Pathophysiology and Pathogenesis of Diabetic Retinopathy

Diabetic retinopathy is traditionally viewed as a disease of the retinal blood vessels, although there is increasing evidence that retinal neurons and glial cells are also affected. This article describes the changes in the diabetic retina that precede the development of clinical diabetic retinopathy, including changes in the rate of retinal blood flow, alterations in the electroretinogram and breakdown of the integrity of the blood-retinal barrier. The long term lesions of diabetic retinopathy are characterised by a complex array of vasodegenerative changes that lead directly to areas of retinal ischaemia. This frequently triggers the onset of macular oedema and/or the proliferative stages of diabetic retinopathy with risk of visual impairment and blindness. Neurodegeneration has also been reported in the retina during both human and experimental diabetic retinopathy, although presently it remains unclear to what extent such changes contribute to visual loss in diabetic retinopathy.

Early functional changes in the diabetic retina

Alterations in retinal blood flow

The earliest suggestion that retinal perfusion may be altered in diabetes came from studies in the 1930’s which showed that the calibre of retinal vessels is increased in diabetes. Direct evidence for changes in retinal perfusion during diabetes came nearly 40 years later with the development of techniques for measuring blood flow from fluorescein angiograms[1].

Various techniques have been used since then to measure blood flow in the retina of individuals with diabetes, including the blue-field entoptic technique[2], laser Doppler flowimetry[3] and laser speckle imaging[4].

Despite some discrepancies between studies, in general individuals with a short duration of diabetes (less than 5 years) show a narrowing of the retinal arteries and retinal blood flow is reduced[5]. As the disease progresses, however, the retinal arteries begin to dilate and retinal blood flow increases in proportion to the degree of retinopathy[6].

Changes in retinal blood flow appear to reflect poor glycaemic control, since retinal perfusion is inversely correlated with levels of glycated haemoglobin in both Type 1 and Type 2 diabetic patients.

Studies into the causes of changes in blood flow during the various stages of diabetic retinopathy will be crucial to devising new ways to regulate this abnormality. This is important because although haemodynamic changes in diabetes have been widely documented, there is at present little evidence to suggest that such alterations contribute to pathology.

Electroretinogram changes

Electroretinography is an electrophysiological technique that allows measurement of retinal neuronal function in response to light stimulation. Different protocols of light stimulation are used, each designed to test the functioning of various cell types in the retina, including the rod and cone photoreceptors, inner retinal neurons (amacrine and bipolar cells) and ganglion cell activity.

The application of electroretinogram studies to diabetes has indicated that inner retinal dysfunction is commonly present in individuals with diabetes prior to the onset of clinically observable retinopathy[7]. This becomes exacerbated during progression to the non-proliferative and proliferative phases of the disease with concurrent changes in outer retinal function[8].

In addition to electroretinography, psychophysical studies of patients with diabetes also suggest early functional alterations in the neural retina, including subtle changes in colour vision, contrast sensitivity, dark adaptation and glare recovery[9].

Blood-Retinal Barrier Dysfunction

The retina is protected from blood-borne products by the inner blood-retinal barrier, which is formed by the intra-retinal microvasculature and by the outer blood-retinal barrier, formed from the retinal pigment epithelium. The capillary endothelium and the retinal pigment epithelium are both characterised by tight junctions between adjacent cells which effectively limit the passage of solutes, proteins and fluids from the choroidal and intra-retinal blood supplies[10].

During diabetic retinopathy, the inner blood-retial barrier can become compromised and blood constituents may leak into the retinal neuropile. This has been visualised in a number of ways including fundus imaging, angiography and magnetic resonance imaging. Breakdown of the blood-retinal barrier in diabetes can result in diabetic macular oedema, the commonest cause of blindness in diabetes[11].

Although macular oedema can develop at many stages of diabetic retinopathy, it is especially common during the later phases of the disease[12]. In animal models of diabetic retinopathy, breakdown of the blood-retinal barrier has been shown after short-term diabetes, often as early as 4 weeks. How this acute-phase phenomenon relates to longer-term diabetes in patients is unknown, but there is strong evidence that potent vasopermeability agents such as VEGF play a key role[10].

It is important to note that the outer blood-retinal barrier is also compromised during diabetes. This is probably in response to retinal pigment epithelium dysfunction leading to leakage into the outer retina from the choriocapillaries and impaired fluid clearance by the retinal pigment epithelium. It is likely that vasopermeability and impaired fluid clearance are intimately linked and, together, contribute to diabetic macular oedema. Left untreated, macular oedema can lead to photoreceptor atrophy and irreversible loss of central vision. More recently, it has been suggested that swelling of the Muller glia also contribute to impaired clearance and retinal oedema during diabetic retinopathy[13].

Long term lesions of diabetic retinopathy

Basement membrane thickening

The vascular basement membrane not only provides structural support for retinal endothelial cells and pericytes, but also influences their growth, function and survival. Thickening of vascular basement membranes is one of the first histopathological changes seen in both human and experimental diabetic retinopathy[14]. Such changes are thought to arise as a result of the upregulation of membrane components (e.g., fibronectin, collagen IV and laminin) and/or reduced breakdown by proteolytic enzymes[15].

Two-way communication between endothelial cells and pericytes is thought to play a crucial role in their maintenance and survival[16], and as such, it has been suggested that basement membrane thickening may contribute to accelerated vascular cell death and vessel instability in the diabetic retina[14].

Capillary degeneration

Figure 1. Low magnification image of a trypsin digest preparation showing widespread loss of arterial smooth muscle cells (arrows) and regions of capillary acelluarity (dashed box). From: Curtis TM and Gardiner TA (2012) Ocular blood flow in diabetes: contribution to the microvascular lesions of diabetic retinopathy. In Ocular Blood Flow Eds Leopold Schmetterer and Jeffrey Kiel. Chapter 15, Part 4, 365-387
Figure 1. Low magnification image of a trypsin digest preparation showing widespread loss of arterial smooth muscle cells (arrows) and regions of capillary acelluarity (dashed box). From: Curtis TM and Gardiner TA (2012) Ocular blood flow in diabetes: contribution to the microvascular lesions of diabetic retinopathy. In Ocular Blood Flow Eds Leopold Schmetterer and Jeffrey Kiel. Chapter 15, Part 4, 365-387
Degeneration of retinal capillaries underlies the progressive ischaemia that occurs in diabetic retinopathy and is an almost universal finding in post-mortem retinas from diabetic animals and patients with long-term diabetes[14](Fig 1).

In trypsin digest preparations, acellular capillaries are manifest as empty tubes of basement membrane devoid of an endothelial lining and abluminal pericytes. Through careful comparison of fluorescein angiograms with retinal trypsin digest preparations, it is evident that acellular capillary beds correspond clinically with areas of retinal non-perfusion[17].

A key component of capillary degeneration during diabetic retinopathy is the loss of pericytes. While these mural cells are present in all vascular beds, they are uniquely critical in the retina where they occur at a one to one ratio with endothelial cells[18]. The selective loss of pericytes from the capillary wall is a histopathological hallmark of diabetic retinopathy, [19]and dropout of these cells is evidenced by the presence of so-called “pericyte ghosts” on histological specimens.

Although not widely appreciated, there is also significant loss of vascular smooth muscle cells in retinal arterial vessels during diabetes, both in patients and animal models[14](Fig 1). Loss of these cells has important implications for tissue perfusion and hydrostatic pressure within the capillary beds with impact on endothelial cell damage and vasopermeability. The exact basis of retinal pericyte and vascular smooth muscle cell death in diabetes has yet to be fully elucidated but may be related to an assortment of high glucose-triggered biochemical insults (see Pathogenesis of Diabetic Retinopathy) coupled with the limited ability of these cells to repair and renew themselves[20].

Microaneurysms

Capillary microaneurysms are normally the first clinically recognisable feature of diabetic retinopathy. Microscopically, microaneurysms are present as ‘balloon-like’ outpouchings of the capillary wall and are often associated with large areas of downstream capillary degeneration[14].

At an ultrastructural level, early stage microaneurysms show extensive accumulation of inflammatory cells[21]. The recruitment of inflammatory cells may damage the endothelium, and it is apparent that later-stage microaneurysms are invariably without an endothelial lining[21].

Microaneurysms eventually become sclerosed and consequently these lesions may appear and then disappear in the retina of diabetic patients over time[22]. Capillary hypertension, microvascular BM thickening, endothelial proliferation, thrombus formation and pericyte dropout have all been implicated as causal or contributory factors to microaneurysm formation[20].

Neurodegeneration & Glial dysfunction

As outlined above, dysfunction and degeneration of the retinal vasculature is a hallmark of diabetic retinopathy. However, in recent years there has been strong clinical and pre-clinical evidence to indicate that retinal neuronal degeneration also occurs as diabetes progresses[23].

Recent research has been conducted in rodents at various stages of diabetes and they indicate a range of neural retina abnormalities ranging from neurotransmitter changes to overt loss of retinal ganglion cells, amacrine cells or even photoreceptors[24]. Glial cells interface closely with the retinal neurons and in particular the Müller glia over-produce glutamate which may contribute to excitotoxicity and eventual depletion of retinal neurons.

In summary, the scientific literature strongly suggests that neural and glial abnormalities are an important factor in diabetic retinopathy. There is a highly complex interplay between neurons, glia and vascular components of the retina and diabetes is likely to profoundly alter the function of these cell interactions. As this evidence accumulates, diabetic retinopathy may become viewed more accurately as a disease of the neuro-vascular unit, resulting in degenerative pathology of many key cells in the retina, each of which is vital for normal function of this delicate tissue.

Pathogenesis

At present, our understanding of the molecular and cellular mechanisms through which diabetes leads to the onset and progression of retinopathy remains incomplete. Studies over the past decade, however, have provided strong evidence that high glucose-driven metabolic dysfunction and the induction of chronic, low-grade inflammatory signalling in the retina play an important role.

Metabolic dysfunction in diabetic retinopathy

A number of seemingly independent biochemical mechanisms may explain the detrimental consequences of cell and tissue exposure to high glucose. These include enhanced flux through the polyol pathway, increased involvement of the hexosamine pathway, accumulation of advanced glycation end-products (AGEs) and de novo synthesis of diacylglycerol causing over-activation of several isoforms of protein kinase C (PKC)[25].

Recent evidence links several mechanisms leading to a common overproduction of mitochondrial superoxide[25]. This free radical reduces the activity of glyceraldehyde phosphate dehydrogenase (GAPDH) and serves to divert upstream metabolites away from glycolysis and into the four glucose-driven signalling pathways above.

Benfotiamine, a transketolase inhibitor which diverts hexose metabolism to the pentose pathway, has been shown to block several of these high glucose-induced pathogenic pathways and inhibit the development of diabetic retinopathy in animal models[26]. Large-scale clinical trials are required to establish if this drug can provide benefits for patients with diabetic retinopathy.

Inflammation in diabetic retinopathy

Inflammation is a protective response of the host against exogenous and endogenous insults. During diabetes, high glucose levels, various oxidized or glycated lipoproteins such as AGEs[27], ALEs[27], and oxidized LDL[28] pose low levels of threat to tissue cells. Circulating immune cells and vascular endothelial cells are constantly exposed to the noxious microenvironment in diabetes. As a result, a low grade of inflammatory response (para-inflammation)[29]may occur.

Diabetes-induced inflammation predominantly appears to involve the innate immune system. Experimental studies have shown that high glucose can induce monocyte activation through upregulation of cell surface toll-like receptors[30]. AGEs can cause neutrophil activation and dysfunction via the RAGE pathway. Furthermore, monocytes and macrophages express a variety of scavenger receptors, and oxidized LDL may affect monocyte activation through these receptors.

Neutrophil, monocyte[31] and platelet activation has been detected in diabetic patients, which is believed to be responsible for the abnormal leukocyte-endothelial interaction (leukostasis) observed in both patients and animal models of diabetes[32]. Leukostasis is a sign of microvascular chronic inflammation in the early stages of diabetes, and has been postulated to be a factor of retinal endothelial cell death in diabetes[33]. This type of microvascular chronic inflammation is accepted as contributory to capillary occlusion, breakdown of the blood-retinal barrier and degenerative vascular pathology in diabetic retinopathy [32]. Microvascular chronic inflammation may therefore underlie the pathophysiology of microaneurysms and capillary non-perfusion seen in diabetic retinopathy.

Studies from numerous animal models have shown that extra-vascular inflammation also exists in diabetic eyes. Microglia and Muller cell activation have been observed in diabetic retina in rats and mice in the absence of clinical pathologies[34]. Complement activation has also been detected in diabetic eyes.

The cause of extra-vascular inflammation in the diabetic eye remains ill-defined. Plasma proteins and other circulating growth factors may cross the blood-retinal barrier and leak into the retina parenchyma from damaged vessels, and may induce various inflammatory responses. In addition, oxidized- or glycated-lipoproteins formed in situ may also activate retinal resident immune cells.

Complement activation in diabetic retinopathy may also be related to systemic failure of its endogenous inhibitors. Reduced expression or impaired function of complement regulators, including CD55, CD59, and DAF, has been observed in diabetes as a result of nonenzymatic glycation[35].

The precise role of the extra-vascular inflammation in various pathological changes seen in diabetic retinopathy remains illusive, but this may contribute to retinal microvascular damage. Pericyte cell death is known to be related to oxidized LDL-initiated inflammatory responses and complement activation[36]. VEGF released from activated Muller cells is believed to play an important role in blood-retinal barrier breakdown in DR. It may also contribute to retinal neuronal damage in diabetic eyes, although direct evidence supporting this is lacking.

Advanced stages such as proliferative diabetic retinopathy are defined by the development of retinal neovascularization, fibrovascular epiretinal membranes, and related complications including vitreous hemorrhage and tractional retinal detachment. Both systemic and local inflammation, although at low levels, have been detected in patients with diabetic retinopathy.

The plasma levels of high-sensitive C-reactive protein are significantly higher in such patients, as compared to healthy controls or diabetes without advanced retinopathy. The systemic neutrophil count is increased in PDR patients[37], and the cells express higher levels of myeloperoxidase and produce more hydrogen peroxide compared to cells from healthy controls. Monocytes from diabetes patients are activated and express higher levels toll-like receptors[38].

In the eye, higher levels of inflammatory cytokines such as IL-8, CCL2, have been detected in the vitreous from patients with proliferative retinopathy[39]. Increased ICAM-1 and LFA-1 expression was observed in human epiretinal membranes removed from PDR patients. Furthermore, CD11a/CD11c+ or HLA-DR+ monoyctes and CD3+ T cells were also detected in epiretinal membranes obtained from PDR patients[40].

During diabetes, in addition to hyperglycaemia, various noxious molecules can induce inflammation. Inflammatory responses in diabetic retinopathy are diverse, and different inflammatory pathways are initiated at different stages of disease, and are involved in different types of pathology.

Whilst compelling evidence supports the detrimental role of inflammation in diabetic retinal vasculopathy, and patients suffering from diabetic macular oedema can benefit from anti-inflammatory therapy (e.g.intravitreal steroid injection), the role of inflammation in diabetic retinal neuropathy remains poorly-defined. Further studies on the detailed inflammatory pathways involved in different stages of diabetic retinopathy and the role of inflammation in retinal neuronal degeneration will be crucial for further developing more specific immuno-therapy for diabetic retinopathy.

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