Immunogold study of non-collagenous matrix components in normal and exfoliative iris.

The present investigation was undertaken to determine if some of the components of exfoliation material in iris tissue were unique to exfoliation or were part of normal iris architecture. Eleven normal iris specimens and 10 exfoliative iris specimens were processed for cryoultramicrotomy and London resin white embedding. Immunogold electron microscopy was used to investigate the fine structural distribution of amyloid P component, elastin, entactin, fibronectin, gp115, and vitronectin in normal iris and their association with exfoliation material. Exfoliation material was positive for amyloid P component and possibly gp115, neither of which were present in normal iris tissue. Elastin and fibronectin were present in the normal iris stroma but were not associated with exfoliation material. The distribution of amyloid P component in the vessel lumen and wall led to the conclusion that amyloid P is a serum contaminant. The presence of gp115 in exfoliation material represents the synthesis of a component novel to the iris vascular cell synthetic repertoire.     

Immunogold localization of type IV collagen and laminin in the aging human outflow system

Tissue from the outflow system of six surgically enucleated aged eyes was used for an ultrastructural immunocytochemical study of the distribution of laminin and type IV collagen. The immunogold technique provided precise localization of laminin beneath lining endothelial cells of the inner wall of Schlemm's canal. Laminin labelling was absent in the trabecular beams. Type IV collagen was found in the basement membranes of the trabecular beams and in fine filamentous basement membrane material in the cribriform layer. Electron dense plaques in the cribriform layer labelled positively for laminin in the outer coarse fibrillar zone but not in the electron dense core. Long-spacing collagen was negative for type IV collagen.     

层黏连蛋白体外对人虹膜色素上皮细胞的影响

目的:研究不同浓度的层黏连蛋白对体外培养的人虹膜色素上皮细胞(IPE)的作用.方法:采用酶辅助的显微分离法对成人眼进行IPE的原代和传代培养,观察其形态;以不同浓度的层黏连蛋白(0,1,5,10mg/L)处理第3代IPE细胞,MTT法作出生长曲线,免疫荧光法检测细胞角蛋白1(CK1)表达的变化.结果:原代培养的IPE细胞内色素丰富,而传代培养后色素显著减少;低浓度层黏连蛋白(5mg/L)明显促进IPE细胞增殖(0.187 vs 0.151,0.236 vs 0.176,0.245 vs 0.215,0.261vs 0.238,0.279 vs 0.254;P＜0.05);高浓度(10mg/L)明显抑制细胞增殖(0.121 vs 0.151,0.081 vs 0.176,0.094 vs0.215,0.065 vs 0.238,0.078 vs 0.254;P＜0.05);CK1染色均为阳性,高浓度层黏连蛋白(10mg/L)使IPE细胞聚集,CK1表达明显增强.结论:层黏连蛋白对IPE细胞具有双重作用,低浓度的层黏连蛋白适于IPE细胞的体外培养.     

Immunocytochemical localisation of collagens (I–V) in the human iris

In this study we investigated the distribution of collagen types I–V in the human iris at the fine-structural level using cryoultramicrotomy and London Resin White plastic embedding. Collagen type I was shown to be present in the basement membrane of iris vessels, in contrast to type III, which was absent; both types I and III were present in the iris stroma. Collagen type IV was a major component of basement membranes of vascular cells, myoepithelial cells, fibroblasts and epithelial cells. Types II and V were absent. Both cryo and plastic embedding techniques produced closely comparable results. Supported by the WH Ross Foundation for the Prevention of Blindness     

Immunocytochemical localisation of collagens (I–V) in the human iris

In this study we investigated the distribution of collagen types I–V in the human iris at the fine-structural level using cryoultramicrotomy and London Resin White plastic embedding. Collagen type I was shown to be present in the basement membrane of iris vessels, in contrast to type III, which was absent; both types I and III were present in the iris stroma. Collagen type IV was a major component of basement membranes of vascular cells, myoepithelial cells, hbroblasts and epithelial cells. Types II and V were absent. Both cryo and plastic embedding techniques produced closely comparable results.Supported by the WH Ross Foundation for the Prevention of Blindess     

Effects of aging on fluorescein leakage in the iris and angle in normal subjects.

PURPOSE: To determine the presence of fluorescein leakage from the iris and angle in normal subjects, and how it is affected by aging. METHODS: The subjects were 92 normal volunteers and patients with senile cataract who ranged in age from 20 to 93 years and were free from any systemic or ocular diseases. Fluorescein iris and angle photography and color iris and angle photography were performed using a goniolens and a photo slit lamp. RESULTS: Radial ciliary body vessels were found in 22 eyes (24%), radial iris vessels or trabecular vessels in 7 eyes (8%), and circular ciliary band vessels were seen in 4 eyes (4%). Goniovessels were found in 8 of 30 eyes (27%) of those under 50 years of age. No significant difference in the incidence of goniovessels was found between those over and under age 50 (P < .01). In the pupillary margin, fluorescein leakage was seen in 1 of 30 eyes (3%) in the age group under 50 years, whereas leakage was found in 30 of 62 eyes (48%) in the age group over 50 years. In the anterior chamber angle, leakage was seen in 4 of 30 eyes (13%) under age 50 years, and in 38 of 62 eyes (61%) over age 50 years (P < .05). CONCLUSIONS: These findings suggested that the incidence of leakage of fluorescein from the pupillary margin and anterior chamber angle tends to increase with age. Thus, when leakage of fluorescein in angle and iris is observed, it is important to consider the physiological changes resulting from aging.     

Immunogold localization of actin in developing photoreceptor cilia of normal and rds mutant mice

Recent immunocytochemical studies have localized actin to the distal cilium of mature vertebrate photoreceptors. Since this is the site where the ciliary plasma membrane evaginates to form new outer segment discs, the results suggest that an actin-mediated contractile mechanism or cytoskeletal network might regulate some aspect of outer segment disc morphogenesis. In the present study, immunoelectron microscopy was used to localize actin to the developing cilia of normal and rds mutant mouse photoreceptors. In normal mice, actin could not be localized to newly projecting cilia, but an actin-rich domain was demonstrated within the distal, bulbous ending of elongated cilia just prior to outer segment development. These results suggest that actin is not important for ciliary growth, but that it may be necessary for the subsequent differentiation of an outer segment. In the rds mutant mouse, there is an absence of outer segment formation, although cilia appear to develop normally. Rhodamine phalloidin staining of cryostat sections demonstrated a normal F-actin distribution within the rds retina. Utilizing immunogold labeling of developing rds photoreceptors, actin was localized to the distal, bulbous ending of elongated cilia. This result indicates that actin is situated within its normal domain in rds cilia.     

Synthesis of hyaluronan by normal and wounded rabbit iris

Background: Endogenous hyaluronan has been found in different tissues in the normal and traumatized eye. However, the main source, the biological aspects and the full potential role of hyloronan are still unclear. Methods: Hyaluronan production was studied both in organ culture and in vivo, using a double-label protocol with [³⁵S]sulfate and [³H]glucosamine. Results: [³H]glucosamine and [³⁵S]sulfate were incorporated into hyaluronan and sulfated glycosaminoglycans in normal and in traumatized iris tissue in organ culture and in vivo. There was low relative hyaluronan synthesis in vivo, only 2% of total incorporated [³H]glucosamine in normal irides. Increased relative incorporation of [³H]glucosamine into hyaluronan was seen after operative trauma to iris tissue both in vivo and in vitro. Conclusion: Our findings demonstrate synthesis of hyaluronan by normal and traumatized iris. The iris seems to be the most important source of aqueous hyaluronan. Received: 7 October 1999 Revised: 31 January 2000 Accepted: 31 January 2000     

Immunogold localization of chondroitin 6-sulfate in the interphotoreceptor matrix of normal and RCS rats

Localization of chondroitin 6-sulfate (6S) in the interphotoreceptor matrix (IPM) of both normal and RCS rats with inherited retinal dystrophy has been carried out using light and electron microscopic immunogold cytochemistry. In the normal rat, 6S antibody labeling was found in highest concentration at the apical surface of the retinal pigment epithelium (RPE) and between adjacent photoreceptors near their basal inner segment-outer segment junction. In the apical zone, label was localized in the IPM and toward the distal portions of the RPE apical processes. In the basal zone, label was found in the IPM and near the outer plasma membranes of inner and outer segments. Interstitial labeling, between the shafts of outer segments, occurred at much lower concentration than in the apical and basal zones. In all zones, the extent of labeling appeared to be space-dependent; it was most abundant where extracellular spaces were large, and little was present where adjacent cell membranes were contiguous. In the dystrophic rat, apical zone labeling of the RPE apical processes was minimal; instead, the relatively small amount of label that was present was localized primarily in the IPM and within encapsulated spaces of membranous whorls of debris. Label was most concentrated in the basal zone, primarily associated with the IPM within abundant interphotoreceptor spaces, and near the plasma membranes of disorganized inner and outer segments. In isolated eyecups and neural retinas, some labeling persisted after extensive buffer rinses which suggests that chondroitin 6-sulfate is a somewhat insoluble component of the IPM in the rat retina.     

Immunogold localization of myosin in the photoreceptor cilium.

The photoreceptor outer segment (OS) develops from the distal end of a nonmotile cilium and exhibits a continuous renewal of its disc structures throughout life. Immunocytochemical studies have localized an actin-rich domain to the ciliary axoneme at the base of the OS, and recent ultrastructural studies of detergent-extracted retinas have demonstrated actin filaments at this site. These filaments are believed to participate in the formation of new outer segment discs. In this study, rat neural retinas were treated with the detergent saponin, fixed with aldehydes, and embedded in London resin white resin for immunoelectron microscopy. In thin tissue sections, actin filaments were observed within the appropriate ciliary domain and these filaments could be positively stained with anti-actin antibodies. When this tissue was immunogold labeled with anti-myosin antibodies, myosin also was found in this locale. The results of this study demonstrate that saponin-extracted retinas can be used for immunoelectron microscopy studies of the photoreceptor cytoskeleton. In addition, the presence of myosin within the actin-filament-containing portion of the cilium suggests that an actomyosin contractile mechanism may play a role in outer segment disc morphogenesis.     

Effects of aging on fluorescein iris and angle photography in normal subjects]

In the present study we took the fluorescein iris and angle photography of normal eyes and studied them according to different age groups. The range of age in 108 eyes of 92 subjects was from 20 to 93 years. They were free from any systemic diseases or ophthalmic diseases which would cause iris rubeosis. Fluorography was carried out using a Mizuno Trabeculens to determine the leakage points of fluorescence from the iris and the angle. In the angle there were gonio vessels which revealed no leakage of fluorescein. They were observed in 8 of 30 eyes (27%) for subjects in the fifth decade of life or less and in 24 of 78 eyes (31%) for subjects aged 50-90. No significant difference was seen between the two age groups. As to variations of gonio vessels, radial ciliary body vessel was found in 23 eyes, radial iris vessel or trabecular vessel in 10 eyes, and circular ciliary band vessel in 7 eyes. In the pupillary margin no leakage was seen in 30 eyes of 30 cases in the fifth decade of life or less, while leakage was found in 33 of 78 eyes, 42 of 62 cases over the fifth decade of life. In the angle leakage was seen in 4 of 30 eyes, 13 of 30 cases in the fifth decade of life or less, while leakage was observed in 43 of 78 eyes over the fifth decade of life. These results suggested that leakage of fluorescence from the pupillary margin, the angle or both increases with age.(ABSTRACT TRUNCATED AT 250 WORDS)     

荧光血管造影用于国人棕色虹膜新生血管诊断的初步观察

目的观察国人虹膜新生血管（NVI）患者的虹膜荧光血管造影（IFA）表现,与裂隙灯检查对比,探讨IFA在NVI诊断上的临床应用价值。方法对国人40例（40眼）正常者和27例（28眼）各种病因继发NVI患者及24例（27眼）可疑NVI患者,进行了裂隙灯、虹膜彩照和IFA检查。结果正常对照组IFA不显影,呈色素遮蔽荧光,部分（22.2%）大于60岁正常老年人瞳孔缘可见轻微荧光素渗漏。所有NVI患者（100%）IFA显示瞳孔缘和虹膜表面出现小团状、细线状或不规则交叉网状强荧光,充盈迅速,伴有不同程度的荧光素渗漏。对于部分（11.1%）严重眼底缺血患者,IFA可提前于裂隙灯发现NVI。结论IFA在国人棕色虹膜病变特别是NVI的诊断中有应用价值,能够更敏感地显示NVI的形态和位置,并判断其活动性,为NVI的诊断及疗效判断提供了客观依据。     

Natural killer-cell lymphoma of the iris with a normal fundus

BACKGROUND: Intraocular lymphoma is a relatively uncommon malignancy, rarely presenting with exclusively anterior segment findings in a normal fundus. CASE: A 38-year-old male, previously diagnosed with nasal lymphoma, currently in complete remission after chemotherapy and radiotherapy, presented with blurring of vision in his left eye, which initially responded to local steroid therapy. However, the patient developed resistance to steroid therapy and developed nodular masses in the left iris. Ultrasound biomicroscopy revealed diffuse thickenings leading to a complex and uneven shape of the left iris. An iris biopsy was done which led to the diagnosis of natural killer(NK)-cell lymphoma. Radiation therapy was instituted which led to rapid resolution of both the nodular masses and iritis. CONCLUSION: This is a case of malignant lymphoma presenting as metastasis to the iris without involvement of the chorioretinal tissues. When encountering a patient with iritis resistant to the steroid therapy, clinical ophthalmologists should consider metastatic malignancy in the differential diagnosis.     

Natural killer-cell lymphoma of the iris with a normal fundus

BackgroundIntraocular lymphoma is a relatively uncommon malignancy, rarely presenting with exclusively anterior segment findings in a normal fundus.CaseA 38-year-old male, previously diagnosed with nasal lymphoma, currently in complete remission after chemotherapy and radiotherapy, presented with blurring of vision in his left eye, which initially responded to local steroid therapy. However, the patient developed resistance to steroid therapy and developed nodular masses in the left iris. Ultrasound biomicroscopy revealed diffuse thickenings leading to a complex and uneven shape of the left iris. An iris biopsy was done which led to the diagnosis of natural killer (NK) -cell lymphoma. Radiation therapy was instituted which led to rapid resolution of both the nodular masses and iritis.ConclusionThis is a case of malignant lymphoma presenting as metastasis to the iris without involvement of the chorioretinal tissues. When encountering a patient with iritis resistant to steroid therapy, clinical ophthalmologists should consider metastatic malignancy in the differential diagnosis.     

Ultrastructural localization of collagen IV, fibronectin, and laminin in the trabecular meshwork of normal and glaucomatous eyes.

PURPOSE: To determine whether differences in the ultrastructural characteristics or composition of the basement membranes of the trabecular lamellae and Schlemm's canal exist in normal eyes and eyes with primary open-angle glaucoma (POAG). Basement membranes play key roles in the attachment of the overlying trabecular cells and Schlemm's canal cells. METHODS: Electron microscopy used in conjunction with immunogold labeling was used to examine the ultrastructure of the basement membranes in the trabecular meshwork and to determine the presence of collagen IV, laminin, and fibronectin in 6 normal eyes and 6 eyes with POAG. To determine which cells in the meshwork synthesized these molecules in situ hybridization was studied in an additional 8 normal eyes. RESULTS: No distinctive ultrastructural changes were found in the basement membranes of glaucomatous eyes, whether early or advanced disease, when compared with normal eyes. Label for all three proteins was present in the basement membranes of the trabecular lamellae, Schlemm's canal, and in scattered patches within the juxtacanalicular tissue. Laminin and fibronectin were most abundant in the periphery of the sheath material surrounding the elastic tendons in the juxtacanalicular tissue. In contrast to previously published light microscopic studies, no increase in fibronectin was found in glaucoma. Regions of the basement membrane of the canal underlying giant vacuoles were similar to regions without giant vacuoles in both appearance and labeling. In situ hybridization revealed that mRNA for all three proteins was present in most trabecular cells throughout the meshwork; no regional differences in cellular labeling within were observed. CONCLUSION: The ultrastructural characteristics and immunogold labeling of basement membranes were similar in normal and glaucomatous eyes; no additional structures were labeled in POAG eyes that were not also labeled in normal eyes. Label of the patches of amorphous fibrogranular material within the juxtacanalicular tissue suggests it is basement membrane in origin, while the sheath material which is known to accumulate in POAG was not heavily labeled and does not appear to be basement membrane in origin.     

Histochemical studies on the glycosaminoglycans in the normal and glaucomatous iris of human eyes

Summary In 70 irisspecimens of normal and glaucomatous eyes of all age groups (2–80 years), the changes in the amount and composition of acidic glycosaminoglycans (GAG) were studied using the CEC method of Scott and Dorling (1965) with Alcian blue and MgCl₂ with and without hyaluronidase or chondroitinase digestion. The staining intensity was measured in estimated arbitrary units. Chondroitin sulfates were found within the iris vessel walls and the anterior border layer. Evidence was found indicating that a certain amount of hyaluronic acid and hyaluronidase-resistant GAG is present in these structures.In the aging iris, there was a tendency toward a decrease in alcianophilia within the perivascular sheaths and an increase in alcianophilia in the anterior border layer. The age-correlated decrease in hyaluronidase-resistant Alcian blue staining of the vessel wall was much more moderate in glaucomatous than in normal eyes, which therefore showed a higher content of hyaluronidase-resistant GAG (probably keratan sulfate or heparan sulfate) in old age.The described changes could be of some importance to the process of maturation of the collagenous fibers located within the perivascular sheaths of the iris vessels. The differences observed between normal and glaucomatous eyes may be related to the still unknown pathogenetic process of this disease.

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Zusammenfassung Bei 70 Iridektomiestückchen von normalen und glaukomatösen Augen verschiedener Altersgruppen (2–80 Jahre) wurden die alternsabhängigen Veränderungen der sauren Glykosaminoglykane (GAG) histochemisch mit Hilfe der CEC-Methode von Scott und Dorling (1965) bei zusätzlicher Verdauung mit Hyaluronidase und Chondroitinase neu untersucht. Die jeweiligen Färbeintensitäten wurden mit Hilfe von Schätzwerten quantitativ zu erfassen versucht. In den Gefäßscheiden sowie in der vorderen Grenzschicht wurden reichlich Chondroitinsulfate nachgewiesen. Daneben fanden sich auch Hinweise auf das Vorkommen von Hyaluronsäure und Hyaluronidase-resistenten GAG.In der alternden Iris besteht eine Tendenz zur Verminderung der Alcianophilie in den Gefäßscheiden sowie einer Vermehrung der Alcianophilie in der vorderen Grenzschicht. Die alternsabhängige Abnahme der Hyaluronidase-resistenten Alcianblaufärbung in der Gefäßwandung war im glaukomatösen Augen wesentlich geringer als in normalen Augen. Letztere zeigten daher im hohen Alter einen höheren Gehalt an Hyaluronidaseresistenten GAG (eventuell Keratansulfat oder Heparansulfat).Die beschriebenen Veränderungen könnten für die Kollagenfaserreifung in den Gefäßscheiden der Irisgefäße wichtig sein. Die Unterschiede zwischen normalen und Glaukomaugen hängen möglicherweise mit dem Entstehungs mechanismus des Glaukoms zusammen.