Design-Based Stereology and Binary Image Histomorphometry in Nerve Assessment

The role of Clemex software in this study

Clemex Vision was used to detect nerves separately from background non-nerve profiles. The software was able to measure the area of each component — myelin (blue) and axon (magenta). Combining the two colours into another binary plane, the software could also measure the total fibre width and the area of axon + myelin. Adding other measurement parameters, such as circular diameter, aspect ratio, or roughness, is easily done within the software.

Authors: Daniel A. Hunter, Deng Pan, Matthew D. Wood, Alison K. Snyder-Warwick, Amy M. Moore, Eva L. Feldman, Susan E. Mackinnon, Michael J. Brenner

Abstract

Background: Stereology and histomorphometry are widely used by investigators to quantify nerve characteristics in normal and pathological states, including nerve injury and regeneration. While these methods of analysis are complementary, no study to date has systematically compared both approaches in peripheral nerve. This study investigated the reliability of design-based stereology versus semi-automated binary imaging histomorphometry for assessing healthy peripheral nerve characteristics.

New Method: Stereological analysis was compared to histomorphometry with binary image analysis on uninjured sciatic nerves to determine nerve fibre number, nerve area, neural density, and fibre distribution.

Results: Sciatic nerves were harvested from 6 male Lewis rats, aged 8–12 weeks, for comprehensive analysis of 6 nerve specimens. From each animal, sciatic nerve specimens were fixed, stained, and sectioned for analysis by light and electron microscopy. Both histomorphometry and stereological peripheral nerve analyses were performed on all specimens by two blinded and independent investigators, who quantified nerve fibre count, fibre width, density, and related distribution parameters.

Comparison with existing methods: Histomorphometry and stereological analysis provided similar outcomes in nerve fibre number and total nerve area. However, the light microscopy — but not electron microscopy — stereological analysis yielded higher nerve fibre area compared to histomorphometry or manual measurement.

Conclusion: Both methods measure similar fibre number and overall nerve fibre area; however, stereology with light microscopy quantified higher fibre area. Histomorphometry optimizes throughput and comprehensive analysis but requires user thresholding.

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