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whereas in glaucomatous eyes either the signal is not received by all RGCs at the same time or they respond differentially to the signals (Laquis et al., 1998).

RGC death may have two modes. One is the direct insult of a primary pathological process in glaucoma and the second is the effect of dying RGCs on the surrounding RGCs that lead to death of normal uninjured cells, which is termed secondary degeneration (Levkovitch-Verbin et al., 2001). Convincing evidence of secondary degeneration in glaucoma is lacking. Specific initiators of apoptosis in glaucoma may include blockage of axonal transport leading to neurotrophin depletion (Quigley et al., 1979), glutamate excitotoxicity (Dreyer at al., 1996), antibodies to heat-shock proteins (Tezel and Yang, 2004), ischemia (Osborne et al., 1999), and nitric oxide synthase (NOS) upregulation with reactive oxygen species formation (Neufeld et al., 1999).

Clinical relevance of optic neuropathy

The relationship between elevated IOP and glaucomatous optic neuropathy has been extensively studied. Elevated IOP remains the etiological factor toward which all current therapeutic efforts are directed, although other approaches are now being entertained (Levin, 2003; Osborne et al., 2004).

The prevailing hypothesis regarding the mechanism of elevated IOP mediated optic nerve damage includes compression of optic axons at the lamina cribrosa, ischemia in the lamina cribrosa (Kolker and Hetherington, 1983), and expression of NOS in the optic nerve head (Shareef et al., 1999; Liu and Neufeld, 2001). Animal models became a necessity to study the pathology of the disease process and the effect of pharmacological intervention on death cascade. The resemblance between primate and human glaucoma stimulated the use of primates for research during the last couple of decades (Quigley and Hohman, 1983). However, economical and ethical issues led to a decrease in use of primates and partial replacements by rodents in glaucoma research. Rats have anatomical similarities with primates, regarding

the anterior segment blood supply and aqueous humor drainage (Morrison et al., 1995). No specific model yet exists that has direct homology to human glaucoma. However, the rat model of elevated IOP developed in our laboratory (Shareef et al., 1995) compares closely with the secondary form of human glaucoma and this animal model has been replicated in several laboratories. In a recent study, Urcola et al. (2006) compared three experimental approaches to induce elevation of IOP in rats: injection of latex microspheres in the anterior chamber, injection of latex microspheres plus hydroxypropyl methyl cellulose in the anterior chamber, and episcleral venous occlusions. Although RGCs death in temporal and spatial order was similar using all methods, the episcleral venous occlusion method was preferred by these authors as it had fewer complications to the anterior chamber.

In order to obtain massive RGC death, optic nerve transection or IOP elevation has been used to study the apoptotic pathway (Quigley et al., 1979; Berkelaar et al., 1994; Garcia-Valenzuela and Sharma, 1996). In adult rats, 90% of RGCs die within the first 2–3 weeks after optic nerve transection. The apoptotic RGC death after axotomy and/or elevated IOP has been established in rat (Garcia-Valenzuela et al., 1993, 1994, 1995; Berkelaar et al., 1994) as well as in monkey (Quigley et al., 1995). The induction of apoptosis in RGCs can also be initiated by ischemia/ reperfusion, withdrawal of trophic factors, and radiation. Apoptosis is the mechanism through which superfluous, ectopic, damaged, or mutated cells are eliminated. RGC survival is enhanced by intravitreal injection of neurotrophic factors (Mey and Thanos, 1993), such as brain-derived neurotrophic factor (BDNF) (Krueger-Naug et al., 2003), ciliary neurotrophic factor (CNTF) (van Adel et al., 2003), and NT3/NT4 (Peinado-Ramon et al., 1996). Numerous studies showed the survival effect of neurotrophic factors on axotomized RGCs (reviewed in Chaudhary and Sharma, 2001). A combination of trophic factors, such as glia-derived neurotrophic factor (GDNF) and BNDF, leads to further attenuation of RGC death in 75% of injured RGCs surviving up to 2 weeks following axotomy (Yan et al., 1999). Induction of

CNTF through viral vector has resulted in increased survival and regeneration of injured RGCs (van Adel et al., 2003).

In the past decade or so, great strides have been made in understanding the molecular cascades of RGCs apoptosis in experimental glaucoma; however, many questions remain unanswered. No single mechanism under one experimental condition has been elucidated to account for the apoptosis. Why some RGCs die first and why others survive for a prolonged exposure of elevated IOP stress remains a mystery. Rescuing RGCs by intervening in the death cascade has been partially successful in ameliorating cell death in experimental glaucoma. However, the functional consequences of the rescued RGCs remain unclear.

Is there a remodeling of retinal circuitry?

Extensive evidence has emerged in the past decade that some retinal diseases whose progression has been correlated with photoreceptor degeneration lead to the remodeling of retinal circuitry (reviewed in Jones et al., 2005; Marc et al., 2006). The loss of photoreceptors and, therefore, the glutamatergic loss to the bipolar cells lead to retinal remodeling apparent in migration of neurons, retinal rewiring, and creation of microneuromas. These authors reported Mu¨ller cell hypertrophy and axonal sprouting in ganglion, bipolar, and amacrine cells as a consequence of photoreceptor death. The evidence of neuronal plasticity in the adult retina is real.

Plasticity of RGCs has been the focus of earlier studies where neurons react to lesions by structural reorganization of somas, axons, and dendrites. During development, RGC morphology is influenced by either increasing or decreasing cell densities in the retina using experimental manipulations (Rappaport and Stone, 1983; Peichl and Bolz, 1984; Eysel et al., 1985; Kirby and Chalupa, 1986; Leventhal et al., 1988; Bahr et al., 1992). In glaucomatous eyes the densities of RGCs change. We have shown earlier that following increase in the IOP and subsequent death at the rate of 3% per week, the soma diameter of remaining ganglion cells became significantly larger in all cell

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types. Ganglion cell types I and III showed the increase in the area of dendritic field (Ahmed et al., 2001). Rodent data are surprising as the primate glaucomatous eye showed no increase in RGC soma or dendrites (Weber et al., 1998, 2000). On the contrary, Weber and Harman (2005) concluded that ganglion cells in the glaucomatous eye retain normal intrinsic electrical properties yet they have decreased dendritic field sizes. Some controversies still exist, for example, Smith et al. (1993) had proposed earlier that visual deficit in long-term glaucoma results from ganglion cell loss and not reduction in the functional capacity of the surviving neurons. One could conclude that plasticity in RGCs persisted in adult rats.

In the glaucomatous eye of the primates, data to date suggest that following loss of RGCs there is continuous atrophy of the surviving cells that leads to visual dysfunction. Data accumulated in the past several years point to transneuronal changes resulting either in the loss of some and/or reduction of other soma sizes of the lateral geniculate nucleus (LGN). No studies exist as yet showing similar changes in the rodent visual system.

Functional assessment of visual changes in glaucoma patients was made possible by recent studies of Duncan et al. (2007) using functional magnetic resonance imaging (fMRI) method. The spatial pattern of activity in the glaucomatous eye was correlated on a retinotopic map with that of the fellow eye. The fMRI signals in visual I area of the human cortex showed a loss of visual function that correlated nicely with the loss of visual field in the eye. The fMRI technique has opened a new vista to quantify glaucomatous changes in the neural activity. The fMRI method allows assessment of the changes in the visual brain centers following damaged retina; however, it is not clear whether this method can assess changes in the early stages of glaucoma before the death of either RGCs or the changes in the function of RGCs where dendrites have begun to shrink yet the axons are still properly connected to the visual centers. This problem is further compounded by the fact that fMRI studies in macular degeneration patients with losses of foveal vision show extensive activity in the cortical foveal area (Ngugen et al., 2004a, b; Baker et al., 2005). These studies point to

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the extensive reorganization of visual terminal areas that would account for the results. It is reasonable to assume that in macular degeneration some foveal retinal cells survived and these RGCs expanded their projections in the cortex. It is further conceivable that the parafoveal RGC axons invaded the territory in the foveal area of the cortex. Either possibility will lead one to believe that many limitations persist in assessing the early functional changes in glaucomatous eyes using fMRI methods.

Optic nerve and RGC damage produces changes in the visually evoked potentials in patients with glaucoma, as the scotoma enlarges with the progression of the disease. The enlargement of the visual field deficits is due to an increase in dysfunction or dying RGCs in the focal areas of the retina. In order to determine the functional consequences of RGC pathology in glaucomatous eyes, visual receptive fields were mapped onto the superior colliculus contralateral to the experimental eye and in control eye in elevated IOP rats (King et al., 2006).

Retinotopic organization in the normal rat was generated by recording from specific locations on the contralateral superior colliculus. Visual receptive field sizes at each recording site on the superior colliculus were in the order of 5–151. Essentially, the nasal visual field projects to the contralateral rostral superior colliculus and the temporal visual fields project to the caudal tectum, whereas the

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dorsal visual field projects to the medial and the ventral visual field projects to the lateral edges of the tectum. This topographic pattern is highly ordered (Fig. 1).

To assess the effects of localized scotoma, we first created a small lesion in the retina via the insertion of a 22-gauge needle into the nasal retina. We then mapped electrophysiologically the retinotopic projection to the contralateral superior colliculus. A well-demarcated silent area in the contralateral temporal visual field was obtained (Fig. 2). An electrophysiological mapping of visual field projections in a rat that had elevated IOP (28–30 mmHg) was generated. Normal pressure before surgery and in the control eye was 16 mmHg. Following the IOP elevation via episcleral venous occlusion, the animal was allowed to recover and maintain at a 12-h light and 12-h dark cycle. IOP was measured once a week for up to 4 weeks and was within 25–27 mmHg immediately prior to recording. We assumed that RGC death would be in the range of 12–15% in 5 weeks following IOP elevation. In normal animals, the sizes of receptive fields recorded at their terminal on the superior colliculus are in the range of 5–151 of the visual space. However, in these experiments with elevated IOP for up to 5 weeks, some receptive fields on the peripheral visual area were in the range of 25–301 of the visual space. The visual field map was not interrupted. All electrode locations provided visually evoked responses

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without any silent area that may correspond to the retinal scotomas. The enlarged receptive fields were definable. The recordings were done in a mesopic luminance level. The dots and numbers on the left superior colliculus indicate electrode positions, 200 mm apart. The receptive fields on the right visual field map are of various sizes; some enlarged receptive fields overlap with each other. These were noticed primarily in the dorsal areas of the visual field (Fig. 3).

These results indicated that although loss of the RGCs continued, the visual field scotoma was not apparent in the early period following elevation of IOP. Additionally, larger receptive fields on the periphery may represent the early signs of altered geometry of the retina as large RGCs die in the periphery of the retina in early stages of glaucoma (Shareef et al., 1995). It would be tempting to assume that following the death of larger cells on the periphery, the remaining ganglion cells

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expanded their axonal arbors in the tectum leading to the enlarged receptive fields. This may represent the earliest changes in visual receptive fields in the tectum following death of selective RGCs.

Expansion of visual receptive fields in glaucoma

As pointed out above, the sprouting of axons and dendrites in other retinal cells has been reported in adult mammals (Peichl and Bolz, 1984; Lewis et al., 1998). The glaucomatous eye’s retinotopic map onto the superior colliculus was recorded in order to assess the project patterns of the remaining RGCs following significant death of RGCs due to elevated IOP (King et al., 2006). IOP was elevated in the eye via episcleral venous occlusion (Garcia-Valenzuela et al., 1995) and it remained elevated to 150% of the control eye for the duration of the experiment. The sizes of the visual receptive fields recorded from the superior colliculus, contralateral to the glaucomatous eyes, were significantly large (e.g., 236 degree2 for normal vs. 849 degree2 for the glaucomatous eye). This increase in area of visual receptive fields was proportional to both the degree and the duration of IOP elevation. Scotomas due to RGCs loss, although present in the eye following prolonged cell death, were undetectable due perhaps to the massive overlap of visual receptive fields in the superior colliculus generated by the remaining RGCs (Fig. 4).

The ramifications of the above-described studies are the following:

1.Change in the dendritic arbor of RGCs in glaucomatous eyes (Ahmed et al., 2001) cannot solely describe the compensatory effect of the changes in the receptive fields in the colliculus. Ahmed et al. (2001) reported changes in only two cell types in the retina whose dendritic arbors expanded following death of RGCs. Type I and III cells expand their dendritic fields for up to 6 weeks following IOP elevation, whereas the soma diameter of all three cell types was enlarged. These observations stand in contrast to that

seen in the primate retina. Experimentally induced RGC death by partial optic nerve cut or other means leads to the vacated sites. This reduction in the density of RGCs in cats led to the increases in soma sizes of the remaining ganglion cells (Rappaport and Stone, 1983; Kirby and Chalupa, 1986). Observations of Rousseau et al. (1999) in adult rat showing the compensatory soma size changes following 50% RGC death supported our observations in glaucomatous rats (Ahmed et al., 2001). It is important to keep in mind that following partial optic nerve crush, cells that are destined to die (within 2 weeks) show soma swelling. The remaining cells increase their soma size similar to physiological parameters of recovery of function (Schmitt et al., 1996). It is therefore conceivable that early increase in soma sizes may represent the cells that are destined to die. It is clear that surviving RGCs have differential ability to adapt in glaucomatous retina. Many questions remain unanswered, e.g., (a) what are the differences between primate and rat retina in glaucomatous eye; (b) are the increases in dendritic arbor owing to new synaptic connections created by vacant bipolar and amacrine cells in rat and not in primates; (c) what are the differential time sequences of RGC death; (d) what are the functional consequences of remaining RGCs?

2.The relationship of duration and magnitude of IOP elevation showed a significant positive correlation between percentages of receptive field size increases in glaucomatous animal (Fig. 4D). This correlation becomes evident after 6 months of sustained elevated IOP. A greater increase in visual receptive field size correlates reasonably well with a greater increase in the IOP. When the IOP level increases over 50–60%, a definite increase in the visual receptive field is encountered. Hence, an increase in the visual receptive field may represent a very long-term effect of the remaining ganglion cells in the glaucomatous eyes in which ganglion cell axons try to compensate for the loss of the fields by occupying a larger than normal territory of

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the colliculus. Since these changes represent the long-term effect in elevated IOP rats, one wonders how much it may take to manifest similar changes in primate glaucomatous animals.

3.The magnification factor, or the degree of visual field per millimeter square of the tectal surface, is variable in the rat superior colliculus. The comparison of the recorded

visual receptive field from the experimental and mirror image in the control tecta provided the best avenue to measure the relative changes. At every point recorded on the experimental tectum, the size of the visual receptive field was significantly larger. These data suggest that every tectal point in the experimental animal independent of the magnification factor showed an increase in