Morphological Regeneration and Functional Recovery of Neuromuscular Junctions after Tourniquet-Induced Injuries in Mouse Hindlimb

Abstract

Tourniquet application and its subsequent release cause serious injuries to the skeletal muscle, nerve, and neuromuscular junction (NMJ) due to mechanical compression and ischemia-reperfusion (IR). Monitoring structural and functional repair of the NMJ, nerve, and skeletal muscle after tourniquet-induced injuries is beneficial in exploring potential cellular and molecular mechanisms responsible for tourniquet-induced injuries, and for establishing effective therapeutic interventions. Here, we observed long-term morphological and functional changes of the NMJ in a murine model of tourniquet-induced hindlimb injuries. Unilateral hindlimbs of C57/BL6 mice were subjected to 3 h of tourniquet by placing an orthodontic rubber band, followed by varied periods of tourniquet release (1 day, 3 days, 1 week, 2 weeks, 4 weeks, and 6 weeks). NMJ morphology in the gastrocnemius muscle was imaged, and the endplate potential (EPP) was recorded to evaluate NMJ function. In NMJs, nicotinic acetylcholine receptor (nAChR) clusters normally displayed an intact, pretzel-like shape, and all nAChR clusters were innervated (100%) by motor nerve terminals. During 3 h of tourniquet application and varied periods of tourniquet release, NMJs in the gastrocnemius muscle were characterized by morphological and functional changes. At 1 day and 3 days of tourniquet release, nAChR clusters retained normal, pretzel-like shapes, whereas motor nerve terminals were completely destroyed and no EPPs recorded. From 1 to 6 weeks of tourniquet release, motor nerve terminals gradually regenerated, even reaching that seen in sham mice, whereas nAChR clusters were gradually fragmented with prolongation of tourniquet release. Additionally, the amplitude of EPPs gradually increased with prolongation of tourniquet release. However, even at 6 weeks after tourniquet release, the amplitude of EPPs did not restore to the level seen in sham mice (13.9 ± 1.1 mV, p < 0.05 vs. sham mice, 29.8 ± 1.0 mV). The data suggest that tourniquet application and subsequent release impair the structure and function of NMJs. Morphological change in motor nerve terminals is faster than in nAChR clusters in NMJs. Slow restoration of fragmented nAChR clusters possibly dampens neuromuscular transmission during the long phase following tourniquet release.

Dexamethasone Protects Against Tourniquet-Induced Acute Ischemia-Reperfusion Injury in Mouse Hindlimb

“…Tourniquet-induced mouse hindlimb IR is a mature animal model and has been used in our previous studies (Tran et al, 2011 , 2012 ; Tu et al, 2017 ; Zhang et al, 2017 ). Briefly, under anesthesia with a cocktail consisting of 100 mg/kg ketamine and 10 mg/kg xylazine, given as an intraperitoneal injection (10 ml/kg body weight), the tourniquet in unilateral hindlimb (left hindlimb) was done for 3 h by setting an orthodontic rubber band at the hip joint, using a McGivney hemorrhoidal ligatorer.…”

“…The gastrocnemius muscle contractile force was measured in five sham, five tourniquet-induced IR, and five tourniquet-induced IR + Dex mice as described previously (Tu et al, 2017 ; Zhang et al, 2017 ). Briefly, under anesthesia (800 mg kg −1 urethane and 40 mg kg −1 chloralose, a mixing solution of 20% urethane and 1% chloralose, 4 ml kg −1 , i.p.…”

NMJ maintenance and repair in aging

“…These are diseases that invariably compromise the function and health of motor neurons, muscle fibers and PSCs. The NMJ also falls apart in conditions that affect tissue homeostasis, such as diabetes, vascular exclusion and nerve injuries [4–7]. Additionally, the morphological integrity and function of the NMJ is intimately linked to sympathetic nerves that innervate skeletal muscles, which are also affected by a variety of diseases and injuries [8].…”

When motor unit expansion in ageing muscle fails, atrophy ensues

“…Whereas a recent study suggests neuromuscular junction alterations (e.g. fragmentation of the acetylcholine receptor cluster on the muscle fibre) do not occur with ageing in human muscle (Jones et al 2017), the results of this recent study are confounded by the source material which largely came from the amputated limbs of individuals with a history of peripheral vascular disease, noting that ischaemia-reperfusion is known to cause neuromuscular junction degeneration (Tu et al 2017;Wilson et al 2018). Indeed, other studies of neuromuscular junction morphology with ageing in human skeletal muscle (Oda, 1984;Wokke et al 1990) have observed similar fragmentation patterns to that seen in rodent models of ageing (Valdez et al 2012).…”

“…), the results of this recent study are confounded by the source material which largely came from the amputated limbs of individuals with a history of peripheral vascular disease, noting that ischaemia–reperfusion is known to cause neuromuscular junction degeneration (Tu et al . ; Wilson et al . ).…”