CONE PHOTORECEPTOR DEGENERATION IS WIDESPREAD IN NORMAL AGED AND AMD RETINAS

Elizabeth J. Shelley1,2, Jan M. Provis1,2 and Michele C. Madigan3

1RSBS, The Australian National University; 2ARC Centre of Excellence in Vision Science; 3Save Sight Institute, University of Sydney

Purpose: To investigate the pathology status of photoreceptors in the human central retina of normal, aged and AMD retinas. Methods: Eyes (9 of >75 years; 1 of 34 years) were collected with informed consent through the Lions Sydney Eye Bank, fixed in 2% paraformaldehyde, rinsed and the fundus photographed. The retina and RPE were dissected whole, flattened, and embedded in gelatin. A gelatin block (~8 x 10 mm) including the macula was frozen and sectioned at 10 or 14 µm. One in every 60 or 43 sections was stained using haematoxylin and eosin for histopathology and two adjacent series were double immunolabeled with antibodies to rhodopsin and long-medium wavelength sensitive (L/M) opsin. Selected sections were double immunolabeled with antibodies against synaptic vesicle protein 2 and vesicular glutamate transporter 1 (vGluT1). Results: By histopathology, 5 retinas had changes consistent with AMD, 4 eyes were histologically normal and 1 had an unspecified degeneration. All retinas (10/10) showed evidence of widespread photoreceptor degeneration, particularly in the cone population. This included cone prolapse into the subretinal space; opsin expression in the cell membrane, axon and pedicle; axonal swelling and beading; loss of synaptic markers; and loss of outer and inner segments. Rod pathology was evident surrounding overt histopathological lesions and the optic nerve head, and included rhodopsin expression in the soma and spherule. Conclusion: We identified evidence of pan-retinal degeneration of photoreceptors in a small sample of normal, aged and AMD retinas. Cone pathology was more varied and more extensive than rod pathology.

CAN LIGHT MANAGEMENT GIVE HOPE TO RETINAL DEGENERATION SUFFERERS? RESULTS FROM THE P23H-3 TRANSGENIC RAT SAYS SO

Krisztina Valter, Juliet Fisher, Diana K. Kirk, Jonathan Stone

CNS Stability and Degeneration Group, research School of Biological Sciences, ARC Centre of Excellence in Vision Science, ANU, Canberra

Aim: Present study is aimed to establish if light management can modify the progress of the degeneration in the rhodopsin mutant transgenic rat. Methods: P23H-3 transgenic rats were used. All animals were raised in cyclic ambient light (12h light/dark) with the light phase at either 5lux or 40lux conditions. One group of animals was raised to P(ostnatal day)120 at 5lx (scotopic control). Another one was raised to P120 in 40lx light (mesopic control). Two other groups were raised in either of these conditions to P30 and then transferred to the other light level (S/M or M/S group). Full field ERG both with single and double flash paradigms were performed and tissue samples were collected at P30, 80 and 120. TUNEL labelling and retinal layer thickness measurements were used to follow cell loss and immunohistochemistry to observe structural changes in retinal cells with time at all lighting conditions. Results: Mesopic light exposure decreased both a- and b-wave amplitudes of the ERG and increased photoreceptor cell death when compared to scotopic-reared animals. Photoreceptor outer segment length decreased and synaptic vesicle concentration was also reduced following exposure. After light restriction (M/S group), both the a- and b-wave amplitudes increased after the transfer from mesopic to scotopic light conditions. By the end of our observation time (P120), retinal function in this group was not significantly different from the scotopic control group. Photoreceptor outer segment length and synaptic vesicle concentration also increased close to scotopic control levels. Conclusion: Light restriction can slow the progression of degeneration in this strain.

DIFFERENCES BETWEEN MOUSE STRAINS IN THE VUNERABILITY OF PHOTORECEPTORS TO HYPEROXIA: SEARCH FOR THE GENETIC BASIS

Yuan Zhu1,3, Krisztina Valter1,3, Ruth Arkell2 and Jonathan Stone1,3

1CNS Stability and Degeneration Group; 2Molecular Genetics & Evolution Group, Research School of Biological Sciences; 3ARC Centre of Excellence in Vision Science, The Australian National University

Purpose. Hyperoxia is known to induce photoreceptor degeneration in the C57BL/6J mouse strain, but not in the Balb/c strain. The aim of the present study is to explore the genetic regulation of this strain difference in photoreceptor vulnerability. Human orthologues of gene(s) identified in the mouse genome contributing to the strain difference should be important targets for further research. Methods. C57BL/6J and Balb/c mice were crossed to generate the heterozygous F1 strain. F1 mice were then backcrossed to Balb/c to generate an N1 strain. At the age of P (postnatal date) 70-200, C57BL/6J, Balb/c, F1 and N1 mice were exposed to 75% oxygen or room air for 2 weeks. After euthanasia, eyes were fixed and processed for phenotype analysis and 1cm tail of each animal was collected for genetic mapping. TUNEL labelling and analysis of the layered structure of the retina were used to define the phenotype of an animal. Results. After 2w hyperoxia, sections of C57BL/6J retina showed an average of ~25 TUNEL+ cells/mm with significant variation between individuals. They also clearly demonstrated a thinning of the outer nuclear layer (ONL), assessed as the ratio of the thickness of the ONL to inner nuclear layer (INL). Both TUNEL frequency and ONL thinning were more prominent in inferior retina. In Balb/c mice, sections had an average of ~5 TUNEL positive cells/mm and no significant thinning of ONL was observed. F1 mice showed an average of ~20 TUNEL positive cells/mm while N1 mice had ~ 7 TUNEL positive cells/mm, with significant variation in this measure of vulnerability. Conclusions. The phenotypic differences between C57BL/6J and Balb/c were confirmed. The range of photoreceptor vulnerability within the N1 group provides a basis for genetic analysis, to localise the genes regulating this parameter.

PROTECTIVE MECHANISMS IN THE RETINA: COMPARISON OF THE PROTECTION INDUCED BY LIGHT STRESS AND BY HYPEROXIA

Yuan Zhu, Krisztina Valter, Jan Provis and 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 2601, Australia

Purpose: Previous studies have shown that bright light can precondition the retina, upregulating protective mechanisms which increase resistance to subsequent light challenge. The present study was designed to test whether preconditioning with another photoreceptor-lethal stimulus, hyperoxia, can provide similar protection against light-induced death of photoreceptors. We also tested whether condition with bright light is protective against hyperoxia-induced damage. Methods: Protection against light-induced damage was tested in the BALB/c mouse, which is light-sensitive; protection against hyperoxia-induced damage was tested in the C57BL/6J strain, which is hyperoxia-sensitive. Balb/c and C57BL/6J mice were born and raised in dim cyclic light (12h 5 lux, 12h dark). At age P (postnatal day) 79-165, mice of each strain were divided into two groups. One group of Balb/c was placed in 75% O2 for 2w and the other group was maintained in normoxia. Both groups were then exposed bright (1000 lux) cyclic light for 1w. For the C57BL/6J mice, one group was exposed to bright light (1000 lux) for 5-7 days while the other group was maintained in dim light, before both groups placed into 75% O2 for 2w. Eyes from these animals were collected and cryosectioned. TUNEL labelling, gene expression assay and immunohistochemistry analysis were carried out. Results: So far, TUNEL assay showed that light preconditioning decreased the rate of photoreceptor death induced in the C57BL/6J retina by hyperoxia, by ~50%. By contrast, hyperoxic preconditioning did not cause a significant decrease in the rate of photoreceptor death induced in the BALB/c retina by bright light. In C57BL/6J retinas, bright cyclic light exposure did not increase the expression of stress-related molecules including GFAP, FGF-2 and CNTF. Conclusions: This is the first demonstration that light preconditioning can have general effects; i.e. it can protect the retina from a different form of stress, such as hyperoxia. The mechanism by which light-conditioning protects C57BL/6J photoreceptors from hyperoxic damage is not known, but appears not to involve FGF-2 or CNTF. The failure of hyperoxic conditioning to protect BALB/c photoreceptors from light damage suggests that hyperoxic stress does not induce the same protective mechanisms at light stress. It is possible that the retinal mechanism for protection against bright light is active and evolved than the protective mechanisms evoked by hyperoxia.