Histomorphometry in Peripheral Nerve Regeneration: Comparison of Different Axon Counting Methods

Histomorphometry quantitatively evaluates nerve regeneration. Total myelinated fiber count (TMFC) is most accurately obtained manually across full nerve cross-sections, but most researchers opt for automated, sampled analysis. Few of the numerous techniques available have been validated. The goal of this study was to compare common histomorphometric methods (full manual [FM], sampled manual [SM], and sampled automatic [SA]) to determine their reliability and consistency.

Twenty-four rats underwent sciatic nerve (SN) repair with 20mm isografts; SNs distal to the graft were analyzed. TMFC was manually determined in each full cross-section. Counts were also extrapolated from sampled fields, both manually and automatically with ImageJ software. Myelinated fiber diameter, axon diameter, and myelin sheath thickness were measured manually in full and sampled fields; G-ratio was calculated. Repeated-measures MANOVA, Spearman correlation, and Wilcoxon signed-rank tests were performed. A systematic review of histomorphometry in rat SN repair was performed to analyze the variability of techniques in the literature.

FM TMFC was 13,506 ± 4,217. Both sampled methods yielded significantly different TMFCs (SM:14.4 ± 13.4%, P< 0.001; SA:21.8 ± 44.7%, P = 0.037). All three methods strongly correlated with each other, especially FM and SM (r_s = 0.912, P< 0.001). FM fiber diameter, axon diameter, and myelin sheath thickness did not differ from SM (P = 0.493, 0.209, and 0.331, respectively). 65% of papers used sampling; 78% utilized automated or semi-automated analysis. Software, sampling, and histomorphometric parameters varied widely.

SM and SA analysis are reliable with standardized, systematic sampling. Transparency is essential to allow comparison of data; meanwhile, researchers must be cognizant of the wide variety of methodologies in the literature.