Outcome measures of peripheral nerve regeneration

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Summary

Animal models of nerve compression, crush, and transection injuries of peripheral nerves have been subject to extensive study in order to understand the mechanisms of injury and axon regeneration and to investigate methods to promote axon regeneration and improve functional outcomes following nerve injury. Six outcome measures of regenerative success including axon and neuron counts, muscle and motor unit contractile forces, and behavior are reviewed in the context of nerve injury types, crush, transection and nerve repair by direct coaptation, or transection and repair via a nerve graft or conduit. The measures are evaluated for sciatic, tibial, common peroneal, femoral, single nerve branches such as the soleus nerve, and facial nerves. Their validity is discussed in the context of study objectives and the nerve branch. The case is made that outcome measures of axon counts and muscle contractile forces may be valid during the early phases of axon regeneration when regenerating sprouts emerge asynchronously from the proximal nerve stump and regenerate towards their denervated targets. However, care must be taken especially when experimental interventions differentially affect how many neurons regenerate axons and the number of axons per neuron that sprout from the proximal nerve stumps. Examples of erroneous conclusions are given to illustrate the need for researchers to ensure that the appropriate outcome measures are used in the evaluation of the success of peripheral nerve regeneration.

Introduction

Peripheral nerve injuries vary widely in extent and severity. They result from birth trauma, accidents, and acts of violence all of which are common and often debilitating (Kouyoumdjian, 2006, Borschel and Clarke, 2009, Muir, 2010). Peripheral nerves contain myelinated motor and sensory axons as well as unmyelinated sensory and autonomic axons. The neurons regenerate their axons after injury and the Schwann cells within the denervated nerve pathways support the regenerating axons and remyelinate the large ones (Fu and Gordon, 1997, Zochodne, 2008). Despite this capacity for repair, functional outcomes after nerve injuries in human patients are frequently disappointing, the outcomes varying widely depending on extent and severity of the injuries and the distance and time required for axons to regenerate (Lundborg, 2000, Lundborg and Rosen, 2007, Sulaiman et al., 2010, Pfister et al., 2011). Axon regeneration is slow, progressing at speeds of 1 and 3 mm/day in humans and animals, respectively (Gutmann et al., 1942, Sunderland, 1947, Holmquist et al., 1993). A progressive regression in regenerative capacity of the neurons and growth support of denervated Schwann cells account for deterioration of regenerative capacity with time and distance (Fu and Gordon, 1995a, Fu and Gordon, 1995b, Fu and Gordon, 1997).

Human nerve injuries are classified depending on the extent of the injury. Three types of nerve fiber injury and subsequent sparing or loss of nerve continuity were the basis for the first clinical classification scheme (Seddon, 1943). The injuries are nerve compression with or without demyelination (neurapraxia), axon transection with both perineurium and epineurium remaining intact (axonotmesis), and nerve transection (neurotmesis) where the continuity of the epineurium is disrupted. In these injuries, recovery depends on remyelination, axon regeneration through the original endoneurial pathways without surgical reunion of nerve stumps, and surgical reunion of proximal and distal nerve stumps to encourage regeneration into the disrupted endoneurial tubes. Intact endoneurial tubes contain the denervated Schwann cells that guide regenerating axons back to their former targets. However, the disruption of these tubes after nerve transection severely compromises the direction taken by the regenerating axons with random reinnervation of former endoneurial tubes and denervated targets (Young, 1949, Haftek and Thomas, 1968, Brushart and Mesulam, 1980a, Thomas et al., 1987).

The second major system of nerve injury classification is that of Sunderland, which expands upon that of Seddon by dividing nerve injuries into five different degrees of injury severity (Sunderland, 1978). A first-degree injury is similar to Seddon's neurapraxia, while a second-degree injury is equivalent to axonotmesis. Third-degree injuries occur when the endoneurium is disrupted, but both the perineurium and epineurium remain intact. Recovery from these injuries is variable, ranging from poor to complete, depending on the degree of intrafascicular fibrosis. In a fourth-degree injury, all neural and internal supporting elements are interrupted but the epineurium remains intact. These injuries are associated with spontaneous recovery, although rarely, and usually require surgical intervention to excise the injured area and surgical repair to improve prognosis. The fifth-degree injury is similar to Seddon's neurotmesis with complete transection of the nerve separating it into both a proximal and distal stump. Recovery after this injury is not possible without surgical intervention and treatment.

In practical terms, surgical repair of injured nerves is performed by coaptation under conditions where proximal and distal nerve stumps can be opposed without undue tension (Lundborg, 2004). Otherwise, nerve grafting is essential to bridge the gaps between the nerve stumps, a topic of wide experimentation in light of the limited supply of nerve for autografting (transplanting a nerve within an individual). Many different materials for artificial conduits have been explored over the years, many of which have been supplanted by newer and improved materials. This topic is beyond the scope of this review but has been reviewed in depth recently (Pfister et al., 2011).

This review specifically concerns measurement of outcomes of nerve regeneration in experimental paradigms of peripheral nerve injury that correspond with clinical injuries of compression, crush, and transection injuries. These are considered in the context of the problems of time and distance and of misdirection of regenerating axons. The peripheral nerve of study and the outcome variables that have been measured vary widely. Frequently these have been chosen based on the rationale of the studies. However, most frequently the rat sciatic nerve has been chosen as a large nerve for study using anatomical and behavioral outcome measures such as axon counts and the sciatic functional index (SFI), respectively (Table 1). Our review focuses on the validity of outcome measures for the sciatic nerve and its branches in the hindlimb, the femoral nerve in the thigh, and the facial nerve. Outcome measures should be chosen according to the research question being addressed although this is not always the case. We make the case for choosing peripheral nerves that serve functional groups of muscles for study. An example is the choice of the common peroneal (CP) and tibial (TIB) branches of the sciatic nerve rather than the sciatic nerve itself as these hindlimb nerve branches supply physiological flexor and extensor muscle groups below the knee that flex and extend the ankle joint. Moreover, under conditions where muscle reinnervation is a relevant outcome measure, nerve branches may be the relevant choice.

Section snippets

Peripheral nerve degeneration and regeneration after injury

The myelinated axons of motoneurons and large sensory neurons and the non-myelinated smaller nerves of sensory and autonomic neurons are bundled into fascicles within the connective tissue layer of the epineurium. Each fascicle is surrounded by the perineurium (the level of the blood-nerve barrier) and each nerve fiber is contained within the endoneurium. Peripheral nerve injuries can involve damage to any one of these tissue layers.

Injury that disrupts axon continuity to the cell body triggers

Outcome measures of recovery in experimental peripheral nerve injury

Animal models of nerve injury are generally of 3 main types: compression, crush, or transection injuries that correspond with the 3 main injury types of Seddon's classification scheme (Table 1). Compression injury is generally induced by mechanical compression of hindlimb nerves to damage myelin but not the axons, usually permitting full recovery (Sunderland type I injury). A loose cuff placed around the sciatic nerve results initially in deterioration of the blood-nerve barrier with formation

Conclusions

There are several outcome measures that have been and are currently being used to evaluate regenerative success after peripheral nerve crush and transection injuries that are repaired by surgical apposition of nerve stumps or via an autograft/conduit. The most frequently used axon counts and muscle contractile forces may provide reasonable views of nerve regeneration during the period of axon outgrowth when regenerating axons ‘stagger’ across the injury site in an asynchronous manner to reach

Acknowledgements

Our grateful thanks to the CIHR for their generous grant support (to TG) of our work on nerve regeneration, to Neil Tyreman for his gracious help with the figures, and to Alex Bilbily for his contributions during the summer of 2010.

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