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Histomorphometry in Peripheral Nerve Regeneration: Experimental Comparison of Different Axon Counting Methods
Lynn M Orfahli, BM1, Majid Rezaei, DDS, MSc1, Audrey Crawford, BS1, Michael J Annunziata, BS1, Carlos Xavier Ordenana, MD1, Brian Alexander Figueroa, MD1, Vahe Fahradyan, MD1, Jerry Silver, PhD2, Antonio Rampazzo, MD, PhD1 and Bahar Bassiri Gharb, MD, PhD1, (1)Cleveland Clinic Foundation, Cleveland, OH, (2)Case Western Reserve University, Cleveland, OH

Introduction: Histomorphometry is a common tool to quantitatively evaluate outcomes of peripheral nerve regeneration by measuring axonal parameters such as count, diameter, and myelination. Manual measurement across the entire nerve cross-section is the most accurate technique of histomorphometric analysis, but is extremely labor intensive. Thus, most researchers have opted for analysis of just a sample of the cross-section. Currently, automated sampled analysis is the most common method. However, no study has been performed to compare the accuracy of these techniques, making it difficult to compare results across the literature.

Materials and Methods: Digitized toluidine blue-stained slides of 24 rat sciatic nerves distal to a 20 mm nerve isograft were utilized. Three blinded researchers manually counted total myelinated fibers in each full cross-section. Total myelinated fiber counts were also extrapolated from sampled fields representing 20% of the cross-sectional area. Sampled counts were performed both manually and automatically with the software ImageJ (National Institutes of Health). Myelinated fiber diameter (FD), axon diameter (AD), and myelin sheath thickness (MTh) were measured manually in the full cross-sections and the sampled fields. ANOVA, Post-hoc Tukey, and paired T-tests were performed.

Results: Results are expressed in mean ± standard deviation. Total manual axon count was 13,506 ± 4,217. Sampled axon counts were significantly higher than total manual (manual sampled: 15,316 ± 4,613, p<0.001; automated: 16,297 ± 7,733, p=0.037). All three methods showed strong, significant correlation with each other (Manual full vs. manual sampled: r2=0.936, p<0.001; manual full vs. automated: r2=0.607, p=0.002; manual sampled vs. automated: r2=0.691, p<0.001). Total manual FD, AD, and MTh were 5.54 ± 1.14 µm, 3.57 ± 0.72 µm, and 0.98 ± 0.29 µm, respectively. They did not differ from sampled measurements (p=0.772, p=0.497, and p=0.101, respectively).

Conclusion: While manual count of sampled nerve sections differs in accuracy from full manual analysis, it produces highly correlated, reliable results when using standardized and systematic sampling methods. Automated data should be regarded with more caution as correlation was not as high. However, the significant correlation indicates it is an acceptable technique if manual sampled analysis is not possible. These methods are only three of the many techniques reported in the literature. Ergo, researchers must be cognizant of this wide variety of techniques and exercise caution when comparing data between studies.

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