The Impact of Motor Axon Misdirection and Attrition on Behavioral Deficit Following Experimental Nerve Injuries

Abstract

Peripheral nerve transection and neuroma-in-continuity injuries are associated with permanent functional deficits, often despite successful end-organ reinnervation. Axonal misdirection with non-specific reinnervation, frustrated regeneration and axonal attrition are believed to be among the anatomical substrates that underlie the poor functional recovery associated with these devastating injuries. Yet, functional deficits associated with axonal misdirection in experimental neuroma-in-continuity injuries have not yet been studied. We hypothesized that experimental neuroma-in-continuity injuries would result in motor axon misdirection and attrition with proportional persistent functional deficits. The femoral nerve misdirection model was exploited to assess major motor pathway misdirection and axonal attrition over a spectrum of experimental nerve injuries, with neuroma-in-continuity injuries simulated by the combination of compression and traction forces in 42 male rats. Sciatic nerve injuries were employed in an additional 42 rats, to evaluate the contribution of axonal misdirection to locomotor deficits by a ladder rung task up to 12 weeks. Retrograde motor neuron labeling techniques were utilized to determine the degree of axonal misdirection and attrition. Characteristic histological neuroma-in-continuity features were demonstrated in the neuroma-in-continuity groups and poor functional recovery was seen despite successful nerve regeneration and muscle reinnervation. Good positive and negative correlations were observed respectively between axonal misdirection (p<.0001, r2=.67), motor neuron counts (attrition) (p<.0001, r2=.69) and final functional deficits. We demonstrate prominent motor axon misdirection and attrition in neuroma-in-continuity and transection injuries of mixed motor nerves that contribute to the long-term functional deficits. Although widely accepted in theory, to our knowledge, this is the first experimental evidence to convincingly demonstrate these correlations with data inclusive of the neuroma-in-continuity spectrum. This work emphasizes the need to focus on strategies that promote both robust and accurate nerve regeneration to optimize functional recovery. It also demonstrates that clinically relevant neuroma-in-continuity injuries can now also be subjected to experimental investigation.

A systematic review of animal models for experimental neuroma

“…Morphological analysis is the most commonly used method, usually performed in light, 18,22,24,26,27,29,45,46,32,34,50,36,38,43,44,49,19,20,47,39,49 electron, 10,30,32,50,36,40 fluorescence 19,37,38,40 or confocal laser microscopy. 20,43,44 Data on the level where biopsies of the nerve and the neuroma are taken and analysed are heterogeneous and often missing, preventing appropriate comparison of results: Chim et al 16 Haematoxylin and eosin 16,18e20,36,50,37,40,49 and toluidine blue 10,18,22,32,45,46,24,19,25,27,30,38,43,…”

“…20,43,44 Data on the level where biopsies of the nerve and the neuroma are taken and analysed are heterogeneous and often missing, preventing appropriate comparison of results: Chim et al 16 Haematoxylin and eosin 16,18e20,36,50,37,40,49 and toluidine blue 10,18,22,32,45,46,24,19,25,27,30,38,43,44 are the most commonly used stains; some studies also use Masson's thricome, 10,20,37 which also allows the labelling of the connective tissue. 70 This is sometimes combined with immunolabelling, the most common targets being S100 protein, 16,25e27,43 neurofilament 200 (NF200), 10,25,30,37,38 neuronal growthassociated protein (GAP)-43, 20,24,43,44 macrophages (ED-1), 20,27,28,43 nerve growth factor (NGF) 27 and calcitonin gene-related peptide (CGRP). 24,28,30 Double staining has also been reported to identify axons of different origin.…”

“…36,39 The most studied morphologic parameters are nerve cross-sectional area, 16,24,26,43e47 the ratio of neural to connective tissue, 16,26,51 myelinated axon count, 24,45e47 neuroma cross-sectional area, 45e47 neuroma size, 20,32,36 scar and regenerative nerve thickness, 19,20 axon and myelin fibre diameter and area. 38 In a recent paper, Yan et al 10 proposed the weight ratio of neuroma/normal nerve segment, which allows to normalise the results, making their comparison easier.…”

“…The use of forelimb models in rabbits is more frequent. 45e47 Some models use more than one nerve of the forelimb, 45e47,53 the hindlimb, 17,21,35,38,42 or both. 33 A neuroma model on the facial nerve has also been described, but it is far less popular.…”

“…28 Some authors also compare different mechanisms of injury in the same animal model. 37,38,40,41,44 Although other types of injuries are well reproducible, crush injuries are difficult to standardise in terms of pressure applied and subsequent response, making comparison of results difficult. Moreover, nerve crush injuries in rats always correspond to Sunderland's grades 2 and 3, and they are likely to undergo spontaneous recovery; thus, crush should not be considered as the method of choice for NIC models.…”