Neurodegeneration, Neuropeptides, and Diabetic Retinopathy

INTRODUCTION

Diabetic retinopathy (DR) is the leading cause of blindness in working-age individuals in developed countries [1]. The tight control of blood glucose levels and blood pressure is essential in preventing or arresting DR development. However, the therapeutic objectives are difficult to achieve, and in consequence, DR appears in a high proportion of patients. When DR appears, laser photocoagulation remains as the main tool in the therapeutic armamentarium. The objective of laser photocoagulation is not to improve visual acuity but to stabilize DR, thus preventing severe visual loss. When laser photocoagulation is indicated in time, the risk of blindness is reduced by 90% in the following 5 years, and the loss of visual acuity is reduced in 50% in those patients with macular edema [2]. However, timely indication is often passed, and therefore, the effectiveness of laser photocoagulation in current clinical practice is significantly lower. In addition, laser photocoagulation destroys a part of the healthy retina, and in consequence, side effects such as loss in visual acuity, impairment of both dark adaptation and color vision, and visual field loss may appear. Vitreoretinal surgery could be indicated in advanced stages of DR (i.e., hemovitreous, retinal detachment). However, this therapeutic option requires a skillful team of ophthalmologists, is expensive, and fails in more than 30% of cases. With this scenario, it seems clear that new treatments based on greater physiopathological knowledge of DR are needed.

DR has been classically considered to be a microcirculatory disease of the retina due to the deleterious metabolic effects of hyperglycemia per se and the metabolic pathways triggered by hyperglycemia (polyol pathway, hexosamine pathway, DAG-PKC pathway, advanced glycation end products [AGEs], and oxidative stress). However, before any microcirculatory abnormalities can be detected in ophthalmoscopic examination, retinal neurodegeneration is already present. In other words, retinal neurodegeneration is an early event in the pathogenesis of DR which antedates and participates in the microcirculatory abnormalities that occur in DR [3, 4]. Therefore, the study of the mechanisms that lead to neurodegeneration will be essential for identifying new therapeutic targets in the early stages of DR.

NEURODEGENERATION AS AN EARLY EVENT

IN THE PATHOGENESIS OF DR

The concept of neurodegeneration as an early event in the pathogenesis of DR was first introduced by Barber et al. [5]. These authors observed that 1 month after inducing diabetes in rats by using streptozotocin, there was a high rate of apoptosis (TUNELpositive cells) in the neuroretina without a significant apoptosis in endothelial cells. In the same paper, the authors found a higher rate of apoptosis in the neuroretina from diabetic donors in comparison with nondiabetic donors, even in the case of a diabetic donor without microvascular abnormalities. These findings have been further confirmed in experimental models. In addition, it has been demonstrated that, apart from apoptosis, another of the features of retinal neurodegeneration is glial activation [3–7]. Our research group has been able to demonstrate that both apoptosis and glial activation occur in the retina of diabetic patients and precede microvascular abnormalities [8, 9] (Fig. 1). This is important because the experimental model in which these findings had

Fig. 1. Comparison of the two key elements of neurodegeneration (glial activation and apoptosis) between a representative case of diabetic patient without DR and a nondiabetic subject. As can be seen, neurodegeneration is higher in the retina from the diabetic donor. (A) Glial activation in the human retina. Glial fibrillar acidic protein (GFAP) immunofluorescence (green) from a nondiabetic donor (left panel) and a diabetic donor (right panel). (B) Apoptosis in the human retina. Upper panel: nondiabetic donor (a: propidium iodide, b: TUNEL immunofluorescence). Low panel: diabetic donor (c: propidium iodide, d: TUNEL immunofluorescence). RPE retinal pigment epithelium; ONL outer nuclear layer; INL inner nuclear layer; GCL ganglion cell layer. The bar represents 20 mm.

been observed was the rat with streptozotocin-induced diabetes (STZ-DM). Streptozotocin is a potent neurotoxic agent and is able to produce neural degeneration. Therefore, neurodegeneration (apoptosis + glial activation) observed in rats with STZ-DM could be due to STZ itself rather than the metabolic pathways related to diabetes. However, our observation that these changes also occur in the retina of diabetic patients and the further demonstration that they are also present in retinal explants cultured with a media

with a high content of AGEs [10] clearly demonstrate that neurodegeneration is a crucial pathogenic factor of DR.

Neuroretinal damage produces functional abnormalities such as the loss of both chromatic discrimination and contrast sensitivity. These alterations can be detected by means of electrophysiological studies in diabetic patients with less than 2 years of diabetes duration, that is before microvascular lesions can be detected in ophthalmologic examination. In addition, neuroretinal degeneration will initiate and/or activate several metabolic and signaling pathways which will participate in the microangiopathic process, as well as in the disruption of the blood-retinal barrier (a crucial element in the pathogenesis of DR). Nevertheless, these metabolic pathways remain to be characterized.

The mechanisms involved in DR neurodegeneration are poorly understood. In addition, it is unknown which of the two primordial pathological elements (apoptosis or glial activation) is the first to appear and is, in consequence, the primary event. Nevertheless, it seems that these diabetes-induced changes occur in the early stages of DR, and that they are closely related.

IN VIVO EXPERIMENTAL MODELS TO STUDY

RETINAL NEURODEGENERATION IN THE SETTING

OF DIABETIC RETINOPATHY

Since neurodegeneration is an early event in the pathogenesis of DR, it is not necessary to use animal models with microangiopathic lesions in the eye such us Torii or GotoKakizaki rats. The experimental model currently used to study retinal neurodegeneration in DR is the rat with streptozotocin-induced diabetes (STZ-DM). In this model, electroretinographic abnormalities are present 2 weeks after inducing diabetes, and the presence of neural apoptosis and glial reaction can be clearly detected 1 month after starting diabetes.

Retinal ganglion cells (RGCs) are the earliest cells affected and with the highest rate of apoptosis [11]. However, an elevated rate of apoptosis has been also observed in the outer nuclear layer (photoreceptors) [12] and in the retinal pigment epithelium (RPE) [13].

As commented above, the interpretation of the results of retinal neurodegeneration in STZ-DM rats is hampered by the neurotoxic effect of STZ. It is worthy of mention that pathological changes to the brain after intraventricular injection of STZ are very similar to the neurodegeneration reported in DR [14]. Therefore, it may be advisable to use murine models with a spontaneous development of diabetes or at least experimental models in which diabetes has not been induced by a neurotoxic drug.

Mice have been much less used than rats as experimental models for the study of DR and retinal neurodegeneration. This is because they are more resistant to the STZ effect (mice need 3–5 doses of STZ to induce diabetes, whereas in rats, one dose is sufficient), have lower eyecups, and present a lower degree of lesions in comparison with rats. This relative protection to developing pathological lesions related to diabetes can be partly attributed to lower activity of aldose reductase (polyol pathway) in comparison with rats [7]. Nevertheless, because of its great potential for genetic manipulation, the mouse offers a unique opportunity to study the molecular pathways involved in disease development. Among mice, C57BL/KsJ-db/db is the model that best reproduces the neurodegenerative features observed in patients with DR (Fig. 2). C57BL/KsJ-db/db mice carry a mutation in the leptin receptor gene, and they are a model for obesity-induced