Ординатура / Офтальмология / Английские материалы / Retinal Degeneration Disease_Hollyfield, Anderson, LaVail_1999

fascicles that may run for >100 microns in addition to elaboration of new “tufts” of IPL (microneuromas) that form outside the boundaries of the normal stratification of the IPL. These microneuromas are populated with synaptic contacts corruptive of normal visual processing (Marc et al., 2003). Finally, migration of adult neuronal phenotypes throughout the vertical axis of the retina is observed with all cell classes participating. It is believed that in order to maintain normal gene expression, neurons will sprout processes to seek lost glutamatergic signaling. Failing to achieve synaptic contact may result either in cell death or cellular soma migration to other regions of the retina. Amacrine cells are commonly observed translocating to the ganglion cell layer with ganglion cells also migrating up into the inner nuclear layer.

The work with naturally occurring human, and natural and genetically engineered animal models has revealed plasticity and retinal neural remodeling as the typical response to apparent sensory deafferentation. Therefore, while light damage has long been recognized as a way to “kill” photoreceptors, this study was designed to use the light damage methodology (LD) to assess the nature and scope of plasticity in the neural retina in response to deafferentation in an environmental rather than a genetic model of deafferentation.

2. METHODS

Over 90 albino Sprague-Dawley rat retinas were exposed to light (Organisciak et al., 1998) of varying durations, pre-adaption states, circadian phases and survival times. Posteuthanasia, enucleated eyes were rapidly fixed in glutaraldehyde, resin-embedded and thin sections (250 nm) were serially probed with IgGs generated against aspartate, glutathione, glutamate, glutamine, glycine, GABA, and taurine, key retinal metabolites and cell specific markers. Primary immunohistochemical labeling was followed by silver intensification with a secondary goat anti-rabbit IgG adsorbed to 1 nm gold particles and visualized with silver intensification (Kalloniatis and Fletcher, 1993). All images of immunoreactivity were captured as 8-bit greyscale images and registered to <250 nm root-mean-square error. Computational molecular phenotyping (Marc and Jones, 2002) was then employed to identify neurons. EM overlay was employed to identify signatures at the ultrastructural level (Marc and Liu, 2000).

3. RESULTS

In the LD model, Müller cells undergo dramatic transformations in areas with complete rod and cone loss. In these regions, plasticity ensues as evidenced by neuronal migration via formation of hypertrophic MC columns throughout the axis of the retina, with some neurons migrating from the retina into the remnant choroid. Synaptic remodeling is demonstrated with neurites engaging in novel, corrupt circuitry via GABAergic, glycinergic and glutamatergic synapses. Within 14 days of even a brief, 3 hr LD treatment, focal photoreceptor loss was accompanied by irregular 2-4 fold increases in RPE glutamine and rod aspartate levels, perhaps presaging cell death. The onset of MC remodeling (formation of a fibrotic glial seal in regions of extensive rod/cone cell death) is accompanied by a dramatic >10-fold increase in MC glutamine. This occurs only in MCs engaged in seal formation; MCs a mere 0.1 mm away are normal (Figure 57.1). By 60 days post exposure, most

Figure 57.1. Early in the degenerative process, Müller cells exhibit large increases in glutamine concentration (often >10-fold increases) in areas where photoreceptor death is complete allowing Müller cells to begin seal formation.

photoreceptors have died and the glial seal has become complete in those areas with complete photoreceptor loss. Small microneuromas have begun to form, originating from sprouting bipolar cells and amacrine cells. Aditionally, fluid channels begin to form, likely originating from the Müller cell seal walling off of the neural retina from the vascular choroid. This seal likely impairs transretinal water flow (Bringmann et al., 2004), resulting in the formation of fluid channels or cysts. Those bipolar cells that have not sprouted and found targets to contact have begun the process of dying. For the most part however, at 60 days post exposure the normal lamination of the IPL is intact and most populations of cells other than the photoreceptors appear in approximately normal numbers. At approximately 120-240 days post-LD, when both neuronal migration on hypertrophic MC columns and synaptic remodeling are initiated, other more dramatic changes ensue. Synaptic remodeling is evinced by neuropil arising from new neurites in the remnant distal retina containing GABAergic, glycinergic, and glutamatergic synapses in novel circuits (Figure 57.2). Distal migration of MC nuclei, MC hypertrophy and disorganization of the inner nuclear layer, including cell loss, match remodeling processes in advanced genetic forms of retinal degeneration, including human retinitis pigmentosa. Neuronal migration throughout the axis of the retina is common. All classes of neurons participate including glycinergic amacrine cells migrating into the ganglion cell layer and ganglion cells can be observed migrating into the inner nuclear layer. By 240 days post-LD, the RPE has been obliterated, the vascular choroid has been compromised and there is extensive emigration of MCs and neurons from the neural retina proper into the remnant choroid, similar to that described by Sullivan et al. (Sullivan et al., 2003) for the aged ambient-LD rat. These neurons posess signatures unchanged from their signatures in the retina proper.

Figure 57.2. A 300 day old rat retina harvested 240 days after light damage at 60 days of age immunohistochemically labeled for GABA. Elaboration of microneuromas outside the normal lamination of the IPL are seen composed of tangles processes labeling for GABA, glutamate and glycine from amacrine cells, horizontal cells, bipolar cells and ganglion cells (Jones et al., 2003). Also observed in this image is the beginning formation of an aqueous fluid channel.

Confirming the exit from the neural retina proper required ultrastructural analysis. Therefore, electron microscopy (EM) with light microscopy overlay (Marc and Liu, 2000) was employed to demonstrate emigration of cells with mature neuronal phenotypes through Brüch’s membrane. Figure 57.3 shows one such neuron: a GABAergic neuron that has completely passed through a small hole in Brüch’s membrane demonstrating adult neuronal phenotypes remain stable when migrating through the retina and into the remnant choroid.

4. CONCLUSIONS

All insults that kill photoreceptors represent sensory deafferentations that trigger retinal remodeling akin to CNS plasticities, including neuronal loss, growth of new neurites, formation of new synapses, and reorganization of the neuronal and glial somatic positions. These data show LD that models exhibit plasticity that mirrors pathology observed in other models of retinal degeneration. LD is a fast, effective trigger of large-scale remodeling (perhaps due to the high temporal coherence of the insult) and enables study of circuitry defects emergent from remodeling.

Sparing of the ventral retina allows for a “built in” control, allowing us to compare within the same preparation both normal and remodeled portions of tissue. Furthermore, the LD model, with the possible exception of the conditional genetic knockouts, is the only model in which we know there are no developmental abnormalities with respect to circuitry and genetics throughout development making the LD model possibly the best model available for Age Related Macular Degeneration (AMD) and AMD like disorders. Cells outside the boundaries of the neural retina have escaped. Furthermore, they appear to have normal amacrine and bipolar cell signatures indicating their metabolic status appears to be stable.

Figure 57.3. An electron micrograph of a GABAergic amacrine cell (confirmed by CMP EMoverlay (Marc and Liu, 2000)). Bruch’s Membrane has been breached allowing neurons to escape the remnant neural retina into the choroid.

Many of these neurons have rewired and apparently have established some form of connectivity keeping them alive. Furthermore, the glial cells are also emigrating, leaving an abandoned retina which, for all intents and purposes may be dead. Rescue at this point is impossible.

These data show light-damaged models exhibit plasticity that mirrors pathology observed in other models of retinal degeneration. We suggest that all insults resulting in loss of photoreceptors represent sensory deafferentations, triggering retinal remodeling akin to CNS plasticities, including neuronal loss, growth of new neurites, formation of new synapses, and reorganization of neuronal and glial somatic positions and finally, adult neurons can migrate without first de-differentiating.

5. REFERENCES

Bringmann A, Reichenbach A, Wiedemann P. 2004. Pathomechanisms of cystoid macular edema. Ophthalmic Res 36:241-249.

Jones BW, Baehr W, Frederick JM, Marc RE. 2001. Aberrant remodeling of the neural retina in the GHL transgenic mouse. In: Invest Ophthalmol Vis Sci.

Jones BW, Watt CB, Frederick JM, Baehr W, Chen CK, Levine EM, Milam AH, LaVail MM, Marc RE. 2003. Retinal remodeling triggered by photoreceptor degenerations. Journal of Comparative Neurology 464:1-16.

Kalloniatis M, Fletcher EL. 1993. Immunocytochemical localization of the amino acid neurotransmitters in the chicken retina. J Comp Neurol 336:174-193.

Li ZY, Kljavin IJ, Milam AH. 1995. Rod photoreceptor neurite sprouting in retinitis pigmentosa. Journal of Neuroscience 15:5429-5438.

Marc RE, Jones BW. 2002. Molecular phenotyping of retinal ganglion cells. Journal of Neuroscience 22:412-427.

410 B.W. JONES ET AL.

Marc RE, Jones BW, Watt CB, Strettoi E. 2003. Neural Remodeling in Retinal Degeneration. Prog Ret Eye Res 22:607-655.

Marc RE, Liu W. 2000. Fundamental GABAergic amacrine cell circuitries in the retina: nested feedback, concatenated inhibition, and axosomatic synapses. Journal of Comparative Neurology 425:560-582.

Milam AH, Li ZY, Cideciyan AV, Jacobson SG. 1996. Clinicopathologic effects of the Q64ter rhodopsin mutation in retinitis pigmentosa. Invest Ophthalmol Vis Sci 37:753-765.

Milam AH, Li ZY, Fariss RN. 1998. Histopathology of the human retina in retinitis pigmentosa. Prog Ret Eye Res 17.

Organisciak DT, Darrow RM, Barsalou L, Darrow RA, Kutty RK, Kutty G, Wiggert B. 1998. Light history and age-related changes in retinal light damage. Invest Ophthalmol Vis Sci 39:1107-1116.

Park SJ, Kim IB, Choi KR, Moon JI, Oh SJ, Chung JW, Chun MH. 2001. Reorganization of horizontal cell processes in the developing FVB/N mouse retina. Cell Tissue Res 306:341-346.

Strettoi E, Pignatelli V. 2000. Modifications of retinal neurons in a mouse model of retinitis pigmentosa. Proc Natl Acad Sci 97:11020-11025.

Strettoi E, Pignatelli V, Rossi C, Porciatti V, Falsini B. 2003. Remodeling of second-order neurons in the retina of rd/rd mutant mice. Vision Res 43:867-877.

Strettoi E, Porciatti V, Falsini B, Pignatelli V, Rossi C. 2002. Morphological and functional abnormalities in the inner retina of the rd/rd mouse. Journal of Neuroscience 22:5492-5504.

Sullivan R, Penfold P, Pow DV. 2003. Neuronal migration and glial remodeling in degenerating retinas of aged rats and in nonneovascular AMD. Invest Ophthalmol Vis Sci 44:856-865.

Zrenner E. 2002. Will Retinal Implants Restore Vision? Science 295:1022-1025.

are entrained by light, but can also be entrained by temperature17 and feeding.18 Circadian rhythms underlie many physiological processes that can lead to a stress tolerant or susceptible environment. There are several prominent circadian regulated physiological processes in the retina. Melatonin and dopamine are synthesized in a circadian manner or in response to light-dark cues, and may be involved in entraining the circadian cycle. They are the most obvious candidates for involvement in increased retinal susceptibility to light. Phototransduction protein levels are also important candidates as they are directly involved in modulating the retinas response to light. A fairly new candidate is metabolic activity of the retina. Increased metabolic activity results in decreased pH levels which may also play a key role as it has been shown to be involved in inducing apoptosis.19 Any or all of these factors may be involved in the circadian dependent susceptibility to light damage. This review covers four important candidates that may play a role in circadian dependent susceptibility to light damage. A complete review of all the factors that may be involved in this phenomenon is beyond the scope of this paper.

2. MELATONIN

Melatonin produced by the pineal is thought to act as an endocrine hormone, entraining overall circadian rhythms.20 In the retina, photoreceptor cells produce melatonin where it acts as a paracrine hormone.21,22 The highest levels of melatonin are present during the night with levels decreasing after light onset.23 Studies have reported that the Mel1b and Mel1c melatonin receptor RNA or protein are expressed in Xenopus retina24,25and that the melatonin MT1 receptor protein is expressed in human retina.26 It has been suggested that the function of melatonin expression at night is to increase the sensitivity of the visual system, and in this way facilitate dark adaptation.27 Exogenous melatonin applied to the retina has been shown to increase light induced retinal damage.28 In contrast, the application of the melatonin receptor antagonist luzindole prevents light induced damage.29 These results suggest that melatonin binding to receptors in the retina results in the increased retinal susceptibility to light damage. However, melatonin has also been shown to be a direct free radical scavenger, and to act as an indirect antioxidant by inducing endogenous antioxidative enzymes.30 This creates a paradox as to the role that melatonin plays in retinal susceptibility to light damage. Although melatonin has been shown to increase sensitivity through receptor binding, oxidative stress also plays a key role in cell loss. The observation that melatonin has antioxidative properties would suggest that it would decrease susceptibility to light damage. To date, the role of melatonin in LIRD is unclear. A study by Wiechmann31 examined melatonin induced gene expression changes by microarray analysis. 14 genes were found to change in expression in the retina, and 17 genes changed in expression in the RPE. Among the genes that changed in expression were gamma crystallin, CREB protein, CED 6, and NGF induced transcription factor.31 Further examination of these genes may help clarify melatonin function in the retina.

3. DOPAMINE

Dopamine is synthesised in the amacrine or interplexiform cells of the retina depending on the species.32 Studies have shown that in a number of species there is a higher con-

58. CIRCADIAN DEPENDENT SUSCEPTIBILITY TO LIGHT INDUCED RETINAL DAMAGE 413

centration of dopamine during the light phase of the light cycle than in the dark phase.32,33 Studies with the chick retina have shown rhythmic changes in dopamine levels for several days in chicks kept in constant darkness, and an increase in dopamine levels in chicks that are exposed to light during the dark phase of the light cycle.33 Photoreceptor inner segments have D2 receptors34 and receive paracrine input from the dopamine producing cells. Dopamine has been shown to induce apoptosis in a number of cultured neuronal and nonneuronal cell types including chick embryo sympathetic neurons and rat PC12 cells.35,36 However it’s role in retinal survival or cell loss is unclear.

4. PHOTOTRANSDUCTION PROTEINS

A number of studies have shown that many proteins involved in phototransduction have a light-dependent expression patterns. However many of the observed changes tend to be species specific. The following section will only address the mouse and rat retina.

Opsin mRNA in the mouse retina is highest just before light onset.37 Whole eye rhodopsin protein levels in the rat retina are higher at night than in the day in both the dark reared and cyclic light reared rat.16 However the levels are significantly higher in only cycliclight reared rats.16 Therefore differences in rhodopsin levels are not likely to be responsible for the difference in light susceptibility in dark-reared rats. It has been suggested that there may be a difference in rhodopsin regeneration between night and daytime.38 However, it was found that RPE65 protein levels, and rhodopsin regeneration kinetics, did not change between night and day.38 It has also been suggested that rhodopsin phosphorylation patterns, which are a measure of the activity of the protein, may vary at different times of the day.39 However, it was again found that the phosphorylation patterns do not change between night and day.39

Transducin is a G protein, activated by rhodopsin, which propagates the visual signal.40 Alpha-transducin mRNA levels in rat retina are highest immediately after lights on, and alpha-transducin protein is highest in the inner segments during the day, until it is transported to the outer segments at night.41 The presence of transducin in the outer segments may increase the sensitivity of the photoreceptor rod cells to light exposure at night.

Recoverin is involved in the recycling of activated rhodopsin. An increase in the length of time that rhodopsin is in the activated state has been suggested to increase LIRD in the ‘equivalent to light’ hypothesis.42 Recoverin delays the termination of phototransduction by inhibiting rhodopsin kinase activity in photoreceptor cells.40 Recoverin mRNA and protein levels in the rat retina are high during the light period, and then decrease sharply after the onset of darkness. Levels again increase throughout the dark period, with a sharp increase in protein levels late at night, approximately 4 hours before light onset.43 These results suggest that low recoverin levels at night may increase sensitivity to light exposure. However the sharp increase in levels before light onset makes this uncertain.

5. METABOLISM

Significant differences in the metabolic activity exist in the retina at different times of the day. Increased metabolism appears to take place at night in several species including the rabbit44 and goldfish retina.45 Several studies have shown an in vitro decrease in the extra-

cellular pH (pHo) of the retina at night. The change in pHo appears to occur at dusk, when a rapid drop is observed. After dusk pHo remains stable, until lights on, when it again increases.46 The extracellular pH of the in vitro rabbit retina varies within the retinal layers, and is the lowest at the outer limiting membrane, which is located near the inner segments of photoreceptor cells. Because the pHo is lowest at the outer limiting membrane, photoreceptor cells are likely the primary source of acid production in the retina.46

It was also observed that removing glucose from the solution surrounding the retina, and thereby inhibiting energy metabolism, lead to a decrease in acid production and a reduction in the difference of pH between the retina and the surrounding solution.43 Inhibition of ATP utilization, through the inhibition of the Na+/K+ ATPase also reduced acid production. Therefore the observed change in pH levels was likely due to an increase in metabolism, and an increase in proton production at night. The observed change in pHo between night and day is not due to an increase in glycolysis over oxidative phosporylation, as both processes occur at the same proportion during the day and night.43 There is evidence that cellular acidification can initiate apoptosis. It has been shown that cellular acidification increases susceptibility of cells to heat damage.47 It has also been shown that neutrophils in tissue culture that undergo spontaneous apoptosis have an increase in acid production before the onset of cell death.19 These studies suggest that cellular pH levels can affect cell response to subsequent stress stimuli, and cellular acidification can lead to an induction of apoptosis. Therefore the increased acidification of the retina at night may lead to an increased susceptibility to a stress signal, such as intense light exposure.

6. CONCLUSION

The retinal environment changes in response to differing physiological requirements during the night and day. These changes may lead to subsequent differences in response to light exposure. Previous research has established that the retina is more susceptible to light damage during the dark phase of the light cycle. Although many circadian changes in hormone, protein, and gene expression levels have been documented, the mechanism underlying the circadian difference in susceptibility is not understood. Current studies examining the molecular environment of the retina at different times of the day using array screening may help elucidate some of the processes that result in the difference in retinal susceptibility. Preliminary results from our lab suggest that there are a number of genes that are differentially expressed at different times of the day that have not been characterized as such previously (unpublished results). An understanding of the conditions under which the retina is susceptible or resistant to retinal damage may help us understand the mechanism underlying cell death in retinal eye diseases.

7. ACKNOWDELGMENTS

This work was supported by NSERC (PW, RG); RP Foundation Fighting Blindness (PW); Alberta Ingenuity (RG); NIH grant EY-01959 (DTO); and M Petticrew Springfield, OH (DTO). We thank the Foundation Fighting Blindness for the travel award provided to RG to attend this meeting.

58. CIRCADIAN DEPENDENT SUSCEPTIBILITY TO LIGHT INDUCED RETINAL DAMAGE 415

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