Expression of muscarinic and adrenergic receptors in normal human conjunctival epithelium

PURPOSE: To study the presence of muscarinic and alpha- and beta-adrenergic receptors in a normal human conjunctival epithelial (IOBA-NHC) cell line. METHODS: Neurotransmitter receptors were determined in IOBA-NHC cells by flow cytometry, immunofluorescence, and Western blot analysis. Antibodies to M(1)-, M(2)-, and M(3)-muscarinic and to alpha(1A)-, alpha(1B)-, alpha(1D)-, alpha(2A)-, alpha(2B)-, alpha(2C)-, beta(1)-, beta(2)-, and beta(3)-adrenergic receptor subtypes were used. Different culture media were tested, including the addition of tumor necrosis factor (TNF)-alpha and/or interferon (IFN)-gamma. Normal human conjunctiva biopsy specimens and rat tissues were used in control experiments. RESULTS: By immunofluorescence microscopy, all receptor subtypes, except the alpha(2C)-adrenergic receptor, were detected in control biopsy specimens. By flow cytometry, the M(2)- and M(3)-muscarinic receptors and alpha(1A)-, alpha(1B)-, alpha(1D)-, alpha(2A)-, alpha(2B)-, alpha(2C)-, beta(1)-, and beta(3)-adrenergic receptors were detected intracellularly and in cell membranes of the IOBA-NHC cells. M(1)-muscarinic and beta(2)-adrenergic receptors were detected only intracellularly, but were mobilized to the cell membrane when cholera toxin and hydrocortisone were omitted from the culture medium. Confocal microscopy detected the M(2) and M(3)-muscarinic and alpha(1A)-, alpha(2A)-, alpha(2B)-, beta(1)- and beta(2)-adrenergic receptor subtypes. Western blot analyses showed bands for all receptors. M(2)-muscarinic and alpha(1B)- and alpha(2B)-adrenergic receptors expression was upregulated when cells were treated with the proinflammatory cytokines IFN gamma and/or TNF alpha. CONCLUSIONS: The IOBA-NHC cell line maintained expression of the neurotransmitter receptors expressed in normal human conjunctival epithelium. A proinflammatory medium upregulated expression of some receptors. Although the functional state of these receptors is unknown, these findings justify further use of the IOBA-NHC cell line to study the neural component of conjunctival inflammation.     

Human trabecular meshwork cell responses induced by bimatoprost,travoprost, unoprostone,and other FP prostaglandin receptor agonist analogues

PURPOSE: To determine the functional agonist potencies of the intraocular pressure (IOP)-lowering prostaglandin F (FP)-class prostaglandin (PG) analogues (e.g., travoprost, latanoprost, bimatoprost, and unoprostone isopropyl ester) in human trabecular meshwork (h-TM) cells, by using phosphoinositide (PI) turnover and intracellular Ca(2+) ([Ca(2+)](i)) mobilization, and to confirm the FP nature of these receptors by using an FP receptor antagonist, 11beta-fluoro-15-epi-15-indanyl-PGF(2alpha) (AL-8810). METHODS: FP-receptor-mediated PI turnover and [Ca(2+)](i) mobilization were measured in h-TM cells by determining the accumulation of [(3)H]-inositol phosphates ([(3)H]-IPs) by anion-exchange chromatography and real-time fluorescence imaging, respectively. RESULTS: Various PG analogues concentration-dependently stimulated production of [(3)H]-IPs in h-TM cells with the following agonist potencies (median effective concentration; EC(50)): travoprost acid (EC(50) = 2.4 nM) > cloprostenol (EC(50) = 4.5 nM) > (+/-)-fluprostenol (EC(50) = 10.8 nM) > latanoprost acid (EC(50) = 34.7 nM) > bimatoprost acid (EC(50) = 112 nM) > PGF(2alpha) (EC(50) = 120 nM) > unoprostone (UF-021; EC(50) = 3280 nM) > S-1033 (EC(50) = 4570 nM; all n = 3-9). Prodrug derivatives of these compounds exhibited the following potencies: travoprost (isopropyl ester; EC(50) = 89.1 nM) > latanoprost (isopropyl ester; EC(50) = 778 nM) > bimatoprost (amide; EC(50) = 1410-6940 nM). Travoprost acid, PGF(2alpha,) unoprostone, and S-1033 were tested in addition for [Ca(2+)](i) mobilization and found to have rapid and dose-dependent effects. The FP receptor-selective antagonist AL-8810 antagonized the (+/-)-fluprostenol-induced PI turnover in these cells (K(i) = 2.56 +/- 0.62 micro M) as well as that induced by bimatoprost and acids of latanoprost and travoprost. The agonist and antagonist potencies of the PG analogues from the PI turnover assays in h-TM cells correlated well with PI turnover data obtained from the cloned human ciliary body FP receptor (r = 0.92; P < 0.0001). CONCLUSIONS: The pharmacology of the h-TM cell FP-receptor-mediated PI turnover and [Ca(2+)](i) mobilization was defined using numerous synthetic (FP-selective) PG agonist analogues and an FP receptor antagonist, AL-8810. Bimatoprost, travoprost, latanoprost, unoprostone isopropyl ester, and their respective free acids were shown to be FP agonists in the h-TM cells.     

Interaction of GABA receptor/channel rho(1) and gamma(2) subunit

PURPOSE: To determine whether protein-protein and functional interactions can occur between gamma-aminobutyric acid (GABA)(A) receptor/channels gamma(2) subunit and the retina-specific GABA(C) rho(1) subunit. METHODS: Protein-protein interaction was characterized by immunocoprecipitation of these subunits in brain and spinal cord with anti-gamma(2) subunit antibody and by Western blot analysis with anti-rho(1) subunit antibody. The rho(1) and gamma(2) subunits were detected in the adult rat brain and spinal cord lysates that had been previously precipitated with the specific antibodies against the rho(1) and gamma(2) subunits, respectively. A two-microelectrode voltage clamp was used to measure GABA-induced currents in oocytes. In addition, a yeast two-hybrid system was used to detect the interactions of these subunits in vivo. RESULTS: Based on yeast transformed with the N-terminal fragment of the gamma(2) subunit (gamma(2)-N'), the N-terminal fragment of the rho(1) subunit (rho(1)-N'), and the full-length rho(1) subunit, the protein-protein interaction of the GABA(A) gamma(2) subunit and the GABA(C) rho(1) subunit was found in yeast grown in triple-dropout medium (deficient in Leu, Trp, and His) and expressing the LacZ reporter gene. Interaction of the rho(1) and gamma(2) subunits was investigated by functional studies in which gamma(2) (gamma(2)-N' with 837 bp) and rho(1) cRNAs were coinjected in Xenopus oocytes. In studies of the functional interaction, after injection of the gamma(2) subunit mutant cRNA containing a N-terminal fragment, GABA-induced rho(1) originated currents declined to 16% of the control level of homooligomeric rho(1) current. This inhibitory effect of coexpressing gamma(2) subunit mutants with rho(1) subunit on the rho(1)-originated current in oocytes was dose dependent. In addition, coexpression of the GABA rho(1) and gamma(2) subunits in oocytes altered pharmacologic properties of the homooligomeric receptor/channel formed by rho(1) or gamma(2) subunits. Further evidence was provided by results obtained with specific antibodies showing that the rho(1) subunit was coimmunoprecipitated with the gamma(2) subunit from the retina, brain, and spinal cord. CONCLUSIONS: The results indicate that protein-protein and functional interactions can occur between the GABA(A) gamma(2) subunit and the GABA(C) rho(1) subunit. Therefore, the functional role of GABA receptor/channels in the brain, retina, and spinal cord is more diversified because of the possible assembly between the GABA(A) gamma(2) subunit and GABA(C) rho(1) subunit.     

Diverse Regulation of Retinal Pigment Epithelium Phagocytosis of Photoreceptor Outer Segments by Calcium-Independent Phospholipase A(2), Group VIA and Secretory Phospholipase A(2), Group IB

Purpose: To investigate the roles of the phospholipases A(2) (PLA(2)) subtypes, iPLA(2)-VIA and sPLA(2)-IB in retinal pigment epithelium (RPE) phagocytosis of photoreceptor outer segments (POS) and to explore a possible interaction between sPLA(2)-IB and iPLA(2)-VIA in the RPE. Methods: To explore the role of iPLA(2)-VIA in RPE phagocytosis of POS, experiments with iPLA(2)-VIA vector transfection, iPLA(2)-VIA(-/-) knockout (KO) mice, and iPLA(2)-VIA inhibition by bromoenol lactone (BEL) were done. Exogenous addition of sPLA(2)-IB was used to investigate the role of sPLA(2)-IB in RPE phagocytosis. A Luciferase Reporter Vector containing the iPLA(2)-VIA promoter was used to study the effects of sPLA(2)-IB on the iPLA(2)-VIA promoter. Results: ARPE-19 and primary mouse RPE cells transfected with iPLA(2)-VIA showed increased phagocytosis. Phagocytosis was reduced in primary mouse RPE inhibited with BEL and in RPE from KO mice. Exogenous addition of enzymatically active and inactive sPLA(2)-IB reduced phagocytosis in ARPE-19 and primary mouse RPE cells. Finally, sPLA(2)-IB did not seem to affect the iPLA(2)-VIA promoter. Conclusion: The present study confirms the involvement of iPLA(2)-VIA in efficient RPE phagocytosis of POS, while exogenously added sPLA(2)-IB decreases phagocytosis regardless of enzymatic activity. No apparent interaction between iPLA(2)-VIA and sPLA(2)-IB was found.     

Ocular biometry in occludable angles and angle closure glaucoma: a population based survey

Aim: To compare ocular biometric values in a population based sample of eyes with occludable angles, angle closure glaucoma, and normal subjects. METHOD: 2850 subjects from a population based glaucoma prevalence study underwent complete ocular examination including indentation gonioscopy. Ocular biometry was performed in all subjects classified to have occludable angles (n = 143); angle closure glaucoma (n = 22), and a random subgroup of 419 normal subjects. Ocular biometry readings between the groups were compared and statistically analysed using "t," "z," and Mann-Whitney U tests. RESULTS: The mean age among subjects with occludable angles (54.43 (SD 9.53) years) and angle closure glaucoma (57.45 (8.5) years) was significantly higher (p<0.001) than normal subjects (49.95 (9.95) years). Axial length was shorter (p<0.001) in the occludable angle group (22.07 (0.69) mm) compared to the normal group (22.76 (0.78) mm). Anterior chamber depth (ACD) was shallower (p<0.001) among subjects with occludable angles (2.53 (0.26) mm) than normal subjects (3.00 (0.30) mm). Lens thickness (LT) was greater (p<0.001) in people with occludable angles (4.40 (0.53) mm) compared to normal subjects (4.31 (0.31) mm). No significant difference was noted in axial length, ACD (p = 0.451), and LT (p = 0.302) between angle closure glaucoma and occludable eyes. CONCLUSION: South Indian eyes with angle closure glaucoma and occludable angles seem to have significantly shorter axial lengths, shallower anterior chambers and greater lens thickness compared to the normal group.     

Effect of diabetes mellitus on corneal biomechanics and measurement of intraocular pressure

Purpose: To determine whether corneal hysteresis (CH) and corneal resistance factor (CRF) are altered in diabetes and whether these parameters are related to HbA1c. Methods: One randomly chosen eye of 35 healthy subjects and 31 patients with diabetes was examined. Patients with diabetes were divided into group 1 with HbA1c <7% (n?=?14) and group 2 with HbA1c ≥7% (n?=?17). CH and CRF were measured using ocular response analyzer (ORA); central corneal thickness (CCT) using ultrasound pachymetry; increased intraocular pressure (IOP) using Goldmann tonometer (IOP(GAT) ), Pascal dynamic contour tonometer (IOP(pasc) ), and ORA (IOP(cc) ). As CH and CRF are dependent on IOP and CCT, they were adjusted for IOP and CCT resulting in CH(corr) and CRF(corr.) Results: Mean HbA1c was 5.44?±?0.46% in healthy subjects, 6.00?±?0.78% in diabetic group 1, 8.58?±?2.44% in group 2. CH(corr) (p?=?0.071) and CRF(corr) (p?=?0.067) were not statistically significantly different between healthy subjects and diabetic group 1, but significantly lower in healthy subjects compared to diabetic group 2 [CH(corr) (p?=?0.031), CRF(corr) (p?=?0.029)]. IOP(pasc) (p?=?0.012), IOP(GAT) (p?=?0.032) and HbA1c (p?=?0.0001) were statistically significantly different between healthy subjects and all patients with diabetes (groups 1?+?2), but not age, sex and CCT. Over all patients with diabetes, CH(corr) (p?=?0.012, R(2) =?0.197) and CRF(corr) (p?=?0.008, R(2) =?0.217) were correlated to HbA1c but not in healthy subjects [CH(corr) (p?=?0.931, R(2) =?0.0001), CRF(corr) (p?=?0.837, R(2) =?0.001)]. Conclusion: In poorly controlled diabetics, CH(corr) and CRF(corr) are significantly higher compared with those of the healthy subjects and patients with well-controlled diabetes. In diabetes, CH(corr) and CRF(corr) are correlated to HbA1c, suggesting that the biomechanical properties of the cornea are altered depending on the glucose control.     

急性中心性浆液性脉络膜视网膜病变视网膜外层的SD-OCT表现

目的 探讨急性中心性浆液性脉络膜视网膜病变(central serous chorioretinopathy,CSC)视网膜外层组织的频域相干光断层扫描(spectral domain optical coherence tomography,SD-OCT)视网膜外层组织表现特征。设计回顾性病例系列。研究对象首次发病、荧光素眼底血管造影检查(FFA)表现为单眼单个明确渗漏点(LP)的急性CSC患者55例(55眼)。方法 分析FFA和SD-OCT同步检查图像对应LP及黄斑中心凹、视网膜神经感觉层脱离(NSRD)区域的视网膜色素上皮(RPE)光反射带及视网膜外层组织形态结构。主要指标SD-OCT中色素上皮层脱离(PED)形态特征、视网膜外层组织形态结构特征。结果 41眼(74.5%)LP表现为PED,其中拱形PED(DSPED)15眼(27.3%),扁平不规则PED(FIPED)26眼(47.2%)。与DSPED相比,FIPED更多见于墨渍状渗漏(χ²=11.116,P=0.001)和黄斑中心无血管区外(χ²=4.432,P=0.035)。黄斑中...     

首次发生急性原发性房角关闭患者发作眼与对侧眼前节生物学参数的对比分析

目的　采用光学相干断层扫描比较首次发生急性原发性房角关闭（ａｃｕｔｅｐｒｉｍａｒｙａｎｇｌｅｃｌｏｓｕｒｅ,ＡＰＡＣ）患者的发作眼与对侧可疑原发性房角关闭（ｐｒｉｍａｒｙａｎｇｌｅｃｌｏｓｕｒｅｓｕｓｐｅｃｔ,ＰＡＣＳ）眼的眼前节生物学参数差异并分析发病相关危险因素。方法　收集２０１３年８月至２０１４年８月于北京协和医院眼科就诊的首次单眼发生ＡＰＡＣ且对侧眼为ＰＡＣＳ的患者共计３０例（发作眼３０眼,对侧眼３０眼）,采用光学相干断层扫描仪测量双眼的中央角膜厚度、瞳孔直径、中央前房深度、晶状体拱高、前房宽度、距离巩膜突５００μｍ和７５０μｍ处的房角开放距离（ａｎｇｌｅｏｐｅｎｄｉｓｔａｎｃｅ,ＡＯＤ５００、ＡＯＤ７５０）及小梁网虹膜间面积（ｔｒａｂｅｃｕｌａｒｉｒｉｓａｒｅａ, ＴＩＳＡ５００、ＴＩＳＡ７５０）、巩膜突角度。采用配对ｔ检验比较双眼各参数之间的差异,条件Ｌｏｇｉｓｔｉｃ回归分析发病相关因素。结果　对侧眼组与发作眼组的中央角膜厚度分别为（５２２．７±３１．３）ｍｍ和（５５７．３±４２．７）ｍｍ,瞳孔直径分别为（２．９０±１．１９）ｍｍ和（３．７８±１．２９）ｍｍ,晶状体拱高分别为（１．０８±０．３６）ｍｍ和（１．２４±０．４２）ｍｍ,发作眼组均较大（均为Ｐ＜０．０１）;中央前房深度分别为（１．８１±０．４５）ｍｍ和（１．６４±０．４４）ｍｍ,ＡＯＤ５００分别为（０．０９７±０．０６５）ｍｍ和（０．０５９±０．０３９）ｍｍ,ＡＯＤ７５０分别为（０．１５７±０．１００）ｍｍ和（０．１２０±０．０６８）ｍｍ,ＴＩＳＡ５００分别为（０．０３６±０．０２１）ｍｍ２和（０．０２０±０．０１６）ｍｍ２,ＴＩＳＡ７５０中位数分别为０．０６５ｍｍ２和０．０４１ｍｍ２,巩膜突角度分别为１１．１３°±６．９２°和６．６８°±４．４３°,发作眼组均较小（均为Ｐ＜０．０５）。条件Ｌｏｇｉｓｔｉｃ回归分析结果显示晶状体拱高的增加与急性房角关闭具有强相关性（ＯＲ＝４０．２５９［１．０２１,１７７９．１９３］,Ｐ＝０．０１４）。结论　晶状体拱高的增加是ＡＰＡＣ发作眼与对侧ＰＡＣＳ眼相比最突出的危险因素,可能在ＰＡＣＳ发展成为ＡＰＡＣ的过程中起关键作用。     

Experimental study on antiviral activity of silicone oil in vitro

OBJECTIVE: To study the antiviral activity of silicone oil against herpes simplex virus-1 (HSV-1) in vitro. METHODS: (1) TCID(50) (Tissue culture infectious dose 50% endpoint) of HSV-1 was titrated. (2) The Hela cells were placed in a 96-well plate. (3) The virus was diluted by 100TCID(50), 50TCID(50), 30TCID(50), 10TCID(50), and 1TCID(50) individually. (4) Each of the five different concentrations of virus was inoculated into 16 wells. Of the 16 wells, 0.1 ml silicone oil was added to eight of them as the experimental group, and the other eight wells were used as controls. (5) Sixteen additional wells were added: silicone oil and maintenance media were added to eight wells, and only maintenance media to the other eight wells. RESULTS: (1) The cytopathic effect (CPE) of wells inoculated with 30TCID(50) combined with silicone oil was significantly less than that of the viral control at 32 hours (P < 0.01), and the same results occurred in group 10TCID(50) combined with silicone oil at 45 hours (P < 0.01) and group 1TCID(50) combined with silicone oil at 51 hours (P < 0.01). (2) In the viral control, the cells in 30TCID(50), 10TCID(50) and 1TCID(50) had pathological changes at 45, 58 and 58 hours respectively. (3) The cells of either the viral control group or group 50TCID(50) and 100TCID(50) had pathological changes at 32 hours (P > 0.05). CONCLUSION: Silicone oil has an antiviral function against HSV-1. The antiviral effect of silicone oil is correlated with concentration of virus.     

Biphasic intraocular pressure response to calcitonin gene-related peptide.

PURPOSE: To examine the effects of calcitonin gene-related peptide (CGRP) on intraocular pressure (IOP). METHODS: IOP was periodically measured in rabbits treated with intravitreal injection (20 ael) of either: 1) CGRP (10(-4) approximately 10(-7) M) into one eye; 2) CGRP (10(-4) or 10(-6) M) into both eyes 30 min after intravitreal administration of CGRP-(8-37) (10( -3) M), a CGRP1 receptor antagonist, into one eye; 3) CGRP (10(-4 ) or 10(-6) M) into one eye 30 min after intravenous administration (200 mg/kg) of either Nw-nitro-L-arginine methyl ester (L-NAME), a nonselective inhibitor of nitric oxide synthase (NOS), or aminoguanidine (AG), a selective inhibitor of inducible NOS; or 4) CGRP (10(-4) or 10(-6) M) into one eye along with intraperitoneal indomethacin (50 mg/ kg at -1 and 4 h). RESULTS: CGRP (10(-4) and 10(-5) M) produced a biphasic IOP response, which consisted of an early ocular hypertensive phase and a subsequent sustained hypotensive phase. While CGRP (10(-6) M) yielded only a profound IOP reduction. CGRP-(8-37) significantly inhibited the IOP elevation induced by CGRP (10(-4) M) and completely abolished the IOP reduction induced by CGRP (10(-6) M). The IOP increase induced by CGRP (10(-4) M) was completely abolished by L-NAME, but not affected by AG. The IOP reduction induced by CGRP (10(-6) M) was not affected by L-NAME. Indomethacin did not significantly affect the IOP responses to CGRP. CONCLUSIONS: CGRP dose-dependently produces a biphasic IOP response, which is mediated by CGRP1 receptors. It is suggested that constitutive NOS is involved in the early ocular hypertensive response.     

Paediatric uveitis: a Sydney clinic experience

PURPOSE: The aim of this study was to retrospectively review uveitis cases at The Children's Hospital at Westmead, Sydney, since its inception in 1997 to 2001, including patients presenting at the Camperdown, Sydney, campus between 1989 and 1997 attending Westmead for further care. Comparison is made with international centres. METHODS: Information was obtained from medical records. RESULTS: Forty patients (53 eyes) presented, of whom 23 (57.5%) were female and 17 (42.5%) were male (mean age 6.7 years). Of 53 eyes, 35 (66%) had anterior uveitis, three (5.7%) intermediate uveitis, seven (13.2%) posterior uveitis and eight (15.1%) panuveitis. Twenty-seven (67.5%) patients had disease unilaterally and 13 (32.5%) bilaterally. Twenty-four (60%) cases were idiopathic. Seven (17.5%) cases were associated with juvenile rheumatoid arthritis, three (7.5%) with herpes zoster, two (5%) with herpes simplex, two (5%) with toxocara, one (2.5%) with toxoplasma, and one (2.5%) with ulcerative colitis. Complications included cataract in 14 (26.4%) eyes; band keratopathy in four (7.5%) eyes; macular scarring in three (5.7%) eyes; and glaucoma in four (7.5%) eyes. Last measured acuity was 6/6 for 19 (35.8%) eyes, < or =6/18 for 15 (28.3%) eyes and <6/60 for eight (15.1%) eyes. CONCLUSIONS: Despite small numbers, the comparisons of this study with some international studies, and its contrasts with other studies, are due to similarities and differences amongst these studies with respect to factors of referral bias, and the aetiological basis of disease.     

Specialized protective role of mucosal glutathione in pigmented rabbit conjunctiva

PURPOSE. To investigate mechanisms of H(2)O(2)-induced reduction in rates of active ion transport (I(sc)) across the pigmented rabbit conjunctival tissue and the protective role afforded by mucosal glutathione (GSH). METHODS. Changes in I(sc) and specific binding properties of ouabain were evaluated in a modified Ussing chamber setup, using conjunctival tissues freshly excised from pigmented rabbits. Effective concentrations of H(2)O(2) at which 50% of I(sc) was inhibited (IC(50)) were determined for the mucosal and serosal instillation of the agent. The rate of exogenous H(2)O(2) consumption in the mucosal and serosal bathing fluids was estimated. Mucosal 8-Br cAMP at 3 mM, serosal bumetanide at 0.5 mM, and both mucosal and serosal bathing of the conjunctiva with Na(+)-free bicarbonated Ringer's solution (BRS) were used to estimate contributions of conjunctival ion transport mechanisms in I(sc) changes elicited by mucosal H(2)O(2) at IC(50). Specific binding of (3)H-ouabain to the serosal side of the conjunctiva was estimated in the presence of mucosal or serosal H(2)O(2) to assess the role of functional Na(+)/K(+)-ATPase pumps in H(2)O(2) injury. The effect of mucosally instilled GSH and other reductive and nonreductive agents on possible restoration of oxidant-induced decrease in conjunctival I(sc) was also determined. RESULTS. Mucosal and serosal H(2)O(2) decreased conjunctival I(sc) gradually in a dose-dependent manner. The mucosal IC(50) of H(2)O(2)was 1.49 +/- 0.20 mM, whereas the serosal IC(50) was estimated at 10.6 +/- 2.0 micro M. The rate of H(2)O(2) consumption from mucosal fluid was six times faster than that from serosal fluid. Conjunctival tissues pretreated with mucosal H(2)O(2) at IC(50) retained approximately 50% of their maximum 8-Br cAMP-dependent increases in I(sc). Serosal bumetanide did not further reduce the I(sc) beyond the initial 70% decrease caused by mucosal H(2)O(2). When conjunctiva was bathed with Na(+)-free BRS on both the mucosal and serosal sides, before or after addition of mucosal H(2)O(2), the combined effects were additive, decreasing I(sc) by up to 95% to 99%. Mucosal, but not serosal, GSH or reduced L-glutathione mono-ethyl ester (GSH-MEE) superfusion of conjunctival tissues pre-exposed to mucosal H(2)O(2) at IC(50) recovered to 60% to 80% of the initial pre-H(2)O(2) I(sc) after approximately 100 minutes. The specific binding of (3)H-ouabain to the serosal side of the tissue was inhibited by 85% in the presence of mucosal or serosal treatment with H(2)O(2) at their respective IC(50) values. Pretreatment for 60 minutes with either 5 mM GSH, 2 mM GSH-MEE, or 0.1 mM ebselen, when instilled into the mucosal fluid, resulted in 30%, 45%, or 55% reductions, respectively, in ouabain binding after exposure to mucosal H(2)O(2) at IC(50). Furthermore, mucosal posttreatment with 10 mM GSH or 5 mM GSH-MEE of conjunctival tissues pre-exposed to mucosal H(2)O(2) resulted in a 30% recovery of the ouabain-binding level above that observed in tissues exposed to 1.5 mM H(2)O(2) alone on the mucosal side. By contrast, the decrease in conjunctival I(sc) or in the ouabain-binding level elicited by serosal H(2)O(2) at IC(50) was irreversible. CONCLUSIONS. A higher mucosal IC(50) of [H(2)O(2)] on conjunctival I(sc) corresponds to the faster consumption of exogenous H(2)O(2) from mucosal bathing fluid. In addition, actively secreted GSH by conjunctival epithelial cells may help reduce the injury by mucosally applied H(2)O(2). Injury by H(2)O(2) may directly affect vital membrane components (e.g., Na(+),K(+)-ATPase) involved in active ion transport across conjunctiva. Mucosal protection by GSH (or its analogues) of active conjunctival ion transport may be useful in maintaining the physiological functions of conjunctiva under oxidative stress.     

Astigmatismus nach perforierender Keratoplastik

Purpose

The aim of this study was a retrospective analysis of postkeratoplasty astigmatism and best corrected visual acuity (BCVA) in patients following penetrating keratoplasty (PK) and a comparison of three suturing techniques.

Patients and methods

In this retrospective analysis penetrating keratoplasty (PK) was carried out on 150 eyes with 3 suturing techniques: single running (SR), double running (DR counterclockwise) and interrupted (IR) sutures. Of the eyes 37 (24.7%) underwent PK with SR sutures, 81 eyes (54%) with DR sutures and 32 eyes (21.3%) had IR. PK for Fuchs?? dystrophy was used on 46 eyes (30.7%), on 33?eyes (22%) for keratoconus, on 12 eyes (8%) for herpetic keratitis and on 7 eyes (4.6%) for pseudophakic bullous keratopathy. For trephination a guided trephine system (GTS) was used in 44%, rotortrepan in 46.6% and best trepan in 5.3%. Postkeratoplasty astigmatism and best corrected visual acuity (BCVA) were evaluated 1, 4, 12 and 24 months after surgery (all sutures removed). Subjective and objective refractions and corneal topography were performed to assess astigmatism. The Kolmogorov-Smirnov test (95% significance) was used to evaluate statistical significance.

Results

Mean topographic astigmatism 4 months (12 months/2 years) after keratoplasty was 4.9?dpt (5.3/4.1, n=4) for SR, 4.2?dpt (4.0/5.3) for DR and 9.7?dpt (n=7) (4.9, n=8/6.8, n=2) for IR suturing techniques. Mean objective astigmatism 4 months (12 months/2 years) after PK was 5.9?dpt (4.1, n=7/5.0, n=3) for SR, 3.4?dpt (4.5/4.98) for DR and 8.0?dpt (n=3) (6.9, n=4/7.4, n=2) for IR sutures. Mean refractive cylinder 4 months (12 months/2 years) after keratoplasty was 4.5?dpt (3.9/4.9) for SR, 3.2?dpt (3.3/3.6) for DR and 6.2?dpt (3.7/4.7) for IR suturing. Mean BCVA 4 months (12 months/2 years) was 0.3 (0.3/0.4) for SR, 0.3 (0.4/0.5) for DR and 0.3 (0.4/0.4) for IR sutures. BCVA 4 months (12 months/2 years) after PK (GTS only) reached 0.3 (0.3/0.5) for SR and 0.3 (0.4/0.6) for DR suturing.

Discussion

Topographic and objective astigmatisms were highest for the IR suturing technique. Topographic astigmatism and refractive cylinder were less in the DR (compared to SR) group 4 and 12 months after surgery (statistically significant). After suture removal (2 years after PK) refractive cylinder was still lower for DR compared to SR but there was no statistical difference between DR and SR regarding topographic and objective cylinders. For the interpretation of these data it should be emphasized that due to the retrospective character of this analysis the number of patients in the subgroups is decreasing with time and as a consequence single (strongly deviating) measurements can have a more powerful impact on the outcome in the individual subgroups.     

过氧化氢对人晶状体上皮细胞内细胞质膜微囊蛋白的影响

目的探讨过氧化氢（H2O2）对人晶状体上皮细胞的细胞质膜微囊蛋白（CP）及磷酸化CP-1分布与表达的影响。方法给予人晶状体上皮细胞（SRA01／04）不同浓度及不同时间点的H2O2刺激。用激光共聚焦显微镜和免疫荧光显微镜观察CP及磷酸化CP-1的分布。通过免疫印迹试验观察CP表达水平的变化以及磷酸化CP—1的表达。结果通过激光共聚焦显微镜和荧光显微镜,观察到人晶状体上皮细胞的质膜和细胞质内含有丰富的CP;当给予细胞H2O2刺激后,细胞质内CP的分布增多;当刺激时间达到1h,细胞膜被破坏,但仍可观察到CP的分布情况。此外,H2O2的刺激可以使CP-1发生磷酸化。免疫印迹试验发现,随着H2O2作用时间的延长和刺激浓度的升高,细胞质膜和细胞总蛋白质的CP表达水平呈下调趋势。结论H2O2刺激人晶状体上皮细胞后,CP重新分布并可能破坏了细胞质膜微囊（caveolae）的结构,使得细胞的CP表达下调。细胞质膜微囊和CP可能在人晶状体上皮细胞中具有重要作用。     

光动力疗法联合玻璃体内注射雷珠单抗治疗湿性年龄相关性黄斑变性后黄斑区中心视野的改变

目的　评价光动力疗法（ｐｈｏｔｏｄｙｎａｍｉｃｔｈｅｒａｐｙ,ＰＤＴ）联合玻璃体内注射雷珠单抗治疗湿性年龄相关性黄斑变性（ａｇｅ-ｒｅｌａｔｅｄｍａｃｕｌａｒｄｅｇｅｎｅｒａｔｉｏｎＡＭＤ）后患者黄斑区中心视野的变化情况。方法　本研究纳入湿性ＡＭＤ患者２６例（２８眼）,每例患者予以１次维替泊芬联合ＰＤＴ后３ｄ再给予玻璃体内注射雷珠单抗治疗,然后根据患者治疗后１个月、３个月、６个月的最佳矫正视力（ｂｅｓｔｃｏｒｒｅｃｔｅｄｖｉｓｕａｌａｃｕｉｔｙ,ＢＣＶＡ）、Ｈｕｍｐｈｒｅｙ１０-２中心视野检查、黄斑中心凹厚度（ｃｅｎｔｒａｌｆｏｖｅａｌｔｈｉｃｋｎｅｓｓ,ＣＦＴ）、眼底荧光血管造影（ｆｕｎｄｕｓｆｌｕｏｒｅｓｃｅｉｎａｎｇｉｏｇｒａｐｈｙ,ＦＦＡ）及吲哚青绿血管造影（ｉｎｄｏｃｙａｎｉｎｅｇｒｅｅｎａｎｇｉｏｇｒａｐｈｙ,ＩＣＧＡ）的渗漏情况决定是否需要再次玻璃体内注射雷珠单抗。结果　联合治疗后１个月、３个月、６个月,湿性ＡＭＤ患者的ＢＣＶＡ明显提高,分别为０．２２±０．０７、０．２６±０．０７、０．２４±０．０８（Ｐ＝０．０２９、０．００４、０．０１５）,而治疗前ＢＣＶＡ为０．１２±０．０９;ＣＦＴ显著降低,分别为（２６４．０９±２８．４０）μｍ、（２７３．４５±２４．８９）μｍ、（２８６．５４±２６．３９）μｍ（均为Ｐ＝０．０００）,而治疗前ＣＦＴ为（４５８．２７±２７．３８）μｍ;中心视野１０°范围内的平均敏感度（ｍｅａｎｓｅｎｓｉｔｉｖｉｔｙ,ＭＳ）增加了,分别为（２４．３３±３．２０）ｄＢ、（２４．５４±３．２５）ｄＢ、（２６．８９±３．４６）ｄＢ（Ｐ＝０．０１５、０．０１０、０．０００）,而治疗前的ＭＳ为（２０．２５±３．１２）ｄＢ;４°范围内ＭＳ增加了,分别为（２３．６６±４．２３）ｄＢ、（２３．４０±４．４９）ｄＢ、（２５．９３±４．４３）ｄＢ（Ｐ＝０．０２４、０．０３４、０．００１）,而治疗前４°范围内ＭＳ是（１９．４９±４．６６）ｄＢ;视野平均缺损（ｍｅａｎｄｅｖｉａ-ｔｉｏｎ,ＭＤ）降低了,分别为（－７．６０±３．２４）ｄＢ、（－７．７２±３．５１）ｄＢ、（－６．２９±３．２１）ｄＢ（Ｐ＝０．００８、０．０１０、０．００１）,而治疗前ＭＤ为（－１１．９８±３．１８）ｄＢ。模式标准差增加了,分别是５．４４±１．５３、５．７８±１．８２、６．８８±１．６５（Ｐ＝０．０１３、０．００４、０．０００）,而治疗前模式标准差为３．３４±１．０４。所有的数据均提示黄斑区的功能有好转。此外,ＢＣＶＡ和ＣＦＴ（ｒ＝－０．２９７,Ｐ＝０．００５）、ＣＦＴ和中心视野１０°范围内的ＭＳ（ｒ＝－０．３８５,Ｐ＝０．０００）、ＣＦＴ和视野的ＭＤ（ｒ＝－０．１９３,Ｐ＝０．０３３）两两之间存在一定的负相关关系。结论　黄斑区中心Ｈｕｍｐｈｒｅｙ视野指数的改变可用于评估ＰＤＴ联合玻璃体内注射雷珠单抗治疗湿性ＡＭＤ后患者视功能的改变。     

Objective assessment of aberrations induced by multifocal contact lenses in vivo.

PURPOSE: This study examined the effects of a soft multifocal progressive contact lens on individual Zernike coefficients describing the aberrations of the eye. METHODS: Ocular wavefront aberrations (OWA) of 10 subjects not wearing contact lenses were quantified using a Shack-Hartmann aberrometer and were repeated after lens insertion (two lenses,+2 diopters [D], and -2D distance power, order of insertion was randomized). All data were captured and stored on computer. Each coefficient of the Zernike polynomials representing, coma, spherical aberration (SA), and the fifth-order aberrations were evaluated for each OWA. Pupil size was monitored using an infrared device. RESULTS: After subjecting the data to various permutations, the following relationships and chief findings were detected: (1) Comparing coefficients with and without lenses, significant correlations for coma (Z(1)3 [rho,theta], P=0.0056; -2D and P=0.0399, +2D); SA (Z(0)4 [rho,theta], P=0.0006; -2D; and P=0.0061,+2D). Fifth-order aberration (Z(-1)5 [rho,theta], P= 0.0029,+2D). (2) With the -2D lens, the average root-mean-square (RMS) value for the SA coefficient Z(4)4 (rho,theta) and fifth-order Z(-3)5 (rho,theta) increased (P=0.045 and 0.0392, respectively). (3) With the +2D lens, the average RMS value for fifth-order coefficient Z(1)5(rho,theta) increased (P=0.0278). (4) Coma (Z(1)3 [rho,theta] coefficient) correlated with pupil size (P<0.05). Initial mean (+/-SD) pupil size (mm) was 3.05 (0.499), and this did not change significantly. CONCLUSIONS: Z(1)3(rho,theta) and Z(0)4(rho,theta) coefficients may be useful objective markers of success or failure for such contact lenses. The -2D lens had a more predictable effect on SA compared with the +2D lens design. The opposite occurs when considering the effects on the higher fifth-order order aberrations.     

Effects of endothelin receptor antagonists on anterior chamber inflammation induced by intravitreal injection of endothelin-1.

To investigate the role of endothelin receptors (ET(A) and ET(B)) in the inflammatory reaction induced by endothelin-1 (ET-1), the time course of changes in aqueous protein concentration (APC) after the intravitreal injection of ET-1 (10(-4), 10(-5) M) was measured using a laser flare-cell meter in pigmented rabbit eyes. The effects of pre-treatment with specific ET(A) receptor antagonist (97-139) (10(-1), 10(-2), 10(-3), 10(-4) M), specific ET(B) receptor antagonist (BQ-788) (1.6 x 10(-3) M), and vehicle solution were assessed. The influence of ET(A)receptor antagonist pre-treatment on aqueous prostaglandin E(2) and leukotriene B(4) concentrations was also evaluated. As a result, pre-treatment with ET(A) receptor antagonist blocked the APC increase induced by 10(-4) M ET-1 in a dose dependent fashion, while BQ-788 did not suppress the inflammatory reaction. The injection of ET-1 increased aqueous prostaglandin E(2) concentration, which was inhibited by pre-treatment with ET(A) receptor antagonist. Aqueous leukotriene B(4) concentration was not affected by ET-1 nor ET(A) receptor antagonist. In conclusion, ET(A) receptor mediates the increases in aqueous protein and prostaglandin E(2) concentration induced by ET-1 injection, and this inflammatory reaction occurs via the cyclooxygenase pathway of arachidonic acid cascade.     

Characterization of adenosine receptors in bovine corneal endothelium

Previous studies indicated that adenosine can increase [cAMP](i) and stimulate fluid transport by corneal endothelium. The purpose of this study was to determine which adenosine receptor subtype(s) are expressed and to examine their functional roles in modulating [cAMP](i), [Ca(2+)](i) and effects on Cl(-) permeability in corneal endothelium. We screened bovine corneal endothelium (BCE) for adenosine receptor subtypes by RT-PCR and immunoblotting, and examined the effects of pharmacological agents on adenosine stimulated Cl(-) transport, [cAMP](i) and [Ca(2+)](i). RT-PCR indicated the presence of A(1) and A(2b) adenosine receptors, while A(2a) and A(3) were negative. Western blot (WB) confirmed the presence of A(2b) ( approximately 50 kDa) and A(1) ( approximately 40 kDa) in fresh and cultured BCE. Ten micromolar adenosine increased [cAMP](i) by 2.7-fold over control and this was inhibited 66% by 10 microm alloxazine, a specific A(2b) blocker. A(1) activation with 1 micromN(6)-CPA (a specific A(1) agonist) or 100 nm adenosine decreased [cAMP](i) by 23 and 6%, respectively. Adenosine had no effect on [Ca(2+)](i) mobilization. Indirect immunofluorescence localized A(2b) receptors to the lateral membrane and A(1) to the apical surface in cultured BCE. Adenosine significantly increased apical Cl(-) permeability by 2.2 times and this effect was nearly abolished by DMPX (10 microm), a general A(2) blocker. Adenosine-induced membrane depolarization was also inhibited by 33% (n=6) in the presence of alloxazine. Bovine corneal endothelium expresses functional A(1) and A(2b) adenosine receptors. A(1), preferentially activated at <1 microm adenosine, acts to decrease [cAMP](i) and A(2b), activated at >1 microm adenosine, increase [cAMP](i).     

Pharmacological characterization of human ciliary muscle adrenoceptors in vitro

The in vitro pharmacological characteristics of adrenoceptors of the human ciliary muscle were investigated. Tissue was obtained from 30 eyes used previously for corneal transplantations which had been enucleated 6-24 hr after death. Experiments were performed within 2 days of enucleation. Strips of the meridional and circular portion of the ciliary muscle were attached to a tension gauge in an organ bath and the effect of drugs added to the perfusion medium was monitored isometrically. The muscle was precontracted with physostigmine (10(-5) M) and acetylcholine (10(-5) M). The non-selective beta adrenoceptor agonist isoproterenol (10(-6)-10(-3) M) caused a dose-related relaxation of the ciliary muscle, an effect which was completely inhibited by the non-selective beta adrenoceptor antagonist timolol (10(-5) M), while the beta 1 adrenoceptor antagonist betaxolol (10(-5) M) had no effect. The beta 2 adrenoceptor agonist salbutamol (10(-6)-10(-3) M) produced a dose-related relaxation of the ciliary muscle, an effect which was completely blocked by the beta 2 adrenoceptor antagonist L1 32-468 (10(-5) M). The non-selective alpha adrenoceptor agonist noradrenaline (10(-6)-10(-3) M) also caused a dose-related relaxation of the ciliary muscle. The non-selective alpha adrenoceptor antagonists phentolamine (10(-5) M) and thymoxamine (10(-5) M) and the alpha 1 adrenoceptor antagonist prazosin (10(-5) M) partially blocked the response to noradrenaline, while the alpha 2 adrenoceptor antagonist idazoxan (10(-5) M) and timolol (10(-5) M) had no effect. The alpha 1 adrenoceptor agonist phenylephrine (5 X 10(-6)-5 X 10(-3) M) caused a dose-dependent relaxation in five out of 12 isoproterenol-sensitive muscle strips. Further, it was not possible to block the phenylephrine-induced relaxation with thymoxamine (10(-5) M). The alpha 2 adrenoceptor agonist clonidine (10(-6)-10(-3) M) had no effect. No qualitative difference between drug effects on the meridional and circular ciliary muscles was observed. We conclude from these data that beta 2, and most probably alpha 1, adrenoceptors are present on both the meridional and circular portions of the ciliary muscle of the human eye.