98 Sensory and motor nerve conduction studies were performed to exclude large fiber involvement. Surface recording electrodes with standard placement were used to measure compound muscle action potential, sensory nerve action potential, and nerve conduction. Motor nerve conduction was assessed for peroneal and tibial nerves and sensory nerve conduction was assessed for the sural nerve. Quantitative Sensory Testing QST measurements were performed on both feet. Cold and warm detection thresholds with the method of limits, thermal sensory limen, paroxysmal heat sensations, cold and heat pain thresholds were performed according to the standard operating procedures described by the German Research Network on Neuropathic Pain (DFNS).29 Normative values published by the DFNS group were used as cutoff values.30 Patients with clinical signs, normal nerve conduction studies and at least two QST abnormalities, were defined as established SFN.31 Corneal Confocal Microscopy The Heidelberg Retinal Tomograph 3 (HRT3) with Rostock Corneal Module (Heidelberg Engineering GmbH, Dossenheim, Germany) was used to image the corneal subbasal nerve plexus. A set up piece of +12 dpt. was used with an x63 objective lens. Using this set up, 384x384 pixels images of 400x400 µm cornea tissue were generated. To obtain high quality imaging, a widely available protocol32 for CCM imaging was used. Analysis of Images The software package CCMetrics was developed at the University of Manchester for manual analysis of nerve tracing.18 Analysis was performed according to a standardized protocol.13 CCMetrics calculated the average CNFL from the selected images. CNFL was defined as the total length of all nerve fibers in the image (in units mm/mm2).18 ACCMetrics was also developed at the University of Manchester for automatic nerve tracing analysis. It was based on the same tracing criteria as CCMetrics.20 Although those programs could also calculate other parameters, NeuronJ was limited to calculating CNFL. Therefore, the other parameters were considered beyond the scope of this study, with the exception of the NFA calculated with ACCMetrics. The semi-automatic nerve tracing software package NeuronJ, a freely available plug-in module for ImageJ (Biomedical Imaging Group, version 1.4.3), was developed at Erasmus Medical Center, Rotterdam, the Netherlands.19 Manual initiation at the beginning of a nerve was required for nerve tracing; the algorithm then calculated the optimal tracing path. When low resolution of a nerve resulted in a failed tracing, manual corrections were possible. The number of highlighted pixels was the output of this program. Since the resolution and image size were known, simple math allowed converting the number of pixels to length in mm and vice versa. NFA FIJI was a custom developed macro for FIJI.21 After several filtering steps described in the original paper33, it quantifies the total number of pixels of a nerve plexus in an image. NFA is a two-dimensional metric that provides additional information about the full spectrum of variation in the nerve plexus. NFA FIJI results were compared with ACCMetrics NFA and with data from another study.21 This previous study defined a relationship between NFA FIJI and CNFL calculated with ACCMetrics. We reproduced that calculation, and in addition, we defined a relationship between CCMetrics and NFA FIJI, as well as between NeuronJ and NFA FIJI. Because ACCMetrics, CCMetrics and NeuronJ were expected to correlate, similar non-linear relations with NFA FIJI were expected. Three images from the center of the cornea of both eyes were used for manual (CCMetrics18) and semi-automatic (NeuronJ19) analysis. The selection was based on one image with lower, one with moderate and one with higher nerve fiber density, for both eyes. The number of images was decreased for manual and semi-automatic analysis to maintain an acceptable workload. As a results, 6 images 6 103 6
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