Hyperglycemia-Induced Abnormalities in Rat and Human Corneas: The Potential of Second Harmonic Generation Microscopy

Abstract

BackgroundSecond Harmonic Generation (SHG) microscopy recently appeared as an efficient optical imaging technique to probe unstained collagen-rich tissues like cornea. Moreover, corneal remodeling occurs in many diseases and precise characterization requires overcoming the limitations of conventional techniques. In this work, we focus on diabetes, which affects hundreds of million people worldwide and most often leads to diabetic retinopathy, with no early diagnostic tool. This study then aims to establish the potential of SHG microscopy for in situ detection and characterization of hyperglycemia-induced abnormalities in the Descemet’s membrane, in the posterior cornea.Methodology/Principal FindingsWe studied corneas from age-matched control and Goto-Kakizaki rats, a spontaneous model of type 2 diabetes, and corneas from human donors with type 2 diabetes and without any diabetes. SHG imaging was compared to confocal microscopy, to histology characterization using conventional staining and transmitted light microscopy and to transmission electron microscopy. SHG imaging revealed collagen deposits in the Descemet’s membrane of unstained corneas in a unique way compared to these gold standard techniques in ophthalmology. It provided background-free images of the three-dimensional interwoven distribution of the collagen deposits, with improved contrast compared to confocal microscopy. It also provided structural capability in intact corneas because of its high specificity to fibrillar collagen, with substantially larger field of view than transmission electron microscopy. Moreover, in vivo SHG imaging was demonstrated in Goto-Kakizaki rats.Conclusions/SignificanceOur study shows unambiguously the high potential of SHG microscopy for three-dimensional characterization of structural abnormalities in unstained corneas. Furthermore, our demonstration of in vivo SHG imaging opens the way to long-term dynamical studies. This method should be easily generalized to other structural remodeling of the cornea and SHG microscopy should prove to be invaluable for in vivo corneal pathological studies.

Determination of collagen fibril size via absolute measurements of second-harmonic generation signals

“…The Descemet's membrane is located in the posterior part of the cornea, near the endothelium, and is mainly composed of nonfibrillar collagens that form centrosymmetrical networks and do not exhibit any SHG signal. However, sparse collagen fibrils were recently evidenced in the Descemet's membrane of diabetic rat corneas, as well as other hyperglycemia-induced structural corneal abnormalities [22][23][24] . Figure 5a,b display ex vivo frontal SHG images of these fibrils in an intact cornea.…”

“…Corneas from a 1-year-old Goto-Kakizaki rat, a spontaneous model of type 2 diabetes, were studied in the framework of a project about hyperglycemia-induced corneal abnormalities 23,24 . This study was performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Research Vision.…”

“…Ultrathin frontal sections were stained with uranyl acetate and lead citrate and observed with TEM (TEM CM10, Philips). Alternatively, ultrathin transverse sections were stained the same way for TEM imaging and compared with SHG imaging of serial thin sections 23 .…”

High throughput second harmonic imaging for label-free biological applications

“…The latter has been used to determine the microtubule organization in neurons [23,24]. SHG studies have also been performed to aid medical diagnostic purposes [25][26][27][28][29] and medicine development [30,31]. Voltage sensitive membrane specific markers have permitted for single site time dependent optical recordings of membrane potential activity [32][33][34][35][36][37].…”

Detection of back-reflected SHG from corneal histological sections

“…The corneal stroma accounts for about 90% of the all corneal thickness [4], making the cornea a primary target for SHG imaging. SHG imaging has already been proved useful to describe corneal modifications caused by keratoconus [5] and induced hyperglycemia [6]. In combination with other multiphoton microscopy imaging techniques, it has also been used to characterize in vivo the human skin [7] and the state of Alzheimer's disease and cancer [8].…”