Validation of a More Reliable Method of Eye Drop Self-Administration.

SIGNIFICANCE: We propose an alternative method for eye drop self-administration. Similar IOP reductions were found with this method compared with clinician instillation. The alternative method of self-administration potentially benefits patients who have trouble successfully instilling drops. PURPOSE: The purpose of this study was to validate the efficacy of an alternative method of drop instillation. METHODS: This study is a randomized controlled crossover clinical trial. Thirty participants were recruited. A drop of 0.5% timolol maleate was instilled into subject's eye on two separate visits. On one visit, eye drop instillation was by a trained clinician, and on the other, self-instillation using an alternative method was used. The order was randomly chosen. Intraocular pressure was measured before drop instillation and 2 hours after drop instillation. The investigator was masked during measurement, and an observer recorded the IOP measurements. RESULTS: Mean ± SD IOP measurement before 0.5% timolol maleate instillation measured 13.89 ± 2.29 mmHg. An average reduction 3.75 ± 2.36 mmHg was found with clinician administration, and an average reduction of 3.32 ± 2.31 mmHg was recorded with the new method. No significance was found in IOP reduction between two groups P < .45. Percent reduction was 25.17 ± 16.21% and 24.38 ± 16.31% in clinician instillation and alternative instillation method group, respectively. No significant difference was found. This percentage reduction was similar to previously reported studies. No reported cases of eye infection or irritation were found in any case, within a 3-month follow-up period. CONCLUSIONS: We have proposed a more reliable method for instillation that provides a larger area for instillation and lessen the risk of contamination and patient's fear for eye drops. Similar efficacy was found compared with that of having a clinician directly administer the drop. This alternative method could potentially benefit patients who require topical eye drop therapy and result in increased compliance.

Consensus recommendations for trabecular meshwork cell isolation, characterization and culture.

Cultured trabecular meshwork (TM) cells are a valuable model system to study the cellular mechanisms involved in the regulation of conventional outflow resistance and thus intraocular pressure; and their dysfunction resulting in ocular hypertension. In this review, we describe the standard procedures used for the isolation of TM cells from several animal species including humans, and the methods used to validate their identity. Having a set of standard practices for TM cells will increase the scientific rigor when used as a model, and enable other researchers to replicate and build upon previous findings.

Pilocarpine-induced flare is physiological rather than pathological.

An elevated aqueous humor protein level (aka flare) has always been considered to represent a pathological breakdown of the blood-aqueous barrier (BAB), regardless of the etiology. Recent studies in humans, using magnetic resonance imaging (MRI) to directly observe BAB kinetics in the posterior chamber of the human eye in-vivo, showed that pilocarpine-induced flare resulting from administration of a single drop of pilocarpine is not the result of breakdown of the BAB in the ciliary body. These MRI studies could not confirm whether pilocarpine caused an increase in iris vascular permeability. In the current studies we completed combined cell-flare meter and intravascular tracer studies, using intravenous horseradish peroxidase (HRP) in rabbits. One hour after receiving 3% pilocarpine in one eye, pupil size significantly decreased and aqueous flare significantly increased in pilocarpine-treated eyes. Light and electron microscopy demonstrated no leakage across either the iris vascular endothelium or the non-pigmented ciliary epithelium in either pilocarpine-treated or control eyes. One animal received HRP directly after pilocarpine to control for a transient increase in permeability before the peak flare response occurred. No leakage was found in the ciliary body or iris of this animal. Additional animals received topical pilocarpine in one eye but after 1 h they were sacrificed without tracer studies. Uveal tissues from these animals were used to assess the distribution of non-HRP protein in the ocular anterior segment and to assess the amount of elutable protein in the iris stromas of both treated and untreated eyes. Immunohistochemistry confirmed the presence of a reservoir of protein in the iris stroma. Analysis of elutable total protein from the iris stroma of pilocarpine-treated and control eyes showed significantly less total elutable protein in pilocarpine-treated eyes. Eyes with the greatest percent change in pupil size (i.e. the strongest miosis) correlated with lowest amounts of residual protein in the iris stroma. The tracer studies confirmed recent MRI studies in humans showing that the source of pilocarpine-induced flare is not disruption of the ciliary epithelial barrier. Extending this work, the current studies also showed no pilocarpine-induced leakage from the iris vasculature. The elutable protein experiments suggested that a primary source of pilocarpine-induced flare was extrusion of a portion of the reservoir of protein in the iris stroma. Taken together, these studies strongly suggest that not all clinically observable flare results from breakdown of the BAB.

Similar hydrodynamic and morphological changes in the aqueous humor outflow pathway after washout and Y27632 treatment in monkey eyes.

Our previous studies in bovine eyes demonstrated that the structural correlate to the increase in outflow facility after either Rho-kinase inhibitor Y-27632 (Y27) treatment or washout appeared to be separation between the juxtacanalicular tissue (JCT) and inner wall (IW) of the aqueous plexus, the bovine equivalent of Schlemm's canal (SC). While these findings suggest that Y27 and washout may increase outflow facility through a similar mechanism, the anatomy of bovine outflow pathway differs considerably from both the human and monkey outflow pathway; however, only the human eye does not exhibit washout. In light of this, we compared the effects of Y27 and washout on outflow facility, hydrodynamic patterns of outflow, and the morphology of the IW and JCT in monkey eyes, given that their anatomy is closer to human eyes. Twelve freshly enucleated monkey eyes were used in this study. Eyes were perfused with Dulbecco's PBS containing 5.5 mM glucose (GPBS) to establish a baseline facility at 15 mmHg. Four eyes were perfused for a short-duration (30 min) as a control, 4 eyes for a long-duration (180 min) to induce washout, and 4 eyes with GPBS+50 µM Y27 for 30 min. All eyes were then perfused with fluorescent microspheres (0.5 µm; 0.002%) to label the hydrodynamic patterns of outflow and then perfusion-fixed. Confocal images of frontal sections were taken along the IW of SC. The total length (TL) and the tracer-decorated length (FL) of the IW were measured to calculate the average percent effective filtration length (PEFL = FL/TL). Sections with SC were examined by light and electron microscopy. The TL of the IW and the length exhibiting separation (SL) in the JCT were measured to calculate the average percent separation length (PSL = SL/TL). Outflow facility increased 149.2% (p < 0.01) from baseline after washout during long-duration perfusion, and 114.9% (p = 0.004) after Y27 treatment, but did not change significantly after short-duration perfusion in control eyes (p = 0.46). Distribution of the tracer labeling appeared punctate along the IW of control eyes, while a more uniform pattern was observed after washout and Y27 treatment. PEFL in washout (83.4 ± 2.1%) and Y27 treated eyes (82.5 ± 1.6%) was 3.4-fold larger compared to controls (24.2 ± 4.2%, P < 0.001). The JCT appeared distended with loss of connections between JCT cells and between JCT cells and their extracelluar matrix in eyes with washout or after Y-27 treatment. PSL in the JCT was 2.3-fold larger in washout eyes (77.4 ± 3.3%) and 2.2-fold larger in Y27 treated eyes (75.2 ± 5.3%) versus controls (33.5 ± 5.3%, p = 0.001). Significant positive correlations were found between outflow facility and PEFL, facility and PSL and between PEFL and PSL. Our data demonstrated that similar hydrodynamic and morphological changes occurred in the aqueous humor outflow pathway of monkey eyes after induction of washout and Y27 treatment. Both Y27 and washout increase outflow facility by redistributing aqueous outflow through a larger area in the JCT. These hydrodynamic changes are likely driven by morphologic changes associated with a decrease in cell-cell and cell-matrix connections in the JCT.

A review of acquired blepharoptosis: prevalence, diagnosis, and current treatment options.

Blepharoptosis (ptosis) is among the most common disorders of the upper eyelid encountered in both optometric and ophthalmic practice. The unilateral or bilateral drooping of the upper eyelid that characterises ptosis can affect appearance and impair visual function, both of which can negatively impact quality of life. While there are several known forms of congenital ptosis, acquired ptosis (appearing later in life, due to a variety of causes) is the predominant form of the condition. This review summarises the prevalence, causes, identification, differential diagnosis, and treatment of acquired ptosis. Particular attention is paid to the differential diagnosis of acquired ptosis and emerging treatment options, including surgical and pharmacologic approaches.

Loss of AND-34/BCAR3 expression in mice results in rupture of the adult lens.

PURPOSE: AND-34/BCAR3 (Breast Cancer Anti-Estrogen Resistance 3) associates with the focal adhesion adaptor protein, p130CAS/BCAR1. Expression of AND-34 regulates epithelial cell growth pattern, motility, and growth factor dependence. We sought to establish the effects of the loss of AND-34 expression in a mammalian organism. METHODS: AND-34(-/-) mice were generated by homologous recombination. Histopathology, in situ hybridization, and western blotting were performed on murine tissues. RESULTS: Western analyses confirmed total loss of expression in AND-34(-/-) splenic lymphocytes. Mice lacking AND-34 are fertile and have normal longevity. While AND-34 is widely expressed in wild type mice, histologic analysis of multiple organs in AND-34(-/-) mice is unremarkable and analyses of lymphocyte development show no overt changes. A small percentage of AND-34(-/-) mice show distinctive small white eye lesions resulting from the migration of ruptured cortical lens tissue into the anterior chamber. Following initial vacuolization and liquefaction of the lens cortex first observed at postnatal day three, posterior lens rupture occurs in all AND-34(-/-) mice, beginning as early as three weeks and seen in all mice at three months. Western blot analysis and in situ hybridization confirmed the presence of AND-34 RNA and protein in lens epithelial cells, particularly at the lens equator. Prior data link AND-34 expression to the activation of Akt signaling. While Akt Ser 473 phosphorylation was readily detectable in AND-34(+/+) lens epithelial cells, it was markedly reduced in the AND-34(-/-) lens epithelium. Basal levels of p130Cas phosphorylation were higher in AND-34(+/+) than in AND-34(-/-) lens epithelium. CONCLUSIONS: These results demonstrate the loss of AND-34 dysregulates focal adhesion complex signaling in lens epithelial cells and suggest that AND-34-mediated signaling is required for maintenance of the structural integrity of the adult ocular lens.

Demonstration of an anterior diffusional pathway for solutes in the normal human eye with high spatial resolution contrast-enhanced dynamic MR imaging.

PURPOSE: The present studies were conducted to determine whether a diffusional pathway for solutes exists from the ciliary body stroma to the anterior chamber of the human eye. The existence of such a pathway has been demonstrated in rabbits and monkeys, but such a pathway in humans would necessitate a shift in the physiological paradigm of the blood-aqueous barrier. METHODS: Seven normal human volunteers (five men, two women; age range, 27 to 59 years) underwent nine dynamic T1-weighted, spin-echo MR imaging studies, using intravenous, gadolinium-based contrast agents. RESULTS: In all cases, signal intensity rose rapidly in the ciliary body. In all subjects, there was a measurable latent rise in signal strength (enhancement) in the anterior chamber. Signal enhancement typically occurred in the angle of the anterior chamber earlier, and to a greater degree, than within the center of the chamber. Increased signal within the posterior chamber was significantly less than in the anterior chamber, with measured increases probably attributable to volume averaging. CONCLUSIONS: These findings are consistent with the existence of an anterior diffusional pathway in the human eye. The model warrants further testing.

The mechanism of increasing outflow facility during washout in the bovine eye.

PURPOSE: To investigate the relationship between outflow facility and separation between the inner wall of the aqueous plexus and the juxtacanalicular connective tissue (JCT) during washout in the bovine eye. METHODS: Facility was recorded during 3 hours of anterior chamber perfusion at 15 mm Hg in eight pairs of bovine eyes. One eye of each pair was then lowered to 0 mm Hg for 1 hour, whereas the fellow eye was kept at 15 mm Hg. After a brief perfusion at 15 mm Hg, both eyes were perfusion fixed and processed for electron microscopy. Micrographs of the inner wall were analyzed for separation from the JCT. To study the role of cellular adhesion between the inner wall and JCT, 12 additional pairs were perfused with integrin-binding peptide (RGD: Arg-Gly-Asp) or sham control peptide (RGE: Arg-Gly-Glu) at 2 micro M to 2 mM, before IOP was reduced. RESULTS: During the first 3 hours, facility increased in both eyes because of "washout." However, after 1 hour of 0 mm Hg, facility decreased by 13% (P < 0.006), whereas facility increased by 20% (P < 0.001) in the fellow eyes maintained at 15 mm Hg. Two types of separation were observed between the inner wall and JCT: cell-matrix separation between the endothelial cell and basal lamina and matrix-matrix separation between the basal lamina and JCT. A significant positive correlation (P = 0.042) was found between the degree of matrix-matrix separation and the change in outflow facility after 1 hour of 0 mm Hg. Compared with RGE control, RGD had no apparent effect on outflow facility (P > 0.35) or on the change in outflow facility after 1 hour at 0 mm Hg (P > 0.15). CONCLUSIONS: The increase in outflow facility that occurs during washout in the bovine eye is reversible and correlates with the degree of separation between the basal lamina of the inner wall endothelium and the JCT. Therefore, adhesions tethering the inner wall to the JCT may be important ultrastructural features involved in the regulation of aqueous humor outflow resistance.

A contemporary concept of the blood-aqueous barrier.

This review traces the evolution of the concept of the blood-aqueous barrier (BAB) during the past 20 years. The Classical model simply stipulated that the tight junctions of the iris vasculature and ciliary epithelium excluded plasma proteins from the aqueous humor (AH). It failed to reconcile the presence of AH protein levels equal to 1% of that found in plasma. Moreover, models of barrier kinetics assumed that the processes of AH secretion and plasma protein entry were directly linked. Thus, elevations of AH protein levels could only be explained by a pathological breakdown of the BAB. Over the last 20 years it has been shown that the plasma proteins in normal AH by-pass the posterior chamber entirely. Instead, these proteins diffuse from the capillaries of ciliary body stroma, into the iris stroma and then into the anterior chamber. This creates a reservoir of plasma-proteins in the iris stroma that is not derived from the iris vessels. This reservoir is prevented from diffusing posteriorly by tight junctions in the posterior iris epithelium. The one-way valve created by the pupil resting on the anterior lens capsule, combined with the continuous, forward flow of AH through the pupil, prevents protein reflux into the posterior chamber. Importantly, in the new paradigm, secretion of AH and the entry of plasma proteins into AH, are semi-independent events. This opens the possibility that AH protein levels could increase in the absence of breakdown of the BAB. Clinical consequences of this new paradigm of the BAB are discussed.

The mechanism of increasing outflow facility by rho-kinase inhibition with Y-27632 in bovine eyes.

Rho-kinase inhibitor Y-27632 (Y-27) affects actomyosin cytoskeletal networks and has been shown to significantly increase outflow facility (C) in enucleated porcine and rabbit eyes, as well as in vivo monkey eyes without obvious toxicity. The mechanisms underlying these responses remain largely unknown. In this study, we investigate how Y-27 affects aqueous humor C, the hydrodynamic patterns of outflow, and the morphology of the inner wall (IW) and juxtacanalicular connective tissue (JCT). 12 bovine eyes were perfused at 15 mmHg with Dulbecco's PBS containing 5.5 mM glucose (DPBS) to establish stable baseline C. The anterior chamber was exchanged and perfused with DPBS containing 50 microM Y-27 in 7 eyes, while 5 eyes received DPBS alone. Eyes were then perfused with DPBS containing fluorescent microspheres (0.5 microm; 0.002% v/v) at a fixed volume to deliver equivalent amounts of tracer to label the hydrodynamic filtration patterns. All eyes were perfusion-fixed with Karnovsky's fixative. Radial and frontal sections were prepared in all quadrants and confocal images were taken along the IW of the aqueous plexus (AP). The total length (TL) and filtration length (FL) of the IW were measured in > or =16 images/eye, and the average percent effective filtration length (PEFL=FL/TL) was calculated. Sections with AP were processed and examined by light and electron microscopy. The TL of the IW and length exhibiting JCT/IW separation (SL) were measured in > or =16 micrographs/eye, and the average percent separation length (PSL=SL/TL) was also calculated. After Y-27 treatment, C increased from 1.54+/-0.34 (+/-SEM) to 2.36+/-0.54 microL/min per mmHg (58.2+/-18.9%) while control eyes changed from 1.67+/-0.41 to 1.71+/-0.39 microl/min per mmHg (6.0+/-9.3%) and the percent changes between the Y-27-treated and control eyes were significant (p=0.03). Control eyes showed segmental distribution of tracer in the trabecular meshwork tending to cluster near collector channel ostia, whereas Y-27-treated eyes showed a more uniform pattern and extensive labeling along the IW. In Y-27-treated eyes, PEFL was 3-fold larger (57.6+/-3.7%) compared to control eyes (18.2+/-4.5%; p<0.001). Light microscopic examination revealed that, with Y-27, the IW and JCT were significantly distended compared to control eyes, with discernible separation between the IW and JCT. PSL was 2.8-fold larger in Y-27-treated eyes (59.3+/-3.6%) than in controls (20.8+/-2.0%; p<0.001). A significant positive correlation was found between PEFL and PSL (p=0.003) suggesting that as connectivity between the JCT and IW decreases the available area for aqueous humor drainage increases along the AP. Our study also demonstrates a significant positive correlation between C and the PSL (p=0.01), a finding identical to what we reported to occur during the "washout" effect in bovine eyes. Our data suggests the structural correlate to the increase in C and PEFL after Y-27-treatment is physical separation between the JCT and IW. The increase in C after Y-27-treatment may share a similar mechanism comparable with the washout effect that occurs in bovine eyes. Overall, these findings support our hypothesis that JCT/IW connectivity influences local outflow hydrodynamics that regulate C, and suggest that strategies targeting JCT/IW connectivity are promising anti-glaucoma therapies to reduce IOP.

T(1) and T(2) measurements of the fine structures of the in vivo and enucleated human eye.

PURPOSE: To measure T(1) and T(2) of the fine structures of the in vivo eye. MATERIALS AND METHODS: Involuntary saccades make it difficult to obtain artifact-free images. Using a method recently reported (Bert et al, Acad Radiol 2006;12:368-378), near artifact-free spin-echo images were obtained. Both an isolated enucleated eye and eight human subjects were studied at 1.5 T. Spin-echo variable TR/TE data was acquired for T(1)/T(2) determination. Average relaxation times were calculated two ways. First, an arithmetic average over different subjects was computed. Second, all data was normalized using the fitted amplitudes of each data set and pooled to obtain a single least squares fit. RESULTS: In vivo T(1)/T(2) (msec) are: arithmetic average T(1), T(2), normalized data T(1), T(2). Anterior chamber: 6233 +/- 979, 468 +/- 149, 5053 +/- 1052, 450 +/- 49. Ciliary body: 1916 +/- 184, 80 +/- 7, 2038 +/- 114, 76 +/- 3. Chorioretina: 1717 +/- 500, 72 +/- 25, 1511 +/- 230, 78 +/- 3. Extraocular muscle: 1581 +/- 646, 41 +/- 7, 1470 +/- 231, 41 +/- 1. Iris: 3334 +/- 989, 163 +/- 63, 3376 +/- 338, 153 +/- 10. Lens cortex: 1712 +/- 466, 93 +/- 36, 1413 +/- 177, 100 +/- 5. Lens nucleus: 1133 +/- 40, 26 +/- 3, 1138 +/- 47, 25 +/- 0.4. Optic nerve: 1906 +/- 301, 68 +/- 16, 1805 +/- 244, 71 +/- 2. Posterior chamber: 7915 +/- 4897, 241 +/- 14, 3323 +/- 2154, 251 +/- 38. Vitreous humor: 5768 +/- 1190, 756 +/- 804, 4855 +/- 1846, 390 +/- 8. CONCLUSION: In vivo T(1) and T(2) for many of the fine structures of the human eye have been measured.

Comparative studies between species that do and do not exhibit the washout effect.

Ocular perfusion studies from all non-human species performed to date consistently demonstrate a perfusion-volume-dependent increase in aqueous outflow facility known as the "washout" effect. However, this "washout" effect does not occur in human eyes. We have recently documented that, in bovine eyes, the washout associated increase in facility correlates with the extent of physical separation between the juxtacanalicular connective tissue (JCT) and the inner wall endothelium lining the aqueous plexus (the bovine equivalent of Schlemm's canal). We hypothesize that if washout truly correlates with inner wall/JCT separation then this separation should not occur in human eyes that do not exhibit the washout effect, even after prolonged perfusion. Eight enucleated human and eight bovine eyes were used in this study. Aqueous humor outflow facility was measured at 15 mmHg for long-duration (3 h) or short-duration (30 min to 1 h) perfusion (n=4 for each group). All eyes were perfusion-fixed at 15 mmHg, and examined morphologically with both light and electron microscopy. In bovine eyes, outflow facility increased 81% (p=0.049) from 1.06 +/- 0.06 microl/min per mmHg (mean+/-SEM) at baseline to 1.92 +/- 0.30 microl/min per mmHg after 3 h due to washout. The pre-fixation outflow facility in long-duration eyes (1.92 +/- 0.30 microl/min per mmHg) was 2-fold greater than pre-fixation facility in short-duration eyes (0.92 +/- 0.11 microl/min per mmHg; p=0.0387). In human eyes, washout was not observed; baseline outflow facility was similar between both groups (0.18 +/- 0.02 vs. 0.25 +/- 0.08 microl/min per mmHg; p=0.518); however, pre-fixation outflow facility in long-duration eyes showed a 40% decrease compared to baseline outflow facility in those same eyes (p=0.017, paired Student's t-test). In bovine eyes, significant expansion and rarefaction of the JCT and inner wall/JCT separation was much more prevalent in long-duration eyes, and data from all bovine eyes revealed a correlation between the extent of inner wall/JCT separation and the absolute value of outflow facility measured immediately prior to fixation (p=0.0024) as well as the washout-induced increase in outflow facility (p=0.0006). In human eyes, no significant morphologic differences were observed between long- and short-duration perfusion, with no observed change in inner wall/JCT separation or expansion between the two groups. Morphologic analysis revealed that the previously described "cribriform plexus" of elastic-like fibers was far more extensive in the JCT of human eyes, appearing to form numerous connections to the inner wall endothelium. The cribriform plexus appears to function as a mechanical tether that maintains inner wall/JCT connectivity in human eyes by opposing hydrodynamic forces generated during perfusion, potentially explaining the lack of washout in humans.

Pilocarpine's effects on the blood-aqueous barrier of the human eye as assessed by high-resolution, contrast magnetic resonance imaging.

The purpose of this research was to reassess the effects of topical pilocarpine on the integrity of the blood-aqueous barrier, using high resolution, magnetic resonance imaging, and the standard intravenous contrast agent gadolinium dimeglumine. It has long been known that topical pilocarpine gives rise to an increase in protein levels in the anterior chamber of the eye. This protein scatters light and is referred to clinically as 'flare'. Prior studies concluded that pilocarpine-induced flare resulted from disruption of the blood-aqueous barrier. These studies relied upon indirect methods that precluded direct visualization of the posterior chamber of the eye. Five normal, human volunteers (age 22-40) received a single drop of 3% pilocarpine in one eye, following baseline measurements of pupil size and anterior chamber 'flare'. These measurements were repeated every 15 min for 45 min. The subject was then positioned in the magnet and the eye that received pilocarpine was taped closed and covered with a 3 in.-diameter receive-only surface coil. The open contralateral eye focused on a target to maintain fixation of the imaged eye. A baseline image of the eye was obtained and the contrast agent was administered intravenously. A series of additional images was obtained during the following 60 min to track the movement of contrast material from the bloodstream into the tissues and compartments of the eye. Percent enhancement was calculated from selected regions of interest in the images, including the ciliary body, and the anterior and posterior chambers. Within the 45 min after administration of pilocarpine, pupil size in mm decreased from (mean+/-s.d.) 5.7+/-1.5 to 2.5+/-0.5 (p=0.0106). During this period, average flare/s.d. (photons msec-1) increased from 3.7+/-1.1 to 12.5+/-4.7 (p=0.0151). In all cases, MRI images showed rapid enhancement of the ciliary body, followed by a progressive increase in signal in the anterior chamber but not the posterior chamber. These studies confirm that topical pilocarpine gives rise to 'flare' in the anterior chamber. But the lack of enhancement in the posterior chamber strongly suggests that the presence of this added protein in the anterior chamber is not the result of increased permeability of the blood-aqueous barrier of the ciliary body. These studies also introduce the novel concept that not all clinically observed flare is the result of blood-aqueous barrier compromise.

Co-localization of junction-associated proteins of the human blood--aqueous barrier: occludin, ZO-1 and F-actin.

Recent studies in mouse and rabbit eyes have begun to identify the molecular constituents of tight and adherens junctions that represent the structural equivalent of the blood--aqueous barrier (BAB). These species are commonly used as experimental models to examine the pathobiology of anterior uveitis, an inflammatory condition in which the junctions of the BAB are compromised. Because it was unclear whether major molecular elements of the junctions in these species were the same as those in humans, the goal of this study was to determine if the junction related proteins ZO-1 and occludin are present in normal human ciliary epithelium and iridial vascular endothelium. To determine their presence, sections of human anterior uvea were probed in 14 normal, human, eyebank eyes immunolabelled with antibodies to ZO-1, and occludin, and confocal microscopy was used to examine them. Phalloidin staining for F-actin was also assessed. ZO-1 and occludin were both localized along the apico-lateral surfaces of the non-pigmented ciliary epithelium and the interendothelial clefts of iris blood vessels. In both locations, the distribution of occludin was more focal than seen for ZO-1. ZO-1 was also found along the apical surfaces between the pigmented and non-pigmented ciliary epithelial cell layers. The distribution of these proteins supports the notion that occludin is more specifically associated with tight junctions than is ZO-1 in the normal human BAB. No change in this distribution was found with increasing age. These data are consistent with findings reported previously in rabbit ciliary epithelium and iridial vascular endothelium, indicating the relevance of experimental induced uveitis studies in rabbit, as a model of BAB breakdown in human uveitis.

A new view of the human trabecular meshwork using quick-freeze, deep-etch electron microscopy.

Conventional transmission electron microscopic (TEM) images of the juxtacanalicular tissue (JCT) region of the trabecular meshwork (TM) show flow passages that are much too large to generate significant outflow resistance. The goal of the current study was to use quick-freeze/deep-etch (QF/DE), a technique that better preserves extracellular matrices, to determine if extracellular matrix material fills all the apparently 'open-spaces' that were observed using conventional TEM. Normal adult human eyes were fixed by immersion or under flow at pressures of 15 or 45 mmHg. The TM and the inner wall of Schlemm's canal (SC) were examined using QF/DE and compared to the observations using conventional TEM. The structure of the TM, as seen using QF/DE, showed much greater three dimensional ultrastructural detail than was seen with conventional TEM. Open space was confirmed to be present between the trabecular beams. Although significantly more extracellular matrix was observed in the JCT region using QF/DE than by conventional TEM, some micron-sized open-spaces were still present immediately beneath the inner wall of SC. Consistent with prior reports, the basement membrane of the cells lining the inner wall of SC exhibited discontinuities but the basement membrane as seen by QF/DE was much more elaborate and complex than was evident in conventional TEM of immersion-fixed eyes. This basal lamina became less continuous with increasing perfusion pressure.QF/DE, although difficult and very labor-intensive when used to examine the TM, offers several clear advantages over conventional methods of tissue preparation for ultrastructural study. Although a more complex and less open extracellular matrix structure was seen in the JCT using QF/DE compared with conventional TEM, some open-spaces, similar in size to those seen by TEM, were still observed in this region. The continued presence of such open-spaces in QF/DE images suggests that either the JCT may not generate a significant fraction of outflow resistance in normal eyes or even QF/DE is not sensitive enough to preserve and identify all the extracellular matrix in the JCT region of the TM.SC appears to exhibit a discontinuous basal lamina like lymphatic channels but, like venules, exhibits a wide lumen and continuous endothelium. Because of these features, and the presence of continuous tight junctions that typify continuous capillaries, but neither lymphatic channels nor venules, SC has traditionally been described as a vessel sui generis. Our observations of perfusion-induced changes in the basal lamina of the inner wall of SC suggest that the discontinuous basal lamina underlying the inner wall of SC may not represent the normal expression of the vessel but may simply be a consequence of the way in which giant vacuoles and their pores give rise to outflow.