Comparison of Biodegradable Conduits within Aged Rat Sciatic Nerve Defects (2008)

Sir: We read with interest the article by Clavijo-Alvarez et al.1 published in the May 2007 issue of the Journal. These authors should be congratulated for addressing a key issue in peripheral nerve surgery, namely, the influence of age on functional recovery expectations of elderly patients. However, we would like to point out two main shortcomings in the experimental protocol adopted in this study. In our opinion, these shortcomings hinder the results obtained and the conclusions drawn by these authors. The first shortcoming is related to the 3-month end-point, which, as the authors also discuss in their article, is too short to allow the assessment of functional recovery after repair of a 1-cm-long defect of the rat sciatic nerve. Strasberg et al.,2 in their comparative assessment of strain differences in rat tibial nerve regeneration, found that functional recovery is poor at postoperative week 13 in five strains of adult male rats. Thus it is not surprising that the authors found that no group was able to recover function at week 12 in an aged model. Yet the majority of sciatic rat nerve repair studies adopt a much longer evaluation time (see Table 2 in the article by Dijkstra et al.3), so considering the expected delay in nerve healing of older animals, at least a 5-month end-point should have been used in this study. The second and most critical shortcoming is related to the method used for quantitative assessment of nerve regeneration. Luxol blue myelin stain is rather unusual in nerve repair studies and appears to be much less reliable than the accepted standard in nerve quantitative morphology, namely, toluidine blue staining of resin semithin sections after fixation in osmium tetroxide, which works as an excellent stain for myelin sheaths (Fig. 1). This procedure, besides being the most widely used, has also been validated against the possible sources of bias of nerve regeneration studies4; thus the employment of an alternative method needs to be motivated and validated by the authors. Yet the images provided in the authors’ Figure 5 confirm the limitations of Luxol stains for nerve regeneration investigation, since it is very difficult to recognize myelinated areas. (Actually, most of the structures indicated by the arrows in Fig. 5 seem to correspond to cell nuclei rather than myelin sheaths.) Further doubts on the reliability of the automatic procedure for measuring myelinated areas that was utilized in this study arise from the observation that the authors still found a 6 percent myelinated area in the distal nerve segment of the negative group in which, by contrast, no more myelin sheaths should be detectable 3 months after a nerve transection not followed by surgical repair (Wallerian degeneration).Fig. 1.: Micrograph of a regenerated rat sciatic nerve embedded in resin after fixation in 1.5% osmium tetroxide. The 2-μm-thick semithin section was stained using Richardson’s toluidine blue method for 3 minutes. Note how osmium tetroxide postfixation labels the myelin sheaths of nerve fibers, making it possible to easily count and measure them (magnification, 1000×).In conclusion, we believe that Clavijo-Alvarez et al. have raised a very important issue that needs to be addressed further with adequate experimental protocols. Yet the high percentage of limb autotomy, which represents an important limitation of Sciatic Function Index calculation, as the authors also recognize in their article, suggests that future studies might be better addressed using forelimb nerve experimental models, which have several practical advantages in comparison to hindlimb nerve models.5–7 DISCLOSURE The authors have no financial interest in any of the information disclosed in this communication. Stefano Geuna, M.D. Pierlugi Tos, M.D. University of Turin San Luigi Hospital Orbassano, Italy