Ординатура / Офтальмология / Английские материалы / Mechanisms of the Glaucomas_Shields, Tombran-Tink, Barnstable_2008

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VI

MODELS OF GLAUCOMA

INTRODUCTION

The human eye offers limited value in studying the mechanisms of the glaucomas, as most tissues for histologic and other in vitro investigations are obtained rather late in the course of the glaucomatous process. As a result, the observations may not provide insight into the early mechanisms of the process, and it is not always clear whether the observed changes are part of the earlier alterations in the disease process or are a secondary result of those earlier changes. Hence, the value of animal models of glaucoma.

There are a number of spontaneous forms of glaucoma throughout the animal kingdom, but these too are of limited value in understanding the mechanisms of elevated intraocular pressure (IOP) in humans, because the specific mechanisms of those glaucomas do not closely mimic the more common forms of glaucoma in the human eye. They do, however, provide an opportunity to study the mechanisms of glaucomatous optic neuropathy that results from the elevated pressure. Likewise, artificially induced models of elevated IOP have their value in studying the response of the retinal ganglion cells and optic nerve to the pressure, as well as in evaluating new pressure-lowering drugs and surgical procedures and also in assessing potential IOP-independent neuroprotective drugs.

models also permit one to study the disease process over a relatively shorter time period than that which occurs naturally.

Clinically, most cases of glaucoma are characterized by higher than “normal” (>21 mmHg) IOP, structural changes in the appearance of the optic disc, and a progressive loss of vision. Although elevated IOP no longer is considered a reliable predictor of the disease, it remains a critical risk factor; patients with unilateral elevation of IOP that is secondary to other eye disorders develop glaucoma in the eye with elevated pressure, and animals with experimentally induced elevations of IOP display anatomical and physiological changes characteristic of human glaucoma (9–11). For this reason, and because of the high degree of similarity between the human and the non-human primate eye and central visual pathway (12), much of our current understanding of POAG derives from monkey models of pressure-induced glaucoma.

BACKGROUND

The earliest models of non-human primate glaucoma evolved from the observations of acute elevations in IOP in humans following cataract surgery, and were based on intraocular injections of the proteolytic enzyme alpha chymotrypsin (13–16). The drug, however, produced highly variable responses, depending on the dose and region of the eye into which it was applied. Although anterior chamber injections had little effect on IOP, application of the enzyme to the posterior chamber often produced sudden (within 2–4 h) and severe (>61 mmHg) elevations in pressure. Intravitreal injections showed mixed results, and often were accompanied by retinal hemorrhages, severe photoreceptor degeneration, and complete loss of the electroretinographic response. These results suggested that the substrate underlying IOP elevation in this primate model resided primarily in the posterior chamber of the eye and that the drug has direct, dose-dependent, degenerative effects on the neural retina and vasculature that are independent of those common to simple, pressure-induced, glaucoma (e.g., optic disc cupping, excavation of the nerve head, primary degeneration of retinal ganglion cells, and a progressive loss of visual responsiveness). In a follow-up study, Lessell and Kuwabara (15) noted that, in addition to producing corneal lesions and dislocation of the lens, posterior chamber injections of alpha chymotrypsin also result in significant atrophy of the ciliary body, suggesting that the most likely cause of elevated IOP in this primate model is blockage of the anterior chamber drainage channels by drug-induced lysates of the ciliary body. Such atrophy, however, is uncommon even in advanced cases of human glaucoma.

In an attempt to overcome the various difficulties associated with the alpha chymotrypsin model (rapid and severe elevation of IOP, lens subluxation and perforation, corneal edema, pupillary dilation, ciliary body atrophy, retinal hemorrhaging, direct retinal degeneration) and to develop a primate model more similar to human POAG, Gaasterland and Kupfer (17) examined the possibility of generating experimental glaucoma in monkeys by laser scarification of the trabecular meshwork (TM) located in the anterior chamber of the eye. As described later, this complex structure is intimately involved with the fluid dynamics of the primate eye, and thus the regulation of IOP. Using a goniolens and slit lamp equipped with an argon laser, they applied approximately 200 laser “burns” (50 μm spot size; 0.2–0.5 s duration; 0.4–0.8 W

power) to the full circumference of the mid-TM of both eyes in five rhesus monkeys. This scarring resulted in a significant decrease (80–90%) in the outflow facility of aqueous humor from each of the laser-treated eyes, and elevated IOP in seven of them. In all cases, at least two laser treatments were needed to achieve a sustained elevation in pressure, and the final levels varied greatly, ranging from 16 to >50 mmHg. The laser-treated eyes also showed mild–moderate levels of post-treatment inflammation, which often took up to 2 weeks to resolve. Nevertheless, unlike the alpha chymotrypsin model, the IOP elevations in the laser-treated animals developed over several days to weeks, with mean pressures in the 30to 40-mmHg range. In addition, the laser-treated eyes showed a selective loss of retinal ganglion cells, thinning of the optic nerve fiber layer, cupping of the optic nerve head (ONH), and posterior bowing of the lamina cribrosa (LC), features highly characteristic of chronic, pressure-induced, glaucoma in humans.

Aiming to refine the monkey laser model, Quigley and Hohman (18) systematically investigated IOP levels in cynomolgus monkey eyes following different combinations of laser treatment duration, power, and number of application spots. On the basis of the criterion that a continuous rise in IOP above 22 mmHg for 1 month constituted a sustained elevation of IOP, they determined that the normal monkey TM can absorb about 50 joules of energy before the level of damage leads to a sustained elevation in IOP. Using laser treatment durations of 0.5–1.0 s and total energy delivered levels of 60–90 joules, Quigley and Hohman (18) were able to reliably produce mean IOP levels of 35–45 mmHg that were sustained for 4–11 months. Furthermore, in many cases, the elevation in IOP was achieved with a single treatment session.

Taken together, the work of Gaasterland and Kupfer (17) and Quigley and Hohman (18) set the foundation for the use of argon laser photocoagulation as a means for generating pressure-induced experimental glaucoma in non-human primates. More recently, Wang et al. (19) have demonstrated that similar results can be achieved in the monkey using a high-power diode laser. Although the lasered-primate approach has the advantage of being relatively quick to perform, it is not without limitations. In addition to the more costly and rigorous animal-handling procedures needed to work with non-human primates, the technique also requires access to expensive and highly specialized ophthalmic equipment and skilled personnel. Moreover, it remains the case that most eyes require at least two or more laser sessions before a sustained elevation in IOP is achieved, and each treatment yields an inflammatory response that may last from a few days to a week or more. Furthermore, laser burns that encroach on the iris and/or ciliary muscle often result in eyes with asymmetric, semi-responsive pupils and hyphema. To date, there also remains no standard protocol for reliably producing specific target pressures. This is due mainly to the high degree of variability common among laser-treated eyes, even those receiving the same treatment paradigm. IOP also may vary considerably from day to day or week to week; however, this is not necessarily atypical, because glaucoma often is marked not only by higher than normal mean levels of IOP but also by an increase in the frequency and magnitude of naturally occurring pressure spikes (20–26).

Although the monkey laser model of experimental glaucoma has been used most extensively, it is important to note that non-laser-based approaches also have been

APPLICATIONS OF THE PRIMATE MODEL OF GLAUCOMA

The eye is an intricate organ, the function of which requires complex interactions among several different components. With respect to IOP and glaucoma, much of the focus has been on the TM in the anterior chamber of the eye, the LC region of the ONH, the ganglion cell layer of the retina and associated optic nerve, and the retinal vasculature. In addition, considerable effort has been made toward the development of non-invasive methods for detecting the onset and monitoring the progression of glaucomatous neuropathy. In the following sections, we outline briefly the knowledge gained from examining each of these regions using the primate model.

Trabecular Meshwork

The TM is a multi-layered, sponge-like tissue located in the anterior chamber angle of the eye. The uveal (inner) and corneoscleral (middle) layers of the TM consist primarily of well-defined collagen beams covered with endothelial-like cells and surrounded by extracellular matrix (ECM) material. The cribiform (juxtacanalicular) layer represents the outermost region of the meshwork. Unlike the uveal and corneoscleral portions, this region does not contain collagenous beams, but rather consists of elongated cells interspersed within the ECM and arranged in layers. Adjacent to, and interconnected with, the cribiform layer is a monolayer of endothelial cells that form the inner wall of Schlemm’s canal (SC) (33,34). This venous drainage channel encircles the eye and is continuous with the intrascleral plexus of veins. The TM and canal of Schlemm represent the primary route by which aqueous humor, the blood-derived fluid that bathes the anterior and posterior chambers of the eye, is returned to the vascular system. The aqueous circulation has two important functions. First, it provides nutrients and removes metabolites for the avascular lens, cornea, and anterior chamber angle tissues, and second, through resistance to its outflow through the TM–SC pathway, it generates an IOP of about 15 mmHg, which helps maintain the eye’s normal size and shape. Because of this relation between aqueous outflow resistance and IOP, considerable work has focused on the TM and its possible role in glaucoma. In particular, these studies have demonstrated that most of the resistance to aqueous outflow resides within the cribiform layer and/or inner wall of SC (35,36). This is due primarily to the relatively large, low-resistance, intertrabecular spaces found within the uveal and corneoscleral regions of the meshwork, and which are absent from the cribiform area. Light and electron microscopic studies in which the anterior chambers of monkey eyes were perfused with labeled particles have demonstrated the presence of both transcellular and paracellular routes through the endothelial wall (37–40). Furthermore, it has been shown that the different components of these routes thought to be involved in aqueous transfer (“giant vacuoles,” transendothelial “pores,” and paracellular channels) are responsive to changes in IOP; vacuole size and density, as well as paracellular channel size, increase with increased levels of IOP, thus facilitating aqueous outflow (38–46). Although the relation between the endothelial wall pores and the IOP is less clear, a recent study using human eyes has determined that the inner wall pore density in glaucomatous eyes is only one-fifth that found in normal eyes (46). The authors conclude that this reduction would be sufficient to explain the decrease in outflow facility seen clinically in glaucoma.

Because of their critical position within the TM, and wide range of functional capabilities (phagocytosis, ECM regulation, prostaglandin, and cytokine secretion), a number of studies have focused on the trabecular cells themselves. In particular, these studies have examined the effect that different cytoskeletal drugs, especially those affecting actin, have on the regulation of aqueous humor dynamics (47–52). In general, these drugs were found to increase aqueous outflow, probably by altering cell shape and cell–cell interactions within the TM, and thus “relaxing” the structural integrity of the drainage tissues. Other studies have indicated that TM cells contain receptors for, and are influenced by, a number of different growth factors, including transforming growth factor beta (TGF-beta), a pluripotent cytokine, although their roles in glaucoma have yet to be defined (53–58).

Despite intense scrutiny, the primary site of aqueous outflow resistance within the primate eye remains undefined. Most likely this is because IOP regulation within the cribiform and inner wall regions of the TM depends on a combination of complex interactions among the different cells, their structural organization, and the level, content, and turnover rate of ECM materials (see 59 for Review). The recent development of a monkey anterior segment organ culture system (60), as well as adenoviral transfection and protein transduction techniques aimed at modifying selectively the structural and functional properties of TM cells, should provide new and important information concerning these relations, and their roles in aqueous humor dynamics and IOP regulation (61–64).

Although most aqueous outflow occurs through the “conventional” route through the TM, it is important to note the presence of a uveoscleral pathway through the ciliary body and sclera (37,65). This route, which is relatively pressure insensitive compared with the trabecular pathway (66), has received considerable attention because of its sensitivity to prostaglandins (67–69). Studies using the primate model of glaucoma have demonstrated that prostaglandin F2-alpha (PGF2 ) and 8-iso prostaglandin E2 (8-iso PGE2) lower IOP by increasing uveoscleral outflow. In addition, they also have been instrumental in demonstrating the enhanced effect of applying these compounds together, or in combination with other IOP-regulating medications shown previously to either increase outflow facility or reduce aqueous production (70–76). Although the mechanism by which prostaglandins effect an increase in uveoscleral outflow remains undefined, recent immunohistochemical studies in monkeys with experimental glaucoma suggest that prostaglandin-induced upregulation of matrix metalloproteinases (MMPs) within the uveoscleral pathway might be involved (77). MMPs are proteolytic enzymes known to degrade ECM material. These results are consistent with other monkey studies showing that PGF2 leads to a decrease in collagen within the uveoscleral pathway (78), and thus presumably a decrease in resistance to aqueous outflow through this pathway.

Lamina Cribrosa/Optic Nerve Head (LC/ONH)

The expanded optic cup and excavated optic disc of the glaucomatous eye reflects a reduction of neural tissue, as well as pressure-induced compression, displacement, and reorganization of the LC, the perforated support structure within the ONH through which the optic nerve fibers must pass to exit the eye (4,79–83). The primate LC contains at least five types of collagen, a highly organized pattern of elastin fibers,

basement membranes, and proteoglycans (84–87). Together, these ECM components provide the LC with its unique strength, flexibility, and elasticity, and their close spatial relations have been well documented at both the light and the electron microscopic levels. In brief, the normal LC is characterized by parallel-running collagen and elastin fibers that lie in close apposition, creating the highly organized fibroelastic beams of the cribiform plates, as well as the channels through which the optic nerve axon bundles pass. By contrast, the glaucomatous ONH has a highly disorganized appearance, owing to glial hyperplasia, a decrease in elastin and collagen density, and extensive fragmentation and dissociation of the individual elastin and collagen fibers that give the laminar beams a “wavy” appearance (see Fig. 2; 86,89–92). The affects that elevated IOP has on the structure and biomechanical properties of the ONH and adjacent tissues have been examined extensively by Burgoyne and colleagues (92–97). These elegant studies have demonstrated that both permanent and hypercompliant deformation of the load-bearing tissues of the LC and anterior scleral canal wall are early characteristics of the glaucomatous ONH.

Although much of the structural abnormalities can be attributed to excess stress to individual support fibers (93–97), more recent work suggests that pressure-induced activation of ONH astrocytes also plays a significant role in LC remodeling in glaucoma. In particular, Hernandez et al. (98) have shown that ONH astrocytes are capable of de novo elastin synthesis when subjected to increased levels of hydrostatic

Fig. 2. Electron microscopic comparisons of the lamina cribrosa in the normal and glaucomatous optic nerve head. In the normal optic nerve head (A, B), the collagen and elastic fibers are highly organized. By contrast, these ECM components show a more disorganized appearance in the glaucomatous optic nerve head (C, D), resulting in a decrease in the structural integrity of the optic nerve head (ONH) [(A, C) Republished with permission of the Association for Research in Vision and Ophthalmology from Hernandez M.R. (1992) Ultrastructural immunocytochemical analysis of elastin in the human lamina cribrosa, Investig. Ophthalmol. Vis. Sci. 33(10); permission conveyed through Copyright Clearance Center; B, D Reproduced/Amended with permission of the BMJ Publishing Group from Quigley et al. (1991). Alterations in elastin of the optic nerve head in human and experimental glaucoma, Br J Ophthalmol 75(9); permission conveyed through BMJ Publishing Group, Ltd.].

pressure. This new elastin is abnormal, however, and as such it is not incorporated properly into the LC, resulting in elastotic degeneration of the ECM. That this represents a response to elevated IOP, and not simply the resultant nerve injury, has been demonstrated by Pena et al. (83), where they used the primate model of experimental glaucoma to compare elastin expression in eyes with pressure-induced optic nerve injury versus optic nerve section. Additional human and monkey studies have indicated that, when activated by elevated IOP, ONH astrocytes also are capable of releasing a number of different factors, including nitric oxide and tumor necrosis factor alpha, which can be neurotoxic (99–101). As noted previously with respect to the TM, the ONH of the glaucomatous eye also shows increased levels of MMPs (102,103). MMP-induced ECM regulation in both the TM and the LC also appears to occur as a result of changes in the level and expression of TGF-beta associated with the resident cells of each region (104–107). Although the exact roles of these different factors remain undefined, it is reasonable to assume that the significant remodeling that takes place as part of the disease process has a negative impact on the biomechanical properties of the lamina, reducing its ability to resist additional pressure fluctuations, and increasing the potential for optic nerve injury.

Optic Nerve and Retina

Primate models of experimental glaucoma have been used extensively to study the effects that elevation of IOP has on the optic nerve and retina. With respect to the optic nerve, Lambert et al. (108) were the first to demonstrate, based on the accumulation of axonal materials, that the primary site of injury in glaucoma is the optic nerve at the level of the scleral LC. This result later was confirmed by Anderson and Hendrickson (109) following injection of tritiated leucine into the vitreal chamber of non-human primate eyes, and has since been demonstrated in several other studies using light and electron microscopy, alone or in combination with different neuronal tracers (17,27,109–115). Additional work in humans and monkeys have shown that the pattern of nerve degeneration often is not uniform, but instead shows a bias toward greater damage in the superior and inferior quadrants of the nerve (4,113,115). These results are consistent with morphological studies showing an asymmetry in the density of connective tissue within the primate ONH; compared to the nasal and temporal poles, the superior and inferior regions of the nerve head have larger openings for the passage of ganglion cell axons and they contain less connective tissue, suggesting that these regions provide less structural support during periods of elevated IOP (4,116,117).

Although the mechanism(s) underlying optic nerve injury in glaucoma remain equivocal, at least three, non-mutually exclusive, theories have been suggested: mechanical, vascular, and biochemical. In the mechanical theory, pressure-induced compression and distortion of the laminar sheets and pores of the LC result in shearing and compressive forces that act directly on the ganglion cell axons. Support for this theory comes from the many studies noted previously in which elevation of IOP has been shown to produce an accumulation of intracellular material within the optic nerve fibers at the level of the scleral LC. With respect to the vascular theory, there is growing evidence that, due to deformation of the LC, ocular blood flow within the glaucomatous ONH also might be inadequate, and that optic nerve ischemia could be