Outcome measures 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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