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Neurodegeneration is an early event in diabetic retinopathy: therapeutic implications
  1. Rafael Simó,
  2. Cristina Hernández,
  3. on behalf of the European Consortium for the Early Treatment of Diabetic Retinopathy (EUROCONDOR)*
  1. CIBERDEM (CIBER de Diabetes y Enfermedades Metabólicas Asociadas) and Diabetes and Metabolism Research Unit, Vall d'Hebron Institut de Recerca (VHIR), Universitat Autónoma de Barcelona, Barcelona, Spain
  1. Correspondence to Dr Rafael Simó, Diabetes and Metabolism Research Unit, Vall d'Hebron Institut de Recerca, Pg. Vall d'Hebron 119-129, Barcelona 08035, Spain; rsimo{at}ir.vhebron.net

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Diabetic retinopathy (DR) is the most common complication of diabetes and remains the leading cause of blindness among working-age individuals in developed countries. Population-based studies suggest that approximately one-third of the population with diabetes have signs of DR and approximately one-tenth have vision-threatening stages of retinopathy such as diabetic macular oedema and proliferative retinopathy.1–3 Healthcare costs for patients with DR are almost double those in patients without DR.4 ,5 Notably, average healthcare costs increase considerably with the severity of DR, which suggests that preventing the progression of DR may alleviate the economic burden related to this complication of diabetes.6

Current treatments for DR (laser photocoagulation, intravitreal corticosteroids, intravitreal anti-vascular endothelial growth factor (VEGF) agents and vitreoretinal surgery) are applicable only at advanced stages of the disease and are associated with significant adverse effects.7 ,8 Therefore, new pharmacological treatments for the early stages of the disease are needed.

Neurodegeneration: an early event in the pathogenesis of DR

DR has been classically considered a microcirculatory disease of the retina. However, there is mounting evidence to suggest that retinal neurodegeneration is an early event in the pathogenesis of DR, which participates in the microcirculatory abnormalities that occur in DR.9–22 The main features of retinal neurodegeneration (apoptosis and glial activation) have been found in the retinas of donors with diabetes without any microcirculatory abnormalities appearing in ophthalmoscopic examinations performed during the year before death.23–25 Therefore, a normal ophthalmoscopic examination does not exclude the possibility that retinal neurodegeneration is already present in the diabetic eye (figure 1).

Figure 1

Esquematic representation of diabetic retinopathy (DR) development. The metabolic pathways triggered by hyperglycaemia, and hyperglycaemia itself, lead to DR but a period of at least 5 years is required before DR can be diagnosed under ophthalmoscopic examination. The first stage that can be seen is background retinopathy or non-proliferative DR (NPDR; which is constituted by microaneurysms, microhaemorrhages and exudates). From this stage the natural history of the disease can follow two directions that do not exclude the other. One of them is the development of clinically significant macular oedema, in which the most important pathogenic element is the breakdown of the blood–retinal barrier. This pathway is more frequent in patients with type 2 diabetes. The other direction is towards proliferative DR (PDR), which is more frequent in type 1 diabetes. In this setting capillary occlusion plays an essential role generating an imbalance between angiogenic and anti-angiogenic factors, which finally stimulates neovascularisation (the hallmark of proliferative DR). However, even before non-proliferative DR can be detected in the ophthalmological examination, retinal neurodegeneration does exist. At the bottom is a representative case of a type 2 donor with diabetes without any abnormality in the funduscopy performed in the preceding year before death. Left panel: immunostaining with glial fibrillary acidic protein showing glial activation. Right panel: TUNEL immunostaining showing a high rate of apoptosis. AGE, advanced glycation end-products; DMO, diabetic macular oedema; PEDF, pigment epithelial derived factor; PKC, protein kinase C; VEGF, vascular endothelial growth factor. This figure is only reproduced in colour in the online version.

Diabetes increases apoptosis in neurons, especially in the retinal ganglion cell layer.22 This loss of neural cells results in a reduction in the thickness of the retinal nerve fibre layer.26–28 It should be noted that this thinning of the ganglion cell layer has been found in patients with diabetes with no or only minimal DR.27–29

Neural apoptosis is accompanied by reactive changes in glial cells, the most representative being those occurring in Müller cells, the predominant type of macroglial cell. Müller cells normally do not express glial fibrillary acidic protein (GFAP) but in diabetes they show an aberrant expression of GFAP.30 Because Müller cells produce factors capable of modulating blood flow, vascular permeability and cell survival, and their processes surround all the blood vessels in the retina it seems that these cells play a key role in the pathogenesis of retinal microangiopathy in the diabetic eye.

Functional abnormalities

Neuroretinal damage produces functional abnormalities such as the loss of both chromatic discrimination and contrast sensitivity. These alterations can be detected by means of electrophysological studies in patients with diabetes with less than 2 years of diabetes duration, which is before microvacular lesions can be detected under ophthalmological examination.31–35 In addition, characteristic abnormalities in the electroretinogram (ERG) such as a reduction in the amplitude and a delay in the latency of the oscillatory potentials have been found in both patients with type 1 diabetes and rats without any evidence of microvascular abnormality.32–35 Noteworthy ERG abnormalities in the diabetic retina are not only due to apoptosis of retinal neurons. In this regard, glial activation has been involved in the ERG abnormalities observed in patients with diabetes.36 In addition, there are functional abnormalities related to glucose control that are partly reversible and, therefore, do not necessarily implicate cellular loss or major structural damage.37

The use of multifocal ERG (mfERG) has provided compelling evidence suggesting a direct link between neural dysfunction and vascular abnormalities in DR. In this regard, it has been shown that a delayed mfERG implicit time predicts the development of early microvascular abnormalities.38–42

Finallly, it has recently been reported that patients with diabetes without structural microvascular abnormalities have a reduced vasodilation response to flicker light stimulation.43 ,44 Furthermore, it has been suggested that flicker light-induced vasodilation is mediated primarily by ganglion cells whose function is strongly correlated with the ERG pattern.45

All these findings suggest that neuronal apoptosis precedes overt vascular dysfunction. However, it could be argued that this is due to the higher sensitivity of the tools used for assessing neural damage (ie, mfERG, flicker light stimulation) than those used to assess vascular damage (ie, fluorescein angiography). Therefore, specific research addressed at examining with appropriate methodology the temporal sequence of neural and vascular abnormalities in DR is needed.

Pathogenic mechanisms involved in retinal neurodegeneration

The excitotoxicity mediated by glutamate accumulation, the downregulation of neuroprotective factors synthesised by the retina, the increase of oxidative stress and neuro-inflamation are significant pathogenic factors accounting for neurodegeneration in the diabetic eye.

Glutamate excitotoxicity

The main neurotoxic metabolite involved in diabetic retinal neurodegeneration is glutamate. Glutamate is the major excitatory neurotrasmitter in the retina and is involved in neurotransmission from photoreceptors to bipolar cells and from bipolar cells to ganglion cells. Elevated levels of glutamate in the retina have been found in experimental models of diabetes,46–48 as well as in the vitreous fluid of patients with diabetes with proliferative DR.49 ,50 The reasons why diabetes facilitates the extracellular accumulation of glutamate include: (1) Increase of glutamate production by glial cells due to the loss of the Müller cell-specific enzyme glutamine synthetase, which converts glutamate to glutamine.46 ,47 (2) Reduction in the retinal ability to oxidise glutamate to α-ketoglutarate.45 (3) Impairment of glutamate uptake by the glial cells.51 These elevated concentrations of extracellular and synaptic glutamate in the retina lead to the so-called ‘excitotoxicity’ in which the excess of glutamate stimulation causes an uncontrolled intracellular calcium response in postsynaptic neurons that results in cell death.50 The excitoxicity of glutamate is the result of overactivation of ionotropic glutamate receptors, mainly α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) and N-methyl-d-aspartame (NMDA), which have been found to be overexpressed in streptozotocin-induced diabetic rats.52 ,53 There are at least two mechanisms involved in glutamate-induced apoptosis: a caspase-3-dependent pathway and a caspase-independent pathway involving calpain and mitochondrial apoptosis-inducing factor.54

Loss of neuroprotective factors

The retina synthesises neuroprotective factors that counteract the deleterious effects of neurotoxic factors involved in neurodegeneration. The loss of these neuroprotective factors or the reduction of their effectiveness is essential for the development of retinal neurodegeneration. Among the neuroprotective or neurotrophic factors pigment epithelial derived factor (PEDF), somatostatin (SST) and erythropoietin seem to play an essential role.

PEDF is a potent neuroprotective and anti-angiogenic factor that is downregulated in DR.55 PEDF protects neurons from glutamate-mediated neurodegeneration and it has recently been reported that PEDF increases the expression of glutamine synthetase in the early phase of experimental DR.56 Intraocular gene transfer of PEDF significantly increases neuroretinal cell survival after ischaemia–reperfusion injury and excessive light exposure. These promising results suggest that enhancing the expression and function of PEDF can be a therapeutic target in DR. The neuroprotective role of PEDF, however, has not been examined in models of diabetes, a fact that should encourage further studies on the potential therapeutic role of PEDF.

SST acts as a neuromodulator and angiostatic factor in the retina and it is mainly synthesised by retinal pigment epithelium (RPE).23 ,57 ,58 The amount of SST produced by the human retina is significant as can be deduced by the strikingly high levels found in vitreous fluid.59–61 Apart form SST, SST receptors are also expressed in the retina,58 thus suggesting an autocrine action. In DR there is a downregulation of retinal revels of messenger RNA levels of SST, and a dramatic decrease of SST has been found in the vitreous fluid of patients with diabetes.23 ,59–61 These findings lead to the suggestion of replacement treatment by intravitreal injections of SST or SST analogues as a new therapeutic approach in DR.62 We have recently found that the decrease in SST production by the retina is associated with retinal neurodegeneration.23 In this regard, it should be noted that SST and SST analogues administered intravitreally protect the retina from AMPA-induced neurotoxicity.63

Erythropoietin and its receptor are expressed in the human retina.64 ,65 Erythropoietin is mainly produced by RPE and is upregulated in DR.64–67 This is in agreement with the elevated concentrations of erythropoietin found in the vitreous fluid of patients with diabetes (∼30-fold higher than plasma and ∼10-fold higher than in individuals without diabetes).64 The consequences of erythropoietin overexpression in DR remain to be elucidated, but the bulk of the available information points to a protective effect rather than a pathogenic effect, at least in the early stages of DR. In addition, erythropoietin is a potent physiological stimulus for the mobilisation of endothelial progenitor cells and, therefore, it could play a relevant role in regulating the traffic of circulating endothelial progenitor cells towards injured retinal sites.68 In this regard, the increase in intraocular synthesis of erythropoietin that occurs in DR can be contemplated as a compensatory mechanism to restore the damage induced by the diabetic milieu. In fact, exogenous erythropoietin administration by intravitreal69 or intraperitoneal injection70 ,71 in early diabetes may prevent structural vascular and neural damage in streptozotocin-induced diabetic rats. Nevertheless, in advanced stages the elevated levels of erythropoietin could enhance the effects of VEGF, thus contributing to neovascularisation and, in consequence, worsening proliferative DR.68 Therefore, endogenous erythropoietin might act as a double-edged sword in the pathogenesis of DR.

Other neuroprotective factors such as insulin, neuroprotectin D1, brain-derived neurotrophic factor, glial cell-line-derived neurotrophic factor, ciliary neurotrophic factor, nerve growth factor and adrenomedullin might also be involved in the neurodegenerative process that occurs in DR, but specific studies on this issue are still needed. A low expression and content of interstitial retinol-binding protein (IRBP) has recently been reported in the retinas from donors with diabetes at very early stages of DR, and this downregulation was associated with retinal neurodegeneration.25 The essential role of IRBP in the visual cycle and as a supplier of fatty acid to the photoreceptors points to IRBP replacement as a serious new target for DR treatment.

Finally, it should be mentioned that VEGF, apart from leading to an increase of permeability and a proliferation of vascular endothelial cells, is a well recognised neurotrophic and neuroprotective factor. In this regard, a dose-dependent decrease in ganglion cells has been reported following the injection of an antibody that blocks all VEGF isoforms in rats.72 These findings could have clinical implications because, so far, clinical trials using anti-VEGF treatment have focused only on studying the systemic side effects but not the incidence of retinal neurodegeneration, such as retinal atrophy or RPE degeneration. Therefore, more studies are needed to fill the scientific gap regarding the long-term effects of anti-VEGF therapy, especially in populations with diabetes.73

Oxidative stress

Oxidative stress plays an essential role in the pathogenesis of DR.74 ,75 In fact, the retina is the only neural tissue that has a direct and frequent exposure to light. These results in the photo-oxidation of many lipids, especially polyunsaturated fatty acids (which are mainly located in the photoreceptor outer segments) and cholesterol esters, and these oxidised lipids become extremely toxic to retinal cells.76 In DR, this problem is aggravated by the increase of oxidative stress and lipid peroxidation associated with diabetes.

There are several pharmacological studies showing that reducing oxidative stress may be an effective approach to slow neurodegeneration in experimental DR.77 ,78 In addition, it has been demonstrated that deficit of ascorbic acid (AA) is the main metabolic fingerprint of vitreous fluid of proliferative DR patients in a 1H-NMR metabonomic approach.79 AA is a potent antioxidant, participates in neuropeptide production and inhibits VEGF. Therefore, the consequences of the low intraretinal levels of AA will be an increase of oxidative stress, an impairment of neuropeptide production (thus contributing to neurodegeneration), and an increase of angiogenesis (thus favouring neovascularisation). Consequently, it seems reasonable to design strategies to increase AA levels in the diabetic retina.

Neuroinflammation

A complex milieu of dysregulated pro-inflammatory factors occur in the diabetic retina, and while retinal microglia and infiltrating monocytic cells probably make an important contribution, there is also strong evidence that Müller glia show inflammation-linked responses when exposed to the diabetic milieu.11 ,80 Genomic assessment of the whole retinas of diabetic rats identified increased expression of the inflammatory genes CCL2, ICAM-1, STAT3, CCR5 and CD44,81 and Müller cells isolated from diabetic rats had increased expression of several genes associated with immune function and inflammation.82 In addition, it has recently been demonstrated that activation of the receptor for advanced glycation end-products plays a key role in hyperglycaemia-induced activation of Müller glia and downstream cytokine production in the context of DR.83

The mechanism by which these cytokines may contribute to neural apoptosis is not clear but may involve the induction of excitotoxicity, oxidative stress, or mitochondrial dysfunction.22

Cross-talk between neurodegeneration and vascular abnormalities

Diabetes causes apoptosis of neural and vascular cells in the retina. There is thus good reason to define DR as a form of chronic neurovascular degeneration. However, the relationship between apoptosis of vascular and neural cells is unclear, with the possibility that the loss of these different classes of cells occurs over different timeframes and possibly by unrelated mechanisms.22

There are several pieces of evidence suggesting that retinal neurodegeneration participates in early microvascular changes that occur in DR such as the breakdown of the blood–retinal barrier (BRB), vasoregression and neurovascular coupling impairment.16–21 41–44 ,83

It has recently been reported in diabetic rats that the excitotoxicity induced by glutamate upregulates VEGF production, thus favouring BRB breakdown.84 In this regard, it has recently been reported that the attenuation of retinal NMDA receptor activity by brimonidine (an α-2 adrenergic receptor agonist) results in a marked decrease in vitreoretinal VEGF and the inhibition of BRB breakdown in diabetic rats.20

The relationship between neurodegeneration and vasoregression has recently been reported by Feng et al,19 who have characterised a transgenic rat with a defect in a cilia gene that mimics the specific neurodegenerative features observed in DR. Interestingly, in this model primary neuronal degeneration is followed by vasoregression (the vascular hallmark of the early stages of DR). In addition, activated microglial cells close to the vessels undergoing vasoregression seem to play an essential role in this process.21

Neurovascular coupling is the process that enables the retina to regulate blood flow in response to neural activity.83 Flicker light stimulation is an original stimulus to the retina that has been used to investigate this process because it induces increases in neural activity and leads to retinal arterial and venous dilation because of the release of vasodilating factors, especially nitric oxide, from neural cells and endothelial cells.85 ,86 Flicker-induced retinal diameter change has been shown to deteriorate early in patients with diabetes.43 ,86 ,87 However, little is known about the mechanisms underlying the neurovascular coupling impairment related to retinal neurodegeneration.

Therapeutic implications

Given the essential role of neurodegeneration in the pathogenesis of DR, it is reasonable to hypothesise that therapeutic strategies based on neuroprotection will be effective in preventing or arresting DR development. In fact, several neuroprotective drugs have been successfully used in experimental models of DR, but clinical trials have not yet been conducted.

When the early stages of DR are the therapeutic target, it would be inconceivable to recommend an aggressive treatment such as intravitreal injections. The use of eye drops has not been considered an appropriate route for the administration of drugs aimed at preventing or arresting DR because of the general assumption that they do not reach the retina. However, there is emerging evidence to show that many drugs are able to reach the retina in pharmacological concentrations, at least in animal models,88 and the neuroprotective effects of the topical administration of brimonidine and nerve growth factor have already been reported in experimental models.89–91 These findings open up the possibility of developing topical therapy in the early stages of DR.92 ,93 In this regard a multicentric, phase II–III, randomised controlled clinical trial (EUROCONDOR-278040) to assess the efficacy of brimonidine and SST administered topically to prevent or arrest DR has been approved by the European Commission in the setting of the FP7-HEALTH.2011.

Potential impact and new perspectives

Duration of diabetes, glycaemic and blood pressure control only partly explain the risk of DR development and progression.94–96 There is thus a need to identify patients at risk of the development of non-proliferative DR that goes beyond the conventional measures. The use of mfERG, which is able to detect functional abnormalities even before any microvascular abnormality can be detected under ophthalmoscopic examination will permit us to do this. However, whereas the usefulness of mERG as a research tool for monitoring neurodegeneration has been clearly proved, its effectiveness as a screening method in a large population remains an open question. In addition, standardisation processes will be required before encouraging their widespread use in clinical practice.97–99 In addition, it could be argued that this new proposed screening method for detecting DR would be too expensive for healthcare systems. At this point, it should be stressed that the majority of the recommended diabetes interventions provide both health benefits and a good use of healthcare resources.100 In addition, targeting prevention is usually more cost-effective than assuming the considerable costs related to laser photocoagulation, vitrectomy or intravitreal anti-VEGF therapy, not to mention the social costs due to legal blindness. Nevertheless, in order to obtain accurate information on this issue, a subsequent pharmacoeconomic study would appear to be warranted.

In conclusion, neuroprotective drugs could open up a new strategy for the treatment of early stages of DR. However, clinical trials to determine the effectiveness of a non-invasive route to administer these drugs, as well as a standardisation of such methods of monitoring as mfERG and frequency domain optical coherence tomography, are needed. Meanwhile, ophthalmologists and physicians treating patients with diabetes should be aware of the potential usefulness of these drugs and work together in future research. Only such coordinated action, as well as competent strategies targeting prevention, will be effective in reducing the burden and improving the clinical outcome of this devastating complication of diabetes.

Appendix I

Members of the EUROCONDOR Consortium

R Arce (CAIBER, Spain), F Bandello (Scientific Institute San Raffaele, Italy), D Burks (Centro de Investigaciones Principe Felipe, Spain), J Cunha-Vaz (Association for Innovation and Biomedical Research on Light and Image, Portugal), C Egan (Moorfields Eye Hospital, UK), J García-Arumí (Vall d'Hebron University Hospital, Spain), J Gibson (University of Aston, UK), SP Harding (University of Liverpool, UK), S Karadeniz (International Diabetes Federation, Europe Region, IDF Europe), G Lang (University of Ulm, Germany), P Massin (Hôpital Lariboisière-APHP, France), E Midena (Univeristy of Padova, Italy), B Ponsati (BCN Peptides, Spain), M Porta (University of Turin, Italy), P Scanlon (Cheltenham General Hospital, UK), R Simó (Vall d'Hebron Research Institute, Spain), AK Sjolie (Odense University Hospital, Denmark), and AM Valverde (Instituto de Investigaciones Biomédicas Alberto Sols, Spain).

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