Invited Discussion

The authors investigate the potential of a reverse end-to-side repair ot facilitate nerve regeneration. They conclude that while an end-to-side repair will not support regeneration into a normal nerve, it will regenerate in an end-to-side fashion into a denervated nerve.

In order to conclude that this reverse end-to-side repair actually works, two things must be ruled out. First, has spontaneous regeneration from the proximal peroneal nerve contaminated the distal peroneal nerve? The authors buried the proximal and distal nerve stumps to minimize the potential for contamination, but no technique was employed to prove that contamination did not occur. One way to answer this question would be to label axons regenerating into the distal peroneal nerve stump with a retrograde labeling agent such as horseradish peroxidase, Fast Blue, or Fluoro Ruby. The presence of tracer could then be evaluated at the spinal cord level. Labeling in the sacral spinal cord segments would confirm tibial nerve-derived fibers whereas increased labeling in the lower lumbar spinal cord would strongly suggest the presence of contamination from the proximal peroneal nerve stump. Labeling of the uninjured contralateral peroneal or tibial nerves would serve as an internal control and would direct the region of the spinal cord requiring sectioning.[1] [2]

Alternatively, this experiment could be performed in transgenic mice constitutively expressing a fluorescent protein in their axons. We have followed the paths of enhance yellow-fluorescent protein (EYFP)-labeled axons in mice following a small epineurial injury, have noted distal injury to the donor nerve and redirected regenerating units in this model. As shown in Figure [1], randomly regenerating branches can be traced from the epineurotomy and can travel > 1 cm in the absence of any form of nerve repair. This model clearly illustrates how a proximal stump could contaminate a distal repair site, and could serve as a means for ruling out such contamination in future studies.

Figure 1 Randomly regenerating axons 3 weeks following epineurotomy of the mouse tibial nerve. Axons travel in a random fashion along the biceps femoris and gastrocnemius muscle without the benefit of a nerve graft or neurorrhaphy. Image taken from a double transgenic mouse overexpressing enhanced yellow fluorescent protein (EYFP) in its peripheral nerves and glial-derived neurotrophic factor (GDNF) under the control of the glial fibrillary acidic protein promoter (GFAP) of the neuroglia. Callibration bar denotes 5 mm. ^*denotes the epineurotomy site. > denotes the paths of randomly regenerating axons over a > 1cm course in a living mouse using fluorescence microscopy, z-motor, CoolSnap Monochromic CCD, and MetaMorph version 6.2 software.

Secondly, if retrograde staining was done and showed that indeed the nerves in the distal peroneal nerve were labeling back into the tibial motor neuron pool, the question is whether or not the regeneration seen in the distal nerve is entering the distal peroneal nerve from the side of it in the reverse end-to-side fashion or is just tracking up proximally to find the cut proximal end of the nerve and then going down the nerve in the regular fashion from proximal to distal. We would suspect that the regeneration seen in the distal nerve is either coming from the peroneal nerve in a proximal direction and then turning down the peroneal nerve and regenerating distally (Fig. [2]). One way to try to control this would be compare the results that Isaacs et al. achieved in this study against a situation where the end-to-side was places as far distally as possible and the transection on the peroneal nerve was made as far proximally as possible so that it would be much more difficult for the tibial nerve to regenerate proximally and then turn and regenerate into the distal nerve. An alternative technique to answer this question would be to divide the peroneal nerve above the end-to-side repair just prior to doing electrical studies.

Figure 2 The proposed end-to-side entry of tibial nerve axons into the peroneal nerve is shown as (A). Axons located in distal peroneal nerve stump may have been derived from contamination from the proximal peroneal nerve stump (B) or a rerouting of axons from the tibial nerve (C), rather than truly entering in an end-to-side fashion.

Experience from doing the initial classical end-to-side studies has shown us that the rat and mouse have great potential to contaminate the distal nerve from a proximal transected nerve stump, and that without retrograde tracing, it is not possible to tell where the nerves are coming from and whether they are sensory or motor.[3]

In summary, this is an interesting concept, but we are not convinced that the data supports their conclusion that denervated muscle can be successfully neurotized using a reverse end-to-side neurorrhaphy.

REFERENCES

• 1 Tarasidis G, Watanabe O, Mackinnon S E, Strasberg S R, Haughey B H, Hunter D A. End-to-side neurorrhaphy resulting in limited sensory axonal regeneration in a rat model.  Ann Otol Rhinol Laryngol. 1997;  106 506-512
• 2 Tarasidis G, Watanabe O, Mackinnon S E, Strasberg S R, Haughey B H, Hunter D A. End-to-side neurorraphy: a long-term study of neural regeneration in a rat model.  Otolaryngol Head Neck Surg. 1998;  119 337-341
• 3 Myckatyn T M, MacKinnon S E. A review of research endeavors to optimize peripheral nerve reconstruction.  Neurol Res. 2004;  26 124-138