Retinal Adaptation to Changing Glycemic Levels in a Rat Model of Type 2 Diabetes

Citation: Johnson LE, Larsen M, Perez M-T ( Retinal Adaptation to Changing Glycemic Levels in a Rat Model of Type 2 Diabetes Leif E. Johnson 0 Michael Larsen 0 Maria-Thereza Perez 0 Bang V. Bui, Univeristy of Melbourne, Australia 0 1 Department of Ophthalmology, Glostrup Hospital , Glostrup , Denmark , 2 University of Copenhagen, Faculty of Health and Medical Sciences , Copenhagen , Denmark , 3 Department of Clinical Sciences, Division of Ophthalmology, Lund University , Lund , Sweden Purpose: Glucose concentrations are elevated in retinal cells in undiagnosed and in undertreated diabetes. Studies of diabetic patients suggest that retinal function adapts, to some extent, to this increased supply of glucose. The aim of the present study was to examine such adaptation in a model of type 2 diabetes and assess how the retina responds to the subsequent institution of glycemic control. Methods: Electroretinography (ERG) was conducted on untreated Zucker diabetic fatty (ZDF) rats and congenic controls from 8-22 weeks of age and on ZDFs treated with daily insulin from 16-22 weeks of age. Retinal sections from various ages were prepared and compared histologically and by immunocytochemistry. Principal Findings/Conclusions: Acute hyperglycemia did not have an effect on control rats while chronic hyperglycemia in the ZDF was associated with scotopic ERG amplitudes which were up to 20% higher than those of age-matched controls. This change followed the onset of hyperglycemia with a delay of over one month, supporting that habituation to hyperglycemia is a slow process. When glycemia was lowered, an immediate decrease in ZDF photoreceptoral activity was induced as seen by a reduction in a-wave amplitudes and maximum slopes of about 30%. A direct effect of insulin on the ERG was unlikely since the expression of phosphorylated Akt kinase was not affected by treatment. The electrophysiological differences between untreated ZDFs and controls preceded an activation of M uller cells in the ZDFs (up-regulation of glial fibrillary acidic protein), which was attenuated by insulin treatment. There were otherwise no signs of cell death or morphological alterations in any of the experimental groups. These data show that under chronic hyperglycemia, the ZDF retina became abnormally sensitive to variations in substrate supply. In diabetes, a similar inability to cope with intensive glucose lowering could render the retina susceptible to damage. - Diabetes affects today approximately 347 million people worldwide, 90% of whom have the type 2 form (http://www. who.int/mediacentre/factsheets/fs312/en/). These figures are conservative since a significant number of people with type 2 diabetes are typically not diagnosed until several years after the onset of the disease. A major complication affecting a number of patients is diabetic retinopathy, which is clinically characterized by retinal vascular abnormalities such as microaneurisms, hemorrhages and neovascularization, eventually leading to visual loss (see reviews [1], [2]). There is, however, a delay of years or decades between the onset of diabetes and the development of microangiopathy. It is now believed that diabetic retinopathy is not, at least initially, a primary vascular disorder but that protracted damage to neuronal and glial components of the retina could be involved. This notion is supported by the demonstration of early subclinical anomalies, such as abnormal oscillatory potentials of the electroretinogram [3], which may reflect alterations that eventually contribute to microvascular retinopathy [1], [2]. In the retina, glucose uptake is not dependent on insulin and therefore intracellular glucose levels rise and fall with systemic glycemia [4], [5]. In diabetes, this is a confounding factor when trying to link abnormal retinal function to retinal disease. Specifically, functional abnormalities such as impaired dark adaptation can be reversed simply by raising blood glucose from normoglycemia to the patients habitual glycemic level [6], [7]. These anomalies may reflect mere adaptations to abnormal conditions rather than irreparable damage to the retina. Evaluations of retinal performance should therefore take extant and historic glycemia into account. In patients with diabetes, we have recently demonstrated protracted adaptation to normalized glycemia with a delay of 4 to 12 months [8]. We postulated that this may be a critical period during which the retina is more susceptible to developing microvascular damage, which in some patients manifests as an early worsening of diabetic retinopathy after institution of improved metabolic control [9]. To better characterize the dynamics of retinal adaptation in diabetes, one would need to examine the retina during defined periods of extended hyperglycemia and subsequently normalized glycemia. In the present study, we examined by electroretinography (ERG) the retinal function of the Zucker diabetic fatty (ZDF) rat. The ZDF rat is a model of type 2 diabetes that lives for months without severe weight loss and some of the other complications often seen in other diabetes models and therefore can be used to facilitate such studies. ZDF rats carry a leptin receptor defect (ZDF-Leprfa) [10]. They begin to develop hyperglycemia at 57 weeks of age and insulin resistance at 710 weeks [11]. We found in these rats that the responses of photoreceptors to light stimuli were unexpectedly higher when compared to controls while the inner retina developed functional impairment. Lowering glycemia with insulin halted the progression of some inner retinal abnormalities. However, it also reduced the photoreceptoral responses, revealing an inability to quickly re-adapt metabolism, which correlates with some aspects of clinical findings. Ethics Statement Experiments were approved by the Supervisory Authority on Animal Testing of Denmark (Dyreforsgstilsynet; permit 2007_561-1401) and the Ethical Committee on Animal Experiments in Malmo/Lund, Sweden (Malmo/Lunds Djurfo rso ksetiska Namnd; permit M79-09) and animals were treated according to the recommendations of the Association for Research in Vision and Ophthalmology. All electrophysiological studies were performed under ketamine/xylazine anesthesia, and all efforts were made to minimize suffering. Animals Male ZDF and ZDF congenic control rats (Lean) of 713 weeks of age were purchased from Charles River Laboratories (Sulzfeld, Germany) and were kept until an age of 16 to 43 weeks. Animals were maintained under a 12-hour light (#140 lux)/12 hour dark cycle and provided with food (Purina 5008 rat chow; International Product Supplies Ltd., London, England) and water ad libitum. A subset of ZDF rats was given daily insulin (ZDF-i) from 16 weeks of age, whereas the others were kept untreated (ZDF). Blood sampled from the lateral tail vein was used to measure glucose concentrations using a handheld glucometer (OneTouch UltraEasy; LifeScan Inc., Milpitas, CA (...truncated)