Multimodal Highlighting of Structural Abnormalities in Diabetic Rat and Human Corneas.

PURPOSE This study aimed to highlight structural corneal changes in a model of type 2 diabetes, using in vivo corneal confocal microscopy (CCM). The abnormalities were also characterized by transmission electron microscopy (TEM) and second harmonic generation (SHG) microscopy in rat and human corneas. METHODS Goto-Kakizaki (GK) rats were observed at age 12 weeks (n = 3) and 1 year (n = 6), and compared to age-matched controls. After in vivo CCM examination, TEM and SHG microscopy were used to characterize the ultrastructure and the three-dimensional organization of the abnormalities. Human corneas from diabetic (n = 3) and nondiabetic (n = 3) patients were also included in the study. RESULTS In the basal epithelium of GK rats, CCM revealed focal hyper-reflective areas, and histology showed proliferative cells with irregular basement membrane. In the anterior stroma, extracellular matrix modifications were detected by CCM and confirmed in histology. In the Descemet's membrane periphery of all the diabetic corneas, hyper-reflective deposits were highlighted using CCM and characterized as long-spacing collagen fibrils by TEM. SHG microscopy revealed these deposits with high contrast, allowing specific detection in diabetic human and rat corneas without preparation and characterization of their three-dimensional organization. CONCLUSION Pathologic findings were observed early in the development of diabetes in GK rats. Similar abnormalities have been found in corneas from diabetic patients. TRANSLATIONAL RELEVANCE This multidisciplinary study highlights diabetes-induced corneal abnormalities in an animal model, but also in diabetic donors. This could constitute a potential early marker for diagnosis of hyperglycemia-induced tissue changes.

[1]  T. Chikama,et al.  Abnormal light scattering detected by confocal biomicroscopy at the corneal epithelial basement membrane of subjects with Type II diabetes , 2001, Diabetologia.

[2]  W. Green,et al.  Immunohistochemistry and electron microscopy of early-onset fuchs corneal dystrophy in three cases with the same L450W COL8A2 mutation. , 2006, Transactions of the American Ophthalmological Society.

[3]  W. Webb,et al.  Nonlinear magic: multiphoton microscopy in the biosciences , 2003, Nature Biotechnology.

[4]  Wei Gao,et al.  Mechanism-based disease progression modeling of type 2 diabetes in Goto-Kakizaki rats , 2011, Journal of Pharmacokinetics and Pharmacodynamics.

[5]  Gereon Hüttmann,et al.  Imaging corneal crosslinking by autofluorescence 2‐photon microscopy, second harmonic generation, and fluorescence lifetime measurements , 2010, Journal of cataract and refractive surgery.

[6]  B. Cho,et al.  Differences in corneal thickness and corneal endothelium related to duration in Diabetes , 2006, Eye.

[7]  Y. Akimoto,et al.  Elevated expression of O-GlcNAc-modified proteins and O-GlcNAc transferase in corneas of diabetic Goto-Kakizaki rats. , 2003, Investigative ophthalmology & visual science.

[8]  Jia Yin,et al.  Corneal complications in streptozocin-induced type I diabetic rats. , 2011, Investigative ophthalmology & visual science.

[9]  H. Taylor,et al.  Corneal epithelial basement membrane changes in diabetes. , 1981, Investigative ophthalmology & visual science.

[10]  T. Chikama,et al.  Development of an Instrument for Measurement of Light Scattering at the Corneal Epithelial Basement Membrane in Diabetic Patients , 2007, Japanese Journal of Ophthalmology.

[11]  R. S. Wall,et al.  Ageing of the human corneal stroma: structural and biochemical changes. , 1992, Biochimica et biophysica acta.

[12]  C. Baudouin,et al.  Principles of Confocal In Vivo Microscopy , 2006 .

[13]  Y. Akimoto,et al.  Change in Long-Spacing Collagen in Descemet's Membrane of Diabetic Goto-Kakizaki Rats and Its Suppression by Antidiabetic Agents , 2008, Experimental diabetes research.

[14]  Karsten Plamann,et al.  Multimodal nonlinear imaging of the human cornea. , 2010, Investigative ophthalmology & visual science.

[15]  M. Lahav,et al.  Ultrastructural Changes in Corneas of Diabetic Patients: An Electron-microscopy Study , 2000, Cornea.

[16]  J. Moss,et al.  The composition of wide-spaced collagen in normal and diseased Descemet's membrane. , 1996, Current eye research.

[17]  R. O. Schultz,et al.  Diabetic keratopathy. , 1981, Transactions of the American Ophthalmological Society.

[18]  F. Behar-Cohen,et al.  Microglia/macrophages migrate through retinal epithelium barrier by a transcellular route in diabetic retinopathy: role of PKCζ in the Goto Kakizaki rat model. , 2011, The American journal of pathology.

[19]  A. Bialasiewicz,et al.  Corneal Endothelium in Patients with Diabetes Mellitus: A Historical Cohort Study , 2009, European journal of ophthalmology.

[20]  N. Thomas,et al.  Endothelial Cell Loss and Central Corneal Thickness in Patients With and Without Diabetes After Manual Small Incision Cataract Surgery , 2011, Cornea.

[21]  Christophe Baudouin,et al.  Atlas of Confocal Laser Scanning In-vivo Microscopy in Ophthalmology , 2006 .

[22]  Hyman A. Carrel,et al.  Decreased density of corneal basal epithelium and subbasal corneal nerve bundle changes in patients with diabetic retinopathy. , 2006, American journal of ophthalmology.

[23]  J. González-Méijome,et al.  Two single descriptors of endothelial polymegethism and pleomorphism , 2010, Graefe's Archive for Clinical and Experimental Ophthalmology.

[24]  R. Nagaraj,et al.  Advanced Maillard reaction and crosslinking of corneal collagen in diabetes. , 1995, Biochemical and biophysical research communications.

[25]  C. Hamlin,et al.  Apparent Accelerated Aging of Human Collagen in Diabetes Mellitus , 1975, Diabetes.

[26]  W. Green,et al.  Advanced glycation end products and receptors in Fuchs' dystrophy corneas undergoing Descemet's stripping with endothelial keratoplasty. , 2007, Ophthalmology.

[27]  S. Horiuchi,et al.  Advanced glycation end products in Descemet’s membrane and their effect on corneal endothelial cell , 2001, Current eye research.

[28]  I. Gipson,et al.  Altered epithelial-basement membrane interactions in diabetic corneas. , 1992, Archives of ophthalmology.

[29]  Alan W. Stitt.  The Maillard Reaction in Eye Diseases , 2005, Annals of the New York Academy of Sciences.

[30]  M. Brownlee.  Biochemistry and molecular cell biology of diabetic complications , 2001, Nature.

[31]  N. Pritchard,et al.  Corneal markers of diabetic neuropathy. , 2011, The ocular surface.

[32]  M. Araie,et al.  Advanced glycation end products in diabetic corneas. , 2000, Investigative ophthalmology & visual science.

[33]  Teruo Nishida,et al.  Second-harmonic imaging microscopy of normal human and keratoconus cornea. , 2007, Investigative ophthalmology & visual science.

[34]  Yuji Nagai,et al.  Corneal Epithelial Barrier Function in Diabetic Patients , 2004, Cornea.

[35]  Chen-Yuan Dong,et al.  Multiphoton fluorescence and second harmonic generation imaging of the structural alterations in keratoconus ex vivo. , 2006, Investigative ophthalmology & visual science.

[36]  D. DuBois,et al.  Adipose Tissue Deficiency and Chronic Inflammation in Diabetic Goto-Kakizaki Rats , 2011, PloS one.

[37]  M. Lahav,et al.  Collagen Pleomorphism in Descemet's Membrane of Streptozotocin-induced Diabetic Rats: An Electron Microscopy Study , 2000, Cornea.

[38]  E. Davidson,et al.  Changes in corneal innervation and sensitivity and acetylcholine-mediated vascular relaxation of the posterior ciliary artery in a type 2 diabetic rat. , 2012, Investigative ophthalmology & visual science.

[39]  C. Murphy,et al.  Non-enzymatic glycation in corneas from normal and diabetic donors and its effects on epithelial cell attachment in vitro. , 2003, Optometry.

[40]  Cheng Zhang,et al.  Fuchs corneal dystrophy: aberrant collagen distribution in an L450W mutant of the COL8A2 gene. , 2005, Investigative ophthalmology & visual science.

[41]  Josef Bille,et al.  Second harmonic generation imaging of collagen fibrils in cornea and sclera. , 2005, Optics express.

[42]  Jin Sook Kim,et al.  Involvement of advanced glycation end products, oxidative stress and nuclear factor-kappaB in the development of diabetic keratopathy , 2011, Graefe's Archive for Clinical and Experimental Ophthalmology.

[43]  Chung-May Yang,et al.  In vivo confocal microscopic findings of corneal wound healing after corneal epithelial debridement in diabetic vitrectomy. , 2009, Ophthalmology.

[44]  C. Baudouin,et al.  Comparative Anatomy of Laboratory Animal Corneas with a New-Generation High-Resolution In Vivo Confocal Microscope , 2006, Current eye research.

[45]  Kaori Suzuki,et al.  Expression and function of receptors for advanced glycation end products in bovine corneal endothelial cells. , 2003, Investigative ophthalmology & visual science.

[46]  A. Boulton,et al.  Corneal confocal microscopy detects improvement in corneal nerve morphology with an improvement in risk factors for diabetic neuropathy , 2011, Diabetic medicine : a journal of the British Diabetic Association.

[47]  J. Moss,et al.  Descemet's membrane in the iridocorneal-endothelial syndrome: morphology and composition. , 1995, Experimental eye research.

[48]  Chen-Yuan Dong,et al.  Multiphoton autofluorescence and second-harmonic generation imaging of the ex vivo porcine eye. , 2006, Investigative ophthalmology & visual science.

[49]  Gaël Latour,et al.  In vivo structural imaging of the cornea by polarization-resolved second harmonic microscopy , 2011, Biomedical optics express.

[50]  R. O. Schultz,et al.  Diabetic corneal neuropathy. , 1983, Transactions of the American Ophthalmological Society.

[51]  G. Bretthauer,et al.  Image reconstruction of the subbasal nerve plexus with in vivo confocal microscopy. , 2011, Investigative ophthalmology & visual science.

[52]  A. Yoshida,et al.  Corneal advanced glycation end products increase in patients with proliferative diabetic retinopathy. , 2001, Diabetes care.

[53]  Bruce J Tromberg,et al.  Selective corneal imaging using combined second-harmonic generation and two-photon excited fluorescence. , 2002, Optics letters.

[54]  T. Chikama,et al.  Delayed wound closure and phenotypic changes in corneal epithelium of the spontaneously diabetic Goto-Kakizaki rat. , 2007, Investigative ophthalmology & visual science.