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

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Fig. 1. Direct and indirect damage signals caused RGC death in glaucomatous eye.

neurons or surrounding supporting cells (11). Because RGCs and the optic nerve do not regenerate, damage to the nerve and RGCs can result in permanent loss of vision. Thus, understanding the precise mechanism(s) of glaucomatous-induced neural and optic nerve degeneration would be crucial to design strategy for treating glaucoma.

Although glaucoma is no longer viewed simply as elevated IOP that damages the optic nerve, at present, treatment of glaucoma is directed at lowering IOP to prevent progression of glaucomatous optic neuropathy (12). Accumulating evidence of pressure-independent causes of glaucomatous optic neuropathy has led to the recognition that lowering IOP alone is insufficient for the long-term preservation of visual function (13). Regardless of the primary trigger of optic nerve and RGC degeneration in glaucoma, the disease often continues to progress even when the risk factor is relieved. Thus, an innovative therapeutic approach is required to prevent progression of glaucomatous optic neuropathy and preserve vision, likely through direct neuroprotection and stimulation of regeneration of the optic nerve and RGCs. Understanding the cellular and molecular mechanisms controlling RGC and optic nerve degeneration and regeneration will be essential for future development of efficacious therapies for glaucoma.

DIVERSE CELLULAR EVENTS ASSOCIATED WITH GLAUCOMA

Optic Nerve Degeneration and RGC Loss in Glaucoma

Clinically, glaucoma is characterized by optic neuropathy with cupping of the optic nerve head that is due to the loss of RGC axons and distortion of the supporting tissue in the lamina cribrosa (see Fig. 2) (14). The initial damage of glaucoma begins at the level of lamina cribrosa, a sieve-like structure with stacks of connective tissues that aligned to form cribriform plates with channels through which RGC axons pass (5,14). RGC axons, once leave the cell bodies, run centripetally along the innermost layer in the retina, and then turn about 90° at the optic nerve head through the lamina cribrosa to form the optic nerve (see Fig. 2). Damage to these axons at the lamina cribrosa causes a retrograde degeneration of RGCs that leads to an irreversible loss of vision.

Fig. 2. Structural relationships between RGCs, the optic nerve, and lamina cribrosa under a normal condition or elevated IOP. (A) RGC axons run centripetally along the innermost layer of the retina and turn 90 degree at the optic nerve head through a sieve-like structure, called lamina cribrosa, where is supported primarily by astrocytes and microglia. Under the normal condition, astrocytes typically extend many thin processes; resting microglia bear thin and simple ramifications. (B) In glaucoma, elevated IOP induces stress at the lamina cribrosa, where it causes activation and morphological remodeling of astrocytes and microglia and, subsequently, RGC axon degeneration. Reactive astrocytes display cell body hypertrophy; activated microglia extend short and complicated remifications.

IOP evaluation is an important causative factor for optic neuropathy in glaucoma (15). The relationship between elevated IOP and glaucomatous optic neuropathy has been extensively studied. In general, glaucoma is broadly divided into two types, normal-tension glaucoma and high-tension glaucoma, and optic nerve damage can occur throughout the entire range of IOP levels. Despite the individual variability, the tendency toward optic nerve damage increases with increasing IOP (16). The fact that treatment of patients whose IOP level is within the normal range (normal-tension glaucoma) to lower IOP is beneficial suggests that individual susceptibility to the IOP levels may play a decisive role in glaucoma (17). Accumulating evidence indicates that genetic factors contribute critically to individual susceptibility to the disease or IOP levels (18,19). The current prevailing view is that glaucoma is a disease of optic neuropathy in which IOP exceeds the level that can be tolerated by an individual optic nerve.

Elevated IOP damages RGCs by inducing cell apoptosis (2,20,21). When axon damage occurs, it initiates signaling cascades of cell apoptosis in RGC soma and causes cell death (22). Recent studies in experimental glaucoma reveal that RGCs undergo morphologic changes before cell death. RGC soma volume is reduced with corresponding reductions in the size of axons and dendritic trees (23). Similar to that occurring in various neurodegenerative diseases in the brain and spinal cord, axons, dendrites, and synapses often degenerate well before the cells die (24). There is increasing evidence that these neuronal changes contribute to the production of clinical symptoms and signs associated with the disease. The retina and RGCs are no exception to this. Initiation of compartmentalized and autonomous programs that regulate axon and dendritic degeneration, likely, will affect the physiologic behavior of RGCs and are of critical importance in the pathophysiology of glaucoma (25). Studies

of these processes, as well as understanding the mechanisms that lead to RGC death in glaucoma, are equally important for a complete understanding of this complex disease.

Responses of Non-Neuronal Cells of the Retina

The optic nerve head and lamina cribrosa, where optic neuropathy initiates in glaucoma, are rich in supporting cells, including astrocytes, microglia, and blood vessels and undergo marked remodeling and distortion (see Fig. 2). Astrocytes are star-shaped cells that support and modulate neuronal activity by releasing neurotrophic factors (NTFs) and controlling the concentration of neuroactive substances, such as neurotransmitters. Astrocytic scar is a hallmark in CNS injury (26), in which astrocytes become reactive, undergoing cellular hypertrophy, hyperplasia, and upregulating the expression of intermediate filament proteins, glial fibrillary acidic protein (GFAP), and vimentin (27,28). Much of the remodeling in the lamina cribrosa in glaucoma is due to the activity of reactive astrocytes. Accumulating evidence has now pointed to a detrimental rather than beneficial effect of reactive astrocytes to neuronal survival and axon regeneration or repair (29–31). In particular, production of certain extracellular matrix macromolecules, for example, elastin and tenancin, by reactive astrocytes after the development of glaucoma is believed to generate an inhospitable microenvironment to RGCs and their axons and contribute to the progression of the disease (32). Astrocytes of the lamina cribrosa are directly responsible for RGC axon degeneration in glaucoma, probably by producing excessive amount of neurotoxic agents (33).

Microglial cells, normally appear as small stellate cells with fine ramified processes when are quiescent, are another cellular component in the lamina cribrosa that may participate in the pathogenetic process of glaucoma (34). These cells are positive for CD45 and HLA-DR and can act as both regulator and executor of autoimmune diseases (35). Upon injury, microglia become activated and display amoebial shape. Following moderate to severe damage to the optic nerve head in glaucomatous eye, activated microglia cluster in the lamina cribrosa (34,36). Along the optic nerve, where there is a defect in blood–retinal barrier, activated microglia form linear arrays near microcapillary vessels (37–39). It is proposed that activation of microglia may be responsible for the destruction of blood–retinal barrier that results in local changes in vascular perfusion and contributes to the damage of RGC axons in the disease (40).

Both astrocytes and microglia are present not only in the optic nerve head, but also in the retina, and may participate in disease development in the retina. In particular, Müller cells, the major glial cells of the retina with cellular processes spanning across the inner and outer retinal layers, are considered a specific form of astrocytes in the retina. In general, neurodegeneration in glaucoma goes through two phases: (i) direct damage to RGC and axons, caused by elevated IOP, and (ii) indirect damage when a direct assault is accompanied by a loss of RGCs adjacent to cells originally damaged. Retinal glial dysfunction in glaucoma can play a critical role in the secondary neuronal damage in glaucoma, likely by releasing free radicals because of the activation of astrocytes and microglia and inflammation (34,41). It is now believed that this secondary damage is the major cause of RGC loss in glaucoma (9,10); thus, finding ways to control glial cell responses and prevent or limit the secondary progressive loss of RGCs is essential for the future therapeutic intervention for glaucoma.

Neurodegeneration in the Higher Visual Centers

Glaucomatous damage is not limited in the retina and optic nerve; it extends from RGCs to the vision centers in the brain (42). Axons of RGCs collectively form the optic nerve, which convey the visual information from eye to their vision center in the brain through a specialized structure called synapse (see Fig. 3). The majority of RGC axons connect with neurons in their relay center between the eye and the visual cortex, the lateral geniculate nucleus (LGN) (43). The LGN neurons are divided into three groups, magnocellular (M), parvocellular (P), and koniocellular (K) neurons, according to their size. In the CNS, neurons survive and function depending on a supply of NTFs released by target cells they connect with (44,45). Injury-induced death of primary

Fig. 3. Schematic representation of the visual pathways that connect RGCs to the LGN and primary visual cortex. (A) Retinal ganglion cell axons pass through the optic chiasm to reach their relaying center, the LGN, where neurons carry visual signals received from eye to the primary visual cortex. RGC axons from the nasal retina of both eyes cross the optic chiasm and continue to innervate the LGN in the contralateral brain, while RGC axons from the temporal retina remain on the same side and reach the ipsilateral brain targets, as it is shown. (B) RGC axons connect with LGN neurons through a specialized structure called synapse. A synapse consists of three elements: presynaptic terminal, postsynaptic terminal, and synaptic cleft. Presynaptic vesicles, which contain neurotransmitters, neuropeptide, and NTFs, are gathered in presynaptic terminals. When an electrical impulse arrives, synaptic vesicle membranes fuse with presynaptic terminals, cause the release of neurotransmitters and other molecules in the synaptic vesicle into the synaptic cleft which bind with corresponding receptors that are localized on the postsynaptic terminals. This results in the activation of signaling events and opening of ion channels in the postsynaptic terminals and begins to transmit the visual signal from the postsynaptic terminal of relay neurons to the visual cortex.

neurons often leads to deprivation of target-derived trophic factors, and consequently, degeneration of target neurons, and even secondary or tertiary neurons, a process being identified as trans-synaptic degeneration. Survival of higher order neurons in the LGN and primary visual cortex are also known to depend on the afferent input from the retina, and eye enucleation triggers trans-synaptic degeneration in the higher visual centers (46). Similar process likely will happen following glaucomatous-induced RGC degeneration.

Recent evidence reveals that persistent elevation of IOP induces significant shrinkage and loss of RGC targets, including M, P, and K neurons in the LGN. This transsynaptic neurodegeneration in the LGN increases as the severity of optic nerve damage increases (47–49). In experimental glaucoma of non-human primate, loss of P and K inter-neurons in LGN correlates with RGC loss or elevation of IOP (47,50). In particular, ongoing ocular hypertension induces a loss of K neurons before the death of RGCs (51), suggesting that degeneration of the LGN K neurons can happen very early in glaucoma and may serve as an early sign of the disease.

Varying degrees of RGC loss also induce neuronal changes in the visual cortex, the secondary target of RGC axons (42,52). The relay neurons in the LGN specifically project and innervate the primary visual cortex that is divided into six layers (layers I–VI). M and P neurons innervate the sub-layer of layer IV, IVCa, and IVCb, respectively, whereas K neurons innervate layers II and III of the visual cortex. After 1 year of elevated IOP, changes are observed in the metabolic activity of neurons in the main cortical target of the LGN, the V1 primary visual cortex (53). At the middle to late stage of glaucoma, a significant amount of neuronal loss in the primary visual cortex occurs. Just as loss of lower order neurons can trigger trans-synaptic neurodegeneration of RGCs, so death of neurons in the higher visual centers, the LGN and primary visual cortex, may also lead to interruption of trophic support to RGCs. This in turn may increase the susceptibility of these cells to glaucoma-induced insults to the eye. Thus, studies of brain changes in glaucoma may provide new insights into the pathology of glaucomatous damage and disease progression as well as new therapeutic avenue to treat blindness. In addition, early treatment for neurodegeneration in the higher visual centers may help to slow down the visual loss or even restore vision in glaucoma.

MOLECULAR MECHANISMS ASSOCIATED

WITH GLAUCOMATOUS NEURONAL DAMAGE

Identification of cellular and molecular changes associated with glaucoma is an essential step toward the understanding of the pathogenesis of glaucoma and the development of therapeutic strategies. RGC death is an important issue in glaucoma; yet, the molecular basis underlying this neurodegeneration is not fully understood. It is generally agreed that in glaucoma, a rise in IOP and the resultant impaired blood flow and local energy deficit initiate an insult to the optic nerve head that directly affects all cell types within the structure, including the blood vessels, astrocytes, microglia, and RGC axons. This damage then generates secondary injury to RGCs, which, once occurs, leads to irreversible loss of vision. A number of mechanisms have been implicated in glaucoma-induced RGC death (see Fig. 1). These include (i) IOP-related mechanical

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glucose supply that leads to a decline in the level of adenosine triphosphate (ATP) and failure of the Na+-K+-ATPase pump. Consequently, this causes an accumulation of intra-axonal Na+ because of leakage through Na+ channels and simultaneous depletion of axoplasmic K+ that induces membrane depolarization, followed by an influx of Na+ and reversal of Na+/Ca++ exchanger, probably by opening voltage-dependent Ca++ channels. Eventually, this leads to intracellular Ca++ overload and initiation of apoptotic signaling events in RGCs and their axons (59).

Elevated IOP is also known to disrupt axonal transport at the level of lamina cribrosa that obstruct the supply of NTFs to the optic nerve and RGCs to contribute to cell loss in glaucoma (2,5,6). All neurons contain a self-destructive mechanism or a phenomenon of programmed cell death, and loss of trophic support, such as due to interruption of retrograde axonal transport, can lead to the initiation of this “suicide” program within individual neurons (60). One such factor that appears to be of particular importance to RGCs is brain-derived neurotrophic factor (BDNF). During development, RGCs require target-derived BDNF to survive (61,62). Moreover, BDNF has been shown to retard RGC loss after retinal hypoxia (63) and optic nerve transaction (64,65). Delivery of BDNF by vitreous injection of recombinant protein or using viral vector to achieve continuous synthesis of the protein increases RGC survival in cat or rat models of experimental glaucoma (6,66). Candidate trophic factors for RGCs now include a variety of molecules. For example, basic fibroblast growth factor (bFGF) (67), ciliary neurotrophic factor (CNTF) (68,69), glial-derived neurotrophic factor (GDNF) (70), nerve growth factor (NGF) (71), and insulin-like growth factor (IGF) (72) have all been reported to delay RGC death following optic nerve trauma. These observations support the idea of neurotrophic deprivation as a cause of RGC death in glaucoma.

Glial Reaction and Nitric Oxide

Glial activation is thought to be another important factor contributing to RGC degeneration in glaucoma. Increased expression of reactive astrocyte or Müller glial marker GFAP in the retina is an early event in glaucoma. In experimental models of glaucoma, upregulation of GFAP can be observed in the optic nerve head as early as 4 days after the induction of elevated IOP, before the widespread RGC apoptosis (73). Increasing evidence suggests that reactive astrocytes and Müller glia produce cytokines, reactive oxygen species, and nitric oxide (NO) to cause neuronal damage.

Tumor necrosis factor alpha (TNF- ) is a proinflammatory cytokine produced by reactive astrocytes. Expression of TNFis drastically upregulated after neural injury in the brain. Its excessive synthesis after trauma has been correlated with poor treatment outcomes, and its inhibition is usually accompanied by reduced brain damage (74–76). It is now known that binding of TNFto its receptor, such as TNFR-1, activates caspase cascades and leads to cell apoptosis (77,78) (see Fig. 1). TNF- -induced death of neurons is thought to participate in the pathogenesis of several CNS degenerative diseases, such as multiple sclerosis and autoimmune encephalomyelitis (79). Previous evidence suggests that elevated IOP and ischemia, two common stress factors in glaucomatous eyes, induce expression of TNFin the retina. TNFis secreted by retinal glial cells in culture, when are exposed to elevated hydrostatic pressure

(80,81). Expression of TNFR-1 is prominent in neuronal tissue and is increased in the glaucomatous eyes. These observations support that TNFmay be a neurotoxic agent that is produced by reactive astrocytes and Müller glia and contribute to retinal neural damage in glaucoma.

Increased production of NO is proposed to serve a major glial cell-mediated apoptotic stimulus in glaucoma (see Fig. 1). NO is a free radical that diffuses freely and readily enters the cells, interacting with superoxide to form a highly destructive protein called peroxynitrite (82). Under normal conditions, NO is a potent vasodilator that regulates blood pressure (83), but it is also known to be involved in various forms of neurodegenerative diseases or damage, such as multiple sclerosis (84), Parkinson’s disease (85), and stroke (86). Excessive levels of NO trigger the death of neurons, including RGCs (87). To date, conclusive evidence demonstrating a significant role of NO in the pathogenesis of human glaucoma has not been established.

Data of immunohistochemistry that label NO synthase (NOS), enzymes synthesizing NO, are supportive to the involvement of NO in glaucoma. NO is synthesized from arginine by NOS. Three NOS have been reported so far: neuronal NOS (nNOS), macrophage NOS or inducible NOS (iNOS), and endothelial NOS (eNOS). In particular, iNOS and nitrotyrosine, a marker of NO-mediated cell destruction, have been found in the optic nerve head of glaucoma patients but not in that of normal age-matched patients (34,88). Most iNOS are present in astrocytes at the glaucomatous lamina cribrosa of optic nerve head (89). Interestingly, an elevated hydrostatic pressure induces iNOS in human astrocytes in cultures (90), suggesting that induction of iNOS can be a stress-response of astrocytes to the elevated IOP. Expression of iNOS in astrocytes can be induced by activation of epidermal growth factor receptor during IOP elevation (91) or in inflammation, by IFN-gamma/IL-1 through the stimulation of the p38 MAPK pathway (33). Both pathways require activation of NF-kappaB (33,92). Suppressing iNOS production or blocking its downstream signaling events decreases NO production and, hence, delays glaucoma-induced RGC degeneration (34,93). In a rat model of glaucoma, administration of aminoguanidine, a specific iNOS inhibitor, significantly reduced RGC loss without affecting the levels of IOP (34). The data suggest that induction of iNOS and increasing the production of NO in laminar cribrosa may generate a local destructive effect on RGC axons and likely contributing to RGC apoptosis in glaucoma.

Inflammation, Aberrant Immunity, and a Role of Heat Shock Proteins

Evidence of studies over the last decade has pointed to a role for humoral immune responses in the pathogenesis of glaucoma (94,95). This mechanism has long been suspected to mediate RGC damage and disease progression, particularly in patients with normal-tension glaucoma. It is proposed that autoimmune damage to the optic nerve may be induced directly by autoantibodies or indirectly by “mimicking” autoimmune responses to sensitizing antigens in the retina, which in turn leads to neural injury (see Fig. 1). Autoantibodies directed against retinal and optic nerve head antigens have been detected from the sera of glaucoma patients (76,96). A likely hypothesis is that immune responses directed against these antigens may weaken the extracellular support in the lamina cribrosa and enhance optic nerve cupping and result in secondary