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

mRNA extracted from tissues is first converted to cDNA, after which the expression pattern of the specimen can be analyzed using DNA microarrays. Computed cDNA and oligonucleotide microarray hybridization profiling systems allow rapid and comprehensive analysis of gene expression patterns. There are now several reports of the gene expression pattern in various experimental glaucoma models—patterns obtained by microarray analysis.

Miyahara et al. (39) investigated gene expression patterns in the cynomologous monkey glaucoma model induced by repeated argon laser photocoagulation of the trabecular meshwork. To our knowledge, this is the first report of systematic studies of gene expression in experimental glaucomatous retina. These investigators used human microarray chips that contained 9182 genes and found only 0.7% of the gene expression to be changed. Among the transformed genes, Akt, a ubiquitously expressed anti-apoptotic gene, was down-regulated, which could be related to the apoptosis of RGCs noted in glaucomatous retina. Up-regulated genes were immuno-related genes, including HLA (MHC)-DR , HLA-DO , and complement components. They also found that GFAP was up-regulated in Muller cells and astrocytes, and that ceruloplasmin was up-regulated in Muller cells, reflecting glial activation in the retina.

Using the rat glaucoma model, which is the most commonly used animal model of glaucoma, Ahmed et al. (40) reported a comprehensive survey of early and late changes in the retina in response to elevated IOP. Their model was induced by hypertonic saline injection into the episcleral vein, and rat microarrays containing approximately 8000 rat oligonucleotides were used. Early expression changes were found in 31 genes, and late changes were found in 81 genes. Of the 81 genes altered in the late stage, 74 were increased and 7 were decreased. Representative genes up-regulated by high IOP in this experimental model included GFAP, MHC molecules, complement components, and ceruloplasmin. Approximately half of the gene expression changes were involved in glial activation, the neuro-inflammatory response, or apoptosis. The down-regulated genes included multiple crystalline genes.

In the mouse model, Steele et al. (41) investigated retinal gene expression in the DBA/2J mouse glaucoma model, which spontaneously develops IOP elevation. They used a mouse microarray chip, which represents more than 39,000 transcripts, and noted significant changes in 68 genes: 32 genes were up-regulated and 36 genes were down-regulated. Among the up-regulated genes, notable ones were immune-related, and included complement factors, lipocalin2, and chitinase 3-like 1, as well as genes related to glial activation, including ceruloplasmin and GFAP. Because the DBA/2J mouse strain lacks complement component C5 and consequently has a modified immunerelated reaction (42), the significance of expression changes in the immuno-related genes in this glaucoma model remains to be investigated. Among the down-regulated genes, multiple crystalline genes are included, similar to the report on the rat model (40) mentioned above.

Considering these reports together, the expression changes detected by the microarray system involve genes related to glial activation, neuro-inflammation, apoptosis, and lens proteins. (see Table 1) In glaucomatous retina, glial cells are known to be activated and to release inflammatory mediators, including complements and cytokines (43). These data suggest that, in response to high IOP, glial cells,

including macroglia and microglia, are activated and, consequently, immuno-related genes are activated. Lens-related genes, including crystalline genes, are down-regulated in glaucoma models, although the significance of these observations is not yet clear.

In conclusion, these gene expression changes in glaucoma models may represent a shared mechanism of the retinal response to elevated IOP and may indicate novel therapeutic targets in neuroprotection of the ocular system.

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V

OCULAR TISSUE

AND PSYCHOPHYSIOLOGICAL RESPONSES

IN GLAUCOMA

INTRODUCTION

Elevated intraocular pressure is a hallmark of glaucoma. The effects of pressure on ocular tissue is complex but can be approached using the tools of biomechanics. An understanding of the stresses and strains on ocular tissue and of the tissue responses is essential to understand the ways in which various classes of drugs can reduce the damage in glaucoma. With our increased knowledge of the structure of the optic nerve head and the lamina cribosa has come a better understanding of the ways in which pressure changes can induce alteration in blood flow, the retinal ganglion cell axons, and the glia of the optic nerve and optic nerve head.

An understanding of ocular biomechanics can give rise to an understanding of the structural changes that occur in glaucoma. What is still a matter of debate is how structural damage affects visual function. The magnitude of the correlation between structural damage and loss of visual function is, to some extent, a function of the measures of visual function. An increasing number of studies are employing a multivariate analysis that combines both structural and functional measures. The hope is that such approaches will lead to better diagnosis of the disease, better control of treatment, and ultimately, better outcomes for the patients.

a physiologically important shear stress is the frictional force exerted by flowing blood on vascular endothelium (8). If the force instead acts perpendicular to the surface, then the resulting stress is known as a normal stress. Pressure is a normal stress.

2.Strain is a measure of deformation and can be defined as the fractional elongation of a cell or tissue divided by the undeformed length of that cell or tissue. As is the case for stress, one can speak about shear and normal strains, depending on whether the tissue deformation is tangential to or aligned with the undeformed length.

Stresses and strains are related to one another, and in fact, for the simplest possible case of a linearly elastic (Hookean) material they are linearly proportional to one another. More generally, the amount of strain present is a function of applied load (forces) and the effective structural stiffness of the tissues, which is determined by tissue geometry and material properties (9). Although they are related, stress and strain are fundamentally different quantities that are not interchangeable. For example, a stiff tissue such as sclera can support a large stress while undergoing little strain, whereas a compliant tissue such as the retina can undergo a large strain in response to only a small stress.

It is important to note that stress cannot be directly measured, felt, or observed; in fact, all man-made stress-measuring devices (e.g., pressure gauges) actually use a sensing element whose force-induced deformation is measured and then converted to a stress reading. So far as we know, in living systems, it is only deformation of cells and molecules (in the form of stretch, compression, and shear) that produces a biological signal (4). This means that, except at very high pressures that far exceed those occurring in the eye, exposure of cells or tissues on rigid substrates to hydrostatic pressure is a very poor model for what happens in the eye, because such approaches fail to replicate the molecular and cellular deformation that appears to be an essential step in mechanobiological sensing. This can be made clearer by recalling that the total pressure acting on an object is the sum of the atmospheric pressure (nominally 760 mmHg, but variable depending on location and weather conditions) and the so-called gauge pressure. As is the case for all physiological pressure readings, IOP is measured and reported as a gauge pressure. Reporting only the gauge pressure is appropriate because the atmospheric pressure acts uniformly throughout tissues and therefore produces only minuscule deformations on aqueous-based cells and tissues, thanks to the fact that water is nearly incompressible. On the contrary, experiments that claim to observe a mechanobiological effect because of modest hydrostatic pressures in the absence of any deformation (e.g., when cells are cultured on a rigid substrate) must, per se, be measuring an effect of total pressure (atmospheric + gauge). If such effects were real, a sure-fire cure for glaucoma would be to move to locations with lower atmospheric pressures, such as Denver, where the pressure is about 130 mmHg lower than at sea level. As far as the authors are aware, Denver is not a glaucoma-free city.

BIOMECHANICS OF AQUEOUS HUMOR DRAINAGE

It is clear that aqueous humor drainage is a biomechanical phenomenon. Aqueous leaves the eye by a pressure-driven process that does not rely on active transport through the trabecular meshwork (10,11). The trabecular meshwork is exposed to a

very dynamic biomechanical environment as it deforms (12) in response to changing IOP, for example, because of the ocular pulse (13) and the action of the ciliary muscle during accommodation (14–16). The lining endothelium of Schlemm’s canal forms unusual structures (giant vacuoles, pores) that are pressure-dependent (17–19).

One of the earliest works to consider the biomechanics of aqueous humor drainage was that of McEwen (20) who considered the trabecular meshwork as a simplified flow resistor consisting of a single equivalent channel. More recent models have treated the meshwork as a porous material whose interstices are filled with glycosaminoglycan and glycoprotein gel-like material (21,22). These models predict that there are sufficient glycoconjugates present within the apparently open spaces of the juxtacanalicular tissue (JCT) to generate the observed outflow resistance in normal eyes (21). Johnson et al. (23) coupled this “gel-filled” meshwork model with an analysis of flow through the inner wall of Schlemm’s canal to show how the hydrodynamic characteristics of the inner wall could influence flow through the JCT and effectively increase the hydrodynamic resistance of the JCT, a phenomenon termed the “funneling effect.” Recent experimental studies provide some support for this phenomenon (24), suggesting that the maintenance of normal outflow facility depends on a hydrodynamic interaction between the inner wall endothelium of Schlemm’s canal and JCT. The important implication is that dysregulation at either site could potentially lead to the ocular hypertension that is characteristic of primary open-angle glaucoma (POAG).

The biomechanics of Schlemm’s canal itself are quite interesting. It has long been known that the caliber of the canal changes acutely in response to IOP (12,25), and investigators had hypothesized that collapse of the canal could play a role in ocular hypertension. Interestingly, measurements indicate that Schlemm’s canal is smaller in the anterior–posterior direction in glaucomatous eyes compared with that in normal eyes (26). The caliber of arterial vessels is known to be regulated by shear stress because of flowing blood, and recent work suggests that a similar mechanism could be operating in Schlemm’s canal, in which canal caliber is sensitive to shear stress exerted by flowing aqueous humor (27). However, modeling by Kamm and Johnson (28), in which the canal was treated as a collapsible channel, suggested that Schlemm’s canal collapse cannot be a primary cause of ocular hypertension. On the contrary, dilation of the canal by viscocanalostomy, in combination with trabecular bypass, is predicted to lower IOP appreciably (29).

Homeostatic Control of IOP

Although our focus is naturally on those individuals who develop ocular hypertension and are thus at elevated risk of developing glaucoma, it is of interest to consider the opposite perspective: over their lifespan, most people are able to maintain an essentially constant IOP (30–32), or one that increases by at most only several mmHg1 (33). This strongly suggests the existence of a feedback loop for IOP control. Considering that aqueous humor secretion rate is essentially independent of IOP except at very high pressures (34), this feedback loop must operate locally within the trabecular meshwork

1 Here, we do not consider diurnal variations, but instead speak about the long-term trend in average IOP.

Fig. 1. Homeostatic response of the trabecular meshwork to a change in perfusion flow rate. Human anterior segments were cultured (45,46) by infusion of media at 2.5 μl/min until intraocular pressure (IOP) had stabilized, after which media flow rate was increased to 5 μl/min (see arrow at 80 h in the above graph). The resulting IOP initially increased by about twofold, but then gradually returned to baseline values (solid circles). By collecting perfusate leaving the eye, it was possible to demonstrate increased gelatinase A (MMP-2) activity in the trabecular meshwork during this time (open squares). From Bradley et al. (36).

to alter outflow resistance so as to maintain a target IOP value. It would be enormously helpful if we could definitively show that such a loop exists and could understand its mechanism of action, because ocular hypertension could result as a malfunction of such a loop.

There is evidence supporting the existence of such a feedback loop. For example, when perfused human anterior segments are subjected to a sudden increase in flow after baseline equilibration, there is a transient increase in facility that tends to lower pressure toward the baseline value (35,36). Associated with this effect, there are changes in gene expression profiles in the trabecular meshwork (37), with up-regulation of genes whose products are involved in vascular permeability, extracellular matrix (ECM) remodeling, and cytoskeletal reorganization (38,39). Bradley and colleagues (36) have specifically investigated how the matrix metalloproteases (MMPs) and their regulators, the tissue inhibitor of metalloproteinases (TIMPs), are affected by mechanical stimuli. They demonstrate a remarkable up-regulation of gelatinase A (MMP-2) after elevation of media inflow rate in cultured human anterior segments. Importantly, this up-regulation was broadly consistent with the time course of pressure renormalization in the anterior segment (see Fig. 1). These important experiments suggest that ECM materials can influence outflow resistance, that the amount of such materials is under active control, and that modulation in the amount of such materials can help regulate IOP.

These reports are generally consistent with results that show that trabecular meshwork cells are mechanosensitive (40–44). For example, cyclic stretch of cultured TM cells and perfused anterior segments led to an increase in IL-6 secretion, and direct