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Laura Kowalczuk, Gaël Latour, Michèle Savoldelli, Jean-Louis Bourges, Marie-Claire Schanne-Klein, Karsten Plamann, Francine F. Behar-Cohen; Correlative Imaging Analysis Of Rats And Human Corneas: The Potential Of Second-harmonic Generation Microscopy. Invest. Ophthalmol. Vis. Sci. 2012;53(14):128. doi: https://doi.org/.
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We aimed to better characterize previously highlighted hyperglycemia-induced abnormalities in the Descemet’s membrane (DM) and to evaluate diagnostic technology by correlating in vivo confocal microscopy (CM) with second-harmonic generation (SHG) microscopy and transmission electron microscopy (TEM).
A corneal confocal microscope was first used to generate in vivo images at the DM level in Goto-Kakizaki (GK) rats (n=6) and in non-diabetic Wistar rats (n=4). Ex vivo corneal observations were then performed with a multiphoton microscope (MPM) equipped with detection channels in the forward and backward directions. Characteristic corneas were analyzed by TEM. The MPM was finally improved to perform in vivo corneal observations of anesthetized rats. Human corneas from diabetic (n=3), hypertensive (n=4) and control (n=2) donors provided by the French Eye Bank were also involved in the study.
In GK ratcorneas, in vivo CM highlighted abnormal hyper-reflective deposits in the DM. These abnormalities exhibited intense SHG signals under the MPM, suggesting the presence of non centro-symmetric structures. TEM analysis showed actually abnormal long-spacing collagen in the DM anterior part. In vivo SHG imaging was performed successfully in GK rat corneas. Ex vivo analysis of human corneas finally showed that similar SHG+ structures are produced in the DM of diabetic patients, and also in the corneas of patients with other metabolic diseases.
We demonstrated for the first time that long-spacing collagen produced in the DM of diabetic subjects can be detected using SHG imaging with enhanced contrast as compared to CM. It demonstrates that in vivo multiphoton imaging is relevant to investigate corneal abnormalities.
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