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.

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.

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.

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.

Effects on the surrounding tissues and morphological changes of components after implantation of PMMA and heparin surface modified PMMA intraocular lens in rabbit eyes.

The aim of this study was to evaluate the cellular response and morphological changes of cells on the intraocular lens(IOL) implanted over a course of time and to identify the basic mechanism of IOL adaptation to tissue reaction in the implanted eye by comparing polymethylmethacrylate (PMMA) IOL with heparin surface modified PMMA IOL. ECCE using Healon was done in 36 eyes of 36 rabbits. A heparin surface modified IOL was implanted in 18 eyes (Group I), while PMMA IOL was implanted into another 18 eyes (Group II). Corneal thickness and endothelial cell density were measured for 3 months. Postoperatively, the eyes were enucleated, and a cytopathologic examination of the cells on the surface of the IOL and their ultrastructural changes were observed with light and scanning microscope at various points of time. The findings of this present study suggested that heparin surface modified PMMA IOL reduced the degree of endothelial cell damage, postoperative tissue reaction, and pigment deposits on the surface of the IOL. These were statistically significant. The most important cell was considered to be the macrophage for the adaptation of IOL in the eye which gradually changed into a fibroblast-like cell, giant cell and finally disappeared after forming an acellular membrane on the IOL.

Simultaneous bilateral diffuse melanocytic uveal hyperplasia.

A 52-year-old woman noted loss of vision in August 1984. Clinical examination disclosed iris cysts and ciliary body cysts, macular edema, and uveal nevi. Cataract extraction and pressure-lowering operations were required in both eyes because of a tumor-induced angle-closure glaucoma. Vision, however, progressively decreased to light perception in each eye. Both eyes were finally enucleated because a malignant melanoma could not be ruled out, though iris tissue obtained in 1985 suggested a nevuslike process. Histologic study indicated a bilateral uveal hyperplasia. Results of light and electron microscopy, immunologic studies, and suspension cell culture suggested that the uveal hyperplasia was more likely a melanoma of low malignancy than a nevuslike process. We could not detect an extraocular primary tumor and assumed that this condition constituted an oncogenic syndrome.

Disobutamide: a model agent for investigating intracellular drug storage.

Disobutamide, a bis tertiary amine (pKa1 = 8.6; pKa2 = 10.2) cationic amphiphilic compound, and a putative cardiac antiarrhythmic drug induced clear cytoplasmic vacuoles in dogs and rats. Ultrastructurally, the vacuoles were membrane-bound vesicles containing primarily electron-lucent material. Some concentric lamellar bodies indicative of phospholipidosis were also present. Although numerous vacuoles were seen in one-year toxicity studies in dogs and rats, there was no apparent evidence of necrosis, inflammation, atrophy, hypoplasia, hyperplasia, or metaplasia. Clinical signs or laboratory findings indicative of functional impairment were also not apparent. The picture of the vacuolation in vivo was one of storage. In cultured cells vacuoles were shown to be storage sites for disobutamide and specifically in distended vesicles of the cytoplasmic acidic compartments, such as lysosomes, endocytic, and probably transport vesicles. Storage of the drug in acidic vesicles is compatible with the dibasic nature of the cationic moiety of disobutamide. The intrinsic cell chemicals which accumulate in the vacuoles along with disobutamide remain unknown. Disobutamide may be a useful agent for defining experimentally the borderline between physiologic limits (normal function) and toxicity (functional impairment) in the condition of intracellular drug storage abnormalities and for advancing knowledge of storage mechanisms.

The role of scanning electron microscopy in ophthalmic science.

The eyeglobe is one of the classical domains of Scanning Electron Microscopy (SEM) in biology as it exposes several inner and outer surfaces. Both corneal and conjunctival epithelia towards the tear film as well as the corneal endothelial cells facing the anterior chamber may be accurately evaluated. The architecture of the angle and particularly the morphology of the Schlemm's canal inner wall are clarified by SEM more than by TEM serial reconstruction. The surfaces of the iris and ciliary body, the zonula and the choroidal vessel arrangement are described in great detail. Three distinct types of membrane anchoring devices are demonstrated among the lens fibers. SEM impressively describes the retina, but it has not yet added any new information as to previous observations in a more conventional way. SEM plays a fundamental role in teaching ocular anatomy and physiology as it makes more comprehensive the interrelationships among different structures. In addition, it represents a proper structural approach of the clinician who is familiar with the three-dimensional observations obtained by means of biomicroscopy, ophthalmoscopy and fluorescein angiography. Therefore, SEM application should be further spread and possibly joined to immunocytochemistry, in order to obtain a more dynamic and functional analysis of the eye.

Kinetics of phagocytosis in the normal canine iridocorneal angle.

Microspheres of 3 different sizes were infused separately into the eyes of dogs with normotensive and hypertensive intraocular pressures. Latex spheres (0.5 or 1.0 micron) or 3.0 micron plastic spheres were added to Ringer's solution with 6% gelatin. Initially, this mixture was injected into the anterior chamber of dogs with intraocular pressures of 20, 50, or 75 mm of Hg. After 10, 20, 30, 60, or 90 minutes had elapsed, the dogs were euthanatized and the gelatin was hardened. Tissues were subsequently studied by light and transmission electron microscopies. Phagocytosis of the 0.5 and 1.0 micron spheres by trabecular cells was first detected within 10 minutes and within 20 minutes for the 3.0 microns spheres. Migration of cells in the corneoscleral trabecular meshwork was observed after 30 minutes. Phagocytosis was less active at hypertensive pressures and had larger sphere sizes. Microspheres in the uveal trabecular meshwork were ingested mostly by macrophages and polymorphonuclear leukocytes.

Glaucoma in Sturge-Weber syndrome.

Trabeculectomy specimens from three eyes with Sturge-Weber syndrome were examined histopathologically. Changes in the trabecular meshwork-Schlemm's canal system were similar to findings in old age and in primary open-angle glaucoma. Two mechanisms for glaucoma are theorized. In cases with buphthalmos and congenital glaucoma, the chamber angle is often anomalous, as in other types of congenital glaucoma. In later onset juvenile cases, the chamber angle more often appears normal. A premature aging of the trabecular meshwork Schlemm's canal complex, as shown by us histopathologically, is a primary cause of juvenile glaucoma. It is suggested that both mechanisms relate to the abnormal hemodynamics of episclera and chamber angle, due to persistence of Streeter's primordial vascular plexus.

Mapping, quantitative distribution and origin of substance p- and VIP-containing nerves in the uvea of guinea pig eye.

VIP- and substance P-like immunoreactivities were found in considerable concentrations (VIP: 17.3 +/- 4.8 pmol/g, mean +/- SEM; substance P:11.1 +/- 1.8 pmol/g) in the uveal portion of the guinea pig eye. Immunocytochemistry localised these two regulatory peptides to nerve fibres found principally in a plexus in the iris (substance P) and in an extensive network surrounding the blood vessels of the choroid (VIP). A remarkable anatomical demarcation of the two types of peptide-containing nerves was established by the staining of substance P-containing nerves, which stops at the level of the ciliary body. This uveal area is known to be involved in the ocular responses to nociceptive stimuli. At the ultrastructural level, immunoreactivity for both peptides was localised to distinct subpopulations of p-type nerves, distinguishable by the size of their large dense-cored vesicles. Those immunoreactive for VIP were significantly larger (p less than 0.0005) than those immunoreactive for substance P (95 +/- 7 nm and 82 +/- 9 nm respectively; mean +/- SD). Interruption of the trigeminal pathway produced a remarkable decrease of substance P immunoreactivity in the anterior portion of the uvea (9.1 +/- 1.5 pmol/g, mean +/- SEM, control; 5.3 +/- 1.3 pmol/g, denervated), but not of VIP immunoreactivity in the choroid. Following colchicine treatment, VIP-immunoreactive neuronal cell bodies were localised in the choroid. The separate anatomical localisations and distributions of the two uveal peptides appear to be related to their different origins and functional roles in the response of the eye to noxious stimuli.

Exfoliation glaucoma: a quantitative perfusion and ultrastructural study.

The aqueous outflow systems of both eyes from a patient with well documented exfoliation syndrome were studied postmortem by quantitative perfusion and scanning and transmission electron microscopy. This provided a rare opportunity to correlate clinical and postmortem findings. the right eye was phakic with early glaucoma. The left eye was aphakic with moderately severe glaucoma prior to the cataract surgery. The eyes were fixed at a perfusion pressure of 25 mm Hg. In both eyes the spaces of the corneoscleral trabecular meshwork were open and mostly free of exfoliation material. The trabecular cells appeared normal and contained no exfoliation material. Failure to take up exfoliation material contrasts with the known tendency of these cells to take up pigment and other particles. The main pathology involved destruction of Schlemm's canal and accumulation of exfoliation material in the juxtacanalicular region of both eyes. The changes in Schlemm's canal and the juxtacanalicular region appeared sufficient to account for increase in resistance to aqueous outflow.

An ultrastructural study of melanocytomas (magnocellular nevi) of the optic disk and uvea.

We examined by light and electron microscopy five melanocytomas from four patients. Two types of cells were observed in each tumor. The predominant cell type in most of the tumors studied consisted of plump polyhedral nevus cells that contained numerous giant melanosomes. These cells showed advanced differentiation. They appeared to be metabolically inactive and to have been the cause of the heavy pigmentation and benign nature of these tumors. The second variant of melanocytoma cells were smaller spindle-shaped cells that were lightly pigmented. Other morphologic features of these cells such as high nucleus-cytoplasm ratio, prominent nucleolus, nuclear membrane infolding, numerous mitochondria, prominent rough endoplasmic reticulum, and free ribosomes, indicated a metabolically active cell, which may explain the infiltrating behavior of these tumors.