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Histomorphometry in Nerve Regeneration: Experimental Comparison Of Different Axon Counting Methods
Majid Rezaei, Audrey Crawford, Michael Annunziata, Carlos Ordenana, Lynn Orfahli, Brian Figueroa, Jerry Silver, Antonio Rampazzo, Bahar Bassiri Gharb
Case Western Reserve University - Cleveland Clinic
2019-02-15
Presenter: Lynn Orfahli
Affidavit:
I certify that the material proposed for presentation in this abstract has not been published in any scientific journal or previously presented at a major meeting.
Director Name: Bahar Bassiri Gharb, MD, PhD
Author Category: Medical Student
Presentation Category: Basic Science Research
Abstract Category: General Reconstruction
Background: Histomorphometry is the most common method to evaluate outcomes of peripheral nerve regeneration. It is simple to implement and provides a quantifiable, objective assessment. However, practical choice of measurement methods can remarkably affect results and there is no universally accepted method.
Methods: Digitized toluidine-stained slides of 34 rat sciatic nerves distal to a 20 mm nerve graft (5 mm proximal to trifurcation) were utilized. All myelinated axons in each nerve section were manually counted. Total axon counts were also extrapolated from manually- and software-counted (ImageJ, National Institutes of Health, USA) sample nerve sections representing 20% of total area. Fiber (FD) and axon (AD) diameters, myelin thickness (MTh), and G-ratio were measured manually throughout.
Results: Total axon counts in the distal sciatic nerves were 12,503±4,195 with mean FD and MTh of 5.4±1.1 µm and 0.9±0.2 µm, respectively. Manual axon counts extrapolated from sampled nerve sections were significantly higher than the manual count of the entire section (13,819±4,688; p=0.000). Counts from both methods were highly correlated (r^2=0.936, p=0.000). ImageJ counts were also significantly different from total counts (p=0.005), but did not correlated with the total manual counts. FD, AD, and MTh were not different between groups (p=0.253, p=0.111, p=0.6).
Conclusion: Total manual axon count remains the most accurate method. Manual count of sampled nerve section can produce highly correlated, reliable counts when using standardized and systematic sampling methods.
