Developing motoneurons depend on target contact for survival. Injury to the sciatic nerve in neonatal rats leads to extensive motoneuron death resulting in permanent loss of muscle function (Lowrie et al. 1987). Injury-induced motoneuron death occurs via a combination of apoptosis and necrosis (Li et al. 1998). These processes are mediated by a family of cystine proteases known as calpains (Wang 2000). In this study we examined the effect of treatment with leupeptin, a calpain inhibitor, on the survival and function of motoneurons following neonatal nerve injury.
In 3-day-old rat pups, the sciatic nerve was crushed under halothane anaesthesia (5% halothane in 1.5 l of O2 per minute) and sterile conditions and a silicon implant containing leupeptin (140 µg mg-1) was applied directly to the spinal cord. Twelve weeks after injury, animals were anaestheisised and prepared for in-vivo assessment of hindlimb muscle function. The distal tendons of both soleus and extensor digitorium longus (EDL) muscles were attached to force transducers and the sciatic nerve was exposed for stimulation. Following assessment all animals were humanely killed. Data are presented as means ± S.E.M. and results were compared statistically using a Mann-Whitney test (P < 0.05).
Neonatal nerve injury is known to result in a loss of motor units and a reduction in force of both soleus and EDL muscles. However, in those animals treated with leupeptin, the reduction in muscle function following nerve injury was significantly reduced. Both soleus and EDL have more motor units, are stronger and the change in muscle phenotype induced by nerve injury is prevented. Thus, the maximum tetanic tension (MTT) of the leupeptin-treated soleus muscle was 71.7 ± 10.1 % (n = 5) of control compared to only 39.2 ± 7.1 % (n = 5) of control in the untreated group. In EDL the MTT of leupeptin-treated animals was 55.6 ± 10.6 % (n = 5) of control compared to 34.1 ± 2.1 % (n = 5) in the untreated group. Leupeptin treatment also significantly improved motor unit survival so that 12 weeks after injury 20.8 ± 1.40 (n = 5) motor units survive compared to only 14.6 ± 1.21 (n = 5) in the untreated group. Furthermore, the transformation of muscle fibre phenotype that occurs in EDL muscles following nerve injury was prevented by treatment with leupeptin. Following nerve crush at 3 days EDL becomes very fatigue resistant and the fatigue index (FI) decreases sharply. Thus, unoperated control EDL muscles have a FI of 0.83± 0.04 (n = 11) whereas following nerve crush at 3 days, the FI drops to 0.38± 0.14 (n = 5). However, treatment with leupeptin significantly improves the FI to 0.73 ± 0.10 (n = 5), which is similar to that observed in control muscles.
Thus, treatment with leupeptin prevents the reduction in muscle function that otherwise occurs as a consequence of nerve injury, possibly by inhibition of calpains.