小鼠骨骼肌挫伤愈合过程中神经肌肉接头降解及再生与损伤时间的关系
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摘要
目的研究小鼠骨骼肌挫伤愈合过程中神经肌肉接头(neuromuscular junction,NMJ)降解及再生的形态学变化,探讨NMJ降解及再生与损伤时间的相关性。方法将50只健康成年雄性小鼠随机分为10组,其中9组为实验组,1组为对照组。应用免疫荧光染色技术,以高分子量神经丝蛋白(neurofilament proteinH,NF-H)标记神经丝,突触囊泡蛋白(synaptophysin,Syn)标记突触前膜,乙酰胆碱受体(acetylcholine receptor,AChR)标记突触后膜,检测小鼠骨骼肌挫伤后不同时间点NMJ再生的形态学变化。结果骨骼肌挫伤后挫伤交界区NMJ突触前膜及神经丝开始降解,3d左右降解完全,之后逐渐再生,至21d左右再生大致完成,基本达到对照组水平。突触前膜和后膜数量之比呈现先下降后上升、最终与对照组水平相当的趋势。结论小鼠骨骼肌挫伤修复过程中,挫伤交界区NMJ的形态学变化与损伤时间具有相关性,有望成为法医学骨骼肌损伤时间推断的生物学指标之一。
Objective To investigate the morphological changes in the degeneration and regeneration of neuromuscular junctions(NMJ) during the repair of mouse skeletal muscle contusion and discuss the correlation between the degeneration and regeneration of NMJ and wound age. Methods A total of 50 healthy adult male mice were randomly divided into 10 groups, including 9 experimental groups and1 control group. Immunofluorescent staining was applied, and neurofilament was marked with neurofilament protein-H(NF-H), presynaptic membrane was marked with synaptophysin(Syn), presynaptic membrane was marked with acetylcholine receptor(AChR). Morphological changes of NMJ regeneration at different time points after mouse skeletal muscle contusion were detected. Results The neurofilament and presynaptic membrane of NMJ at the junction of contusion zones began to degrade after contusion, and completed degradation at about 3 d post-injury. Then they gradually regenerated, roughly completing the regeneration at about 21 d and basically reaching the control group level. The ratio of presynaptic membrane quantity to presynaptic membrane quantity showed a trend of decreasing then rising and finally reaching the control level. Conclusion During the repair of mouse skeletal muscle contusion, the morphological changes and wound age of the NMJ at the junction of contusion zones have a close correlation, which is expected to be one of the biological indicators for forensic skeletal muscle wound age estimation.
Objective To investigate the morphological changes in the degeneration and regeneration of neuromuscular junctions(NMJ) during the repair of mouse skeletal muscle contusion and discuss the correlation between the degeneration and regeneration of NMJ and wound age. Methods A total of 50 healthy adult male mice were randomly divided into 10 groups, including 9 experimental groups and1 control group. Immunofluorescent staining was applied, and neurofilament was marked with neurofilament protein-H(NF-H), presynaptic membrane was marked with synaptophysin(Syn), presynaptic membrane was marked with acetylcholine receptor(AChR). Morphological changes of NMJ regeneration at different time points after mouse skeletal muscle contusion were detected. Results The neurofilament and presynaptic membrane of NMJ at the junction of contusion zones began to degrade after contusion, and completed degradation at about 3 d post-injury. Then they gradually regenerated, roughly completing the regeneration at about 21 d and basically reaching the control group level. The ratio of presynaptic membrane quantity to presynaptic membrane quantity showed a trend of decreasing then rising and finally reaching the control level. Conclusion During the repair of mouse skeletal muscle contusion, the morphological changes and wound age of the NMJ at the junction of contusion zones have a close correlation, which is expected to be one of the biological indicators for forensic skeletal muscle wound age estimation.
引文
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[21]TU H,ZHANG D,CORRICK R M,et al.Morphological regeneration and functional recovery of neuromuscular junctions after tourniquet-induced injuries in mouse hindlimb[J].Front Physiol,2017,8:207.
[2]张小红,刘季,李娜,等.Nrf2蛋白在骨骼肌损伤修复中的时序性表达及作用[J].法医学杂志,2018,34(1):18-22.
[3]孙俊红,朱细燕,张小红,等.大鼠骨骼肌损伤修复中FZD4时序性变化及其应用[J].中国法医学杂志,2016,31(5):444-447.
[4]于天水,官大威,赵锐,等.大鼠骨骼肌损伤后PMN、MNC及FBC百分率与损伤时间关系[J].法医学杂志,2014,30(3):166-168.
[5]于天水,马建伟,官大威,等.大鼠骨骼肌损伤后不同时间中性粒细胞及巨噬细胞比率的变化[J].中国法医学杂志,2015,30(1):16-19.
[6]YU T S,LI Z,ZHAO R,et al.Time-dependent expression of MMP-2 and TIMP-2 after rats skeletal muscle contusion and their application to determine wound age[J].J Forensic Sci,2016,61(2):527-533.
[7]官大威,赵锐,王林林.法医学损伤时间推断:过去、现在与未来[J].法医学杂志,2019,35(2):131-135.
[8]TIAN Z L,JIANG S K,ZHANG M,et al.α7nAChR is expressed in satellite cells at different myogenic status during skeletal muscle wound healing in rats[J].J Mol Histol,2015,46(6):499-509.
[9]TIAN Z L,JIANG S K,ZHANG M,et al.Detection of satellite cells during skeletal muscle wound healing in rats:Time-dependent expressions of Pax7and MyoD in relation to wound age[J].Int J Legal Med,2016,130(1):163-172.
[10]SOBOTKA S,CHEN J,NYIRENDA T,et al.Outcomes of muscle reinnervation with direct nerve implantation into the native motor zone of the target muscle[J].J Reconstr Microsurg,2017,33(2):77-86.
[11]YUAN A,RAO M V,VEERANNA,et al.Neurofilaments and neurofilament proteins in health and disease[J].Cold Spring Harb Perspect Biol,2017,9(4):a018309.
[12]RUDOLF R,DESCHENES M R,SANDRI M.Neuromuscular junction degeneration in muscle wasting[J].Curr Opin Clin Nutr Metab Care,2016,19(3):177-181.
[13]TINTIGNAC L A,BRENNER H R,RüEGG MA.Mechanisms regulating neuromuscular junction development and function and causes of muscle wasting[J].Physiol Rev,2015,95(3):809-852.
[14]于天水,官大威,程子惠,等.大鼠骨骼肌钝挫伤分级模型的建立[J].法医学杂志,2008,24(3):168-171.
[15]CESCON M,GREGORIO I,EIBER N,et al.CollagenⅥis required for the structural and functional integrity of the neuromuscular junction[J].Acta Neuropathol,2018,136(3):483-499.
[16]CONFORTI L,GILLEY J,COLEMAN M P.Wallerian degeneration:An emerging axon death pathway linking injury and disease[J].Nat Rev Neurosci,2014,15(6):394-409.
[17]LIU W,CHAKKALAKAL J V.The composition,development,and regeneration of neuromuscular junctions[J].Curr Top Dev Biol,2018,126:99-124.
[18]WILSON R J,DRAKE J C,CUI D,et al.Mitochondrial protein S-nitrosation protects against ischemia reperfusion-induced denervation at neuromuscular junction in skeletal muscle[J].Free Radic Biol Med,2018,117:180-190.
[19]DUREGOTTI E,ZANETTI G,SCORZETO M,et al.Snake and spider toxins induce a rapid recovery of function of botulinum neurotoxin paralysed neuromuscular junction[J].Toxins(Basel),2015,7(12):5322-5336.
[20]MAGILL C K,TONG A,KAWAMURA D,et al.Reinnervation of the tibialis anterior following sciatic nerve crush injury:A confocal microscopic study in transgenic mice[J].Exp Neurol,2007,207(1):64-74.
[21]TU H,ZHANG D,CORRICK R M,et al.Morphological regeneration and functional recovery of neuromuscular junctions after tourniquet-induced injuries in mouse hindlimb[J].Front Physiol,2017,8:207.