Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis

Summary

In this report we describe a method to crush mouse sciatic nerve. This method uses readily available hemostatic forceps and easily and reproducibly produces complete sciatic nerve crush. In addition, we describe a method to prepare muscle whole mounts suitable for analysis of nerve regeneration after sciatic nerve crush.

Abstract

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative response mounted by PNS neurons thereby possibly illuminating the failures of CNS regeneration¹. Sciatic nerve crush (axonotmesis) is one of the most common models of peripheral nerve injury in rodents². Crushing interrupts all axons but Schwann cell basal laminae are preserved so that regeneration is optimal^3,4. This allows the investigator to study precisely the ability of a growing axon to interact with both the Schwann cell and basal laminae⁴. Rats have generally been the preferred animal models for experimental nerve crush. They are widely available and their lesioned sciatic nerve provides a reasonable approximation of human nerve lesions^5,4. Though smaller in size than rat nerve, the mouse nerve has many similar qualities. Most importantly though, mouse models are increasingly valuable because of the wide availability of transgenic lines now allows for a detailed dissection of the individual molecules critical for nerve regeneration^{6, 7}. Prior investigators have used multiple methods to produce a nerve crush or injury including simple angled forceps, chilled forceps, hemostatic forceps, vascular clamps, and investigator-designed clamps^8,9,10,11,12. Investigators have also used various methods of marking the injury site including suture, carbon particles and fluorescent beads^13,14,1. We describe our method to obtain a reproducibly complete sciatic nerve crush with accurate and persistent marking of the crush-site using a fine hemostatic forceps and subsequent carbon crush-site marking. As part of our description of the sciatic nerve crush procedure we have also included a relatively simple method of muscle whole mount we use to subsequently quantify regeneration.

Protocol

1. Animal Subjects

1.1. Treatment

1. All animal procedures should be performed with the approval of the local Institution's Animal Care and Ethics Committee and in accordance with the Use and Committee and National Institutes of Health guidelines, with measures taken to minimize pain and discomfort.
2. Our mice were housed under temperature-controlled conditions on a 12-hour reverse light and dark cycle, while fed mouse chow and water ad libitum.
3. For studies of adult regeneration, mice should be at least 6 weeks of age when the sciatic nerve crush is performed. This age is beyond the time at which pruning of polyneura....

Discussion

We have presented a method to obtain a reliably complete sciatic nerve crush with precise marking of the crush site. As previously mentioned, sciatic nerve crush is a common model of peripheral nerve injury in mice and rats. Although each method of crush has its advantages and disadvantages, we found this method produced a complete crush that was easily marked with a minimum of special equipment (e.g. special clamps, etc).

Crush Methods

The most common instrument use.......