Ординатура / Офтальмология / Английские материалы / The Retina and its Disorders_Besharse, Bok_2011

Blood–Retinal Barrier

J Cunha-Vaz, AIBILI, Coimbra, Portugal

ã 2010 Elsevier Ltd. All rights reserved.

Glossary

iBRB – The inner blood–retinal barrier is a situation of restricted permeability established at the level of the retinal vessels, between the blood and retinal tissue, by the tight junctions (zonula occludents) between neighboring retinal endothelial vessels and the retinal endothelial cells themselves.

oBRB – The outer blood–retinal barrier is a situation of restricted permeability established at the level of the retinal pigment epithelium, the blood, and the retinal tissue, by the tight junctions (zonula occludents) between neighboring retinal pigment epithelial cells and the retinal pigment epithelial cells themselves.

Retinal leakage analyzer – A method developed to perform localized measurements of blood–retinal barrier fluorescein leakage using a confocal scanning laser ophthalmoscope modified to obtain fluorescence measurements in the vitreous.

Tight junctions – Tight junctions are specialized junctions uniting neighboring cells by fusion of the outer leaflets of their cell membranes (zonulae occludentes) thus obliterating the intercellular space fusion and restricting paracellular diffusion.

Uveitis – Uveitis is inflammation of the uvea, which is the vascular layer of the eye sandwiched between the retina and the sclera. The uvea extends toward the front of the eye and consists of the iris, choroid layer, and ciliary body.

Vitreous fluorometry – A method developed to measure the fluorescence resulting from the presence of fluorescein in the vitreous after intravenous administration. It is a direct indicator of the permeability of the blood–retinal barrier to fluorescein.

The entire eye must function as the organ for vision and is organized with two major goals: normal function of the visual cell and the need to maintain ideal optical conditions for the light to access the visual cells, located in the back of the eye.

The blood–ocular barriers play a fundamental role in the preservation and maintenance of the appropriate environment for optimal visual cell function (Figure 1).

The blood–ocular barriers include two main barrier systems: the blood–aqueous barrier and the blood–retinal

barrier (BRB) (Figure 2), which are fundamental to keep the eye as a privileged site in the body by regulating the contents of its inner fluids and preserving the internal ocular tissues from variations which occur constantly in the whole circulation. The blood–ocular barriers must not only provide a suitable, highly regulated, chemical environment for the avascular transparent tissues of the eye, but also serve as a drainage route for the waste products of the metabolic activity of the ocular tissues.

One of these barriers, the BRB, similar to the blood–brain barrier (BBB), is particularly tight and restrictive and is a physiologic barrier that regulates ion, protein, and water flux into and out of the retina.

It is also important to realize that once inside these barriers there are no major diffusional barriers between the extracellular fluid of the retina and adjacent vitreous; nor does the vitreous body itself significantly hinder the diffusional exchanges between the posterior chamber and the retinal extracellular fluid. This means that the functions of both barriers, blood–aqueous barrier and BRB, influence each other and must work in equilibrium.

Blood–Retinal Barrier

The presence of an intact BRB is essential for the structural and functional integrity of the retina and in clinical conditions where BRB breakdown occurs vision may be seriously affected.

The BRB consists of inner and outer components (inner BRB (iBRB) and outer BRB (oBRB)) and plays by itself a fundamental role in of the microenvironment of the retina and retinal neurons.

The BRB regulates fluids and molecular movement between the ocular vascular beds and retinal tissues and prevents leakage into the retina of macromolecules and other potentially harmful agents (Figure 3).

The iBRB is established by the tight junctions (TJs) (zonulae occludentes) between neighboring retinal endothelial cells. These specialized TJs restrict the diffusional permeability of the retinal endothelial layer to values in the order of 0.14 10 5 cm s 1 for sodium fluorescein. The retinal endothelial layer functions as an epithelium and in this way is directly associated with its differentiation and with the polarization of the BRB function. This continuous endothelial cell layer, which forms the main structure of the iBRB rests on a basal lamina that is covered by the processes of astrocytes and Mu¨ller cells. Pericytes are also present, encased in the basal lamina, in

close contact with the endothelial cells but do not form a continuous layer and, therefore, do not contribute to the diffusional barrier. Astrocytes, Mu¨ller cells, and pericytes are considered to influence the activity of the retinal endothelial cells and of the iBRB by transmitting to endothelial cells regulatory signals indicating the changes in the microenvironment of the retinal neuronal circuitry.

The oBRB is established by the TJs (zonulae occludentes) between neighboring retinal pigment epithelial (RPE) cells. The RPE is composed of a single layer of RPE cells that are joined laterally toward their apices by

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Figure 1 Schematic drawing of the blood–ocular barriers and main fluid movements. RET, retina; PC, posterior chamber; AC, anterior chamber.

TJs between adjacent lateral cell walls. The RPE resting upon the underlying Bruch’s membrane separates the neural retina from the fenestrated choriocapillaries and plays a fundamental role in regulating access of nutrients from the blood to the photoreceptors as well as eliminating waste products and maintaining retinal adhesion. The metabolic relationship of the RPE apical villi and the photoreceptors is considered to be critical for the maintenance of visual function.

In both, iBRB and oBRB, the cell TJs restrict paracellular movement of fluids and molecules between blood and retina, and the endothelial cells and RPE cells actively regulate inward and outward movements. As a result, the levels in the blood plasma of aminoacids or fatty acids fluctuate over a wide range while their concentrations in the retina remain relatively stable.

Inner Blood–Retinal Barrier

Retinal endothelial cells

The endothelial cells of retinal capillaries are not fenestrated and have a paucity of vesicles. The function of these endothelial vesicles has been described as endocytosis or transcytosis that are receptor mediated. Pinocytotic residues are selectively decreased in the BRB endothelial cells. Receptor-facilitated transport mechanisms are used to move materials across the BRB (Figure 4). Channelfacilitated transport using transmembrane proteins is another mechanism for diffusion of specific substrates across the BRB. The glucose transporter Glut 1 is a good example, supplying the neuronal tissue with necessary glucose. Disruption of the iBRB in pathological conditions

is associated with increased vesicle formation and disrupted endothelial membranes. These alterations may develop before opening of the TJs is detected on ultrastructural examination.

Retinal endothelial TJs

TJs or zonula accludentes of the retinal vascular endothelium are formed by fusion of the outer leaflets of adjacent endothelial cell membranes and were described for the first time in the retinal vessels in 1966.

The TJ obliterates the interendothelial space and confers highly selective barrier properties to the capillaries. Diffusion of molecules from the lumen to the tissue is significantly restricted by TJ.

These endothelial junctions have, like in the brain capillaries, extremely high electrical resistance, 1000–3000 ohm cm2.

The TJ complex contains at least 40 proteins composing transmembrane and internal adapter proteins that regulated paracellular flux. Transmembrane proteins that make up the TJ are occludins, claudins, and junctional adhesion molecules (JAMs). Occludin is a 65-kDa protein and its changes correlate with permeability changes

Astrocytes

Figure 3 Pathways for solute movements across the inner blood–retinal barrier (retinal endothelial cells): (a) normal; (b) mechanisms of breakdown of iBRB.

making it a likely candidate in regulating the opening and closing of the TJ. Claudins regulate small charged molecules and ion permeability. JAM is part of a family of proteins and is associated with adhesion molecules.

Numerous adapter proteins localize just below the membrane and act as TJ organizers and cytoskeleton anchors.

It is important to realize that TJs are dynamic structures that can be regulated by signal transduction through cyclic AMP levels, tyrosine kinases, etc.

Mu¨ller cells, astrocytes, and pericytes

A close spatial relationship exists between Mu¨ller cells and blood vessels in the retina suggesting a critical role for these cells in the formation and maintenance of the BRB, regulating the functions of barrier cells in the uptake of nutrients and in the disposal of metabolites under normal conditions. Barrier function is also impaired by matrix metaloproteinases (MMPs) from Mu¨ller cells as these MMPs lead to proteolytic degradation of the TJ protein occludin.

Astrocytes originate from the optic nerve and migrate to the retinal nerve fiber layer during retinal vascular development. They are associated closely with the retinal vessels and help to maintain their integrity. Astrocytes are known to increase the barrier properties of the retinal endothelium by enhancing the expression of TJ protein Z0.1 and may moderate TJ integrity. Astrocytes are considered to play an important regulatory role in the function of the BRB.

Finally, the pericytes have been shown to play a role in regulating vascular tone, secrete extracellular material, and being phagocytic. Pericytes are considered to play an accessory role in maintaining the integrity of the iBRB by inducing mRNA and protein expression of occludin and other protein junctions. There is evidence that pericytes interact with the endothelial cells contributing to their modulation.

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Paracellular

transport

Outer Blood–Retinal Barrier

RPE cells

The RPE cells transport water from the subretinal space or apical side to the blood or basolateral side. Therefore, the RPE has the structural properties of an ion-transport- ing epithelium.

RPE cells regulate water content and lactic acid removal generated by the characteristic high metabolic rates in the retina. RPE cells transport water out of the retina and into the choroidal capillary plexus. The force generated by this water flux produces an adhesion force and helps to maintain retinal attachment. Water transport is linked with ion transport, organic anion transport, and other drainage mechanisms.

This outward molecular movement is largely dependent on active ionic transport associated with a relevant high oncotic pressure in the choroid.

RPE cells also have a fundamental role by transporting glucose and retinol, in the appropriate direction, from blood to the photoreceptors.

RPE tight junctions

Paracellular movement of larger molecules is restricted by the TJ between neighboring RPE cells. The paracellular resistance is 10 times higher than the transcellular resistance, classifying the RPE as a tight epithelium. Occludin, claudins, and adapter proteins have been detected at the RPE TJ as in TJ elsewhere. The TJs of the RPE are anchored to the actin cytoskeleton of RPE cells, interact with signaling molecules, and are important for the establishment of cell polarity.

In addition to TJ between RPE cells, the polarized distribution of RPE membrane proteins contributes to the function of the oBRB.

The outer retinal layers are nourished from the blood circulation through the fenestrated capillaries of the choriocapillaris and to subserve this function there is a necessity of a large baso-apical molecular movement from choroid to retina. Waste products of retinal metabolism are transported to the choroid through the oBRB.

Polarity of the outer and inner barriers: TJ modulation

Establishment of cell polarity is a characteristic of a tissue barrier. The endothelial cells of the retinal vessels and RPE develop distinct apical versus basal membrane surfaces. This cell polarity is associated with organization of the cytoskeleton, apical/basal cell membrane proteins, and organization of the junctional complexes between neighboring cells.

It is these TJ protein complexes that allow the establishment of the polarities of the BRB, restricting paracellular diffusion of blood–barrier compounds into the neuronal tissues.

Understanding the normal function of these TJs and the pathological changes that are induced by their alteration resulting in increased permeability is necessary to understand disease progression in retinal diseases such as diabetic retinopathy (iBRB primarily affected) and wet age-related macular degeneration (oBRB primarily affected).

Understanding the role of relevant proteins such as occludins, claudins, and JAMs in BRB physiology and in retinal pathology will certainly contribute to improved management of retinal disease.

Other factors regulating the molecular movement in the eye

Molecular movement from the retinal and choroidal vascular systems into, out, and across them is complex and limited by a variety of other ocular structures. There is a continuous molecular movement of small molecules (mainly water) from the vitreous cavity into the inner retina and through RPE to the choroid.

The major proportion of aqueous humor secreted by the ciliary body from its rich vascular supply provides a bulk flow of fluid through the anterior chamber of the eye but a smaller proportion enters the vitreous cavity where it is largely cleared across the retina and RPE to the choroidal circulation.

Molecular movement from vitreous to choroid is slowed by the cortical vitreous with its high concentration of hyaluronic acid stabilized in a relatively dense type II collagen matrix and the internal limiting membrane (ILM) of the retina. The ILM offers resistance to the diffusion of macromolecules of 148 kDa but allows the passage of smaller molecules. Movement through the retina of molecules that have crossed the iBRB into the retina is largely through the extracellular tissue spaces. Similary, Bruch’s membrane that separates the basal RPE from the fenestrated capillaries appears, in health, to offer little resistance to molecular movement.

The presence of fenestrations in the choriocapillaris allows the passage of even large molecules such as albumin into the extravascular spaces of the choroid. The choriocapillaris, therefore, contributes little to the oBRB.

Molecular movement across the oBRB is, however, probably influenced by the very high rate of blood flow in the choroid. A possible explanation for the high blood flow in the choroid is that there is a need not only to supply oxygen and metabolites to the energy demanding retina and RPE, but also for the rapid removal of waste products of the retinal metabolism into the blood circulation.

Finally, the ciliary body may have a relevant regulatory role in the overall maintenance of the retinal microenvironment. The large surface covered by the ciliary processes, their location where the aqueous and vitreous meet, and the multiple transport functions of the ciliary

epithelium are all factors suggesting an important role for the ciliary body in the regulation of the inner ocular fluids.

The microenvironment of the retina, which closely resembles brain extracellular fluid and is in equilibrium with the vitreous is, therefore, maintained by a variety of facilitated and active transport processes which are located mainly in the iBRB and oBRB with the retinal endothelial cells and RPE playing fundamental roles.

The Blood–Retinal Barrier and Ocular Immune

Privilege

The immune response has developed and evolved to protect the organism from invasion and damage by a wide range of pathogens. With time, the immune system has developed destructive responses that are specific for pathogens as well as tissues. Such tissue injury might, however, have a devastating effect on the function of an organ such as the eye that needs to maintain optical stability.

The existence of ocular immune privilege is dependent upon multiple factors such as immunomodulatory factors and ligands, regulation of the complement system within the eye, tolerance promoting antigen-presenting cells (APCs), unconventional drainage pathways, and, with particular relevance, the existence of the blood–ocular barriers.

The blood–ocular barriers provide a relative sequestration of the anterior chamber, vitreous, and neurosensory retina from the immune system and create the necessary environment for the existence of ocular immune privilege. The evolution of immune privilege as a protective mechanism for preserving the function of vital and delicate

organs such as the eye has resulted in a complex system with multiple regulatory safeguards for the control of both innate and adaptative immurity. The consequences of inadvertent bystander tissue destruction by antigenmonspecific inflammation can be so catastrophic to the organ or host that a finely tuned regulatory system is needed to ensure the integrity of the ocular tissues and maintain optical relationships.

There are also several lines of evidence that points to immunosuppressive functions in the BRB cells, RPE, and retinal endothelial cells. These immunosuppressive effects are apparently due to the secretion of a variety of soluble factors, such as cytokines and growth factors.

Clinical evaluation of the blood–retinal barrier

Fluorescein angiography, an examination procedure performed routinely in the ophthalmologist’s office, permits a dynamic evaluation of local circulatory disturbances and identifies the sites of BRB breakdown (Figure 5). It is, however, only semi-quantitative and its reproducibility depends on the variable quality of the angiograms.

Vitreous fluorometry was developed as a method capable of quantification of both inward and outward movements of fluorescein across the BRB system in the clinical setting. Protocols were devised, tested, and dedicated instrumentation developed.

With the development of vitreous fluorometry methodologies, a large number of clinical and experimental studies demonstrated convincingly the major role played by alterations of BRB in posterior segment disease.

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In clinical situations, alterations of the BRB have been measured in pathologies of the RPE, aged-related macular degeneration, and macular edema, as well as in hypertension and diabetes. The clinical use of vitreous fluorometry, however, has declined because it offers only an overall measurement over the posterior role and because at the time of its development there were no drugs available for stabilizing the BRB. Nowadays, vitreous fluorometry is mostly used in experimental research and in drug development.

More recently, retinal leakage mapping has been introduced to identify the sites of BRB breakdown. Further developments of this methodology based on confocal scanning laser ophthalmology (SLO-Retinal Leakage Analyzer) associated with improved optical coherence tomography imaging are expected to contribute to earlier diagnosis of BRB alterations in retinal disease as well as improved testing of the effect of new drugs that are now becoming available for treatment of retinal disease.

Blood–retinal barrier and macular edema

Macular edema is the result of an accumulation of fluid in the retinal layers around the fovea, contributing to vision loss by altering the functional cell relationship in the retina and promoting an inflammatory reparative response.

Macular edema is only a nonspecific sign of ocular disease and not a specific entity. It should be viewed as a special and clinically relevant type of macular response to an altered retinal environment, in most cases associated with an alteration of the BRB. It occurs in a wide variety of ocular situations such as uveitis, trauma, intraocular surgery, vascular retinopathies, hereditary dystrophies, diabetes, age-related macular degeneration, etc.

The increase in water content of the retinal tissue that characterizes macular edema may be initially intracellular or extracelullar. In the first case, also called cytotoxic edema, there is an alteration of the cellular ionic distribution.

In the second case, more frequent and clinically more relevant, the extracellular accumulation of fluid is directly associated with an alteration of the BRB. In this later situation, the protective effect of the BRB is lost and the Starling law applies. When there is breakdown of the BRB, any changes in the equilibrium between hydrostatic and oncotic pressure gradients across the BRB contribute to further water movements and progression of the macular edema.

It is also of great relevance to keep in mind that the BRB cells, retinal endothelial cells, and retinal pigment epithelial cells, are both the target and producer of ecosanoids, growth factors, and cytokines. Breakdown of the BRB leading to situations of macular edema may be mediated by locally released cytokines and induction of an inflammatory reparative response creating the conditions for further release of cytokines, growth factors, etc.

Macular edema is also one of the most serious consequences of inflammation in the retinal tissue. Inflammatory cells can alter the permeability of the TJs that maintain the iBRB and oBRB. Cell migration may occur primarily through splitting the junctional complex or through the formation of channels or pores across the junctional complex.

Macular edema has particular relevance for its frequency in diabetic retinopathy. Leukocyte adhesion to retinal vessels and breakdown of the BRB appear to be mediated by nitric oxide (NO). NO upregulates intercellular adhesion molecule-1 (ICAM-1) and promotes the downregulation of TJ protein expression.

Relevance of BRB to Treatment of Retinal Diseases

When administered systemically, drugs must pass the BRB to reach therapeutic levels in the retina. Drug entrance into the retina depends on a number of factors, including the plasma concentration profile of the drug, the volume of its distribution, plasma protein binding, and the relative permeability of the BRB. To obtain therapeutic concentrations within the retina, new strategies must be considered such as delivery of nanoparticles, chemical modification of drugs to enhance BRB transport, coupling of drugs to vectors, etc.

The BRB must be considered as a dynamic interface that has the physiological function of specific and selective membrane transport from blood to retina and active efflux from retina to blood for many compounds, as well as degradative enzymatic activities.

From the viewpoint of drug delivery, designing drugs (including peptides) with greater lipophilicity to enhance BRB permeability seems to be an easy approach. However, such a strategy would not only increase the permeation into tissues other than the retina, but also decrease the bioavailability due to the hepatic first pass metabolism in the case of oral administration. Accordingly, for the development of retina-specific drug delivery systems for neuroactive drugs the most effective approach is to utilize the specific transport mechanisms active at the BRB. That would mean designing drugs that mimic the substrates to be taken by particular transporters or receptors existing in the BRB.

Eye drops are generally considered to be of limited benefit in the treatment of posterior segment diseases. Newer pro-drug formulations that achieve high concentrations of the drug in the posterior segment may have a role in the future. Meanwhile, periocular injection is one modality that has offered mixed results.

Finally, the last years have seen a generalized and surprising safe utilization of intravitreal injections, a form of administration that circumvents the BRB. Steroids and a variety of anti-VEGF drugs have been administered

through intravitreal injections to a large number of patients without significant side effects and demonstrating good acceptance by the patients. Intravitreal injections can achieve high drug concentrations in the vitreous and retina preserving the BRB function and its crucial protective function. At present the major challenge appears to be the need to decrease the number of intravitreal injections which in the case of anti-VEGF treatments are given every 6 weeks to maintain efficacy. The search for safe slow-delivery devices or implantable biomaterials is ongoing but the invasive approach to treat retinal diseases appears to be the only effective way of reaching rapidly therapeutic levels in the retina in the presence of a functioning BRB.

See also: Anatomy and Regulation of the Optic Nerve Blood Flow; Breakdown of the Blood–Retinal Barrier; Breakdown of the RPE Blood–Retinal Barrier; Developmental Anatomy of the Retinal and Choroidal Vasculature; Innate Immune System and the Eye; Macular Edema; Physiological Anatomy of the Retinal Vasculature; Retinal Pigment Epithelial–Choroid Interactions; RPE Barrier.

Further Reading

Cocaprados, M. and Escribano, J. (2007). New perspectives in aqueous humor and secretion and in glaucoma: The ciliary body as multifunctional neuroendocrine gland. Progress in Retinal Eye Research 26: 239–262.

Cunha-Vaz, J. G. (1979). The blood–ocular barriers. Survey of Ophthalmology 23: 279–296.

Cunha-Vaz, J. G., Faria de Abreu, J. R., Campos, A. J., and Figo, G. (1975). Early breakdown of the blood–retinal barrier in diabetes.

British Journal of Ophthalmology 59: 649–656.

Cunha-Vaz, J. G. and Maurice, D. M. (1967). The active transport of fluorescein by retinal vessels and the retina. Journal of Physiology 191: 467–486.

Cunha-Vaz, J. G. and Maurice, D. M. (1969). Fluorescein dynamics in the eye. Documenta Ophthalmologica 26: 61–72.

Cunha-Vaz, J. G. and Travassos, A. (1984). Breakdown of the blood–retinal barriers and cystoid macular edema. Survey of Ophthalmology 28: 485–492.

Cunha-Vaz, J. G., Shakib, M., and Ashton, N. (1966). Studies on the permeability of the blood–ocular barrier. I. On the existence, development and site of a blood–retinal barrier. British Journal of Ophthalmology 50: 411–453.

Kaplan, H. J. and Niederkorn, J. Y. (2007). Regional immunity and immune privilege. In: Niederkorn, J. Y. and Kaplan, H. G. (eds.)

Immune Response and the Eye. Chemical Immubology Allergy vol. 92, pp. 11–26. Basel: Karger.

Lobo, C., Bernardes, R., and Cunha-Vaz, J. G. (1999). Mapping retinal fluorescein leakage with confocal scanning laser fluorometry of the human vitreous. Archives of Ophthalmology 117: 631–637.

Partridge, W. M. (1998). Introduction to the blood–brain barrier: Methodology and pathology. In: Partridge, W. M. (ed.) Introduction to the Blood–Brain Barrier: Methodology, Biology and Pathology, pp. 1–10. New York: Cambridge University Press.

Peyman, G. A. and Bok, D. (1972). Peroxidase diffusion in the normal and laser-coagulated primate retina. Investigative Ophthalmology 11: 35–45.

Philips, B. E. and Antonetti, D. A. (2007). Blood–retinal barrier. In: Joussen, A. M., Gardner, T. W., Kirchhof, B., and Ryan, S. J. (eds.) Retinal Vascular Disease, pp. 139–153. Berlin: Springer.

Rapoport, S. I. (1976). Blood–Brain Barrier in Physiology and Medicine. New York: Raven Press.

Reese, T. S. and Karnovski, M. J. (1967). Fine structural localization of a blood–brain barrier to exogenous peroxidase. Journal of Cellular Biology 34: 207–217.

Shakib, M. and Cunha-Vaz, J. G. (1966). Studies on the permeability of the blood–retinal barrier. IV. Junctional complexes of the retinal vessels and their role on their permeability. Experimental Eye Research 5: 229–234.

Strauss, O. (2005). The retinal pigment epithelium in visual function.

Physiological Review 85: 845–881.

Glossary

Fenestrations – Spaces between vascular endothelial cells that allow free fluid exchange between vessel and tissue. Fenestrations are characteristic of vessels found in tissues that do not have a blood–tissue barrier.

Leukostasis – The adhesion of leukocytes to the vascular endothelium as part of an inflammatory reaction.

Macular edema – Fluid accumulation, due to blood–retinal barrier (BRB) breakdown, in the area of the human or primate retina of highest visual acuity. Tight junctions – Also referred to as zonula occludens, tight junctions are complex arrangements of microfilaments and other proteins that connect retinal vascular endothelial (RVE) or retinal pigment epithelial (RPE) cells and restrict the flow between them. Tight junctions are an integral component of the blood–retinal, blood–brain, or blood–nerve barrier.

Vesicular transport – The nonspecific transcellular transport of fluid and high-molecular-weight molecules from the luminal to the abluminal surface of the vascular endothelium by means of pinocytotic vesicles.

Uveitis – An inflammation of the uvea, or the middle layer of the eye. The uvea consists of three structures: the iris, the ciliary body, and the choroid.

Introduction

The blood–retinal barrier (BRB), which is analogous to the blood–brain barrier, maintains homeostasis in the retina by restricting the entry of blood-borne proteins from the retina and by maintaining strict ionic and metabolic gradients. When this barrier breaks down, excess fluid accumulates in the retina and this can result in macular edema, which is associated with ischemic retinopathies, including diabetic retinopathy (DR) and retinopathy of prematurity (ROP), ocular inflammatory diseases, retinal degenerative diseases, and a variety of other ocular disorders, or following ocular surgery. BRB breakdown can occur at the inner BRB, which is established at the retinal vasculature, at the outer BRB, which consists of the

retinal pigment epithelial (RPE) cells, or at both sites. The BRB is established by the formation of tight junctions between the retinal vascular endothelial (RVE) cells and the RPE cells and a paucity of endocytic vesicles within these cells. The establishment and maintenance of the BRB is regulated by the perivascular astrocytes and pericytes, but the mechanism for this regulation is not entirely clear. Some studies have shown that cell to cell contact is necessary to establish and maintain the BRB, while others provide evidence that a soluble mediator is sufficient. BRB breakdown can result from a disruption of the tight junctions, which are composed of a complex network of junctional proteins, an upregulation of vesicular transport across the RVE or RPE, or by degenerative changes to the barrier-forming cells or to the regulatory cells, the pericytes and glia. In some cases, BRB breakdown is related to identifiable structural defects, such as loss of pericytes, astrocytes, or RPE cells or changes to the vascular endothelial cells, as would be caused by microaneurysm formation. In other cases, where retinal vascular leakage is diffuse, such as in uveitis, or when the leakage is remote from a lesion, such as a surgical wound or tumor, it is clear that diffusible factors are involved. Blood–tissue barriers exist only in the retina, brain, and nerve. Vascular endothelial cells in the choroid and in other tissues are fenestrated (Figure 1(a)), allowing large molecular weight molecules to freely pass from the blood to the tissue, and thus do not have a barrier function.

Tight Junctions

Tight junctions or zonula occludens consist of complex arrangements of over 40 proteins in the peripheral cytoplasm and apical plasma membrane that connect RVE or RPE cells and restrict flow between them (Figure 1(b)). Occludin and the claudins (over 24 isoforms), which form the junctional strands and are believed to constitute the backbone of the tight junction, span the plasma membrane and bind junctional proteins in adjacent cells. Zonula occludens proteins 1, 2, and 3 (ZO-1, -2, and -3) are intracellular proteins that associate with the cytoplasmic surface of the tight junctions and organize the complex. The binding of ZO-1 to occludin establishes the tight junction. Other integral components of the junctional complex are the junctional adhesion molecules, tricellulin, cingulin, 7H6, and symplekin. A breach of the tight junctions (Figure 1(c)) can result from an alteration in the content of the junctional proteins, their redistribution, or their phosphorylation.

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Figure 1 (a) Choroidal vessels are fenestrated (arrow) and, therefore, do not form a blood–tissue barrier.

(b) A morphologically closed tight junction (arrow) in a normal retinal vessel. Note close apposition of vascular endothelial cells and an intact junctional complex. (c) A morphologically open tight junction (arrow) in a retinal vessel. The space between

the vascular endothelial cells allows vascular leakage through the junction.

Occludin content at the tight junction is higher in cells that have a tighter barrier and decreased occludin correlates with increased BRB permeability, but occludin knockout mice appear to form functional tight junctions, so the association is complex and not simply regulated by occludin. Increased occludin phosphorylation is also associated with increased BRB permeability. Altered expression of claudins can lead to changes in selectivity of the junctions and claudin-5 appears to be particularly important for maintenance of a functional tight junction.

Adenosine, prostaglandin E1 (PGE1), interleukin-1b (IL-1b), tumor necrosis factor-a (TNFa), and vascular endothelial growth factor (VEGF) appear to be capable of causing a morphological and functional opening of the RVE tight junctions. A significant number of interendothelial cell tight junctions appeared open along their entire length within 6 h of intravitreal injection of each agent into rabbits with TNFa showing the greatest effect (35.6% of the interendothelial cell junctions appeared open, morphologically). The effect of PGE1 on tight junctions appeared to be transient, that of VEGF and IL-1b were partially reversible by 24 h, and the effect of the adenosine agonist, N-ethylcarboxamidoadenosine was not reversible by 48 h. The demonstration of immunoreactive albumin, which would normally be confined to the lumens of vessels with a blood–tissue barrier, along the entire length of these junctions, from the luminal to the abluminal surface, suggests that they are also functionally open (Figure 2(c)).

Vesicular Transport

Since the tight junctions restrict the flow of molecules across the BRB, a series of pumps, channels, and transporter molecules are necessary to transport specific essential molecules from the blood to the retina. The nonspecific transport of high molecular weight molecules and fluids across the RVE by way of pinocytotic vesicles (Figure 2(a)) or caveolae is referred to as vesicular transport (Figure 2(b)) and serves as a transcellular means of BRB breakdown. This mechanism appears to be the predominant means for BRB compromise associated with VEGF-A-induced hyperpermeability in monkeys and in DR in humans, rats, and rabbits.

In addition to causing the opening of interendothelial cell tight junctions in the retina, adenosine, PGE1, IL-1b, TNFa, and VEGF also promote the formation of pinocytotic vesicles in RVE cells and the distribution of albumincontaining intraendothelial vesicles across the entire RVE cell and at both the luminal and abluminal surfaces suggests that active vesicular transport is occurring. Although infrequently seen, the vesiculo-vacuolar organelle, which is associated with VEGF in the vascular endothelium of tumors, was also evident in the RVE of VEGF-treated rabbits, but not monkeys, and is likely to play a role in VEGF-mediated vascular permeability. The effect of these mediators on the outer BRB is less clear.

Role of Inflammation

Inflammation has been associated with BRB breakdown in DR, choroidal neovascularization (CNV) associated with age-related macular degeneration, aging, ocular inflammatory disease, and the administration of pro-inflammatory molecules. The increased adhesion of leukocytes to

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Figure 2 (a) Immunocytochemical staining for endogenous albumin shows the formation of pinocytotic vesicles (arrows) on the luminal surface of vascular endothelial cells in a 7 month galactosemic rat. Immunoreactive albumin is contained within the formed vesicles (top right). (b) Immunocytochemical staining for albumin demonstrates albumin filled vesicles throughout the cytoplasm of the vascular endothelial cells of

a rabbit. The presence of these vesicles and the positive staining for albumin in the extracellular matrix (left) suggests that vesicles are actively transporting serum proteins across the endothelium and extruding their contents at the abluminal surface as a means of transcellular BRB breakdown.

(c) Immunocytochemical staining for albumin along the entire length of the interendothelial cell junction (arrow) and in the basal lamina indicates that there is vascular leakage through the junction.

endothelial cells in the retina is associated with increased expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), CD18, and other adhesion molecules, which are upregulated by VEGF and other pro-inflammatory molecules in DR and other ocular disorders, and appear to be regulated, at least in part, by protein kinase C (PKC). Diabetic CD18 and ICAM-1 knockout mice have significantly fewer adherent leukocytes than diabetic mice with normal CD18 and ICAM-1 and the decreased leukostasis is associated with fewer damaged endothelial cells and reduced BRB breakdown, supporting the role of adhesion molecules in increased inflammation and the correlation of an inflammatory response with endothelial cell damage and permeability. It is not clear whether the same molecules that facilitate leukostasis also mediate BRB breakdown or if this is attributable to molecules secreted by the recruited leukocytes, or both, but there appears to be a direct correlation between increased leukostasis and vascular permeability in the retina and pro-inflammatory molecules, such as TNFa and IL-1b, are among the most potent inducers of BRB breakdown. Leukocyte adhesion to the diabetic vascular endothelium can promote endothelial apoptosis and inhibition of leukocyte adhesion to the retinal vessels can not only prevent endothelial degeneration, but also reduce the diabetes-associated loss of pericytes, which support the vascular endothelium and help to confer BRB integrity. Inflammation can also alter the distribution of astrocytes and their ensheathment of retinal vessels, leading to alterations in BRB integrity. Leukocytes have also been shown to cause a downregulation and redistribution of tight junctional proteins, which leads to a disruption of tight junctions and a transient breakdown of the BRB during retinal inflammation.

Molecular Mechanisms

The induction of BRB breakdown is a complex process that is mediated, not by a single factor, but by the interaction of multiple factors operating through different receptors and signaling pathways. The list of molecules that have been identified as playing a role in BRB breakdown, which is by no means all-inclusive, includes VEGF, hypoxia-indicible factors-1 and -2 (HIF-1 and -2), placental growth factor (PlGF), TNFa, IL-1b, platelet-activating factor, adenosine, histamine, prostaglandins (PGE1, PGE2, and PGF2a), platelet-derived growth factors A and B (PDGF-A and -B), insulin-like growth factor-1 (IGF-1), ICAM-1, VCAM-1, P-selectin, and E-selectin. The key will be to determine what the initiating event is and which events are parts of the resulting cascade. By targeting the appropriate molecules, subsequent events leading to BRB failure may be blocked.

The various isoforms of VEGF and PlGF are members of the VEGF family. VEGF-A, a potent inducer of vascular