Search

Background: Cytomegalovirus (CMV) can cause a wide panel of ocular infections. The involvement of CMV as a cause of anterior uveitis in the immunocompetent patient is recent and remains poorly understood., Objective: To investigate the presence of CMV in anterior uveal tissues of immunocompetent corneal donors., Study Design: We collected aqueous humor, iris, and ciliary body from both eyes of 25 donors died at the Limoges University Hospital between January 2020 and July 2021. CMV serology was determined for all patients from post-mortem blood sample. Ocular tissues were split in 2 fragments for qPCR and 2 for histological analysis. CMV genomes copies were quantified by Multiplex qPCR after DNA extraction., Results: 16 of 25 patients (64%) displayed positive CMV serology, with a median age of 67 years. Viremia was positive in 3 of 16 (19%) CMV-positive patients. No CMV DNA copies were found from the aqueous humor samples. CMV DNA was detected in iris and ciliary body of 28 of 32 eyes of seropositive donors, and 5 of 18 eyes of seronegative donors. The median viral copy number [IQR] was 2.41 × 10 2 [8.91 × 10 1 - 1.01 × 10 3 ] copies/1 × 10 6 cells in the CMV-positive group and 0.00 [0.00 - 3.54 × 10 2 ] copies/1 × 10 6 cells in the CMV-negative group (p<0.001). Histology and immunohistochemistry did not reveal any CMV lesions from any sample., Conclusion: CMV DNA was found in iris and ciliary body of immunocompetent seropositive patients, but also, although less frequently, from seronegative donors. These results highlight mechanisms of infection, latency and reactivation of CMV in ocular tissues., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Mesectodermal leiomyoma is a benign tumor of smooth muscle of the ciliary body, which is derived from the neural crest., Clinical Case: We report the case of a 35-year-old Mexican woman with visually impaired and blurred vision of the right eye of 2 months duration. The clinical and imaging presuntional diagnosis was adenoma of the non pigmented epithelium of the ciliary body and it was surgically resected. Microscopically, the tumor was composed of cells with round nuclei and scant cytoplasm without atypia or mitosis, arranged in a fibrillary background. The immunohistochemical markers for vimentin, muscle specific actin, smooth muscle actin and calponin were strongly positive in the cytoplasm of the neoplastic cells, while for glial fibrillary acidic protein and S-100 protein were negative in the same cellular population., Conclusions: Mesectodermal leiomyoma of the ciliary body is benign tumor of smooth muscle extremely rare in this location. Until now, there are just 25 previous reported cases in the literature and, the main differential diagnosis is uveal malignant melanoma, therefore some eyes were enucleated. The ultrabiomicroscopy, A and B-scan imaging studies are useful in the evaluation, however, is mandatory the microsocpic examination with routine and histochemical stains as well as the use of immunohistochemical markers such as vimentin, specific muscle actin, smooth muscle actin andcalponin to stablish the smooth muscle origin of this neoplasm, and rule out other malignant neoplams such as malignant melanoma.

A rapid, selective and sensitive method for quantification of latanoprost free acid in rabbit aqueous humor (AH) and ciliary body (CB) using reverse phase-high performance liquid chromatography coupled with electrospray ionization (ESI)-mass spectrometry/mass spectrometry has been developed and validated. Quantification in AH and CB was achieved by stable isotope dilution employing tetra-deuterated analog of latanoprost free acid, used as internal standard. Sample preparation was based on protein precipitation with methanol in AH, and on liquid extraction with a mixture of ethyl acetate and isopropanol 60:40 (v/v) in CB. Elution was achieved on an octylsilica (C8) column, using an isocratic elution method. Detection was performed on a triple quadrupole mass spectrometer, using ESI in positive ion selected reaction monitoring mode. Calibration curves were linear in the validated concentration ranges of 10-160 ng/mL in AH and 80-1280 ng/g in CB. The accuracy and precision values, obtained from three different sets of quality control samples, each analyzed in triplicate on three different days, were within the generally accepted criteria for analytical methods (< 15%). The limit of detection was 30.66 pg/mL in AH and 237.75 pg/g in CB. The assay proved to be accurate and precise when applied to the in vivo study of latanoprost free acid in rabbit AH and CB after single administration of an eye drops containing latanoprost., (Copyright © 2011 John Wiley & Sons, Ltd.)

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.

A rapid, sensitive and selective method for the simultaneous quantification of carteolol and dorzolamide in rabbit aqueous humor (AH) and ciliary body (CB) has been developed and validated using reversed phase-high performance liquid chromatography (RP-HPLC) with isocratic elution coupled with atmospheric pressure chemical ionization mass spectrometry/mass spectrometry (APCI-MS/MS). The analytes and nadolol (used as internal standard, IS) were purified from AH by protein precipitation. The sample preparation from CB was based on a two steps extraction procedure at different pH, utilizing a liquid-liquid extraction with a mixture of ethyl acetate, toluene and isopropanol 50:40:10 (v/v) at pH 8, followed by a second extraction with ethyl acetate at pH 11. The combined organic extracts were then back extracted into 0.1% aqueous trifluoroacetic acid (TFA). The accuracy and precision values, calculated from three different sets of quality control samples analyzed in sestuplicate on three different days, were within the generally accepted criteria for analytical methods (<15%). The assay proved to be accurate and precise when applied to the in vivo study of carteolol and dorzolamide in rabbit AH and CB after single administration of an eye drops containing both drugs., (2010 Elsevier B.V. All rights reserved.)

We report here the development and validation of an LC/MS/MS method for the rapid and accurate quantitation of brimonidine in ocular tissues and fluids using brimonidine-d(4) as an internal standard (IS). Brimonidine was extracted from retina, iris/ciliary body, and vitreous humor samples with an acetonitrile:water (1:1) solution followed by sonication and vortexing. Aliquots of aqueous humor, iris/ciliary body, retina, and vitreous humor samples were diluted with acetonitrile containing IS and were separated on a reverse-phase HPLC column under isocratic conditions. Brimonidine (m/z transition: 292-->212) and the internal standard (m/z transition: 296-->216) were analyzed via multiple-reaction monitoring (MRM) in the positive electrospray mode on a 4000 Q TRAP instrument. The total analysis time for each sample was less than 2.0 min. The calibration curves for brimonidine (1-1000 ng/mL) were constructed using a linear regression with 1/x(2) weighing. The lower limit of quantitation for brimonidine was 1.0 ng/mL for aqueous humor, 10 ng/g for iris/ciliary body, 12.5 ng/g for retina, and 1.6 ng/g for vitreous humor. Intra-day and inter-day estimates of accuracy and precision were within 15% of their nominal values indicating that the method is reliable for quantitation of brimonidine in ocular tissues and fluids.

Purpose: Interstitial cells of Cajal were identified in the gastrointestinal tract of several species, with close relation to the enteric nervous system. Since it was recognized that interstitial cells of Cajal express the gene product of c-kit, we performed immunohistochemistry for c-kit protein in ciliary muscle specimens of monkeys' eyes., Methods: Eight eyes from four adult male new world monkeys (Cebus apella) were studied. After blocking endogenous peroxidase activity and nonspecific protein binding, 1:100 dilution of mouse monoclonal antibody against c-kit human oncoprotein was applied to tissues. Antigen-antibody reaction was visualized using the avidin-biotinylated horseradish peroxidase complex in each slide., Results: We observed some groups of fusiform c-kit expressing cells located amongst muscle bundles of the ciliary muscle. Other pigment cells and mast cells were also observed., Conclusion: C-kit expressing cells observed in the ciliary muscle of Cebus apella, showed no similarity to melanocytes or mast cells and they could be associated with their gastrointestinal interstitial cells of Cajal counterpart.

A case of adenoma of the non-pigmented ciliary epithelium with smooth muscle differentiation is reported. This uncommon ocular tumor affected a 36-year-old woman, and had caused decreased visual acuity and a total cataract. Ultrasound biomicroscopy disclosed an associated persistent hyperplasic primary vitreous (PHPV). Sectoral cyclectomy with removal of the mass and intracapsular cataract extraction were performed. The tumor was diffusely positive for vimentin, smooth muscle actin, NSE, and S-100, focally for CD68 and Melan-A, and was negative for desmin, EMA, HMB-45, and CD99. Occasional cells reacted for cytokeratin. The proliferation index, as assessed by Ki-67, was below 10%. The overlying non-neoplastic ciliary epithelium was positive for vimentin, NSE, and S-100. Myofilaments are not totally unexpected in ciliary adenomas; however, such a diffuse and strong positivity for smooth muscle actin, as in the present case, has only been observed in one case before, but should be considered in the differential diagnosis of these neoplasms.

Primary uveal-tract neoplasms are extremely rare in childhood; the most common lesions found are melanocytic. We report here the case of a 7-year-old girl who underwent enucleation of the right eye with clinical suspicion of choroid melanoma as a result of a ciliary body mass that extended to the posterior chamber. Histologically, the neoplasm featured spindle cell morphology, atypia, and mitoses. The tumor expressed smooth muscle alpha actin, pan-actin HHF-35, and desmin, whereas immunohistochemistry for melanocytic markers, such as S-100, Melan-A, and HMB-45, was negative. Based on these features, the diagnosis of leiomyosarcoma of the ciliary body was firmly established. Although several leiomyomas have been reported in the literature, there are only 2 previously reported cases of primary leiomyosarcoma of the uveal tract. Immunohistochemical expression of muscle proteins allowed distinction from the most common melanocytic tumors arising in this location.

Case Report: We report a rare clinical case of unilateral ciliary body teratoid medulloepithelioma presented first with infantile cataract, subsequently masquerading as chronic granulomatous anterior uveitis, followed by appearance of a tumour over the iris surface., Comments: Diagnosis of the tumour in the early stages allows proper management and avoids enucleation.

Purpose: To determine whether there is an association between dietary omega-3 (omega-3) fatty acid intake, age, and intraocular pressure (IOP) caused by altered aqueous outflow., Methods: Sprague-Dawley rats were fed either omega-3-sufficient (omega-3(+)) or omega-3-deficient (omega-3(-)) diets from conception. The diets had 7% lipid content. The omega-3(+) diet contained safflower, flaxseed, and tuna oils (5.5:1.0:0.5), and the omega-3(-) diet contained safflower oil only. Intraocular pressure was measured at 5 to 40 weeks of age under light anesthesia (omega-3(+), n = 39; omega-3(-), n = 48). Aqueous outflow was determined at 45 weeks in a subgroup of animals (omega-3(+), n = 15; omega-3(-), n = 22) using pulsed infusion. Ciliary body tissues (n = 6 per group) were assayed for fatty acid content by thin-layer and gas-liquid chromatography in both diet groups., Results: Animals raised on omega-3(+) diets had a 13% decrease in IOP at 40 weeks of age (13.48 +/- 0.32 mm Hg vs. 15.46 +/- 0.29 mm Hg; P < 0.01). When considered as a change in IOP relative to 5 weeks of age, the omega-3(+) group showed a 23% decrease (P < 0.001). This lower IOP in the omega-3(+) diet group was associated with a significant increase (+56%; P < 0.001) in outflow facility and a decrease in ocular rigidity (-59%; P < 0.001). The omega-3(+) group showed a 3.3 times increase in ciliary body docosahexaenoic acid (P < 0.001)., Conclusions: Increasing dietary omega-3 reduces IOP with age because of increased outflow facility, likely resulting from an increase in docosanoids. This indicates that dietary manipulation may provide a modifiable factor for IOP regulation. However, further studies are needed to consider whether this can modify the risk for glaucoma and can play a role in treatment of the disease.

The phospholipids present in uveal (iris/ciliary body and choroid) and retinal bovine ocular melanosomes were identified using mass spectrometry. Similar phospholipid content is found for the two types of uveal melanosome, with sphingomyelin being the major species. Significant differences are found between the uveal and retinal melanosome. Glycerophosphoethanolamine (GPEtn) is the major species in the retinal pigment epithelium (RPE); 93% of the GPEtn contain polyunsaturated fatty acids, notably docosahexanoic acid and arachidonic acid, in the sn-2 position. RPE melanosomes also contain detectable quantities of glycerophosphoserine and glycerophosphate; these species were not detected in the uveal samples. While the structural and functional roles of melanosomal lipids largely remain to be determined, these different lipid compositions reported herein offer new insights into the roles of melanosomes in the different ocular tissues.

Caveolae are flask-shaped invaginations of the plasma membrane found in many cell types. Caveolae play a role in lipid transport, endocytosis, signal transduction, and cell transformation. Expression of caveolin-1, the principal component of caveolae and a regulator of caveolae-dependent signaling and endocytosis, was investigated in lens epithelial cells and lens fiber cells in wild-type (wt) and SPARC-null mice and normal human donors in vivo and in vitro. RT-PCR, immunofluorescence and immunoblot analyses of human and murine ocular tissues revealed that caveolin-1 was expressed in the corneal epithelium, corneal endothelial cells, and blood vessels of iris, ciliary body and retina, but minimal in the normal lens epithelia or fiber cells of murine and human lens. In contrast, caveolin-1 was significantly up-regulated in mesenchymal-transdifferentiated lens epithelia in SPARC-null cataract lenses. In addition, lens epithelial cells from primary culture or from cultures of immortalized lens epithelial cell lines expressed significant amounts of caveolin-1. The lens epithelial cells expressed epidermal growth factor (EGF) receptor and were responsive to EGF-mediated cell proliferation, but they did not show EGF-dependent caveolin-1 tyrosine phosphorylation. Caveolin-1 might have a role in the process of epithelial-mesenchymal transdifferentiation (EMT) in the lens, the most common cause of vision loss in human secondary cataracts.

Background: Topical nitric oxide-releasing dexamethasone (NCX1021) may avoid the negative effects of dexamethasone phosphate., Aims: To obtain more information on the role of nitric oxide in glaucoma and to compare a nitric oxide-releasing dexamethasone with dexamethasone phosphate with regard to intraocular pressure (IOP) and ocular haemodynamics in an experimental rabbit model., Methods: Six rabbits were treated with dexamethasone phosphate 0.1% in the right eye and with NCX1021 in the left eye for 5 weeks. The parameters considered were IOP, nitric oxide marker levels in aqueous humour, ocular haemodynamics of ophthalmic artery (by means of colour Doppler imaging), expression of endothelial nitric oxide synthase (eNOS)in ciliary processes and histology of ciliary bodies., Results: Dexamethasone increased IOP levels, NCX1021 did not. Nitrite and cyclic guanosine monophosphate levels in aqueous humour were lowered by dexamethasone and increased by NCX1021. Resistivity index of the ophthalmic artery was increased, eNOS expression was reduced and ciliary bodies showed histological lesions in dexamethasone-treated eyes, not in NCX1021-treated ones., Conclusions: NCX1021 may avoid the IOP increase, impairment of ocular blood flow and the morphological changes in the ciliary bodies possibly induced by corticosteroid treatment.

Previously we described the release of hr44 from the ciliary epithelium to coincide with the loss of the late endosomal/lysosomal marker protein CD63 in mildly inflamed rat eyes. We showed that both proteins are released with microvesicles into the supernatant of cultured retinal pigmented epithelial cells (ARPE-19). Here we wish to determine whether there is a concomitant loss of fas-ligand (FasL) in vivo and whether ocular epithelial cells have secretory lysosomes similar to T cells, from where FasL and hr44 could derive. FasL plays an important role in immunity, immune cell homeostasis and in the maintenance of immune privilege in the eye. However the mode of release of FasL from ocular epithelial cells or its activity in the eye is not fully understood. In normal rat eyes, FasL was detected in the epithelia of the iris and ciliary body and in the anterior region of the retinal pigmented epithelium. FasL is expressed constitutively and is associated with vesicular structures in the normal ciliary epithelium but is not detectable in the ciliary epithelium of inflamed eyes. In contrast, the posterior RPE, which under normal conditions is negative for FasL and hr44 showed strong staining for both molecules in areas adjacent to sub-retinal inflammatory infiltrates. Immunofluorescence and Western blot analysis indicated that cultured ARPE-19 cells express both the soluble and membrane form of FasL. The intracellular concentration of FasL was significantly increased in cells grown in presence of interferon (INF)-gamma. The microvesicles released by cultured ARPE-19 cells and previously shown to be positive for hr44 and CD63 are also positive for membrane FasL. Expression of a recombinant fluorescent construct of FasL together with immuno-staining for CD63 demonstrated that FasL localises to the endocytic compartment of ARPE-19 cells and of melanoma cells (positive control). In cells with lysosomes devoid of specialised secretory functions (e g. HeLa cells) recombinant FasL localised to the cell membrane, demonstrating that RPE cells have secretory lysosomes. We suggest that ocular epithelial cells release soluble FasL and the membrane form of FasL with vesicles. Both forms may contribute in different ways to the effectiveness of the ocular immune response and immune privilege.

The prereceptor regulation of glucocorticoids (GCs) by 11beta-hydroxysteroid dehydrogenase type-1 (11beta-HSD1), a bidirectional isozyme that interconverts active (cortisol) and inactive (cortisone) GCs, is an established determinant of GC function in tissues such as liver, adipose and bone. Although the therapeutic use of GCs is abundant in ophthalmic practice, where GC interactions with nuclear receptors modulate gene transcription, the prereceptor regulation of endogenous cortisol is not well described in ocular tissues. Recent descriptive studies have localised 11beta-HSD1 to the human corneal epithelium and non-pigmented epithelium (NPE) of the ciliary body, indicating a link to corneal epithelial physiology and aqueous humour production. In this study, we characterise the functional aspects of the autocrine regulation of GCs in the anterior segment of the rabbit eye. Using our in-house generated primary antibody to human 11beta-HSD1, immunohistochemical analyses were performed on paraffin-embedded sections of whole New Zealand white albino rabbits, (NZWAR) eyes. As in human studies, 11beta-HSD1 was localised to the corneal epithelium and the NPE. No staining was seen in the albino 'pigmented' ciliary epithelium. Specific enzyme assays for oxo-reductase (cortisone-->cortisol) and dehydrogenase (cortisol-->cortisone) activity indicated predominant 11beta-HSD1 oxo-reductase activity from both the intact ciliary body tissue (n=12, median 2.1 pmol/mg per h and range 1.25-2.8 pmol/mg per h; P=0.006) and primary cultures of corneal epithelial cells (n=12, median 3.0 pmol/mg per h and range 1.0-7.4 pmol/mg per h, P=0.008) compared with dehydrogenase activity (median 1.0 pmol/mg per h and range 0.5-2.0 pmol/mg per h; median 0.5 pmol/mg per h and range 0.25-1.9 pmol/mg per h respectively). These findings were supported by expression of 11beta-HSD1 protein as visualised by Western blotting of ciliary body tissue and immunocytochemistry of corneal epithelial cells. Reduction of corneal epithelial cell proliferation was seen after primary cultures were co-incubated with cortisol and cortisone. 11beta-HSD1 activity was not demonstrated in naïve conjunctival fibroblasts or corneal stromal keratocytes. Our results indicate that the distribution of 11beta-HSD1 in the rabbit resembles that of the human eye and activates cortisone to cortisol in both corneal and uveal tissues. The NZWAR provides a suitable in vivo model for the further evaluation of 11beta-HSD1 activity in the eye, especially its role in corneal epithelial and ciliary body physiology.

1 In the bovine ciliary muscle, stimulation of muscarinic receptors with carbachol (CCh) opens two types of non-selective cation channels (NSCCS and NSCCL) with widely different unitary conductances (100 fS and 35 pS). Here we examined the dependence of the activity of NSCCS on the agonist (CCh) concentration by whole-cell voltage clamp in freshly isolated bovine ciliary muscle cells. We also examined the sensitivity of CCh-evoked NSCCS currents to several muscarinic receptor antagonists. 2 The voltage clamp experiments were carried out using Ba2+ as the charge carrier, as this divalent cation is the most permeant for NSCCS of the alkali and alkaline earth metal ions hitherto examined, whereas it is relatively impermeant to NSCCL. For the dose-activation relationship obtained, the apparent dissociation constant K was estimated to be 0.5 +/- 0.2 microm (n = 31), a value of an order of magnitude smaller than the one reported for CCh-evoked NSCCL currents in our previous experiments. 3 In the dose-inhibition experiments we observed that the CCh-evoked NSCCS currents were inhibited by the muscarinic antagonists with the following potency sequence: atropine approximately 4-DAMP >> pirenzepine > AF-DX116, indicating that the activation of NSCCS by CCh is mediated by an M3 muscarinic receptor. 4 We have previously shown by reverse transcriptase-polymerase chain reaction that the bovine ciliary muscle contains mRNAs for several transient receptor potential channel homologues (TRPC1, TRPC3, TRPC4 and TRPC6) which are attracting attention as molecular candidates for receptor-operated NSCCs. In the present experiments, we succeeded in visually identifying these TRPCs in the plasma membrane of cultured bovine ciliary muscle cells by immunofluorescence microscopy.

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.

Context: Molecular analyses indicate that periodic acid-Schiff (PAS)-positive (laminin-rich) patterns in melanomas are generated by invasive tumor cells by vasculogenic mimicry. Some observers, however, consider these patterns to be fibrovascular septa, generated by a stromal host response., Objective: To delineate differences between vasculogenic mimicry patterns and fibrovascular septa in primary uveal melanomas., Design: Frequency distributions, associations with outcome, and thicknesses of trichrome-positive and PAS-positive looping patterns were determined in 234 primary uveal melanomas. Sequential sections of 13 additional primary uveal melanomas that contained PAS-positive/trichrome-negative looping patterns were stained for type I and type IV collagens, laminin, and fibronectin. Real-time quantitative polymerase chain reaction was performed on RNA from cultured uveal melanoma cells for the expression of COL1A1, COL4A2, and fibronectin., Results: Trichrome-positive loops were encountered less frequently than PAS-positive loops (10% vs 56%, respectively). Death from metastatic melanoma was strongly associated with PAS-positive (P < .001) but not with trichrome-positive (P = .57) loops. Trichrome-positive loops were significantly thicker than PAS-positive loops (P < .001). The PAS-positive patterns stained positive for laminin, type I and type IV collagens, and fibronectin. Type I collagen was detected within melanoma cells and focally within some PAS-positive patterns. Real-time quantitative polymerase chain reaction revealed 3-fold, 25-fold, and 97-fold increases, respectively, in expression of COL4A2, fibronectin, and COL1A1 by invasive pattern-forming primary melanoma cells compared with poorly invasive non-pattern-forming cells., Conclusions: Fibrovascular septa are rare and prognostically insignificant in uveal melanomas, whereas vasculogenic mimicry patterns are associated with increased mortality. Type I collagen, seen focally in some vasculogenic mimicry patterns, may be synthesized by tumor cells, independent of a host stromal response.

Background: Bcl-2 protooncogene alterations are involved in tumorigenesis and may have prognostic ramifications., Aims: To investigate normal ocular structures and choroidal melanoma for: (1) Bcl-2 protein expression (semiquantitative staining values: SI, staining intensity; PP, percentage of positive cells; and IRS, immunoreactivity score) and (2) any associations between the staining values and clinicopathological features in these lesions., Materials/methods: Bcl-2 protein expression was analysed in 24 choroidal melanomas using immunoperoxidase staining methods., Results: Bcl-2 protein expression was seen in corneal epithelium, lens epithelium, the ciliary body, and retinal cells. In these structures, the mean (SEM) values were: 1.1 (0.1), 1.6 (0.3), 1.1 (0.1), and 2.3 (0.3), respectively, for SI; 1.6 (0.2), 1.7 (0.1), 1.7 (0.2), and 1.7 (0.2) for PP, respectively; and 1.9 (0.4), 2.7 (0.5), 1.9 (0.1), and 4.0 (0.8), respectively, for IRS. Based on Bcl-2 immunoreactivity, the lesions were divided into two groups. The first group comprised 12 tumours with Bcl-2 expression. Bcl-2 expression was significantly higher in this group compared with normal ocular structures (1.5 (0.1) v 2.8 (0.2), 1.7 (0.1) v 3.5 (0.1), and 2.6 (0.3) v 9.3 (0.9) for mean (SEM) SI, PP, and IRS scores, respectively; p = 0.00). The second group comprised 12 tumours lacking Bcl-2 protein expression. There was no significant correlation between Bcl-2 protein expression and most of the clinicopathological features of these lesions., Conclusions: Bcl-2 protein expression is altered in choroidal melanomas.

The porcine eye serves as a model to study various functions of the aqueous outflow system. To compare these data with the primate eye, a detailed investigation of the distribution of contractile properties and of the innervation of the outflow region was conducted in the porcine eye. In all quadrants of the anterior eye segment, elastic fibres connected the ciliary muscle (CM) with the well-developed scleral spur (ScS) and also partly with the corneoscleral trabecular meshwork (TM) and the loops of the collecting outflow channels. Immunohistochemistry with antibodies against smooth muscle alpha-actin revealed intense staining of the CM and some myofibroblasts in the ScS and outer TM. In addition to a few cholinergic and aminergic nerve fibres in the outflow region, numerous substance P- and calcitonin-gene related peptide-positive nerve fibres and nerve endings were found near the outflow loops of the porcine TM. Although the porcine CM serves rather as a tensor choroideae muscle than as a muscle for accommodation, the innervation and morphology of the collecting outflow channel loops and of the expanded TM between the ScS and the cornea showed close similarities to the primate eye.

Recently discovered endogenous opioid peptides such as nociceptin are known to modulate neurotransmitter release of primary afferent neurons (especially substance P, SP) and they have also been demonstrated in peripheral nerve fibres. The aim of this study was to investigate the opioid peptidergic innervation of the anterior eye segment and to compare it with the innervation pattern of SP in order to shed light on the functional relationship between these peptides. Anterior eye segments of 20 rat eyes were cut in a tangential plane and the sections stained with antibodies against SP, nociceptin, nocistatin, endomorphin 1 and 2, leu-enkephalin and met-enkephalin. Sections of the spinal cord or brain were used as positive controls. Numerous SP-immunoreactive nerve fibres were found in the conjunctiva, cornea, episclera, trabecular meshwork, iris and ciliary body. A weak staining for met-enkephalin and leu-enkephalin could only be found in the iris and anteriormost ciliary body. Nerve fibres immunoreactive for nociceptin, nocistatin, and endomorphin 1 or 2 could not be detected in any part of the anterior eye segment. It is tempting to speculate that the opioid peptidergic innervation of the anterior ciliary body may play a role in the modulation of intraocular inflammation.

Nitrergic nerve fibres of intrinsic and extrinsic origin constitute an important component of the autonomic innervation in the human eye. The intrinsic source of nitrergic nerves are the ganglion cells in choroid and ciliary muscle. In order to obtain more information on the origin of extrinsic nitrergic nerves in the human eye, we obtained superior cervical, ciliary, pterygopalatine and trigeminal ganglia from six human donors, and stained them for neuronal nitric oxide synthase (nNOS) and nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-D). In the superior cervical ganglia, nNOS/NADPH-D-positive varicose axons were observed whereas perikarya were consistently negative. Fewer than 1% of perikarya in the ciliary ganglia were labelled for nNOS/NADPH-D. The diameter of nNOS/NADPH-D-positive ciliary perikarya was between 8 and 10 microm, which was markedly smaller than the diameter of the vast majority of negative perikarya in the ciliary ganglion. More than 70% of perikarya in the pterygopalatine ganglia were intensely labelled for both nNOS and NADPH-D. In trigeminal ganglia, 18% of perikarya were nNOS/NADPH-D-positive. The average diameter of trigeminal nNOS/NADPH-D perikarya was between 25 and 45 microm. Pterygopalatine and trigeminal ganglia are the most likely sources for extrinsic nerve fibres to the human eye.

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.

The purpose of the present study was to compare the localization of prostaglandin E(2) receptor subtypes in normal human and mouse ocular tissues. Paraffin embedded sections of normal human and mouse (129 Sv/Ev) eyes were treated with EP(1), EP(2), EP(3) and EP(4) specific antibodies and subsequently incubated with Alexa Fluor secondary antibody (Ex/Em=555/571) to detect the presence of EP receptor proteins. Fluorescence of the localized antibodies was visualized in a Carl Zeiss Microscope (Axiovert 200) and photographed using Carl Zeiss Axiocam camera. In mice EP(1) and EP(3) receptor subtypes were only moderately expressed, EP(3) receptor expression being almost negligible. In human cornea and iris ciliary body, EP(1) and EP(3) receptors were prominently expressed. EP(4) receptor was expressed moderately in human and mouse ocular tissues. EP(2) receptor was the most prominently and abundantly expressed receptor in both human and mouse ocular tissues. It is concluded that the pattern of the distribution of EP receptor subtypes in the ocular tissues are similar in human and mouse. Thus, 129 Sv/Ev strains of mice would make an appropriate animal model for studying the ocular pathophysiological roles of prostaglandin receptor agonists.

The objective of this study was to determine the cellular localization of P2Y(2) receptor gene expression in rabbit and primate ocular tissues using the technique of non-isotopic in situ hybridization. Fresh frozen whole eye from a New Zealand White rabbit and whole eye and eyelid from a rhesus macaque were cut into 5 microm thick sections and mounted onto glass slides. In situ hybridization was performed on ocular cryosections using digoxigenin-labeled P2Y(2) receptor riboprobes. Alkaline phosphatase-conjugated anti-digoxigenin antibody was used to localize riboprobe hybridization, which was subsequently visualized by staining with a precipitating alkaline phosphatase substrate. Cytoplasmic staining indicative of antisense riboprobe hybridization to P2Y(2) receptor mRNA was observed in the palpebral and bulbar conjunctival epithelium, including goblet cells, the corneal epithelium, and in meibomian gland sebaceous and ductal cells. Staining was also observed in both layers of the ciliary body epithelium, subcapsular epithelium of the lens, and corneal endothelium. In the posterior eye, staining was observed in various layers of the retina, including ganglion cell, inner nuclear, inner segment and retinal pigment epithelium layers, in the optic nerve head, and in a variety of structures within the choroid. No specific staining of sense riboprobe was seen on any of the ocular structures. These results demonstrate that the P2Y(2) receptor gene is expressed in a variety of ocular cells types and suggest that P2Y(2) receptors are associated with diverse physiological functions throughout the eye.

WE-14 is derived from the cell-specific posttranslational processing of chromogranin A (CgA) in subpopulations of neuroendocrine cells and neurons. Region- and site-specific chromogranin A, pancreastatin and WE-14 antisera were employed to study the generation of WE-14 in porcine ocular tissues. No chromogranin A or pancreastatin immunostaining was detected in ocular tissue. Immunohistochemistry detected WE-14 immunostaining in a network of nerve fibre bundles and nerve fibres throughout the limbus, cornea, iris and ciliary body with sparse nerve fibres detected throughout the choroid and sclera. Retinal analysis detected intense WE-14 immunostaining in large ovoid cells in the ganglion cell layer with weak immunostaining in a population of small cells in the inner nuclear layer; weak immunostaining was detected within the fibre layers in the inner plexiform layer. Quantitatively, the highest WE-14 tissue concentration was recorded in aqueous retinal and corneal extracts with lower concentrations in the sclera, choroid and anterior uveal tissues. Chromatographic profiling resolved a minor chromogranin A-like immunoreactant and a predominant immunoreactant co-eluting with synthetic human WE-14. This is the first study to demonstrate that WE-14 is generated in neuronal fibres primarily innervating the anterior chamber and in select cell populations in the retina.

A case of rare tumor of the iris and ciliary body in a 24-year old woman is presented, which was diagnosed as adenoma of the nonpigmented ciliary body epithelium. The diagnosis was confirmed immunohistochemically.

A 37-year-old woman had a mass in her left ocular globe. Uveal melanoma was suspected and enucleation was performed. Microscopically, the lesion proved to be a typical case of mesectodermal leiomyoma of the ciliary body. According to some authors, the peculiar neural appearance of this tumor could be the reflection of its probable origin from mesectodermal smooth muscle. Immunohistochemical analysis showed reactivity for muscle-specific actin and negativity for desmin, S-100 protein, HMB-45, EMA, and GFAP. Our results do not support the proposed neuroectodermical origin of this tumor, since coexpression of muscular and neural markers was not observed.

Purpose: To describe detailed phenotypic and genotypic analysis of a pleomorphic adenocarcinoma of the ciliary epithelium (CE)., Case Report: An 86-year-old white woman developed an enlarging mass protruding from her previously eviscerated left eye 2 months postoperatively. Based on light and ultrastructural microscopy, the final diagnosis was a pleomorphic adenocarcinoma of the ciliary epithelium (CE)., Discussion: Cell proliferation indices confirmed the unusually rapid growth rate of this tumor; the peridiploid DNA content might explain the relatively low incidence of distant metastases. An imbalance of the chromosome 6 was also found by Comparative Genomic Hybridization (CGH).

Purpose: The isolation and analysis of human ocular RNA is problematic due to variables such as rapid degradation, tissue composition, and melanin contamination. The purpose of this work was to optimize an extraction protocol for the isolation of intact total RNA from a variety of diverse human ocular tissues and to employ RT-PCR to assess the expression of mRNA coding for all eight prostanoid receptors., Methods: Total RNA was extracted from human iris, ciliary body, choroid, and retina using an RNeasy(R) Midi Kit. Total RNA was extracted from human cornea, sclera, and optic nerve using Tri-Pure(R) Isolation Reagent. 1.0 microgram of total RNA was reverse transcribed into cDNA and subsequently amplified by PCR (35 cycles) using primers designed against each of the human prostanoid receptor cDNAs. PCR products were analyzed by gel electrophoresis and endonuclease digestion., Results: The total yield and quality of RNA derived from each tissue varied according to tissue composition and the isolation method employed. RT-PCR analysis revealed that each tissue expressed all prostanoid receptor mRNAs, however, 50 cycles of PCR was required to visualize FP receptor expression in scleral tissue. In all cases, prostanoid receptor mRNA expression was significantly lower than in human nonpregnant myometrium, which was used as the positive control., Conclusions: The different cellular composition of each ocular tissue ultimately dictated the methodology to be employed for the isolation of total RNA. Thus, two extraction protocols were optimized for the isolation of intact high quality RNA from a variety of human ocular tissues. The identification of all prostanoid receptor mRNAs in a diverse set of human ocular tissues suggests potential mechanisms for prostanoid-based therapeutics aimed at IOP reduction and stimulates speculation as to additional physiological and or pathophysiological roles mediated by prostanoids.

Objective: The purpose of this study was to investigate the uveitogenic activity of autoantigen in the bovine iris-ciliary body., Methods: Urea soluble fraction of boveine melanin associated antigen (USF-BMAA) was isolated from the bovine iris-ciliary body biochemically and determined by SDS-PAGE as well as amino acid analysis. Lewis rats and F344 rats were immunized with USF-BMAA emulsified with equal volume complete Freud's adjuvant and Bordetella pertussis., Results: A strongly stained protein band was observed in the USF-BMAA by SDS-PAGE whose molecular weight is approximate 64 000. Amino acid analysis of USF-BMAA showed that it contains 17 kinds of amino acids with high content of Glu, Leu and Asp. The experimental melanin associated antigen-induced uveitis (EMIU) was successfully incited in both eyes of the Lewis and F344 rats. The inflammation was mainly located in the anterior uvea, and spontaneously recovered. Mild focal choroiditis was present in the rats with severe lesion. However, the inflammation was not observed in the retina and pineal gland., Conclusions: USF-BMAA may be the major part of autoantigen of the uveal tract with uveitogenic activity. Unlike experimental autoimmune uveoretinitis (EAU) which is incited by the retinal soluble antigen (S-Ag) in the rats, no involvement of the retina and pineal gland is found in EMIU.

Recent studies in mouse and rabbit eyes have begun to identify the molecular constituents of tight and adherens junctions that represent the structural equivalent of the blood--aqueous barrier (BAB). These species are commonly used as experimental models to examine the pathobiology of anterior uveitis, an inflammatory condition in which the junctions of the BAB are compromised. Because it was unclear whether major molecular elements of the junctions in these species were the same as those in humans, the goal of this study was to determine if the junction related proteins ZO-1 and occludin are present in normal human ciliary epithelium and iridial vascular endothelium. To determine their presence, sections of human anterior uvea were probed in 14 normal, human, eyebank eyes immunolabelled with antibodies to ZO-1, and occludin, and confocal microscopy was used to examine them. Phalloidin staining for F-actin was also assessed. ZO-1 and occludin were both localized along the apico-lateral surfaces of the non-pigmented ciliary epithelium and the interendothelial clefts of iris blood vessels. In both locations, the distribution of occludin was more focal than seen for ZO-1. ZO-1 was also found along the apical surfaces between the pigmented and non-pigmented ciliary epithelial cell layers. The distribution of these proteins supports the notion that occludin is more specifically associated with tight junctions than is ZO-1 in the normal human BAB. No change in this distribution was found with increasing age. These data are consistent with findings reported previously in rabbit ciliary epithelium and iridial vascular endothelium, indicating the relevance of experimental induced uveitis studies in rabbit, as a model of BAB breakdown in human uveitis., (Copyright 2002 Elsevier Science Ltd.)

The human NBC1 gene encodes two electrogenic sodium-bicarbonate cotransport proteins, pNBC1 and kNBC1, which are candidate proteins for mediating electrogenic sodium-bicarbonate cotransport in ocular cells. Mutations in the coding region of the human NBC1 gene in exons common to both pNBC1 and kNBC1 result in a syndrome with a severe ocular and renal phenotype (blindness, band keratopathy, glaucoma, cataracts, and proximal renal tubular acidosis). In the present study, we determined the pattern of electrogenic sodium-bicarbonate cotransporter protein expression in rat eye. For this purpose, pNBC1- and kNBC1-specific antibodies were generated and used to detect these NBC1 protein variants by immunoblotting and immunocytochemistry. pNBC1 is expressed in cornea, conjunctiva, lens, ciliary body, and retina, whereas the expression of kNBC1 is restricted to the conjunctiva. These results provide the first evidence for extrarenal kNBC1 protein expression. The data in this study will serve as a basis for understanding the molecular mechanisms responsible for abnormalities in ocular electrogenic sodium-bicarbonate cotransport in patients with mutations in the NBC1 gene.

11beta-hydroxysteroid dehydrogenase (11beta-HSD) regulates local actions of corticosteroids at glucocorticoid and mineralocorticoid receptors. Corticosteroids are thought to play important roles in ocular function. However, mechanisms of intraocular corticosteroid action are still unclear. Therefore, in this study, we examined the immunohistochemical localization of 11beta-HSD type 1 (11beta-HSD1), 11beta-HSD type 2 (11beta-HSD2), mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) in human ocular tissues from patients (6 months to 78 years of age; n = 10) retrieved from surgical pathology files. Both 11beta-HSD2 and MR immunoreactivity was detected only in non-pigmented epithelium of the ciliary body, but was undetectable in cornea, lens, iris, retina, choroid and sclera, in all the cases examined. GR was detected in all cell types in the human eye. 11beta-HSD1 immunoreactivity was not detected in the human eye in this study. These results suggest that 11beta-HSD2 play an important role in human ocular mineralocorticoid action, such as the production of aqueous humor, in the ciliary body. The widespread expression of GR suggests that glucocorticoids may play an important role in the function and homeostasis of the human eye.

The purpose of this study was to determine the content of total, free, peptide-bound, protein-bound, soluble- and insoluble collagen hydroxyproline (Hyp) in tissues of bovine eye. The results show that lens had the highest content of free Hyp. This was followed by cornea, retina, iris and aqueous humor. The difference between the Hyp content of lens and iris (p < 0.01) and aqueous humor (p < 0.001) was significant. The peptide-bound Hyp was highest in iris followed by cornea, ciliary body, sclera, lens, aqueous humor and retina. Significant differences (p < 0.001) was observed between the concentration of peptide-bound Hyp of iris and ciliary body, sclera, lens, aqueous humor and retina. Protein-bound Hyp was highest in iris, followed by ciliary body, sclera, cornea, lens, retina and aqueous humor. The difference between the protein-bound Hyp levels of iris and sclera, cornea, lens, retina and aqueous humor was significant (p < 0.001). No peptide-bound and protein-bound Hyp was detected in vitreous humor. Iris had the highest content of total Hyp. This was followed by cornea, ciliary body, sclera, lens, retina, vitreous humor and aqueous humor. The difference in the Hyp content of iris with ciliary body, sclera, lens, retina, vitreous humor and aqueous humor was significant (p < 0.001). Cornea had significantly (p < 0.001) higher content of soluble- and insoluble-collagen Hyp as compared to other tissues. This was followed by ciliary body, sclera, lens, iris and retina. Iris had the highest content of collagen. This was followed by cornea, ciliary body, sclera, lens, retina, vitreous humor and aqueous humor. The difference in the collagen content of iris with ciliary body, sclera, lens, retina, vitreous humor and aqueous humor was significant (p < 0.001).

The intrinsic electrical properties of identified choroid and ciliary neurons of the chick ciliary ganglion were examined by patch-clamp recording methods. These neurons are derived from a common pool of mesencephalic neural crest precursor cells but innervate different target tissues and have markedly different action potential waveforms and intrinsic patterns of repetitive spike discharge. Therefore it is important to determine whether these cell types express different types of plasma membrane ionic channels, and to ascertain the developmental stages at which these cell types begin to diverge. This study has focused on large-conductance Ca(2+)-activated K(+) channels (K(Ca)), which are known to regulate spike waveform and repetitive firing in many cell types. Both ciliary ganglion cell types, identified on the basis of size and somatostatin immunoreactivity, express a robust macroscopic K(Ca) carried by a kinetically homogeneous population of large-conductance (BK-type) K(Ca) channels. However, the kinetic properties of these channels are different in the two cell types. Steady-state fluctuation analyses of macroscopic K(Ca) produced power spectra that could be fitted with a single Lorentzian curve in both cell types. However, the resulting corner frequency was significantly lower in choroid neurons than in ciliary neurons, suggesting that the underlying K(Ca) channels have a longer mean open-time in choroid neurons. Consistent with fluctuation analyses, significantly slower gating of K(Ca) channels in choroid neurons was also observed during macroscopic activation and deactivation at membrane potentials positive to -30 mV. Differences in the kinetic properties of K(Ca) channels could also be observed directly in single-channel recordings from identified embryonic day 13 choroid and ciliary neurons. The mean open-time of large-conductance K(Ca) channels was significantly greater in choroid neurons than in ciliary neurons in excised inside-out patches. The developmental expression of functional K(Ca) channels appears to be regulated differently in the two cell types. Although both cell types acquire functional K(Ca) at the same developmental stages (embryonic days 9-13), functional expression of these channels in ciliary neurons requires target-derived trophic factors. In contrast, expression of functional K(Ca) channels proceeds normally in choroid neurons developing in vitro in the absence of target-derived trophic factors. Consistent with this, extracts of ciliary neuron target tissues (striated muscle of the iris/ciliary body) contain K(Ca) stimulatory activity. However, K(Ca) stimulatory activity cannot be detected in extracts of the smooth muscle targets of choroid neurons.

Previous studies have used conventional electron microscopy and freeze fracture to identify the morphological equivalents of the blood-aqueous barrier in the mammalian eye. These equivalents are the tight junctions that form a part of the apicolateral junctional complex between adjacent non-pigmented ciliary epithelial cells and the tight junctions present between endothelial cells of the iris vasculature. Recent investigations have begun to unravel the molecular assembly of the tight junction and some variability has been found. Our goal in the present study was to probe the ciliary epithelium and iris vascular endothelium of the rabbit eye to determine if certain molecular constituents associated with tight junctions in other tissues are also present as parts of the blood-aqueous barrier. The selected constituents were occludin, ZO-1, and a representative, adherens junction-related cadherin. Immunohistochemical and immunoelectron microscopic methods were used. The results showed that occludin was distributed exclusively at known locations of tight junctions. ZO-1 was also expressed at these locations but its distribution extended beyond that of occludin, along the adjacent membranes. Pan-cadherin was expressed ubiquitously within the ciliary epithelium and negligibly in iris vascular endothelium. Our results demonstrate that occludin and ZO-1 are integral components of the blood-aqueous barrier of the normal rabbit eye.

Recently, it has been shown that the choroid of the duck eye harbours approximately 1,000 intrinsic choroidal neurons positive for vasoactive intestinal polypeptide and neuronal nitric oxide synthase. Their connections and functional significance are largely unknown. This study was performed to establish a typical chemical code for these neurons and to define their targets by using immunocytochemistry and confocal laser scanning microscopy. Almost all intrinsic choroidal neurons coexpressed galanin (GAL), vasoactive intestinal polypeptide (VIP), and neuronal nitric oxide synthase (nNOS)/NADPH-diaphorase. A few stained for GAL and/or nNOS only. Among extrinsic ganglia, GAL/VIP/nNOS coexpressing neurons were only found in the pterygopalatine ganglion where they accounted for approximately 30% of the neuronal population. Thus, GAL/VIP/nNOS-positive nerve fibres around branches of the ciliary artery and within the nonvascular smooth muscle stroma of the choroid may originate mainly from intrinsic neurons and to some extent in a subpopulation of pterygopalatine ganglionic neurons exhibiting the same chemical coding. Close contacts of GAL-positive fibres upon intrinsic choroidal neurons may indicate reciprocal connections between them. Thus, intrinsic choroidal neurons may represent peripherally displaced pterygopalatine ganglion neurons forming a local network for regulation of vascular and nonvascular smooth muscle tone in the duck choroid. They may be integrated in the neuronal circuitry controlling intraocular pressure, choroidal thickness, accommodation, and axial bulbus length., (Copyright 2000 Wiley-Liss, Inc.)

Purpose: Investigations into the biology of resident and infiltrating immune cells in the uveal tract of the rodent eye have been greatly aided by the use of tissue wholemount methods. These methods offer a number of advantages over conventional histological and frozen section techniques. The purpose of this article is to provide a detailed step by step guide to aid others who may wish to use this method., Methods: A detailed description of whole-body perfusion fixation, dissection and isolation of the iris-ciliary body from the anterior segment and the choroid from the posterior segment is provided. In addition, the techniques used to handle whole tissue pieces during single and double immunohistochemical staining protocols, as well as the staining protocols themselves, are described., Results: In refining the techniques described, the author has catalogued a number of frequent problems which compromise immunohistochemical staining results. A troubleshooting guide aimed to help identify the cause of common problems and with some suggested remedies is provided., Conclusions: Although tissue wholemounts are frequently used in retinal research, a similar approach to investigating the components of the uveal tract has only recently been applied. The methods described in this article will provide sufficient detail for other investigators to obtain maximum benefit from this alternative approach and provide an additional technique to assist in their investigations of ocular immunobiology.

Immunoreactivity for ezrin, a membrane-organizing phosphoprotein that tethers actin microfilaments to cell membrane proteins, was evaluated as a polarization marker in the intraocular neuroepithelial cells of vertebrates, especially in the retinal pigment epithelium (RPE). Six fetal human eyes representing the 14th-28th gestational weeks, 9 normal adult eyes, 12 eyes with intraocular tumors, and 26 eyes from 15 other vertebrate species were analyzed by immunohistochemistry using the avidin-biotinylated peroxidase complex (ABC) method and monoclonal antibody (mAb) 3C12 to ezrin. The apical cytoplasm and microvilli of the human RPE always reacted with mAb 3C12, but the basal cytoplasm was labeled in reactive RPE only. In autopsy eyes and if fixation was delayed, ezrin immunoreactivity in RPE was more diffuse. Developing RPE became gradually immunoreactive from the 14th week of gestation onward. The microvilli of the baboon, pig, raccoon dog, cow, and rat RPE cells were likewise labeled, and their basal cytoplasm was variably immunoreactive as well, but the microvilli of the avian RPE did not react with the antibody used. In all six mammals mentioned, both layers of the ciliary epithelium and the anterior iris epithelium reacted for ezrin, and the posterior epithelium was weakly labeled in pig, cow, and rat eyes. Normal peripheral and reactive human retina, and normal baboon, pig, raccoon dog, cow, rat, black grouse, and jay eyes, showed immunoreaction for ezrin in Muller cells, usually in their microvilli. Ezrin is widely found in RPE and anterior segment neuroepithelia of the mammalian eye, in which it may segregate membrane proteins to specific membrane surfaces, especially to the apical microvilli of the RPE, which intimately interact with outer segments of photoreceptor cells. The ezrin gene on human chromosome 6q25-26 is consequently a candidate gene for causing retinal degenerations.

Prostaglandin E2 (PGE2) markedly reduces intraocular pressure (IOP) when applied topically and induces strong relaxation of pre-contracted isolated ciliary muscle through PGE2 receptor. Because the ciliary muscle relaxation reduces IOP by enhancing uveoscleral aqueous outflow, the ciliary muscle where the existence of PGE2 receptors has been demonstrated is thought to be one of the target tissues for PGE2-induced IOP reduction. To investigate the subtypes of PGE2 receptors in the ciliary muscle, the regional distribution of four PGE2 receptor subtypes (EP1, EP2, EP3 and EP4) in the mouse ciliary body was investigated by in situ hybridization using specific probes. Consistent messenger RNA signals for EP1 and EP4 receptors were expressed in the ciliary muscle, although signal levels for these subtypes were less potent as compared with the kidney, which was used as a reference organ. EP2 and EP3 signals were not detected. Stimulation of the EP4 receptor activates adenylate cyclase, which should induce ciliary muscle relaxation. Therefore, the IOP reduction induced by PGE2 analogs may be mediated by the EP4 receptor. In contrast, stimulation of the EP1 receptor is believed to promote intracellular Ca2+ mobilization, and hence should cause ciliary muscle contraction. Thus, the coexistence of EP1 and EP4 receptors in the ciliary muscle suggests that the regulation of ciliary muscle tone by PGE2 is based on a complex mechanism involving multiple receptor subtypes.

A conscious rabbit model with microdialysis sampling of endogenous aqueous humor ascorbate was developed in order to assess the pharmacodynamics of beta-blocker modulation of aqueous humor production. CMA/20 microdialysis probes were implanted in the anterior chamber of each eye of rabbits (n = 6). After a 2 week recovery period, an i.v. bolus of 14C-ascorbate (20 microCi) was administered. Blood samples and aqueous humor microdialysis probe effluent were collected and analysed for endogenous and 14C-ascorbate to estimate the basal rate of ascorbate blood to aqueous humor secretion (Ro). After a 1 hr washout, each rabbit received a series of three doses of 3H-propranolol (750-3000 microg, 16.5 microCi mg(-1)) every 60 min into the lower cul-de-sac of each eye. Probe effluent was analysed for endogenous ascorbate and 3H-propranolol; ascorbate and propranolol in the iris/ciliary body, vitreous and aqueous was determined at the end of the experiment. Nonlinear least-squares regression analysis of the concentration-time profiles for aqueous humor ascorbate was performed to estimate the change in aqueous humor flow. The average basal aqueous humor ascorbate secretion rate was approximately 48/microg hr(-1). Propranolol (1500 microg) produced significant increases in aqueous humor ascorbate, this observation is consistent with a reduction in aqueous humor production (approximately 47%). Analysis of intraocular tissue ascorbate indicated that propranolol inhibited ascorbate secretion at the 3000 microg dose, the highest dose examined in this study; this inhibition was not observed at the 750 microg or 1500 microg doses. Changes in aqueous humor production precipitated by the administration of beta-adrenergic antagonists can be estimated by measuring changes in aqueous humor ascorbate concentrations in the conscious rabbit. Microdialysis sampling of aqueous humor for endogenous ascorbate provides a relevant analytic tool to estimate modulatory effects of anti-glaucoma drugs on aqueous humor production.

Purpose: To determine whether the calcitonin gene-related peptide (CGRP) receptor component protein (RCP), a novel signal transduction molecule, is required for CGRP signaling in the eye and to determine potential ocular sites of CGRP action., Methods: The cDNA for the rabbit ocular RCP homologue was cloned using a combination of reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). Function of the rabbit ocular RCP was assessed using a sensitive oocyte-based assay, which utilizes the protein kinase A (PKA)-sensitive cystic fibrosis transmembrane conductance regulator (CFTR) as a sensor of cAMP formation. RCP expression in the rabbit eye was localized using immunohistochemistry., Results: A 2063-bp cDNA for the rabbit ocular RCP was cloned and sequenced. Expression of the rabbit RCP cDNA confers CGRP responsiveness in a sensitive oocyte-based assay. Antisense oligonucleotides made to the ocular RCP abolishes CGRP responsiveness of ciliary body and iris mRNA in the oocyte-CFTR assay. Localization of RCP protein in the rabbit eye using immunohistochemistry demonstrated RCP immunoreactivity in the ciliary body and iris blood vessels, as well as in layers of the ciliary epithelium., Conclusions: The rabbit ocular RCP appears to be required for signal transduction at ocular CGRP receptors and is localized to sites previously reported to bind CGRP, which affect intraocular pressure and neurogenic inflammation.

The purpose of the present investigation was to compare protein expression in various ocular cells and tissues including the human trabecular meshwork (TM) and the lamina cribrosa (LC). To conduct the comparisons, we primarily utilized autofluorography of one-dimensional (1D) and high resolution, two-dimensional (2D) polyacrylamide gels of proteins from radiolabelled tissues and cultured cells. Results from the investigations indicated that patterns of protein expression from TM and LC were the most similar among the ocular cells and tissues compared.Specifically, these autofluorographic ' fingerprints' indicated that proteins in TM and LC cultured cells and tissue were exceptionally similar (a) in band position and intensity (1D gels) and (b) in spot congruence (2D gels) as compared to other ocular cells and tissues. We conclude that the TM and the LC, two ocular tissues intimately linked to the pathogenesis of primary open-angle glaucoma, display remarkable similarity in protein expression. This finding may have implications for the molecular etiology of glaucoma., (Copyright 2000 Academic Press.)

The presence of the alpha(1)-adrenoceptor subtypes in various parts of the pig and rabbit eyes was investigated using [(3)H]-prazosin radioligand binding. The characterization of the subtypes was achieved by performing competition experiments with various subtype selective drugs. In the pig retina, both alpha(1A)- and alpha(1B)-adrenoceptors were detected and the proportion of sites was 70% alpha(1A)- and 30% alpha(1B)-adrenoceptors, respectively. In the pig iris, ciliary body and choroid, which are melanin-rich tissues, the non-specific binding of [(3)H]-prazosin was too high to detect any of the alpha(1)-adrenoceptor subtypes. However, in the albino rabbit iris, ciliary body and retina both alpha(1A)- and alpha(1B)-adrenoceptors were detected. The proportion of sites in the iris was 60 % alpha(1A)- and 40% alpha(1B)-adrenoceptors, respectively. In the ciliary body and rabbit retina the proportion of sites were 70% alpha(1A)- and 30% alpha(1B)-adrenoceptors. Only the alpha(1A)-adrenoceptor subtype was detected in the rabbit choroid., (Copyright 2000 Academic Press.)

Purpose: This study was undertaken to determine the presence of retina-derived fetuin (RDF) protein and its message in retinal tissues and retinal pigment epithelial (RPE) cells. The techniques utilized in this study included light micros-copy, immunochemistry, Western blot, reverse transcriptase-polymerase chain reaction (RT-PCR) and Southern blot., Methods: Retinal tissues and sections from embryonic, early postnatal and adult normal rats and retinal pigment epithe-lial (RPE) cells from postnatal rats were immunostained for fetuin with a polyclonal fetuin antibody and a peroxidase conjugated-secondary antibody using immunocytochemistry and Western blot analyses. The cDNA generated from RNA isolated from early postnatal rat retinas and RPE was probed with primers for rat fetuin, amplified by PCR and the PCR products were analyzed by Southern blot., Results: Fetuin (RDF) was immunolocalized to cells of the neuroepithelium in retinas of early postnatal rats and most prominently in the nuclei and perinuclear region of cultured neonatal rat RPE cells. In adult retinas, ganglion cells, inner segments of photoreceptor cells, some components of the outer plexiform layer, ganglion cells and optic nerve processes were immunoreactive for the fetuin protein. As shown by Western blot, fetuin (RDF) was higher in embryonic and early postnatal retinas than in late postnatal retinas, indicating that this protein may be developmentally regulated. Using RT-PCR, the message for rat fetuin was demonstrated in the retina and RPE of normal postnatal rats. Southern blot confirmed that the PCR product from the retina and RPE was generated from rat fetuin mRNA as well as from rat liver, the primary source of fetuin., Conclusions: Fetuin, termed retina-derived fetuin (RDF), is reported for the first time in retinal tissues. Fetuin is a cysteine protease inhibitor that may play a role in support of neuronal cell survival during early retinal development and the maintenance of neuronal activity. RDF may interact with other growth factors and cytokines in providing trophic support for neurons and possibly other cells of the developing retina.