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Matthew Scott Yorek, Mark A Yorek, Randy H Kardon; Three-Dimensional Analysis of Corneal Epithelial Nerves in Diabetes. Invest. Ophthalmol. Vis. Sci. 2014;55(13):5527.
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Diabetes mellitus is associated with corneal epithelial manifestations including epithelial erosion, chronic epitheliitis, superficial corneal ulcers and delayed epithelial healing. The long-term complications of diabetic corneal neuropathy include diminished corneal sensation and reduced tear secretion, both symptoms contribute to epithelial manifestations and both symptoms are related to the loss of the corneal innervation. Currently, in vivo corneal confocal microscopy (CCM) is being employed to grade the progression of diabetic neuropathy. This study combines both in vivo corneal imaging and immunohistochemistry with laser scanning confocal microscopy of the cornea in an effort to better understand the neuropathic changes associated with diabetes.
At 12 weeks of age C57Bl/6 mice were treated with streptozotocin (150 mg/kg i.p in saline) to induce diabetes. CCM was performed, corneal nerves were imaged using the Rostock cornea module on the Heidelberg Retina Tomograph confocal microscope. Next, corneas were dissected from the eyes and trimmed around the sclero-limbo region, then processed for immunohistochemistry using various antibodies, and were finally visulized using a Zeiss LSM 710 confocal microscope (Carl Zeiss; Germany). An analysis of corneal nerves was completed and is presented with Imaris software version 7.6.4 X64 (Bitplane; Zurich, Switzerland). Three-dimensional representations of confocal stacks were reconstructued by surface rendering, where a surface was created over the fluorescent staining and this three-dimensional surface was used for quantitation of nerve volume.
Using a three-dimensional analysis of the epithelial nerves we observed a significant decrease in the overall volume of nerve immunofluoresence in diabetic mice when compared to control animals. Control mice maintained a healthy 2.0% nerve volume, while diabetic mice lost 0.5% (P = 0.035) nerve volume after 14 weeks of the disease. This result is in agreement with in vivo CCM data showing a significant reduction (P = 0.016) in nerve density in the same animals.
We observed a reduction of nerve density and nerve volume in the mouse cornea during the progression of diabetes. Our data suggest imaging of corneal nerves may be a useful marker of diabetic neuropathy.
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