Cationic ferritin and segmental flow through the trabecular meshwork

PURPOSE: To determine whether segmental labeling by the tracer molecule cationic ferritin (CF) is indicative of preferential patterns of fluid flow in the trabecular meshwork or of differences in cell and extracellular matrix properties. Nonlabeled regions could indicate no fluid entering that area, insufficient perfusion time, or that the cells and extracellular matrix differ in that region and cannot bind CF. METHODS: Six whole eyes (three normal and three with pseudoexfoliation [PEX]) syndrome were perfused with CF for 30 minutes to 4 hours. Wedges of trabecular meshwork were dissected and some wedges immediately fixed. Adjacent wedges were placed in a CF bath before fixation. Transmission electron microscopy was used to analyze CF labeling. RESULTS: CF increased in the trabecular meshwork with increasing perfusion time. At 30 minutes, CF labeled mainly the uveal and corneoscleral regions. By 4 hours, CF was found diffusely through the meshwork, although a few isolated nonlabeled areas were still present. Wedges immersed in the CF bath showed fewer nonlabeled regions at all time points. Clumps of PEX material labeled more heavily in the periphery than the center, suggesting the clumps were less permeable than surrounding regions. PEX eyes otherwise had similar labeling patterns. CONCLUSIONS: Segmental labeling with CF implies regions of preferential flow exist in the meshwork. With increasing perfusion time, there were fewer nonlabeled regions. CF labeling of most regions of bath-immersed tissue suggests that nonlabeled regions do not differ in the characteristics of the cells, but rather that CF does not reach these regions.     

The resistance of the trabecular meshwork to aqueous humor outflow

A theoretical model is presented that is able to explain for the first time the pressure drop across the trabecular meshwork. The ramified flow paths in the subendothelial region of the trabecular meshwork can be interpretated as a filter bed. Data from transmission electron microscope (TEM) photographs are the starting point of the theoretical consideration. Taking shrinkage of the sections into account, the pressure gradient across the subendothelial region amounts to 0.05 mm Hg. As these canaliculi are coated by a film of glycosaminoglycans (GAGs), the pressure drop is presumably a function of the film thickness. Only film thicknesses of 0.35 m lead to pressure gradients in the experimentally verified magnitude. As the whole filter bed probably does not contribute to the filtration but only about 10%, the pressure drop specified is reached when the GAG coating is 0.25 m. As these values seem to be fairly realistic, it can be concluded that the subendothelial region of the juxtacanalicular meshwork (about 2 m thickness) can be regarded as the locus generis of aqueous humor outflow resistance.     

Pressures in the juxtacanalicular tissue and Schlemm's canal in monkeys.

A micropuncture technique involving the use of microcannulas with tip diameters less than 5 microns was used to measure the pressure in Schlemm's canal and in the meshwork at distances approximately 7 and 14 microns from the inner wall of Schlemm's canal. In one set of experiments where the spontaneous intraocular pressure (IOP) was 12.2 +/- 0.5 cmH2O and the Schlemm's canal pressure (PSc) was 7.6 +/- 0.7 cmH2O, the pressure at 7 microns from the inner wall of Schlemm's canal was found to be 8.9 +/- 0.7 cmH2O and at a distance of 14 microns, 11.0 +/- 0.5 cmH2O--that is, 1.3 +/- 0.2 and 3.4 +/- 0.3 cmH2O respectively, higher than the PSc. In another set of experiments, the spontaneous IOP and PSc were also measured and then the IOP was increased by means of an external reservoir and measured once again. Spontaneous IOP was 16.0 +/- 1.3 cmH2O and the PSc was 11.5 +/- 1.4 cmH2O before the IOP was increased. After the IOP was increased to 20.2 +/- 1.2 cmH2O, the PSc was 11.7 +/- 1.6 cmH2O. When the microcannula was introduced into the juxtacanalicular tissue to locations at about 7 and 14 microns from the inner wall of Schlemm's canal the pressure measured at 7 microns was 16.9 +/- 1.3 and at 14 microns it was 18.9 +/- 1.4 cmH2O--that is, 5.2 +/- 0.8 and 7.2 +/- 1.0 cmH2O respectively, higher than the PSc. The results indicate that at the spontaneous IOP about 75% of the resistance between the anterior chamber and Schlemm's canal is located within 14 microns from the canal with some 50% being located within the region 7 and 14 microns from the canal. After a small increase in IOP, the tissue causing most of the outflow resistance became relocated to a region within 7 microns from the canal.     

Distribution of myocilin and extracellular matrix components in the juxtacanalicular tissue of human eyes

PURPOSE: To examine ultrastructurally the composition of extracellular matrix (ECM) materials, the distribution of myocilin, and the colocalization of myocilin with ECM components in the juxtacanalicular tissue (JCT) of normal human eyes. METHODS: Postembedding immunoelectron microscopic studies were performed with antibodies specific for major ECM components, including fibronectin, laminin, vitronectin, tenascin, elastin, fibrillin-1, microfibril-associated glycoprotein (MAGP)-1, decorin, versican, and five types of collagen (I, III, IV, V, and VI). Hyaluronic acid was localized with the use of biotinylated hyaluronic acid-binding protein. Colloidal gold labeling was also performed using an anti-human myocilin polyclonal antibody. Colocalization of myocilin with ECM components was examined by double labeling, using different-sized gold particles. The possible interaction between myocilin and ECM molecules was evaluated by in vitro binding assays. RESULTS: Amorphous basement membrane-like materials in the JCT were confirmed to be made up chiefly of collagen type IV, laminin, and fibronectin. Elastin was localized to the central core of sheath-derived plaques. Fibronectin, fibrillin-1, MAGP-1, decorin, and type VI collagen were all localized to clusters of the banded material in the sheath surrounding the core, where several types of collagen, glycoproteins, and proteoglycans were also detected. Myocilin was found to associate mainly with the sheath material, overlapping extensively in distribution with fibronectin, fibrillin-1, and MAGP-1 and moderately with decorin and type VI collagen. Its localization was distinct from that of elastin. Interactions of myocilin with molecules such as fibronectin and fibrillin-1 were confirmed biochemically. CONCLUSIONS: This study illustrated ultrastructurally the composition of ECM materials in the JCT of normal human eyes. The key finding was the association of myocilin with microfibrillar architecture in sheath-derived plaques where pathologic changes have been documented to occur in eyes of patients with primary open-angle glaucoma.     

Matricellular proteins in the trabecular meshwork

The trabecular meshwork is one of the primary tissues of interest in the normal regulation and dysregulation of intraocular pressure (IOP) that is a causative risk factor for primary open-angle glaucoma. Matricellular proteins generally function to allow cells to modulate their attachments with and alter the characteristics of their surrounding extracellular matrix (ECM). In non-ocular tissues, matricellular proteins generally increase fibrosis. Since ECM turnover is very important to the outflow facility, matricellular proteins may have a significant role in the regulation of IOP. The formalized study of matricellular proteins in trabecular meshwork is in its infancy. SPARC, thrombospondins-1 and -2, and tenascins-C and -X, and osteopontin have been localized to varying areas within the trabecular meshwork. Preliminary evidence indicates that SPARC and thrombospondin-1 play a role in the regulation of IOP and possibly the pathophysiology of glaucoma. These data show promise that matricellular proteins are involved in IOP dysregulation and are potential therapeutic targets. Further study is needed to clarify these roles.     

Extracellular matrix in the trabecular meshwork

The extracellular matrix (ECM) of the trabecular meshwork (TM) is thought to be important in regulating intraocular pressure (IOP) in both normal and glaucomatous eyes. IOP is regulated primarily by a fluid resistance to aqueous humor outflow. However, neither the exact site nor the identity of the normal resistance to aqueous humor outflow has been established. Whether the site and nature of the increased outflow resistance, which is associated with open-angle glaucoma, is the same or different from the normal resistance is also unclear. The ECMs of the TM beams, juxtacanalicular region (JCT) and Schlemm's canal (SC) inner wall are comprised of fibrillar and non-fibrillar collagens, elastin-containing microfibrils, matricellular and structural organizing proteins, glycosaminoglycans (GAGs) and proteoglycans. Both basement membranes and stromal ECM are present in the TM beams and JCT region. Cell adhesion proteins, cell surface ECM receptors and associated binding proteins are also present in the beams, JCT and SC inner wall region. The outflow pathway ECM is relatively dynamic, undergoing constant turnover and remodeling. Regulated changes in enzymes responsible for ECM degradation and biosynthetic replacement are observed. IOP homeostasis, triggered by pressure changes or mechanical stretching of the TM, appears to involve ECM turnover. Several cytokines, growth factors and drugs, which affect the outflow resistance, change ECM component expression, mRNA alternative splicing, cellular cytoskeletal organization or all of these. Changes in ECM associated with open-angle glaucoma have been identified.     

Ablation of the trabecular meshwork

In an experimental investigation we examined the possibility to create an open pathway between the anterior chamber and Schlemm's canal by excimer laser ablation of the trabecualr meshwork (AT) in enucleated eyes. A quartzfiber was directed through the anterior chamber to the opposite chamber angle. With an energy of 0.3-1.5 mJ and wavelengths of 248 and 308 nm pores were easily made into the trabecular meshwork leading to a direct connection between the anterior chamber and Schlemm's canal. This result was confirmed by histologic examination of the globes. The trabecular meshwork has disappeared completely. The surrounding tissue reveals only minimal thermal effects due to the laser burns. With the same method cyclodialysis and basal iridectomy may be performed. The operation is simple and effects can be placed with great accuracy.     

小梁网干细胞的归巢及其在小梁创伤愈合中的作用

目的 研究胶原蛋白在小梁网干细胞（trabecular meshwork stem cells，TMSC）归巢中的作用以及小梁网（trabecular meshwork，TM）细胞和TMSC在创伤愈合中的相互作用。  设计 实验研究。研究对象 人原代TM细胞和TMSC。方法 分离、培养和传代人TM细胞和TMSC，通过定量RT-PCR和地塞米松诱导方法明确细胞类型；通过细胞贴附实验评价TM细胞及胶原蛋白对TMSC的亲和力；利用划痕损伤实验和时差显微镜观察TMSC分布及其与TM的相互作用。主要指标  TMSC和TM细胞的鉴定、TMSC穿膜数量和划痕损伤实验中TM细胞的迁移状态。 结果TMSC高表达OCT4而TM细胞高表达CHI3L1和MYOC，地塞米松刺激后TM细胞MYOC的表达显著增加。TMSC在含有TM细胞、胶原蛋白、TM细胞＋胶原蛋白的培养皿中的穿膜数分别为（13.1±5.3）个/高倍视野、（22.6±10.1）个/高倍视野、（4.8±2.2）个/高倍视野，均明显高于在单纯细胞培养皿中的穿膜数（3.7±0.5）个/高倍视野（P=0.0009，<0.0001，<0.001）。用AMD3100抑制TMSC中CXCR4的表达后，TMSC在各种状态的迁移数量差异消失。在TM细胞划痕损伤模型中加入TMSC可促进TM细胞的分化和迁移。结论  TMSC可诱导小梁网细胞分裂向受损区域迁移，从而促进组织损伤修复。胶原蛋白在TMSC归巢过程中发挥重要作用。     

蒙古族原发性闭角型青光眼患者小梁细胞凋亡的流式细胞学研究

背景 研究表明,蒙古族原发性闭角型青光眼(PACG)的患病率较汉族人群高3.02倍,了解其发病原因及机制对于蒙古族人群PACG的预防和治疗有积极的意义. 目的 从小梁细胞凋亡的角度探讨蒙古族PACG的发病机制.方法 参照中华医学会眼科分会青光眼组1987年制定的诊断标准纳入在内蒙古自治区人民医院眼科接受治疗的蒙古族PACG患者32例32眼及汉族PACG患者40例40眼,另选取因眼外伤需摘除眼球的蒙古族正常人13例13眼及汉族正常人17例17眼作为对照.术前测量受检眼眼压.所有患者均于常规小梁切除手术时取小梁组织,采用annexinv-FITC/PI双标记染色,在FACS alibur型四色流式细胞仪上进行双参数分析,测定坏死、凋亡和正常细胞的百分率. 结果 蒙古族、汉族PACG组及蒙古族和汉族正常对照组眼压测量值分别为(35.97-±7.11)、(38.70±6.82)、(14.69±2.91)和(13.59±2.91) mmHg(1 mmHg=0.133 kPa),4个组间的总体比较差异有统计学意义(F=106.144,P=0.000),其中蒙古族PACG组和汉族PACG组的眼压均明显高于正常对照组,差异有统计学意义(P＜0.05).蒙古族PACG组、汉族PACG组及蒙古族正常对照组、汉族正常对照组小梁细胞凋亡率分别为(7.14±0.67)％、(5.40±0.69)％、(5.86±0.91)％和(2.29±0.65)％,总体差异有统计学意义(F=174.888,P=0.000),其中蒙古族PACG组小梁细胞凋亡率明显高于汉族PACG组和蒙古族正常对照组,差异有统计学意义(P＜0.05).蒙古族PACG组和汉族PACG组比较、蒙古族正常对照组和汉族正常对照组间小梁细胞的凋亡率比较差异均无统计学意义(P＞0.05).小梁细胞凋亡率随着眼压的升高而增加(b=0.990,F=10.209,P=0.009),直线回归方程为Y=2.788±0.092X.结论 蒙古族PACG人群小梁细胞凋亡率明显高于汉族人群,是否与蒙古族PACG发病率高有关值得进一步研究.     

Stress response of the trabecular meshwork

The trabecular meshwork (TM) is known to be subjected to different types of stress such as mechanical, oxidative, and phagocytic stress. Although short-term exposure to these stresses is expected to elicit adaptive responses, long-term exposure may lead to permanent alterations in the tissue physiology and contribute to the pathologic increase in aqueous humor outflow resistance frequently associated with glaucoma. A fuller understanding of the cell-specific and tissue-specific responses to stress in the TM, including changes in gene and protein expression, signal transduction, and potential pathogenic effects, could lead to novel prevention and therapeutic strategies for glaucoma. This review summarizes the current information available about how the TM responds to mechanical, oxidative, and phagocytic stress, as well as the evidence supporting the role that such responses may have in the alterations of the TM in aging and glaucoma.     

Functional properties of fibronectin in the trabecular meshwork

Fibronectin plays a number of important roles in the extracellular matrix (ECM) including providing structural support and signaling cues for cell survival, migration, differentiation, gene expression, growth factor signaling, and cell contractility. In this review, we examine recent findings about the biological and structural properties of fibronectin and discuss how these properties could contribute to the regulation of aqueous humor (AH) outflow in the trabecular meshwork (TM).     

Innervation of trabecular meshwork

The innervation of the trabecular meshwork of the anterior chamber angle in monkey eyes was studied by transmission and scanning electron microscopy. According to the transmission electron microscopic examination, the nerve terminals in the trabecular meshwork mostly contained dense core vesicles after treatment with 5-hydroxydopamine and thus were identified as the adrenergic nerve terminals. Regarding the mode of ending of the nerve terminals in the trabecular meshwork, it was divided into three types from the positional relationship of the nerve terminals with the endothelial cells in the trabecular meshwork. Type I nerve terminals are naked and they form synaptic contact with endothelial cells of the trabecular sheets of the uveal and corneoscleral meshwork. Type II nerve terminals are located in the trabecular sheets of the uveal and corneoscleral meshwork and in the endothelial meshwork, and partly surrounded by the Schwann cell processes. This type of nerve terminals end as free terminals without a constant relation to endothelial cells of the trabecular meshwork. Type III nerve terminals usually not surrounded by the Schwann cell processes are observed on the free surface of the endothelium of the trabecular sheets facing the aqueous humor. The incidence of these three types of nerve terminals was the highest in type II followed by type III. Type I nerve terminals were very rare. When the morphological characteristics of the trabecular meshwork are considered, the transmitter released from type II and type III nerve terminals enter the aqueous humor and perhaps affects the aqueous outflow area to control the intraocular pressure, as judged from the flow of aqueous humor.(ABSTRACT TRUNCATED AT 250 WORDS)