Development of Protocol for 3D Image Analysis of Intraepidermal Nerve Fibers in Diabetic Mice
Mervak, Colin M.
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Diabetes, and the resulting diabetic neuropathies, is one of the greatest health problems facing Americans today. These neuropathies are characterized by a length-dependent degradation of sensory nerves in the body, leading to sensory loss especially in the peripheral limbs. Currently the diagnosis of diabetic neuropathy is difficult with few valid diagnostic techniques at a clinician’s disposal. Measuring the density of nerve fibers found in the epidermis of diabetes patients has proven to be an effective method of diagnosing diabetic neuropathy, but the current technique relies solely on fiber density counts of 2D images. This study tested the validity of using 3D imaging software as a diagnostic technique for diabetic neuropathy and developed a protocol for the use of said technique in the diagnosis of the disease in a type 2 diabetes mouse model. Mouse footpads were harvested, stained using fluorescent immunohistochemistry, and imaged on a confocal microscope to produce 3D fluorescent images. These images were then analyzed using 3D analysis programs and measurements of nerve volume, nerve branching, and branch volume were quantified. Our data suggest changes in nerve branching behavior that could be linked to the progression of diabetic neuropathy, supporting the hypothesis that this novel method could be an accurate diagnostic technique. Additional studies must be performed in order to further test the protocol, but these results indicate its potential as a reliable tool in the diagnosis of diabetic neuropathies.