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Purpose: Fibrillin-1 and -2 are major components of tissue microfibrils that compose the ciliary zonule and cornea. While mutations in human fibrillin-1 lead to ectopia lentis, a major manifestation of Marfan syndrome (MFS), in mice fibrillin-2 can compensate for reduced/lack of fibrillin-1 and maintain the integrity of ocular structures. Here we examine the consequences of a heterozygous dominant-negative mutation in the Fbn1 gene in the ocular system of the mgΔ lpn mouse model for MFS., Methods: Eyes from mgΔ lpn and wild-type mice at 3 and 6 months of age were analyzed by histology. The ciliary zonule was analyzed by scanning electron microscopy (SEM) and immunofluorescence., Results: Mutant mice presented a significantly larger distance of the ciliary body to the lens at 3 and 6 months of age when compared to wild-type, and ectopia lentis. Immunofluorescence and SEM corroborated those findings in MFS mice, revealing a disorganized mesh of microfibrils on the floor of the ciliary body. Moreover, mutant mice also had a larger volume of the anterior chamber, possibly due to excess aqueous humor. Finally, losartan treatment had limited efficacy in improving ocular phenotypes., Conclusions: In contrast with null or hypomorphic mutations, expression of a dominant-negative form of fibrillin-1 leads to disruption of microfibrils in the zonule of mice. This in turn causes lens dislocation and enlargement of the anterior chamber. Therefore, heterozygous mgΔ lpn mice recapitulate the major ocular phenotypes of MFS and can be instrumental in understanding the development of the disease., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Objective: Determine the immediate post-operative effects of MicroPulse ™ transscleral cyclophotocoagulation (MP-TSCPC) in healthy equine eyes., Animals Studied: Ten adult horses., Methods: MP-TSCPC was performed on sedated horses in 12 eyes (4 groups) using the following parameters (power, duration, duty cycle): (1) 2000 mW, 180 seconds, 31.3%; (2) 3000 mW, 180 seconds, 31.3%; (3) 3000 mW, 270 seconds, 31.3%; and (4) 3000 mW, 270 seconds, 50%. Three additional eyes were left untreated (control). Eyes were monitored clinically until euthanasia (mean = 3 hours post-procedure). Histologic sections were assessed with light microscopy and transmission electron microscopy (TEM)., Results: MP-TSCPC was well tolerated by sedated horses. Adverse effects were only noted in Group 4: ocular hypertension (n = 3/3), conjunctival burns (3/3), aqueous flare (2/3), and a corneal erosion (1/3). Histologic scoring of Group 4 was statistically greater than other treated groups (1-3) and control eyes (P ≤ .021). TEM showed subtle changes to the mitochondria and plasma membrane infoldings of the basilar surface of the nonpigmented epithelium in all treated eyes., Conclusions: MP-TSCPC does not cause immediate post-procedure adverse clinical effects or pronounced morphological changes to the ciliary body, except with the highest laser settings evaluated (power 3000 mW, duration 270 seconds, duty cycle 50%)., (© 2020 American College of Veterinary Ophthalmologists.)

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 βB1-crystallin promoter was used to overexpress Loxl1 in the lenses of βB1-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. βB1-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 βB1-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., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Purpose: To evaluate the influence of prostaglandin (PG) analogues on the ciliary zonular fibers of the crystalline lens using scanning electron microscopy (SEM) of rabbit eyes, and to measure the matrix metalloproteinase (MMP) and tissue inhibitor of metalloproteinase (TIMP) levels of the aqueous humor and crystalline lens treated with topical PG analogues Methods: Fifty eyes from 25 New Zealand white rabbits were divided into five groups of five rabbits each. In the control group, balanced salt solution was administered via the topical route once a day to the eyes. The benzalkonium chloride (BAC) group was treated with 0.02% BAC, the Latanoprost group with 0.005% latanoprost, the Travoprost group with 0.004% Travoprost, and the Bimatoprost group with 0.03% Bimatoprost for 10 months. We examined the ciliary zonular fibers using SEM. We also measured the MMP and TIMP levels of the aqueous humor and crystalline lens., Results: SEM revealed some splitting of zonular fibers in eyes treated with topical PG analogues when compared with the control and BAC groups. The MMP-1 and TIMP-1 levels after treatment with the PG analogues did not differ significantly from the control and BAC groups (P > 0.05). There was no significant difference in MMP-1, MMP-3, TIMP-1, and MMP-1/TIMP-1 levels in the lens among all five groups., Conclusions: PG analogues may induce zonular change in rabbits microscopically. There was no association between zonular changes and the levels of certain types of MMP or TIMP in the aqueous humor or crystalline lens after topical treatment with PG analogues.

Purpose: To investigate intraocular expression of COL7A1 and its protein product type VII collagen, particularly at the accommodation system., Methods: Eyes from 26 human adult donors were used. COL7A1 expression was analyzed in ex vivo ciliary epithelium by microarray. Type VII collagen distribution was examined by Western blot analysis, immunohistochemistry. and immuno-electron microscopy., Results: COL7A1 is expressed by pigmented and nonpigmented ciliary epithelia. Type VII collagen is distributed particularly at the strained parts of the accommodation system. Type VII collagen was associated with various basement membranes and with ciliary zonules. Anchoring fibrils were not visualized., Conclusions: Type VII collagen distribution at strained areas suggests a supporting role in tissue integrity.

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.

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.

Purpose: To elucidate the anatomy of the trabecular meshwork (TM) and its connection to ciliary muscle (CM) tendons with two photon excitation microscopic (TPEM) imaging., Methods: The human aqueous outflow pathway was imaged in an unfixed and nonembeded state by using an inverted TPEM. Laser (Ti:Sapphire) was tuned at 850 nm for emission. Backscatter signals of second harmonic generation (SHG) and autofluorescence (AF) were collected through 425/30-nm and 525/45 emission filters, respectively. Multiple, consecutive, and overlapping image stacks (z-stacks) were acquired to generate three-dimensional data sets., Results: Collagen and elastin structures of the TM were successfully visualized with TPEM. The TM and CM tendons were found to contain both collagen and elastin fibers. What appears to be juxtacanalicular tissue (JCT) was identified by its honeycomb-like appearance in AF images. Tracing CM tendons from their origins and to their insertions revealed that elastin fibers of CM tendons were connected to the elastin network within the trabecular lamellae. The CM tendons converged or diverged along their course, forming intricate networks with the TM. The CM tendon fiber density varied depending on its location within the aqueous outflow pathway with tendons near the JCT found to be the most dense, and in a fine-tooth comb arrangement., Conclusions: By using TPEM imaging, new details of the human aqueous outflow pathway were elucidated. This high-resolution imaging technique revealed the intricate interconnections between the TM and CM tendons.

Purpose: To describe an anteriorly located system of zonular fibres that could be involved in fine-tuning of accommodation., Methods: Forty-six human and 28 rhesus monkey eyes were dissected and special preparations were processed for scanning electron microscopy and reflected-light microscopy. Additional series of frontal and sagittal histological and ultrathin sections were analysed in respect to the origin and insertion of anteriorly located zonules. The presence of sensory terminals at the site of the originating zonules within the connective tissue of the ciliary body was studied by immunohistochemistry. For in-vivo visualization ultrasound biomicroscopy (UBM) was performed on 12 human subjects., Results: Fine zonular fibres originated from the valleys and lateral walls of the most anterior pars plicata that covers the anterior and inner circular ciliary muscle portion. These most anterior zonules (MAZ) showed attachments either to the anterior or posterior tines or they inserted directly onto the surface of the lens. At the site of origin, the course of the MAZ merged into the connective tissue fibres connecting the adjacent pigmented epithelium to the ciliary muscle. Numerous afferent terminals directly at the site of this MAZ-origin were connected to the intrinsic nervous network of the ciliary muscle., Conclusions: A newly described set of zonular fibres features the capabilities to register the tensions of the zonular fork and lens capsule. The close location and neural connection towards the circular ciliary muscle portion could provide the basis for stabilization and readjustment of focusing that serves fast and fine-tuned accommodation and disaccommodation., (© 2015 The Authors Ophthalmic & Physiological Optics © 2015 The College of Optometrists.)

Ciliary body is responsible for humour aqueous production in posterior chamber. Valproic acid (VPA) has been widely used for the treatment of epilepsy and other neuropsychiatric diseases such as bipolar disease and major depression. Oxcarbazepine (OXC) is a new anti-epileptic agent that has been used recently for childhood epilepsies such as VPA. In this study, we aimed to investigate the effects of VPA and OXC treatments used as antiepileptic in ciliary body by electron microscopy. In our study, 40 Wistar rats (21 days old) were divided equally into four groups which were applied saline (group 1), VPA (group 2), OXC (group 3) and VPA + OXC (group 4). The as-prepared ocular tissues were characterized by transmission electron microscopy (TEM) technique in scanning and transmission electron microscopy (SEM-TEM) (Carl Zeiss EVO LS10). The results confirmed that VPA caused dense ciliary body degeneration. Additionally, ciliary body degeneration in group 4 was supposed to be due to VPA treatment. Ciliary body damage and secondary outcomes should be considered in patients with long-term VPA therapy.

Using light microscopy, immunohistochemistry and electron microscopy, the structural organization of interstitial spaces and vessels of the ciliary body of the human eye (n = 5) were studied. The ciliary body was found to contain wide interstitial spaces--tissue clefts bound by collagen fibers and fibroblasts. Organ-specific lymphatic capillaries were also demonstrated in the ciliary body. According to the present findings and the lymphatic region concept, the first 2 elements of the lymphatic region of the eye were described: tissue clefts--prelymphatics and lymphatic capillaries of the ciliary body. The third element of the lymphatic region are the lymph nodes of the head and neck.

Purpose: To determine the connections between the ciliary muscle (CM), trabecular meshwork (TM), and Schlemm's canal (SC) and their innervations that allows CM contraction (by pilocarpine) to influence conventional outflow in mice., Methods: Sequential sections and whole mounts of murine corneoscleral angles were stained for elastin, α-smooth muscle actin (αSMA), vesicular acetylcholine transporter (VAChT), neuronal nitric oxide synthase (nNOS), vasoactive intestinal peptide (VIP), and tyrosine hydroxylase (TH). Elastic (EL) fibers between the CM, TM, and SC were examined in ultrathin, sequential sections from different planes. The effect of pilocarpine (100 μM) on conventional outflow facility was measured by perfusion of enucleated mouse eyes., Results: The mouse TM contains a three-dimensional (3D) net of EL fibers connecting the inner wall of SC to the cornea anteriorly, the ciliary body (CB) internally and the choroid and CM posteriorly. The CM bifurcates near the posterior TM, extending outer tendons to the juxtacanalicular tissue and inner wall of SC and internal connections to the lamellated TM and CB. Ciliary muscle and lamellated TM cells stain with αSMA and are innervated by VAChT-containing nerve fibers, without TH, VIP, or nNOS. Pilocarpine doubled outflow facility., Conclusions: Mouse eyes resemble primate eyes not only by their well developed SC and TM, but also by their 3D EL net tethering together the TM and SC inner wall and by the tendinous insertion of the CM into this net. The increase in outflow facility following cholinergic stimulation in mice, as in primates, supports using mice for studies of aqueous humor dynamics and glaucoma., (Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.)

Aquatic mammals underwent morphological and physiological adaptations due to the transition from terrestrial to aquatic environment. One of the morphological changes regards their vision since cetaceans' eyes are able to withstand mechanical, chemical, osmotic, and optical water conditions. Due to insufficient information about these animals, especially regarding their sense organs, this study aimed to describe the morphology of the Humpback whale (Megaptera novaeangliae) eyeball. Three newborn females, stranded dead on the coast of Sergipe and Bahia, Brazil, were used. Samples were fixed in a 10% formalin solution, dissected, photographed, collected, and evaluated through light and electron microscopy techniques. The Humpback whale sclera was thick and had an irregular surface with mechanoreceptors in its lamina propria. Lens was dense, transparent, and ellipsoidal, consisting of three layers, and the vascularized choroid contains melanocytes, mechanoreceptors, and a fibrous tapetum lucidum. The Humpback whale eyeball is similar to other cetaceans and suggests an adaptation to diving and migration, contributing to the perception of differences in temperature, pressure, and lighting., (Copyright © 2014 Wiley Periodicals, Inc.)

Purpose: Structures of the aqueous humor drainage tract are contractile, although the tract is not entirely composed of muscle. We characterized the mouse aqueous drainage tract by immunolabeling contractile markers and determined whether profiling these markers within the tract distinguished its key structures of the trabecular meshwork (TM) and ciliary muscle (CM)., Methods: Enucleated eyes from pigmented C57BL/6 (n=8 mice) and albino BALB/c (n=6 mice) mice were processed for cryo- and formalin-fixed paraffin-embedded sectioning. Immunofluorescence labeling was performed for the following: (a) filamentous actin (using fluorescence-conjugated phalloidin), representing a global contractile marker; (b) α-smooth muscle actin (α-SMA), caldesmon, and calponin, representing classic smooth muscle epitopes; and (c) nonmuscle myosin heavy chain, representing a nonmuscle contractile protein. Tissue labeling was identified by confocal microscopy and analyzed quantitatively. Hematoxylin and eosin staining provided structural orientation., Results: A small portion of the TM faced the anterior chamber; the rest extended posteriorly alongside Schlemm's canal (SC) within the inner sclera. Within the drainage tract, filamentous actin labeling was positive in TM and CM. α-SMA and caldesmon labeling was seen primarily along the CM, which extended from the anterior chamber angle to its posterior termination beyond the SC near the retina. Low intensity, patchy α-SMA and caldesmon labeling was seen in the TM. Myosin heavy chain immunoreactivity was primarily found in the TM and calponin was primarily observed in the CM. C57BL/6 and BALB/c comparison showed that pigment obscured fluorescence in the ciliary body., Conclusions: Our strategy of profiling contractile markers distinguished mouse aqueous drainage tract structures that were otherwise indistinguishable by hematoxylin and eosin staining. The mouse TM was seen as an intervening structure between SC, a part of the conventional drainage tract, and CM, a part of the unconventional drainage tract. Our findings provide important insights into the structural and functional organization of the mouse aqueous drainage tract and a basis for exploring the role of contractility in modulating aqueous outflow.

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.

An elevated aqueous humor protein level (aka flare) has always been considered to represent a pathological breakdown of the blood-aqueous barrier (BAB), regardless of the etiology. Recent studies in humans, using magnetic resonance imaging (MRI) to directly observe BAB kinetics in the posterior chamber of the human eye in-vivo, showed that pilocarpine-induced flare resulting from administration of a single drop of pilocarpine is not the result of breakdown of the BAB in the ciliary body. These MRI studies could not confirm whether pilocarpine caused an increase in iris vascular permeability. In the current studies we completed combined cell-flare meter and intravascular tracer studies, using intravenous horseradish peroxidase (HRP) in rabbits. One hour after receiving 3% pilocarpine in one eye, pupil size significantly decreased and aqueous flare significantly increased in pilocarpine-treated eyes. Light and electron microscopy demonstrated no leakage across either the iris vascular endothelium or the non-pigmented ciliary epithelium in either pilocarpine-treated or control eyes. One animal received HRP directly after pilocarpine to control for a transient increase in permeability before the peak flare response occurred. No leakage was found in the ciliary body or iris of this animal. Additional animals received topical pilocarpine in one eye but after 1 h they were sacrificed without tracer studies. Uveal tissues from these animals were used to assess the distribution of non-HRP protein in the ocular anterior segment and to assess the amount of elutable protein in the iris stromas of both treated and untreated eyes. Immunohistochemistry confirmed the presence of a reservoir of protein in the iris stroma. Analysis of elutable total protein from the iris stroma of pilocarpine-treated and control eyes showed significantly less total elutable protein in pilocarpine-treated eyes. Eyes with the greatest percent change in pupil size (i.e. the strongest miosis) correlated with lowest amounts of residual protein in the iris stroma. The tracer studies confirmed recent MRI studies in humans showing that the source of pilocarpine-induced flare is not disruption of the ciliary epithelial barrier. Extending this work, the current studies also showed no pilocarpine-induced leakage from the iris vasculature. The elutable protein experiments suggested that a primary source of pilocarpine-induced flare was extrusion of a portion of the reservoir of protein in the iris stroma. Taken together, these studies strongly suggest that not all clinically observable flare results from breakdown of the BAB., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

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.

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.

Purpose: To search for proprioceptive nerve terminals in human ciliary muscle., Methods: In 48 human donor eyes, histologic and ultrathin sections cut in different planes and wholemounts of the ciliary muscle were studied. Immunohistochemical staining with antibodies against pan-neuronal antigens and antigens reported as markers for sensory terminals in other organs was performed., Results: Among the markers for proprioceptive terminals, only calretinin was present in the ciliary body. Calretinin-immunoreactive (IR) nerve terminals surrounded the posterior and reticular ciliary muscle tips and their elastic tendons. Terminals in that region contained mitochondria and neurofilaments. At the anterior tips larger terminals with numerous membrane-filled vesicles were located between the muscle fibers. The most elaborate network of calretinin-IR nerve fibers was present in the ground plate covering the circular muscle portion. Here calretinin-IR neurons with morphologic features of mechanoreception were present. Within the circular muscle portion numerous calretinin-IR ganglion cells were found. Their processes were connected to the calretinin-IR network but also surrounded ciliary muscle cells and NADPH-diaphorase-positive ganglion cells., Conclusions: These morphologic findings indicate that there are proprioreceptors in the ciliary muscle that morphologically and presumably functionally differ at different locations. At the posterior muscle tips, the receptors could measure stretch of the tendons, whereas the large receptor organs located at the anterior muscle tips morphologically resemble mechanoreceptors measuring shear stress. The presence of the numerous intrinsic nerve cells indicates that contraction of the circular muscle portion can be modulated locally via a self-contained reflex arc.

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.

Purpose: To investigate the potential toxicity of the vehicle used for triamcinolone acetonide (TA) to the cornea, lens, ciliary body, and retina of pigmented rabbits., Methods: Forty chinchilla rabbits (40 eyes) were divided into four groups: group A (control group) eyes received an intravitreal injection of 0.1 ml sterile saline solution; group B eyes received 0.1 ml (1.3 mg) TA plus vehicle; group C eyes received 0.1 ml (1.3 mg) TA alone (vehicle eliminated); group D eyes received 0.1 ml vehicle only. Intraocular pressure (IOP) was measured pre-injection and at 1, 7, 14, 30, and 90 days post-injection. Scotopic and photopic electroretinograms (ERG) were examined pre-injection and 7, 30, and 90 days post-injection. Animals were sacrificed 7, 30, and 90 days post-injection. Eyes were enucleated and examined by light microscopy (LM) and electron microscopy (EM)., Results: The IOP of groups B and C was higher than that of other groups on days 1, 7, and 14 post-injection (p < 0.05). ERG amplitudes of groups B and D were lower than those of other groups on days 7 and 30 post-injection (p < 0.05). Histopathological sections indicated morphologic changes in the ciliary body, lens, and retina of eyes in groups B and D., Conclusions: Vehicle used for TA is toxic to the lens, ciliary body, and retina of pigmented rabbit eyes after injection of intravitreal TA.

Purpose: Conditional inactivation of connexin43 (Cx43) in the pigmented epithelium of the mouse eye results in a reduction in aqueous humor production and complete loss of the vitreous chamber. It was proposed that gap junctions between pigmented and nonpigmented epithelia of the ciliary body are critical for the production of the aqueous humor. To form such junctions, Cx43 in the pigmented epithelium must interact with connexin(s) present in the adjacent cells of the nonpigmented epithelium. The importance of Cx43 expression in the nonpigmented epithelium for the establishment of gap junctions and the regulation of intraocular pressure was tested., Methods: To inactivate Cx43 in the nonpigmented epithelium of the mouse eye, a mouse line was crossed with a floxed Cx43 locus (Cx43(flox/flox)) and a transgenic mouse line expressing cre recombinase under the control of the Pax6alpha promoter. General eye structure was evaluated by light microscopy, gap junctions were analyzed by electron microscopy, and intraocular pressure was directly assessed with micropipettes., Results: In Pax6alpha-cre/Cx43(flox/flox) mice, Cx43 was partially inactivated in the nonpigmented epithelium of the ciliary body and iris. Animals developed dilatations between the pigmented and nonpigmented epithelia and displayed a significant reduction in intraocular pressure. However, gap junctions between the ciliary epithelial layers were decreased but not eliminated., Conclusions: Cx43 expression in the nonpigmented epithelium of the ciliary body contributes to the formation of gap junctions with the cells of the pigmented epithelium. These gap junctions play a critical role in maintaining the physical integrity of the ciliary body epithelium. Although the partial loss of Cx43 from the nonpigmented epithelium was correlated with a measurable drop in intraocular pressure, possible changes in Cx43 in the aqueous outflow pathway may provide an additional contribution to the observed phenotype.

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.

Purpose: The structures of the ocular anterior segment responsible for aqueous humor secretion and absorption have been well characterized in mammals. However, the underlying molecular and cellular mechanisms that regulate aqueous humor flow have remained elusive. Experimental analysis in Danio rerio, the zebrafish, is providing mechanistic insights into many cellular processes relevant to normal human physiology and disease. To facilitate studies on the molecular and cellular mechanisms of aqueous humor dynamics using this species, the authors have characterized the anatomy of aqueous secretion and outflow in adult zebrafish eyes., Methods: Analyses by light and transmission electron microscopy, coupled with molecular tracers of fluid flow, were used to identify and study the sites of aqueous humor secretion and absorption in adult zebrafish eyes., Results: Zebrafish eyes show aqueous humor secretion primarily from the dorsal ciliary region and outflow through a ventral canalicular network that connects with an aqueous plexus and veins of the choroidal rete., Conclusions: Vectorial flow of zebrafish aqueous humor is in contrast to that in mammals in which secretion and absorption of aqueous humor are circumferential around and through the iridocorneal angle. However, local anatomy and ultrastructure of the tissues and cells specialized for aqueous humor dynamics in zebrafish show conservation with that of mammals. These observations suggest that zebrafish can serve as a useful genetic model to help understand the regulation and cellular basis of normal and abnormal aqueous humor dynamics in humans.

The morphology of the trabecular meshwork in three types of open angle glaucoma: primary open angle glaucoma (POAG), corticosteroid-induced glaucoma and pigmentary glaucoma (PG) are described. Ageing is one major risk factor for development of POAG. It is assumed that preexisting age-related changes of the trabecular meshwork (TM) play a role for the development of increased outflow resistance and intraocular pressure (IOP) in various types of glaucoma. These age-related changes in the TM develop concomitant with that of presbyopia. Therefore the functional relationship between ciliary muscle (CM) and TM and the age-related changes in morphology of the outflow system are described first. One main finding in the ageing TM concerns changes of the elastic fiber network and the anterior elastic tendons of the CM. There is an increase in thickness of the sheath of the elastic fibers. Cross-sections through these fibers with their sheath appear as extracellular plaques and were therefore termed "sheath derived plaques" (SD-plaques). Morphologically, the TM changes in POAG resemble that of the ageing TM, but in POAG there is a significant increase in SD-plaques compared to age-matched controls. This increase is due to fine fibrils and other components of the extracellular matrix (ECM) that adhere to the sheaths of the elastic fibers and their connections to the inner wall endothelium. In POAG eyes there is also a marked loss of TM cells, at places leading to fusion and thickening of trabecular lamellae. In steroid-induced glaucoma there is also an increase in fine fibrillar material in the subendothelial region of SC. In contrast to POAG eyes these fibrils do not adhere to the sheath of the elastic fibers but are deposited underneath the inner wall endothelium. The main finding in steroid-induced glaucoma is an accumulation of basement membrane-like material staining for type IV collagen. These accumulations are found throughout all layers of the TM. In pigmentary glaucoma loss of cells was more prominent than in POAG eyes. Presumably, this cell loss occurs after overload of TM cells with pigment granules. Denuded TM lamellae fuse and the TM collapses. In the subendothelial region of these collapsed TM areas an increase in ECM presumably due to underperfusion was observed. At other places SC was occluded and the cribriform region appeared disorganized. In most parts of the circumference of the eye, the TM cells contained pigment granules. Occlusion of TM spaces by pigment granules or cells loaden with pigment was not seen in eyes with PG.

In contrast to most mammalian species, the ciliary processes in the mouse eye form an irregular pattern. Different strains were studied using scanning electron microscopy. The ciliary processes of C57BL/6J animals showed quadrant-specific characteristics: in the superior quadrant, large and radial oriented processes were present. In the inferior quadrant, the processes were small but still mainly radial oriented. In the temporal quadrant, the processes showed a radial, longitudinal course, some being L-shaped. In the nasal quadrant, few processes were oriented longitudinal. In DBA/2 animals, the processes were shorter and the radial orientation less developed. NMRI animals showed the shortest processes with no increase towards the superior quadrant. Additionally we investigated age-related changes in the ciliary processes of Pde6b(rd1) mice, which develop retinal degeneration. In C57BL/6J mice, the ciliary body shape, size and architecture was comparable between 3 and 10 months of age, but showed a mild shortening of the pars plicata in the temporal, inferior and nasal quadrants in animals older than 20 months of age. The parameters of the ciliary body in 3 months old Pde6b(rd1) mice were comparable to those of age-matched C57BL/6J mice. Pde6b(rd1) mice 10 months old revealed significant shortening of the total width of the ciliary body and of the length of ciliary processes in all quadrants. The shape and architecture of the ciliary processes remained preserved.

Background/aims: To evaluate the safety of suramin compared with mitomycin-C (MMC) as an adjunctive agent in trabeculectomy by determining its ciliary body toxicity at predetermined effective dosages in rabbit eyes., Methods: Thirty-two New Zealand albino rabbits received either suramin (200, 300, 400, or 800 mg/ml) or MMC (0.2, 0.3, 0.4, or 0.8 mg/ml) injections subconjunctivally in the right eye. Enucleations were performed on the 1st, 3rd, 7th and 28th day. Untreated left eyes were injected with balanced salt solution and served as controls. The injection-exposed ciliary body specimens were processed to be investigated under the light microscope and transmission electron microscope., Results: There was no pathologic abnormality in specimens under light microscopy. The morphologic evaluation with transmission electron microscopy showed severe changes in structure, except for eyes treated with 200 mg/ml suramin and 0.2 mg/ml of MMC. These changes were more prominent in eyes exposed to MMC, and appeared earlier compared to suramin-treated eyes., Conclusions: Suramin 200 mg/ml and MMC 0.2 mg/ml seem to be comparatively nontoxic to the ciliary body of the rabbit eye. Concentrations higher than these values caused severe damage., ((c) 2008 S. Karger AG, Basel.)

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.

Purpose: In this study, biochemistry, molecular biology, immunohistochemistry, and electron microscopy techniques were used to examine whether versican, which is known to bind fibrillin-1, interacts with fibrillin-1 in the ciliary body and vitreous, and whether the versican in this complex binds to hyaluronan., Methods: The new polyclonal antibodies against the amino and carboxyl termini of versican were raised and characterized. The mRNA expression levels of versican and fibrillin-1 were analyzed by RT-PCR and real-time PCR, and their protein levels were evaluated by Western blot analysis and immunohistochemistry. Isolation of versican bound to fibrillin-1-containing microfibrils from ciliary bodies was performed by extraction studies. Slot-blot analyses and rotary shadowing electron microscopy were applied to identify versican associated with fibrillin-1-containing microfibrils after gel filtration chromatography and density gradient centrifugation., Results: The newly prepared polyclonal antibodies recognized amino and carboxyl termini of chicken versican. Versican, principally V0 and V1, was found to be securely bound to fibrillin-1-containing microfibrils, forming a major hyaluronan-binding structure in the ciliary nonpigmented epithelium. In addition, Western blot analysis revealed two cleaved complexes, the carboxyl-terminal end of versican bound to fibrillin microfibrils and the amino terminal end of versican bound to hyaluronan in the vitreous body., Conclusions: Fibrillin-1, versican, and hyaluronan form a unique complex in the ciliary nonpigmented epithelium, and two cleavage products of this complex were shown to exist in the vitreous body. This newly clarified fibrillin-versican-hyaluronan (FiVerHy) complex and its cleavage products may be indispensable for the physiological properties important to the ciliary body and vitreous.

Zonular fibers (ZF) play an important role in accommodation. With the rapid increase over the last decade of the oldest part of the population in industrialized countries, age-dependent loss of accommodation became an increasingly important problem. It appeared therefore interesting to review old and recent literature on ZF, their composition, structure and pathological alterations. By comparing former and recent reports it appeared to us, that several previous reports were not sufficiently taken in consideration for the understanding of the rheological properties of ZF. Elastin and proteoglycans-glycosaminoglycans were reported previously as constituents of ZF. Their presence besides fibrillin, the major constituent, helps to explain the rheological properties of these fibers, and especially their elasticity and its age- and pathology-dependent decline. Our review points also to some of the major problems, which remain to be addressed by future experiments.

Objective: To examine the functional anatomy of the ocular circulation in four bottlenose dolphins (Tursiops truncatus) and five melon-headed whales (Peponocephala electra)., Procedure: Eyes were obtained postmortem from whales that died while in captivity and/or were found beached. Specimens from whales were investigated using scanning electron microscopy (SEM) of vascular corrosion casts and histology. The thermal characteristics of live dolphin eyes were measured using an infrared imaging system., Results: The whale eye receives its primary blood supply from the ophthalmic rete, which extends just behind the eyeball. The ophthalmic rete diverges from the basilar rete and the cervical rete via the posterior thoracic artery. The iris and ciliary processes are supplied by iridic arteries via the major arterial circle that is located around the iris edge. The retinal vessels show the holangiotic type. Choroidal arteries run in parallel arrays so as to interdigitate the densely packed choroidal veins. The venules in the conjunctival fold and palpebral conjunctiva form a well-developed venous plexus. Thermographic examination revealed that the eye shows a substantially higher degree of thermal emission than adjacent skin areas., Conclusions: The cetacean eye is characterized by a unique vascular pattern and multivessel plexuses, which are quite different from those of terrestrial mammals. This suggests that the ocular vasculature might function as a thermoregulatory system so that the appropriate operating temperature for the photoreceptors can be maintained in a deep and cold aquatic environment. The distinctive plexuses in the orbit might also be for pooling blood in the eye to conserve oxygen during dives. The ophthalmic rete might play a role in a pressure-damping effect on cetacean ocular circulation as well.

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.

Background: The objective of this study is to investigate the acute histological effects of transscleral cyclophotocoagulation and endoscopic cyclophotocoagulation on the ciliary body and other structures of porcine eyes compared with untreated controls., Methods: Transscleral cyclophotocoagulation and endoscopic cyclophotocoagulation were performed on porcine eyes. Detailed histological evaluations were performed with light and scanning electron microscopy of treated eyes and compared with untreated controls., Results: Histological changes were observed with both light and scanning electron microscopy for all treated tissues. Tissue treated with transscleral cyclophotocoagulation showed pronounced tissue disruption of the ciliary body muscle and stroma, ciliary processes, and both pigmented and non-pigmented ciliary epithelium. Endoscopic cyclophotocoagulation-treated tissue exhibited pronounced contraction of the cilliary processes with disruption of the ciliary body epithelium, with less architectural disorginization and sparing of the ciliary body muscle. The sclera was not affected by either laser treatment., Conclusion: The endoscopic cyclophotocoagulation treatment caused less damage to the ciliary body compared with the transscleral cyclophotocoagulation when evaluated by light and scanning electron microscopy. Compared with transscleral cyclophotocoagulation, endoscopic cyclophotocoagulation appears to be a more selective form of cyclophotocoagulation resulting in less tissue disruption while achieving the goal of destroying ciliary body epithelium.

The pathogenesis of glaucomatic illnesses is poorly understood. An increase in ocular pressure can be caused by an increase in the secretion of aqueous humour or a reduction in its outflow. In the elderly, outflow is reduced while at the same time less aqueous humour is produced. This balance is easily disturbed, so that age represents a risk factor for glaucoma in addition to increased ocular pressure. Therapeutic possibilities involve, on the one hand, reducing the secretion of aqueous humour, for example using, beta blockers, carbonic anhydrase inhibitors and clonidine. On the other hand, aqueous humour outflow can also be influenced by drugs. Conventional outflow is increased by the administration of miotics. The uveoscleral outflow can be increased by prostaglandin derivates. Drugs which only influence trabecular outflow are not yet available. Future therapeutic possibilities involve new aspects of the pathophysiology, e.c. the use of growth factors, free radical scavenging enzymes and choroidal blood flow.

Aim: To study the histological effects of trans-scleral cyclophotocoagulation (TCP) and endoscopic cyclophotocoagulation (ECP) on the ciliary body and other structures collected at autopsy and to compare with untreated controls., Materials and Methods: TCP and ECP were performed on human eyes at autopsy. Detailed histological evaluations were perfomed using light microscopy and scanning electron microscopy on treated eyes and compared with untreated controls., Results: Histological changes were observed with both light microscopy and scanning electron microscopy for all treated tissues. Tissue treated with TCP showed pronounced tissue disruption of the ciliary body muscle and stroma, ciliary processes, and both pigmented and non-pigmented ciliary epithelium. ECP-treated tissue exhibited pronounced contraction of the ciliary processes with disruption of the ciliary body epithelium, sparing of the ciliary body muscle and less architectural disorganisation. The sclera was not affected by either laser treatment., Conclusions: ECP treatment caused less damage to the ciliary body compared with TCP when evaluated by light microscopy and scanning electron microscopy. Compared with TCP, ECP seems to be a more selective form of cyclophotocoagulation, resulting in less tissue disruption while achieving the goal of destroying ciliary body epithelium.

Summary The aim of this study was to describe and localize the intercellular junctions in the ora serrata region of albino and pigmented rabbit eyes. Eyes of albino and pigmented rabbits were fixed and processed for transmission electron microscopy. Light and electron microscope examination was carried out on semithin and ultrathin sections. The ora serrata region showed adherens, gap and tight junctions in the retinal and ciliary margins of albino and pigmented rabbit eyes. In the retinal margin, zonulae adherens between Müller cells and photoreceptors are associated with tight junctions. In the ciliary margin, epithelial cells are joined by adherens, gap and tight junctions localized between apical and apicolateral cell membranes. Tight junctions appear as zonulae occludens in the non-pigmented apicolateral cell membranes and as tight focal junctions between pigmented and non-pigmented apical cell membranes. Between the ciliary and retinal margins there are adherens and tight focal junctions which attach pigmented apical cell membranes to adjacent cells. There were no differences in the distribution of intercellular junctions between albino and pigmented rabbits.

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.

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.

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.

Objective: To examine the angioarchitecture of the ciliary body in the West Indian manatee (Trichechus manatus), through the use of three-dimensional reconstruction., Procedure: Specimens from West Indian manatee were preserved in 10% buffered formalin, embedded in paraffin, serial sectioned and stained by Masson trichrome for light microscopic three-dimensional reconstruction and evaluation., Results: The network of blood vessels in the ciliary processes of the West Indian manatee is fed by the major arterial circle that lies mostly near the base of the iris. The branching arterioles give rise to a capillary-sinusoidal bed that extends internally along each process, emptying into two sets of veins, one being elevated. The elevated and nonelevated veins join posteriorly before emptying into the choroidal venous system., Conclusions: The angioarchitecture of the ciliary body of the West Indian manatee is clearly unique when compared to those previously examined in land mammals. Three-dimensional reconstruction of paraffin sections is an effective means to evaluate vascular patterns in ocular specimens, especially those unavailable for corrosion casting.

There are species-related anatomical differences in the ciliary muscle of the avian eye. The arrangement of muscular fibers in the avian eye is not well defined. To clarify this situation, we studied the architecture of ciliary muscle of Gallus domesticus by light and scanning electron microscopy (SEM). Our results showed the existence of three main muscular groups that we defined as anterior, posterior, and intermediary. These muscle divisions correspond to the description of the ciliary muscle as previously stated by Crampton (1813), Brucke, and Muller (1856). The striated fibers have a meridian orientation. The anterior and posterior muscular groups are inserted in the sclera, around the Schlemm's canal wall and ciliary process stroma. The vitreal intermediary muscle has fibers inserted in Schlemm's canal wall and ciliary process stroma. The framework of these muscular fibers may according to its insertions participate in the visual accommodation mechanism and outflow of the aqueous humor system.

We have studied the vasculature of the retina, ciliary processes and choroid in the North American raccoon (Procyon lotor), a nocturnal mammal, using light and scanning electron microscopic examination of corrosion casts. We carried out an identical study in the crab-eating macaque (Macaca fascicularis), which forages only during the daytime, in order to compare the ocular vasculature with that of nocturnal mammals. Our observations in raccoons demonstrated a photoreceptor layer associated with rich lymph and a poorly vascularized retina. The meridian region of the eye, which lies in the horizontal plane and pass around the optic disc, had a markedly sparse capillary network. This horizontal sparse vascular band may correspond to a visual streak. Ciliary process capillaries were delicate, and formed a well-developed and compact network. Choriocapillaries were quite thin and formed a coarse capillary network. This contrasted with the dense retinal and well-extended choroidal capillary networks noted in the macaques. Our findings suggest that the sparse retinal capillary network in raccoons is extremely beneficial for photon capture, thereby allowing the raccoon to see well at night, as the retinal vessels restrict the inflow of photons toward the photoreceptors. The well-developed lymph probably compensates for the sparse retinal capillaries and choriocapillaries and nourishes the retina in the nocturnal raccoon.

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.

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.

Purpose: To systematically study the anterior segment morphometric measurements by ultrasound biomicroscopy (UBM) in a large population of normal eyes, to evaluate their intra-observer reproducibility. Two new measurements are proposed and also tested concerning their intra-observer reproducibility., Methods: One hundred ninety eyes of 101 patients with normal ophthalmic examination were studied by UBM. Anterior chamber depth (ACD) along with 11 different morphometric parameters that characterize the anterior segment morphometry were measured in the superior, nasal, inferior and temporal meridians. All measurements were performed twice with a minimum interval of four weeks between them., Results: There were no statistical significant differences between the first (M1) and second (M2) measurements and regarding each studied parameter except for 2 variables in 2 meridians in the right eyes (OD) and 2 variables of one meridian in the left eyes (OS). Even in these cases, the largest difference between M1 and M2 was less than 0.006 mm, which was not clinically significant. The new tested parameters also presented a good intra-observer reproducibility., Conclusion: This study confirmed the good intra-observer reproducibility of the variables that characterize anterior segment morphometry by UBM.

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.

The aim of the present paper was to reexamine fine structural characteristics and glycogen topochemistry of ciliary processes in small laboratory mammals (hamsters, guinea-pigs and mice). A two-layered epithelium continuously covered all ciliary processes. The epithelium consisted of inner nonpigmented and outer pigmented cells whose apices faced each other. They were linked by desmosomes and tight junctions. Basal cell aspects showed extensively interdigitating processes adjacent to the inner (rarely also outer) basal lamina. The ciliary process core was made up of reticular fibers, few fibrocytes, and capillaries with or without fenestrations. No glycogen particles were found in the ciliary epithelium using the PA-TSC-SP procedure.

Histopathological findings of ciliary epithelium and muscle were discussed in the inflammatory conditions that are induced in endophthalmitis, blood-aqueous barrier destruction, cyclocryotherapy and cyclophotocoagulation. The clinical results were also discussed regarding the reduction of aqueous production and uveoscleroplasty which are introduced by the experimental cyclophotocoagulation at the pars plicata/pars plana. Tissue destruction by inflammatory reaction occurred in not only the ciliary body but also in the retina if the endophthalmitis had progressed to the point of severity. The post-inflammatory reactive hypertrophy of nonpigmented and pigmented ciliary epithelium was mild at pars plicata and the anterior portion of the pars plana. On the other hand, it was severe at the posterior portion of the pars plana and the ora serrata, and it was clear that the cyclitic membrane developed into proliferative vitreoretinopathy. Adult pig cultured nonpigmented ciliary epithelium showed proliferated tissue resembling the cyclitic membrane. These results show that nonpigmented ciliary epithelium has a strong proliferative activity as much the same as fibroblasts. The destruction of the blood-aqueous barrier was produced by corneal perforation, paracentesis, prostaglandin E1 (PGE1) subconjunctival injection, and hyperosmotic agent intraophthalmic artery injection. The localization of the tissue destruction was made clear at the beginning portion of the ciliary process and the posterior part of the pars plana. The repetitive instillation of latanoprost and cyclosporin A eye drops showed the same localization of the tissue damage. These results suggest that the beginning portion of the ciliary process is vulnerable to inflammatory agents. Cyclosporin A might produce a toxic reaction on nonpigmented ciliary epithelium, and latanoprost destroys the blood-aqueous barrier. Cyclocryotherapy produces severe necrosis of the pigmented ciliary epithelium and melanocytes, and atrophy of pigmented ciliary epithelium and ciliary muscle, and the proliferation of nonpigmented ciliary epithelium follows. Because cyclocryotherapy is a blind therapy, hypotony and phthisis bulbi will occur if the pars plicata is damaged severely, and the intraocular pressure (IOP) decrease will not be achieved if pars plana is damaged locally. Finally, we demonstrated that it is difficult to predict the therapeutic outcome of cyclocryotherapy. Cyclophotocoagulation of the pars plicata and the pars plana produced severe necrosis of pigmented ciliary epithelium and melanocytes, and atrophy of pigmented ciliary epithelium and ciliary muscle, and the proliferation of nonpigmented ciliary epithelium followed. Cyclitic membrane was developed from the proliferation of nonpigmented ciliary epithelium depending on the severity of photocoagulation. From these experiments the complete destruction of the pars plicata might result in for hypotony and phthisis bulbi. The reduction of aqueous production for angle-closure glaucoma was achieved by moderate cyclophotocoagulation of the pars plicata. On the other hand, moderate cyclophotocoagulation of the pars plicata for open-angle glaucoma produced the obstruction of uveoscleral aqueous outflow which compensated for the reduction of aqueous production. From these results we suggested that moderate cyclophotocoagulation of the pars plana for open-angle glaucoma might be necessary to effect the increase of uveoscleral aqueous outflow. Moderate and severe cyclophotocoagulation of the transit area between the pars plicata and the pars plana might bring about reduction of aqueous production and the increase of uveoscleral aqueous outflow.

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.