Experimental Study On Treatment Of Denervated Skeletal Muscle Atrophy By Transplantation Of Muscle Satellite Cell In Rabbit

Objective: To establish a model of rabbit gastrocnemius muscle atrophy through denervation,to extract and transplant muscle satellite cells to the atrophied muscle,and to observe the effect of muscle satellite cells on delaying skeletal muscle atrophy after transplantation.Methods:(1)Rabbit skeletal muscle satellite cell separation,extraction and culture:Select rabbits within 1 week of birth,remove skeletal muscles from the limbs,digest with a collagenase and pancreatic enzyme mixture,then through two differential attachment methods to get the original generation of muscle satellite cells,continue to culture these cells for transplantation.(2)Animal grouping and modeling: Choose 35 healthy rabbits,male or female,2.0 ~ 2.5 Kg weight,randomly divide them into normal group(normal,n=5),model group(model,n=10),control group(control,n=10),and transplantation group(transplant,n=10).No procedures were done to the normal group,the other three groups were further divided into two subgroups as 4-week group and 8-week group.In the experimental groups,the left hind leg popliteal fossa was exposed under aseptic conditions.The tibial nerve was cut and the cutting ends were fixed to the deep fascia.(3)Transplantation of muscle satellite cells: The left gastrocnemius of the transplantation group was injected with 0.5 m L muscle satellite cells which were labeled with CM-Di L marker whereas the control group was injected with equal volume of culture medium in the same location.(4)Morphological observation of the gastrocnemius muscle and muscle wet weight measurement: Four and eight weeks after transplantation,bilateral gastrocnemius muscles were removed from each group respectively and muslce wet weights were measured.A tissue block was removed from the muscle belly and saved for follow-up studies.(5)The gastrocnemius muscle fiber cross-sectional area was measured on H&E stained paraffin sections.(6)The muscles' motor end plates were visualized through Kamovsky-roots staining on frozen sections.(7)Bcl-2 and Bax protein expression were detected via Western blot.(8)The t test and single factor variance analysis were used for statistical analysis.Results:(1)By mixing enzyme digestion method and two differential adherent method extraction,round shaped original muscle satellite cells were obtained.They continued to develop into long spindle adherent cells and gradually merged into multinucleated cells.(2)Compared with the normal group,model group gastrocnemius muscles' wet weight was obviously to reduce and cross-sectional area decreased after 4 weeks or 8 weeks of denervation.The difference was statistically significant(P<0.05).The motor end plate shape change is not obvious between the two groups in 4 weeks,both of which appeared brownish and with clearly outlines.At eight weeks,the motor endplate of the normal group still showed brown distribution,while the model group displayed disappeared motor endplate and obvious cytolysis.(3)At 4 weeks and 8 weeks in both control group and model groups,muscle wet weight,muscle fiber cross-sectional area and the Bcl-2 and Bax protein expression were not significantly different(P> 0.05).At the same time the motor endplate of the two groups showed the same changes,with brownish appearance at 4weeks and obvious disappearance with cytolysis at 8 weeks.(4)Compared with the model group,the transplantation group had less reduction in muscle wet weight and muscle fiber cross-sectional area,increased Bcl-2 protein expression,and decreased Bax expression(P<0.05).At 4 weeks,the motor endplate change is not obvious between the two groups,whereas at 8 weeks,the model group motor endplate disappeared while the transplantation group still had observable motor endplate.However,the edges of which were blurry.(5)At4 weeks and 8 weeks,clearly marked muscle satellite cells can be observed under the fluorescence microscope in the transplantation group but fluorescence intensity reduced with the time.Conclusion:(1)A rabbit model of skeletal muscle atrophy can be successfully established by excising the tibial nerve.(2)Skeletal muscle wet weight and muscle fiber cross-sectional area were obviously decreased,motor endplate reduced,and structure damaged after denervation.(3)Muscle satellite cell transplantation to denerved muscle can delay muscle atrophy accompanied by Bcl-2 protein expression increase and that of the Bax downgrade.