Cytoskeletal filament typing of human corneal endothelial cells.

Flat mounts of human corneal endothelial cells (HCECs) were examined immunohistochemically by using a wide assortment of monoclonal antibodies against the five classes of intermediate filaments (IFs) and actin and myosin. HCECs showed uniform immunostaining with monoclonal antibodies against the 40-kD (CK 19) and 45-kD (CK 18) cytokeratin (CK). Only part of the endothelial cells reacted with monoclonal antibodies against the 52-kD (CK 8) and 54-kD (CK 7) cytokeratin polypeptides and with monoclonal antibodies against vimentin. Monoclonal antibodies against the low- and middle-molecular-mass neurofilament proteins produced positive staining of all HCECs. No positivity was obtained with antibodies against desmin or glial fibrillary acidic protein. In addition, positive immunostaining with monoclonal antibodies against actin and slow myosin demonstrate that these proteins form part of the cytoskeleton of HCECs. The results of this study show that immunostaining of flat cell preparations is very useful for studies on HCECs. HCECs display an unusual combination of cytokeratin IFs and neurofilaments, together with vimentin, and are heterogeneous with respect to their IF makeup. These findings are discussed in relation to the presumed origin of HCECs.     

Cytoskeleton and tissue origin in the anterior cynomolgus monkey eye

We studied cytoskeletal proteins and other markers for embryologic origin in the outflow pathways of the aqueous humor, cornea, sclera, and ciliary muscle of the cynomolgus monkey. The corneal endothelium and trabecular cells stained with markers for vimentin, smooth muscle cell α-actin, F-actin, spectrin, vinculin, and talin. The endothelium of Schlemm's canal stained with markers for vimentin, spectrin, and F-actin. These results suggest that trabecular cells are a kind of myofibroblast and support the belief that the endothelial cells of Schlemm's canal are vascular in origin. Fibrillary staining with antibodies to vimentin, spectrin, neurofilament protein, and glial acid fibrillary protein was observed along and between the ciliary muscle cells. Cells in the deep sclera adjacent to the supraciliary space stained with antibodies to smooth muscle α-actin, α-vinculin, talin, and desmin. These cells may anchor ciliary muscle cells into the sclera or may be developmental remnants of ciliary muscle cells. Leu 19 immunoreactivity was found in the corneal endothelium, in all trabecular cells, in ciliary muscle cells, and in keratocytes and fibroblasts in the superficial part of the cornea and sclera. All of these cells are therefore likely to express neural cell adhesion molecules indicating neuroectodermal origin.     

Changes in the monkey outflow apparatus at graded levels of intraocular pressure: a qualitative analysis by light microscopy and scanning electron microscopy

The outflow apparatus of 14 rhesus monkeys was studied by light and scanning electron microscopy in an experiment designed to investigate the influence of graded intraocular pressure on the trabecular meshwork and the canal of Schlemm in vivo. With increasing pressure the morphological alterations included progressive distention of the outer meshwork and an increase in vacuolation in the endothelial lining layer. It was concluded that vacuolation in the trabecular wall lining endothelium was a pressure-dependent phenomenon and that excessively high pressures cause occlusion of the canal by the distention of the outer part of the meshwork and prolapse of the endothelium. It is therefore advisable in all morphological studies on the outflow apparatus to regulate pressure and so avoid results which may possibly be misleading.     

Clinical and morphological studies in a case of congenital glaucoma combined with an ocular-dental-digital dysplasia (author's transl)]

Clinical and morphological studies were undertaken to characterize a case of congenital glaucoma combined with an ocular-dental-digital dysplasia in a two year old patient. Light and electromicroscopic investigations of a surgically removed trabecular specimen were carried out. A trabeculotomy had been performed at this exact site 10 months prior to removal. The existence of a canal of Schlemm was demonstrated. The regular trabecular meshwork and trabecular region was replaced by fairly dense scar tissue, containing rudimentary intertrabecular spaces. Proliferating corneal endothelial cells covered the wound area up to the uveal part of the trabecular region. The morphological findings correlated well with the clinical observation of continued raised intraocular pressure despite trabeculotomy. It is concluded that, contrary to the situation in adult onset glaucoma, the strong proliferative tendency of juvenile tissue has a marked influence on the postoperative efficacy of the trabeculotomy.     

Localization of nitric oxide synthase isoforms in porcine ocular tissues.

PURPOSE: To determine by immunohistochemistry the distribution of different nitric oxide synthases (NOS) isoforms in the porcine eye. METHODS: By light microscopy (immunofluorescence), porcine ocular tissues were investigated using monoclonal antibodies against neuronal NOS (nNOS; NOS I), endothelial NOS (ecNOS; NOS III), and macrophage NOS (macNOS; NOS II). RESULTS: Specific nNOS immunoreactivity was detected along the apical cytoplasmic portions of the non-pigmented and pigmented ciliary epithelial cells, in the endothelial lining of the corneoscleral meshwork and the uveal cords of the iridocorneal angle tissue, as well as in the photoreceptor layer of the sensory retina. Immunoreactivity for ecNOS was confined to the vascular endothelium of the vessels of the conjunctiva, iris, ciliary body, retina, choroid and optic nerve. A mild immunostaining for macNOS was present in the cytoplasm of some non-pigmented ciliary epithelial cells. CONCLUSIONS: The predominant localization of nNOS in ciliary epithelial and trabecular endothelial cells suggests a possible involvement of nNOS in both the production and outflow of aqueous humor in porcine eyes.     

Neovascular tissue in the intertrabecular spaces in eyes with neovascular glaucoma.

AIMS/BACKGROUND: To investigate histological changes in the trabecular meshwork in eyes with neovascular glaucoma. METHODS: Light and electron microscopic studies were carried out on the trabecular meshwork of three enucleated eyes with neovascular glaucoma. The presence and distribution of factor VIII in the trabecular meshwork was assessed using the ABC method. RESULTS: Peripheral anterior synechiae covering the trabecular meshwork were detected in two eyes, which would explain the rise in intraocular pressure. In the third the angle was not completely closed by peripheral anterior synechiae. The spaces between the trabecular beams were lined by a single layer of vascular endothelium, and were filled with red blood cells in this patient. Factor VIII was positively stained in the endothelial cells, lining both these spaces and Schlemm's canal. A basal lamina and microfibrils were detected just beneath the newly formed vascular endothelial cells. CONCLUSION: The neovascular tissue found in the trabecular spaces might be one of the factors responsible for intraocular pressure elevation in eyes with neovascular glaucoma.     

Short-pulsed neodymium-YAG laser trabeculotomy. An in vivo morphological study in the human eye

The in vivo response to short-pulsed Nd-YAG laser damage to the trabecular meshwork has not been studied in the human eye. The nature of the response will determine the potential efficacy of this treatment for glaucoma. We have investigated short-pulsed laser trabeculotomy lesions created in the trabecular meshwork of four human eyes within 18 hr prior to enucleation for intraocular melanoma. Scanning electron micrographs showed irregular craters (150-300 micron diameter) in the trabecular meshwork surrounded by trabecular beams which were splayed towards the anterior chamber. The adjacent damage to trabecular and corneal tissues was characterized by denudation of endothelial cells and deposition of debris. Light and transmission electron micrographs of the edge of the trabeculotomy lesions revealed fragmentation of the endothelial cells and splitting of the trabecular beams. Preservation of normal morphology was noted in the deeper tissues within 50 micron of the edge of the crater. Neutrophils were present within 20 min of laser treatment whilst macrophages characterised the inflammatory response at later stages. Perforation of the canal of Schlemm was only obtained with lesions in the middle of the trabecular meshwork but not with lesions placed more anteriorly.     

Funktionelle Morphologie der Abflusswege des Kammerwassers und ihre Veränderungen beim Offenwinkelglaukom

Aqueous humor exits the eye through the trabecular and uveoscleral outflow pathways. Under normal conditions intraocular pressure is maintained in the trabecular outflow pathways in which aqueous humor passes through the trabecular meshwork into Schlemm’s canal. Intraocular pressure is generated through an outflow resistance in the juxtacanalicular region which consists of juxtacanalicular tissue and the inner wall endothelium of Schlemm’s canal. The resistance of this region is under the influence of two contractile systems, the anterior longitudinal portion of the ciliary muscle and the contractile myofibroblast-like cells in the trabecular outflow pathways. Resistance is lowered through contraction of the ciliary muscle or relaxation of the contractile cells in the trabecular outflow pathways. In primary open angle glaucoma, resistance in the juxtacanalicular region is abnormally high. The cause of the increase is related to an increased activity in transforming growth factor beta and connective tissue growth factor signaling. The cells of the trabecular meshwork outflow pathways are stimulated to form a stronger contractile phenotype involving both an increase in the actin cytoskeleton and the surrounding fibrillar extracellular matrix. As a result there is an increase in cellular tone in the trabecular outflow pathways leading to an increase in rigidity and outflow resistance.     

Retinoblastoma: messenger RNA for interphotoreceptor retinoid binding protein.

Surgically excised retinoblastomas from 14 patients (age range nine months to two years) were assessed by immunocytochemistry for the expression of photoreceptor-specific proteins and neuronal and glial cell markers. Adjacent tissues were examined for messenger RNA expression of interphotoreceptor retinoid-binding protein (IRBP) using Northern blots. For immunocytochemical stains (ABC method), monoclonal and polyclonal antibodies included S-Ag, rhodopsin, neuron specific enolase (NSE), glial fibrillary acidic protein (GFAP), IRBP, neural adhesion molecule (N-CAM), and rod and cone specific transducin (TR alpha and TC alpha). Histopathology revealed mostly poorly differentiated tumors with necrosis and lack of Flexner-Wintersteiner rosettes. Immunocytochemical staining showed focal IRBP expression in one of the tumors and S-antigen in two cases. Immunoreactivity with rhodopsin was negative. N-CAM, a neural adhesive protein which appears to be involved in the regulation of adhesive interaction during neuronal differentiation, was positive except in two cases. All tumors showed immunoreactivity with NSE, whereas GFAP staining was limited to the perivascular glial tissue confirming the essential neuronal nature of retinoblastoma cells. TC alpha was detected in all tumors and TR alpha in one case. Messenger RNA for IRBP was detected in tumors in which IRBP immunoreactivity could not be detected.     

Gene transfer to trabecular meshwork endothelium via direct injection into the Schlemm canal and in vivo toxicity study

PURPOSE: Our aim was to investigate the efficiency of adenoviral gene transfer via direct injection into the Schlemm canal ex vivo in human donor eyes and to examine the effect of human MMP-3 transgene expression in a rat model in vivo. METHODS: A viscocanalostomy-like operation was performed and adenoviral vector encoding for MMP-3 and green fluorescent protein was injected into human Schlemm canal or rat anterior chamber. RESULTS: Transgene expression was high in trabecular meshwork endothelium in human donor eyes. In vivo, adenovirus caused dose-dependent inflammation. CONCLUSIONS: Direct injection of adenoviral vectors into the Schlemm canal has potential in glaucoma treatment.     

Pathological study of cases with secondary open-angle glaucoma due to sarcoidosis

PURPOSE: To investigate the cause of secondary open-angle glaucoma due to sarcoidosis. DESIGN: Observational case series studied by histological methods. METHODS: Seven trabeculectomy specimens from six patients with secondary open-angle glaucoma due to ocular sarcoidosis (trabeculectomy group) and anterior parts of seven autopsy eyes from four patients (autopsy eye group) diagnosed as confirmed sarcoidosis were processed for light and transmission electron microscopy. Pathological changes of outflow routes were investigated. RESULTS: Granulomata were found in three eyes of the trabeculectomy group and in three eyes of the autopsy eye group. Part of the Schlemm canal was occluded and replaced by fibrotic tissue and it became narrow in four eyes of the trabeculectomy group and three eyes of the autopsy eye group. The spaces of the trabecular meshwork appeared wide, even in the area close to granulomata and peripheral anterior synechia of the iris. Infiltration of lymphocytes, monocytes, and macrophages around the Schlemm canal was found in all eyes of the trabeculectomy group and in four eyes of the autopsy eye group. The infiltration of these cells was observed not only in the inner wall, but also in the posterior outer wall of the Schlemm canal and the collector channels. CONCLUSION: "Schlemm canalitis" is proposed from the results of inflammatory cell infiltration around the wall of the canal. The occlusion of the Schlemm canal by granulomata or fibrotic tissue replacement of the canal may play an important role in secondary open-angle glaucoma due to sarcoidosis.     

Individual factors influencing trabecular morphology in glaucoma patients undergoing filtration surgery

PURPOSE: Epidemiologic studies have shown that various lifestyle characteristics are statistically associated with the chronic open-angle glaucomas. This study was designed to investigate the influence of individual factors on the light-microscopic morphology of the trabecular meshwork in open-angle glaucomas. METHODS: Quantitative computer-assisted topographic analysis of the trabecular meshwork was performed in meridional sections of 80 trabeculectomy specimens from patients with primary open-angle (n = 36), exfoliative (n = 30) and pigment-dispersion (n = 14) glaucoma. Measurements included inner wall length of the, central thickness of the trabecular meshwork, and compactness of the Schlemm canal and trabecular meshwork. Morphologic data were correlated with individual patient data including age, duration of the disease, maximum intraocular pressure, cup-disc ratio, refraction, height, weight, body mass index, a simple morbidity index, previous surgery, and number of topical antiglaucomatous medications used. RESULTS: Inner wall length of the Schlemm canal was significantly lower in eyes with previous filtering surgery (P = 0.03), but not in eyes with a high number of topical medications (P = 0.17). There was a significant tendency for the inner wall length of the Schlemm canal to be shortened in patients where high maximum intraocular pressure was combined with long-term glaucoma (P = 0.027). Body mass index did not differ significantly between patients with primary open-angle, exfoliative, and pigment-dispersion glaucoma and showed no correlation with the quantitative data of the meshwork. The morbidity index correlated well with body mass index (0.0006) and age (P < 0.0001). CONCLUSION: Contrary to findings of experimental mice studies, we found no indication that glaucoma patients with lower body mass index have a larger lumen of the Schlemm canal than patients with a higher body mass index. Although caution should be used when interpreting data from trabeculectomy studies, there is a certain probability that a history of previous filtering surgery and of a long-term high intraocular pressure will be associated with a shortening of the Schlemm canal.     

Pharmacologic disruption of Schlemm's canal cells and outflow facility in anterior segments of human eyes

PURPOSE: To determine the effect of disruption of Schlemm's canal cells on outflow facility. Pharmacologic agents that weaken the cytoskeleton or interfere with integrin binding may allow targeted disruption of the cells lining Schlemm's canal because of the transmural pressure gradient the cells face as aqueous passes into the canal. METHODS: Anterior segments of human eyes were placed in perfusion organ culture, and either single or sequential doses of H-7 or RGD peptide were added. Fellow eyes received vehicle or RGE peptide. Eyes were fixed and examined by light and electron microscopy. RESULTS: Both agents caused a partial loss of the endothelial lining of Schlemm's canal cells without disruption of trabecular cells in other regions. H-7 significantly increased outflow facility after single or sequential doses, with moderate cell loss of both the inner and outer wall canal cells: 20.0% +/- 10.5% of the width of the canal versus 5.2% +/- 3.7% in control meshworks (P = 0.05). No significant correlation between the amount of canal cell loss and outflow facility was found. RGD was associated with a variable loss of canal cells but did not change outflow facility. CONCLUSIONS: Pharmacologic disruption of Schlemm's canal cells appears possible. H-7 increased outflow facility, causing a partial loss of the endothelial lining of Schlemm's canal. A simple relationship between canal cells and outflow facility was not found; canal cells probably interact with the extracellular matrix in influencing outflow facility.     

Isolation and characterization of a mouse monoclonal antibody against human corneal endothelial cells.

In vitro cultivation of human corneal endothelial cells (HCEC) is associated with loss of typical cobblestone-like appearance during successive passages. Thus far morphology was the sole criterion for the cell's endothelial nature. Mouse monoclonal antibodies (mabs) to human corneal endothelial cells were raised using standard immunization and hybridoma isolation procedures. The specificity of mabs for human corneal endothelial cells was tested in comparison to other endothelial cell types, to fibroblasts, corneal keratocytes and to human retinal pigmented epithelial cells. In addition immunofluorescence or immunoperoxidase staining was performed with frozen tissue sections of human corneas and with various other human tissues. The mab 9.3.E reacts with cultured human corneal endothelial cells, but not with cultured human fibroblasts and human keratocytes. In frozen sections selective positivity of corneal endothelium in contrast to negativity of the other corneal cell types was confirmed. In investigated extraocular tissues positivity was observed in smooth muscle cells including related cells (i.e. Ito and mesangial cells) and in Schwann's cells and adipocytes, but apparently not in vascular endothelial cells. The mab is human-specific and binds to a protein with a molecular weight of 130 kDa mainly accumulating along cell membranes. A mouse monoclonal antibody against human corneal endothelial cells was established in vitro and was shown to be capable of differentiating corneal endothelial cells from other corneal cell types, especially from corneal keratocytes. It is, however, not cornea-specific, but also reacts with certain extraocular cell types.     

原发性开角型青光眼小梁细胞的体外培养及其生物学特性

背景原发性开角型青光眼（POAG）是一种常见致盲性眼病,其特点是房水外流阻力增加导致眼压增高。位于房水外流通道的小梁网调节房水的外流,因此研究小梁网细胞的生物学特性有着重要的意义。目的探讨POAG小梁细胞体外培养的方法及其生物学特性。方法经小梁切除术收集8例开角型青光眼患者患眼的带小梁网的深层巩膜组织块进行体外原代和传代培养,用鼠抗人层黏连蛋白（LM）单克隆抗体、兔抗人纤维连结蛋白（FN）单克隆抗体、鼠抗人神经元特异性烯醇化酶（NSE）单克隆抗体进行免疫组织化学检测以对传代细胞进行鉴定,在透射电子显微镜下对传代细胞的超微结构进行观察,并将传代小梁细胞的生物学特性与本研究组前期培养的正常小梁细胞进行比较。结果组织块培养10d左右,可见细胞从其边缘向外生长。传代细胞在4d内处于对数生长期,其后进入平台期,第7天细胞基本融合。第3代POAG小梁细胞及正常人眼小梁细胞中可见FN、LM和NSE均呈阳性表达,证实传代细胞为小梁细胞,而空白对照组细胞未见FN、LM和NSE表达。第3代POAG小梁细胞和正常小梁细胞中FN的A450值分别为0．354±0．06和0．26±0．01,LM的A450值分别为0．34±0．03和0．25±0．02,差异均有统计学意义（FN：t=14．446,P=0．001;LM：t=9．346,P=0．001）。与正常小梁细胞比较,第3代POAG小梁细胞表面的微绒毛、细胞质的溶酶体及吞噬小泡含量减少。结论采用组织块培养法可成功在体外培养POAG小梁细胞,该研究结果为研究青光眼的发病机制提供了细胞学基础。     

Demonstration of acid mucopolysaccharides in the trabecular meshwork of the Rhesus monkey.

The ability to demonstrate AMPS in the trabecular region in the normal eye of the Rhesus monkey was shown to be critically dependent upon technical variation. Staining the fixed specimen prior to dehydration and embedding permits the uniform demonstration of AMPS in the trabecular region of the Rhesus monkey and shows it to be hyaluronidase-sensitive. Electron microscopy using the modified technique shows the reaction products to be present within the trabecular band, the intertrabecular spaces, and the canal of Schlemm. More impressive distribution was seen in the basement membrane of trabecular endothelium intimately related to the cell wall and in the ground substance and basement membrane of the endothelium of the inner wall of the canal Schlemm. The technique is also successful in the human eye and suggests a greater abundance of trabecular AMPS in open-angle glaucoma.     

Pressure-dependent changes in nuclei and the process origins of the endothelial cells lining Schlemm's canal.

Monkeys eyes were fixed with glutaraldehyde in vivo at positive intraocular pressure of 35 or 25 mm Hg and compared with eyes fixed without a positive pressure gradient, with the use of light microscopy and transmission and scanning electron microscopy. The entire endothelial lining of the inner wall of Schlemm's canal ballooned or distended toward the external wall of the canal at positive intraocular pressure. Characteristic nuclear shapes were identified and appeared to result from the increased pressure forcing the lining away from the meshwork opposed by a restraining or anchoring effect of cytoplasmic processes attached to the subendothelial cells and trabecular meshwork. Without positive intraocular pressure endothelial cell nuclei were rounded, with many folds and notches in the nuclear membrane and were not deformed by their cytoplasmic processes. These findings suggest that the cells may be capable of elastic recoil or contraction.     

In situ immunohistochemical analysis of cell adhesion molecules on human corneal endothelial cells.

Interaction of leucocytes with human corneal endothelial cells (HCECs) can be observed in several clinicopathological conditions, such as uveitis, keratitis, and corneal graft rejection. Since leucocyte-endothelial cell interactions involve various adhesion receptors we have analysed the expression and distribution pattern of the neural cell adhesion molecule (NCAM), the intercellular adhesion molecule-1 (ICAM-1), the vascular cell adhesion molecule-1 (VCAM-1), the endothelial leucocyte adhesion molecule-1 (ELAM-1), and the cluster of differentiation antigen-44 (CD44) on flat preparations of normal and organ-cultured HCECs. NCAM and ICAM were constitutively expressed on HCECs whereas VCAM-1, ELAM-1, and CD44 were absent from normal HCECs. However flat mounts of HCECs from organ-culture preserved corneas showed a mosaic-like distribution pattern of VCAM-1 and ELAM-1 positive cells and garland-like clusters of CD44 positive cells. We suggest that modulation of ELAM-1, VCAM-1, and CD44 expression on HCECs may contribute to the regulation of leucocytes-HCECs interaction in the case of anterior segment inflammation.     

Pulsed neodymium-YAG laser trabeculotomy: energy requirements and replicability.

Short pulsed laser trabeculotomy has been shown to reduce intraocular pressure in patients with primary open angle glaucoma. This study seeks to determine the energy levels required to produce a fistula into the canal of Schlemm for four different Q-switched neodymium-YAG lasers. The laser was fired at fixed human trabecular meshwork specimens at a range of energy settings for each laser and the characteristics and replicability of the lesions produced were analysed. Energy levels between 3 and 5 mJ were sufficient to produce fistulae into the canal of Schlemm with an approximately 50% success rate for each instrument.