nervous tissues, but the neurotrophin that is most commonly linked with ganglion neurones is brainderived neurotrophic factor (BDNF). BDNF is released by target neurones in the brain. Ganglion cell axons will synapse with these neurones, take up the neurotrophin, and axonally transport it back in a retrograde manner to the retina. During development, after the first BDNF arrives in the retina in this way, all ganglion cells become dependent upon its presence in order to survive. Thus, if anything occurs to compromise the transport of BDNF to the retina, ganglion neurones are thought to die, mainly by apoptosis.

This fact can be supported experimentally: removal of the ganglion cell axonal brain target (superior colliculus) from neonatal Wistar rats results in a rapid loss of ganglion cells [11]. Transection of the adult rat optic nerve also leads to degeneration of 80% or more of the ganglion cells. This is presumed to occur through the physical effects of ganglion cell axotomy and the associated cessation in retrograde transport of neurotrophins. Application of BDNF or neurotrophin-4/5 to the retina under these conditions can significantly prevent ganglion cell degeneration and indeed can promote regeneration of the axons intraretinally [60]. In an elegant study by Castillo et al. [7], conditioned medium from astrocytes that had been genetically modified to produce elevated levels of BDNF was added to rat retinal cultures. In such cultures, the excessive levels of BDNF led to a significant (15fold) survival of Thy-1 immunoreactive ganglion cells as compared with control cultures.

14.6.5 Inflammation (Fig. 14.5)

By hypothesizing that ganglion cells die by apoptosis in glaucomatous optic neuropathy, one would be led to conclude that there would be no associated inflammation, since this type of cell death involves phagocytic destruction of the cellular corpses by surrounding or invading phagocytes. Indeed, clinical evidence appears to support this notion. The disappearance of ganglion cell bodies and a thinning of the inner retina in glaucoma 211 appear not to be associated with oedema or inflammation. Neufeld and colleagues [36], for example, could find no evidence for any

inflammatory response associated with glaucoma. He showed that the level of inducible cyclooxygenase (COX)-2-immunoreactivity in the optic nerve of glaucoma eyes was unaffected. COX enzymes are involved in inflammatory responses by influencing the biosynthesis of eicosanoids. Interestingly, we have shown that when NMDA is injected into the rat eye, COX-2 mRNA is upregulated, suggesting that inflammation may in fact be associated with excitotoxicity in vivo.

The generation of nitric oxide (endotheliumderived relaxing factor) is often associated with both a stimulation of ionotropic glutamate receptors and with inflammation. Injection of the nitric oxide donor S-nitroso-N-acetyl-dl-penicillamine (SNAP, final concentration, 200 nmol) into the vitreous humor of albino rabbits causes marked degenerative changes that include death of large numbers of ganglion cells [41]. These data show that ganglion cells are susceptible to attack by nitric oxide, and if this compound is generated by invading macrophages or microglia during a posttraumatic inflammatory response, then ganglion cell layer degeneration will result.

Microglia exist in the optic nerve head in significant amounts, and these cells become activated when the optic nerve is transected [32] or when this region is subjected to ischemia. Microglial cells have now been shown to release nerve growth factor (NGF) in vitro which can directly kill retinal neurones [20]. Thus, it is possible that during ischemia, ganglion cells can be induced to die not only by excessive release of substances from retinal neurones but also through a release of mediators such as NGF, from microglia. It seems likely that the release of substances from microglia is associated with inflammation. Thus, if ischemia is associated with glaucoma, as seems likely, the suggestion is made that some degree of retinal inflammation will actually result.

14.6.6 Role of Mitochondria (Fig. 14.6)

It is expected that if cells gradually accumulate calcium as during ischemia [25], then mitochondrial failure will result. This is because mitochondria will sequester intracellular calcium as levels become elevated [37]. This leads to

mitochondrial membrane depolarization and a consequent production of reactive oxygen species [25]. Mitochondrial dysfunction has been reported to occur as a result of ischemia, excitotoxicity, inflammation, oxidative stress, and loss of neurotrophic support resulting in cell death.

Apoptosis appears to be responsible for retinal ganglion cell death during development and to a lesser extent after axotomy or ischemia [40]. Mitochondria have been implicated in controlling signal transduction pathways in apoptosis, mainly due to their retaining a preserved intact morphology throughout the death process. This relates to the disease process in Leber’s hereditary optic neuropathy (LHON), where genetic mutations in mitochondrial complex I lead specifically to gradual ganglion cell death by apoptosis [6]. Although no reports exist that describe mitochondrial dysfunction leading to ganglion cell atrophy on a large scale or as a result of glaucoma, this would be expected should such organelles malfunction.

It has been suggested that apoptosis can be triggered by the opening of mitochondrial megachannels, in the process of mitochondrial permeability transition (MPT) [74]. The link between MPT and the activation of the cytosolic proteolytic cascade resulting in apoptotic death is complicated, but, for example, it has been reported that a soluble mitochondrial protein, apoptosisinducing factor (AIF), is released through the open mitochondrial megapores to cause nuclear apoptosis. This can be blocked by overexpression of Bcl-2, which is a protein found associated with the outer mitochondrial membrane.

As stated previously, optic nerve transection leads to apoptotic degeneration of retinal ganglion cells. In transgenic mice that overexpress the Bcl-2 gene, approximately 65% of ganglion cells survive this insult up to 3.5 months following the insult. In contrast, non-transgenic control mice had less than 10% of their original number of ganglion cells remaining after this period of time. Similar mice that are transgenic for Bcl-2 do not lose as many neurones during normal developmental ganglion cell death: they have up to 50% more of these neurones. These mice also have a much reduced infarct volume subsequent to ischemia induced by middle cerebral artery occlusion. Thus, the Bcl-2 protein, which protects against

the destructive effects of mitochondrial failure, can prevent the type of ganglion cell death that is thought to be associated with glaucoma.

14.7Neuroprotection Specifically Related to Glaucoma

The cause of visual loss in glaucoma is due to death of retinal ganglion cells. Their differential demise with time is proposed to be elicited by a combination of ischemia to their axons in the optic nerve head region and chemicals released from activated microglia and astrocytes, as well as light impinging on their intra-axonal mitochondria. Clearly, reducing the amount of light entering the eye is one way forward. Also, drugs targeted to reduce the release of chemicals from activated glia, the impact of ischemia to the optic nerve head region (vasoconstrictors) and the negative effects of raised extracellular chemicals to ganglion cells are required. It is unlikely however that a single substance with a defined mode of action will achieve all these aims. It is therefore proposed that for neuroprotection in glaucoma to become a reality, a spectrum of drug actions is required which might be achieved by a cocktail of defined substances or drugs that display multiple mechanisms of actions. Such drugs should be taken orally to allow penetration to the retina with minimum side effects. Present research suggests that extracts from variety of naturally occurring substances like green tea and Ginkgo [34, 57] have characteristics that might be exploited for such use.

Acknowledgement NNO acknowledges with thanks the support (Cátedra de Biomedicina) from the Fundación BBVA in Spain.

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