![]() Sets of parameters were measured for both structures: area, perimeter, mean radius and Gratio. Finally, the myelin sheaths were detached by subtraction of the axons from the nerve fiber. Next, the nerve fiber area was distinguished on an inverted input image by the Distance-B method with Kapur thresholding. Axons were identified with adaptive Otsu thresholding. This software pipeline was designed to automatically identify three sets of objects: primary objects–axons secondary objects–nerve fibers and tertiary objects–myelin sheaths. Processed images were uploaded into CellProfiler for automated axon and myelin sheath identification and measurement. Images were uploaded in self-design image processing macro in FIJI that included Contrast Limited Adaptive Histograph Equalization of the original image (CLAHE), 16-bit conversion, and another CLAHE (converted image as a mask). Preanalytical image processing began with artifact removal in GIMP (non-neural elements). Semithin sections were stained with toluidine blue and captured at 40× magnification. Other steps included: wash in 0.1M cacodylate, postfixation in solution of 4% osmium tetroxide in 0.2M cacodylate buffer, dehydration and embedding in Poly/Bed812. Rat sciatic nerve fragments were harvested and fixed in a solution of 2.5% glutaraldehyde and 0.1M cacodylate buffer. Our research presents a novel method based on digital image analysis in free and open software. Manual methods are prone to human error, inaccurate and time-consuming. Quantitative analysis of the components in nerve section is especially useful in neuroregeneration research. Spaces in between are filled with parenchyma and vessels. Peripheral nerves are composed of nerve fibers: axons surrounded by a myelin sheath. ![]() Orlowski Memorial Hospital, Warsaw, Poland Wlodarski, MD, PhD 1ġDepartment of Research Methodology, Laboratory of Center for Preclinical Research, Medical University of Warsaw, Warsaw, Poland, 2Department of Histology and Embryology, Laboratory of Center for Preclinical Research, Medical University of Warsaw, Warsaw, Poland, 3Department of Plastic and Reconstructive Surgery, Center of Postgraduate Medical Education, Prof. Paskal, MD 1 Piotr Pietruski, MD, PhD 3 Pawel K. ![]() Wiktor Paskal, MD 1 Micha? Kopka, Medical Student 1,2 Adriana M. An Automated Peripheral Nerve Image Analysis Protocol For Axon And Myelin Sheath Determination And Evaluation ![]()
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