to probe thatthere are whether or not the expression of the gelatinase A and B after the nervecrushing injuried meanwhile the role of them.Method:we exposed the hibateral sciatic nerves of the rats by means ofexperimental modes,one side was being retained for sixty seconds,the other was justbeing exposed for same time to be the nomal contrl,so we established the rats'ischiadic nerve crushing injuried models.Method 1.All the rats were allocated into five groups,each group was sixrats.They were weighed and intraperitoneally injected the keanaesthesiad to gotanaesthesia,the dosage was 150mg/kg.The for legs were fixed on the operating-table,shaving the waist,buttocks and thighs,sterilized with povidone iodine,spread thesterilitas gauze.Incised an oblique cut on the bilateralis buttocks and extended to thepost of the femoris.Splited open the gluteus,biceps femoris,semi-membrane andsemitendinosus,exposed the bilateral sciatic nerves.The nerves were ringed clamp forsixty seconds just about 8mm far from the inferior border of musculus piriformis,theleft side was the experimental group and the other side was the normal group andmarked the operative and exposed regions with suture silks.All the rats of normalgroups was cut open to just expose for the same time as that the experimentalgroups'.The gluteus and skins were sutured with 0/1 silks.When they were awake,sentback to the breeding room and gave the animal full diets to breed under thecoordinate conditions.The six rats were sacrificed randomly after the nerve crushinginjury on the first day,three day,one week,two weeks and four weeks,infused 0.01MPBS to draw the materials from the bilateral sciatic nerves,utilized the assay methodof gelatinase enzymogram to mensurate the change of activity of the matrix metalloproteinase 2/9;Method 2.All 15 rats were allocated to three groups,one day,seven day and 4weeks,each group was five rats.The operation method was same to the anteriorexperiment. The five rats were sacrificed randomly after the nerve crushing injury onthe first day,one week and four weeks,infused 0.01 M PBS to draw the materials fromthe bilateral sciatic nerves,the incised nerve length was 5-8mm,OTC imbeded and putinto the container filled 4℃isopentane liquid immediately,immerged the liquidnitrogen about two minutes,then put into 25℃freezing mictotome and incided thenerve into 10mm thick frozen section portaitly,pasted them on glass slide,air dryingunder the room temperature.The specimen which needed in situ zymograms wasdropwised straightly reaction buffer.The specimen that needed immunohistochemicalstains was conserved in -20℃refrigerator.We observed the expressive sites andfunctions of matrix metalloproteinase in the injured nerve axon.Those specimens thatneeded GFAP and CSPG immunohistochemical imbedded in paraffin routinely andcut sheet to comprehend the possible relations among CSPG,scar tissue and thegelatinases.Result:1.Variety classes gelatinases were expressed after the rats' ischiadic nervecrushing injury,that was gelatinase A and B.The result of the enzymogram showed thenormal rat ischiadic nerve disply the expression of gelatinase A but not B.Theexpression of gelatinase B after injured 24 houres extremely higher, contradictory thegelatinase A slightly increased post traum and highest on seven days afterinjured. Western blotting inspected the strap were gelatinase A and B.2.The results of the in situ zymograms and immunohistochemical showednot only gelatinase can be expressed in borderline of the injured area but also betaken on negative correlation to the CSPG in the injured area.Conclusion:1.Not only the macrophagocyte, Schwann cells et al cytokines existed aroundinjured nerve axon,but also the gelatinases participated in the regernaeration andreparation of the rat's sciatic nerve injured.2. Macroscopicly the reparation of the nerve was the process of regeneration anddegeneration of the myelin sheath,in micro it was the result that masses of cell factors regulated them syntheticly.3.There were chondroitic acid proteoglycan in the center of the injured nerve,thegelatinase maybe degradated them and promoted the regenerative process.