Core Messages

•Ischemia occurs in many ocular diseases when the blood delivery does not meet the cellular energy demands of the tissue. Ischemia has characteristics associated with hypoxia/anoxia but hypoxia/ anoxia does not imply ischemia.

•It is proposed that a cause for the initiation of glaucoma to the retina is a mild ischemic insult to the optic nerve head region. In contrast, a complete loss of blood supply occurs in ischemic optic neuropathy.

•In glaucoma, ganglion cell mitochondria are affected by lack of an optimum blood supply resulting in oxidative stress so making them susceptible to additional insults like light. A more drastic reduction of optic nerve head blood supply will result in a complete loss of ganglion cell mitochondrial function as in ischemic optic neuropathy.

•A mild reduction of blood supply to dividing retinal glial cells results in them

N.N. Osborne, Ph.D., D.Sc.

Fundación de Investigación Oftalmológica, Avda. Doctores Fernández-Vega 34, E-33012 Oviedo, Asturias, Spain

Nuffield Laboratory of Ophthalmology, Oxford University,

Headley Way, Oxford OX3 9DU, UK e-mail: neville.osborne@eye.ox.ac.uk

becoming activated releasing substances to support and enhance neuronal function. However, as glaucoma progresses the continuous release and accumulation of such substances in the extracellular space might reach high levels so becoming toxic to the already fragile ganglion cells.

•It is therefore suggested that as glaucoma progresses the combined elevated levels of substances released from glial cells together with light impinging on ganglion cell intra-axonal mitochondria triggers their demise at different times. Thus, the receptor profile and number of mitochondria associated in individual ganglion cells might be major factors at determining when they die after initiation of the disease.

14.1 Introduction

The word ischemia was coined by Virchow, who combined the Greek iskho, meaning “I hold back,” with háima, meaning “blood”. Hence, ischemia refers to a pathological situation involving an inadequacy (not necessarily a complete lack of) blood flow to a tissue, with failure to meet cellular energy demands. Ischemia should be distinguished from anoxia (a complete lack of oxygen) and hypoxia (a reduction in oxygen): ischemia always has a component of

Fig. 14.1 Diseases associated with the retina where ischemia is implicated. Ischemia can be global affecting all blood supplies to the retina or focal where defined blood supplies

associated with different parts of the retina and/or optic nerve head. Moreover, ischemia can be complete or incomplete as well as being associated with reperfusion or not

hypoxia/anoxia, but hypoxia/anoxia does not imply ischemia. For example, the retina may become hypoxic at high altitudes, producing loss of vision, but it is not ischemic. Similarly, anemia (generally a reduction, rather than complete absence of hemoglobin) is always a component of ischemia but not vice versa.

Ischemia deprives a tissue of three requirements: oxygen, metabolic substrates and removal of waste products. The loss of these requirements will initially lower homeostatic responses and, with time, will induce injury to the tissue. If withheld for a sufficiently long time, the tissue will die (an infarct). The molecular events that accompany ischemia and exactly what constitutes a “sufficiently long time” vary and depend on different factors. The retina being a thin tissue surrounded by the vitreous humor is therefore more resistant than deeper brain structures to a persistent complete reduction of blood flow.

Ischemia plays a part in a number of ocular pathologies (Fig. 14.1). For example, occlusion of central retinal vein or artery is associated with acute retinal ischemia and rapid visual loss [23].

In other conditions which gradually develop, such as diabetic retinopathy and glaucoma, chronic retinal ischemia (not a complete but a reduced blood supply) is a pathogenic feature of the disease [10, 34, 72]. Ischemic challenges also contribute to the pathogenesis of less prevalent conditions to the retina, such as sickle cell retinopathy, radiation retinopathy, and retinopathy of prematurity [24, 61]. Moreover, ischemic challenges also occur in the aging retina which probably results in neuronal loss [70]. The complexity of mechanisms involved in causing ischemic damage to central nervous tissue is suggested by the variety of ways by which the process can be attenuated experimentally (Table 14.1).

14.2Retinal Ischemia Basic Mechanisms

Mammalian retinal ischemia results in irreversible morphological and functional changes. These are the consequence of depleted ATP stores, due to deprivation of both glucose and oxygen, though

Table 14.1 This table documents the number of ways by which insults of ischemia to central nervous tissue can be attenuated in various animal studies

Maintain retinal energy supply

Decrease extracellular glutamate levels

Block excitotoxic depolarization (e.g., blockade of NMDA, AMPA/kainate receptors)

Activate inhibitory receptors (e.g., GABA, glycine receptor agonists)

Block calcium entry (e.g., voltage-sensitive calcium channels)

Block axonal sodium channels

Inhibit influx of chloride ions

Enhance intracellular glutamate catabolism

Inhibit free radical production

Inhibit nitric oxide production

Prevent lipid peroxidation

Prevent apoptotic cell death pathways

Block mitochondrial permeability transition

Prevent inflammation

Prevent oxidative stress

Compensate for compromised axonal flow of neurotrophins (e.g., BDNF, NGF, NT-4/5)

Growth factors (e.g., bFGF, EGF, CNTF)

Induction of heat shock proteins

transient loss of these substrates is not immediately lethal. The cell death is the result of an extremely complex (not completely understood) cascade of biochemical responses initiated by energy failure. The tissue damage and functional deficits that follow periods of transient ischemia reflect the combined effects of several, often interrelated, pathophysiological pathways. These result in drastic changes in ion movements, neurotransmitter levels, and metabolites.

It is useful to consider that stroke lesions consist of a densely ischemic focus, the ischemic core, surrounded by a better perfused area, the ischemic penumbra [27]. Cells in the focus are usually doomed unless reperfusion is quickly instituted. In contrast, penumbral cells may remain viable for several hours and can be saved by reperfusion or by drugs that prevent the infarction extending into the penumbral zone [27].

During the past decade, a considerable amount of experimental work has been devoted to the elucidation of the mechanisms of ischemic neuronal injury, but there is still much debate over

the underlying processes causing the injury. One area of interest is the role of glutamate and aspartate, whose extracellular concentrations increase markedly during ischemia [12]. It is generally accepted that glutamate release during the early phase of brain ischemia triggers events leading to irreversible injury not only in those areas in which oxygen supply is critically reduced but also in regions of seemingly less disturbed energy metabolism, i.e. the penumbra of focal ischemia. However, there is still much controversy over the extent of their role in the pathophysiology of neural ischemia. Moreover, neuronal bodies (gray matter) and axons (white matter) are not affected in precisely the same way by ischemia [53].

It is important to remember that much of the work studying ischemic neuronal and axonal damage has been carried out on tissues derived from defined brain regions, so care is important when drawing comparisons with what happens in the retina. In particular, the way photoreceptors respond to ischemia in light and dark conditions may be unique. Moreover, the thinness of the retina, its well-defined blood systems and the large glycogen supply associated with the Müller cells provide the tissue with a unique energy supply when compared with the brain, so making the response of tissues to ischemia not the same.

14.3Oxidative Stress

Oxidative stress is imposed on cells as a result of one of three factors: (1) an increase in oxidant generation, (2) a decrease in antioxidant protection, or

(3) a failure to repair oxidative damage. Cell damage is induced by reactive oxygen species (ROS). ROS are either free radicals, reactive anions containing oxygen atoms or molecules containing oxygen atoms that can either produce free radicals or are chemically activated by them. Examples are hydroxyl radical, superoxide, hydrogen peroxide and peroxynitrite. The main source of ROS in vivo is aerobic respiration, although ROS are also produced by peroxisomal b-oxidation of fatty acids, microsomal cytochrome P450 metabolism of xenobiotic compounds, stimulation of phagocytosis by pathogens or lipopolysaccharides, arginine

Fig. 14.2 Production and reactions of nitrogen-derived radicals. (a) Nitric oxide synthase (NOS) produces nitric oxide radicals in the conversion of l-arginine to l-citrul- line. This compound acts as an important homeostatic modulating agent under physiological conditions via its vasodilator, antioxidant, antiplatelet, and antineutrophil actions. In the event that superoxide radicals are present (·O2−), usually as a result of rapid tissue reoxygenation subsequent to an ischemic event, peroxynitrite is formed which rapidly decomposes to highly reactive oxidant species that can cause tissue injury. Under physiological conditions, there is a critical balance between cellular concentrations of NO, ·O2− and superoxide dismutase

activity which favor NO production. In pathological conditions such as reperfusion following an ischemic event, the formation of ONOO− is favored. The latter compound can be rapidly detoxified if it is combined with reduced glutathione (GSH) to form S-nitrosoglutathione (GSNO), but this depends upon the cellular antioxidant defense system being functional, and this is generally overwhelmed during tissue reperfusion. (b) Reactions and formation of nitric oxide radicals as a result of ischemia-reperfusion. The combined production of nitric-oxide-derived radicals and failure of cellular antioxidant defense will lead to widespread macromolecular damage and cell death

metabolism and tissue-specific enzymes. Under normal conditions, ROS are cleared from the cell by the action of superoxide dismutase (SOD), catalase or glutathione (GSH) peroxidase. The main damage to cells results from the ROS-induced alteration of macromolecules such as polyunsaturated fatty acids in membrane lipids, essential proteins and DNA. Additionally, oxidative stress and ROS have been implicated in retinal ischemic disease states.

14.4The Role of Free Radicals

in Retinal Ischemia (Fig. 14.2)

Many cascades generated by glutamate and glucose/oxygen deprivation result in the formation of free radicals [51], and it has been proposed that free radicals are important mediators in damage caused by retinal ischemia [3, 46]. Reperfusion

injury after ischemia appears paradoxical, but oxygen-derived and other free radicals are principally formed when reduced compounds, which accumulate during ischemia, are reoxidized. There is evidence that this free radical burst, produced during the early stage of reperfusion, overwhelms normal cellular antioxidant defense mechanisms, causing oxidative stress and a variety of types of tissue injury [21].

There are many ways in which free ROS can be formed during ischemia-reperfusion, but the burst of superoxide radicals (·O2−) which occurs during the early stage of reperfusion is thought to occur by the following pathway. During ischemia, degradation of ATP leads to the formation of hypoxanthine, and increases in intracellular calcium in neurones activate the Ca2+-dependent protease calpain. Calpain converts xanthine dehydrogenase into xanthine oxidase, and upon reperfusion, the latter enzyme oxidizes the accumulated

radical formation. Although there is no direct evidence for increased free radical formation from neutrophils, blocking leukocyte accumulation has been shown to afford protection to the ischemic retina [73].

Oxygen-derived free radicals cause extensive cellular damage in the brain. One of the main mechanisms by which this occurs is by attacking unsaturated fatty acids, which leads to lipid peroxidation of membranes. This will result in loss of membrane fluidity, cell swelling, oedema, and feed-forward production of more free radicals. Many additional mechanisms of damage have been ascribed to free radicals generated during ischemia-reperfusion, including attacking sulphhydryl protein bonds, which leads to the destruction of amino acids and polypeptide chains; fragmentation of DNA molecules, which leads to activation of poly(ADP-ribose) polymerase; activation of cytokines and NF-kB, which are likely to be instrumental in upregulations of iNOS and COX-2 and subsequent release of glutamate; and effects on Ca2+ homeostasis [28]. Interestingly, a clear association between neuronal cell death and formation of free radicals and lipid peroxides in the retina subjected to ischemia-reperfusion has only recently been demonstrated [8]. In all of these studies, ischemia-reperfusion induced free radical formation, lipid peroxidation, and neuronal injury and were largely preventable by administration of free radical scavengers [8, 63] and a novel metal chelate [1].

14.5Ischemia in Glaucoma

(Figs. 14.3 and 14.4)

Glaucoma is defined as an optic neuropathy associated with characteristic changes in the optic nerve [54, 55]. Patients suffer a gradual loss of

vision that can span a 30–40-year period, with retinal ganglion cells slowly, but constantly, dying. The disease is often associated with raised intraocular pressure, but this does not occur in approximately one sixth of all glaucoma patients [55]. Thus, glaucoma can be viewed as a neurodegenerative disease of the retinal ganglion cells.

It has been proposed that in most cases of glaucoma, ischemia initiates the disease by affecting solely components in the optic nerve head of the retina [17, 47]. In this particular region of the eye, minute blood vessels exist, and when intraocular pressure is increased, perfusion pressure in these vessels is reduced [19, 39]. In laboratory studies, it can be shown that a sustained elevation of intraocular pressure causes glaucoma-like pathology in animals [46]. We also know that various risk factors often associated with glaucoma subjects (e.g., hemorrhaging, inability to autoregulate, hypotension, hypertension) relate to the vasculature [17–19]. Thus, a body of evidence suggests that the initiation of glaucoma is caused by an ischemic or other metabolic insult to the optic nerve head region. It should be noted that ischemia is defined here as a pathological situation involving a degree of inadequacy (but not a lack) in the blood flow with a subsequent failure to meet appropriate cellular energy demands. Moreover, it is possible that at least in some glaucoma patients, ischemia to the optic nerve head is not constant but intermittent, and this would result in additional oxidative stress, reperfusion injury [46]. Fluctuations in intraocular pressure causing changes of blood flow to the optic nerve head would theoretically cause both ischemia and reperfusion injury.

It has been proposed that ischemia to the optic nerve head initiates a process that eventually results in ganglion cells dying at different rates [17, 45, 47] in glaucoma. Tissue components

Fig. 14.4 Hypothetical cascade of events that lead to ganglion cells dying at different times during the lifetime of a glaucoma patient. The initial insult in the optic nerve head region affects all components (astrocytes, microglia, Müller cell end feet, ganglion cell axons, lamina cribrosa) resulting in ganglion cells existing at a reduced energetic state and an elevation of various substances (glutamate,

TNFa, NO, endothelin, d-serine, potassium) in the extracellular space caused by activated astrocyte and microglia cells. These substances together with light impinging on mitochondria in ganglion cell axons eventually cause apoptotic insults to the energetically compromised ganglion cells at different times, depending on the axonal length and receptor profile of individual ganglion cells

Release of a number of substances e.g., TNF-α, NO, endothelin, glutamate, serine, potassium

Into the extracelluar spaces of the retina.

Müller cells gradually become unable to clear extracellular spaces from substances released from activated astrocytes and microglia cells

Light insults to ganglion cell mitochondria.

Ganglion cells at a reduced energetic state but still able to transmit information to the brain

Elevated levels of substances (e.g., TNF-α, NO endothelin, glutamate, serine, potassium) in

extracellular spaces are toxic in particular to the ganglion cells.

Ganglion cells die at different rates by apoptosis