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Morphological regeneration and functional recovery of neuromuscular junction after ischemic/reperfusion injury induced by tourniquet in mouse hind limb
Author(s) -
Tu Huiyin,
Zhang Dongze,
Muelleman Robert L,
Wadman Michael C,
Li YuLong
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.1022.4
Subject(s) - neuromuscular junction , tourniquet , hindlimb , skeletal muscle , neuromuscular transmission , acetylcholine receptor , medicine , ischemia , reperfusion injury , crush injury , anatomy , motor nerve , anesthesia , biology , neuroscience , receptor , surgery
Background Tourniquet‐induced ischemia and subsequent reperfusion cause serious acute ischemia‐reperfusion (IR) injury in the neuromuscular junction (NMJ) and skeletal muscle. These injuries are associated with permanent neurological deficiency, and long‐term skeletal muscle contractile dysfunction, even complete limb paralysis. Monitoring structural and functional repair of the NMJ and skeletal muscle after tourniquet‐induced IR is beneficial to exploring the mechanisms responsible for tourniquet‐induced IR injury and finding effective therapeutic interventions. Here, we observed long‐term morphological and functional alterations of the NMJ in tourniquet‐induced hindlimb IR. Method Unilateral hindlimb of C57/BL6 mice was subjected to 3 hours of ischemia by placing an orthodontic rubber band and followed different periods of reperfusion (1 day, 3 days, 1 weeks, 2 wekks, 4 weeks, and 6 weeks). At each time point of reperfusion, the gastrocnemius muscle was isolated for measurement of the NMJ morphology and function. Motor nerve terminals and nicotinic acetylcholine receptors (nAChRs) in the NMJ were measured by immunofluorescence staining. The end plate potential (EPP) was recorded by the intracellular recording technique. Results In the NMJ of sham mice, the structure of the nAChR clusters remained intact pretzel‐like shape and motor nerve terminals innervated the nAChR clusters to form synapses for signal transmission. The number of fragments in one nAChR cluster was 1.83 ± 0.07 and all nAChR clusters were innervated by motor nerve terminals (100%). The amplitude of EPPs was 29.84 ± 1.02 mV. At 1 day and 3 days of reperfusion, the motor nerve terminals disappeared, the AChR clusters kept normal shape, and no EPPs were recorded. From 1 week to 6 weeks of reperfusion, the motor nerve terminals gradually reinnervated (46.88%, 88.05%, 99.2%, and 100% for 1, 2, 4, and 6 weeks of reperfusion, respectively). However, the AChR clusters were gradually fragmented and the number of fragments in one nAChR cluster increased with longer period of reperfusion (2.92 ± 0.14, 5.50 ± 0.30, 8.94 ± 0.67, and 6.88 ± 0.50 for for 1, 2, 4, and 6 weeks of reperfusion, respectively; p<0.05 vs. sham). The amplitude of EPPs also increased gradually with longer period of reperfusion, but the amplitude of EPPs at 6 weeks of reperfusion didn't restore towards the level seen in sham mice (13.92 ± 1.11 mV, p<0.05 vs. sham). Conclusion Tourniquet‐induced ischemia and subsequent reperfusion impaired the NMJ. The morphological change of motor nerve terminals was faster than that of the nAChR clusters. The slow recovery of fragmented nAChR clusters could attenuate neuromuscular transmission.