An improved electrophysiological method to study peripheral nerve regeneration in rats

https://doi.org/10.1016/j.jneumeth.2009.05.017Get rights and content

Abstract

After restitution of motor function the grasping test alone is insufficient to figure out any further differences of axonal nerve regeneration of the median nerve in rats. To avoid this problem we developed a standardized electrophysiologic method for testing median nerve regeneration. Threshold, latency, compound muscle action potentials (CMAP) and velocity of neuromuscular transduction were recorded in 54 rats 20 weeks post-operatively. Animals of group 1 served as control group, no transection of the median nerve was carried out. Animals of groups 2 and 3 underwent either primary nerve coaptation or autologous nerve grafting after transection of the median nerve. To ensure validity of the method additional correlation between all parameters was investigated.

Reliable electrophysiological results were observed in all animals. As expected, group 1 animals showed lowest threshold and latency and highest CMAP levels. Transection of the median nerve and additional nerve repair leads to significant increase of threshold and latency as well as reduction of CMAP. Furthermore, animals of group 3 showed higher levels for threshold and latency and reduced CMAP levels compared with animals of group 2. The grasping test alone could not demonstrate these slight differences 20 weeks post-operatively. Additionally, we observed strong correlations between threshold, latency and CMAP using the Spearman correlation ranking. We describe the usage of motor neurography as a reproducible and valid tool which should be mandatory for detailed analysis of regeneration in the rat median nerve model.

Introduction

Despite the overall superior nerve regeneration of rodents compared to that of humans, the rat remains the most commonly used animal model to study the regeneration of peripheral nerves (Sinis et al., 2005, Guntinas-Lichius et al., 2005, Robinson and Madison, 2005, Tos et al., 2004, Varejao et al., 2001). Although the vast majority of peripheral nerve repair procedures in clinical practice occur in the upper limb, the most commonly used nerve injury model is that of the sciatic nerve in the rat (Fujiwara et al., 2007, Worthington, 1969).

In the middle of the 1990s Bertelli and Mira published a median nerve model with the corresponding function test, called the grasping test (Bertelli and Mira, 1995). The usage of this median nerve model provides the obvious advantages of less distress for the animals, a decreased burden of animal care and a simple and easy to handle way to assess the functional nerve regeneration. Furthermore, the rat brachial plexus and its terminal branches bear close similarity to the human one and the grasping test provides a tool for the measurement of functional recovery that is relatively easy to perform. At last a current study from Ibrahim and co-workers had confirmed that section of 4 dorsal roots (C6 to T1) leads to consistent loss of grasping (Ibrahim et al., 2009).

Thus, the rat median nerve model has gained increasing popularity. Studies of end-to-side neurorraphy, regeneration specificity, regeneration effectiveness, axon pruning and strategies for nerve graft or nerve transduction have been conducted using this model (Bertelli et al., 2004, Fujiwara et al., 2007, Ronchi et al., 2009, Sinis et al., 2005, Sinis et al., 2006, Sinis et al., 2008).

In the median nerve model the process of peripheral nerve regeneration can be evaluated with various methods, including the grasping test, histomorphometry and muscle weight (Sinis et al., 2005). The most commonly used evaluation test remains the grasping test, with focus on functional regeneration. However, due to the superior nerve regeneration of rats, several months after treatment slight differences in nerve regeneration could not be figured out by using the grasping test alone (Sinis et al., 2005).

In the clinical setting as well as in other animal models electrophysiologic testing is one of the most popular methods for studies of nerve injury, axonal regeneration and peripheral neuropathy. By this way a quantitative and qualitative evaluation of nerve regeneration is possible without sacrificing the rat.

Even if neurography testing assures a high level of information concerning the quality of axonal nerve regeneration, it remains less cited in the median nerve model of the rat. Reasons for this lack are high technical and financial efforts as well as recurrent difficulties in producing reliable results.

We try to underline the importance of electrophysiologic testing in the median nerve model of the rat for evaluation of axonal recovery after restitution of functional regeneration and discuss the pitfalls that inhibit valid and reliable assessment.

To these aims, we describe our standardized method of electrophysiology in the median nerve model of the rat, then demonstrate electrophysiologic results in animals with primary nerve coaptation as well as nerve transplantation and prove the reliability of the method.

Section snippets

General considerations

In electrophysiology several parameters are also used in clinical practice. Each parameter has its own field of information. The latency time is one parameter in neurography, which includes the neuromuscular transmission up to time of the muscle depolarization. This parameter is similar to distal motor latency, which is used in clinical diagnostics (Bischoff et al., 2008). It provides information about the quality of the fastest conducting nerve fibres (Bischoff et al., 2008). Compound muscle

General observations

All animals survived the operation and the duration of the experiment. They showed normal behaviour in eating and drinking.

Functional results

All operated animals underwent functional assessment of regeneration using the grasping test.

Animals with end-to-end-coaptation (group 2) show a faster development of nerve regeneration than animals with transplantation (group 3), but after 16–18 weeks animals of both groups demonstrated similar strength. After 17 post-operative weeks the force/weight-quotient is about 1.00

Discussion

The median nerve model has become a frequently used animal model for studying the regeneration of peripheral nerves in the rat (Ronchi et al., 2009). Unfortunately, to date no universally accepted method for evaluation of neurography for axonal nerve regeneration is documented in literature (Wang et al., 2008).

The reason for this lack is the huge surgical and technical effort in neurographic measurement which is necessary to produce reliable results. However, the great advantage of the

Acknowledgement

The support of Dr. C. Meisner, Institute for Medical Biometry, University of Tübingen, is gratefully acknowledged.

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