REVERSAL OF ROD AND CONE STRUCTURAL AND FUNCTIONAL LOSS IN THE P23H-3 RETINA BY MANAGEMENT OF AMBIENT LIGHT
Vicki Chrysostomou1, Sally Stowe1, Krisztina Valter1, Nigel Barnett2 and Jonathan Stone1
1Research School of Biological Sciences, The Australian National University; 2School of Biomedical Sciences, University of Queensland
Purpose: This study tests whether cones in the rhodopsin-mutant transgenic P23H-3 retina are damaged by ambient light, and whether restricting ambient light exposure allows repair of the function and structure of damaged cones. Methods: P23H-3 rats were raised to adulthood in scotopic cyclic (12h 5 lux, 12h dark) ambient light. Some were transferred to photopic conditions (12h 300 lux, 12h dark) for 1 week, and then returned to scotopic conditions for up to 5 weeks. Photoreceptor function was assessed by amplitude of the dark-adapted flash-evoked electroretinogram (ERG), using a two-flash paradigm to isolate the cone response. Outer segment structure was demonstrated by immunohistochemistry for rod and cone opsins, and by electron microscopy. Results: Exposure of dim-reared rats to photopic ambient light for 1 week reduced the rod a-wave, rod b-wave and cone b-wave by 45-55%, and caused shortening and disorganisation of rod and cone outer segments. Restoration of scotopic conditions for 2-5 weeks allowed partial recovery of the rod a-wave (63%), rod b-wave (78%) and cone b-wave (87%), and regrowth and re-organisation of rod and cone outer segments. Conclusions: Although the P23H-3 transgene is a mutation in a gene expressed specifically in rods (rhodopsin), cones are rapidly damaged by the same modest levels of ambient light that damage rods. Restricting light to scotopic levels allows the partial restoration of the structure and light responsiveness of cones as well as rods. This study emphasises the dependence of cones on rod function, and the capacity or cones to repair their structure and resume function.
A ROLE FOR SAFFRON IN THE PROTECTION OF PHOTORECEPTORS FROM LIGHT DAMAGE?
Juliet A. Fisher1,3, Tina Salmon1,3, Silvia Bisti2,3 and Krisztina Valter1
1Research School of Biological Sciences, The Australian National University, Australia; 2Universita del L’Aquila, L’Aquila, Italy; 3ARC Centre of Excellence in Vision Science
AIM: Saffron has long been known to have medicinal and culinary properties. In this study, the protective effect of saffron on photoreceptors following light damage was investigated. METHODS: Young adult Sprague Dawley rats were fed saffron daily at a rate of 1 mg/kg for 3 weeks. The rats were then exposed to 1000 lux continuous light for varying lengths of time (0, 1, 3, 6, 12, 24 or 36 hours). Following light exposure, eyes were collected, fixed in 4% paraformaldehyde, cryoembedded and sectioned. TUNEL technique was used to detect cells undergoing apoptosis and measurements of the retinal cell layers were taken to determine total cell loss. Immunohistochemistry using a range of antibodies to study oxidative stress and tissue damage was performed to determine the integrity of the retina. RESULTS: A significant difference in the number of TUNEL positive cells was observed between saffron treated and untreated animals after 3 to 24 hours light exposure. The number of apoptotic cells observed was much reduced in sections from the rats which had been fed saffron. Staining for markers of oxidative damage such as acrolein was observed to be much greater in untreated animals than treated after 6 hours light exposure or greater. GFAP staining was also much greater in untreated sections than treated with the difference detectable following 3 hours of light exposure. CONCLUSION: Oral treatment of rats with saffron for 3 weeks prior to high intensity light exposure has been shown to decrease cell loss, oxidative stress and tissue damage indicating a protective effect on the retina.
THE IDENTIFICATION OF MOLECULAR MARKERS INVOLVED IN PRIMATE RETINAL VASCULAR PATTERNING
Peter Kozulin1, Riccardo C. Natoli1, Michele C. Madigan2, Keely M. Bumsted O’Brien3, and Jan M. Provis1
1Research School of Biological Sciences, Australian National University, ARC Centre of Excellence in Vision Science; 2Save Sight Institute, University of Sydney; 3University of Auckland, Auckland, New Zealand
Purpose: Eph receptors and their ligands, the ephrins, play a role in angiogenesis as well as providing guidance cues to outgrowing retinotectal axons. In this study we investigated the expression and distribution of Ephs and ephrins that might be associated with the definition of the foveal avascular zone (FAZ) in developing primate retina. Methods: The expression and distribution of Eph-A5, -A6, -A7, -B1, and ephrins B2 & A5 was investigated by RT-PCR. Digoxigenin-labeled riboprobes were prepared from RT-PCR products using RNA extracted from 17 weeks gestation (wg) human retina. These riboprobes were used to localize expression of mRNA in foetal primate retinas by in situ hybridisation. In addition, data indicating differential levels of expression of these genes in central vs. peripheral retina from quantitative PCR analysis was compared with data obtained from Affymetrix GeneChip® microarray analysis. Results: RT-PCR revealed the expression of the Ephs/ephrins in 17wg retina. Under optimal in situ hybridisation conditions, no sense labelling was observed in developing primate retinas. Eph-A6 was expressed in a gradient in the ganglion cell layer (GCL) and nerve fibre layer (NFL) with peak expression in the innermost part of the GCL/NFL at the incipient fovea. The level of expression tapered off in the peripheral retina and centrally, in the outer GCL. The high-to-low, central-to-peripheral expression pattern corresponds with the microarray finding of a 3.2 fold change up-regulation of Eph-A6 in the foveal region compared with the surrounding retina (p<0.01). Conversely, microarray analysis showed a down-regulation of expression for three Eph-A6 ligands (ephrin-A1, -A2 & -A4) in the foveal region. This finding was verified using quantitative PCR. Conclusions: The distribution of Ephs and ephrins is consistent with a role in retinal vascular patterning and definition of the fovea and FAZ in the primate retina.
UNDERSTANDING THE LATER STAGES OF RETINAL DEGENERATIONS: TOXICITY OF HYPEROXIA IN THE MOUSE RETINA
Riccardo Natoli, Jan Provis and Jonathan Stone
Research School of Biological Sciences, Australian National University; Centre of Excellence in Vision Science
The precise mechanism underlying hyperoxia-triggered cell death is not yet fully understood, although it is known that an increase in oxygen causes an increase in free radicals and superoxides. These free radicals and their associated cascades lead to the attack of fatty acid residues, DNA and other biological structures. The photoreceptors of the retina are particularly vulnerable to oxygen toxicity due to the lack of autoregulation in the choriocapillaris, their principal source of oxygen. In the late stages of retinal degeneration, the absence of autoregulation renders surviving photoreceptors susceptible to hyperoxia, because of the reduced photoreceptor population, the resultant reduction in oxygen usage and hence, increased P02 in outer retina. To study the effects of sustained hyperoxia and thus mimic the later stages of retinal degenerations, we exposed adult C57Bl mice (3 – 6 months) to 75% oxygen for a period of 0, 3, 7 and 14 days. Following oxygen treatment ERG recordings were performed to evaluate retinal function. Animals were sacrificed and eyes removed, and either fixed embedded and sectioned for immunohistochemistry and TUNEL, or used for RNA extraction and gene expression studies (Affymetrix GeneChip® and Real-time PCR). Many of the modulated genes are involved in free radical scavenging including superoxide dismutase and glutathione which are up-regulated after 7 days as well as genes involved in stabilisation of the retina (which are up-regulated ahead of photoreceptor cell death (3 days). Since hyperoxia is a feature of probably all photoreceptor degenerations, understanding the cells’ responses to hyperoxia may be important in devising ways of stabilizing the degenerating retina.
IDENTIFYING FACTORS THAT DEFINE THE FOVEAL AVASCULAR ZONE OF PRIMATE RETINA
Jan M Provis1, Keely Bumsted O’Brien2, Riccardo Natoli1, Peter Kozulin1 and Michele C Madigan3
1Research School of Biological Sciences, The Australian National University; 2Department of Optometry and Vision Sciences, University of Auckland; 3Department of Clinical Ophthalmology, University of Sydney.
The fovea centralis, or ‘fovea’ is a depression in the inner surface of the retina associated with high resolution vision in a variety of species of fish, reptiles and birds, and in simian primates. There is considerable evidence now indicating that in humans, formation of a normal fovea is dependent on the prior definition of an avascular zone in central retina. This zone is centered on the rod free region of the photoreceptor mosaic, at the intersection of the horizontal and vertical retinal meridia. To identify genes involved in specification of the foveal avascular zone of human retina, we extracted RNA from 5 mm biopsies of the developing foveal region (‘fovea’), corresponding nasal retina (‘nasal’), and the remaining ‘surround’ of two 19 week gestation foetuses. These samples (6 in total) were hybridized to Affymetrix GeneChips® (HG-U133 Plus 2). Data from each of the biopsy positions was pooled for further analysis. Results confirmed lower levels of expression of FGF2 in foveal compared with other retinal locations, and higher levels of expression of EphA6, by ISH. We also identified several novel genes showing significant regulation in the foveal samples which have been confirmed by quantitative PCR, including members of the forkhead family of transcription factors, leucocyte differention markers involved with activation of immune processes, and other genes associated with axon guidance and cell differentiation. We are presently developing riboprobes to investigate the expression patterns of these genes of interest, and their potential significance will be discussed.
PARTIAL RECOVERY OF RETINAL STRUCTURE AND FUNCTION FOLLOWING EXPOSURE TO DAMAGING LIGHT
Matt Rutar, Juliet Fisher, Krisztina Valter
Research School of Biological Sciences & ARC Centre of Excellence in Vision, The Australian National University, ACT, 2601
AIM: To investigate retinal cell damage and recovery following exposure to bright light in the albino rat retina. METHODS: Young adult albino Sprague Dawley rats were exposed to bright (1000 lux) continuous light for 24 hours, then returned to dim lit (5 lux) conditions. At set time points after light exposure (0, 1, 3, 7, 14, and 21 Days), eyes were examined. Full field ERGs were recorded, and tissue was collected and fixed in 4% paraformaldehyde then cryoembedded and sectioned at 16mm. Selected sections were immunolabeled with antibodies to follow structural changes in the retina. Additional sections were used to investigate cell death via TUNEL labeling, and retinal cell layer measurements. RESULTS: Initial time points showed reduced retinal function, upregulation of stress related factors, and a gradual decrease in photoreceptor outer segment length. There was an immediate increase in cell death in the ONL, which was followed by a significant thinning of the ONL after 3 days. We identified a region of the retina in the superior mid-periphery, which displayed more damage than other regions of the retina (hotspot). In later recovery time points, there was evidence of structural remodeling of the inner retina, and a gradual recovery in retinal function and structure, except in the hotspot. CONCLUSION: Following damage induced by bright continuous light, our data demonstrates a partial recovery in both retinal function and structure. Recovery was not observed in the area of the hotspot.
REVERSAL OF CONE FUNCTIONAL LOSS IN THE P23H-3 RETINA BY MANAGEMENT OF AMBIENT LIGHT
Vicki Chrysostomou, Krisztina Valter, Jonathan Stone
CNS Stability and Degeneration Group, Research School of Biological Sciences,ARC Centre of Excellence in Vision Science,The Australian National University, Canberra, ACT, Australia
Purpose: Our group has recently reported a recovery of function in the retina of the rhodopsin-mutant P23H-3 rat in response to the management of ambient light. Here, we extend the study to specifically examine the effect of light restriction on cone photoreceptors in the degenerative retina. Methods: Heterozygous transgenic P23H-3 rats, a model of autosomal dominant retinitis pigmentosa, were raised to young adulthood in scotopic light (12 h 5 lx ‘daylight’, 12 h dark). The brightness of the day part of the cycle was increased to 300 lx (low end of the daylight range) for 1 week and then reduced to 5 lx for 5 weeks. Using a double flash protocol, the electroretinogram (ERG) was used to assess cone function after 1 week of 300 lx light exposure and after 2 and 5 weeks recovery in dim light. Results: Exposure of dim-reared rats to 300 lx cyclic light for 1 week reduced the cone-associated ERG b-wave. Returning animals to 5 lx conditions for 2-5 weeks resulted in substantial recovery of the cone b-wave amplitude from 57% to 77% of control values. Conclusions: The degenerative P23H-3 retina shows significant recovery of cone function in response to a reduction in ambient light. These findings suggest that, in humans suffering visual loss due to comparable genetic mutations, light management should improve retinal function. Specifically, improvement should be seen in daytime and high-acuity vision that is mediated by the cone system.