Role of aquaporin water channels in eye function

The aquaporins (AQPs) are a family of more than 10 homologous water transporting proteins expressed in many mammalian epithelia and endothelia. At least five AQPs are expressed in the eye: AQP0 (MIP) in lens fiber, AQP1 in cornea endothelium, ciliary and lens epithelia and trabecular meshwork, AQP3 in conjunctiva, AQP4 in ciliary epithelium and retinal Müller cells, and AQP5 in corneal and lacrimal gland epithelia. This cell-specific expression pattern suggests involvement of AQPs in corneal and lens transparency, intraocular pressure (IOP) regulation, retinal signal transduction, and tear secretion. Indeed, humans with mutant AQP0 develop cataracts. Mice lacking AQP1 have reduced IOP and impaired corneal transparency after swelling, and mice lacking AQP4 have reduced light-evoked potentials by electroretinography. There is evidence for impaired cellular processing of AQP5 in lacrimal glands of humans with Sjogren's syndrome. AQPs facilitate fluid secretion and absorption in the eye, and hence are involved in the regulation of pressure, volume and tissue hydration. Pharmacological alteration of AQP function may provide a new approach for therapy of glaucoma, corneal edema, and other diseases of the eye associated with abnormalities in IOP or tissue hydration.     

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

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

大鼠视网膜神经网膜上神经营养因子的表达

目的　探讨神经生长因子 (N GF)、神经营养因子 3 (N T- 3 )及脑源性神经营养因子 (BDN F)在大鼠神经视网膜上的表达与分布。方法　 4 0 g· L- 1多聚甲醛灌注成年 Wistar大鼠 ,断头取眼球 ,放入 4 0 g· L- 1多聚甲醛 -蔗糖 (3 0 0 g· L- 1)固定液中过夜。运用免疫组化方法定性测定视网膜神经网膜上 N GF、BDN F、N T- 3的表达与分布。结果　 NG F、BDN F、NT - 3在 W istar大鼠视网膜神经网膜内均有表达 ,而且 3种神经营养因子主要分布于神经节细胞层和内核层 ,NT- 3主要分布于细胞核内 ,而N GF和 BDN F主要分布于细胞浆内。结论　 W istar大鼠视网膜神经网膜内含有丰富的神经营养因子 N GF、BDN F、N T- 3。     

Benzodiazepine receptors in the eye.

Central and peripheral benzodiazepine receptors were localized in the rat, monkey, and human eye by in vitro autoradiography. Central benzodiazepine binding sites, visualized with 3H-R015-1788, were enriched in the inner plexiform layer in all three species. Binding sites also were present in the nerve fiber layer, the ganglion cell layer, and in portions of the inner nuclear layer. Peripheral benzodiazepine binding sites, visualized with 3H-PK-11195, were found in the corneal epithelium and endothelium, iris, ciliary epithelium, trabecular meshwork, and throughout the retina. Binding sites for 3H-PK-11195 also were present in the retinal pigment epithelium and choriocapillaris areas and retinal vascular structures.     

Functions of aquaporins in the eye

The aquaporins (AQPs) are integral membrane proteins whose main function is to transport water across cell membranes in response to osmotic gradients. At the ocular surface, AQP1 is expressed in corneal endothelium, AQP3 and AQP5 in corneal epithelium, and AQP3 in conjunctival epithelium. AQPs are also expressed in lens fiber cells (AQP0), lens epithelium (AQP1), ciliary epithelium (AQP1, AQP4) and retinal Müller cells (AQP4). Mutations in AQP0 produce congenital cataracts in humans. Analysis of knockout mice lacking individual AQPs suggests their involvement in maintenance of corneal and lens transparency, corneal epithelial repair, intraocular pressure (IOP) regulation, retinal signal transduction and retinal swelling following injury. The mouse phenotype findings implicate AQPs as potential drug targets for therapy of elevated IOP and ocular disorders involving the cornea, lens and retina. However, much research remains in defining cell-level mechanisms for the ocular AQP functions, in establishing the relevance to human eye disease of conclusions from knockout mice, and in developing AQP-modulating drugs.     

Distribution of glucocorticoid and mineralocorticoid receptors and 11beta-hydroxysteroid dehydrogenases in human and rat ocular tissues

PURPOSE: The administration of glucocorticoids as topical or systemic medications may lead to the development of ocular hypertension through the induction of morphologic and biochemical changes in the trabecular meshwork leading to a reduction in the facility of aqueous outflow. Glucocorticoids exert their physiological effects by binding to and activating glucocorticoid and mineralocorticoid receptors. The activity of glucocorticoids is critically regulated at a prereceptor level by the two isozymes of 11beta-hydroxysteroid dehydrogenase. The purpose of this study was to determine the distribution of glucocorticoid target receptors and the isozymes of 11beta-hydroxysteroid dehydrogenase (11 beta-HSD) that regulate the activity of glucocorticoids at a prereceptor level in human and rat ocular tissues. METHODS: Horizontal sections of normal adult human and rat eyes were cut and hybridized with 35S-labeled cRNA probes specific for the glucocorticoid receptor, mineralocorticoid receptor, and 11beta-HSD types 1 and 2 using in situ hybridization. Immunohistochemical analysis of glucocorticoid and mineralocorticoid receptors using monoclonal antibodies was carried out on rat eye tissue sections. Whole rat eyes were homogenized and the activity of 11beta-HSD types 1 and 2 in the eye assessed as the percentage conversion of tritiated corticosterone to tritiated 11-dehydrocortico-sterone when corticosterone was added to the homogenate. RESULTS: In the rat ocular tissues mRNAs encoding glucocorticoid receptor, mineralocorticoid receptor, and 11beta-HSD types 1 and 2 were detected in nonpigmented ciliary epithelium, trabecular meshwork, corneal epithelium and endothelium, and anterior lens epithelium. Immunohistochemistry confirmed the presence of glucocorticoid and mineralocorticoid receptors at these sites. Activity of both isozymes of 11beta-HSD was demonstrated in homogenized rat eyes (percentage conversion of tritiated corticosterone to 11-dehydrocorticosterone; mean +/- SD, 11beta-HSD 1 = 15% +/- 5.3%, 11beta-HSD 2 = 7.9% +/- 2.8%). In both human and rat eyes, expression of mRNAs encoding glucocorticoid receptor and 11beta-HSD type 1 was high in the trabecular meshwork and lens epithelium, whereas expression of mRNAs encoding the mineralocorticoid receptor and 11beta-HSD type 2 was high in nonpigmented ciliary epithelium and corneal epithelium and endothelium. CONCLUSIONS: Glucocorticoid target receptors and the enzymes regulating glucocorticoid activity at these receptors are present in mammalian ocular tissues, which regulate aqueous humor formation and outflow. Alteration in the number or affinity of receptors or in the activity of regulatory enzymes may alter the susceptibility of certain individuals to the effects of glucocorticoids on intraocular pressure.     

Autoradiographic mapping of the glucose transporter with cytochalasin B in the mammalian eye

The anatomic localization of the glucose transport protein in the eyes of rats, rabbits, baboons, marmosets, and humans with [3H]cytochalasin B using in vitro autoradiography showed high densities of glucose carrier densities. These densities were seen in the ciliary body, especially ciliary processes, iris, retina, and in some species, the trabecular meshwork and lens. In the lens, specific [3H]cytochalasin B-binding sites were mainly concentrated in the lens nucleus. Lower concentrations were found in the cortex. During aging, glucose transporter concentration increases up to the age of 8 yr in the marmoset lens nucleus, but decreases in the cortex and retina. Moderate amounts of carrier are located in the corneal endothelium and epithelium. The enrichment of glucose carrier protein in the trabecular meshwork suggests a high metabolic activity and a possible relationship in the regulation of intraocular pressure.     

Expression patterns of cytochrome P4501B1 (Cyp1b1) in FVB/N mouse eyes

Homozygous and compound heterozygous mutations in cytochrome P4501B1 (CYP1B1) cause primary congenital glaucoma (PCG) in humans. It is hypothesized that developmental anomalies of the trabecular meshwork prevent appropriate drainage of the aqueous humor and cause PCG in human patients. In this report, we studied the expression patterns of Cyp1b1 in the eye of albino FVB/N mouse at different developmental stages. We isolated a cDNA fragment corresponding to the 3'untranslated region (3'UTR) of the Cyp1b1 gene by PCR and used it to make an (35)S-labelled riboprobe for in situ hybridization. We found that Cyp1b1 is expressed in both anterior and posterior segments of the eye. Anteriorly, the expression is confined to the ciliary body, most likely in the outer/pigmented ciliary epithelial cells. Cyp1b1 mRNA can be detected in these cells at postnatal day 4 (P4) and the expression continues into adulthood. Surprisingly, no above-background levels of Cyp1b1 mRNA were found at or around the trabecular meshwork at all the stages we examined. In the posterior region of the embryonic day 15 (E15) eye, Cyp1b1 is expressed in the retinal neuroepithelium and in the tissues surrounding the optic nerve, but not in the optic nerve itself. In the P7 retina, Cyp1b1 mRNA is found in the inner nuclear layer. Based on our finding that Cyp1b1 is expressed in the developing and mature ciliary body of the mouse eye, we speculate that mutation in this gene can directly contribute to the abnormal elevation of the intraocular pressure (IOP) in the PCG patients or indirectly affect the aqueous outflow by disrupting the proper development of the trabecular meshwork in these patients.     

Expression of the antigens recognized by monoclonal antibodies to human amnion (GB4, GB9, GB11) on the rabbit eye during embryonic development

Three monoclonal antibodies to human amnion (GB4, GB9 and GB11) which recognized well-defined structures of the adult rabbit eye were used to study the embryonic eye development. The reactivity of GB4 could be identified on the migrating neural crest cells under the corneal epithelium at 2 weeks of gestation. At 3 weeks of gestation, the conjunctival epithelium, corneal epithelium and corneal endothelium reacted with GB4. In the newborn rabbit, the corneal epithelium was reactive with GB9 instead of GB4; corneal endothelium was positive with GB4 at this stage, but gradually became negative in the adult rabbit; the subcapsular epithelium of the lens was recognized by GB4 when it changed from pseudostratified to simple cuboidal epithelium. During early embryonic life, GB11 was positive on the external retinal layer of the optic cup and lens vesicle. In the adult, GB11 reacted only with the pigmented epithelium of the ciliary processes. The results of this study demonstrated that the expression of antigens recognized by GB4, GB9 and GB11 could be mapped according to the epithelial differentiation of the rabbit eye during embryogenesis.     

Localization of myocilin/trabecular meshwork--inducible glucocorticoid response protein in the human eye

PURPOSE: To study distribution and cellular localization of myocilin/trabecular meshwork-inducible glucocorticoid response protein (TIGR) in the human eye. METHODS: A peptide antibody against a portion of the myosin-like domain of myocilin/TIGR was developed. Different ocular tissues from three human donors were investigated by one- and two-dimensional gel electrophoresis and Western blot analysis. Immunohistochemistry was performed on 25 human eyes enucleated because of posterior choroidal melanoma and on 7 normal human donor eyes. RESULTS: By Western blot analysis, a band at approximately 57 kDa was visualized in cornea, trabecular meshwork, lamina cribrosa, optic nerve, retina, iris, ciliary body, and vitreous humor. By immunohistochemistry, immunoreactivity for myocilin/TIGR was observed in cells of the corneal epi- and endothelium and extracellularly in the corneal stroma and sclera. In the trabecular meshwork, cells of the uveal and corneoscleral meshwork were stained, as was the cribriform area directly adjacent to Schlemm's canal. Positive staining was seen in cells of the ciliary epithelium, ciliary muscle, lens epithelium, and in stromal and smooth muscle cells of the iris. Throughout the entire vitreous body, fine filamentous material was positively labeled. In the retina, staining was seen along the outer surface of rods and cones, in neurons of the inner and outer nuclear layer, and in the axons of optic nerve ganglion cells. Optic nerve axons were stained in the prelaminar, laminar, and postlaminar parts of the nerve. In the region of the lamina cribrosa, astrocytes in the glial columns and cribriform plates were positively labeled. CONCLUSIONS: Myocilin TIGR is expressed in almost every ocular tissue. Depending on the respective tissue, it is observed extra- or intracellularly. The presence of myocilin/TIGR in optic nerve axons and lamina cribrosa astrocytes indicates that the trabecular meshwork might not be the only target of abnormal myocilin/TIGR in GLC1A-linked open-angle glaucoma.     

TRPV4在眼病理生理功能中的研究进展

瞬时受体电位香草醛受体4(TRPV4)是一种非选择性阳离子通道，负责感知细胞肿胀、温度、机械牵张、剪切应力和渗透压的变化，通过调节跨膜钙信号进而影响基因表达、细胞形态和细胞骨架等构建。TRPV4在全身广泛表达。在眼内，TRPV4在角膜、晶状体、睫状体、小梁网和视网膜等组织均有功能性表达。本文就TRPV4在眼内各个组织中的表达及生理病理功能方面进行阐述。随着TRPV4在眼部病理生理功能中的深入研究，TRPV4在角膜损伤修复、青光眼及视网膜血管生成方面可能成为潜在的新兴药物靶点，但仍需进一步的深入研究。     

二肽基肽酶II在正常大鼠眼球各组织中的免疫表达

目的:探讨二肽基肽酶II(dipeptidyl peptidaseII,DPPII)在正常大鼠眼球各组织中的免疫表达。方法:应用抗生物素蛋白-生物素-过氧化物酶复合体法,检测DPPII在正常大鼠眼球各组织中的免疫表达。结果:发现DPPII在角膜上皮细胞、角膜基质层、角膜内皮细胞、睫状体无色素上皮细胞、虹膜色素上皮细胞、晶状体上皮细胞、晶状体纤维、视网膜神经纤维层、神经节细胞、视网膜色素上皮细胞中免疫染色均呈阳性。结论:DPPII存在于角膜上皮细胞、角膜基质层、角膜内皮细胞、睫状体无色素上皮细胞、虹膜色素上皮细胞、晶状体上皮细胞、晶状体纤维、视网膜神经纤维层、神经节细胞、视网膜色素上皮细胞。     

Herpes simplex virus-mediated gene delivery to the rodent visual system

PURPOSE: To determine the types of cells in the visual system of the mouse and rat that can express a transgene delivered by an attenuated replication competent Herpes simplex virus-1 (HSV-1) vector. METHODS: C57/BL6 x BALB/C mice and Albino rats were treated with 1 x 10(7) pfu of the HSV-1 ribonucleotide reductase mutant (hrR3) expressing the Escherichia coli lacZ gene. The hrR3 virus was delivered by topical application to the cornea, intravitreal (IV) injection, intracameral injection (IC), or stereotactic injection into the visual cortex (VC). At specified times postinfection, animals were killed and tissues were removed, fixed, sectioned, and stained with X-gal or hematoxylin and eosin for histochemical and histopathologic examination. RESULTS: Topical delivery after corneal scarification in both mouse and rat resulted in lacZ expression in 25% of the corneal epithelial cells and 25% of the retinal pigment epithelium (RPE) cells. Topical application without concurrent scarification also resulted in transgene delivery to 20% of the RPE cells of the rat but not the mouse. IV injection resulted in expression primarily in RPE cells, with up to 100% of the cells expressing lacZ in the mouse and rat. Other cells expressing the transgene included ciliary body (CB) and optic nerve cells. Up to 25% of the retinal ganglion cells in the rat expressed lacZ, but only rarely in mice. IC delivery in rats resulted in expression in trabecular meshwork, CB cells, RPE, and iris epithelium. Injection into area 17 of the rat VC resulted in efficient labeling of the VC neurons and neurons in the lateral geniculate nucleus without any evident pathology or inflammation. Neither inflammation nor disease pathology was observed in either the mouse or rat after any route of delivery. CONCLUSIONS: It was demonstrated that the hrR3 HSV-1 virus can deliver a functioning gene to several cell types in the eye and neurons in the VC and that the virus can move via retrograde transport to nuclei that project to the VC.     

细胞因子对视网膜神经元保护和诱导作用的研究进展

近年来细胞因子在视网膜功能重建中的作用已初见端倪。研究发现,脑源性神经营养因子、睫状神经营养因子、碱性成纤维细胞生长因子及神经营养素4可以防止视网膜光感受器细胞变性的发生,增强光感受器细胞损伤后的修复,促进视网膜神经节细胞的发育,刺激神经节细胞轴突的再生。同时,晶状体上皮源性生长因子及人羊膜上皮细胞分泌的细胞因子也对神经节细胞有保护效应。而神经营养素-3在视网膜的作用性质还有待更多的研究。（中华眼科杂志,2005,41：861-864）     

钠尿肽受体A（NPR-A）在小鼠角膜和晶状体发育过程中的表达

目的　检测钠尿肽受体A（natriuretic peptide receptor A,NPR-A）在不同鼠龄小鼠角膜和晶状体内的表达,探讨其在小鼠眼发育过程中的作用。方法　选用C57BL/6转基因小鼠,收集从 E14到P90小鼠眼球标本120只,采用40 g·L^-1多聚甲醛灌注固定后,石蜡包埋切片,采用免疫组织化学方法对NPR-A在角膜和晶状体中的表达进行免疫荧光检测。结果　在E16,NPR-A高表达于角膜上皮细胞中,并持续至成年;其在角膜基质层和内皮细胞的表达也始于E16,而在P14之后,NPR-A表达随鼠龄的增加逐渐减弱。此外,NPR-A在P0小鼠晶状体上皮细胞膜内被检测到,并持续高表达至成年。在E16,由晶状体后壁上皮细胞生成的初级纤维开始高表达NPR-A,但随着鼠龄的增长和纤维结构的改变而逐渐减弱,直到P90消失。结论　NPR-A可能参与了角膜和晶状体生长和发育,并且对维持角膜上皮细胞的增生和修复以及晶状体的通透性具有重要作用。     

Cystatin C in the anterior segment of rat and mouse eyes

PURPOSE: Cystatin C is a mammalian cysteine protease inhibitor. This study describes the localization of cystatin C in the anterior segment of normal rat and mouse eyes. Cysteine proteases play an important role in protein degradation (e.g. of photoreceptor outer segments in the retinal pigment epithelium) and the balance between these proteases and their specific inhibitors is therefore of great interest. METHODS: Cells containing cystatin C were identified by immunohistochemistry and quantified by ELISA. Messenger RNA levels were analysed by quantitative real-time polymerase chain reaction. RESULTS: Cystatin C is present at biologically significant levels in the corneal epithelium, endothelium and stromal keratinocytes, lens epithelium, epithelial cells in the ciliary processes, aqueous humour and iris stromal cells. In the rat anterior segment, the highest cystatin C concentrations were found in the ciliary epithelium. CONCLUSIONS: Cystatin C is present in several cell types and is probably locally produced. The inhibitor is likely to be an important regulator of cysteine proteases in the retinal pigment epithelium, ciliary epithelium, aqueous humour, lens epithelium and in the corneal endothelium and epithelium.