American Society for Peripheral Nerve

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Introducing New Software Programs for the Counting of Nerve Axons and the Determination of Mean Axon Area
Chase Tobin, BS; Ziyi Wang, BS; Lin-Ling Zhang, MD; Michael Agresti, MS; Hani Matloub, MD; Ji-Geng Yan, MD, PhD
Medical College of Wisconsin, Milwaukee, WI, USA

Purpose: The purpose of this work was to design a method using computer software to improve the speed and accuracy of axon counting and the calculation of mean axon area of a peripheral nerve cross section.

Materials and Methods: Two software programs, ImageJ (FIJI distribution) and Paint.net, were used to design the new analysis methods. Multiple photographs of a nerve cross section, taken at 200x magnification, were “stitched” together within the ImageJ software to produce one complete image (Figure 1). Paint.net was used to crop the photograph (Figure 2) which allowed the removal of large histological content outside the fascicle that are not axons, such as epineurium and perineurium, as well as blood vessels inside the fascicle. ImageJ was then used to perform the quantitative analysis. Within ImageJ, the cropped photograph was converted to a binary image (Figure 3). The original “stitched” image was then opened within ImageJ which permitted calculation of the area of 10 small and 10 large axons. The average area of each group was determined. The program could then produce a new digital image of the axons within the cross section (Figure 4) and calculate the mean axon area and number of axons.

     

Results: Our results indicated that using the software greatly improves the quantitative analysis. After familiarization, the analysis can be performed in 20 minutes on average depending on the amount of cropping required and the size of the cross section. The images and values obtained were consistent and enhance the efficiency of data collection dramatically. Also, these methods can be used to determine the number of degenerated myelinated nerve fibers (calcified spots).

Conclusions: The new nerve fiber counting method we developed combines two software programs and provides a standardized approach for the calculation of mean axon area and numbers of nerve fibers with consistent accuracy and precision as well as reduces both human error and the time required to determine the parameters of interest.


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