Telocytes and stem cells in limbus and uvea of mouse eye.

The potential of stem cell (SC) therapies for eye diseases is well-recognized. However, the results remain only encouraging as little is known about the mechanisms responsible for eye renewal, regeneration and/or repair. Therefore, it is critical to gain knowledge about the specific tissue environment (niches) where the stem/progenitor cells reside in eye. A new type of interstitial cell-telocyte (TC) (www.telocytes.com) was recently identified by electron microscopy (EM). TCs have very long (tens of micrometres) and thin (below 200 nm) prolongations named telopodes (Tp) that form heterocellular networks in which SCs are embedded. We found TCs by EM and electron tomography in sclera, limbus and uvea of the mouse eye. Furthermore, EM showed that SCs were present in the anterior layer of the iris and limbus. Adhaerens and gap junctions were found to connect TCs within a network in uvea and sclera. Nanocontacts (electron-dense structures) were observed between TCs and other cells: SCs, melanocytes, nerve endings and macrophages. These intercellular 'feet' bridged the intercellular clefts (about 10 nm wide). Moreover, exosomes (extracellular vesicles with a diameter up to 100 nm) were delivered by TCs to other cells of the iris stroma. The ultrastructural nanocontacts of TCs with SCs and the TCs paracrine influence via exosomes in the epithelial and stromal SC niches suggest an important participation of TCs in eye regeneration.

Dysfunctional uveoscleral pathway in a rat model of congenital glaucoma.

UNLABELLED: The purpose of the study was to investigate the presence of the uveoscleral pathway in the normotensive rat (NR) and in a rat model of congenital glaucoma (CGR). We injected the fluorescent tracer 70-kDa dextran rhodamine B in the anterior chamber of four NRs and four CGRs. At 10 and 60 minutes after injection, rats were euthanized by CO2 inhalation and eyes were enucleated. Cryosections were prepared and analyzed using fluorescent microscopy. Hematoxylin-eosin staining and electron microscopy of the anterior chamber angle (ACA) were performed. At 10 minutes after injection, fluorescent tracer was detected in the iris root and ciliary processes of NRs and CGRs. At 60 minutes, NRs showed prominent signal in the suprachoroidal, whereas, in the CGRs, tracer was barely detectable. Histology of the anterior chamber revealed the presence of an open ACA and electron microscopy confirmed the normal structure of the ciliary body in CGRs. CONCLUSIONS: Our results document the presence of an uveoscleral pathway in the normotensive rat. The rat model of congenital glaucoma shows severe impairment of the uveoscleral pathway, suggesting that alterations of the uveoscleral outflow might play a role in the pathogenesis of CG.

Histological findings of uveal capillaries in rabbit eyes after multiple intravitreal injections of bevacizumab.

PURPOSE: To evaluate the potential toxicity of multiple intravitreal injections of bevacizumab on the uveal capillaries of rabbit eyes. MATERIALS AND METHODS: Nine eyes of nine rabbits that received single intravitreal injections of bevacizumab (IVB) constituted the single IVB group, while nine eyes of nine rabbits that received three injections of IVB, with an interval of 28 days between injections, constituted the repeat IVB group. Seven eyes of seven rabbits constituted the control group. The rabbits in the single and repeat IVB groups were sacrificed 7 and 28 d after the single and third IVB injection, respectively. Uveal specimens were compared between groups after immunohistochemical staining. Ultrastructural findings were evaluated by electron microscopy. Control group rabbits were sacrificed 7 d after saline injection. Clinical examination and fundus fluorescein angiography were performed at baseline, 7 d after the first injection, and after the last injection. RESULTS: Differences in the CD31-positive areas of the iris, ciliary body and choroid 7 d after IVB were not statistically significant among the single IVB, repeat IVB and control groups (p = 0.0749, p = 0.7237 and p = 0.7346, respectively; analysis of variance). Endothelial cell fenestrations (ECFs) in the choriocapillaris and ciliary body observed by electron microscopy on day 7 in the single and repeat IVB groups were decreased by 50% (p < 0.0001) and 33% (p < 0.0001), respectively, in both IVB groups compared with those in the control group. However, ECFs observed on day 28 in both groups were comparable to those observed in the control group. CONCLUSIONS: Single IVB and repeated IVB did not have any effect on normal vessel endothelium density as per immunohistochemical findings. Ultrastructural findings revealed that IVB transiently decreased the ECFs in the choriocapillaris and ciliary body.

Morphology of the long and short uveal nerves in the human eye.

The aim of this study was to examine the architecture of the uveal nerves in the sclera and suprachoroid of human eyes. Eyes from 17 adult human donors were investigated. The uveal nerves in different regions (retrobulbar, intrascleral, suprachoroidal, pars plana) were prepared and studied by light and electron microscopy. In addition, immunohistochemistry was performed for various neuronal markers. The long uveal nerves showed a characteristic suprachoroidal location with no branches supplying the choroid. It was found that typically they are composed of myelinated (75%) and non-myelinated (25%) nerve fibres. They mainly contain aminergic and sensory nerve fibres. A separate set of cholinergic non-myelinated nerve fibre bundles runs parallel with these long uveal nerves. The short uveal nerves supply the suprachoroidal nerve plexus with approximately 13% of their nerve fibres. The nerves and the branches supplying the choroid appear as mixed nerves containing sympathetic, parasympathetic and sensory axons. This study therefore provides new information about the quantity, type and distribution of myelinated and non-myelinated nerve fibres in the posterior uvea of the human eye.

Amylin competes for binding sites of CGRP in the chamber angle and uvea of monkey, cat, and pig eye.

Calcitonin gene-related peptide (CGRP) binding sites have been identified previously in the eyes of monkey, cat, pig, and guinea pig. In this study, the ability of cat, human, and rat amylins to displace the binding of CGRP in the anterior part of the eye of monkey, cat, and pig was studied. The location and displacement of 125I-hCGRPalpha by amylins as concentrations of 1-1000 nM were studied in cryosections by autoradiography. In the monkey eye, cat and rat amylins were able to compete for the binding sites of CGRP in ciliary muscle and ciliary processes. In the cat eye, cat and human amylins clearly displaced CGRP binding from ciliary muscle, ciliary processes, iris, and chamber angle. Furthermore, rat amylin clearly displaced CGRP binding from ciliary muscle and ciliary processes. In the pig eye, cat, human, and rat amylins competed for the binding sites of CGRP in ciliary muscle, ciliary processes, iris, and limbal conjunctiva. Specific amylin receptors or the possible physiological role of amylin in the eye have not hitherto been reported. It seems, however, that amylin can bind to ocular CGRP receptors and thus probably plays a role in the regulation of the same functions as CGRP, (e.g., aqueous humor outflow).

Additional cell damage after transpupillary thermotherapy in choroidal malignant melanoma.

The aim of this study was to evaluate the effectiveness of transpupillary thermotherapy (TTT) in generating tumour necrosis by light and electron microscopy, as well as to evaluate additional cell damage in the area directly adherent to the necrotic zone. Four eyes of four patients diagnosed with intraocular malignant melanoma of the uvea were treated experimentally with diode laser TTT. In all cases a standard technique was used. All eyes were enucleated: one eye the day after TTT, two eyes 2 days after TTT, and one eye 6 weeks after TTT. Immediately after enucleation the eyes were immersed in standard Karnovsky's fixative with cocodylate buffer and prepared for light and electron microscopy. In the treated area of all four melanomas we found a dense band of necrotic tissue (zone A) consisting of an amorphous mass of dead cells sharply demarcated from the rest of the neoplastic tissue. Next to this zone was a more eosinophilic and also sharply demarcated band (zone B) that consisted of similar but less intensive changes. In the next band (zone C), marked injury to the cellular membrane and subcellular structures were seen on electron microscopy. The next band (zone D) consisted of changes mainly observed only within the cytoplasm of neoplastic cells and significantly less intensive than those in zone C. Outside zone D tumour cells that were normal in appearance were seen. No scleral alterations induced by heat were found. We concluded that after TTT the cytotoxic effect gradually decreases in proportion to the distance from the central point of the diode laser spot, with additional cell damage in the area adjacent to the necrotic zone. The interval between TTT and enucleation had no influence on the histological results.

How does nonpenetrating glaucoma surgery work? Aqueous outflow resistance and glaucoma surgery.

Histologic, experimental, and theoretical studies of the aqueous outflow pathways point toward the juxtacanalicular region and inner wall of Schlemm's canal as the likely site of aqueous outflow resistance in the normal eye. At least 50% of the aqueous outflow resistance in the normal eye and the bulk of the pathologically increased resistance in the glaucomatous eye resides in the trabecular meshwork and the inner wall of Schlemm's canal. The uveoscleral, or uveovortex, pathway, which accounts for perhaps 10% of the aqueous drainage in the healthy aged human eye, can become a major accessory route for aqueous drainage after pharmacologic treatment. Surgeries designed to incise or remove the abnormal trabecular meshwork of glaucoma address the pathologic problem of the disease. Surgeries that unroof Schlemm's canal or expand the canal, such as viscocanalostomy, probably cause inadvertent ruptures of the inner wall and juxtacanalicular tissue, thus relieving the abnormal outflow resistance of glaucoma. This review is a summary of current thought on the pathophysiology of aqueous outflow resistance in glaucoma and, in light of this, provides an interpretation of the mechanism of pressure reduction created by these new surgeries.

Interaction between inflammatory cells and heparin-surface-modified intraocular lens.

PURPOSE: To investigate the interaction and adherence of inflammatory cells to a heparin-surface-modified intraocular lens (HSM IOL). SETTING: Department of Ophthalmology, Tokyo Medical University Hospital, Tokyo, Japan. METHODS: Splenic mononuclear leukocytes from rats with experimental autoimmune uveitis were cultured with the optic of an HSM IOL for 96 hours. The number of adherent cells on the HSM IOL surface was measured with and without the addition of interphotoreceptor retinoid-binding protein and concanavalin A (ConA) to the culture medium. The adherent cells were observed under a light microscope or a scanning electron microscope. RESULTS: Interphotoreceptor retinoid-binding protein and ConA increased the number of adherent cells on the HSM IOL relative to the control. Adherent cells on the HSM IOL were small and round, considered to be mainly lymphocytes. CONCLUSION: Activated lymphocytes tended to adhere to the surface of the HSM IOL.

Mast cell heterogeneity in the human uvea.

To identify chymase- and tryptase-positive mast cells in the human uvea, and to study their associations with different types of resident uveal cells, uveal specimens from 24 human donor eyes were cryosectioned in sagittal and tangential planes. Enzyme histochemical staining of chymase was combined with immunohistochemical staining for tryptase, detected with the APAAP method. Fluorescence immunohistochemistry was performed with antibodies against c-kit, alpha smooth muscle actin, protein gene product (PGP) 9.5, CD45, and HLA-DR. In different uveal compartments, the total amounts of mast cells were calculated and the distributions of chymase and tryptase were quantified. All uveal mast cells were c-kit and CD45 positive and HLA-DR negative. No association existed between mast cells and actin-containing cells. Only a few mast cells were in close association with PGP 9.5-labeled nerve fibers. In the choroid, most mast cells were located in the inner central part (mean density = 48.9/mm(2)), and contained both chymase and tryptase (96%). The ciliary muscle contained numerous mast cells (mean density = 33.7/mm(2)), many of them tryptase positive but chymase negative (63%). In the pars plana, a high number of chymase-positive, tryptase-negative mast cells were found (20%). In the iris only a few mast cells were present. Although the choroid contains the most common subtype of mast cells, a unique situation concerning the distribution of chymase and tryptase is present in the anterior uveal tissues. A possible role for these cells in the special immunological situation of the anterior eye chamber merits further investigation.

Blood-retinal barrier (BRB) breakdown in experimental autoimmune uveoretinitis: comparison with vascular endothelial growth factor, tumor necrosis factor alpha, and interleukin-1beta-mediated breakdown.

Experimental autoimmune uveoretinitis (EAU) induced in Lewis rats by immunization with S-antigen is a model of human uveitis. By using immunocytochemical staining for albumin, relatively minor blood-retinal barrier (BRB) breakdown was initially shown in the peripheral retina 8 days after immunization and in the posterior retina by 10 days. Albumin extravasation appeared to occur by opening of the retinal vascular endothelial (RVE) and the retinal pigmented epithelial (RPE) tight junctions, by transendothelial vesicular transport, and by permeating damaged RVE cells. Each of three anti-inflammatory agents reduced or delayed autoimmune-mediated cell destruction but did not eliminate any particular route of extravasation. Vascular endothelial growth factor (VEGF), tumor necrosis factor alpha (TNF alpha), and interleukin-1beta (IL-1beta) are intimately associated with the development of EAU and are capable of causing BRB dysfunction. A high percentage of RVE tight junctions appeared open ultrastructurally after intravitreal injection of VEGF (26.7%), TNF alpha (35.6%), or IL-1beta (22.1%) compared with saline-injected control (11.4%) or normal, untreated rabbits (4.1%). Heat treatment abolished the effect of IL-1beta on the BRB but only partially reduced the effect of VEGF. By 24 hr after injection, the effect of TNF alpha had reversed, but that of IL-1beta had not; VEGF-mediated BRB dysfunction was partially reversible. In addition, albumin-filled vesicle-like structures were seen in the RVE cytoplasm following treatment with each mediator. This study shows that VEGF, TNF alpha, and IL-1beta each cause BRB breakdown by opening tight junctions between RVE cells and possibly by increasing transendothelial vesicular transport. Each of these agents may contribute to BRB breakdown in EAU and in patients with uveitis.

Morphologic evidence for a preferential storage of tissue plasminogen activator (t-PA) in perivascular axons of the rat uvea.

The uveal layer is thought to hold the largest stores of tissue plasminogen activator (t-PA) within the eye. However, the uveal cell types that contain and could release t-PA to contiguous tissues and fluids have not been clearly identified. In the present study the general distribution pattern of t-PA antigen in fresh rat iris and choroid tissue was determined by immunofluorescence in preliminary light microscopic (LM) cryosections. Transmission electron microscopic (TEM) immunogold localization was then used to detect specific cellular and subcellular sites of t-PA antigen. The primary antibody was rabbit anti-mouse t-PA IgG. The immunofluorescence in preliminary LM cryosections of both tissues was most intense over discrete linear and cross-sectioned structures that resembled the contours of axon bundles. This impression was strengthened when silver impregnation highlighted similar structures in separate sections of the same tissue samples. TEM immunogold labeling of thin sections then confirmed that the t-PA antigen was confined to the axoplasm of both myelinated and unmyelinated perivascular nerve fibers in both the iris and choroid. Gold particles were not observed over axonal membranes, myelin sheaths, Schwann cells, retinal pigment epithelium or vascular endothelial cells. Ultrathin TEM cryosections of the iris showed a localization of some particles over structures that resembled tubules and vesicles within the axoplasm, but not over mitochondria. The axonal location of t-PA was shown by the co-localization of t-PA with an antibody against rat neurofilaments. The typical axon morphology that enclosed the t-PA particle markers in all TEM sections also indicated an axonal location. Separate TEM sections were processed with conventional fixatives and stains to highlight the typical uveal axon morphology, which also confirmed the identity of perivascular axons as the sites of t-PA localization. Affinity of the primary antibody for rat t-PA was shown by an inhibition ELISA against rat uveal tissue extracts and by the inhibition of t-PA activity in aqueous humor. An amidolytic assay was used to quantify t-PA activity. Possible explanations for the preferential immunolocalization of t-PA antigen to the axoplasm of uveal nerve terminals and the need for additional functional studies to confirm a putative neural t-PA synthesis are discussed.

Corrosion casts of the suprachoroidal space and uveoscleral drainage routes in the pig eye.

Eyes from pigs were studied by corrosion casting technique. Batson's mixture No. 17 (methyl methacrylate) was injected through a sclerotomy into the suprachoroidal space. Following polymerisation of the injected mixture the surrounding tissue was dissolved with 10% natrium hydroxide. Macroscopic and scanning electron microscopic findings in casts of the suprachoroidal space are presented. The outer (scleral) surface of the casts was rough, with a fish-scale-like appearance caused by fine lamellas between the sclera and choroid, running from the inner layers of the sclera traversing the space anteriorly to the choroid. Frequently, irregular, Y-shaped branches deriving from the outer surface of the casts were observed. They corresponded to the perivascular space of the vortex veins and represent a possible uveoscleral drainage route for aqueous humour.

Corrosion casts of the suprachoroidal space and uveoscleral drainage routes in the human eye.

Human cadaver eyes were studied by corrosion casting technique. Batson's mixture No. 17 (methyl methacrylate) was injected through a sclerotomy into the suprachoroidal space. Following polymerisation of the injected mixture the surrounding tissue was dissolved with 10% natrium hydroxide. Macroscopic and scanning electron microscopic findings in casts of the suprachoroidal space are presented. Due to transscleral drainage of resin from the suprachoroidal space, different types of branches deriving from the outer (scleral) surface of the casts were found. Some of these branches corresponded to the perivascular spaces of both ciliary vessels and vortex veins. In addition, we found branches probably representing channels deriving directly from the suprachoroidal space, communicating with the intrascleral venous plexus. Such channel systems have previously not been described, and their possible relation to the uveoscleral drainage of aqueous humour is discussed.

Immunocytochemical localization of prostaglandin E2 receptor subtypes in porcine ocular tissues. I. Uveal tissues.

Polyclonal antibodies were raised against 15-residue sequences in the carboxyl terminal region of mouse EP1, EP2, and EP3 subtypes. The selected sequences are well conserved in different species. Using the antibodies, the localization of the receptor subtypes in porcine uveal tissues was investigated by immunoperoxidase reaction (by light microscopy) and immunogold labeling (by electron microscopy). EP1 immunoreactivity was found in ciliary nonpigmented epithelium and iris muscles (both sphincter and dilator). EP2 was localized to ciliary nonpigmented epithelium and muscle, iris sphincter muscle, and trabecular meshwork. EP3 immunoreactivity was detected in all uveal tissues examined.

Localization and characterization of major histocompatibility complex class II-positive cells in the posterior segment of the eye: implications for induction of autoimmune uveoretinitis.

PURPOSE: To identify potential antigen-presenting cells in the choroid and retina of the normal rat eye, with a view to proposing a role for such cells in the induction and perpetuation of experimental autoimmune uveoretinitis, a model of human uveoretinal inflammation. METHODS: Immunohistochemical and electron microscopic studies using a panel of monoclonal antibodies were performed on frozen sections of the perfused-fixed normal Lewis rat eye, choroid whole mounts, and cytospin preparations of cells harvested from choroid/ciliary body explant cultures. In addition, time-lapse video recordings of migratory uveal tract cells in culture were taken. RESULTS: No major histocompatibility complex class II-positive cells were found in the normal Lewis rat retina. However, at least three populations of potential antigen-presenting cells were found in the uveal tissues of the eye: classical dendritic cells expressing high levels of major histocompatibility complex class II antigen; resident dendritiform macrophages, which were negative for major histocompatibility complex class II antigen, but expressed specific macrophage markers (ED2); and blood-borne macrophages (ED1) that had emigrated from the vasculature into the tissue compartment. In addition there were small numbers of cells expressing novel markers such as markers usually found only on macrophage subsets in splenic tissue (ED3) and a recently described marker for veiled dendritic cells (OX62). Dendritic cells and resident dendritiform macrophages closely interacted with each other and with tissue cells, particularly retinal pigment epithelial cells. CONCLUSIONS: The posterior uveal tract is richly populated with classical dendritic cells expressing constitutive high levels of major histocompatibility complex class II antigen. There are also several types of macrophages with the potential to modulate immune responses in the posterior segment. Interactions among these cells and with resident tissue cells such as retinal pigment epithelial cells are probably central to the initiation of (auto)immune responses in the posterior segment of the eye.

Report of a case of sympathetic ophthalmia with special regard to ultrastructural examination.

A case of sympathetic ophthalmia (SO) is reported. A long lasting stable result was obtained for this patient treated basically with traditional Chinese medicine. His exciting eye was investigated under light and transmission electron microscopes. Prominent granulomatous lesions in the choroid, Dalen-Fuchs nodules (DFNs) and disruption of outer and inner basement membrane of Bruch's membrane under DFNs are found, plasma cells are not few and melanocytes and retinal pigment epithelial cells are possibly the target cells. In various cells, nuclear bodies (NBs) are ubiquitous and sometimes multiple in an individual cell nucleus. Microtubule-like structures are present inside and outside the NBs and parallel lines composed of relatively uniform high electron dense granules as lattice-like structures can be seen. It was surmised that a virus induced autoimmune process might be involved in the pathogenesis of SO.

Studies of human uveal melanocytes in vitro: isolation, purification and cultivation of human uveal melanocytes.

PURPOSE: To develop the methods for isolation and cultivation of human uveal melanocytes (UM) from adult donor eyes. METHODS: After removal of the pigment epithelium, the uvea was pretreated in trypsin solution at 4 degrees C overnight, incubated at 37 degrees C with trypsin for 1 hr, then incubated with collagenase for 3 hr. Released cells were collected each hour during the incubation and cultured with F12 medium supplemented with fetal bovine serum, basic fibroblast growth factor, isobutylmethylxanthine and cholera toxin. Contaminant cells were eliminated by adding a selective cytotoxic agent, geneticin, when necessary. RESULTS: These methods provide pure melanocyte cultures with high cell yields, good viability, and rapid growth rates. UM isolated and maintained using these methods can be passaged 23 times for a period of 7 mo for more than 35 population doublings. This is comparable to results obtained with cultured neonatal dermal melanocytes and exceeds results obtained with adult dermal melanocytes cultured in media supplemented with phorbol ester, isobutylmethylxanthine, and cholera toxin. CONCLUSION: A method for isolation and cultivation of UM has been developed that yields satisfactory results. Cultured UM may be useful in in vitro studies of UM physiology and may allow development of in vitro models of the pathogenesis of uveal malignant melanoma.

Uveoscleral permeability to intracamerally infused ferritin in eyes of rabbits and monkeys.

The permeability of the uveoscleral outflow pathway from the anterior ocular chamber was examined in rabbit and monkey eyes using anionic ferritin as a tracer. Ferritin, infused intracamerally, had ready access to the choroidal interstitium, and the degree of penetration was generally correlated with the time and pressure relationships during infusion. In both species, there were accumulations of tracer in intercellular spaces at the lamina fusca, but tracer was also present in the sclera. Thus, in contrast to the situation in the eyes of hamsters, the uveoscleral outflow pathway in the eyes of rabbits and monkeys includes the choroidal connective tissue and allows passage of relatively large molecular weight substances.

[Study on the influence of silicone oils of various viscosities on anterior eye structures]. / Badanie wplywu oleju sylikonowego o róznej lepkosci na struktury przedniego odcinka galki ocznej.

Intraocular silicone oil tamponade plays important role in the treatment of complicated retinal detachments. Aim of present study was an investigation of the effects of different silicone oil fluids on the anterior chamber structures. Standard silicone oils with 100, 990, 3690 mPa.s viscosity and highly purified silicone oil with viscosity 5000 mPa.s were used. Corneal silicone oil impregnation was observed in corneas which were in contact with silicone oil with 100 mPa.s. viscosity. Silicone oil impregnation of uveal structures was observed in all cases after the 4 months follow-up. "Posterior collagenous layer" was observed subendothelial in eyes injected with various silicone oil fluids including highly purified one. Results of our investigation show that use of highly purified silicone oil does not allow to reduce the side effects on ocular structures caused by emulsification or a barrier to the nutrition and exchange of products of metabolism provided by silicone oil bubble.

Morphologic correlations with fluorophotometric data from monkey eyes with anterior uveitis.

Acute anterior uveitis was induced in monkeys by unilateral intravitreal injection of 1.0 ng of Escherichia coli endotoxin. Twenty-four hours later, each animal received an intravenous injection of 250 mg/kg body weight of fluoresceinated horseradish peroxidase (F-HRP), and fluorophotometric measurements were taken for 90 min. The animals were killed, and both eyes were processed for HRP demonstration. In the anterior chamber aqueous humor of normal control eyes, F-HRP concentrations were less than 0.002 mg/ml at 90 min. The F-HRP concentration was elevated consistently in the endotoxin-injected eyes; however, the magnitude of the effect varied. By fluorophotometry, inflamed eyes fell into two distinct groups. At 90 min, most had an anterior chamber F-HRP concentration of 0.014-0.06 mg/ml, although others had 0.39 mg/ml. In the latter group, an appreciably shorter latency was observed between the time of tracer injection and its detection in the anterior chamber. Aqueous humor protein concentrations, although highest in the most F-HRP-permeable eyes, followed more of a continuum in their distribution and identified less clearly the subpopulations seen by fluorophotometry. Normal eyes had no tracer leakage across either the ciliary epithelial or iris vascular endothelial barriers. All inflamed eyes had HRP leakage across the ciliary epithelium, but the subpopulation of eyes with shorter latencies and higher F-HRP concentrations by fluorophotometry also had iris vascular leakage.