Invited ReviewFunctional evaluation of peripheral nerve regeneration in the rat: walking track analysis
Introduction
Despite recent advances in microsurgical techniques and the understanding of nerve regeneration, functional recovery following repair of transected peripheral nerves often remains poor (Millesi, 1990, Fu and Gordon, 1997, Meyer et al., 1997). The clinical results of peripheral nerve repair remain disappointing, with loss of muscle function, impaired sensation and/or painful neuropathies (Evans et al., 1999). The poor functional outcome after peripheral nerve repair has motivated a better understanding of the molecular and cellular events surrounding nerve regeneration (Mackinnon, 1989, Madison et al., 1992, Borkenhagen et al., 1998).
In the biological sciences, a number of animal models have been developed in order to study peripheral nerve regeneration. The experimental model of choice for many neuroscientists remains the rat. It provides an inexpensive source of mammalian nervous tissue of identical genetic stock that is easy to work with and well studied (Mackinnon et al., 1984, Mackinnon et al., 1985b, Bain et al., 1989, Meek et al., 1999). The sciatic nerve shows an equivalent capacity for regeneration in rats and subhuman primates (Mackinnon et al., 1985a).
The rat sciatic nerve model is a widely used model for the evaluation of motor as well as sensory nerve function at the same time (Dellon and Mackinnon, 1989, Shen and Zhu, 1995, Dijkstra et al., 2000). Traditional methods of assessing nerve recovery following peripheral nerve injury and repair, such as electrophysiology and histomorphometry, while universally employed in neural regeneration experiments, do not necessarily correlate with return of motor and sensory functions (Dellon and Mackinnon, 1989). Therefore, extrapolation of the electrophysiological parameters, including the nerve conduction velocity, peak action potential amplitude and the area of the compound action potential may lead to inappropriate interpretation of return of function (Rosen and Jewett, 1980, Kanaya et al., 1996). The withdrawal response after application of a small electric current on the most lateral aspects of the footsole is an easy and simple method, but can only assess return of sensory function after peripheral nerve damage (De Koning et al., 1986). With axon count and degree of myelination studies it is not possible to know if the axon reaches the appropriate target organ or not (Rosen and Jewett, 1980, Mackinnon et al., 1991, Kanaya et al., 1996).
Several investigators have taken the triceps surae muscle from the experimental and control sides and weighed them, obtaining the conservation muscle mass ratio (Kobayashi et al., 1997, Terris et al., 1999). However, adipose tissue and fibrosis seen in denervated muscles can increase their weight (Kanaya et al., 1992, Kanaya et al., 1996). Muscle contractile force measurement is a sensitive and reliable method of assessing the function of individual muscles (Kanaya et al., 1992, Shen and Zhu, 1995, Mackinnon, 1996). On the other hand, it provides no specific information on integrated motor function (Urbanchek et al., 1999).
Using a confining walkway to analyse rat gait, Hruska et al. (1979) developed a novel and relatively simple method to study animal models of neurological diseases, that cause dysfunctions in locomotion. Their analysis of the normal walking pattern in rats indicated that free, spontaneous locomotion in the animals is highly consistent and readily quantifiable. A standard battery of neurobehavioral tests, including gait analysis, providing a sensitive and reliable technique for detecting, quantifying, and differentiating various ataxic syndromes, was proposed by Jolicoeur et al. (1979). Newby-Schmidt and Norton (1981) designed a new method for recording chick footprints to assess motor function in the chicken.
In 1982, De Medinaceli et al. (1982) designed a quantitative method of analysing the sciatic nerve function in rats, known as the sciatic function index (SFI), based on several measurements of the footprints made on X-ray film. Many investigators have used the SFI as an assessment of hind limb function after sciatic nerve lesions and repair, which can be quantified reliably and easily determined by gait analysis through footprints (Carlton and Goldberg, 1986, Bain et al., 1989, Wong and Mattox, 1991, Hare et al., 1992, Hirasé et al., 1992, Hall and Van Way, 1994, Ansselin et al., 1997, Babovic et al., 1998).
Section snippets
Walking tracks
Several strains of Rattus norvegicus have been used to obtain walking tracks. The animal is placed in a walking pathway ending in a darkened cage (De Medinaceli et al., 1982, Brown et al., 1989, Maeda et al., 1993, Maeda et al., 1999, Strasberg et al., 1996, Meek et al., 1999). All rats are first allowed two or three conditioning trials, during which they often stop to explore the corridor, thereafter they walk steadily to the darkened cage (De Medinaceli et al., 1982, Meek et al., 1997) (Fig. 1
Analysis of walking tracks
Several measurements are taken from the footprints (Fig. 4): (i) distance from the heel to the third toe, the print length (PL); (ii) distance from the first to the fifth toe, the toe spread (TS); and (iii) distance from the second to the fourth toe, the intermediary toe spread (ITS). All three measurements are taken from the experimental (E) and normal (N) sides. (Bain et al., 1989, Brown et al., 1989). Several prints of each foot are obtained on each track.
The SFI was originally developed by
Toe spread
Hasegawa (1978) reported that the use of the distances between the first and the fifth digits and between the second and fourth digits of the rat's hind paw provide a useful method to evaluate the functional recovery after sciatic nerve crushing. As noted by De Medinaceli et al. (1982), when there is a serious lesion, such as sciatic nerve transection, determining the toe spread from the walking track alone can be very difficult. In this case, they employed an arbitrary value of 6 mm for the
Limitations of the SFI
Since its development by De Medinaceli's group in 1982, the validity of the SFI has been questioned by several investigators. Sometimes several trials are required to obtain the most representative prints for analysis (Bain et al., 1989, Shenaq et al., 1989, Hare et al., 1992). It is also crucial to observe the rat's progress along the walking pathway: when the animal is placed for the first time in the corridor, they often stop, pressing the entire footpad and heeldown, creating a false,
Conclusions and future perspectives
Selection of appropriate evaluation methods is crucial when measuring experimental nerve recovery. In 1982, De Medinaceli first reported that the SFI could be used to evaluate total lower limb function, including nerve, muscle and joint function in rats. Since then, many investigators have used walking track analysis as an assessment of global function recovery after sciatic nerve injuries or repair methods.
The use of walking track analysis provides a non-invasive method of assessing the
Acknowledgements
This research was supported by a grant from the Ministry of Education-PRODEP III (Educational Development Program for Portugal) and co-financed by the European Social Fund. We gratefully acknowledge the efforts of Professor P.H. Robinson, MD, PhD, FRCS for his careful reading of the manuscript.
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