American Society for Peripheral Nerve

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A Comparison of Histological Methods for Evaluating Axon Density in Peripheral Nerve – a Rat Sciatic Nerve Model
Shai Luria, MD; Ariel Kerpel, MD; Ido Volk, MD; Avraham Cohen, MD
Orthropaedic Surgery, Hadassah-Hebrew University Medical Center, Jerusalem, Israel

Background: A key method in evaluation of nerve regeneration is the evaluation of axon density in nerve cross sections. Formal quantification of axons is classically performed by toluidine blue staining even though this technique is considerably time-consuming and more expensive then immunohistochemical or immunofluorescence techniques that are used extensively in neuropathology. Immunofluorescence staining may have the additional advantages of differential axon count as well as fluorescent techniques of automated axon count.

Our hypothesis was that using immunofluorescent stains, evaluation of axon density is as accurate as Toluidine Blue staining.

Methods: Seven wild type rats underwent a unilateral crush injury of the sciatic nerve. The nerves were harvested at either 1 or 3 weeks post insult, including segments proximal and distal to the injury site and from the contralateral (uninjured side, designated control). Each segment was divided into two parts and fixated either for the toluidine blue (TB) staining or for 2 types of immunofluorescent staining with axon-specific antibodies (anti-NeuroFilament (NF) and anti-Protein-Gene-Product 9.5 (PGP)). Manual axon count was performed and compared between the groups using Pearson as well as Spearman’s rho correlation measures.

Results: We found a high correlation between the immunofluorescent stains and the toluidine blue for all segments including those distal to the injury. Comparing NF with TB, yielded a Pearson’s coefficient of 0.992 (p<0.001) for proximal, 0.857 (p=0.029) for distal and 0.977 (p=0.001) for control segments. Comparing PGP with TB yielded a Pearson coefficients of 0.767 (p=0.044), 0.915 (p=0.011) and 0.968 (p<0.001) for proximal, distal and control segments, respectively.

Discussion: We found that immunofluorescent staining resulted in comparable estimation of axon counts in peripheral nerve, as the toluidine blue stain, regardless of the injury. Immunofluorescence techniques may be a more affordable tool for the analysis of peripheral nerve injury and regeneration.

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