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Reorganization of the muscle endplate structures is an important parameter for the study of posttraumatic neuromuscular recovery. The aim of this study was to investigate the changes and the distribution of the acetylcholine receptors (AChRs) in flexor digitorum sublimis muscle after 30 days of denervation in the rat forelimb experimental model. In young male rats, the median and ulnar nerves of the right forelimb were surgically transected and a 1-cm-long segment was removed to avoid spontaneous regeneration. Along the postoperative, the presence of complete functional loss was assessed by the grasping test. After 30 days, rats were sacrificed and flexor digitorum sublimis muscles of both limbs were explanted. The muscles were analysed by light microscopy, to assess the degree of muscle atrophy, and by immunofluorescence after rodhamine-conjugated alpha-bungarotoxin incubation to investigate the reorganization of endplates. The occurrence of muscle denervation was established, prior to sacrifice, by complete loss of the grip function. Light microscopy showed that 30-day denervation is sufficient to induce severe muscle fiber atrophy. Fluorescence analysis at low resolution showed that background fluorescence was higher in denervated muscles possibly because of the presence of extrajunctional AChR. At higher resolution, the endplates were clearly visible as ribbon-like structures. In control fibres, AChR formed a compact and bright structure while in denervated samples it appeared more diffuse and dimmer. Quantitative analysis showed that endplate area was larger in denervated muscles than in control samples. A corresponding decrease in fluorescence intensity was observed after subtracting the basal fluorescence. In conclusion, results of the present study demonstrate that 30 days of denervation induce severe atrophy in rat flexor digitorum sublimis muscle that is accompanied by significant changes in acetylcholine receptor density and distribution. These results also suggest that the rat median nerve denervation experimental model can be an excellent approach for the study of the progression of endplate re-organization after muscle denervation, and reinnervation, considering also its relatively low impact on animal well being in comparison to other experimental models.

Over the last five years, we have used the rat forelimb model for investigating neuromuscular recovery after microsurgical nerve reconstruction of median and ulnar nerves by end-to-side neurorrhaphy and muscle-vein-combined tubulization (using both straight and Y-shaped guides). The outcome of nerve repair at different postoperative times was assessed by functional, morphological and biomolecular analysis. Results showed that both end-to-side and tubulization repair of rat median and ulnar nerves led to successful axonal regeneration along the severed nerve trunk as well as to a partial recovery of the lost function as assessed by grasping test. Biomolecular analysis by means of reverse transcription polymerase chain reaction (RT-PCR) demonstrated early overexpression during nerve regeneration of the gliotrophic factor NRG1 and two of its receptors: erbB2 and erbB3. Finally, our experience also suggests that the rat forelimb experimental model is particularly appropriate for the study of microsurgical reconstruction of major mixed nerve trunks. Furthermore, since the forelimb model is less compromising for the animal, it should be preferred to the hindlimb model for many research purposes.

The experimental investigation of nerve regeneration after microsurgical repair is usually carried out in rats, rather than mice, because of the larger sized peripheral nerves. Today however, the availability of genetically modified mice makes the use of this laboratory animal very intriguing for investigating nerve regeneration at a molecular level. In this study we aimed to provide a standardization of the experimental model based on microsurgical direct repair, by 12/0 suture, of the left median nerve in adult male mice. Postoperative recovery was regularly assessed by the grasping test. At day-75 postoperative, regenerated median nerve fibers were analyzed by design-based quantitative morphology and electron microscopy. Yet, sections were immuno-labelled using two axonal antibodies commonly employed for rat nerve fibers. Results indicated that functional recovery begun at day-15 and progressively increased reaching values not significantly different from normal by day-50. Quantitative morphology showed that, at day-75, the number of regenerated nerve fibers was not significantly different in comparison to controls. In contrast, differences were detected in fiber density, mean axon and fiber diameter and myelin thickness which were all significantly lower than controls. Immunohistochemistry showed that axonal markers commonly used for rat nerves studies are effective also for mouse nerves. Similar to the rat, the mouse median nerve model is superior to sciatic nerve model for the minimal impact on animal well-being and the effectiveness of the grasping test for motor function evaluation. The main limitation is the small nerve size which requires advanced microsurgical skills for performing 12/0 epineurial suturing.