Methods to evaluate functional nerve recovery in adult rats: walking track analysis, video analysis and the withdrawal reflex
Introduction
Severe traumas often lead to peripheral nerve damage. In the most severe cases complete transection may occur, which needs surgical intervention. Primary suturing of the nerve ends is only possible when no gap exists between the nerve ends, or else tension will occur. This causes disruption of the microvascularisation (Ogata and Naito, 1986) and adversely affects functional nerve recovery (De Medinaceli et al., 1997). In such cases, gaps have to be bridged. The most widely used method for the reconstruction of a peripheral nerve gap is an autologous nerve graft. The donor nerve usually is a section of a nerve, which is considered to be functionally less important. This method has some disadvantages (De Medinaceli and Seaber, 1989, Hall and van Way, 1994) and therefore alternative techniques have been studied. Materials as veins, biodurable- and biodegradable nerve guides have been used to replace the nerve graft. These materials function as a scaffold for nerve regeneration.
For the evaluation of these different therapeutical strategies, rats are often used. Frequently, sciatic nerve lesions are made, gaps are bridged by different materials and recovery is measured by morphological, electrical and functional methods. However, the functional aspects of nerve recovery, are generally only poorly correlated to electrophysiological and histomorphometrical data (De Medinaceli, 1990, Munro et al., 1998). An additional disadvantage of these methods is that they do not allow recovery to be followed in a longitudinal fashion. Therefore, it is important to use quantifiable, non-invasive and reproducible methods for the evaluation of functional nerve recovery.
Several methods of assessing sensory- and motor nerve recovery in experimental research have been designed. Most methods for the evaluation of sensory nerve recovery, including the pinch test and measurements of conduction velocity, are invasive and not applicable in longitudinal studies (Zeng et al., 1994). The use of the non-invasive withdrawal test, originally described by De Koning et al. (1986), on the other hand, provides a reliable method for evaluating the sensory nerve recovery. The analysis of a rat’s walking pattern by recording their footprints is a well-established and widely employed method for the assessment of motor nerve recovery after nerve injury (De Medinaceli et al., 1982, Bain et al., 1988, Brown et al., 1989, Dellon and Mackinnon, 1989, Hare et al., 1992, Shen and Zhu, 1995, Meek et al., 1996). Since its first description by De Medinaceli et al. (1982), modifications have been developed which focused on refining the method and further quantification of aspects of the footprints (Carlton and Goldberg, 1986, Bain et al., 1988, Bain et al., 1989, Hare et al., 1992). In the original method by De Medinaceli et al., photographic paper was used to obtain footprints. Later, photographic paper and developer have been replaced by white paper and block printing paint (Johnston et al., 1991, Walker et al., 1994, Shen and Zhu, 1995), water-soluble paint (Dellon et al., 1994), diluted black poster paint (Zellem et al., 1989) or fingerprint powder (Hruska et al., 1979). The most recent modification is the use of Xerox paper saturated with bromophenol blue, which changes from orange to dark blue when contacted with the moisturized hindfeet of the rat (Lowdon et al., 1988). Problems arise, however, when rats mutilate their toes and particularly, when contractures of the paws occur. A promising method therefore seems to be to record walking patterns on video (Westerga and Gramsbergen, 1990, Walker et al., 1994, Lin et al., 1996).
The aim of our study was to investigate different methods to evaluate functional nerve recovery in rats. For the measurement of global sensory nerve recovery we evaluated the withdrawal test and for motor nerve recovery we compared three different methods to record walking tracks for the evaluation of the sciatic function index. We chose photographic paper in combination with developer on the rat’s feet, normal paper in combination with finger paint and we also recorded and quantified walking patterns from video-recordings. Measurement of the sciatic function index using video analysis has never been reported before.
Section snippets
Materials and methods
In total, 29 adult male Wistar rats were studied, weighing approximately 250 g. In 18 rats, an autologous nerve graft was implanted (group A) and in five rats the sciatic nerve was crushed (group B). In addition we studied a group of six control rats (group C), which were not operated. All experimental procedures had been approved by the Ethics Committee of the Medical Faculty of the University of Groningen, The Netherlands (FDC 2009).
Results
After a recovery period of a few days, all rats in group A were and remained in a good condition. The recovery period after the crush lesion in group B lasted even shorter. However, even after 2 weeks signs of automutilation in the paws were noted. In group A one rat chewed the lateral side of its operated foot. Three weeks after the operation a second rat showed signs of automutilation which after 7 weeks resulted in the loss of the third toe. In the weeks thereafter two other rats mutilated
Discussion
The aim of our study was to compare different methods to evaluate functional nerve recovery in rats. For the recovery of sensory nerves we applied and evaluated the withdrawal test and for motor nerve recovery we compared different methods to record walking tracks.
Transection of the sciatic nerve generally might be followed by a good recovery of the withdrawal response after electrical stimulation of the footsole (Meek et al., 1999). Similar results were described in crush-lesioned animals by
Acknowledgements
The assistance of H.L. Bartels in the operations is greatly appreciated. The authors gratefully acknowledge Carolien Kooijman, Fester Klok and Bert Otten for their help in the preparation of the figures.
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