Role of timing in assessment of nerve regeneration

Abstract

Small animal models are indispensable for research on nerve injury and reconstruction, but their superlative regenerative potential may confound experimental interpretation. This study investigated time-dependent neuroregenerative phenomena in rodents. Forty-six Lewis rats were randomized to three nerve allograft groups treated with 2 mg/(kg day) tacrolimus; 5 mg/(kg day) Cyclosporine A; or placebo injection. Nerves were subjected to histomorphometric and walking track analysis at serial time points. Tacrolimus increased fiber density, percent neural tissue, and nerve fiber count and accelerated functional recovery at 40 days, but these differences were undetectable by 70 days. Serial walking track analysis showed a similar pattern of recovery. A "blow-through" effect is observed in rodents whereby an advancing nerve front overcomes an experimental defect given sufficient time, rendering experimental groups indistinguishable at late time points. Selection of validated time points and corroboration in higher animal models are essential prerequisites for the clinical application of basic research on nerve regeneration.

Figure 1

Schematic representation of the experimental design. Group sizes are indicated in italics. Animals were sacrificed 40, 46, and 70 days postoperatively.

Figure 2

Mean print length factors of surviving animals in Groups I-III plotted over 70 days. Animals treated with tacrolimus appear to normalize and stabilize more quickly than those left untreated or treated with cyclosporine-A. Differences were not statistically significant.

Figure 3

Representative photomicrographs of the toluidine blue-stained sections 5-mm distal to nerve allografts harvested at 40 and 70 days at ×400 magnification. By 40 days postoperatively, treatment with (A) Tacrolimus increased nerve regeneration compared with (B) Cyclosporine-A and (C) untreated allografts, while at 70 days, differences between (D) tacrolimus, (E) Cyclosporine-A, and (F) untreated groups could not be distinguished from one another. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Figure 4

Mean histomorphometric parameters for groups I–III harvested at time points of 40, 46, and 70 days postoperatively. Tacrolimus-treated animals demonstrated significantly greater nerve regeneration than Cyclosporine-A treated or untreated animals at 40 days (* denotes a significant difference at P < 0.05 in comparisons against untreated group, error bars indicate standard deviation).