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Summary In view of the lack of theoretical information, end-to-side neurorrhaphy is a frequent object of experimental interest. End-to-side neurorrhaphy is based on collateral sprouting of an intact axon. The quantitative assessment of collateral sprouts sent by an intact motor and sensory axon was the goal of the present study. End-to-side neurorrhaphy of the distal stump of transected musculocutaneous nerve (MCN) with intact ulnar nerve (UN) was performed in a rat model. Collateral sprouts were quantitatively evaluated by counting of motoneurons and DRG neurons following their retrograde labeling by Fluoro-Ruby and Fluoro-Emerald applied to the UN and MCN, respectively. The results suggest a comparable capacity of both intact sensory and motor axons to send collateral sprouts into a denervated nerve stump. The ratio of sensory/motor neurons, the axons of which reinnervated distal MCN stumps, was very similar to that of intact UN (6.500 and 6.747, respectively), but different from intact MCN (5.029). This suggests that the pruning process occurred to balance the collateral sprouts at a ratio of sensory/motor neurons for the donor UN, but not according to the number of sensory and motor bands of Bungner available in the distal stump of the MCN. The present experimental study confirms end-to-side neurorrhaphy as a suitable method of nerve reconstruction.

In our experimental paradigm we sutured the distal stump of a transected musculocutaneous nerve to an intact ulnar nerve of the rat in an end-to-side fashion. We demonstrated the formation of collateral sprouts from intact afferent and motor axons by application of one type of molecule conjugated by two different fluorophores (Fluoro-Ruby and Fluoro-Emerald). Fluoro-Ruby and Fluoro-Emerald were applied distal to end-to-side suture into fresh cut ends of the ulnar and musculocutaneous nerves, respectively. Formation of collateral sprouts was evidenced by findings of mixed (a yellow to orange color) fluorescence labeling of spinal motoneurons and dorsal root ganglia neurons. Colocalization of two different tracers retrogradely transported to the neurons was verified by the individual green and red fluorescence profiles analyzed by means of the computer-assisted image-analyzing system. Our results unequivocally demonstrate that a nerve stump attached to an intact nerve can induce collateral sprouting of both afferent and motor axons.

Summary The acellular nerve graft was utilised to restore a functional reinnervation of the biceps brachii muscle from the motoneuron pool of the cervical spinal cord. The musculocutaneous nerve stump was sutured to an acellular nerve graft, the opposite end of which was inserted into the cervical spinal cord cranial to the avulsed C5 ventral root. The acellular nerve graft was repopulated by Schwann cells heavily immunostained for NGFr within 90 days. The Schwann cells migrating from the nerve stump reached the spinal cord grey matter, where they stimulated the motoneurons to send axonal sprouts. The functional reinnervation of the biceps brachii muscle was assessed by means of the behavioural (grooming) test and EMG, the presence of myelinated and unmyelinated axons was demonstrated by light and electron microscopy. The axonal reconnection of the musculocutaneous nerve stump was verified by horseradish peroxidase retrograde labelling of the spinal motoneurons. Moreover, the motoneurons on the operated side of the C5 spinal segment displayed increased immunostaining for GAP-43 in comparison to the contralateral side, whereas the pattern of AChE histochemical reaction was similar on both the operated and contralateral side, of the C5 segment 150 days after acellular graft implantation. The regenerated axons bridged a 4-cm long originally acellular nerve graft to reach and reinnervate the biceps brachii muscle. The reinnervation of the neuromuscular junctions was morphologically determined by immunofluorescence for neurofilaments. The number of myelinated axons in the acellular nerve graft was significantly higher than those growing over the cellular graft, but their diameter was smaller. The results of experiments presented here demonstrate functional recovery of the biceps muscle reinnervation through the acellular nerve graft repopulated by migrating Schwann cells. The process of reinnervation by acellular nerve graft is however delayed and worse in comparison with the cellular graft.

Summary The application of cutaneous nerve grafts is accompanied by some disadvantages, including insufficient graft material for the reconstruction of a large mixed nerve. Recently, evacuated muscle autografts have been suggested as possible alternatives to cutaneous nerve grafts. In the present paper we have demonstrated a possible preparation of the evacuated muscle graft using an infiltration of Marcaine. The reinnervation of the distal stump of the rat median nerve was evaluated by morphological and morphometric analysis after application of the muscle acellular grafts prepared by three methods: an ordinary freeze-thawed muscle graft, a Marcaine evacuated muscle graft and a Marcaine treated graft with subsequent freezing and thawing. A comparison of the numbers and diameters of the myelinated axons in the distal nerve stumps revealed very similar conditions for axon regrowth and maturation in Marcaine evacuated and freeze-thawed muscle grafts. The best results with myelinated axon numbers and spectrum of their calibres were obtained when the Marcaine treated graft was repeatedly frozen and thawed. The pre-treatment of the muscle graft by Marcaine prevents it from shrinking and fragmenting, the main disadvantage during freeze-thawing of fresh muscle. The present results demonstrate that infiltration of striated muscles with a myotoxic compound, e. g. Marcaine, with subsequent freezing-thawing is the method of choice for the preparation of an acellular muscle graft used in peripheral nerve reconnection in the experimental model.