Experimental Study On The Relationship Between The Degeneration And Regeneration Of Neuromuscular Junction And Injury Time After A Mouse Skeletal Muscle Contusion Model

Objective:Skeletal muscle injury is very common in forensic practice.Estimating the time of injury using skeletal muscle injury is an important topic in forensic work.The contractile activity of skeletal muscle is regulated by the central nervous system through the transmission of action potentials from motor neurons to muscle fibers.Neuromuscular junctions?NMJs?are specialized sites that connect the ends of motor neuron axons to skeletal muscle and they are specialized chemical synapses.As a synapse NMJ integrity is essential for transducing motor neuron signals that initiate skeletal muscle contraction and it's necessary for skeletal muscle to perform its normal physiological functions.Many experts and scholars have conducted researches on the regeneration of NMJs,mainly focusing on models of ischemia-reperfusion,crush injury,toxin damage,and some of them have also carried out a series of discussions on the regeneration mechanisms,found out many key molecules and pathways involved in this complex process.Some experts have also studied the relationship between NMJs and muscle atrophy caused by genetic diseases and aging,and proved that the dysfunction of NMJs could lead to muscle atrophy and muscle wasting^[1,2].Thus NMJs and skeletal muscle are an inseparable whole.The completion of the repair process of skeletal muscle requires the complete regeneration of NMJs,and only after NMJs being completely regenerated,could the new formed skeletal muscle perform its normal physiological functions.Until now,there is no report of the relationship between NMJs and time after injury found,so this experiment focused on the morphological and molecular biological changes of NMJs regeneration in the healing process of mouse skeletal muscle contusion,to explore the correlation between NMJs regeneration and injury time.Methods:Eighty healthy adult male C57BL/6 mice,weighing 22-25 g,were randomly divided into 10 groups,9 of which were experimental group and 1 group was control group.The left hind limb was hit at 1 cm away from the calcaneus,causing skeletal muscle contusion by an established contusion model.Five in each group were used for morphological examination and three for protein detection.The mice were anesthetized at 3h,6h,12h,1d,3d,5d,7d,14d,21d post injury,and perfused with normal saline.All gastrocnemius muscles were divided into two parts,one was made into paraffin slides for H.E.staining and the other was made into vibrating sections for immunofluorescent staining.The gastrocnemius muscles used for protein detection were quickly frozen in liquid nitrogen immediately after dissection.Then the protein was extracted and detected by Western blot.Antibodies against neurofilament-H for neurofilament,against synaptophysin for presynaptic membranes,against?-Bungarotoxin for postsynaptic membranes in immunofluorescent staining.Antibodies against neurofilament-H for protein neurofilament,against synaptophysin for protein synaptophysin in Western blot.Results:The morphological changes of skeletal muscle and NMJs regeneration are time dependent.3h after injury,there is edema,degeneration,partial muscle fiber rupture at the injury site.The muscle gap is widened,and bleeding could be seen;6h after injury,muscle cells'margins become unclear,polynuclear cells and some monocytes infiltrate into the injury site,and a few NMJs are disintegrated;12h after injury,the cytoplasm of skeletal muscle was further homogenized,the nucleus disappear.Polymorphonuclear cell and monocyte number increases at injury site.The presynaptic membranes disappeared completely and some neurofilaments are segmentally fractured in some NMJs.1d after injury,a large number of inflammatory cells infiltrate into the injury site.The presynaptic membrane components in NMJs remained in a small amount,and the postsynaptic membranes are clear.3d after injury,the infiltrated inflammatory cells are mainly composed of monocytes,and some fibroblasts begin to proliferate.The presynaptic membranes are almost invisible in NMJs,and there are no complete NMJs structures.5-7 days after injury,the number of monocytes decreases,a large number of fibroblasts proliferate,collagen fibers and regenerated skeletal muscle cells could be found.At this time,signs of NMJs regeneration are also observed,neurofilaments reappear,and some presynaptic membranes are successfully regenerated.14d after injury,a large amount of collagen fibers are formed in the contusion area.The necrotic skeletal muscles are replaced by new skeletal muscles and collagen fibers.So far,most of the NMJs are successfully regenerated,remaining a small amount of NMJs without presynaptic membranes.21d after injury,most of the skeletal muscle regeneration is completed,and a small amount of collagen fibers are still visible at the injury site.Almost all of the NMJs observed are in complete structures,which are basically the same as those in the normal control group.Western blot results show that the expression of NF-H and Syn changes at different post-traumatic intervals,but it's not statistically significant after average gray value analysis.The reason may be due to the fact that the protein is extracted from the entire gastrocnemius muscle rather than separately extracted from the damaged area.Conclusion:The morphological changes of NMJs in mice skeletal muscle contusion is time dependent,which could be expected to become one of the biological indicators to estimate wound age in the future.