Ординатура / Офтальмология / Английские материалы / Ocular Disease Mechanisms and Management_Levin, Albert_2010

Clinical background

Glaucoma is a collection of optic neuropathies that exhibit similar clinical phenotypes of thinning of the nerve fiber layer and excavation or cupping of the optic nerve head. Collectively, the glaucomas are a relatively common, but serious, blinding disease that is anticipated to affect nearly 80 million individuals worldwide by the year 2020.1 The most prevalent form of this disease is known as primary open-angle glaucoma (POAG). It is often associated with elevated intraocular pressure (IOP) and has no distinguishing pathology in the angle of the eye, which is defined as the junction between the iris and cornea, and is the location of the aqueous outflow channels that are critical to IOP homeostasis. In addition to POAG, there are several other varieties of this disease. These include normal-tension glaucoma (NTG), which is associated with IOPs at or below the population average, angle closure glaucoma (ACG), and secondary glaucomas resulting from pigment dispersion or pseudoexfoliation, which are associated with the accumulation of debris in the angle leading to obstruction of the outflow pathways and the elevation of IOP. Clearly, there are a variety of factors at play that affect an individual’s susceptibility to the effects of IOP. Not surprisingly, family history of glaucoma is a major risk factor and glaucoma is often considered a complex genetic chronic neurodegenerative disease of the central nervous system (CNS).2

Pathology

The common feature of all forms of glaucoma is the progressive degeneration of the optic nerve and retinal ganglion cells (RGC) in the retina. This loss of RGCs occurs through an apoptotic-like pathway.3,4 In this chapter, we will discuss the current knowledge of ganglion cell death in the context of elevated IOP and damage to the optic nerve. The relationship between IOP and initiation of the disease is not well understood. Although increased IOP is the most important risk factor, the majority of individuals with ocular hypertension will never develop the disease. Alternatively, lowering IOP, which is the only current treatment for glaucoma, is an effective therapy for most forms of the disease, even for people with NTG. Recent findings may suggest new ways to

Retinal ganglion cell death in glaucoma

Heather R Pelzel and Robert W Nickells

help early diagnosis of the disease and provide therapeutic options in addition to lowering IOP.

Etiology

RGCs are CNS neurons that transmit visual signals processed in the retina to the visual centers of the brain. Although the mechanism of insult that initiates apoptosis in the RGCs of a glaucomatous eye is not well understood, there are two predominant theories – mechanical damage of RGC axons in the region of the optic nerve head and vascular disturbances in the optic nerve head leading to ischemia in the retina that directly affects RGCs. In humans, it is thought that mechanical damage occurs at the lamina cribrosa where the axons pass through the laminar plates. Pressure on this series of collagenous plates may cause conformational changes in the pores through which bundles of axons pass, thus leading to compression of the axons. The compression could compromise the transport of small molecules, such as neurotrophins, through the axonal process. The loss of neurotrophic support to neuronal soma likely plays a role in the response of the cell body to axonal damage. A similar pattern of damage is seen in mouse models of glaucoma, but the mouse laminar region does not contain collagenous plates. Instead, bundles of murine axons in the optic nerve are surrounded by sheaths of glial cells.5 Focal damage to these discrete bundles leads to wedge-shaped sectors of RGC loss in the retina (Figure 27.1).

Both mechanical damage and ischemia could lead to activation of the supporting glial cells in the optic nerve. It is well established that glia in the CNS become activated in response to damaging stimuli, and several studies have shown that this is also the case in glaucoma.6–8 For example, Hernandez et al have shown that at least 150 genes are upregulated in astrocytes from a glaucomatous optic nerve head.6 More recently, Johnson et al have shown that glial changes in the early glaucomatous optic nerve head include the upregulation of genes involved in cell proliferation, suggesting that this behavior of glia is one of the earliest events associated with optic nerve head pathology.9 Although it is possible that glial cells are exerting a protective effect on adjacent axons, several studies have suggested that they are triggering the damage to these axons. There are several theories as to how this is accomplished, ranging from direct

Box 27.2  Rules of apoptosis

•Changes in gene expression are an important early event in apoptosis. These changes, which are marked by the downregulation of normal ganglion cell gene expression, take place before the committed stage of apoptosis

•Loss of retinal ganglion cells in mouse models of glaucoma occurs via the intrinsic apoptotic pathway, which must proceed through a stage of mitochondrial dysfunction

•BH3-only proteins mediate the activation of the proapoptotic protein BAX. BAX plays a critical role in mitochondrial dysfunction during apoptosis

•Activation of the caspase cascade enables the cell to autodigest itself and complete the apoptotic process

Selectivity of ganglion cell loss

Some controversy exists over whether or not some ganglion cells are more susceptible to apoptosis in glaucomatous conditions (Box 27.2). Early studies indicated that large ganglion cell and nerve fibers were selectively lost in experimental glaucoma of nonhuman primates and human glaucoma.18–21 In support of these observations, another study showed a selective loss of anterograde axonal transport to the magnocellular layer of the dorsal lateral geniculate nucleus, which is the target area for the largest RGCs.22 A caveat of these early studies is that the conclusions were based only on size comparisons between average cell diameters in unaffected and glaucomatous eyes. A potential confounder of this phenomenon is that damaged RGCs atrophy prior to succumbing to apoptosis. Two different studies compared midget and parasol cell soma size and dendritic features to determine if the larger parasol cells were selectively destroyed.14,23 These studies found that both RGC populations underwent shrinkage and loss in approximately the same proportions. In addition to this, a study by Jakobs et al24 used several labeling methods to identify different subtypes of RGCs in the DBA/2J mouse retina and found that the loss of RGCs was not limited to any particular subtype. A more complete discussion of the process of cell shrinkage is made in a following section.

Intrinsic versus extrinsic apoptosis

Apoptosis occurs via one of two major pathways. The first pathway occurs when the cell senses intracellular stress, hence the name intrinsic apoptosis. The intrinsic pathway relies on control by the members of the Bcl2 gene family and involves deregulation of mitochondrial function leading to activation of a cascade of proteases called caspases, initially triggered by caspase-9. In contrast, the extrinsic pathway is initiated by cell surface signaling following the binding of an extracellular ligand to a “death receptor.” This signal leads directly to the caspase cascade via activation of caspase-8, without involvement from the mitochondria. Although the two pathways can operate independently, there is some crossover. For example, caspase-8 can process the Bcl2 family member, Bid, into its active form, tBid, causing amplification of the cell death process by activation of the intrinsic pathway. Like the majority of neurons, it is believed that RGCs die using the intrinsic pathway of apoptosis (Figure

Figure 27.2  The intrinsic pathway of apoptosis. The apoptotic pathway begins with an unknown intracellular apoptotic signal that triggers several events in the cell. The first is the inhibition of the Na+-K+ ATPase and the activation of an unknown K+ channel. The combination of these two events leads to a decrease in intracellular potassium levels due to the efflux of K+. The other main event that is triggered is a change in normal gene expression. This is the first of four stages in the apoptotic pathway. The second stage involves the activation of downstream effectors. The diagram represents the indirect model of activation of BAX. BAX activation leads to the third stage of mitochondrial dysfunction, which includes a decrease in mitochondrial membrane potential and an increase in mitochondrial membrane permeability. Several molecules, including cytochrome c, are released due to the increase in permeability. Cytosolic cytochrome c allows the formation of the apoptosome with Apaf-1, procaspase-9, and adenosine triphosphate (ATP), which is the beginning of stage 4. Stage 4 continues with the activation of the caspase cascade, leading to activation of other digestive enzymes. All of these activated enzymes act to autodigest the cell and prepare it for phagocytosis.

27.2). The main evidence for this comes from Bax knockout mice. The prevalent theory of Bax function in an apoptotic cell is the creation of a pore in the mitochondrial outer membrane by oligomerized BAX proteins. This pore allows the release of small molecules, such as cytochrome c, that are critical for downstream events in the apoptotic pathway. The absence of Bax function prevents the loss of RGCs in both acute and chronic models of optic nerve damage.13,25

The intrinsic pathway follows a basic temporal order of four stages.26 The first stage involves changes in gene expression, including both the downregulation of normal gene expression and the upregulation of stress response and pro­ apoptotic genes. The second stage is characterized by the activation of downstream effector molecules. These activated molecules lead to the mitochondrial involvement that is indicative of stage 3. The fourth and final stage involves activation of caspases and endonucleases and is triggered by the release of molecules from the mitochondria. Each of these stages of the intrinsic pathway will be discussed in more detail below.

Stage 1: changes in gene expression

As stated above, the first stage in the intrinsic pathway is characterized by changes in gene expression. In all cases of RGC death due to induced or spontaneous glaucomatous conditions, a common response has been a decrease in normal gene expression and an increase in stress and pro­ apoptotic genes. Some of the normal genes whose expression is known to decrease include Thy1, NF-L, BclXL, Fem1c, Brn3b, and TrkB receptor.27–32 Napankangas et al33 have shown that expression of the proapoptotic factors, Bim and possibly Bax, increase after injury. An increase in the expression of a variety of stress response genes has also been shown, including Hsp72,34 alpha and beta crystallins,35 and several iron-regulating genes.36,37 The clinical importance of the downregulation of normal gene expression during this stage in the apoptotic pathway is not clear. As more therapies become available for blocking cell loss, however, a greater understanding of how to reactivate silenced genes is likely to become more relevant. Glimpses of this have been observed in the effects of glaucoma in Bax knockout mice; the RGCs in these mice undergo some changes in gene expression even though there is no somal loss.27 The fact that rescued ganglion cells in Bax knockout mice are affected by early apoptotic events suggests that researchers need to address the difference between mere survival of ganglion cells and the retention of normal function.

Stage 2: downstream effectors

The second stage of the intrinsic pathway involves the activation of downstream effectors, several of which are members of the Bcl2 family. The Bcl2 family is divided into three subfamilies. The pro-survival family, including BCL-2 and BCL-X, and the proapoptotic family, including BAX and BAK, share three conserved BCL homology (BH) domains, termed BH1, BH2, and BH3. The third family, the BH3-only proteins, are also proapoptotic, but are structurally unlike the other two families, except for the conserved BH3 domain.38 The BH3 domain plays the important role of allowing Bcl2 family members to interact with each other via the presence of an amphipathic alpha helix that can bind to a hydrophobic groove formed by the BH1, BH2, and BH3 domains.38 There are two proposed models for how downstream activation occurs.37 In the direct model, BH3-only proteins, termed “sensitizers,” bind BCL-X and displace “activator” molecules. These “activator” molecules, which are also BH3-only proteins, then bind directly to the proapoptotic proteins BAX and BAK. In the indirect model, the BH3-only proteins function only to bind the antiapoptotic proteins and prevent them from contacting and desensitizing the proapoptotic proteins. Most of the current experimental literature points to the indirect model of BAX/BAK activation.39

Stage 3: mitochondrial involvement

Mitochondrial dysfunction is a distinct feature of RGC death in glaucoma. The evidence for this is a loss of the electrochemical gradient across the mitochondrial inner membrane, the generation of reactive oxygen species, and the release of cytochrome c. Many of the changes in mitochondria, particularly the release of cytochrome c, are mediated by the proapoptotic protein, BAX. Although it had been

Pathophysiology

shown that BAX played an important role in the apoptotic loss of RGCs,13,25 it had not been definitively demonstrated that mitochondria were involved until a study done by Mittag et al in a rat model of glaucoma.40 In this work, the mitochondrial membrane potential was determined in rats with chronically elevated IOP and compared between cells in the late stages of apoptosis and cells from unaffected fellow eyes. This study showed that the mitochondrial membrane potential was decreased by approximately 17.5% in cells in the RGC layer of the experimental eye. In a follow-up study, Tatton et al suggest that this decrease in mitochondrial membrane potential implied an increase in mitochondrial permeability, possibly due to the affects of BAX on the permeability transition pore complex.41 However, the predominant theory on mitochondrial permeability during apoptosis is the formation of pores in the outer mitochondrial membrane by BAX oligomerization. An increase in the permeability of the outer mitochondrial membrane is permissive for the release of several factors important to the apoptotic pathway, including cytochrome c and apoptosis initiation factor.41

Stage 4: caspase activation

In the intrinsic pathway, caspase activation requires the formation of an activating complex called an apoptosome. This complex is made up of four components, including cytochrome c, apoptosis-activating factor 1 (Apaf-1), adenosine triphosphate (ATP), and procaspase-9.42 Once all of the components are in place, Apaf-1 provides a scaffold that repositions procaspase-9, allowing it to cleave itself autoproteolytically into the active form (caspase-9). In turn, caspase-9 cleaves caspase-3, which activates endonucleases and other caspases. Each caspase has reportedly specific targets, while the active endonucleases digest DNA within the intact nucleus.43 This latter event is one of the hallmarks of apoptosis, which results in the classic pyknotic nucleus referred to by pathologists, and the ability of researchers to label dying cells with terminal nucleotidyl transferase using the terminal uridine deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) method. Overall, the activation of the caspase cascade results in the autodigestion of the cell itself.

Cell shrinkage

Cell shrinkage, including chromatin condensation and membrane blebbing, occurs late during the cell death pathway, typically as a result of caspase-mediated degradation events (Box 27.3). Several of these morphological changes are considered classic descriptors of this process of cell death. Recent studies, however, have demonstrated that overall cell shrinkage can occur very early and may actually be an intracellular apoptotic trigger.44 RGC shrinkage occurs prior to the appearance of other apoptotic hallmarks in various mouse and primate models of experimental glaucoma, including Bax-deficient RGCs that cannot fully execute the apoptotic program14,23,24 (Figure 27.3). Cell shrinkage appears to have a temporal order with changes in the dendritic arbor and soma occurring concurrently followed by changes in prelaminar (intraretinal) axon diameter.14 Although there are few theories for why this apoptotic cell shrinkage occurs, it could be linked to an efflux of intra­ cellular potassium,44 associated with trophic factor depriva-

tion.11 In fact, it is possible that these two events may be linked, with glial activation as the common stimulus. The fact that somal changes appear to occur prior to any damage to the axonal transport system indicates that damage to RGCs may be detectable before any perimetric loss of vision occurs, and possibly before the cell death pathway is activated. If this is the case, a small window of time exists for the treatment of glaucoma prior to irreversible damage to these neurons. The problem, of course, is detecting these minute changes in ganglion cell appearance in time to treat the damaged cells effectively.

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Future therapies/clinical management

Although it is not currently possible to visualize changes in ganglion cell morphology in the living eye, new retinal imaging technology is closer to turning this feat into reality. Two prospective scenarios for the early detection of glaucoma are emerging from new research. The first involves the use of adaptive optics technology with ophthalmoscopy. Scientists using this method have been able to visualize individual photoreceptors at a high enough resolution to

Box 27.3  Cell shrinkage and apoptosis

•Cell shrinkage is a phenomenon that occurs very early in the apoptotic pathway, and can still occur if the process is blocked in BAX-deficient ganglion cells

•Cell shrinkage appears to be mediated by rapid changes in ion

concentration across the ganglion cell plasma membrane, mediated by changes in voltage-gated K+ channels

•The role of the cell shrinkage phenomenon in the apoptotic program is not well understood, but blocking it has been shown to prevent apoptosis43

•Cell shrinkage may provide early gross morphological changes in the ganglion cells that could be detected using new imaging technologies like adaptive optics

•Blocking cell shrinkage could become an important pharmacologic target for the treatment of glaucoma

identify the spatial arrangement of the different types of cones, as well as measuring capillaries as small as 6 m in diameter.45 Although researchers have not yet been able to produce images of individual ganglion cells, this goal is a main focus of improving this technology. Recently, adaptive

Key references

optics was used to visualize the nerve fiber layer as an individual three-dimensional entity.46 The second emerging technique also involves the use of a confocal scanning laser ophthalmoscope with the apoptotic specific fluorophores bound to dying cells.47 Using this technology, researchers were able to watch the progression of apoptosis in RGCs following optic nerve transection.

As this new retinal imaging technology becomes available, it may be possible to screen individuals for glaucoma before they experience loss of vision or, at the very least, to detect damage to the RGCs prior to soma loss, which could have substantial benefits. The early detection of RGC damage, for example as these cells undergo preapoptotic shrinkage, could be followed up with therapies to block cell death. One promising possibility for blocking cell death is inhibiting BAX, which has been shown to prevent RGC loss in murine glaucoma models.13,25

Although much about the complex pathway of RGC death in glaucoma remains a mystery, the progress in knowledge that has been made in the last 15 years provides the promise that the intricacies of the pathway will some day be elucidated.

2.Wiggs JL. Genetic etiologies of glaucoma. Arch Ophthalmol 2007;125:30–37.

4.Quigley HA, Nickells RW, Kerrigan LA, et al. Retinal ganglion cell death in experimental glaucoma and after axotomy occurs by apoptosis. Invest Ophthalmol Vis Sci 1995;36:774–786.

11.Whitmore AV, Libby RT, John SW. Glaucoma: thinking in new ways – a role for autonomous axonal self-destruction and other compartmentalised processes? Prog Retin Eye Res 2005;24:639–662.

13.Libby RT, Li Y, Savinova OV, et al. Susceptibility to neurodegeneration in a glaucoma is modified by Bax gene dosage. PLoS Genet 2005;1:17–26.

14.Weber AJ, Kaufman PL, Hubbard WC. Morphology of single retinal ganglion cells in the glaucomatous primate retina. IOVS 1998;39:2304–2320.

16.Schlamp CL, Li Y, Dietz JA, et al. Progressive ganglion cell loss and optic nerve degeneration in DBA/2J mice is

variable and asymmetric. BMC Neurosci 2006;7:66.

38.Adams JM, Cory S. The Bcl-2 apoptotic switch in cancer development and therapy. Oncogene 2007;26:1324– 1337.

44.Bortner CD, Cidlowski JA. Cell shrinkage and monovalent cation fluxes: role in apoptosis. Arch Biochem Biophys 2007;462:176–188.

Clinical background

Scarring constitutes the major threat to long-term success after most forms of glaucoma filtration surgery (GFS). Successful modulation of scarring increases the percentage of patients achieving final intraocular pressures (IOPs) that are associated with virtually no glaucoma progression. Anti­ fibrotic agents for inhibition of scarring of trabeculectomy blebs are widely used worldwide, and their use is now well established, although they are linked to severe complications such as leakage, infection, hypotony, and endophthalmitis. These complications may lead to irreversible blindness. In addition, as surgery still fails in some individuals, despite maximal doses of current antifibrotics, more effective and selective therapeutic agents are sought.

Pathology

Hemostasis, inflammation, cell proliferation, and remodeling are the main phenomena observed in wound healing. After injury, formation of fibrin clots takes place with activation of the clotting cascade. This mechanism reduces blood loss. At the same time, neutrophils, macrophages, and lymphocytes are attracted to the region (inflammatory phase). Following these phenomena, the proliferative phase occurs: this comprises re-epithelialization and formation of granulation tissue and involves migration of fibroblasts, keratinocytes, and vascular endothelial cells to the wound region from neighboring tissues. Finally, in the remodeling phase, remodeling of the tissue takes place and involves the formation of scar tissue (Figure 28.1). Many different cell types participate in the healing process, including fibroblasts, keratinocytes, endothelial cells, neutrophils, macrophages, lymphocytes, and mast cells.

Pathogenesis

Inflammation

The grading system used in our long-term Medical Research Council trial showed a good correlation between inflamma-

Stelios Georgoulas, Annegret Dahlmann-Noor,

Stephen Brocchini, and Peng Tee Khaw

tion and long-term outcome (www.blebs.net) (Figure 28.2). This finding agrees with many studies that have reported that inflammatory cells and mediators released during and after surgery stimulate the scarring cascade. Topical steroids applied as part of the routine postoperative management are effective in reducing inflammation.1 Additionally, IOP reduction has been achieved with intrableb triamcinolone acetonate injection at the conclusion of GFS, and this constitutes a relatively safe method for steroid administration.2 Topical nonsteroidal anti-inflammatory drugs may be effective,1 but their use is still controversial.

The use of other agents, including ciclosporin and cyclooxygenase-2 inhibitors, against inflammation has been suggested (Box 28.1).3 For the inhibition of inflammatory cytokines, a novel approach has been attempted with the development of dendrimers: hyperbranched nanomolecules that can be chemically synthesized to have precise structural characteristics. In our in vivo model of GFS, water-soluble conjugates of d(+)-glucosamine and d(+)-glucosamine 6-sulfate with immunomodulatory and antiangiogenic properties applied together enhanced the long-term success of GFS from 30% to 80%.4 This experimental result is far more effective than that seen with conventional steroids (Figure 28.3).

Fibrin and hemostasis

Fibrin constitutes an important part of wound healing. Fibrinolytic agents are effective in lysing blood clots after surgery,5 and, in the short term, these agents may lower IOP. The main side-effects that may deter the use of these agents are an increased risk of prolonged bleeding as well as the fact that fibrin breakdown molecules may have a longer-term stimulatory effect on the induction of scarring (Box 28.2).6

Cytokines, chemokines, and growth factors

Large numbers of growth factors or cytokines are contained in the tissues in a wound and this is the case in GFS and in the aqueous flowing through the bleb (Box 28.3).7 Transforming growth factor-β (TGF-ß) in wound healing has been shown to be more stimulatory than other growth factors and cytokines found in the aqueous.8 TGF-ß may even reverse the effect of mitomycin C (MMC) in vivo.8 Recent finding

Neovascularization (red lines)

Contraction forces (double arrows)

E F

Figure 28.1  Sequence of wound-healing phenomena after glaucoma filtration surgery.

of enhanced expression of TGF-ß-RII receptors in failed blebs indicates the importance of TGF-ß in scarring after GFS.9 Modulation of the activity of growth factors may be a useful therapeutic strategy for the inhibition of fibrosis.

Because TGF-ß in the eye seems to be involved in many pathways that are vital for the scarring process,8 we performed several studies using a variety of biological mechanisms to block TGF-ß activity, including antisense oligonucleotides10 and a human monoclonal antibody against the active form of human TGF-ß2, the predominant isoform in the aqueous (Lerdelimumab, TrabioR, Cambridge Antibody Technology, Cambridge, UK). The theoretical advantage of antibodies includes a self-regulating concept, only working when levels of the target protein are high (Figure 28.4). In an in vivo model of conjunctival scarring, the administration of this antibody significantly improved GFS outcome11 and appeared much less destructive to local tissue than MMC. A pilot clinical study of this antibody in

Box 28.2  Fibrin and hemostasis

Side-effects of anticoagulants and thrombolytic agents

Risk of further bleeding

Fibrin breakdown molecules may induce scarring

GFS demonstrated the absence of significant side-effects, inflammatory reaction, and cystic bleb formation. However, two larger randomized controlled trials have not shown a significant effect on the outcome of GFS.12 Based on the data obtained from an earlier study,11 we believe that the dose used was not sufficient. Further studies from our lab have shown a significantly enhanced effect with a prolonged dosing regimen,13 and the data also suggested an enhanced effect in the GFS outcome when the antibody is combined with intraoperative 5-fluorouracil (5-FU).

Small interfering RNA against TGF-ß receptor II mRNA reduced the production of TGF-ß receptor II, the expression