We examined changes in intraepidermal nerve fibers (IENFs) to differentiate patients with diabetic neuropathy (DN) and neuropathic pain (DN-P) from those with DN without pain (DN-NOP). Punch skin biopsies were collected from the proximal thigh (PT) and distal leg (DL) of normal subjects (NS), patients with type 2 diabetes without evidence of DN (DM), or DN-P and DN-NOP patients. Protein gene product 9.5 (PGP) immunohistochemistry was used to quantify total IENF, and growth associated protein 43 (GAP43) for regenerating IENF. Compared to NS and DM, both DN-P and DN-NOP have reduced PGP+ IENF densities in DL and PT. Although GAP43+ IENF densities were also reduced in DL for both DN-P and DN-NOP, the GAP43+ IENF densities in PT of DN-P remained at the control levels. Higher GAP43/PGP ratios were detected in DN-P compared to DN-NOP in the DL and PT. In parallel, increased numbers of axonal swellings per PGP+ fiber (axonal swelling/PGP) were detected in DN-P compared to NS, DM, and DN-NOP in the DL. These axonal swellings were positive for tropomyosin-receptor-kinase (Trk) A and substance P, suggesting that they are associated with nociception.

In order to develop an efficient, reproducible, and well-tolerated protocol for assessing corneal innervation, 11 normal subjects underwent corneal confocal microscopy (CCM) using a Heidelberg Retinal Tomography III microscope. Five standardized locations were sampled in the left eye and one centrally in the right. The protocol was repeated 1-4 weeks later. A blinded technician measured nerve fiber length (NFL) and tortuosity coefficient (TC). The relationship between image location and NFL and TC was assessed using one-way analysis of variance, and reproducibility determined using relative intertrial variability and intraclass correlation coefficients. NFL reproducibility was maximized by averaging four or more images from the left eye, or one central image from both eyes. TC was less reproducible. CCM is a rapid, well-tolerated, and reproducible method for assessing corneal innervation.

Single confocal images have poor reliability for any of the 4 corneal nerve measurements, and there is no single location on the cornea that has improved reproducibility. Averaging 5 images, from different locations, improves the reproducibility and is essential for obtaining clinically meaningful data.

ObjectiveThis study focused on altered mitochondrial dynamics as a potential mechanism for diabetic peripheral neuropathy (DPN). We employed both an in vitro sensory neuron model and an in situ analysis of human intraepidermal nerve fibers (IENFs) from cutaneous biopsies to measure alterations in the size distribution of mitochondria as a result of hyperglycemia and diabetes, respectively.MethodsNeurite- and nerve-specific mitochondrial signals within cultured rodent sensory neurons and human IENFs were measured by employing a three-dimensional visualization and quantification technique. Skin biopsies from distal thigh (DT) and distal leg (DL) were analyzed from three groups of patients; patients with diabetes and no DPN, patients with diabetes and confirmed DPN, and healthy controls.ResultsThis analysis demonstrated an increase in mitochondria distributed within the neurites of cultured sensory neurons exposed to hyperglycemic conditions. Similar changes were observed within IENFs of the DT in DPN patients compared to controls. This change was represented by a significant shift in the size frequency distribution of mitochondria toward larger mitochondria volumes within DT nerves of DPN patients. There was a length-dependent difference in mitochondria within IENFs. Distal leg IENFs from control patients had a significant shift toward larger volumes of mitochondrial signal compared to DT IENFs.InterpretationThe results of this study support the hypothesis that altered mitochondrial dynamics may contribute to DPN pathogenesis. Future studies will examine the potential mechanisms that are responsible for mitochondrial changes within IENFs and its effect on DPN pathogenesis.

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