Transgenic lysyl oxidase homolog 1 overexpression in the mouse eye results in the formation and release of protein aggregates.

Sequence variants in LOXL1 coding for the secreted enzyme lysyl oxidase homolog 1 (LOXL1) associate with pseudoexfoliation (PEX) syndrome, a condition that is characterized by the deposition of extracellular fibrillar PEX material in the anterior eye and other parts of the body. Since the specific role of LOXL1 in the pathogenesis of PEX is unclear, and an increase in its expression was reported for early stages of PEX syndrome, we generated and studied transgenic mice with ocular overexpression of its mouse ortholog Loxl1. The chicken ßB1-crystallin promoter was used to overexpress Loxl1 in the lenses of ßB1-crystallin-Loxl1 transgenic mice. Transgenic lenses contained high levels of the protein LOXL1 and its mRNA, which were both not detectable in lenses of wildtype littermates. In wildtype mice, immunoreactivity for LOXL1 was mainly seen extracellularly in region of the ciliary zonules. ßB1-crystallin-Loxl1 littermates showed an additional diffuse immunostaining in lens fibers and capsule, and in the inner limiting membrane and retina indicating secretion of soluble LOXL1 from transgenic lenses. In addition, lens fibers of transgenic animals contained multiple distinct spots of very intense LOXL1 immunoreactivity. By transmission electron microscopy, those spots correlated with electron-dense round or oval bodies of 20-50 nm in diameter which were localized in the rough endoplasmic reticulum and not seen in wildtype lenses. Immunogold electron microscopy confirmed that the electron-dense bodies contained LOXL1 indicating aggregation of insoluble LOXL1. Similar structures were seen in the extracellular lens capsule suggesting their secretion from lens fibers. Otherwise, no changes were seen between the eyes of ßB1-crystallin-Loxl1 mice and their wildtype littermates, neither by light microscopy and funduscopy of whole eyes, nor by scanning and quantitative transmission electron microscopy of ciliary epithelium and zonules. At one month of age, intraocular pressure was significantly higher in transgenic mice than in wildtype littermates. No differences in IOP were seen though at 2-5 months of age. We conclude that LOXL1 has a strong tendency to aggregate in the rER when expressed in vivo at high amounts. A similar scenario, involving intracellular aggregation of LOXL1 and secretion of LOXL1 aggregates into the extracellular space, may be involved in the early pathogenetic events in eyes of PEX patients.

Morphology and hydro-sensory role of superficial neuromasts in schooling behaviour of yellow-eyed mullet (Aldrichetta forsteri).

The lateral line system is a mechanosensory organ found in all fish species and located on the skin or in subdermal canals. The basic functional units are superficial and canal neuromasts, which are involved in hydrodynamic sensing and cohesion in schooling fish. Yellow-eyed mullet (Aldrichetta forsteri) are an obligate schooling species found commonly in shallow coastal areas of New Zealand and Australia. Schooling is a fundamental part of their behavioural repertoire, yet little is known about the structure or functionality of the lateral line in this species. We used scanning electron microscopy to characterise the morphology of trunk superficial neuromasts. We then took a multi-sensory approach and conducted behavioural experiments comparing school structure in groups of fish with and without fully functioning lateral lines, under photopic and scotopic conditions. A highly developed hydro-sensing system exists on the trunk of yellow-eyed mullet consisting of superficial neuromasts containing hundreds of hair cells aligned, with respect to their most sensitive axis, in a rostrocaudal direction. Without functioning superficial neuromasts, schooling behaviour was disrupted under both photopic and scotopic conditions and the ability to detect stationary objects decreased. Results highlight the importance of this component of the lateral line system to schooling behaviour.

The effects of VEGF-A-inhibitors aflibercept and ranibizumab on the ciliary body and iris of monkeys.

PURPOSE: To investigate the effects of intravitreal ranibizumab (Lucentis®) and aflibercept (Eylea®) on the ciliary body and the iris of 12 cynomolgus monkeys with regard to the fenestrations of their blood vessels. MATERIALS AND METHODS: Structural changes in the ciliary body and in the iris were investigated with light, fluorescent, and transmission electron microscopy (TEM). The latter was used to specifically quantify fenestrations of the endothelium of blood vessels after treatment with aflibercept and ranibizumab. Each of the two ciliary bodies treated with aflibercept and the two treated with ranibizumab and their controls were examined after 1 and 7 days respectively. Ophthalmological investigations including funduscopy and intraocular pressure measurements were also applied. RESULTS: Ophthalmological investigations did not reveal any changes within the groups. Both drugs reduced the VEGF concentration in the ciliary body pigmented epithelium. The structure of the ciliary body was not influenced, while the posterior pigmented epithelium of the iris showed vacuoles after aflibercept treatment. Ranibizumab was mainly concentrated on the surface layer of the ciliary epithelium, in the blood vessel walls and the lumen of some of the blood vessels, and in the cells of the epithelium of the ciliary body. Aflibercept was more concentrated in the stroma and not in the cells of the epithelium, but as with ranibizumab, also in the blood vessel walls and some of their lumina, and again on the surface layer of the epithelium. Both aflibercept-and ranibizumab-treated eyes showed a decreased number of fenestrations of the capillaries in the ciliary body compared to the untreated controls. On day 1 and day 7, aflibercept had fewer fenestrations than the ranibizumab samples of the same day. CONCLUSIONS: Both aflibercept and ranibizumab were found to reach the blood vessel walls of the ciliary body, and effectively reduced their fenestrations. Aflibercept might eliminate VEGF to a greater extent, possibly due to a higher elimination of fenestrations in a shorter time. Moreover, the vacuoles found in the iris need further research, in order to evaluate whether they carry a possible pathological potential.

Comparison of tight junction protein expression in the ciliary epithelia of mouse, rabbit, cat and human eyes.

Tight junctions in the nonpigmented epithelium (NPE) of the ciliary processes and the iris vascular endothelium form the ocular blood aqueous barrier that prevents leakage of proteins, immune cells and non-immune cells of blood into the anterior chamber. We attempted to determine whether ultrastructural differences in tight junctions reported in earlier studies are reflected in the expression pattern of tight junction proteins (TJP) and whether the TJP in mice, rabbits and cats resemble those of humans. For immunohistochemistry, 10 µm thick cryosections were rehydrated in PBS and fixed in 50 mM ammonium chloride at room temperature. After rinses in PBS, the sections were incubated twice in 0.1% Triton X-100, 10% goat serum, specific primary antibody or in PBS. After rinses in PBS, the sections were incubated in FITC-conjugated secondary antibody. After rinses in PBS, the sections were mounted with Vectashield mounting medium with propidium iodide, examined and photographed using a confocal microscope. The expression patterns of TJP in ocular ciliary epithelium of human, rabbit, cat and mouse were similar. Occludin immunoreactivity was observed as a sharp line along the junction between pigmented epithelium (PE) and NPE, and along the apico-lateral surfaces of NPE. Very light staining of the ciliary stroma was observed in cat and mouse. Claudin-1 was expressed along the entire boundaries of NPE and was more distinct between PE and NPE in rabbit. The ciliary stroma showed faint staining in cat and mouse. ZO-1 showed staining between PE and NPE, and at the adjacent membrane. Moderate staining was seen in PE in cat and mouse, which suggests that claudin-1, occludin and ZO-1 are expressed along the junction between PE and NPE, and the apico-lateral border of NPE. Lack of major difference in the expression patterns among the different species is important for validating the use of rabbit, mouse and cat in studies of intraocular inflammation in humans.

Intravitreal triamcinolone acetonide induced changes in the anterior segment in a pig model of branch retinal vein occlusion.

BACKGROUND: Triamcinolone acetonide (TA) has applications for the treatment of a large range of intraocular vascular diseases. The present study in pigs was performed to investigate histopathological and histochemical changes in the levels of myocilin deposition in the anterior segment in a model of branch retinal vein occlusion (BRVO) after vitreal administration of TA. METHODS: After ophthalmoscopic examination, intraocular pressure (IOP) measurement and fundus photography, a BRVO was created photothrombotically in each eye of six pigs, using argon green photocoagulation. The left eye was then injected intravitreally with 4 mg/0.1 ml TA. After 11 weeks, the eyes were re-examined, animals sacrificed, and eyes enucleated and processed in paraffin and epoxy resin. Immunofluorescence cytochemistry on paraffin sections was performed to localise the distribution of myocilin in the anterior segment and histology by light and transmission electron microscopy on epoxy resin sections on TA-treated and untreated eyes. RESULTS: Histology revealed pathological changes in the TA-treated eye, including swollen mitochondria, layered long endoplasmic reticulum, pleomorphic nuclei, dense fibrillar extracelluar deposits and aggregates of unusual cell inclusions. Myocilin levels were significantly higher in the TA-treated eyes in the trabecular meshwork (p = 0.001), ciliary process (p = 0.011) and iris (p = 0.030) than in the untreated eyes. CONCLUSIONS: This study suggests that increased myocilin synthesis and related ultrastructural changes in the anterior segment after treatment with intravitreal TA in a porcine model of retinal oedema in BRVO may contribute to IOP elevation.

Ultrastructural analysis of the pigment dispersion syndrome in DBA/2J mice.

PURPOSE: To characterise ocular pigment abnormalities associated with iris atrophy in DBA/2J mice as a model for human pigment dispersion syndrome. METHODS: Immunohistochemistry, electron and light microscopy were performed to examine the eyes of DBA/2J mice ranging in age from 2.5 to 18 months old. The focus of our study was the description of the ultrastructural modifications in the irides of DBA/2J mice. RESULTS: The DBA/2J mice presented modifications in the melanosomes in all the pigmented parts of the eye, including the retinal pigment epithelial cells and choroidal melanocytes of the ciliary pigment epithelium. The extracellular matrix of the iris stroma disappeared with ageing. Pigmented cells detached from the iris and migrated into the trabecular meshwork exclusively on the anterior iris surface. These cells were identified as macrophages by immunohistochemistry and electron microscopy. There was no evidence that melanocytes or iris pigment epithelial cells migrated into the trabecular meshwork, but they became more and more depigmented. The aqueous outflow was blocked by pigment-laden cells, but not by cellular debris or melanosomes. No substantial amount of extracellular melanosomes was observed. CONCLUSION: The morphology of melanosomes is aberrant in all pigment cells in the eyes of DBA/2J mice. We conclude that the disease process begins with the transfer of both immature melanosomes from the iris pigment epithelium (IPE) and melanocytes to macrophages, which subsequently migrate into the trabecular meshwork. Accumulating macrophages cause a blockade of the chamber angle. As the disease progresses, the IPE, melanocytes and iris stroma, including blood vessels, disappear, leading to iris atrophy. It is speculated that the loss of these pigment cells is partly caused by reduction of the iris stroma.

Cells previously identified as retinal stem cells are pigmented ciliary epithelial cells.

It was previously reported that the ciliary epithelium (CE) of the mammalian eye contains a rare population of cells that could produce clonogenic self-renewing pigmented spheres in culture. Based on their ability to up-regulate genes found in retinal neurons, it was concluded that these sphere-forming cells were retinal stem cells. This conclusion raised the possibility that CE-derived retinal stem cells could help to restore vision in the millions of people worldwide who suffer from blindness associated with retinal degeneration. We report here that human and mouse CE-derived spheres are made up of proliferating pigmented ciliary epithelial cells rather than retinal stem cells. All of the cells in the CE-derived spheres, including the proliferating cells, had molecular, cellular, and morphological features of differentiated pigmented CE cells. These differentiated cells ectopically expressed nestin when exposed to growth factors and low levels of pan-neuronal markers such as beta-III-tubulin. Although the cells aberrantly expressed neuronal markers, they retained their pigmented CE cell morphology and failed to differentiate into retinal neurons in vitro or in vivo. Our results provide an example of a differentiated cell type that can form clonogenic spheres in culture, self-renew, express progenitor cell markers, and initiate neuronal differentiation that is not a stem or progenitor cell. More importantly, our findings highlight the importance of shifting the focus away from studies on CE-derived spheres for cell-based therapies to restore vision in the degenerating retina and improving techniques for using ES cells or retinal precursor cells.

A comparison of epithelial and neural properties in progenitor cells derived from the adult human ciliary body and brain.

Cells isolated from the ciliary body (CB) of the adult human eye possess properties of retinal stem/progenitor cells and can be propagated as spheres in culture. As these cells are isolated from a non-neural epithelium which has neuroepithelial origin, they may have both epithelial and neural lineages. Since it is the properties of neural progenitor cells that are sought after in a future scenario of autotransplantation, we wanted to directly compare human CB spheres with neurospheres derived from the human subventricular zone (SVZ), which is the best characterized neural stem cell niche in the CNS of adults. The CB epithelium was dissected from donor eyes (n = 8). Biopsies from the ventricular wall were harvested during neurosurgery due to epilepsy (n = 7). CB and SVZ tissue were also isolated from Brown Norwegian rats. Dissociated single cells were cultivated in a sphere-promoting medium and passaged every 10-30 days. Fixed spheres were studied by immunohistochemistry, quantitative RT-PCR and scanning/transmission electron microscopy. We found that both CB and SVZ spheres contained a mixed population of cells embedded in extracellular matrix. CB spheres, in contrast to SVZ neurospheres, contained pigmented cells with epithelial morphology that stained for cytokeratins (3/12 + 19), were connected through desmosomes and tight-junctions and produced PEDF. Markers of neural progenitors (nestin, Sox-2, GFAP) were significantly lower expressed in human CB compared to SVZ spheres, and nestin positive cells in the CB spheres also contained pigment. There was higher expression of EGF and TGF-beta receptors in human CB spheres, and a comparative greater activation of the canonical Wnt pathway. These results indicate that adult human CB spheres contain progenitor cells with epithelial properties and limited expression of neural progenitor markers compared to CNS neurospheres. Further studies mapping the regulation between epithelial and neural properties in the adult human CB spheres are vital to fully utilize them as a clinical source of retinal progenitor cells in the future.

[Ultrastructural evaluation of mitomycin C toxic effects on the ciliary epithelium of normal rabbits eyes with and without aqueous humor suppressant treatment]. / Avaliação ultra-estrutural dos efeitos tóxicos da mitomicina C no epitélio ciliar de olhos de coelhos normais com e sem tratamento hipotensor ocular prévio.

PURPOSE: To evaluate the effects of mitomycin C (MMC) on the internal ciliary epithelium (ICE) of the ciliary body of animals treated with two different aqueous humor suppressants. METHODS: The eyes of sixteen Norfolk albino rabbits divided into four experimental groups were studied. The right eyes (RE) of the four groups received 0.1 ml of MMC (0.5 mg/ml) under the scleral flap. The left eyes (LE) was the control group. Group 1 (G1) did not have any other treatment. To Group 2 (G2) and Group 4 (G4) acetazolamide was administered. To Group (G3) and Group 4 (G4) timolol maleate was administered. ICE was examined by transmission electron microscopy (TEM). RESULTS: The following aspects were observed in all groups, except in G1 LE: cell shrinkage and/or enlargement of intercellular spaces, rarefied mitochondria, clear vesicular structures and electron-dense bodies. The internal limitant membrane showed to be thickened, discontinued and separated in all groups, except in G1 LE and G2 LE. Discharge of cytoplasmatic material was observed only in the groups treated with aqueous humor suppressants. CONCLUSIONS: 1) MMC, acetazolamide and timolol maleate caused morphological alterations in the ciliary epithelium even when used alone. 2) The combination of MMC and acetazolamide caused more alterations than did isolated acetazolamide, but not more than MMC alone. 3) For the other combinations the alterations were similar.

Avaliação ultra-estrutural dos efeitos tóxicos da mitomicina C no epitélio ciliar de olhos de coelhos normais com e sem tratamento hipotensor ocular prévio / Ultrastructural evaluation of mitomycin C toxic effects on the ciliary epithelium of normal rabbits eyes with and without aqueous humor suppressant treatment

OBJETIVO: Avaliar o epitélio ciliar interno (ECI) do corpo ciliar após aplicação de mitomicina C (MMC) sob retalho escleral, em animais tratados com dois tipos de inibidores da produção do humor aquoso. MÉTODOS: Foram estudados ambos os olhos de 16 coelhos divididos em 4 grupos experimentais. Foi realizado retalho escleral em todos os olhos dos animais, mas apenas os olhos direitos (OD) receberam MMC. No grupo 1 (G1) não houve tratamento prévio. Nos grupos G2 e G4 foi administrada acetazolamida e nos grupos G3 e G4 maleato de timolol. O ECI foi examinado à microscopia eletrônica de transmissão (MET). Os olhos esquerdos formaram os grupos controle. RESULTADOS: Em todos os grupos exceto no G1 OE, foram observadas: retração das células e/ou alargamento entre invaginações, mitocôndrias com rarefação, vesículas claras e corpos densos. A membrana limitante interna estava espessada, descontínua ou descolada em todos grupos exceto G1 OE e G2 OE. Foi observada liberação de material citoplasmático apenas nos grupos tratados com inibidores da produção de humor aquoso. CONCLUSÕES: 1- MMC, acetazolamida e maleato de timolol causaram alterações morfológicas no epitélio ciliar mesmo usados isoladamente. 2- A associação MMC e acetazolamida causou mais alterações do que a acetazolamida isoladamente, mas não mais do que a MMC isoladamente. 3- Nas demais associações as alterações foram semelhantes.

PURPOSE: To evaluate the effects of mitomycin C (MMC) on the internal ciliary epithelium (ICE) of the ciliary body of animals treated with two differents aqueous humor supressants. METHODS: The eyes of sixteen Norfolk albino rabbits divided into four experimental groups were studied. The right eyes (RE) of the four groups received 0.1 ml of MMC (0.5 mg/ml) under the scleral flap. The left eyes (LE) was the control group. Group 1 (G1) did not have any other treatment. To Group 2 (G2) and Group 4 (G4) acetazolamide was administered. To Group (G3) and Group 4 (G4) timolol maleate was administered. ICE was examined by transmission electron microscopy (TEM). RESULTS: The following aspects were observed in all groups, except in G1 LE: cell shrinkage and/or enlargement of intercellular spaces, rarefied mitochondria, clear vesicular structures and electron-dense bodies. The internal limitant membrane showed to be thickened, discontinued and separeted in all groups, except in G1 LE and G2 LE. Discharge of cytoplasmatic material was observed only in the groups treated with aqueous humor supressants. CONCLUSIONS: 1) MMC, acetazolamide and timolol maleate caused morphological alterations in the ciliary epithelium even when used alone. 2) The combination of MMC and acetazolamide caused more alterations than did isolated acetazolamide, but not more than MMC alone. 3) For the other combinations the alterations were similar.

Contact transscleral ciliary body photodynamic therapy in pigmented rabbits using verteporfin and diode laser: evaluation of treatment parameters.

PURPOSE: To evaluate the treatment parameters necessary for achieving ciliary body photodynamic damage, enough to significantly reduce IOP, using verteporfin and a diode laser. DESIGN: Animal study. METHODS: The right eye ciliary body of 30 pigmented rabbits was irradiated using verteporfin (Visudyne) and a diode laser. Photosensitizer dose ranged from 0.375 to 2 mg/kg. Three adjacent laser spots were applied 0.5 mm behind limbus at 12 o'clock position using a contact transscleral technique. The laser power was ranging from 10 to 70 mW and the duration of irradiation from 1 to 5 min per spot. The left eyes of the rabbits were used as controls. Animals were sacrificed 24 hours after the procedure and their eyes were evaluated by means of light and electron microscopy. A step-by-step approach was adopted with adjustment of experimental parameters according to histological findings. The end point was to identify the irradiation parameters necessary for induction of photodynamic damage while minimizing thermal damage. Subsequently, 10 more animals were used in order to verify the effectiveness of these irradiation parameters in reducing the intraocular pressure. RESULTS: The therapy parameters that led to photodynamic effect avoiding thermal damage were laser power of 25 mW, irradiation time of 3 min per spot, and verteporfin dose of 1 mg/kg. Transscleral ciliary body irradiation using these parameters resulted in vascular thrombosis of ciliary vessels and in substantial edema, resulting in separation of the two ciliary epithelium layers. These parameters were applied to 4 rabbits, resulting in a mean IOP reduction of 1.8 mmHg +/- 1.2 that lasted for 4 days. An increase of the laser power to 35 mW tested in 6 additional animals, resulted in mean IOP reduction of 2.2 mmHg +/- 1.2, lasting 6 days; some minimal thermal damage was seen with the later settings. CONCLUSION: The combination of verteporfin and 690 nm diode laser is effective for the induction of ciliary body photodynamic damage, which results in significant but temporary IOP reduction, after transscleral PDT in pigmented rabbits. With appropriate parameter selection, intraocular pressure reduction can be achieved while thermal damage is kept to a minimum.

A relação entre o comprimento dos processos ciliares medidos pela biomicroscopia ultra-sônica e a duração, localização e gravidade das uveítes / The relationship between the length of ciliary processes as measured by ultrasound biomicroscopy and the duration, localization and severity of uveitis

OBJETIVO: Determinar a relação entre o comprimento dos processos ciliares com a duração, localização e gravidade das uveítes. MÉTODOS: Foram analisados em estudo prospectivo, 58 indivíduos, incluindo pacientes com diferentes estágios de uveíte e indivíduos sem a doença (112 olhos, sendo 18 normais), no período de agosto de 2001 a agosto de 2002, no Departamento de Uveítes do Cole Eye Institute da Cleveland Clinic Foundation - Ohio - EUA. Todos os pacientes foram referidos para o exame de biomicroscopia ultra-sônica, pelo Departamento de Uveítes após exame oftalmológico de rotina. O aparelho modelo 840 (Zeiss-Humphrey) com transdutor de 50 MHz foi utilizado para análise dos processos ciliares sob anestesia tópica e técnica de imersão. RESULTADOS: Em relação à etiologia das uveítes, a de maior freqüência foi a idiopática (27,8 por cento). As uveítes recorrentes, agressivas e difusas levaram ao significante dano nos processos ciliares. A maior perda significativa na medida dos processos ciliares foi encontrada no quadrante inferior e as maiores medidas foram encontradas em olhos normais e no quadrante temporal. CONCLUSÃO: A biomicroscopia ultra-sônica mostrou ser método útil para avaliar as alterações anatômicas encontradas nos processos ciliares dos pacientes com uveíte. De acordo com estes achados, podemos criar recomendações para futuros trabalhos, que nos ajudem a avaliar a necessidade destes pacientes vir a receber tratamento mais agressivo em qualquer sinal de inflamação, com o objetivo de prevenir futuro dano e eventual hipotonia.

Sphere formation of ocular epithelial cells in the ciliary body is a reprogramming system for neural differentiation.

It is well known that neural stem/progenitor cells of the central nervous system (CNS) can proliferate to form neurospheres (CNS-neurospheres) that are positive for nestin, an intermediate filament for neural progenitors. Retinal stem/progenitor properties were also isolated from the ciliary body (CB) of the eye where, as in the CNS, such stem/progenitors also form spheres and have been considered to expand only via expansion by their proliferation even from the single-cell level (called spheres of pigment cells from the ciliary margin: PCM-spheres). We here found a new and distinct process underlying the growth of CB cell-derived spheres (CB-spheres) that is unlike the mechanism of CNS- and PCM-sphere expansion; this new process is a cell proliferation-independent incorporation of neighbor spheres and cells cultured at high density (200 cells/mul). The majority of cells in CB-spheres consisted of nestin-negative epithelia-like cells and started to express nestin during the course of their expansion by high-density cultivation. The growth of CNS-neurospheres was sensitive to a cell-cycle inhibitor, whereas the growth of CB-spheres was not seriously affected by cell proliferation; rather, the spheres grew by incorporating other CB-spheres and nestin-negative adherent cells, the latter of which started to express nestin and lost the expression of epithelial markers after being incorporated. These results indicate that CB-spheres do not form by the accumulation of neural progenitors but rather by a reprogramming system from epithelia-like cells for neural differentiation, a clearly distinct mechanism from sphere formation by single-cell expansion of retinal stem/progenitor populations.

Investigating the effect of ciliary body photodynamic therapy in a glaucoma mouse model.

PURPOSE: To investigate the morphologic and functional effects of verteporfin ciliary body photodynamic therapy (PDT) in a murine glaucoma model and normal mouse eyes. METHODS: A glaucomatous mouse strain, DBA/2J and a normal control mouse strain (C57BL/6) were used in the study. Verteporfin was injected intravenously at doses of 1.0 (DBA/2J) or 2.0 or 4.0 (C57BL/6) mg/kg. Transscleral irradiation of the ciliary body was performed with light at a wavelength of 689 nm delivered through an optical fiber, with irradiance of 1800 mW/cm2 and fluence of 100 J/cm2. Laser irradiation was applied for 360 degrees of the corneoscleral limbus in C57BL/6 normal mice and for 180 degrees in DBA/2J mice. Retreatment was performed in C57BL/6 normal mice that had been treated with 2.0 mg/kg of verteporfin at post-PDT day 7. One eye of each animal was treated, and the fellow eye served as the control. The morphologic effect of PDT on the ocular structures was assessed by light and electron microscopy. The IOP was measured using an applanation tonometer with a fiber-optic pressure sensor. Surviving retinal ganglion cells (RGCs) in DBA/2J mice eyes were retrogradely labeled with a neurotracer dye at 12 weeks after PDT. RESULTS: In all groups, almost all ciliary body blood vessels in the treated area were thrombosed 1 day after PDT. In DBA/2J mice, ciliary epithelium and stroma were severely damaged 1 day after PDT. The mean IOP in treated eyes was significantly reduced compared with that in the control eyes in all groups. The reduction of mean IOP in DBA/2J mouse eyes persisted for 7 weeks, although the mean IOP in normal mouse eyes treated with 2 or 4.0 mg/kg verteporfin returned to the level of the fellow control eyes by 7 and 17 days after treatment, respectively. The mean number of RGCs in the DBA/2J treated eyes was significantly higher than in control eyes. CONCLUSIONS: Ciliary body PDT resulted in morphologic changes in the ciliary body, significant reduction of IOP, and prevention of ganglion cell loss in a mouse glaucoma model. These results suggest that ciliary body PDT is a more selective cyclodestructive technique with potential clinical application in the treatment of glaucoma.

[Cellular characteristics of cultured non-pigmented ciliary epithelium from adult pigs].

PURPOSE: To compare the reactive proliferation of non-pigmented ciliary epithelial cells in patients with cyclitis, or after cyclophotocoagulation and cyclocryocoagulation, and the cultured non-pigmented ciliary epithelial cells from adult pigs. METHODS: Porcine ciliary epithelial cells were cultured and non-pigmented ciliary epithelial cells were isolated. Detection of DNA synthesis, morphological observation by a phase contrast microscope and a transmission electron microscope, and staining of senescence-associated beta-galactosidase were carried out. RESULTS: The cells proliferated without showing contact inhibition of growth or reconstitution of epithelial morphology. With a decrease of proliferative activity, the cultured cells expressed senescence-associated beta-galactosidase. Although DNA synthesis persisted for a long time, some cells in later culture periods showed morphologically abnormal nuclei or plural nuclei indicating dysfunction of cell division, or apoptotic features. CONCLUSION: The uncontrolled growth and loss of the epithelial nature of non-pigmented ciliary epithelial cells in vitro resembles the process of formation of cyclitic membrane and proliferation of ciliary epithelium after cyclophotocoagulation and cyclocryocoagulation in patients. Observatin of the behavior of cultured non-pigment epithelial cells could aid in understanding the mechanism of cyclitic membrane formation.

Effect of mitomycin C on ciliary body and intraocular pressure with various application depths: an experimental study.

BACKGROUND: To evaluate the effects of mitomycin C (MMC) on intraocular pressure (IOP) and ciliary body via transmission electron microscopy when applied under conjunctiva or different depths of sclera, without performing any filtering surgery. METHODS: Thirty-six eyes of 36 New Zealand albino rabbits were used in this study. MMC was prepared in a concentration of 0.4 mg/mL and 0.05 cc (20 microg) was soaked in preprepared sterile surgical sponges. Six groups each consisting of six eyes were formed and IOP was measured preoperatively. Group 1 was the control group: the superior conjunctiva was opened and only irrigated with balanced salt solution (BSS). In group 2, MMC soaked sponges were applied under the conjunctiva. In groups 3 and 4, a scleral flap of approximately 1/3 scleral thickness was prepared and in groups 5 and 6, and a scleral flap of approximately 2/3 scleral thickness was prepared, all with a standard size of 4 x 4 mm. MMC soaked sponges were applied under these areas for 5 min in eyes in groups 3 and 5 followed by an irrigation of the relevant areas with 10 cc BSS, whereas only irrigation with BSS was done in groups 4 and 6 as control groups. No filtering procedure was performed in any of the eyes. Eyes were enucleated on the 30th day following measurement of IOP and the ciliary body regions were evaluated using transmission electron microscopy. Kruskal-Wallis test was used for the statistical assessment of IOP between groups. RESULTS: The deep scleral flap group (group 5) showed statistically significantly more IOP reduction than both the superficial scleral flap group (group 3; P = 0.004) and the subconjunctival group (group 2; P = 0.002) on postoperative day 30. Electron microscopic evaluation of the surgical groups revealed a wide range of different histopathological effects due to different MMC application methods. The histopathological changes were more evident in the group 5, where MMC was applied under deep scleral flap. CONCLUSIONS: Subscleral application of MMC seems to provide greater IOP decrease than subconjunctival application, possibly caused by a more significant ciliary body toxicity. This may be the beginning of a non-penetrating, easy to perform and safe method to decrease IOP in glaucoma patients, which the authors call 'toxic ciliary ablation surgery'. However, the long-term results and complications must be assessed with further studies.

Reprodutibilidade intra-observador de medidas morfométricas do segmento anterior em olhos normais pela biomicroscopia ultra-sônica (UBM) / Intraobserver reproducibility in anterior segment morphometry of normal eyes using ultrasound biomicroscopy (UBM)

OBJETIVO: Estudar as variáveis que caracterizam a morfometria do segmento anterior usando a biomicroscopia ultra-sônica (UBM), em pacientes com o exame oftalmológico normal, em amostra de tamanho significativo, de forma sistematizada, com o intuito de avaliar sua reprodutibilidade intra-observador. Dois novos parâmetros tiveram sua reprodutibilidade intra-observador também testada. MÉTODOS: Foram examinados 190 olhos de 101 pacientes com exame oftalmológico normal empregando-se a UBM. Em cada olho, além da profundidade da câmara anterior (PCA), onze outros parâmetros que caracterizam a morfometria do segmento anterior foram medidos nos meridianos superior, nasal, inferior e temporal, em dois momentos distintos com intervalo mínimo de quatro semanas entre eles, pelo mesmo observador. RESULTADOS: Não se observaram diferenças estatisticamente significativas entre a primeira e a segunda medida (M1 e M2) de cada parâmetro estudado, exceto quanto a duas variáveis em dois meridianos nos olhos direitos (OD), e duas variáveis em um meridiano nos olhos esquerdos (OE). Mesmo estas diferenças mostraram-se clinicamente não significantes, por serem inferiores a 0,006 mm. Os dois novos parâmetros testados também apresentaram boa reprodutibilidade intra-observador. CONCLUSAO: Este estudo confirmou a boa reprodutibilidade intra-observador das variáveis que caracterizam a morfometria do segmento anterior pela UBM.

Viscocanalostomy in rhesus monkeys.

OBJECTIVE: To examine structural changes and aqueous humor outflow after viscocanalostomy in live normal monkey eyes. METHODS: Viscocanalostomy surgery was performed in 1 eye of each of 4 rhesus monkeys. Outflow facility was determined before and after surgery. All eyes were fixed and examined by light and/or electron microscopy 36 or 63 days postoperatively. RESULTS: Schlemm canal was replaced by scar tissue at the surgical site. The juxtacanalicular zone contained homogeneous material, probably high-molecular-weight 1.4% sodium hyaluronate. The sclera external to Schlemm canal was overhydrated, and remains of a scleral lake were present in 1 animal. Multiple defects were present in the endothelial lining of Schlemm canal inner and outer wall. Fine fibrillar material and sheath-derived plaque material partly bridged the defects. Along the inner wall, aggregations of thrombocytes covered some defects in the endothelial lining of the canal. At 90 degrees to 180 degrees from the surgical site, small and fewer breaks in the inner wall were seen. Postsurgery outflow facility (n = 2) was approximately 30% higher in the treated eye than in the contralateral control, corrected bilaterally for presurgery baseline. CONCLUSIONS: The most likely explanations for the increase in outflow facility in monkeys after viscocanalostomy are focal disruptions of the inner wall endothelium of Schlemm canal and disorganization of the juxtacanalicular zone, resulting in direct communication of juxtacanalicular zone extracellular spaces with the lumen of Schlemm canal. The continuous presence of sodium hyaluronate might prevent repair of these defects by interfering with thrombocyte function. CLINICAL RELEVANCE: In nonhuman primates, viscocanalostomy appears to decrease outflow resistance through persisting focal disruption of the inner wall endothelium and opening of the juxtacanalicular or cribriform region of the trabecular meshwork, the tissue most affected by pathologic changes in primary open-angle glaucoma in humans.

[Histopathologic study on cyclocryotherapy of human and monkey eyes].

PURPOSE: To evaluate the lesion and repair process of the ciliary body and adjacent tissues in glaucomatous human eyes and normal monkey eyes with cyclocryotherapy. METHODS: We used light and electron microscopy to observe the ciliary body and adjacent tissues in five glaucomatous human eyes and five normal monkey eyes which were given cyclocryotherapy. RESULTS: Five absolute glaucomatous eyes were given cyclocryotherapy from one and a half to 3 years before the enucleation. One eye had no coagulated spots on the ciliary process, but five eyes had coagulated spots on the pars plana. The pigment epithelial cells were atrophic or had disappeared, but non-pigment epithelial cells had proliferated by one or two layers. At the lesion of monkey pars plicata cyclocryotherapy up to three months, melanophage phagocytosed pigment epithelium and melanocytes accumulated, although the proliferation of non-pigment epithelial cells was seen. At the lesion of monkey pars plana cyclocryotherapy melanophage accumulation was also marked, although non-pigment epithelial proliferation was greater than in pars plicata cyclocryotherapy. CONCLUSION: As cyclocryotherapy is a blind therapy, it is uncertain and difficult to destroy the ciliary process precisely. The repair process continued up to three months after monkey cyclocryotherapy, but it took up to one year and 6 months after human cyclocryotherapy.

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

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