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Alexander Heisterkamp, Tobias Ehmke, Maria Reichard, Heike Weiss, Simone Baltrusch, Oliver Stachs; In-vivo Confocal and two-photon fluorescence microscopy on the cornea of diabetic and non-diabetic thy1 YFP mice. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2643.
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© ARVO (1962-2015); The Authors (2016-present)
Diabetes mellitus is one of the most widespread diseases worldwide, with neuropathy as one of the major complications. Neuropathy detection is currently undergoing sophistication by applying the confocal laser scanning microscopy (CLSM) on the cornea to detect early neurodegenerative changes. Further laser based nonlinear imaging techniques like two-photon excitation fluorescence microscopy (TPM) are suitable for high resolution imaging at a subcellular scale. TPM allows label-free imaging by second-harmonic generation (SHG), which sensitive to collagen, and by excitation of metabolic molecules like NADH, visualizing cells within cornea.
The corneal nerves of neurofluorescent B6.Cg-Tg(Thy1-YGP)Jrs/J mice were co-examined in vivo with clinical established CLSM and two-photon excitation fluorescence microscopy. The corneal subbasal nerve plexus of healthy (n=3) and diabetic (n=3) age matched thy1-YFP mice was studied under general anesthesia with both methods and the nerve fiber density (NFD) was estimated with NeuronJ. Diabetes mellitus was induced in 9 weeks old mice by injection of streptozotocin (STZ, low dose protocol). The diabetic mice were examined 14 days after STZ injection at a very early stage of diabetes manifestation (6.67+/-1.15 days of duration).
TPM was more sensitive to nerve detection than CLSM (healthy mice with TPM: 24.52 +/- 2.25 mm/mm2 (SEM); CLSM: 16.93 +/- 0.691 mm/mm), thus the NFD values of both techniques correlated well. With both methods it could be shown, that already after this short duration of diabetes the NFD decreases significantly in diabetic mice (TPM: 16.26 +/- 2.26 mm/mm2; CLSM: 11.04 +/- 0.42 mm/mm2). Using TPM, the position and path of nerves within the stroma and the epithelial layer can be visualized by SHG, autofluorescence and YFP excitation.
Confocal laser scanning microscopy and two-photon microscopy are useful tools for in-vivo observation of diabetes related nerve fiber degeneration in the eye with the possibility of further nonlinear label-free detection modalities, like four-wave mixing or third-harmonic generation which might help determining early changes within the cornea of diabetic patients.
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