Heavy metal concentrations in human eyes

PURPOSE: To measure the concentration of toxic heavy metals in the fluids and tissues of human eyes. DESIGN: Laboratory investigation. METHODS: Thirty autopsy eyes of 16 subjects were dissected to obtain the aqueous, vitreous, lens, ciliary body, retina, and retinal pigment epithelium/choroid. Concentrations of lead, cadmium, mercury, and thallium in ocular tissues, ocular fluids, and blood were determined using an inductively coupled plasma-mass spectrometer and expressed as ng/g. Heavy metal concentrations in ocular tissues were compared using a paired t test. RESULTS: Lead and cadmium were found in all of the pigmented ocular tissues studied, concentrating to the greatest extent in the retinal pigment epithelium/choroid (mean, 432 +/- 485 ng/g and 2,358 +/- 1,522 ng/g). Cadmium was found in the retina in all eyes (mean, 1,072 +/- 489 ng/g) whereas lead was found in the retina in 9 (30%) of 30 eyes (mean, 53 +/- 54 ng/g). Trace concentrations of lead and cadmium were detected in the vitreous (mean, 0.5 +/- 1.0 ng/dl and 19 +/- 29 ng/dl), lens (mean, 13 +/- 18 ng/g and 20 +/- 18 ng/g), and blood (mean, 0.5 +/- 1.2 mug/dl and 3.1 +/- 4.1 mug/l) but were not detected in the aqueous. Mercury and thallium were not detected in any ocular tissues or fluids or in the blood. CONCLUSIONS: Lead and cadmium accumulate in human ocular tissues, particularly in the retinal pigment epithelium and choroid. The potential ocular toxicity of these heavy metals and their possible role in eye disease requires further study.     

Transthyretin (prealbumin) in eye structures and variation of vitreous-transthyretin in diseases

PURPOSE: To evaluate the presence of transthyretin (TTR, prealbumin) a protein which binds retinol to retinol-binding protein in various ocular tissues and to study its quantitative changes in the vitreous humor in various diseases. METHOD: Estimation of TTR was done by electrophoresis of 10 mg protein in each sample of tears, aqueous humor, vitreous, retina, and lens by an Imaging Densitometer using prealbumin as the standard. RESULTS: TTR was present in all the eye structures except the lens and tear. The retina and the vitreous had relatively higher amounts of TTR compared with aqueous. The identity of TTR was confirmed by immuno-electrophoresis using anti-human TTR. Two bands in SDS electrophoresis revealed that this protein is a heterodimer. There was a significant decrease in vitreous TTR in diabetes with hypertension and increase in one case each of diabetes with hypertension associated with leukaemia or carcinoma with hepato-splenomegaly. CONCLUSION: Vitreous TTR is probably from retina and retinal pigment epithelium. The level of vitreous TTR is likely to have diagnostic significance in some retinal diseases.     

Identification and quantitation of carotenoids and their metabolites in the tissues of the human eye

There is increasing evidence that the macular pigment carotenoids, lutein and zeaxanthin, may play an important role in the prevention of age-related macular degeneration, cataract, and other blinding disorders. Although it is well known that the retina and lens are enriched in these carotenoids, relatively little is known about carotenoid levels in the uveal tract and in other ocular tissues. Also, the oxidative metabolism and physiological functions of the ocular carotenoids are not fully understood. Thus, we have set out to identify and quantify the complete spectrum of dietary carotenoids and their oxidative metabolites in a systematic manner in all tissues of the human eye in order to gain better insight into their ocular physiology.Human donor eyes were dissected, and carotenoid extracts from ocular tissues [retinal pigment epithelium/choroid (RPE/choroid), macula, peripheral retina, ciliary body, iris, lens, vitreous, cornea, and sclera] were analysed by high-performance liquid chromatography (HPLC). Carotenoids were identified and quantified by comparing their chromatographic and spectral profiles with those of authentic standards.Nearly all ocular structures examined with the exception of vitreous, cornea, and sclera had quantifiable levels of dietary (3R,3'R,6'R)-lutein, zeaxanthin, their geometrical (E / Z) isomers, as well as their metabolites, (3R,3'S,6'R)-lutein (3'-epilutein) and 3-hydroxy-beta,epsilon-caroten-3'-one. In addition, human ciliary body revealed the presence of monohydroxycarotenoids and hydrocarbon carotenoids, while only the latter group was detected in human RPE/choroid. Uveal structures (iris, ciliary body, and RPE/choroid) account for approximately 50% of the eye's total carotenoids and approximately 30% of the lutein and zeaxanthin. In the iris, these pigments are likely to play a role in filtering out phototoxic short-wavelength visible light, while they are more likely to act as antioxidants in the ciliary body. Both mechanisms, light screening and antioxidant, may be operative in the RPE/choroid in addition to a possible function of this tissue in the transport of dihydroxycarotenoids from the circulating blood to the retina. This report lends further support for the critical role of lutein, zeaxanthin, and other ocular carotenoids in protecting the eye from light-induced oxidative damage and aging.     

Immunohistochemical localization of cathepsin D in ocular tissues

Cathepsin D has been believed to play an important role in the catabolism of protein in various tissues. In retinal pigment epithelium, cathepsin D degrades rod outer segments and rhodopsin into glycopeptides. To our knowledge, no reports have described the immunohistochemical localization of cathepsin D in whole ocular tissues. We investigated the reaction of bovine, rat, and human eyes with a polyclonal antibody to cathepsin D from bovine spleen. Cathepsin D immunoreactivity was observed in the cytoplasm of the following cells: epithelium and endothelium of the cornea; keratocytes; pigmented and nonpigmented epithelium of the ciliary body; epithelium and cortex of the lens; epithelium and sphincter and dilator muscles of the iris; Müller cells; ganglion cells and pigment epithelium of the retina; and endothelium of various vessels. Positively stained ocular tissues were believed to have a high activity of protein catabolism. Since cathepsin D was closely associated with phagosomes in retinal pigment epithelium, we concluded that cathepsin D probably contributes to the physiologic degradation of rod outer segments.     

Distribution of epidermal growth factor in rat ocular and periocular tissues

Using a sensitive radioimmunoassay (RIA) specific for rat epidermal growth factor (rEGF), we investigated the presence of rEGF in a variety of rat ocular and periocular tissues. Immunoreactive rEGF (IR-rEGF) was present in tear fluids (25.5 ± 5.8 ng/ml), exorbital lacrimal gland (6.73 ng/g wet weight), intraorbital lacrimal gland (2.80 ng/g wet weight), Harderian gland (1.90 ng/g wet weight), and conjunctiva (0.16 ng/g wet weight). EGF was not detectable in aqueous humor, cornea, iris and ciliary body, lens, or the posterior part of the globe (retina, choroid, and vitreous body). Gel exclusion chromatography and reverse-phase high-performance liquid chromatography revealed that IR-rEGF in the above ocular fluids and tissues was indistinguishable from standard rEGF. Using enzyme-linked immunohistochemistry, rEGF was demonstrated to be localized in the duct epithelial cells of lacrimal glands. These findings reveal that EGF is preferentially localized in the ocular surface and lacrimal apparatus.     

Collagen XVIII/endostatin shows a ubiquitous distribution in human ocular tissues and endostatin-containing fragments accumulate in ocular fluid samples

Background The endostatin domain of type XVIII collagen (ColXVIII) inhibits neovascularization and regulates cell migration and matrix turnover. This study was designed to demonstrate the protein and gene expression patterns of ColXVIII/endostatin in the human eye and to ascertain whether endostatin is detectable in ocular fluid samples.Methods Twenty human eyes enucleated on account of choroidal melanoma were used for immunohistochemical stainings with antibodies against ColXVIII and endostatin. In situ hybridization was used to localize cells responsible for the production of mRNA for ColXVIII. Tear fluid, aqueous humor, and vitreous gel samples were used for Western immunoblotting to detect endostatin fragments in these samples.Results ColXVIII was immunolocalized to almost all ocular structures, namely the basement membranes (BMs) of the corneal and conjunctival epithelia, Descement’s membrane, the anterior border layer and posterior pigmented epithelium of the iris, the BMs of the pigmented and non-pigmented ciliary epithelia, the internal wall of Schlemm’s canal and trabeculae, the ciliary and iris muscle cells, the BMs of the pigment epithelium of the retina, and the internal limiting membrane. Universal expression was seen in the BMs of vascular endothelial cells, and in fibroblasts located in the conjunctiva, the iris, and the ciliary body. Endostatin showed a corresponding pattern, but additional immunostaining was present in the corneal and conjunctival epithelial cells. Most epithelial and mesenchymal cells expressed the mRNA for ColXVIII. Endostatin-containing fragments varying in size were detected in tear fluid, aqueous humor and vitreous gel samples.Conclusions Practically all structures of the human eye contain ColXVIII/endostatin, emphasizing its possible important structural and functional role in the human eye. Furthermore, ocular fluid samples contain endostatin fragments, which may contribute to the antiangiogenic properties of the eye.     

Circadian rhythms in the eye: the physiological significance of melatonin receptors in ocular tissues

Many biological processes display circadian rhythms in activity, which presumably operate to coordinate cellular functions with daily environmental oscillations. The diurnal changes in environmental illumination are conveyed by the retina to the brain to entrain circadian rhythms throughout the body. Many ocular tissues themselves exhibit circadian rhythms of activity to optimize specific processes which require coordination with the light-dark cycle. The circadian signaling molecule, melatonin, is secreted into the circulation from the pineal gland, and is also produced within specific ocular cells such as retinal photoreceptors, ciliary epithelial cells, and perhaps cells of the lens. Melatonin appears to entrain many aspects of the biological clock via activation of specific G-protein-coupled integral membrane melatonin receptors. Melatonin receptors have been identified in many ocular tissues, including the neural retina, retinal pigment epithelium, ciliary body, cornea, sclera, and lens. This review will describe the circadian rhythmicity of some of the functions of these various ocular tissues, and will attempt to correlate these circadian activities with the expression of specific G-protein-coupled melatonin receptors, the role of melatonin in the regulation of circadian activity in ocular tissues, and its potential role in ocular diseases.     

Isolation of a cDNA encoding a glutathione S-transferase (GST) class-pi from the bovine ocular ciliary epithelium.

We have used a polyclonal antiserum to bovine ciliary epithelium, a secretory tissue involved in the formation of aqueous humor, to immunoscreen a directional lambda gt11 Sfi-Not cDNA expression library prepared from bovine ciliary epithelium poly(A)+ RNA. After immunoscreening 6 x 10(5) independent clones, 41 cDNA clones positive for ciliary epithelium were isolated and characterized. About one-third of the positive cDNA clones were found to be identical and to encode a glutathione S-transferase (GST) class-pi. The largest bovine GST cDNA clone isolated, pCN11, contains an open reading frame of 630 bases, encoding a protein of 210 amino acids with a calculated molecular weight of 23,335 Da. The corresponding amino acid sequence showed an overall identity of 85.6% with the human, and 85.2% with the rat and mouse GST class-pi. Northern analysis of bovine ocular tissues revealed that the GST class-pi gene encodes a 0.8-kilobase mRNA which is expressed most abundantly in cornea, ciliary epithelium and retina, and in lower levels in iris and lens. Cell lines derived from non-pigmented or pigmented bovine ciliary epithelium also showed high levels of GST-pi mRNA expression. These results provide additional evidence for differential gene expression of GST class-pi mRNA in various areas of the bovine eye.     

Dynamics of ascorbate in the aqueous humor and tissues surrounding ocular chambers

The level of ascorbate in the aqueous humor and surrounding intraocular structures in enucleated arterially perfused rabbit eye was investigated. The enucleated eye preparation was shown to be capable of secreting ascorbate from the perfusate into the aqueous humor. Ascorbate in the iris, ciliary body and cornea was released into the aqueous humor when the eye was perfused with ascorbate-free solution. Failure to obtain aqueous flow rates from the decay of ascorbate in the anterior chamber was due to the contribution of ascorbate from these ocular tissues during the perfusion. Histochemically, ascorbate was localized in the pigmented epithelial layer in the valleys between the ciliary processes and the pars plana of the ciliary body and in the iris. In the cornea, distinct localization of ascorbate was observed in the endothelium and basal cell layer of the epithelium.     

Distribution of transthyretin in the rat eye

We reported previously synthesis of transthyretin (TTR), or prealbumin, a transport protein for thyroxine and retinol, in the eyes of rats and cows and showed that in the rat eye, TTR mRNA is localized exclusively in the retinal pigment epithelium (RPE). We now demonstrate by immunohistochemistry that TTR has a more widespread distribution in the rat eye than does its mRNA. Intense immunoreactivity for TTR was found in the RPE, ciliary epithelium, iris epithelium, corneal endothelium, optic nerve fiber layer of the retina, and lens capsule. Depending on the method of processing, immunoreactivity of varying intensity was found also in other ocular structures. In particular, the retinal ganglion cells were strongly immunoreactive on frozen sections but not on paraffin sections. Although vitreous humor was not included in the sections of adult rat eye, sections of a 25-mm rat embryo showed intense immunoreactivity in the vitreous humor. Since plasma TTR does not cross Bruch's membrane into the retina, our findings suggest that ocular TTR is synthesized, at least in part, in the RPE and is transported to specific locations within the eye. Although the physiologic role of ocular TTR is unknown, it is possible that it participates in retinol cycling within the eye. The widespread ocular distribution of TTR may account for the occurrence of various forms of ocular amyloidosis in the familial amyloidotic polyneuropathies, a group of dominantly inherited disorders caused by point mutations in the TTR gene.     

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.     

醋酸强的松龙滴眼后在兔眼内的分布

目的观察醋酸强的松龙短期和长期滴眼后在兔眼多种组织中的分布。方法用1g.L-1醋酸强的松龙滴眼液分1d内数次或连续29d长期滴兔眼,分别于滴眼后15min、30min、60min、120min和29d取材,高效液相色谱法测定角膜、房水、晶状体、玻璃体和血液中的激素含量。结果1g.L-1醋酸强的松龙滴眼液滴眼后兔眼不同组织和血液中的激素分布随时间而变化,无论短期或长期连续滴眼在角膜和房水中均获得较高的激素含量,血液和玻璃体中亦有少量进入。短期滴眼后,仅在晶状体赤道和前皮质有少量激素进入,长期局部滴眼时在晶状体各部位均有蓄积。结论1g.L-1醋酸强的松龙局部滴眼后易进入角膜和房水。此结果有助于了解眼部激素治疗和并发症发生的关系。     

外排转运蛋白在眼部给药中的研究动态

外排运转蛋白是一种广泛分布于人体全身组织器官的细胞膜转运蛋白,它可以将进入细胞内的外源性物质,包括药物等转运出细胞外,对细胞起到保护作用.近年研究表明,该蛋白亦分布于角膜、虹膜、睫状体、视网膜神经上皮和视网膜色素上皮(RPE)等眼部组织,每种转运蛋白都有其相对应的特异性底物及抑制剂.外排转运蛋白通过其对药物的外排转运功能影响不同给药途径中药物在眼内的利用和分布.深入研究外排运转蛋白药物的吸收和分布,清除时所起的作用,有助于进一步了解眼部给药的机制和设计给药方略,为临床眼部给药和给药途径的开发提供更多的科学依据.     

Ocular trace metal kinetics and toxicology. I. The distribution of intravitreally injected 67Cu++ within intraocular compartments and its loss from the globe

Radioactive copper (67Cu++) was injected into the center of the vitreous body of rabbits. The relatively rapid initial loss of 67Cu from the vitreous was associated with its accumulation in intraocular tissues. At 24 hr, 20% of the injected 67Cu was found in the retina, representing the highest 67Cu concentration among all ocular tissues, and this high 67Cu concentration was maintained in this tissue throughout the 10-day observation period. Significant amounts of 67Cu were not detected in the aqueous humor at any time. About one-half of the injected 67Cu was lost from the whole globe in 5 days, but the remaining Cu was retained in the globe during the next 5 days. Eyes that received a large dose of CuSO4 in addition to the tracer showed decreased 67Cu activity in the retina and a slight increase in the aqueous humor. Endotoxin-induced ocular inflammation decreased the rate of 67Cu loss from the vitreous, reduced its accumulation by the retina, and increased 67Cu entry into the aqueous humor. It is concluded that 67Cu is retained in the vitreous and the globe due to its binding by, and/or uptake into, intraocular tissues, especially the retina. Cu does not effectively enter the anterior chamber from the vitreous, apparently due to its effective removal by the ciliary processes, thus ruling out the possibility of identifying the existence of Cu-containing intraocular foreign bodies in the posterior segment of the eye by analysis of Cu in the aqueous humor.     

Intraocular penetration kinetics of prednisolone after subconjunctival injection in rabbits

Prednisolone concentrations in cornea, aqueous humor, and vitreous humor and the residual amount in conjunctival tissue were assayed by high-performance liquid chromatography during a 14-hour period after subconjunctival injection of prednisolone sodium succinate in rabbits. Prednisolone was concentrated in the corneal epithelium and reached a peak within 5 min, whereas the peak level of prednisolone in stroma-endothelium was achieved 1 h after the injection. There was an apparent linear binding of prednisolone with the ocular tissue homogenates and fluids except for the vitreous humor. However, the protein binding of prednisolone with vitreous humor showed marked concentration dependency. A pharmacokinetic model involving a rapid conversion to prednisolone from its ester prodrug, first-order transfer to various tissues, and first-order elimination of unbound prednisolone from vitreous humor succeeded in predicting the observed concentration-time profiles of prednisolone in various ocular tissues and fluids after subconjunctival injection at three different doses: 0.1, 1.0, and 10.0 mg/kg. The present model predicted that absorption into precorneal area and epithelium and direct penetration into aqueous humor and vitreous humor are 1.7, 0.1, and 0.2% of the applied dose, respectively, and that almost the entire dose (98%) is absorbed into the systemic circulation, with a half-life of 38 min.     

The lens influences aqueous humor levels of transforming growth factor-β2

Background: Transforming growth factor-beta 2 (TGF-β2) is a pluripotent cytokine which has been suggested to play a number of roles in ocular physiologic and pathologic states. Intraocular fluid (IOF) levels of TGF-β2 are quite high. Although the sources of ocular TGF-β are not completely defined, the retinal pigment epithelium, the epithelium of the ciliary body and trabecular meshwork cells all secrete it. In this study we utilized canine lens and rabbit ciliary pigmented epithelial cell cultures to quantitate the in vitro secretion of TGF-β2. In addition, the effects of aphakia or the presence of cataractous lenses on IOF TGF-β2 levels were determined. · Methods: Lens and ciliary body epithelial cell culture supernatants and aqueous humors were assayed for total TGF-β2 levels by ELISA and bioassay. · Results: TGF-β2 accumulated in the media bathing lens epithelial cell cultures (0.7 ± 0.03 ng/ml at day 2) and ciliary pigmented epithelial cell cultures (0.8 ± 0.06 ng/ml at day 2) in a time-dependent manner. Surprisingly, aqueous humor from aphakic rabbit eyes contained significantly higher levels of TGF-β2 than their contralateral phakic controls. Furthermore, aqueous humor from canine eyes with cataracts also contained significantly higher levels of TGF-β2 than normal eyes. · Conclusions: These results suggest that the lens secretes TGF-β2 and that the presence and status of the lens may influence IOF TGF-β2 levels. Received: 12 August 1996 Revised version received: 18 June 1997 Accepted: 18 July 1997     

两种不同注射方法眼局部注射鼠神经生长因子后兔眼内药物分布

目的 观察兔玻璃体腔注射与球周注射~(125)I-神经生长因子(NGF)后眼部各组织药物含量分布.方法 45只家兔分别于左眼玻璃体腔(A组)和右眼球周(B组)注射~(125)I-NGF 30 μg/100μl.于注药后15、30 min,1、3、6、8、12、24、48 h取眼房水、玻璃体等眼内容物,角膜、巩膜等眼球外壁组织,虹膜睫状体、视网膜、脉络膜等眼球内壁组织进行γ-计数,计算~(125)I-NGF的含量.结果 A组药物在眼内容物及眼球内壁各组织弥散较快,玻璃体内含量呈梯度下降,其它眼内组织含量呈正态曲线变化;房水、虹膜睫状体、视网膜、脉络膜达峰值时间均为给药后3 h,巩膜、角膜分别为6、8 h;B组眼内各组织药物含量呈正态曲线变化,药物达眼内峰值时间较慢,除房水在给药后3 h达峰值,其他组织均在6 h达峰值;A组各组织~(125)I-NGF峰值含量也明显高于B组;A组除玻璃体直接注射导致高药物含量外,视网膜药物含量最高.结论 玻璃体腔注射~(125)I-NGF较球周注射更能在眼内各组织达到高药物含量.     

Immunocytochemical localization of ornithine aminotransferase in rat ocular tissues

Gyrate atrophy of the choroid and retina is a rare inherited form of chorioretinal degeneration due to a deficiency of ornithine aminotransferase (OAT). We localized the enzyme in rat ocular tissues using immunocytochemical procedures. Immunoreactivity was observed in the epithelia of ciliary body, iris, and lens. Retinal pigment epithelium and Müller cells were immunoreactive in the retina. A little immunoreactive product was found in the choroid. Our findings suggested that OAT plays an important role in ornithine metabolism in these ocular tissues.     

新型生物活性物质尾加压素Ⅱ在人眼组织的分布

目的:探讨新型生物活性物质尾加压素Ⅱ(UrotensinⅡ,U-Ⅱ)在人眼组织的分布。方法:将正常新鲜人眼解剖分离出各部分组织并作匀浆后,采用放射免疫测定法检测U-Ⅱ在人眼各组织的分布。结果:U-Ⅱ在人眼各组织均有分布,范围在0.807～32.958ng/g之间,其中以视神经中的含量最高(32.958±1.741ng/g),其后依次为巩膜、虹膜、角膜、睫状体、脉络膜、视网膜、晶状体,玻璃体和房水中的含量最低。视神经中U-Ⅱ含量是房水中的40.4倍。结论:U-Ⅱ广泛分布于人眼各组织,其生理和病理生理作用有待于进一步探讨。