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Review Article

Diabetic Retinopathy: Role of Neurodegeneration and Therapeutic Perspectives

Simó, Rafael MD∗,†; Simó-Servat, Olga MD∗,†; Bogdanov, Patricia PhD∗,†; Hernández, Cristina MD∗,†

Author Information
Asia-Pacific Journal of Ophthalmology: March-April 2022 - Volume 11 - Issue 2 - p 160-167
doi: 10.1097/APO.0000000000000510
  • Open

Abstract

Diabetic retinopathy (DR) is the most frequent complication of diabetes and remains as the leading cause of preventable blindness among working-age individuals in developed countries.1,2 In addition, DR is an independent predictor of both microvascular and macrovascular complications.3,4 Current treatments are addressed to treat advanced stages of the disease when vision has already been significantly affected5 and, therefore, new therapeutic strategies targeting early stages of the disease are needed.

DR has long been considered a microvascular complication of diabetes; however, growing evidence suggests that neurodegeneration is an early event in its pathogenesis.6 In fact, the American Diabetes Association has recently defined DR as a highly specific neurovascular complication.7 In this article, an overview of the main components of neurodegeneration, its key underlying mechanisms, and the more useful experimental models for investigative purposes will be given. In addition, the results of most relevant treatments based on neuroprotection will be summarized.

MAIN COMPONENTS OF NEURODEGENERATION

Neural apoptosis and glial activation are the main components of retinal neurodegeneration induced by diabetes.8 It should be noted that these main features of retinal neurodegeneration have been observed before the detection of overt microangiopathy in both experimental models and in the retina of diabetic donors.9–11

Neural Apoptosis

Retinal ganglion cells and amacrine cells are the first components of the neuroretina in which diabetes-induced apoptosis is detected.8 However, photoreceptors also have an increased apo- ptotic rate and play an important role not only in the neurode- generative process but in triggering microvascular impairment.12–14 Apoptotic death leads to a reduction in the thickness of inner retinal layers and the nerve fiber layer. Optical coherence tomography can detect this morphological change.8,15 Multifocal electroretinography has demonstrated that neurodegeneration results in a delayed P1 implicit time and reduced traces.16 These structural and functional changes have clinical implications regarding such visual deficiencies as decreased hue discrimination, less contrast sensitivity, delayed dark adaptation/adjustment, and abnormal visual fields, leading to a reduced vision-related quality of life.17–19

Glial Activation

Neural apoptosis is accompanied by changes in glial cells known as glial activation or reactive gliosis.8 The Muller cell is a macroglia cell type that is only found in the retina. Muller glia are spindle-shaped and encompass the whole retina from the outer limiting membrane to the retinal ganglion cells. One of the most significant characteristics of reactive gliosis is the overexpression of glial fibrillary acidic protein (GFAP). Unlike retinal astrocytes, Muller cells do not normally express GFAP.8 However, in diabetes, an aberrant expression of GFAP is shown by Muller cells.20 Because Muller cells produce factors capable of modulating blood flow, vascular permeability, and cell survival, and their processes surround all the blood vessels in the retina, these cells have a key role in linking neurodegeneration with retinal microangiopathy in the diabetic eye.8

Apart from macroglia, the microglia are also activated in the diabetic retina.8 Microglial cells are the main resident sentinel immune cells found in the inner part of the retina. They migrate to the subretinal space and release cytokines, thus helping to bring about neuronal cell death.21 Once activated, the microglia begin to increase rapidly and change from a ramified structure, with long and thin processes, to an amoeboid structure with larger cell bodies and thicker and shorter processes.21 Moreover, there is an increase in immunoreactivity, expression of proinflammatory mediators, and migratory and phagocytic activity.22 Activated microglial cells adjacent to the vessels also seem to play an important role in vasoregression, a characteristic feature of early stages of DR.23

Neurovascular Unit Impairment

The neurovascular unit (NVU) in the retina refers to the functional coupling and interdependency of neurons, glia, and the highly specialized vasculature.24 The components of the NVU consist of neurons (ie, ganglion cells, bipolar, amacrine, and horizontal cells), glia (Muller cells, astrocytes, and microglia), and vascular cells (endothelial cells and pericytes).6,25,26 All these components are in intimate communication and maintain the integrity of the inner blood–retinal barrier. Some types of cells, such as glial cells, endothelial cells, and pericytes, can communicated through physical interactions, whereas cells without physical connections communicate at distance through soluble ligands and/or exosomes.24 Given that retinal vasculature lacks autonomic innervation, the circulating hormones and local factors released from endothelial cells and the other components of NVU (neurons and glial cells) play a key role in regulating retinal blood flow.27,28 Glial activation and neuron death contribute significantly to microvascular damage through several mechanisms that have been reviewed elsewhere.8

MOLECULAR MEDIATORS OF NEURODEGENERATION

Inflammation

Inflammation plays a major role in the pathogenesis of DR.29 The enhancement of several proinflammatory cytokines has been detected in ocular tissues of patients with diabetes not only in advanced stages such as proliferative DR and diabetic macular edema but also in early stages of the disease.30–32 In fact, glial activation is a sign of neuroinflammation, which upregulates the expression of acute-phase response proteins and other inflammation-related genes.33 Neuroinflammation plays an essential role in the pathogenesis of DR, and several experimental studies have shown that drugs with capacity to abrogate glial activation have also a beneficial vascular effect by reducing vascular leakage.24

The main inflammatory cytokines involved in DR are IL-1β, TNF-α, monocyte chemotactic protein-1, stromal cell-derived factor-1, and the adhesion molecules (ICAM-1, VCAM-1, sVAP-1).34 IL-1β is a pivotal inflammatory cytokine because in addition to its direct deleterious effect it is able to activate NF- kB, which in turn governs the production of IL-8, monocyte chemotactic protein-1, and TNF-α.35 The main sources of cytokines production are the retinal pigment epithelium and the activated glial cells (microglia and macroglia), but in later stages, even the endothelial cells and neurons contribute to their production.36 At present, little is known regarding the mechanisms involved in neural apoptosis due to proinflammatory cytokines. However, excitotoxicity, oxidative stress, and mitochondrial dysfunction are among the most important mediators.37 The link between the inflammatory response, neurodegeneration, and vascular impairment is graphically summarized in Figure 1.

F1
Figure 1:
Main mechanisms involved in neuroinflammation in the diabetic retina. Retinal pigment epithelium and glial cells (macroglia and microglia) are the main sources of proinflammatory cytokines, which lead to vascular injury and neurodegeneration as a result of NVU impairment.33–36 In addition, inflammation triggers oxidative stress, excitotoxicity, and mitochondrial dysfunction, which also contribute to the impairment of NVU, and the pathways activated by chronic hyperglycemia: polyol and hexosamine pathways, diacylglycerol-protein kinase C pathway, and advanced glycation end-products upregulation.5 NVU indicates neurovascular unit.

Oxidative Stress/Mitochondrial Dysfunction

The retina is particularly susceptible to oxidative stress because of high energy demands and light exposure.38 The increased production of reactive oxygen species (ROS) due to the activation of NADPH oxidase 2 is an early event in the pathogenesis of DR.39,40 One of the integral proteins in the assembly of NADPH oxidase 2holoenzyme, Rac1, is also upregulated and activated in diabetes.40 In addition, upregulation of ROS can lead to increased activation of NF-κB, which, in turn, increased the release of proinflammatory cytokines and nitric oxide.39

Mitochondrial DNA is particularly prone to oxidative damage; this is because of its close proximity to the ROS-generating electron transport chain system and lack of protective histones.41 This mitochondrial dysfunction further increases oxidative stress and contributes to accelerate the apoptosis of retinal vascular cells. In addition to damage to mitochondrial DNA, diabetes also compromises the DNA repair machinery in the retina, further contributing to mitochondrial dysfunction.42

The oral administration of antioxidants43 or genetic overexpression of superoxide dismutases38 has been shown to inhibit the diabetes-induced degeneration of retinal capillaries in animal studies. In addition, early interventional trials with dietary carotenoid supplementation seem to exert neuroprotection and could be envisaged as an adjunct nutraceutical strategy for management of DR.44 Overall, these findings support the essential role of oxidative stress in the pathogenesis of DR.

Proapoptotic Signaling

A myriad of actions mediated by inflammatory mediators, oxidative stress, mitochondrial impairment, and the enhanced pathways due to the chronic hyperglycemia (polyol and hexos- amine pathways, diacylglycerol–protein kinase C pathway, and advanced glycation end-products/ receptor of advanced glycation end-products activation), lead to an imbalance between proapop- totic and survival signaling, favoring the former. Little is known regarding the specific mechanisms orchestrating the activation of the proapoptotic pathways in the neuroretinas of diabetic patients. Abu-El-Asrar et al45 reported the presence of Fas and FasL immunoreactivity in ganglion and glial cells, respectively in diabetic human retinas. Valverde et al46 observed in neuroretinas from diabetic donors with early stages of DR that the apoptotic signals emerging from the death receptor pathway were amplified by the additional activation of the intrinsic apoptotic signaling pathway, resulting in stronger activation of caspase-3. Finally, data from proteomic analysis using retinas from diabetic donors without DR revealed several potential pathways involved in neuroregulation and neuroprotection that merit to be further investigated.47

Autophagy

Autophagy is an intracellular degradation system, essential to eliminate unnecessary cell components, including damaged organelles and proteins. Autophagy is regulated by endoplasmic reticulum stress, oxidative stress, and inflammation-related pathways, and it is increased in the retina of diabetic patients.48 Autophagy may protect retinal cells from oxidative stress49 and from inflammation by suppressing inflammasome activation.50 In this regard, it has been reported that gypenoside-17 prevents early DR by decreasing apoptosis and increasing autophagy in Muller cells in db/db mice.51 However, overexpression of autophagy with lysosome dysfunction may aggravate cell death, including necroptosis and apoptosis, vascular damage, and retinal neovascula- rization.52–54 The underlying mechanism governing this dual effect remains to be fully elucidated.

ANIMAL MODELS

There are excellent comprehensive reviews on the available animal models to study DR.55 In this minireview we will briefly summarize general features and will mainly focus on the most useful models to study retinal neurodegeneration.

The most popular model for the study of diabetes-induced neurodegeneration has been the rat with diabetes induced by streptozotocin (STZ).56–58 Mice have been used much less because they are more resistant to the STZ effect (mice need 3–5 doses of STZ to induce diabetes, whereas in rats 1 dose is enough) and have smaller eyeballs compared to rats. However, it should be noted that diabetes induced by STZ has the inconvenience of the intrinsic neurotoxicity of STZ.59–61 Therefore, it is difficult to dissect what lesions in the neuroretina are due to diabetes or to STZ itself, at least shortly after STZ administration. The db/db mouse (a spontaneous model of obesity-induced type 2 diabetes due to a mutation in the gene encoding the leptin receptor) mimics very well the key characteristics of the neurodegenerative process that occurs in the human diabetic eye.62 Therefore, it seems an appropriate model for investigating the underlying mechanisms of retinal neurodegeneration caused by diabetes and for testing new neuroprotective agents.63–65

Rabbits have a larger eye globe than rodents, but they have a merangiotic retinal circulation (retinal blood vessels confined to a linear horizontal streak on both sides of the optic disc, accompanying streaks of myelinated nerve fibers), whereas humans have an holangiotic pattern of circulation (blood vessels distributed throughout most of the neurosensory retina, supplying the inner retina only). 39 Therefore, although very useful for ocular toxicity preclinical studies, rabbits are not a good model to reproduce DR.

Monkeys have a structural similarity to that of the human eye in which macula is present.56–58 However, as occurs in rodents, they do not develop vascular lesions of advanced stages of DR.58 Their use as animal models for DR has been limited due to their large size, long lifespan, and unavoidable ethical requirements.

Other large animals, such as pigs, dogs, and cats, have larger eyeballs that are easier to handle than rodent eyes. However, their rising price, the unavailability of specific molecular reagents and antibodies, the fact that they have different gene backgrounds than humans, and the difficulty of maintaining them due to their large size, limit their use as DR experimental animal models.56–58

Zebrafish (Danio rerio), a freshwater fish native to South Asia, is a popular aquarium fish widely used in preclinical drug development. Zebrafish is used as DR model given that it has a retinal structure similar to that of the human eye including the main components of the NVU.66 In contrast to rodents, zebrafish have a cone-dominated retina and develop neovascularization. These characteristics, combined with their short lifespan, rapid growth, and ability to breed in large numbers, make them ideal for high-throughput screening, genetic screening, and significantly shorter experimental times.49 However, the lack of similarity in retinal vascular structure between zebrafish and humans, difficult handling due to tiny size, and a lack of reagents for molecular studies are factors responsible for the limited use of zebrafish as a screening model for DR.58 Regarding neurodegeneration induced by diabetes, there are conflicting results that could be attributed to the different methods used to induce diabetes (immersion-induced hyperglycemia, STZ injection, or genetic models),66 as well to the regenerative capabilities of the zebrafish retina.67 In this regard, it should be noted that, unlike mammals, zebrafish are able to regenerate the damaged neuroretina. The Muller cells play a key role in this neuroregenerative process by undergoing a reprogramming event that allows them to divide and generate a retinal progenitor that is multipotent and responsible for regenerating all major retinal neuron types.68

TREATMENT BASED ON NEUROPROTECTION

There are mounting evidence of successful neuroprotective treatments in experimental models but there is little information coming from clinical trials performed in patients with diabetes. A main factor for this scarce information is that only microvascular endpoints but not those related to neurodegeneration have been considered by the regulatory authorities in the development of new drugs for treating Dr. However, new evidence addressed to update the staging system of diabetic retinal disease could change this important issue in the coming years.69

Experimental Studies

There are several theoretical approaches to treat retinal neurodegeneration by targeting the main mechanisms involved in its pathogenesis. In this regard, the blockade of the glutamate signaling pathway, the reduction of neuroinflammation, and the improvement of the neurovascular functions seem as pivotal treatments.5 However, it seems even more effective to abrogate several underlying pathways simultaneously than to limit the action to a single one. Two drugs, fenofibrate and calcium dobesilate, administered by oral route have provided effectiveness in preventing neurodegeneration by targeting various pathways simultaneously.24,63,70–73 The neuroprotection conferred by peptides in retinal disease has been recently comprehensively reviewed by Cervia et al.74 Nutraceuticals can also exert this multipathway effect and their beneficial effects in DR-related models have been recently and extensively reviewed.75

Recent research showing the downregulation of several proteins synthesized by the retina with neurotrophic activity, such as pigment epithelium-derived factor, somatostatin, and glucagon-like peptide 1 (GLP-1), have pointed to their replacement as a reasonable approach for preventing retinal neurodegen- eration.5,24,76 Interestingly, it has been suggested that GLP-1 participates in neurogenesis and, therefore, might be useful as a therapeutic agent for other retinal neurodegenerative diseases.77 However, the systemic administration of these neuropeptides can hardly reach the retina at pharmacological concentrations and, by contrast, could have systemic adverse effects.5 On the other hand, when the early stages of DR are the therapeutic target, it would be inconceivable to recommend an aggressive treatment such as intravitreal injections.5 For all these reasons, the ocular topical route has emerged as an effective therapeutic approach to deliver these neuroprotective factors to the diabetic retina. The most relevant treatments using the topical route for treating diabetes- induced neurodegeneration and early microvascular abnormalities experimental models are displayed in Table 1.

Table 1 - Topical Treatments Successfully Used in Experimental Models of Diabetes for Treating Diabetes-Induced Retinal Neurodegeneration
Animal Model Glial Inflammation Neuronal Apoptosis Oxidative Stress Microvascular Abnormalities
PEDF86 Ins2(Akita) mice + + +
Insulin87 STZ-rats +/− +
NGF88 STZ-rats +
SST89,90 STZ-rats, db/db mice + +
GLP-165,79,91 db/db + + + +
DPP-IVi92 db/db + + + +
Bosentan93 db/db + + +
SOCS194 db/db + + +
Citicoline95 db/db + +
GLP-1 indicates glucagon-like peptide 1; PEDF, pigment epithelium-derived factor; STZ, streptozotocin.

Clinical Trials and Perspectives in Humans

There are few clinical trials aimed to treat retinal neurodegeneration or neurodysfunction induced by diabetes. It has been observed that low dose of oral doxycycline, an antibiotic with anti-inflammatory properties, improves inner retinal function in diabetic patients with severe nonproliferative diabetic retinopathy or non-high-risk proliferative diabetic retinopathy, in a 2-year, randomized, proof-of-concept (n = 30 patients) clinical trial (NCT00511875).78

Fenofibrate has been shown effective in preventing the progression of DR,79,80 and there is some evidence that calcium dobesilate is useful in early stages of DR.81,82 Interestingly, it has been recently shown that fenofibrate is able to increase circulating hematopoietic stem/progenitor cells in diabetic patients with DR.83 Because hematopoietic stem/progenitor cells have vascular properties and have been shown to protect from DR, this could be a mechanism by which fenofibrate exerts its beneficial effects in Dr. However, there is no information regarding the effect of these drugs in preventing or arresting retinal neurodegeneration. Therefore, specific clinical trials aimed at examining the neuroprotec- tive effect of fenofibrate and calcium dobesilate are needed. The promising results of nutraceuticals in animal models should also be tested in appropriate clinical trials.

The problem of systemic administration of neuroprotective agents for treating diabetes-induced retinal neurodegeneration is that they can hardly reach the retina at pharmacological concentrations and, by contrast, could have systemic adverse effects. On the other hand, when the early stages of DR are the therapeutic target, it would be inconceivable to recommend an aggressive treatment such as intravitreal injections.

Topical (eye drops) administration of neuroprotective agents has emerged as a new strategy for treating early stages of DR due to several reasons: (1) This is a noninvasive route that permits self-application by the patient. (2) There is evidence that a lot of drugs are able to reach the retina at pharmacological concentrations, and multiple experimental studies have demonstrated the effectiveness of eye drops for treating early stages of DR in experimental models.64,77,84–93(3) The systemic side effects are minimized when compared with systemic administration. (4) The blood-retinal barrier, a serious limiting factor to reach the retina when systemic administration is used, can be sorted out. In this regard, it has been demonstrated that treatment with eye drops containing citicoline and vitamin B12 for a 36month period improved the macular bioelectrical responses (multifocal electroretinography recordings) in type 1 diabetic patients (n = 20) with nonproliferative diabetic retinopathy (NCT04009980).94 The EUROCONDOR study is the largest study reported till now (n = 449 patients) aimed at treating neurodegeneration. This phase II-III clinical trial provided evidence that 2 neuroprotective drugs (SST and brimonidine) administered by eye drops were able to arrest the progression of retinal neurodysfunction in type 2 diabetic patients.95However, no effect in preventing or arresting microvascular disease was observed. The 2-year short follow-up, the high proportion of patients with no or very mild DR, and the good glycemic control during follow-up could explain these negative findings.24,95 Nevertheless, a substudy showed that topical treatment with either brimonidine- or SST-induced retinal arteriolar and venular dilation in those patients with preexisting early DR.96 Therefore, this study demonstrated for the first time that long-term topical neuroprotection modulates the diabetes-induced microvasculature changes in the clinical arena. In addition, the EUROCONDOR study provided evidence that neurodegeneration does not seem in a significant proportion of patients (around 30%) in whom early stages of microvascular disease already exist.97 This data points to screening retinal neurodysfunction as an essential procedure for selecting those patients in whom neuroprotective treatment might be useful.6

Based on the experimental studies using topical route, the more promising drugs for testing in clinical trials are those with dual effect simultaneously (neuroprotective and vasculotropic) such as pigment epithelium-derived factor,84 GLP-1,64 DPP-IV inhibitors,90 Bosen- tan (competitive antagonist of endothelin-1 receptors),91 and SOCS1-derived peptide.92 DPP-IV inhibitors deserve a further comment because they act by inhibiting the degradation of GLP-1 produced by the neuroretina, and by other not well understood pleiotropic actions unrelated to GLP-1 enhancement. In addition, they are safe, not expensive, and with high stability. All of them are important properties when planning to reach the market.

CONCLUDING REMARKS AND FUTURE PERSPECTIVES

Neurodegeneration plays an important role in the pathogenesis of DR. Its underlying mechanisms are diverse, complex, and with a very intricate cross-talk among them. The successful use of neuroprotective agents to prevent not only neurodegeneration, but also early stages of microvascular impairment in experimental models, has increased the interest to implement this approach in clinical practice. However, it seems that neurodegeneration is not always essential for the development of microvascular disease and, therefore, a careful selection of patients to optimize the costeffectiveness might be recommended. Nevertheless, even in the group of patients in whom neurodegeneration seems to be dissociated from the development and progression of microvascular disease, neuroprotection could be very useful to mitigate the loss of quality of life linked to retinal neurodegeneration. Specific clinical trials aimed at answering these important points seem warranted. Meanwhile, a consensus regarding the best manner to determine the presence of retinal neurodegeneration and its repercussion in the retinal microvasculature is needed.

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Keywords:

diabetic retinopathy; eye drops; neurodegeneration; neurovascular unit; treatment

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