The Contribution of Environment to Impair Re‐Innervation in Age‐Related Failure of Muscle Regeneration Following Injury

Abstract

An age‐related decline in the structure and function of the neuromuscular junction (NMJ) has been hypothesised to play an important role in the loss of muscle mass and force generating capacity that occurs during ageing and to contribute to the incomplete regeneration of skeletal muscle following damage.The aim of this study was to determine the relative levels of innervation of muscle fibres from adult and old wild type (WT) mice and in mice lacking superoxide dismutase (SOD1) SodKO mice prior to and during regeneration following extensive muscle damage induced by barium chloride (BaCl2). Based on the known role of environment as a contributor to the success of muscle regeneration, an additional aim was to test whether the deficits in innervation, muscle mass, and force generation seen in muscles of old mice was evident in muscles transplanted from old donors into adult hosts and vice versa.Male B6.Cg‐Tg (Thy1‐YFP) mice aged 8–10 months (adult) or 24 months (old), male adult (8–10 months) mice lacking SOD1 (C57Bl/6 Sod1KO mice) and male adult SOD1KO mice crossed with Thy1‐CFP (SOD1KO‐CFP) were used for the BaCl2 study. Mice were anaesthestised and the extensor digitorum longus (EDL) muscle was injected with 50ul BaCl2, (1.2% wt/vol in sterile saline). Adult and old WT mice were allowed to recover for up to 60 days. SOD1KO mice were studied prior to‐ and at 28 days post damage, the time point where failure in recovery of contractile force was seen in old WT mice.Male WT adult (6–8 months) and old (22 – 24 months) mice were used for the muscle transplant studies. EDL muscles were transferred to the new host, where the tendons were hen repaired and the nerve from the host implanted into the transplanted muscle. At 28 and 60 days following transplantation, mice were culled and EDL muscles were removed, contractile function measured and NMJ structure examined.For analysis of NMJ structure the post‐synaptic regions of fixed EDL muscles were stained with alpha‐bungarotoxin and Dapi. Multiple images were collected from each muscle and the innervation status of each NMJ was assessed.Data demonstrated that although the 30% full denervation of NMJ structure in association with some reduction in fibre diameter may be responsible for the 35% muscle force deficit that occurred in mice that lack SOD1, the level of denervation could not solely explain the substantial force deficit in muscle from old WT mice following extensive damage and regeneration. In contrast, data support the conclusion that the deficit in force seen in muscles from old WT mice following regeneration from damage is caused by the environment in which the muscle regenerated since transplantation of EDL muscles from either adult or old mice into old mice leads to defective regeneration of those muscles; however, experiments aimed at determining whether the environment impacted the effectiveness of re‐innervation remained inconclusive with substantial disruption evident in all instances.Support or Funding InformationWith thanks to National Institute of Ageing (AG051442) and University of Liverpool for their support.