[Glaucomatous trabeculum: An inflammatory trabeculopathy? [French translation]]. / Trabéculum glaucomateux : une trabéculopathie inflammatoire ?

Glaucoma is an optic neuropathy in which the primary risk factor is increased intraocular pressure (IOP), attributed to increased resistance to trabecular outflow of aqueous humor (AH). This resistance is believed to result from trabecular degeneration secondary to chronic oxidative stress and cellular senescence but may also involve inflammatory mechanisms whose roles are little known. In fact, inflammatory processes play a major role in the pathophysiology of glaucoma to varying degrees, affecting all structures of the eye, including the ocular surface, the anterior and posterior segments, and even the visual pathways of the brain. These processes are thought to result from dysfunction of a regulatory, protective para-inflammation, becoming chronic and harmful in glaucoma. While the mechanisms of the retinal inflammation which accelerates the degeneration of retinal ganglion cells (RGC) as well as the inflammation of the ocular surface aggravated by long-term use of preserved glaucoma eye drops have been described for several years, very little is known about the pathophysiology of trabecular inflammation in glaucoma. The objective of this literature review is to provide a synthesis of knowledge on the roles and mechanisms of inflammation in both the healthy and glaucomatous trabecular meshwork, as well as its role in the pathophysiology of glaucoma. Therefore, after a review of the mechanisms of cellular senescence and oxidative stress - sources of trabecular inflammation, we will approach the study of the expression and roles of the main inflammatory mediators within the trabecular meshwork. Finally, we will discuss current knowledge on the toxicity of glaucoma eye drops and their preservatives on the ocular surface and trabecular meshwork as well as their role in trabecular inflammation.

[The trabecular meshwork: Structure, function and clinical implications. A review of the littérature (French translation of the article)]. / Le trabéculum : structure, fonction et implications cliniques. Une revue de la littérature.

Glaucoma is a blinding optic neuropathy, the main risk factor for which is increased intraocular pressure (IOP). The trabecular meshwork, located within the iridocorneal angle, is the main pathway for drainage of aqueous humor (AH) out of the eye, and its dysfunction is responsible for the IOP elevation. The trabecular meshwork is a complex, fenestrated, three-dimensional structure composed of trabecular meshwork cells (TMC) interdigitated into a multilayered organization within the extracellular matrix (ECM). The purpose of this literature review is to provide an overview of current understanding of the trabecular meshwork and its pathophysiology in glaucoma. Thus, we will present the main anatomical and cellular bases for the regulation of aqueous humor outflow resistance, the pathophysiological mechanisms involved in trabecular dysfunction in the various types of glaucoma, as well as current and future therapeutic strategies targeting the trabecular meshwork.

The trabecular meshwork: Structure, function and clinical implications. A review of the literature.

Glaucoma is a blinding optic neuropathy, the main risk factor for which is increased intraocular pressure (IOP). The trabecular meshwork, located within the iridocorneal angle, is the main pathway for drainage of aqueous humor (AH) out of the eye, and its dysfunction is responsible for the IOP elevation. The trabecular meshwork is a complex, fenestrated, three-dimensional structure composed of trabecular meshwork cells (TMC) interdigitated into a multilayered organization within the extracellular matrix (ECM). The purpose of this literature review is to provide an overview of current understanding of the trabecular meshwork and its pathophysiology in glaucoma. Thus, we will present the main anatomical and cellular bases for the regulation of aqueous humor outflow resistance, the pathophysiological mechanisms involved in trabecular dysfunction in the various types of glaucoma, as well as current and future therapeutic strategies targeting the trabecular meshwork.

An In-Depth View of the Porcine Trabecular Meshwork Proteome.

The house swine (Sus scrofa domestica Linnaeus 1758) is an important model organism regarding the study of neurodegenerative diseases, especially ocular neuropathies such as glaucoma. This is due to the high comparability of the porcine and human eye regarding anatomy and molecular features. In the pathogenesis of glaucoma, the trabecular meshwork (TM) forms a key ocular component in terms of intraocular pressure (IOP) elevation. Thereby, functional TM abnormalities are correlated with distinct proteomic alterations. However, a detailed analysis of the TM proteome has not been realized so far. Since the porcine eye has high potential as a model system to study ocular diseases such as glaucoma, the present study focuses on the in-depth analysis of the porcine TM proteome. By use of a bottom-up (BU) mass spectrometric (MS) platform utilizing electrospray ionization liquid chromatography tandem MS (LC-ESI-MS/MS) considering database-dependent and peptide de novo sequencing, more than 3000 TM proteins were documented with high confidence (FDR < 1%). A distinct number of proteins with neuronal association were revealed. To the best to our knowledge, many of these protein species have not been reported for TM tissue before such as reelin, centlein and high abundant neuroblast differentiation-associated protein AHNAK (AHNAK). Thereby, AHNAK might play a superordinate role in the TM regarding proposed tissue involvement in barrier function. Also, a high number of secretory proteins could be identified. The generated TM proteomic landscape underlines a multifunctional character of the TM beyond representing a simple drainage system. Finally, the protein catalogue of the porcine TM provides an in-depth view of the TM molecular landscape and will serve as an important reference map in terms of glaucoma research utilizing porcine animal models, porcine TM tissues and/or cultured TM cells.

Regulation of intraocular pressure by microRNA cluster miR-143/145.

Glaucoma is a major cause of irreversible blindness worldwide. Elevated intraocular pressure (IOP), which causes optic nerve damage and retinal ganglion cell death, is the primary risk factor for blindness in glaucoma patients. IOP is controlled by the balance between aqueous humor secretion from the ciliary body (CB) and its drainage through the trabecular meshwork (TM). How microRNAs (miRs) regulate IOP and glaucoma in vivo is largely unknown. Here we show that miR-143 and miR-145 expression is enriched in the smooth muscle and trabecular meshwork in the eye. Targeted deletion of miR-143/145 in mice results in significantly reduced IOP, consistent with an ~2-fold increase in outflow facilities. However, aqueous humor production in the same mice appears to be normal based on a microbeads-induced glaucoma model. Mechanistically, we found that miR-143/145 regulates actin dynamics and the contractility of TM cells, consistent with its regulation of actin-related protein complex (ARPC) subunit 2, 3, and 5, as well as myosin light chain kinase (MLCK) in these cells. Our data establish miR-143/145 as important regulators of IOP, which may have important therapeutic implications in glaucoma.

Bevacizumab clearance through the iridocorneal angle following intravitreal injection in a rat model.

Antivascular endothelial growth factor (Anti-VEGF) agents have been widely used for a variety of ocular disorders. The etiology of sustained ocular hypertension following intravitreal administration of anti-VEGF agents is yet to be unraveled. Our study investigates and characterizes the presence of intravitreally injected bevacizumab in the aqueous outflow channels of a rat model. Choroidal neovascularization (CNV) was induced by diode laser photocoagulation to the right eye of twelve Brown Norway rats. Bevacizumab (25 mg/ml) was injected intravitreally after 3 days. Immediately after bevacizumab injection, and 3, 6, 24 and 48 h later, animals were euthanized for immunofluorescence staining. Donkey anti-human IgG labeled with Alexa Fluor(®) 488 was used for bevacizumab immunoreactivity detection. Anti-CD31 antibody was used as a marker for Schlemm's canal endothelial cells. Untreated eyes were used as negative controls. The intensity of the immunostaining was analyzed qualitatively. Bevacizumab immunoreactivity was found in the aqueous outflow channels including the trabecular meshwork and Schlemm's canal immediately after injection, and declined incrementally within the following hours. Forty-eight hours after the injection, no bevacizumab staining was detected in the aqueous outflow channel structures. Our manuscript demonstrates the presence of bevacizumab in the trabecular meshwork and Schlemm's canal structures after intravitreal injection in a CNV induced rat model. Bevacizumab molecules passed through the aqueous outflow channels within 48 h after intravitreal bevacizumab injection.

Imaging the effects of prostaglandin analogues on cultured trabecular meshwork cells by coherent anti-stokes Raman scattering.

PURPOSE: The aim of this study was to nondestructively monitor morphological changes to the lipid membranes of primary cultures of living human trabecular meshwork cells (hTMC) without the application of exogenous label. METHODS: Live hTMC were imaged using two nonlinear optical techniques: coherent anti-Stokes Raman scattering (CARS) and two-photon autofluorescence (TPAF). The hTMC were treated with a commercial formulation of latanoprost (0.5 µg/mL) for 24 hours before imaging. Untreated cells and cells treated with vehicle containing the preservative benzalkonium chloride (BAK; 2 µg/mL) were imaged as controls. After CARS/TPAF imaging, hTMC were fixed, stained with the fluorescent lipid dye Nile Red, and imaged by conventional confocal microscopy to verify lipid membrane structures. RESULTS: Analysis of CARS/TPAF images of hTMC treated with latanoprost revealed multiple intracellular lipid membranes absent from untreated or BAK-treated hTMC. Treatment of hTMC with sodium fluoride or ouabain, agents shown to cause morphological changes to hTMC, also did not induce formation of intracellular lipid membranes. CONCLUSIONS: CARS microscopy detected changes in living hTMC morphology that were validated by subsequent histological stain. Prostaglandin-induced changes to hTMC involved rearrangement of lipid membranes within these cells. These in vitro results identify a novel biological response to a class of antiglaucoma drugs, and further experiments are needed to establish how this effect is involved in the hypotensive action of prostaglandin analogues in vivo.

Two-photon immunofluorescence characterization of the trabecular meshwork in situ.

PURPOSE: To develop an in situ model to study biological responses and glaucoma pathology in the human trabecular meshwork (TM). Characteristic TM cell- and glaucoma-associated markers were localized in situ in relation to the tissue's autofluorescent structural extracellular matrix (ECM) by two-photon excitation fluorescence optical sectioning (TPEF). METHODS: Human donor corneoscleral (CS) tissue containing the intact aqueous drainage tract was incubated with dexamethasone (Dex) or TGF-ß1, and immunostained for epifluorescence (EF) microscopy with antibodies to myocilin and alpha smooth muscle (α-SMA). Separate specimens were labeled for Type-IV collagen and fibronectin. Nuclei were stained with Hoechst 33342. Multimodal TPEF was used to visualize EF, intravital dyes, and autofluorescence (AF) in situ. Three-dimensional (3D) localization of fluorescence within the TM was analyzed using reconstruction software. RESULTS: Autofluorescent beams, perforated sheets, and fibers, consistent with the uveal (UV), CS, and juxtacanalicular (JCT) meshwork, respectively, were captured at different depths of the TM. Type-IV collagen EF distinctly outlined the AF beams in a location consistent with basement membrane. Fibronectin EF showed a diffuse reticular pattern throughout the TM. TGF-ß1-induced α-SMA expression, which was distributed perinuclearly in cells among autofluorescent structures. Dex-induced myocilin expression had both cytosolic and extracellular distributions. CONCLUSIONS: The authors have localized markers that are characteristic of TM cells and relevant to glaucoma pathogenesis in situ using multimodal TPEF without conventional histological embedding and sectioning. Protein expression was inducible in situ and could be analyzed with respect to cells and the ECM within the 3D environment of the human TM.

Trabecular meshwork in normal and pathological eyes.

PURPOSE: The impact of glycosaminoglycans on intraocular pressure in glaucoma patients and in healthy young or aging subjects is explored. MATERIALS AND METHODS: Thirty small autoptic samples were harvested from the tissue localized around the iridocorneal angle of the eye, taking care not to cause aesthetic damage. The samples came from three groups (young, old, and subjects with glaucoma). All samples were divided in two fragments and both were used for morphological and biochemical analyses. Quantitative data were obtained from image analysis to correlate with biochemical values. All results were statistically analyzed. RESULTS: The findings show the following changes of iridocorneal angle are caused by glycosaminoglycans both in aging and in glacoumatous patients: (1) deposition of fibrous granular material and increased electron density of the structures close to the iridocorneal angle; and (2) strong decrease of hyaluronic acid content and increase of sulfated glycosaminoglycans. CONCLUSIONS: Similar to what happens in other tissues in the body, glycosaminoglycans of the human iridocorneal angle undergo physiological and pathological changes. The trabecular meshwork is the structure responsible for the regulation of the aqueous humor outflow that is often altered in primary open-angle glaucoma patients.

Non-housekeeping genes expressed in human trabecular meshwork cell cultures.

PURPOSE: To identify non-housekeeping genes definitively expressed in the human trabecular meshwork (TM). METHODS: Microarray gene expression data on TM cultured cells from four studies were compared. Genes that were queried in at least three studies and assessed to be expressed in at least three studies were considered definitively expressed genes of the human TM. Housekeeping genes were removed from this set of genes. The non-housekeeping TM gene profile was analyzed for pathway enrichment and microRNA targeting, using bioinformatics tools. The results were compared with results of previous non-array based studies. RESULTS: Nine hundred and sixty-two genes were identified as non-housekeeping TM expressed genes. Analysis of these by Kyoto Encyclopedia of Genes and Genomes led to identification of two enriched biologic pathways that achieved a highly significant Bonferroni p-value (p≤0.01): focal adhesion and extracellular matrix (ECM)-receptor interaction. Many of the genes were previously implicated in TM-related functions and the TM-associated disease glaucoma; however, some are novel. MicroRNAs known to be expressed in the trabecular meshwork were predicted to target some of the genes. Ten genes identified here, ALDH1A1 (aldehyde dehydrogenase 1 family, member A1), CDH11 (cadherin 11, type 2, OB-cadherin), CXCR7 (chemokine (C-X-C motif) receptor 7), CHI3L1 (chitinase 3-like 1), FGF2 (fibroblast growth factor 2), GNG11 (guanine nucleotide binding protein [G protein], gamma 11), IGFBP5 (insulin-like growth factor binding protein 5), PTPRM (protein tyrosine phosphatase, receptor type, M), RGS5 (regulator of G-protein signaling 5), and TUSC3 (tumor suppressor candidate 3), were also reported as TM expressed genes in three earlier non-microarray based studies. CONCLUSIONS: A transcriptome consisting of 962 non-housekeeping genes definitively expressed in the human TM was identified. Multiple genes and microRNAs are proposed for further study for a better understanding of TM physiology.

Progenitors for the corneal endothelium and trabecular meshwork: a potential source for personalized stem cell therapy in corneal endothelial diseases and glaucoma.

Several adult stem cell types have been found in different parts of the eye, including the corneal epithelium, conjunctiva, and retina. In addition to these, there have been accumulating evidence that some stem-like cells reside in the transition area between the peripheral corneal endothelium (CE) and the anterior nonfiltering portion of the trabecular meshwork (TM), which is known as the Schwalbe's Ring region. These stem/progenitor cells may supply new cells for the CE and TM. In fact, the CE and TM share certain similarities in terms of their embryonic origin and proliferative capacity in vivo. In this paper, we discuss the putative stem cell source which has the potential for replacement of lost and nonfunctional cells in CE diseases and glaucoma. The future development of personalized stem cell therapies for the CE and TM may reduce the requirement of corneal grafts and surgical treatments in glaucoma.

Cadmium is more toxic on volume bone mineral density than tissue bone mineral density.

It has been showed that Cd induces low areal bone mineral density, but we do not know the effect of Cd on cubic bone density. This study was aimed to investigate the effects of Cd on volumetric bone mineral density (VBMD) and tissue bone mineral density (TBMD) in male rats. Twenty-four Sprague-Dawley male rats were randomly divided into four groups that were given cadmium chloride by subcutaneous injection at doses of 0, 0.1, 0.5, and 1.5 mg/kg body weight for 8 weeks, respectively. Then, microcomputed tomography scanning was performed on the proximal tibia, and region of interest was reconstructed using microview software. The VBMD, bone volume fraction of rats treated with 1.5 mg Cd/kg, were significantly decreased compared to control (p < 0.01). The trabecular numbers of rats exposed to Cd were all significantly decreased relative to control (p < 0.05). The trabecular separation of rats treated with 1.5 mg Cd/kg was obviously increased compared to control (p < 0.01). However, Cd had no obvious influence on TBMD. Cd induced low VBMD but not TBMD; Cd effect on bone may be related with trabecular bone loss but not with trabecular bone demineralization.

Trabecular meshwork and lens partitioning of corticosteroids: implications for elevated intraocular pressure and cataracts.

OBJECTIVE: To determine whether adverse effects such as elevated intraocular pressure and cataracts, which are lower with dexamethasone when compared with fluocinolone acetonide or triamcinolone acetonide, may be explained in part by the differences in drug lipophilicity and partitioning of these drugs into the trabecular meshwork and lens. METHODS: The n-octanol/phosphate-buffered saline (pH 7.4) partition coefficient (log distribution coefficient [D]) and bovine/human ocular tissue partition coefficients were determined for triamcinolone, prednisolone, dexamethasone, fluocinolone acetonide, triamcinolone acetonide, and budesonide at 37°C. RESULTS: The log D of the corticosteroids ranged from 0.712 to 2.970. The ranges of tissue:PBS partition coefficients following drug incubation at 0.4, 2.0, and 10.0 µg/mL were 0.35 to 1.56, 0.30 to 2.12, and 0.30 to 1.95, respectively, for the bovine lens, 0.87 to 4.18, 0.71 to 4.40, and 0.69 to 5.86, respectively, for the human lens, and 2.98 to 9.48, 2.41 to 9.16, and 1.71 to 9.96, respectively, for the bovine trabecular meshwork. In general, tissue partitioning showed a positive correlation with log D. Dexamethasone, with lipophilicity less than triamcinolone acetonide and fluocinolone acetonide, exhibited the least amount of partitioning in the trabecular meshwork and lens among these 3 corticosteroids commonly used for treating diseases at the back of the eye. CONCLUSION: Binding of corticosteroids to the trabecular meshwork and lens increases as drug lipophilicity increases. CLINICAL RELEVANCE: Less lipophilic corticosteroids with limited partitioning to the trabecular meshwork and lens may result in reduced incidence of elevated intraocular pressure and cataracts.

Alterations in microRNA expression in stress-induced cellular senescence.

We investigated miRNA expression changes associated with stress-induced premature senescence (SIPS) in primary cultures of human diploid fibroblast (HDF) and human trabecular meshwork (HTM) cells. Twenty-five miRNAs were identified by miRNA microarray analysis and their changes in expression were validated by TaqMan real-time RT-PCR in three independent cell lines of HTM and HDF. SIPS in both HTM and HDF cell types was associated with significant down-regulation of four members of the miR-15 family and five miRNAs of the miR-106b family located in the oncogenic clusters miR-17-92, miR-106a-363, and miR-106b-25. SIPS was also associated with up-regulation of two miRNAs (182 and 183) from the miR-183-96-182 cluster. Transfection with miR-106a agomir inhibited the up-regulation of p21(CDKN1A) associated with SIPS while transfection with miR-106a antagomir led to increased p21(CDKN1A) expression in senescent cells. In addition, we identified retinoic acid receptor gamma (RARG) as a target of miR-182 and showed that this protein was down-regulated during SIPS in HDF and HTM cells. These results suggest that changes in miRNA expression might contribute to phenotypic alterations of senescent cells by modulating the expression of key regulatory proteins such as p21(CDKN1A) as well as by targeting genes that are down-regulated in senescent cells such as RARG.

Immunohistochemical comparison of differentiation markers on paraffin and plastic embedded human bone samples.

To assess bone pathologies and bone regeneration immunohistochemistry may provide additional information compared to conventional histology. However, the effectiveness of this technique is limited due to tissue fixation, preparation and embedding. For bone tissue the standard immunohistological procedure includes formalin fixation, followed by decalcification and paraffin embedding. This may lead to a badly preserved trabecular bone structure but allows antibody application. Alternatively, methyl-methacrylate (MMA) resin may be used for embedding, thus circumventing the decalcification procedure. In this study immunohistology of typical bone markers was compared using human bone samples fixed either with alcohol or formalin and further decalcified and embedded in paraffin and decalcified or non decalcified samples embedded in Technovit 9100 New(R). On semi-thin sections immunohistochemistry with bone markers osteocalcin, osteonectin, osteopontin, collagen type I and the cellular markers CD34 and CD68 was performed. Independent of the fixative used, Technovit 9100 New embedded non-decalcified bone yielded a stronger immunostaining for all markers when compared to decalcified bone embedded either in methyl-methacrylate or paraffin. In addition there was a better preservation of the trabecular bone morphology. The immunohistochemical results demonstrate that Technovit 9100 New as a low-temperature acrylic resin embedding method can be favoured over paraffin embedding.

Stem cell markers in the human posterior limbus and corneal endothelium of unwounded and wounded corneas.

PURPOSE: The corneal endothelium is a monolayer of cells in the posterior cornea that is responsible for maintaining a clear cornea. Corneal endothelial cells may be induced to divide, but it has been held that they do not divide in the normal cornea of an adult human. Some studies have suggested that a stem cell population for the corneal endothelium exists. This population could give rise to mature corneal endothelial cells and may reside either in the peripheral corneal endothelium or in the adjacent posterior limbus. This study was initiated to demonstrate the presence of such stem cells in the region of the posterior limbus and to show the response of these cells to corneal wounding. METHODS: Unwounded and wounded corneas with their attached limbal sections were analyzed by immunofluorescence for the presence of nestin, telomerase, Oct-3/4, Pax-6, Wnt-1, and Sox-2. Alkaline phosphatase activity was observed with an enzyme-based reaction that produced a fluorescent product. RESULTS: In the unwounded cornea, stem cell markers nestin, alkaline phosphatase, and telomerase were found in the trabecular meshwork (TM) and in the transition zone between the TM and the corneal endothelial periphery (including Schwalbe's line). Telomerase was also present in the peripheral corneal endothelium. When wounded corneas and their attached limbii were tested, the same markers were found. However, after wounding, additional stem cell markers, Oct-3/4 (in the TM) and Wnt-1 (in both the TM and the transition zone), appeared. Moreover, the differentiation markers Pax-6 and Sox-2 were seen. Pax-6 and Sox-2 were also manifest in the peripheral endothelium post-wounding. CONCLUSIONS: Well documented specific stem cell markers were found in the TM and the transition zone of the human posterior limbus. Wounding of the corneas activated the production of two additional stem cell markers (Oct-3/4, Wnt-1) as well as two differentiation markers (Pax-6, Sox-2), the latter of which also appeared in the corneal endothelial periphery. It is suggested that stem cells reside in the posterior limbus and respond to corneal wounding to initiate an endothelial repair process. The stem cells may also contribute to a normal, slow replacement of corneal endothelial cells.

Actin structure in the outflow tract of normal and glaucomatous eyes.

To characterize the in situ distribution of actin in Schlemm's canal endothelium (SCE) and juxtacanalicular tissue (JCT) cells in glaucomatous human eyes, and compare to the distribution in normal eyes. Fresh human eye bank eyes were perfused and fixed at pressure (n=27 normal eyes and 22 confirmed glaucomatous eyes). Schlemm's canal was opened by microdissection and outflow tissues were labelled for confocal microscopy to visualize F-actin, nuclei, laminin and/or CD31. Images were acquired in Z-series from the inner wall of Schlemm's canal, juxtacanalicular tissue and outer corneoscleral meshwork. In normal eyes, inner wall Schlemm's canal endothelial (SCE) cells showed a dense peripheral F-actin band, as previously described. JCT cells showed a more random and amorphous F-actin distribution. In glaucoma eyes, peripheral F-actin bands were less common in inner wall SCE cells; instead, F-actin was more centrally located within the cell and appeared "tangled". These actin tangles were also prominent in JCT cells of glaucoma eyes. Glaucoma eyes also demonstrated structures with features of cross-linked actin networks (CLANs), and more frequent occurrence of punctuate actin concentrations. There was a significant degree of heterogeneity, with some regions from glaucomatous eyes appearing normal and vice versa. F-actin architecture in human outflow pathway cells in situ differs between normal and glaucoma eyes, with glaucomatous tissue showing a more "disordered" actin architecture overall. Some of these changes are likely due to effects secondary to administration of anti-glaucoma medications. Most of the changes that we observed could potentially affect the biomechanical properties of the outflow pathway tissues in glaucoma, but their role in the pathogenesis of ocular hypertension remains unclear.

First look at the effect of overexpression of TIGR/MYOC on the transcriptome of the human trabecular meshwork.

Wild-type TIGR/MYOC is a secreted protein implicated in the development of steroid glaucoma. Mutations in TIGR/MYOC have been linked to some patients who develop elevated intraocular pressure (IOP) and glaucoma. Because there is evidence of some other factors contributing to the TIGR/MYOC causative role in glaucoma, and because substantial increased levels of a particular cellular mRNA and protein might alter expression of other host genes, we began to investigate the effect of TIGR/MYOC overexpression on the transcriptome of human trabecular meshwork cells. We used a recombinant adenovirus carrying wild-type TIGR/MYOC cDNA, primary HTM cells, 300 viral particles per cell and U133 Affymetrix GeneChips. Our results indicate that 2361 out of the 22,284 genes (10.6%) were altered more than two-fold (p

Immunolocalization of CYP1B1 in normal, human, fetal and adult eyes.

CYP1B1 is a cytochrome P450 enzyme implicated in autosomal recessive primary congenital glaucoma (PCG). The mechanism and function of CYP1B1 in the development of the PCG phenotype is unknown. Previously, investigators have reported detection of Cyp1b1 mRNA in the ciliary body and epithelium and neuroepithelium in the developing mouse eye, employing in situ hybridization techniques. Similarly, additional investigators have detected CYP1B1 mRNA in the iris, ciliary body, non-pigmented ciliary epithelial line, cornea, retinal-pigment epithelium, and retina in the human adult eye, using Northern blotting. This study was designed to immunolocalize CYP1B1 protein in the various ocular structures of normal, human fetal and adult eyes. Normal fetal and adult eyes were immunolabeled with a polyclonal antibody against human CYP1B1 using indirect immunofluorescence, and then compared with appropriate controls. The intensity of immunolabeling of the various ocular structures was assessed by qualitative and semi-quantitative techniques. In the anterior segment anti-CYP1B1 immunoreactivity (IR) was detected early in fetal development in the primitive ciliary epithelium. As well, the most intense CYP1B1 IR was in the non-pigmented ciliary epithelium. In addition, CYP1B1 IR was also present in the corneal epithelium and keratocytes, both layers of the iris pigmented epithelium, and retina. However, CYP1B1 IR was absent in the trabecular meshwork in all of the samples. In general, CYP1B1 immunolabeling in the human fetal eyes was more intense when compared to adult eyes. CYP1B1 IR was primarily immunolocalized to the non-pigmented ciliary epithelium and early in fetal development. In addition, CYP1B1 IR was not detected in the trabecular meshwork. These findings suggest that the abnormalities in the development of the trabecular meshwork in PCG may result from diminished or absent metabolism of important endogenous substrates in the ciliary epithelium due to non-functional CYP1B1 enzyme.