Evaluation of therapeutic effectiveness using MR imaging in a rabbit model of anterior uveitis.

Magnetic resonance imaging (MRI) has been used to examine conditions that alter the permeability of the blood-retinal barrier. Our goal was to determine if blood-aqueous barrier permeability could be similarly assessed, because MRI offers the theoretical advantage of providing quantitative data directly from inflamed uveal tissues rather than from the aqueous humor into which the inflammatory reaction spills. As an additional challenge, we sought to use MRI to measure differences between the inflamed uveal tissues of corticosteroid-treated and placebo-treated uveitic eyes. Anterior uveitis was induced in one eye of eight rabbits by subcutaneous injection of Mycobacterium tuberculosis, followed after 10 days with intravitreal challenge. One rabbit of each pair was treated with topical 1% prednisolone acetate while control rabbits were treated with artificial tears. Contrast-enhanced MRI studies were performed prior to uveitis induction, one day after induction and then weekly for at least 2 weeks. MR image data were analyzed to determine percent change in peak enhancement of the ciliary body and anterior chamber. The initial rate of change of enhancement of the anterior chamber was also measured. Extensive contrast agent-induced MR image enhancement of both the anterior chamber and the ciliary processes was measured following the induction of uveitis. More rapid improvement was measured for the 1% prednisolone acetate-treated rabbit eyes (P < 0.001). MR signal enhancement data obtained from the ciliary processes proved to be the most reliable indicator of disease activity in this rabbit model of uveitis. Such data can only be obtained using MRI.

Specific hydraulic conductivity of corneal stroma as seen by quick-freeze/deep-etch.

Previous studies of the hydraulic conductivity of connective tissues have failed to show a correspondence between ultrastructure and specific hydraulic conductivity. We used the technique of quick-freeze/deep-etch to examine the ultrastructure of the corneal stroma and then utilized morphometric studies to compute the specific hydraulic conductivity of the corneal stroma. Our studies demonstrated ultrastructural elements of the extracellular matrix of the corneal stroma that are not seen using conventional electron microscopic techniques. Furthermore, we found that these structures may be responsible for generating the high flow resistance characteristic of connective tissues. From analysis of micrographs corrected for depth-of-field effects, we used Carmen-Kozeny theory to bound a morphometrically determined specific hydraulic conductivity of the corneal stroma between 0.46 x 10(-14) and 10.3 x 10(-14) cm2. These bounds encompass experimentally measured values in the literature of 0.5 x 10(-14) to 2 x 10(-14) cm2. The largest source of uncertainty was due to the depth-of-field estimates that ranged from 15 to 51 nm; a better estimate would substantially reduce the uncertainty of these morphometrically determined values.

The lipid composition of drusen, Bruch's membrane, and sclera by hot stage polarizing light microscopy.

PURPOSE: To detect and identify, in situ, the lipid composition of drusen, diffuse Bruch's membrane deposits, and sclera in aging human eyes using hot-stage polarizing microscopy (HSPM), a method that allows qualitative determination of lipid subtypes within histologic sections based on morphology and melting temperatures of liquid crystals as monitored by birefringence during heating and cooling. METHODS: Full-thickness buttons of the central macula and the periphery of human eyes from 17 patients were fixed in 5% calcium-buffered formalin. Frozen sections were stained with oil red O or Sudan black or were analyzed by HSPM. RESULTS: Birefringent anisotropic droplets ("maltese crosses") with melting characteristics of cholesterol esters were identified within diffuse Bruch's membrane deposits, drusen, and sclera. Deposits that melted from crystal to oil without any maltese cross formation when cooled were present in the sclera and are consistent with triglyceride-rich deposits. Deposits with optical properties consistent with phospholipids were identified in a single aged eye. Eyes from young donors did not show these changes. CONCLUSIONS: HSPM is a valuable technique for evaluating the nature of lipid deposits in aging eyes. Further studies are warranted to determine whether similar changes are also present in eyes with age-related macular degeneration.

Localization of occludin, ZO-1, and pan-cadherin in rabbit ciliary epithelium and iris vascular endothelium.

Previous studies have used conventional electron microscopy and freeze fracture to identify the morphological equivalents of the blood-aqueous barrier in the mammalian eye. These equivalents are the tight junctions that form a part of the apicolateral junctional complex between adjacent non-pigmented ciliary epithelial cells and the tight junctions present between endothelial cells of the iris vasculature. Recent investigations have begun to unravel the molecular assembly of the tight junction and some variability has been found. Our goal in the present study was to probe the ciliary epithelium and iris vascular endothelium of the rabbit eye to determine if certain molecular constituents associated with tight junctions in other tissues are also present as parts of the blood-aqueous barrier. The selected constituents were occludin, ZO-1, and a representative, adherens junction-related cadherin. Immunohistochemical and immunoelectron microscopic methods were used. The results showed that occludin was distributed exclusively at known locations of tight junctions. ZO-1 was also expressed at these locations but its distribution extended beyond that of occludin, along the adjacent membranes. Pan-cadherin was expressed ubiquitously within the ciliary epithelium and negligibly in iris vascular endothelium. Our results demonstrate that occludin and ZO-1 are integral components of the blood-aqueous barrier of the normal rabbit eye.

Interendothelial junctions in normal human Schlemm's canal respond to changes in pressure.

PURPOSE: To determine if changes in the structure and complexity of junctions between endothelial cells lining Schlemm's canal (SC) occur in normal human eyes with changes in perfusion pressure. METHODS: Twelve normal human eyes were either perfusion-fixed (at 15 or 45 mm Hg) or immersion-fixed (0 mm Hg) in modified Karnovsky's fluid. 'Outflow facility was measured continually during the perfusion fixation. The intercellular junctions of the endothelial cells of SC were ultrastructurally examined in thin sections, including serial sections and freeze-fracture replicas. Morphometric data on the number of junctional strands per total length of tight junction were documented and categorized by the number of strands (one, two, or three or more). The length of endothelial cell overlap was measured on thin sections. RESULTS: In freeze-fracture replicas, perfusion-fixed eyes demonstrated less complex junctions. At 15 mm Hg, 18.06% of the total junctional length was represented by three or more strands; at 45 mm Hg, this percentage decreased to 8.59%. In immersion-fixed eyes, 24.17% of the total junctional length was represented by three or more strands. These differences were statistically significant (P < 0.0012). In sections, the amount of endothelial cell overlap, and thus the length of paracellular pathway, was reduced in perfusion-fixed versus immersion-fixed eyes (P < 0.02). Extensive serial sectioning demonstrated that giant vacuoles were formed, either by individual endothelial cells or by two or more adjacent endothelial cells. CONCLUSIONS: When compared with specimens fixed at zero pressure, overlap between endothelial cells of SC is reduced significantly when this cell layer is under conditions of flow similar to those encountered in vivo. The tight junctions between cells of the inner wall of SC become less complex with increasing pressure. Our data suggest that the paracellular pathway into SC in the normal eye is sensitive to modulation within a range of physiologically relevant pressures.

The role of soluble proteins in generating aqueous outflow resistance in the bovine and human eye.

Previous research has shown that wash-out in bovine and primate eyes can be greatly reduced by perfusing with buffer containing 5-15% serum. It was suggested that protein diffusion from the iris root might raise the in vivo protein concentration in the trabecular meshwork to a level much higher than in the anterior chamber. In this study, we investigated the protein concentration in effluent from the outflow pathways in bovine and human eyes, its possible relationship to wash-out, and whether the reduction of wash-out was caused by a bulk protein effect. Bovine and human eyes were placed under silicone oil and perfused with buffer. Outflow facility was continuously determined while effluent was periodically collected from the surface of the eye, and the soluble protein concentration in the effluent was determined. Separate studies were conducted perfusing either albumin or gamma-globulin through bovine eyes. Theoretical models were developed to study the transport of protein into the perfusion fluid. In the bovine eyes, the initial protein concentration in the collected effluent was approximately 1% that of serum, much lower than the 10-15% buffer in serum required to prevent wash-out. Furthermore, the rate of change of outflow facility showed a different dependence on perfused volume than did the protein concentration. Human eyes showed a much higher level of protein in the perfusate, that decayed over a much longer time period. A statistically significant correlation existed between outflow resistance and soluble protein concentration in both bovine and human eyes. However, modelling studies suggested that this correlation might be due to flow resistance setting the flowrate which then determines the protein concentration of the effluent. Separate experiments indicated that the decreased rate of wash-out caused by perfusion of 10-15% serum in buffer was not due to either albumin or gamma-globulin alone. These results suggest that the reduction of wash-out observed in previous studies when serum proteins were perfused through bovine and monkey eyes was not due to the general level of serum proteins but may instead be due to interactions of a particular protein(s).

Hyaluronic acid in the normal and glaucomatous optic nerve.

Eighteen normal human eye-bank eyes (age: 18-81 years), five fetal eyes (16-24 weeks), 11 primary open-angle glaucoma (POAG) eyes (age: 76-89 years), and two Schnabel's cavernous optic atrophy eyes were examined using a biotinylated-hyaluronan binding protein to study the changes in the distribution of hyaluronic acid (HA) in the fetal, adult and glaucomatous optic nerve head. The vitreous body served as a positive control. Sections treated with Streptomyces hyaluronidase were used to confirm specificity. Monoclonal antibodies to myelin basic protein (MBP) and glial fibrillary acidic protein (GFAP) were used as additional controls. In fetal optic nerve, HA was localized in blood vessels, peripapillary sclera and the pial septae in the retrolaminar nerve. No staining was associated with axons. Staining for MBP was negative. In adults, HA was found surrounding the myelin sheaths in the retrolaminar nerve; staining decreased with age. In contrast, HA staining in myelinated peripheral nerves (e.g. ciliaries) remained unchanged with age. HA also was localized to the adventitia of arteries and veins throughout the posterior segment. Compared to age-matched normal eyes, HA staining was virtually absent around myelin sheaths of the retrolaminar nerve in POAG eyes. Similar changes were not found in other HA positive structures. In Schnabel's cavernous optic atrophy. HA was present in increased amount in the atrophic area, but virtually absent in the remaining retrolaminar nerve. HA staining was invariably positive in vitreous, and Streptomyces hyaluronidase treated sections were negative. In adults, staining of MBP was associated with the myelin sheath in the retrolaminar nerve. In contrast to HA, staining of MBP was unchanged with age and in POAG. In Schnabel's atrophy, MBP staining disappeared only in the atrophic area. HA in the retrolaminar optic nerve appears to be associate with the space-filling matrix between myelin sheaths. HA is not present in the axon bundles prior to myelination of the optic nerve. HA in the retrolaminar optic nerve appears to decrease with age and is further reduced in POAG; however, corresponding changes are not found in MBP or in peripheral nerves. Perhaps, decreased amounts of HA is related to a higher susceptibility to elevated intraocular pressure or to optic nerve atrophy. In Schnabel's cavernous optic atrophy, HA is present in increased amount only in the atrophic area while MBP is markedly decreased, suggesting in situ production of HA in areas of optic nerve atrophy.

Cytokeratin expression in normal human bulbar conjunctiva obtained by impression cytology.

PURPOSE: To document the cytokeratin expression patterns in the normal human conjunctival epithelium obtained directly from patients using impression cytology. METHODS: Impression cytology specimens were obtained from normal volunteers using pure nitrocellulose membranes rather than cellulose acetate. The 31 volunteers of both sexes ranged in age from 18 to 79 years. Impression cytology specimens were analyzed for individual cytokeratins by either immunocytochemistry or electrophoresis with immunoblotting using a defined panel of monoclonal antibodies. RESULTS: Using the corroborative methods of immunocytochemistry and electrophoresis with immunoblotting, cytokeratins characteristic of nonkeratinized, stratified (K4 and K13), simple (K8 and K19), and glandular epithelia (K7) were present in the superficial layer (s) of normal human conjunctiva. Cytokeratins typical of keratinized epithelia (K1, K2, and K10) and the keratinization-related proteins filaggrin and involucrin were not expressed in normal conjunctival epithelium. CONCLUSIONS: The normal human conjunctiva demonstrates a unique cytokeratin expression pattern containing cytokeratins characteristic of nonkeratinized, stratified epithelia, as well as others more typical of a simple differentiation pattern, a glandular differentiation pattern, or both. These findings provide a foundation for examining changes in the cytokeratin expression pattern in diseased human conjunctival epithelium using impression cytology.

The non-uniform distribution of albumin in human and bovine cornea.

In our previous studies, we noted a non-uniform distribution of protein tracer preferentially entering the anterior stromal lamellae of the cornea from the limbus. Given other differences reported previously between the anterior and posterior lamellae of the cornea, and the number of corneal disorders in which abnormalities are preferentially confined to either the anterior or posterior lamellae, we were prompted to examine the distribution of albumin in normal human and bovine cornea. The distribution of albumin in bovine and human cornea was studied immunohistochemically. Total soluble protein and albumin in the anterior 1/3 and posterior 2/3 of the central, middle and peripheral cornea of bovine eyes was measured biochemically. To aid in interpreting the findings, a theoretical model was developed based upon the combined effects of diffusive and convective transport. Using immunohistochemical methods, in both bovine and human eyes, intense staining of albumin was found in the anterior 1/3 of the corneal stroma. There was a gradual reduction in staining intensity from the limbus to the central cornea in the anterior corneal stroma. Less staining was found in the posterior 2/3 of corneal stroma. Additionally, a greater concentration of soluble protein and albumin was found in the anterior stroma than in the posterior stroma of the bovine eyes by biochemical analyses. The theoretical model demonstrated that this distribution of protein required a difference in excluded volume fraction between the anterior and posterior stroma and was consistent with a convective flux originating at the limbus and passing through the corneal stroma. The soluble proteins of the bovine and human cornea are preferentially concentrated in the anterior cornea and near the limbus. This distribution is likely due to differences in excluded volume fraction between the anterior and posterior stroma and a small convective flux passing through the cornea.

Contrast-enhanced magnetic resonance imaging confirmation of an anterior protein pathway in normal rabbit eyes.

PURPOSE: Contrast-enhanced proton magnetic resonance imaging (1H MRI) has been used as a quantitative, noninvasive method to corroborate a pathway for the diffusion of plasma-derived protein into the aqueous humor in the normal rabbit eye. METHODS: T1-weighted magnetic resonance images were produced over 1- to 3-hour periods after the intravenous injection of gadolinium diethylenetriamine-pentaacetic acid. RESULTS: Analysis of the images yielded the time dependence of signal enhancements within the areas of interest. The ciliary body showed an immediate sharp increase, followed by a gradual decrease in signal enhancement with time. Although a gradual increase in signal enhancement was found in the anterior chamber, no significant change occurred in the posterior chamber. A similar MRI experiment with an owl monkey produced parallel, though smaller, signal enhancements in the ciliary body and anterior chamber. Again, however, no significant change was found in the posterior chamber. CONCLUSIONS: These results support and extend those of recent fluorophotometric, tracer-localization, and modeling studies demonstrating that in the normal rabbit and monkey eye, plasma-derived proteins bypass the posterior chamber, entering the anterior chamber directly via the iris root.

Freeze-fracture studies of interendothelial junctions in the angle of the human eye.

PURPOSE: The aim of this study was to determine the structure and complexity of the intercellular junctions between trabecular endothelial cells and Schlemm's canal endothelial (SCE) cells as they exist in the normal human eye. Despite the probable relevance of these junctions to aqueous outflow, examination of these junctions has been limited to monkey eyes. METHODS: Human eyes (< 24 hours after death) were fixed by immersion in modified Karnovsky's fluid. Radially oriented tissue-chopper sections (190 microns) were trimmed to contain only the trabecular meshwork, Schlemm's canal, and a narrow strip of the sclera. Specimens were processed for conventional electron microscopy and freeze-fracture. Replicas were produced in a freeze-fracture apparatus operated at -115 degrees C and 10(-7) torr. Thin sections were stained with uranyl acetate and lead citrate. Micrographs were taken on a transmission electron microscope. RESULTS: The trabecular endothelial cells were joined by gap junctions and short discontinuous junctional strands that partitioned predominantly with P-face. The strand pattern varied from short and isolated undulating strands to radiating arrays of short junctional strands. No continuous zonulae occludentes were observed. The SCE cells were joined by continuous tight junctions (tj) composed of discontinuous strands that rarely branched or anastomosed. The tj strands fractured preferentially with the E-face, where they were positioned at the bases of shallow valleys. On the P-face, a complementary system of shallow ridges was observed with few particles at their crests. Often, only a single continuous strand was present for long distances, with occasional short discontinuous strands running parallel to the single strand. Less often, short lengths of remarkably complex junctions, which occasionally branched or anastomosed, were also encountered. When more than one strand was present, numerous free endings and transjunctional pathways, described in monkey eyes as "slit-pores," were evident. CONCLUSION: The overall structure of the junctions between endothelial cells of Schlemm's canal in the human eye was more complex, and thus less permeable, than that reported in the monkey eye. The role of slit-pores and other junctions of SCE and trabecular endothelial cells will require further investigation under conditions of flow and in glaucoma to determine if these junctions change in a manner that might influence outflow resistance.

Hyaluronan in the bovine ocular anterior segment, with emphasis on the outflow pathways.

PURPOSE: It has been postulated that glycosaminoglycans play a role in the regulation of outflow resistance. The purpose of these studies was to localize the distribution of hyaluronan (HA) in the anterior segments of bovine eyes to understand better the possible role of HA in the outflow pathway. METHODS: Eyes from four 2-week-old calves and four 1-year-old cows were examined using a biotinylated-hyaluronan binding protein to localize HA in tissue sections of the anterior segment of bovine eyes. Various fixations and microwave irradiation were compared. The vitreous body in each section served as a positive control. Sections treated with Streptomyces hyaluronidase were used to confirm specificity. RESULTS: No significant difference in distribution of HA was found between various fixations and between calves and cows. HA was found in subconjunctival connective tissue and in intercellular spaces of limbal and conjunctival epithelium, but not in corneal epithelium. Staining was sometimes found on the surface of the corneal epithelium and endothelium, as well as on the conjunctival epithelium. No staining was found within the corneal stroma. There was HA in iris stroma, but not in the root of the iris or ciliary body stroma. HA was present in the anterior, nonfiltering part of the trabecular meshwork (TM) and surrounding collector channels and blood vessels in the sclera; to a lesser extent, it was present in the juxtacanalicular region of the TM. HA was detectable neither within trabecular beams nor filling the intertrabecular spaces. Strong staining was found, however, in the nerve bundles in the angle region. Staining for HA in vitreous was invariably positive, and in Streptomyces hyaluronidase-treated sections it was negative. CONCLUSION: HA was not uniformly distributed in the bovine TM. The distribution of HA in the flow pathway of the aqueous outflow system indicates that only a small fraction of the HA found in biochemical analyses of the bovine meshwork is located in the region where the flow resistance is thought to reside.

Education for entry level competency: teaching strategies.

The goal of this discussion is to summarize the results of the Workshop on Teaching Strategies that was held as part of the Curriculum Conference at Denver, Colorado in July, 1992. Various teaching methods were presented as alternatives to traditional lectures and laboratories; among these problem-based learning (PBL) was the subject of greatest interest. Advantages and disadvantages encountered using PBL in optometric education were presented. The need for a more uniform core curriculum among optometric institutions is emphasized, as well as the need for appropriately trained faculty to teach in the newer curricular areas.

Serum proteins and aqueous outflow resistance in bovine eyes.

PURPOSE: Recent evidence shows that much of the protein in the anterior chamber aqueous humor enters diffusively through the root of the iris. The proximity of the protein entry point to the trabecular meshwork suggests that the protein content of the aqueous humor in the trabecular meshwork might be much higher than previously suggested. The authors were interested in investigating the possible hydrodynamic implications of these proteins. METHODS: Bovine eyes were perfused with concentrations of bovine serum in buffer ranging from 0% to 15% to determine the effect on outflow resistance. Immunohistochemical methods were used on these eyes and unperfused eyes to determine the distribution of albumin in the anterior segment. RESULTS: Preliminary perfusion studies suggested that increasing the concentration of serum in buffer from 0% to 15% decreased the rate of "wash-out" in bovine eyes, with a 15% solution essentially eliminating the wash-out phenomenon. Perfusion of a series of bovine eyes with a total of 5 ml of 15% serum in buffer showed a "wash-out" rate of 0.0498 +/- 0.0428 ([microliters/min/mm Hg]/[ml perfusate]), whereas control eyes perfused with buffer washed-out at a rate of 0.1677 +/- 0.0271 (P < 0.05); a second series of eyes perfused with a total of 10 ml of 15% serum washed-out at a rate of 0.0533 +/- 0.0294, whereas control eyes had a rate of 0.1813 +/- 0.0342 (P < 0.02). Immunohistochemical investigations showed significant quantities of albumin in the outflow pathway of unperfused eyes, whereas perfusion with buffer eliminated this protein; perfusion with 10% to 15% serum in buffer maintained the level of albumin in the outflow pathway similar to that found in unperfused eyes. Use of cuprolinic blue in a critical electrolyte concentration confirmed previous findings that sulfated proteoglycans are not eliminated from the trabecular meshwork during wash-out. CONCLUSIONS: Wash-out in nonhuman species may result from progressive depletion of an anterior segment depot of plasma-derived proteins entering the trabecular meshwork. Modeling studies confirm that plasma-derived proteins in the aqueous humor of the trabecular meshwork can generate a significant fraction of aqueous outflow resistance. The lack of wash-out in human eyes suggests that this system may maintain flow resistance in a fashion fundamentally different from other species.

The Glenn A. Fry Award Lecture 1992: aqueous humor proteins: a key for unlocking glaucoma?

In this report I summarize recently reported studies which establish the existence of a previously undocumented diffusional pathway for plasma-derived proteins from the ciliary body stroma to the anterior chamber of the normal mammalian eye via the iris root. The data support the hypothesis that nearly all of the plasma-derived protein present in the aqueous humor of the anterior chamber arrives via this pathway, thus by-passing the posterior chamber. Additional studies suggest that some of the protein transported via the pathway is shunted directly into the trabecular meshwork and aqueous outflow pathways. Perfusion studies show how the perfused eyes were later immunohistochemically stained to reveal the distribution and qualitative amounts of serum albumin. These studies strongly suggest that the well-known "wash-out" effect, encountered in experimental studies of aqueous outflow, arises predominantly from wash-out of the protein depot that serves as the anterior diffusional pathway for proteins. Taken as a whole, these studies support the hypothesis that protein is a physiologically relevant and important element in the generation of normal aqueous outflow resistance, possibly more important than glycosaminoglycans (GAG's).