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A Comparison of Stereology and Binary Histomorphometry Methodology and Reliability in Peripheral Nerve
Hank Haw Sun, BA; Daniel A. Hunter; Gregory Opara; Michael J. Brenner, MD; Philip J. Johnson, PhD; Susan E. Mackinnon, MD
Washington University School of Medicine, St. Louis, MO, USA
Introduction: Stereology and histomorphometry are widely used by investigators for quantifying peripheral nerve regeneration after nerve injury. Although several of the parameters measured are similar with the two techniques, no study to date has systematically compared these methods in assessment of peripheral nerve. This study investigated the reliability of design-based stereology against semi-automated binary imaging histomorphometry for assessment of peripheral nerve parameters.
Design: Sciatic nerves were harvested bilaterally from 6 male Lewis rats, aged 8-12 weeks. From each animal, one nerve specimen was fixed, stained, and sectioned for electron microscopy, while the contralateral nerve was prepared for analysis under light microscopy. Equal numbers of left and right nerves were included in each group. Histomorphometry and stereology peripheral nerve analysis methods were applied and analysis of the nerves was performed by 2 blinded investigators to quantify nerve fiber parameters of fiber count, fiber width and density.
Results: Both methods proved highly reliable. No significant differences were identified between stereology and binary histomorphometry in accuracy for quantifying nerve fibers counts, fiber width, or density. Histomorphometry required less time per nerve to derive the quantitative parameters. In addition, histomorphometry allowed for more comprehensive analysis of nerve parameters (number of myelinated axons, myelinated fiber diameter, myelin thickness, fiber distributions, myelinated fiber density, and neural debris). Electron microscopic stereology allowed for quantitative analysis of unmyelinated axons, however.
Conclusion: Binary histomorphometry is more powerful than non-biased stereology for comprehensive analyses of peripheral nerve regeneration and may be less time and labor intensive. Nevertheless, stereology may be beneficial for assessing unmyelinated fiber morphology. The methods have similar reliability. Decisions regarding nerve analysis must therefore be based on the needs of each study.
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