Blood–retinal barrier in hypoxic ischaemic conditions: Basic concepts, clinical features and management

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Abstract

The blood–retinal barrier (BRB) plays an important role in the homeostatic regulation of the microenvironment in the retina. It consists of inner and outer components, the inner BRB (iBRB) being formed by the tight junctions between neighbouring retinal capillary endothelial cells and the outer barrier (oBRB) by tight junctions between retinal pigment epithelial cells. Astrocytes, Müller cells and pericytes contribute to the proper functioning of the iBRB. In many clinically important conditions including diabetic retinopathy, ischaemic central retinal vein occlusion, and some respiratory diseases, retinal hypoxia results in a breakdown of the iBRB. Disruption of the iBRB associated with increased vascular permeability, results in vasogenic oedema and tissue damage, with consequent adverse effects upon vision. Factors such as enhanced production of vascular endothelial growth factor (VEGF), NO, oxidative stress and inflammation underlie the increased permeability of the iBRB and inhibition of these factors is beneficial. Experimental studies in our laboratory have shown melatonin to be a protective agent for the iBRB in hypoxic conditions.

Although oBRB breakdown can occur in conditions such as accelerated hypertension and the toxaemia of pregnancy, both of which are associated with choroidal ischaemia and in age-related macular degeneration (ARMD), and is a feature of exudative (serous) retinal detachment, our studies have shown that the oBRB remains intact in hypoxic/ischaemic conditions.

Clinically, anti-VEGF therapy has been shown to improve vision in diabetic maculopathy and in neovascular ARMD. The visual benefit in both conditions appears to arise from the restoration of BRB integrity with a reduction of retinal oedema.

Introduction

The presence of an intact blood–retinal barrier (BRB) is essential for the structural and functional integrity of the retina and in clinical conditions where BRB breakdown occurs vision may be seriously and adversely affected.

The BRB consists of inner and outer components and plays an important role in the homeostatic regulation of the retinal microenvironment. The BRB, like the blood– brain barrier (BBB), controls fluid and molecular movement between the ocular vascular beds and the retinal tissues and prevents leakage into the retina of macromolecules and other potentially harmful agents. The inner BRB (iBRB) is formed by the tight junctions (TJ) between neighbouring capillary endothelial cells (Shakib and Cunha-Vaz 1966) which rest on a basal lamina (Fig. 1) covered by the foot processes of astrocytes and Müller cells (Ashton, 1965, Reichenbach et al., 2007). Pericytes, separated from the endothelial cells by the basal lamina, are the third cell type of relevance to the iBRB. Astrocytes, Müller cells and pericytes are thought to contribute to the proper functioning of the iBRB.

The outer BRB (oBRB) is formed by TJ between cells of the retinal pigment epithelium (RPE) (Cuhna-Vaz, 1976) (Fig.2). The RPE resting upon the underlying Bruch's membrane separates the neural retina from the fenestrated choriocapillaris (Fig. 3) and plays an important role in transporting nutrients from the blood to the outer retina.

The apical surface of the RPE shows long projecting processes that partially envelop the outer segments of the photoreceptors, longer villi ensheathing cone outer segments and shorter villi rod outer segments (Steinberg et al., 1977) (Fig. 3). These villi are involved in the phagocytosis of outer segment discs (Steinberg et al., 1977). The metabolic interaction between these apical villi and the photoreceptors, e.g. exchange of metabolites such as vitamin A (Dowling, 1961) and amino acids (Miller and Steinberg, 1976), is considered to be critical for the maintenance of visual function (Futter, 2006). There are no structural attachments between the RPE and the outer retina but neural cell adhesion molecules expressed on the apical surface of the RPE cells create adhesion between the retina and RPE (Gundersen et al., 1993) although functional factors may be more important (Foulds, 1975, Foulds, 1985, Marmor, 2006). Mechanical separation of the retina from the RPE occurs more readily after death than in the living eye (Zauberman and DeGuillebon, 1972). The strength of the adhesion between RPE apical villi and the outer segments of the retinal photoreceptors is demonstrated in the experimental animal eye in which a partial retinal detachment has been induced. In the detached area, photoreceptor outer segments torn from their inner segments remain embedded within the apical villi of the RPE (Fig. 4).

While inter-RPE cell TJ are important in the control of paracellular movement of fluids and molecules between the choroid and retina, the polarised distribution of membrane proteins in the RPE is also important (Anderson and Van Itallie, 1995, Rizzolo, 1997).

Retinal ischaemia is an important cause of visual impairment and blindness. It has been stressed (Osborne et al., 2004) that at the cellular level ischaemia results in a self-reinforcing destructive cascade involving neuronal depolarisation, calcium influx and oxidative stress initiated by energy failure and glutamatergic excitation.

As the integrity of the BRB is dependent upon the normal functioning of the cells that determine its structure, it is no surprise that hypoxia–ischaemia is associated with BRB breakdown.

Section snippets

Capillary endothelial cells

The blood vessels in the retina are distributed as two plexuses: the inner or superficial plexus located in the nerve fibre and ganglion cell layers, and the outer or deep plexus located at the junction of inner nuclear and outer plexiform layers. In the human there are also radial peripapillary capillaries lying in the superficial nerve fibre layer (Henkind and De Oliveira, 1967).

The endothelial cells of retinal capillaries are not fenestrated (Bernstein and Hollenberg, 1965) and have a

Outer blood–retinal barrier

In health molecular movement across the RPE is both transcellular and paracellular. The outward movement of molecules from the subretinal space involves hydrostatic and osmotic forces largely acting on small molecules travelling through the paracellular inter- RPE cellular clefts and by active transport through the transcellular route (Pederson, 2006). Paracellular movement of larger molecules is restricted by the TJ between neighbouring RPE cells. As with TJ elsewhere, occludin-1, claudins,

Supplementary barriers to molecular movement

Molecular movement from the retinal and choroidal blood vascular systems into, out of and across the retina is complex and limited by a number of relative or absolute barriers to free diffusion. There is a continuous molecular movement of small molecules (mainly water) from the vitreous cavity and inner retina across the retina and RPE to the choroid (Moseley et al., 1984, Alm, 1992). A major proportion of aqueous humour secreted by the ciliary body from its rich vascular supply provides a bulk

Retinal energy requirements

The retina consumes oxygen more rapidly than any other tissue (Cohen and Noell, 1965). Oxidative enzymatic activity is highest in the inner segments of the retinal photoreceptors and in the RPE (Pearse, 1961, Hansson, 1970). The high energy requirements of the RPE stem from synthesis of cellular constituents required for their constant renewal and in the photoreceptors for the replacement of outer segment discs that undergo a rapid daily turnover (Young and Bok, 1969). In both the RPE and in

Hypoxia–ischaemia and the inner blood–retinal barrier

Retinal hypoxia occurs acutely in central retinal artery occlusion (CRAO) and more chronically in ischaemic central retinal vein occlusion. Tissue hypoxia and hyperglycaemia are regarded as principal stimulants of diabetic retinal pathology (Wilkinson-Berka, 2004). Systemic causes of retinal hypoxia include the cardiovascular effects of chronic obstructive airways disease, the ocular ischaemic syndrome associated with arterial obstructive conditions such as carotid artery stenosis (Brown and

Hypoxia–ischaemia and outer blood–retinal barrier

The RPE appears to be resistant to hypoxic–ischaemic insults and even post-mortem the RPE maintains its morphological characteristics for some time after autolysis has destroyed other retinal elements (Johnson and Grierson, 1976).

In a hypoxic adult rat model in which a severe disruption of the iBRB is induced by two hours of breathing a 93%/7% nitrogen/oxygen mixture, no disruption of the oBRB was seen (Kaur et al., 2007). The intercellular spaces between RPE cells were widened but TJ remained

Hypoxia–ischaemia, oxidative stress and blood–retinal barrier

Generation of reactive oxygen species (ROS), resulting in oxidative stress, occurs in many tissues in hypoxia–ischaemia. In ocular pathologies, ROS have been correlated with neovascularisation in diabetic eyes in humans (Augustin et al., 1993) and animals (Armstrong and al-Awadi, 1991), and in eyes with retinopathy of prematurity (Saugstad and Rognum, 1988).

Oxidative stress has also been reported to affect the integrity of the iBRB and oBRB as it is associated with a redistribution of occludin

Hypoxia–ischaemia and tight junctions

Hypoxia has been reported to produce alteration in the TJ proteins claudin-3, ZO-1 and ZO-2, correlating with increased permeability (Witt et al., 2003). A recent study has reported that there was no significant change in the distribution of occludin, ZO-1 and ZO-2 under hypoxic/reoxygenation stress (Torii et al., 2007). In vitro and in vivo experiments have indicated that claudin-5 is a target molecule in hypoxia leading to disruption of the barrier function of neural vasculature (Koto et al.,

Hypoxia–ischaemia, inflammation and blood–retinal barrier

Inflammation occurs in the retina in hypoxic–ischaemic injuries. Retinal hypoxia–ischaemia has been shown to induce the gene expression of monocyte chemoattractant protein (MCP)-1 to attract macrophages to the hypoxic area. The hypoxia-activated macrophagic/microglial release of TNF-α has been reported as a triggering factor activating the production of interleukin (IL)-8, VEGF, basic fibroblast growth factor or MCP-1 in retinal vascular cells and/or glial cells adjacent to microvessels (

Ageing and the blood–retinal barrier

In the ageing eye there is a marked loss of both retinal capillary endothelial cells and pericytes, many of the capillaries being converted into acellular tubes (Fig. 16). There is also a loss of capillaries including those in the central retina so that there is an enlargement of the foveal avascular zone in the elderly eye (Wu et al., 1985). Even in old age, however, loss of retinal capillary endothelial cells and pericytes is not accompanied by retinal oedema or other evidence of iBRB

Clinical features and significance of hypoxia-induced blood–retinal barrier breakdown

As the blood–retinal barriers subserve important functions in the eye it is no surprise that their failure has adverse effects upon the eye and on vision. The functions of the BRBs that are clinically important can be summarised as follows:

  • 1.

    Control of the delivery of nutrients and the removal of waste products of metabolism to and from the retina

  • 2.

    The exclusion of large molecules from the transparent tissues of the eye (especially vitreous) so as to maintain optical transparency

  • 3.

    The maintenance of

Diabetes

There is good evidence that hypoxia plays a significant role in the iBRB breakdown that can occur in the diabetic eye. Breakdown of the iBRB in the diabetic eye can occur early and can be detected by vitreous fluorophotometry before there is any overt sign of diabetic retinopathy (Cunha-Vaz et al., 1978).

The commonest manifestation of iBRB breakdown in the diabetic eye is an increase in retinal volume from an accumulation of extracellular fluid within the retina. Extracellular fluid in the

Outer blood–retinal barrier breakdown related to hypoxia

Where there is a breakdown in the oBRB the commonest clinical manifestation is a localised or more generalised serous retinal detachment the extent of which is dependent upon the extent of oBRB breakdown. As the oBRB is largely constituted by the TJ between neighbouring RPE cells and as the closely related choroidal circulation is greatly in excess of metabolic requirements, hypoxic damage to the oBRB is uncommon unless, as has been suggested, hypoxia plays a role in the development of the

Management of hypoxic–ischaemic blood retinal breakdown

As hypoxia induced BRB breakdown is largely restricted to the iBRB, its management is essentially the management of retinal oedema and the adverse effects this has upon vision.

Apart from measures to improve control of blood sugar levels in diabetes or to correct underlying aetiological factors in retinal vein occlusion the mainstay of treatment for both diabetic macular oedema (DMO) and for the macular oedema that may follow retinal vein occlusion has been laser photocoagulation. This includes

Anti-vascular endothelial growth factor treatment for inner blood–retinal barrier breakdown

It is now accepted that VEGF is a major factor in the genesis of the angiogenesis that characterises CNV in ARMD, the retinal new vessel formation of proliferative diabetic retinopathy or ischaemic retinal vein occlusion and the destructive neovascularisation of the retinopathy of prematurity.

VEGF is undoubtedly the Factor X postulated originally by Michaelson as aetiological in pathological neovascularisation (Michaelson, 1948). When the pivotal role of VEGF in neovascularisation became

Laser treatment of inner blood–retinal barrier breakdown

The mainstay of treatment of CSMO until recently was focal laser photocoagulation in the case of focal iBRB breakdown, and grid laser treatment as originally suggested by Dobree (1970) for diffuse macular oedema.

In one study (ETDRS (Early Treatment of Diabetic Retinopathy Study Research Group), 1987) either focal or grid laser treatment reduced visual loss associated with DMO from 24% in untreated cases to 12% in those treated by laser. Grid laser treatment tended to improve reading vision more

Corticosteroids for hypoxia-induced inner blood–retinal barrier breakdown

Corticosteroids have a specific effect on reducing vascular permeability (Nussenblatt et al., 1996). Steroids have anti-angiogenic, anti-inflammatory, anti-apoptotic, anti-proliferative and anti-oedematous activity (Cunningham et al., 2008). Hypoxia induces an inflammatory response in the retina with increased levels of inflammatory cytokines and macrophage attractants. As corticosteroids are anti-inflammatory, the actions of corticosteroids in conditions of hypoxia induced iBRB breakdown

Summary

In many clinically important conditions including diabetic retinopathy, central or BRVO, and some respiratory diseases retinal hypoxia results in a breakdown in the BRB. Disruption of the iBRB with increased vascular permeability causes vasogenic retinal oedema and tissue damage, with consequent adverse effects upon vision. Factors such as enhanced production of VEGF, NO, oxidative stress and inflammation underlie the increased permeability of the iBRB and inhibition of these factors may be

Abbreviations

  • AQ Aquaporin

  • ARMD Age-related macular degeneration

  • ATP Adenosine triphosphate

  • BBB Blood–brain barrier

  • BRB Blood–retinal barrier

  • iBRB Inner BRB

  • BRVO Branch retinal vein occlusion

  • CMO Cystoid macular oedema

  • CNV Choroidal neovascularisation

  • CRAO Central retinal artery occlusion

  • CRT Central retinal thickness

  • CRVO Central retinal vein occlusion

  • CSMO Clinically significant macular oedema

  • DMO Diabetic macular oedema

  • 2,3-DPG 2,3-Diphosphoglycerate

  • ELM External limiting membrane

  • eNOS Endothelial nitric oxide synthase

Acknowledgements

This study was supported by a research grant (R-181-000-098-112) from the National University of Singapore and a research grant (05/1/35/19/422) from the Singapore Biomedical Research Council. The help provided by Dr Viswanathan Sivakumar in the preparation of the manuscript is gratefully acknowledged. Figs. 3 and 7A are reproduced with permission of the Nature Publishing Group in respect of earlier publication in the Transactions of the Ophthalmological Societies of the United Kingdom or in Eye

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