Fluid transport phenomena in ocular epithelia

This article discusses three largely unrecognized aspects related to fluid movement in ocular tissues; namely, (a) the dynamic changes in water permeability observed in corneal and conjunctival epithelia under anisotonic conditions, (b) the indications that the fluid transport rate exhibited by the ciliary epithelium is insufficient to explain aqueous humor production, and (c) the evidence for fluid movement into and out of the lens during accommodation. We have studied each of these subjects in recent years and present an evaluation of our data within the context of the results of others who have also worked on electrolyte and fluid transport in ocular tissues. We propose that (1) the corneal and conjunctival epithelia, with apical aspects naturally exposed to variable tonicities, are capable of regulating their water permeabilities as part of the cell-volume regulatory process, (2) fluid may directly enter the anterior chamber of the eye across the anterior surface of the iris, thereby representing an additional entry pathway for aqueous humor production, and (3) changes in lens volume occur during accommodation, and such changes are best explained by a net influx and efflux of fluid.     

Acidic fibroblast growth factor distribution in normal human eye and possible implications in ocular pathogenesis

Using a rabbit anti-acidic fibroblast growth factor (anti-aFGF) antiserum, we tried to establish a precise mapping of aFGF localization in normal human ocular structures, from samples obtained by autopsies. Cell cultures of retinal pigment epithelium and ciliary pigment epithelium were also established and immunofluorescence studies were performed after 1 month. Corneal and conjunctival epithelia were strongly positive for anti-aFGF antibodies as well as the subcapsular epithelium of the lens. The cortical fibers were weakly reactive and the lens nucleus negative. A strong intracytoplasmic reactivity was observed in the pigmented and nonpigmented epithelial cells of ciliary processes and pars plana, both ex vivo and in vitro. Retina was brightly positive, mostly in the photoreceptor and plexiform layers. The possible involvement of aFGF in normal eye growth and in various ocular diseases was then discussed.     

Dynamics of ascorbate in the aqueous humor and tissues surrounding ocular chambers

The level of ascorbate in the aqueous humor and surrounding intraocular structures in enucleated arterially perfused rabbit eye was investigated. The enucleated eye preparation was shown to be capable of secreting ascorbate from the perfusate into the aqueous humor. Ascorbate in the iris, ciliary body and cornea was released into the aqueous humor when the eye was perfused with ascorbate-free solution. Failure to obtain aqueous flow rates from the decay of ascorbate in the anterior chamber was due to the contribution of ascorbate from these ocular tissues during the perfusion. Histochemically, ascorbate was localized in the pigmented epithelial layer in the valleys between the ciliary processes and the pars plana of the ciliary body and in the iris. In the cornea, distinct localization of ascorbate was observed in the endothelium and basal cell layer of the epithelium.     

Improved ocular: systemic absorption ratio of timolol by viscous vehicle and phenylephrine

Increasing the ocular absorption of timolol relative to its systemic absorption is important clinically because ophthalmic timolol may cause serious respiratory, cardiac, and central nervous system side effects. The authors evaluated the effects of phenylephrine coadministration and solution viscosity on the aqueous humor:plasma and iris ciliary body:plasma ratios of peak timolol concentrations after ocular application. Timolol eye drops (5 mg/ml, 25 microliters) were administered to the eyes of pigmented rabbits. Coadministered phenylephrine (0.8-8.2 mg/ml) decreased the systemic peak concentrations of timolol significantly. Since ocular absorption of timolol was not affected by phenylephrine, the ocular:systemic concentration ratios were improved four- to fivefold. Phenylephrine slows down the systemic absorption of timolol by constricting the conjunctival and nasal capillaries. The ratios of the aqueous humor:plasma and iris ciliary body:plasma peak concentration of timolol were improved three- to ninefold in the presence of sodium carboxymethylcellulose compared with nonviscous eye drops. The improved ocular penetration is probably due to the longer corneal contact, and the decreased rate of systemic absorption may be caused by the slower spreading of the solution on the nasal mucosa. Compared with timolol eye drops, the ratio of the eye:plasma peak timolol concentrations was improved tenfold by using viscous eye drops with phenylephrine. Systemic concentrations of ophthalmic timolol and possibly related side effects can be decreased when timolol is instilled in a viscous vehicle with a low phenylephrine concentration.     

Localization of alpha 2 receptors in ocular tissues

Alpha 2 adrenergic agonists are used for controlling intraocular pressure (IOP) in the treatment of glaucoma. They have also been shown to be neuroprotective to retinal cells in a variety of injury models. Despite this significance, the localization of the three known alpha 2 adrenergic receptors has not been unequivocally established. The aim of this study was to determine the location of the three alpha 2 adrenergic receptors in ocular tissues using immunohistochemical techniques. New antibodies were generated and their specificity was determined using Western blotting and preadsorption. In the anterior segment of the eye alpha 2A immunoreactivity was located in the nonpigmented ciliary epithelium, corneal, and conjunctival epithelia. Alpha 2B staining was not apparent in these tissues. Alpha 2C immunostaining was present in the membrane of pigmented ciliary epithelium and corneal and conjunctival epithelial cells. In the rat retina, all three receptor subtypes were present but were differentially localized. Alpha 2A was present in the somata of ganglion cell layer and inner nuclear layer somas, alpha 2B was located in the dendrites and axons of most of the neurons as well as glia, while alpha 2C was present in the somata and inner segment of the photoreceptors. In human and monkey retinas, similar pattern of labeling for alpha 2A and 2B receptors were observed, while alpha 2B was additionally present in the membranes of many cell somata in addition to dendrites and axons. Alpha 2C labeling was much weaker but exhibited similar pattern to that observed in the rat. These data provide additional information on the location of the alpha 2 receptors in the anterior portion of the eye and present new information on their specific location in the retina. This offers insights into possible targets for adrenergic agonists in a therapeutic context.     

Transferrin receptor expression by retinal pigment epithelial cells in proliferative vitreoretinopathy.

Immunotoxins directed against a membrane marker of cell proliferation, transferrin receptor, were investigated to inhibit the growth of retinal pigment epithelial (RPE) cells in proliferative vitreoretinopathy (PVR). We undertook an immunocytological study in specimens of vitreous, subretinal fluid, and epiretinal membranes from patients with PVR to address the expression of transferrin receptor by proliferating pigment epithelial cells during the course of PVR and in normal human ocular structures. Thirty four specimens of vitreous and subretinal fluid, as well as seven epiretinal membranes, were immunocytologically examined using monoclonal antibodies to transferrin receptor. They showed a strong expression of this marker by a large majority of the cells in these two periretinal fluids (mean percentages 80 and 91% in vitreous and subretinal fluid, respectively). In contrast, only a few cells within epiretinal membranes were found to express transferrin receptor. In normal human eye sections conjunctival and corneal epithelial cells, subcapsular epithelium of the lens strongly expressed transferrin receptor, whereas RPE cells remained negative to antitransferrin receptor antibodies. A few iris or ciliary pigment epithelial cells reacted weakly. Thus, this study shows that most intravitreal and subretinal fluid proliferating cells strongly express transferrin receptor on their surface. Also confirmed is that immunotoxins to this membrane antigen could constitute potentially useful therapeutic agents in PVR.     

Melatonin receptor mRNA and protein expression in Xenopus laevis nonpigmented ciliary epithelial cells.

Melatonin is an output signal of the circadian clock, and may regulate diurnal rhythms in ocular tissues. A role for melatonin has been suggested in the circadian changes in intraocular pressure (IOP). Changes in IOP may be due partially to changes in the rate of aqueous humor secretion, which is produced by the nonpigmented epithelium of the ciliary body. To examine the mechanism by which melatonin may influence ciliary epithelium function and perhaps the IOP diurnal rhythm, immunocytochemistry with an antibody directed against the Mel(1c) melatonin receptor subtype was performed on sections of Xenopus eyes. Melatonin receptor immunoreactivity was observed in the basolateral regions of the nonpigmented epithelial cells of the ciliary body. Receptor immunoreactivity was also observed in cells of the retina, as has been previously reported. Specific immunoreactivity was not observed in the epithelium of the iris or pigmented ciliary epithelium. In situ hybridization of the Xenopus eye revealed expression of Mel(1c) but not Mel(1b) receptor mRNA in the nonpigmented ciliary epithelium. These results provide evidence that the nonpigmented epithelia of the ciliary body are direct targets for melatonin, and supports previous work that melatonin may influence the rate of aqueous humor secretion by ciliary epithelium, and perhaps the circadian rhythm of IOP.     

Localization of collagen XVIII and endostatin in the human eye

PURPOSE: To investigate the localization of endostatin, a potent angiogenesis inhibitor, and its progenitor collagen XVIII in the human eye. METHODS: Twelve normal human eyes were investigated. Immunohistochemistry of the anterior and posterior eye segment was performed using a polyclonal endostatin and collagen XVIII antibody and a monoclonal collagen XVIII antibody. Specificity of the antibodies was confirmed by Western blot analysis. RESULTS: The antibody against collagen XVIII stained Bowman's membrane, the lens capsule, the trabecular meshwork, and all epithelial and endothelial basal membranes in the anterior and posterior eye segment. In contrast, the antibody against endostatin showed a more distinct staining pattern. Intense intracellular staining for endostatin was present in the lens epithelium and in the non-pigmented epithelium of the ciliary body. Extracellular presence of endostatin could be detected in the lens capsule and all border membranes lining the aqueous humor including the anterior surface of the iris. The choroid was unstained. In the retina, staining was restricted to the inner limiting membrane and to endothelial cells of larger vessels. CONCLUSIONS: Our results show that there is a ring of specifically endostatin expressing structures forming a "barrier" around the anterior chamber and the vitreous. This might physiologically prevent vessels from sprouting into these avascular compartments.     

Ocular histopathology of systemic mucopolysaccharidosis, type II-A (Hunter syndrome, severe)

A case of mucopolysaccharidosis, Type II-A (Hunter syndrome, severe) is described, with emphasis on ocular ultrastructural findings. Single membrane-bound structures containing fibrillogranular and, less commonly, multi-membranous material were found in conjunctival epithelium, pericytes and fibrocytes; corneal epithelium, keratocytes, and endothelium; trabecular endothelium; iris pigmented epithelium, smooth muscle, and fibrocytes; ciliary pigmented and nonpigmented epithelium and fibrocytes; retinal pigment epithelium and ganglion cells; optic nerve astrocytes and pericytes; and sclerocytes. The most striking accumulation was in the nonpigmented ciliary epithelium. These findings are compared with those seen in MPS II-B, and in other systemic mucopolysaccharidoses. The nature and distribution of inclusions are not specific to any one disorder, but help to signal the presence of one of the storage disorders. Distension of corneal keratocytes may play a role in the corneal clouding seen in some of these disorders. The importance of tissue examination, especially conjunctival biopsy, in the diagnosis of storage disorders and in assessment of future modes of therapy for the mucopolysaccharidoses is discussed.     

Distribution of transthyretin in the rat eye

We reported previously synthesis of transthyretin (TTR), or prealbumin, a transport protein for thyroxine and retinol, in the eyes of rats and cows and showed that in the rat eye, TTR mRNA is localized exclusively in the retinal pigment epithelium (RPE). We now demonstrate by immunohistochemistry that TTR has a more widespread distribution in the rat eye than does its mRNA. Intense immunoreactivity for TTR was found in the RPE, ciliary epithelium, iris epithelium, corneal endothelium, optic nerve fiber layer of the retina, and lens capsule. Depending on the method of processing, immunoreactivity of varying intensity was found also in other ocular structures. In particular, the retinal ganglion cells were strongly immunoreactive on frozen sections but not on paraffin sections. Although vitreous humor was not included in the sections of adult rat eye, sections of a 25-mm rat embryo showed intense immunoreactivity in the vitreous humor. Since plasma TTR does not cross Bruch's membrane into the retina, our findings suggest that ocular TTR is synthesized, at least in part, in the RPE and is transported to specific locations within the eye. Although the physiologic role of ocular TTR is unknown, it is possible that it participates in retinol cycling within the eye. The widespread ocular distribution of TTR may account for the occurrence of various forms of ocular amyloidosis in the familial amyloidotic polyneuropathies, a group of dominantly inherited disorders caused by point mutations in the TTR gene.     

Monoclonal antibodies facilitate analyses of ocular development in mice

To clarify molecular fluctuations during ocular development, we prepared monoclonal antibodies named MAb 111 and MAb 23 which bound differentially to the neural retina, pigmented epithelium, iris and ciliary body. In newborn mice, MAb 111 bound to the pigmented epithelium including the retinal pigmented epithelium, the pigmented layer of the ciliary epithelium and the outer layer of the iris epithelium. MAb 111 also bound to the non-pigmented layer of the ciliary epithelium and the inner layer of the iris epithelium. MAb 23 bound to the neural retina, the pigmented layer of the ciliary epithelium and the outer layer of the iris epithelium. Using these two monoclonal antibodies, we performed immunohistochemical analyses on developing mouse eyes and reached the conclusion that the optic cup has already been determined to differentiate into the retina, ciliary body and iris, on the 15th day of gestation, when the ciliary body and iris are not morphologically demarcated.     

Expression of the receptor tyrosine kinases, epidermal growth factor receptor, ErbB2, and ErbB3, in human ocular surface epithelia

PURPOSE: To investigate the distribution and relative level of expression of the receptor tyrosine kinases, epidermal growth factor receptor (EGFR), ErbB2 and ErbB3, in human ocular surface epithelia. METHODS: Immunofluorescent staining was performed to identify expression of the EGFR, ErbB2 and ErbB3 in the corneal, limbal and conjunctival epithelium in tissue sections and impression cytologies taken from normal human eyes. Western blotting was undertaken to confirm the results of immunofluorescent staining. RESULTS: The three receptor tyrosine kinases, EGFR, ErbB2 and ErbB3, were detected in human corneal, limbal and conjunctival epithelia by immunofluorescent staining. Strong staining for the EGFR was observed in the basal epithelial cells at all 3 sites and throughout the corneal epithelium. Minimal or no staining for the EGFR was observed in the superficial conjunctival and limbal epithelia. The strongest staining for ErbB2 and ErbB3 was observed in the superficial ocular surface epithelium. All three receptors were detected in the corneal, limbal and conjunctival epithelium by western blot. CONCLUSION: EGFR, ErbB2 and ErbB3 are expressed by the ocular surface epithelia. EGFR is preferentially expressed by the basal epithelial cells that have the greatest proliferative potential. In contrast, ErbB2 and ErbB3 are preferentially expressed by the superficial differentiated ocular surface epithelia.     

Expression of two molecular forms of the complement decay-accelerating factor in the eye and lacrimal gland.

Complement is present in ocular fluids, but the molecular mechanism(s) restricting its activation to exogenous targets and not to autologous ocular cells are currently unknown. To clarify how this control is achieved, monoclonal antibody (mAb)-based techniques were used to examine the eye, the lacrimal gland, and ocular fluids for the decay-accelerating factor (DAF), a membrane regulatory protein which protects blood cells from autologous complement activation on their surfaces. Immunohistochemical staining of tissue sections revealed DAF antigen on corneal and conjunctival epithelia, corneal endothelium, trabecular meshwork, and retina, as well as on lacrimal gland acinar cells and in adjacent lumens. By flow cytometry, cultures of conjunctival epithelium exhibited the highest DAF levels and levels on corneal epithelium greater than corneal endothelium greater than conjunctival fibroblasts. Biosynthetic labeling of corneal endothelium yielded de novo DAF protein with an apparent molecular weight (Mr) of 75 kD, approximating that of blood cell DAF protein, and digestions of conjunctival epithelium with phosphatidylinositol-specific phospholipase C (PI-PLC), an enzyme which cleaves glycoinositolphospholipid membrane anchors, released approximately 70% of the ocular surface DAF protein similar to leukocyte surface DAF protein. Quantitations of DAF by radioimmunometric assay employing mAbs against two DAF epitopes revealed 325 ng/ml (n = 12), 4.8 ng/ml (n = 10), and 22.0 ng/ml (n = 8) of soluble DAF antigen in tears, aqueous humor, and vitreous humor, respectively. Western blot analyses of the tear DAF antigen revealed two DAF forms, one with an apparent Mr of 72 kD resembling membrane DAF forms in other sites, and a second with an apparent Mr of 100 kD, which is previously undescribed. Since DAF activity is essential physiologically in protecting blood cells from autologous complement attack, the identification of DAF on the ocular surface, intraocularly, in the lacrimal gland, and in tears suggests that DAF-mediated control of complement activation is also required in these locations.     

Detection and identification of endogenous small molecules in ocular tissues by proton nuclear magnetic resonance spectroscopy

Proton nuclear magnetic resonance (nmr) spectra of rabbit ocular tissue homogenates (corneal epithelium, conjunctiva and iris ciliary body) and aqueous humor have been recorded for the first time by incorporation of a spin-spin relaxation reagent and use of the CPMG pulse sequence. A number of endogenous species such as lactate, glucose, alanine and valine have been observed in these ocular homogenates and significant differences have been noted in the distribution of these small molecules in the ocular tissues studied. This technique has been used to study the hydrolysis of acetylcholine by ocular esterases in the iris ciliary body.     

Age-dependent iris abnormalities in collagen XVIII/endostatin deficient mice with similarities to human pigment dispersion syndrome

PURPOSE: Collagen XVIII is expressed in ocular basement membranes (BMs) and inactivating mutations cause Knobloch syndrome, with several ocular abnormalities. In this study we investigated ocular structures in collagen XVIII/endostatin (Col18a1(-/-))-deficient mice to elucidate the role of this extracellular matrix component in the eye. METHODS: Eyes of Col18a1(-/-) and control mice were examined by light and transmission electron microscopy, laser scanning ophthalmoscopy, and fluorescence angiography. Immunohistochemical analysis of neuronal, epithelial, and immune cells in the eye was performed with antibodies against established cell markers. RESULTS: Col18a1(-/-) mice showed a disruption of the posterior iris pigment epithelial (IPE) cell layer with release of melanin granules. The BM of the posterior IPE was attached to the lens and the nonpigmented epithelium of the ciliary body, which was flattened in mutant mice. In aged mutant mice a severe thickening of the stromal iris BM zone was found, and pigmented cells migrated out of the iris and covered the retina along the inner limiting membrane (ILM), sometimes penetrating into the retina. These cells resembled iris clump cells, and immunohistochemistry demonstrated that they were macrophage-like cells. Furthermore, morphologically abnormal retinal vasculature was seen by fluorescence angiography. CONCLUSIONS: The abnormalities in the iris and ciliary body of Col18a1(-/-) mice demonstrate an important role of collagen XVIII for the function of ocular BMs. The absence of this collagen alters the properties of BMs and leads to severe defects in the iris, showing striking similarities to human pigment dispersion syndrome. In addition, loss of collagen XVIII creates changes that allow clump cells to migrate out of the iris. These cells have not been well characterized previously. In the current study we showed that they are macrophage-like cells and are able to penetrate the ILM in mutant mice. The disease mechanism of human pigment dispersion syndrome is not well understood, but Col18a1(-/-) mice may serve as a model and demonstrate the potential importance of alterations in extracellular matrix components in this disease.     

Localization of myocilin/trabecular meshwork--inducible glucocorticoid response protein in the human eye

PURPOSE: To study distribution and cellular localization of myocilin/trabecular meshwork-inducible glucocorticoid response protein (TIGR) in the human eye. METHODS: A peptide antibody against a portion of the myosin-like domain of myocilin/TIGR was developed. Different ocular tissues from three human donors were investigated by one- and two-dimensional gel electrophoresis and Western blot analysis. Immunohistochemistry was performed on 25 human eyes enucleated because of posterior choroidal melanoma and on 7 normal human donor eyes. RESULTS: By Western blot analysis, a band at approximately 57 kDa was visualized in cornea, trabecular meshwork, lamina cribrosa, optic nerve, retina, iris, ciliary body, and vitreous humor. By immunohistochemistry, immunoreactivity for myocilin/TIGR was observed in cells of the corneal epi- and endothelium and extracellularly in the corneal stroma and sclera. In the trabecular meshwork, cells of the uveal and corneoscleral meshwork were stained, as was the cribriform area directly adjacent to Schlemm's canal. Positive staining was seen in cells of the ciliary epithelium, ciliary muscle, lens epithelium, and in stromal and smooth muscle cells of the iris. Throughout the entire vitreous body, fine filamentous material was positively labeled. In the retina, staining was seen along the outer surface of rods and cones, in neurons of the inner and outer nuclear layer, and in the axons of optic nerve ganglion cells. Optic nerve axons were stained in the prelaminar, laminar, and postlaminar parts of the nerve. In the region of the lamina cribrosa, astrocytes in the glial columns and cribriform plates were positively labeled. CONCLUSIONS: Myocilin TIGR is expressed in almost every ocular tissue. Depending on the respective tissue, it is observed extra- or intracellularly. The presence of myocilin/TIGR in optic nerve axons and lamina cribrosa astrocytes indicates that the trabecular meshwork might not be the only target of abnormal myocilin/TIGR in GLC1A-linked open-angle glaucoma.     

Immunomorphological study of the vitreous body and subretinal fluid in vitreoretinal proliferation

Proliferative vitreoretinopathy (PVR) is a major complication of rhegmatogenous retinal detachment. Its pathogenesis remains poorly understood and the accurate nature of the growing cells on both surfaces of the detached retina has not been yet determined. We undertook an immunocytological study on 28 specimens of vitreous or subretinal fluid removed from patients with PVR. Five main types of cells could be identified: heavily pigmented cells, poorly pigmented ones, large totally unpigmented macrophage-like ones, smaller unpigmented cells and lymphocytes. Analysis of intravitreal pigment granules showed two different types of pigmented cells, those with lipofuscin and melanin and those with melanin without any granules of lipofuscin, which could originate from ciliary or iris pigment epithelia. Immunostaining procedures confirmed the epithelial non macrophagic lineage of the intravitreal and subretinal cells. Lymphocytes were only B cells. These results confirm the importance of proliferative process during the course of PVR and shows the involvement of other ocular structures other than the retinal pigment epithelium.