Aqueous Outflow Pathway Research Articles

Ultrastructural alterations in the aqueous outflow pathway of adult buphthalmic rabbits

The aqueous outflow pathway of adult rabbit eyes with congenital glaucoma (buphthalmos) was examined by light microscopy and by scanning and transmission electron microscopy. The morphology of the buphthalmic rabbit aqueous outflow pathway was markedly abnormal when examined at 6 months, 1 yr, and 2 yr displaying apparent loss and/or compression of the iris pillars, dilation of the intertrabecular spaces, loss of endothelial cell-to-cell association and disorganization of trabecular lamellae, and posterior displacement of the aqueous plexus. In addition, the trabecular meshwork lamellae were observed only adjacent to the sclera and the inner portion of the trabecular meshwork was limited to swirls of collagen with scattered cells. These morphological findings suggest that the disease process in the rabbit principally involves an alteration in the differentiation and maintenance of the structural integrity of the trabecular meshwork. The loss of structural support of the buphthalmic trabecular meshwork may be a factor in the wide variation in intraocular pressure and may allow for compression of the trabecular meshwork against the aqueous plexus.

Brain natriuretic peptide-immunoreactive nerves in the porcine eye

Using immunohistochemistry, brain natriuretic peptide (BNP)-like immunoreactive (-LI) nerves localize to the anterior segment of the porcine eye. BNP-LI nerve fibers are visualized mainly in the aqueous outflow pathway, ciliary processes and anterior ciliary muscle. A small number of immunoreactive nerve fibers occur in the anterior iris stroma and in relation to the iris muscles and in the cornea. Given the known ocular effects of atrial natriuretic peptide (ANP) and that BNP and ANP seem to share common receptors, these results suggest an ocular role for peptides of this class independent of delivery by the systemic circulation.

Expression of HLA by the human trabecular meshwork and corneal endothelium

By using immunohistochemical techniques, we demonstrated that HLA class I (A, B and C) and HLA class II (DR), the major histocompatibility antigens in man, are expressed constitutively by cells of the trabecular meshwork/Schlemm's canal system and corneal endothelium, as well as by the conjunctival epithelium. Langerhans cells, vascular endothelium and uvea. Because clinical studies indicate that these antigens are involved in mediating corneal graft rejection and possibly in glaucomatous disease of the eye, the presence of both class I and II HLA in the corneal endothelium and in the trabecular cells has important implications for an understanding of immune disorders in the anterior segment of the eye. The presence of HLA on trabecular cells raises the possibility that these antigens potentiate the recognized role of Langerhans cells at the limbus and that they participate in, and/or regulate, the maintenance and defense of the aqueous outflow pathway. Our findings also open up the possibility of using HLA as a genetic marker in the determination of susceptibility to these disorders in man.

Detection of specific proteins in the aqueous humor in primary open-angle glaucoma

To elucidate the mechanism of increased intraocular pressure in primary open-angle glaucoma (POAG), the protein profiles of aqueous humor obtained from POAG patients were compared with those of cataract patients as a control group. Aqueous humor proteins were analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and detected by the ultrasensitive silver staining technique. In 79% of the samples taken from POAG patients, protein bands of 140,000 or 160,000 daltons were stained, but none were stained from cataract patients. The presence of these protein bands revealed statistically significant differences between the two groups. Protein bands of 140,000 or 160,000 daltons were evenly visible at all ages in POAG patients, and the positivity of bands had no correlation with sex or initial intraocular pressure level. It is possible that the ultrastructural changes of the aqueous outflow pathway in POAG may be related to the changes in the aqueous protein, presence of 140,000 or 160,000 daltons protein bands.

Patterns of aqueous humor outflow in glaucomatous and nonglaucomatous human eyes. A tracer study using cationized ferritin.

We used cationized ferritin, which binds to negatively charged membrane surfaces, as a tracer to delineate the aqueous humor outflow pathway by perfusing it into the anterior chamber of 14 normal human eyes and five with primary open angle glaucoma. In the normal human eyes, diffuse labeling with cationized ferritin was evident throughout the outflow pathway, while in the glaucomatous eyes distinctly different staining patterns were noted. A decorating pattern similar to that seen in normal eyes was observed, as well as apparent areas of underperfusion, suggestive of possible segmental changes in aqueous outflow. These findings may support the hypothesis that primary open angle glaucoma is a segmental disease of the outflow system due to possible regional increases in resistance to aqueous outflow.

Alterations of aqueous humour outflow following argon laser trabeculoplasty in monkeys.

We performed argon laser trabeculoplasty in one eye in each of three cynomolgus monkeys, leaving the contralateral eyes as controls. Four weeks later in both eyes of each monkey we infused cationized ferritin as a tracer of the aqueous outflow pathways for 30-40 minutes before fixation. In the control eyes cationized ferritin was found throughout the conventional aqueous outflow tract, labelling trabecular endothelial cells, both inner and outer walls of Schlemm's canal, and the collecting channels. No permeation of cationized ferritin through continuous cellular layers such as the corneal endothelium was detected. In the lasered spots of the treated eyes cationized ferritin was found to label the cellular sheet covering the trabecular scar at the lasered site, but no tracer was detected within the scar or in Schlemm's canal immediately beneath. In contrast in adjacent non-lasered regions the trabecular cells, the observed juxta-canalicular herniations, and the vacuoles of the inner wall as well as Schlemm's canal itself were extensively labelled. Our findings suggest impermeability to aqueous humour flow through the actual lasered lesion, with shift of flow to adjacent, non-lasered regions. In the normal monkey these regions compensate structurally and functionally in ways that may help to deal with this diverted flow.

An Ultrastructural Study of DNA Precipitation in the Anterior Segment of Eyes with Retinoblastoma

The anterior segment of six infant eyes enucleated for retinoblastoma were studied by light and electron microscopy in an attempt to describe the pattern of DNA precipitation. In only one of these cases was extracellular deposition of DNA in the iris and aqueous outflow pathways detectable by routine light microscopy and Feulgen staining. In two cases, ultrastructural examination was necessary to resolve the presence of small discrete electrondense DNA granules (60–200 nm). In three cases, the DNA deposits were found on the anterior surface of the iris, in the mid-iris stroma around blood vessels, and in the outer layers of the trabecular meshwork around Schlemm's canal. Although the quantity of the material varied in the three cases, the similar pattern of distribution appeared to reflect routes of fluid movement within the eye and sites of permeability barriers at which the passage of larger particulate matter is obstructed. The DNA deposits had an affinity for collagen and basal lamina, especially in the iris. Interestingly, they were not phagocytosed by the trabecular endothelial cells. No DNA could be detected ultrastructurally in the anterior segment of the three remaining eyes also enucleated for retinoblastoma. The authors' findings suggest that DNA deposition in the anterior segment may be a more common phenomenon in retinoblastoma than has been suspected previously from routine histopathologic studies. The findings are discussed in relation to other types of tumor in which DNA coating around blood vessels has been observed. It would appear that tumor necrosis and cell death are crucial factors that may lead to DNA precipitation in a variety of forms.

Establishment of calf trabecular meshwork cell cultures

Trabecular meshwork cell cultures have been grown from excised calf aqueous outflow pathway tissue using two different methods. In one method, tissue explants were placed directly in culture dishes and primary cultures were established by those cells migrating from the tissue. In the second method, a cell suspension was generated by incubating trabecular tissue strips with 0.1% pronase, and the washed cells were used to seed primary cultures. The cell cultures generated by each method appeared to progress through morphological and biochemical changes characteristic of the cell type, although at different respective rates. Comparisons of the cell morphologies, biosynthetic activities, and growth characteristics of the respective subcultures suggested that similar cell populations were obtained by each method. These studies further suggested that sufficient quantities of calf trabecular cells could be grown in culture to permit biochemical and pharmacological studies of trabecular-cell metabolism.

Factors influencing glycosaminoglycan synthesis by calf trabecular meshwork cell cultures

When cell or explant cultures were established from excised calf aqueous outflow pathway tissue and subsequently labeled with radioactive precursors of glycosaminoglycans [( 3H]glucosamine and [35S]sulfate), hyaluronic acid was the predominant glycosaminoglycan produced initially. As the cultures reached confluence, sulfated glycosaminoglycans became more prominent products. Further investigation revealed that the cultured trabecular cells could synthesize all of the glycosaminoglycans found in isolated calf trabecular tissue once a critical cell density had been attained. Increasing concentrations of fetal bovine serum in the culture medium stimulated glycosaminoglycan accumulation without altering the quantitative distribution of the various glycosaminoglycan products. Treatment of confluent monolayers with enzymes capable of degrading extracellular matrix molecules caused marked changes in the distribution of glycosaminoglycans produced by calf trabecular-cell cultures. The results suggest that calf trabecular cell cultures can be used to study the dynamics of extracellular matrix production and turnover under controlled experimental conditions.

The anatomy of the rabbit aqueous outflow pathway.

The morphology of the filtration angle and pathway of aqueous outflow was studied in 10 pigmented rabbits using light and electron microscopic techniques. The following conclusions were arrived at: Numerous pectinate ligaments were found to cross the rabbit filtration angle. However, large spaces between these ligaments allows aqueous access to the peripheral portion of the angle and then to the trabeculum. The drainage of aqueous filtered through the meshwork is served by a plexus of 1 to 4 channels, which contain giant vacuoles along their internal and external walls. A single layer of endothelial cells was found covering all meridians of the entire trabeculum from the termination of the cornea to the filtration angle recess. This layer, here termed the trabecular endothelial layer, is a continuation of the corneal endothelium, and it is accompanied by the posterior limiting lamina (Descemet's membrane) for its full length. The aqueous passes through the trabecular endothelial layer by an intracellular route that possibly requires energy. The presence of the trabecular endothelial layer in the rabbit may explain the variation that is known to exist between human and rabbit in responses to pharmaceutical agents. The physiology of the rabbit trabecular endothelial layer needs to be investigated to determine the suitability of this species as an animal model for the human glaucomatous conditions caused by the presence of such a layer.

Computed tomography of aqueous humour outflow pathways

The anterior chamber of adult Rhesus monkeys ( Macaca mulatta) was perfused with a radioopaque contrast medium (Amipaque; mol. wt 789 daltons) at controlled intraocular pressure (IOP), and its subsequent distribution examined by computed tomography. In the living monkey no contrast medium was detectable outside the anterior chamber, even after prolonged (8 hr) perfusion. However, if the animal was then killed, and the IOP maintained artificially at its previous level, opacity appeared immediately throughout the anterior segment. Subsequent scans showed the contrast medium to diffuse into the anterior orbital tissues, and to move posteriorward through the globe wall and extraocular muscles. None entered the vitreous body and very little progressed behind the point at which the extraocular muscles exited from Tenon's capsule. Elevation of IOP did not appear to accelerate this posterior diffusion. It is inferred that in vivo contrast medium passing into the posterior, non-conventional aqueous drainage pathway is cleared immediately by the circulating blood in the uvea and possibly also the extraocular muscle. Tenon's capsule may provide a barrier to further intra-orbital diffusion.

Aqueous humour turnover and intraocular pressure during menstruation.

Both intraocular pressure and aqueous humour turnover rate were determined at intervals over three months in three females in order to investigate whether a correlation existed between these variables and the menstrual cycle. Not only was there a lack of correlation between intraocular pressure or aqueous humour flow rate and menses but intraocular pressure and aqueous humour flow rate were also not related to each other. If pharmacologically administered doses of progesterone or oestrogen influence intraocular pressure, the present data indicate that the effect is probably mediated through effects on the aqueous outflow pathways.

Trabecular Meshwork Cell Culture in Glaucoma Research:: Evaluation of Biological Activity and Structural Properties of Human Trabecular Cells In Vitro

The propagation of human trabecular cells in culture allows the study of the structural and functional properties of this distinct cell type under reproducible experimental conditions. Human trabecular cells can be effectively grown from dissected explants of trabecular tissue, and the cultured cells can maintain the distinctive ultrastructural features of uncultured trabecular cells through at least five passages in vitro. The trabecular cell possesses a wide range of biochemical and structural properties that may be important for the maintenance of the aqueous outflow pathway. These properties include the growth of trabecular cells as an endothelial monolayer with a nonthrombogenic cell surface, the production of plasminogen activator, avid phagocytosis, and the ability to synthesize glycosaminoglycans, collagen, fibronectin, and other connective tissue elements. The presence of hyaluronidase and other lysosomal enzymes emphasizes that human trabecular cells are capable of metabolizing hyaluronic acid and other extracellular materials. Potential mechanisms of trabecular cell damage in vitro are examined by evaluating the effects of extended passage, peroxide exposure, and laser treatment on cellular morphology.

Aqueous outflow in the beagle. II. Postnatal morphologic development of the iridocorneal angle: corneoscleral trabecular meshwork and angular aqueous plexus.

The morphology and postnatal ontogeny of the major pathway for aqueous outflow in the canine eye has been examined. Normal canine chamber angles of the Beagle at one day, one through ten weeks, three, four and six months, and one and two years of age were used. Angle tissue was prepared and examined by standard techniques for light and electron microscopy. At birth the corneoscleral trabecular meshwork largely consisted of spongy mesenchyme. By the end of the first month the corneoscleral trabecular meshwork developed into a series of longitudinally oriented sheets anteriorly covered by the peripheral extremity of the corneal endothelium. The angular aqueous plexus (trabecular veins), which was formed at birth, was continually separated from the corneoscleral meshwork by an endothelial lining. Transcellular channels interconnected the trabecular veins and the corneoscleral trabecular meshwork. The corneoscleral trabecular meshwork and angular aqueous plexus in the canine angle are similar topographically, structurally and ultrastructurally to that described in other nonprimate and primate angles.

The Conventional Outflow Resistances

The concept of the conventional aqueous outflow pathway (through the trabecular meshwork and other components of the canal's inner wall, along Schlemm's canal to collector channels, and out of the eye through the collector channels) was modeled on tubes with leaky walls and a network of electrical resistances. Analysis of these models suggested experiments to test the models and to assess the magnitude of the resistance of portions of the pathway. We found that the well-known increase in outflow resistance with increased intraocular pressure was, in large measure, the result of blockage of the collector channels, probably by the canal's inner wall. A model of the trabecular meshwork predicted that tension on the scleral spur and trabecular mesh would cause the meshwork to arch over Schlemm's canal. We depressed the lens to generate such tension, and found that the resistance of the inner wall of the canal decreased. In one eye, lens depression also decreased resistance to flow in the collector channels and the canal of Schlemm and prompted the suggestion that when secretory inhibitors are used in glaucoma, cyclo-tonic agents should be used simultaneously to relieve canal collapse and blockage of collector channels by inner canal wall.

Aqueous outflow pathway glycosaminoglycans

The acidic glycosaminoglycan distribution patterns of the aqueous outflow pathway, the iris-ciliary body, and sclera of the New Zealand Red rabbit were identified by analysis of the glycosaminoglycan moieties and by the use of zone electorphoresis. Alcian blue positive bands on cellulose acetate membranes were characterized by a determination of their electrophoretic mobility in two electrolyte systems and by a comparison to the electrophoretic mobility of standard reference glycosaminoglycans. To verify the identity of each band, specific glycosaminoglycan degrading enzymes were used to remove the glycosaminoglycans. Glycosaminoglycan samples equivalent to 5 μg uronic acid were treated with hyaluronate lyase, testicular hyaluronidase, chondroitin AC lyase and chondroitin ABC lyase. The glycosaminoglycans resistant to chondroitin ABC lyase were treated with nitrous acid and tested for solubility in cetyl pyridinium chloride. The scleral distribution pattern was hyaluronic acid, chondroitin sulfate and hybrid dermatan sulfate-chondroitin sulfate. The aqueous outflow pathway and iris-ciliary body distribution patterns were hyaluronic acid, keratan sulfate, heparan sulfate and hybrid dermatan sulfate-chondroitin sulfate.

The existence of smooth muscle adjacent to the Schlemm's canal of the normal albino rat eye.

The existence of smooth muscle adjacent to the rat aqueous outflow pathway was confirmed in this study. The smooth muscles ran parallel to the Schlemm's canal and reached the level of the posterior end of Schlemm's canal. The characteristics of these muscles were similar to smooth muscles in other organs. The existence of smooth muscle in this region depends on the fact that the rat eye lacks the scleral spur and the poorly developed meridional fibers of ciliary muscles come to lie close to the endothelial cells of Schlemm's canal. The innervation and function of these smooth muscles are unknown.

The fate of anterior chamber fluorescein in the monkey eye 1. The anterior chamber outflow pathways

As defined by freeze-dry tissue techniques, there are two distinct routes by which fluorescein leaves the anterior chamber of the living rhesus monkey eye. I. The conventional pathway: Within five minutes of the initiation of fluorescein perfusion through the anterior chamber, the conventional pathway filled. Tracer passed through the trabecular meshwork into Schlemm's canal, and flowed back within scleral and episcleral venous derivatives to enter large veins in the extraocular muscles. II. The uveo-vortex pathway: Fluorescein rapidly moved into the iris stroma and into the anterior part of the ciliary body. The anterior chamber border of the ciliary body is little more than a sparse mesh from which fluorescein entered directly into the substance of the anterior ciliary muscle. Fluorescein penetrated blood vessels in the iris stroma and in the anterior ciliary body from where it could be traced posteriorly to the equatorial choroid and vortex veins. The specific sequential tissue distribution of fluorescein strongly supports the existence of a uveo-vortex pathway for aqueous outflow.

Electron microscopic studies on the postnatal growth of mouse iridocorneal angle

The iridocorneal angle(ICA) occupies an important position in the eye from the viewpoint of the aqueous outflow pathway. There are several studies on the iridocorneal angle formation of the mammalian eyes, such as the rat, monkey and man. However, there is no paper available on that of the mouse. The purpose of this study is to report the developmental process of the iridocorneal angle formation in the mouse eye.Albino mice of various ages from newborn to adult were used in this study. The ages of mice were 3, 5, 7, 9, 10, 11, 14 days old, 1, 6 and 12 months old. At least three individual animals were studied at each developing stage. After decapitation, the eyes were quickly enucleated, and were prefixed in 2. 5% glutaraldehyde in 0. 1 M phosphate buffer (pH 7. 4) for three hours. They were then measured at their diameters before postfixation in 1% osmium tetroxide for two hours, dehydrated in a graded series of ethanol and embedded in Epon with a flat embedding mold to ensure their direction. Ultrathin sections were cut with an LKB Ultrotome, placed on the collodion coated grids and stained with uranyl acetate and lead citrate before observation with a Hitachi HS-9 electron microscope.

Coagulation factor VIII. Localization in the aqueous outflow pathways.

Antihemophilic factor A-von Willebrand factor (AHF-vWF) is a protein involved in the first phase of blood coagulation and in the mechanism of platelet adhesion. The localization of this protein has been studied in the anterior segment of fetal and adult human eye by a direct immunofluorescence method. It was found that AHF-vWF is present in the wall of aqueous outflow pathways but not in that of Schlemm canal. As it has fibrinolytic activity in the endothelium of Schlemm canal, this structure can be expected to be especially well protected against fibrin deposition and occlusion by platelets and thrombi. The ciliary processes of the adult eye had no AHF-vWF activity. Instead, fluorescein isothiocyanate-fluorescence could be seen in the vessel wall of the ciliary processes of the fetal eye. This difference may be connected with the function of aqueous secretion.