In the pathogenesis of diabetic retinopathy, accelerated apoptosis of retinal capillary cells precedes the formation of degenerative capillaries, and increased degenerative capillaries represent one of the features of retinopathy seen in diabetic rodents [32, 33]. Our previous work has shown that the supplementation with antioxidants containing β-carotene in rodent model of diabetic retinopathy protects the retina from the development of degenerative capillaries. Furthermore, we have shown that AREDS-based antioxidants also protect the retina from pathology associated with diabetic retinopathy . Carotenoids are not synthesized in animals, but are generally obtained from the diet. Among this group, α-, β- and γ-carotene, lycopene, lutein and zeaxanthin are some of the most abundant carotenoids in the North American diet, and luetin and zeaxanthin preferentially concentrate in the retina . Diabetes is shown to decrease the macular pigment optical density and also retinal zeaxanthin levels [22, 26]. Here, we show that administration of micronutrients containing carotenoids prevents accelerated loss of retinal capillary cells and the onset of diabetic retinopathy in a rodent model. This is accompanied by protection of the retinal dysfunction, which precedes the capillary cell apoptosis. This nutritional supplementation reduces oxidative stress and damage to the retinal mitochondria, and regulates VEGF and inflammatory mediators increased in diabetes. Thus, the supplementation, which is now being tested for diabetes-related visual dysfunction, appears to have potential to inhibit the development of diabetic retinopathy.
In diabetic retinopathy, increase in capillary cell apoptosis is considered as a surrogate marker which precedes the pathology characteristic of diabetic retinopathy . Data presented here show that the nutritional supplementation protects the retina from both accelerated apoptosis of retinal capillary cells and from the formation of degenerative capillaries.
Although the pathology associated with diabetic retinopathy is observed in the retinal vasculature, several physiologic and functional abnormalities are observed before this histopathology appears. These functional abnormalities are mainly neuronal in origin; and neuronal cell apoptosis can be seen as early as one month after induction of diabetes [34, 35]. Abnormal ERG responses appear before vascular lesions begin to appear; streptozotocin-induced diabetic rats present delayed ERG responses within one month of induction of diabetes , while retinal capillary cell apoptosis and histopathology are not observed till the duration is extended to at least 6 months . Similarly, alterations in multifocal ERG are considered to predict the onset and progression of retinopathy in diabetic patients [37, 38]. Furthermore, recent studies have implicated photoreceptors in the development of early stages of diabetic retinopathy, and the possible mechanism appears to be increase in oxidative stress . The results presented here clearly show that the supplementation with the nutrients, in addition to protecting the retina from vascular abnormalities, also helps in the neuronal function as demonstrated by amelioration of deficits in the amplitudes of both a- and b- waves. These results demonstrate that this supplementation, in addition to protecting the retinal vasculature, also protects the non-vascular cells, including photoreceptors, bipolar, amacrine and Muller cells. Thus, this nutritional supplementation has potential to inhibit abnormalities associated with the early stages of diabetic retinopathy before the capillary cells of the retina begin to die and histopathology starts to appear.
Diabetes increases oxidative stress in the retina and its capillary cells, and impairs the antioxidant defense mechanism. Mitochondrial superoxide radicals are increased, and mitochondria become dysfunctional, their copy numbers are decreased and mtDNA is damaged. The damaged mtDNA results in decreased mtDNA-encoded proteins important in the electron transport chain, and this initiates a continuous cycle of free radicals [1–6]. Carotenoids are powerful antioxidants, and by scavenging free radicals, they protect the cells from the damage caused by free radicals [39, 40]. Antioxidant properties of carotenoids are routinely linked with their beneficial effects on chronic diseases including diabetes, and the uptake of lutein and zeaxanthin in the retina is decreased in diabetes . Furthermore, diets rich in carotenoids have shown protective effects against some of the chronic eye diseases, including age-related macular degeneration [21–23]. Our results suggest that the nutrient supplementation containing carotenoid prevents the development of diabetic retinopathy by protecting mtDNA from undergoing damage, and thus preventing the initiation of the self-propagating cycle. In support, dietary wolfberry supplementation (which contains zeaxanthin) has been shown to protect from decrease in TFAM and mitochondria copy number [41, 42], which the retina experiences in diabetes [29, 43].
VEGF, a hypoxia-induced factor is considered as one of the major growth factors in the development of diabetic retinopathy [13, 14]; Diabetes-induced increase in VEGF plays a pivotal role in the increased cell permeability during the early stages of diabetic retinopathy, and in later stages of the disease VEGF is implicated in the angiogenesis. Antioxidants administration protects the retina from increases in VEGF , and we have shown that zeaxanthin or curcumin supplementation for 2 months in diabetic rats attenuates increase in retinal VEGF . Here, we provide data showing that the long-term administration is also protective, retinal VEGF remains normal in diabetic rats treated with the nutrients. This clearly implies that the carotenoids protect increase in VEGF, both in the early stages of the disease, and also during the later stages of diabetic retinopathy when the capillary cells are being lost and the capillaries are degenerating.
Diabetic retinopathy is also considered as a low-grade chronic inflammatory disease, and sub-clinical inflammation is responsible for many of the vascular lesions seen in patients with diabetic retinopathy. The levels of inflammatory mediators (NF-k B, IL-1β, tumor necrosis factor α, and ICAM-1 etc.) are elevated in the retina in diabetes and leukostasis is increased [11, 12]. NF-k B, a redox-sensitive transcriptional factor, controls the transcription of DNA, and also plays a central role in activating pro-inflammatory genes. Diabetes activates NF-k B in the retina and its vascular cells, and in the pathogenesis of retinopathy, activation of NF-k B is considered to act as pro-apoptotic [18, 45]. Furthermore, activation of NF-k B elevates IL-1β, and IL-1β plays an important role in retinal capillary cell death and the formation of acellular capillaries, the microvascular pathology that is characteristic of retinopathy in diabetes, and the antioxidants, which inhibit the development of diabetic retinopathy in rodent models, also inhibit diabetes-induced increases in retinal IL-1β [17, 31, 46]. We have shown that one of the key events via which inflammation could contribute to the activation of the apoptotic machinery resulting in the development of diabetic retinopathy, could be the damage to the mitochondria [17, 31]. Here, we show that the this supplementation also prevents diabetes-induced activation of NF-k B in the retina. These data further strengthen the hypothesis that the beneficial effects of the nutrients on diabetic retinopathy are mediated via inhibition of both inflammation and mitochondrial damage. However, with the encouraging results of these nutrients on inflammatory mediators and VEGF, the possibility that this supplementation also protects blood-retina barrier, an abnormality which can be seen during early stages of diabetic retinopathy , cannot be ruled out.
In summary, our data demonstrate that the nutritional supplementation, which is now in preclinical trials for maintaining the structure and function of the retina of human subjects with long term diabetes, also protects neuronal cells and vascular cells, and inhibits the development of retinopathy. This is achieved, possibly, via ameliorating increase in inflammatory mediators and maintaining mitochondria homeostasis, thus protecting the retina from the self-propagating vicious cycle of mitochondrial damage. Along with other experimental data demonstrating the beneficial effects of strategies to ameliorate oxidative stress, and prevent/retard diabetic retinopathy [9, 10, 15, 26], supplementation with this micronutrients appears as an inexpensive adjunct therapy to inhibit retinal dysfunction, and the onset of this blinding disease. We believe that with the shortcomings of the clinical studies presenting inconclusive results with the antioxidants [48, 49], results from a controlled clinical trial with this supplementation, which is already being tested for preserving retinal structural and functional abnormalities associated with diabetes, could help diabetic patients inhibit retinopathy, and spare them from losing their vision.