2006 Abstracts Text

Near infrared (NIR) light-emitting diode (LED) arrays have been shown to stimulate mitochondrial respiration and improve functional recovery of the retina and optic nerve after acute toxic injury. The present study was undertaken to test the hypothesis that a brief course of 670 nm LED treatment would improve mitochondrial function, attenuate oxidative stress and improve photoreceptor survival in a rodent model of retinitis pigmentosa, the P23H rats. Methods: Normal SD and P23H rat pups were exposed to 4 joules cm 2 of 670 nm light from a light emitting diode (LED) array or received sham treatment once per day for 5 days from postnatal day (pnd) 16 to 20. At pnd 21, retinae were collected and mitochondrial metabolism was assessed by measurement of cytochrome oxidase activity. The other eye of each animal was immersion fixed in 4% paraformaldehyde, washed, cryoprotected in 15% sucrose and sectioned at 14µm. TUNEL labeling was performed to detect apoptotic cells. Results: In both P23H and SD rats, 670 nm LED treatment increased cytochrome oxidase activity when compared to untreated littermate controls. Treatment with 670 nm light attenuated photoreceptor death by nearly 50% in the P23H rat compared with untreated littermate controls. In SD rats, 670 nm light treatment produced a small, but statistically significant increase in photoreceptor cell death compared with littermate controls. Conclusions: Excess photoreceptor degeneration in the P23H retinal begins just after eye opening, peaks in early postnatal life then slows, but persists into adulthood. Previous studies in our laboratory have demonstrated the neuroprotective and retinoprotective actions of 670 nm light treatment in vitro and in vivo. The present study extends these findings to include protection against photoreceptor degeneration in P23H rats. The results of these studies further support the use of 670 nm light therapy in the treatment of retinal injury and degenerative retinal disease

ROLE OF EPH RECEPTORS AND EPHRINS IN VASCULAR PATTERNING IN DEVELOPING PRIMATE RETINA

The Eph family of receptor tyrosine kinases and their ligands, the ephrins, play an important role in the mediation of neuronal axon guidance and vascular patterning in the developing primate retina. Given the highly specialized pattern of ganglion cell axons and retinal vessels at the incipient fovea, we hypothesize that Ephs and ephrins play a role in establishing the radiating pattern of axons around the incipient fovea and the formation of the foveal avascular zone. Primers were designed for Eph-A5, -A6, -A7, -B1 and ephrins B2 & A5. DIG-labeled riboprobes were prepared from PCR products (Roche DIG RNA labelling kit SP6/T7) using RNA extracted from 17 week gestation human retina. Expression was tested in foetal retinas over a range of ages by in situ hybridisation. Developing macaque and human retinas were embedded in paraffin and sectioned at 9µm. Ephrin-A5 and -B2 were expressed throughout the cell layers, with little or no plexiform layer or nerve fibre layer (NFL) labelling present. EphA5 was expressed predominantly in the ganglion cell layer (GCL). Both Eph-A6 and -B1 were expressed predominantly in the inner retina, in a gradient that was high-to-low, central-to-peripheral. In central retina expression of Eph-A6 and -B1 mRNAs was also higher in the innermost part of the GCL and NFL, and lower in the outer part of the GCL. Specific patterns of localization within the inner retina suggest that Ephs and ephrins may regulate patterning of axons and/or blood vessels in the inner retina, including definition of the foveal avascular area.

EVIDENCE OF WIDESPREAD PHOTORECPTOR PATHOLOGY IN AGE-RELATED MACULAR DEGENERATION (AMD)

The histopathology of AMD includes drusen, laminar deposits, RPE changes, neovascularization, gliosis and photoreceptor degeneration. The histology of regions of the retina unaffected by these features, however, is apparently normal. We examined AMD retinas exhibiting a range of histopathological signs, for indicators of retinal stress in the macula and adjacent regions, using antibodies to GFAP, to detect abnormal expression by Müller cells; rhodopsin, cone arrestin and L/M opsin, to detect changes in expression patterns in photoreceptors that indicate stress or early stages of degeneration; as well as, glutamine synthetase, and retinoid cycle proteins RPE65 and CRALBP. Eyes were collected with informed consent through the Lions Sydney Eye Bank, fixed in 4% paraformaldehyde, rinsed and the fundus photographed. The retina and RPE were dissected out whole, flattened, incubated in 14% gelatin in sucrose-saturated phosphate buffered saline at 37°C for 1-3 hours. The preparation was allowed to set and the macular region cut out as a gelatin block measuring about 8 x 10 mm, frozen and sectioned at 10µm. One in 60 sections was stained using haematoxylin and eosin. Using immunofluorescence, we identified broad areas of retinal stress indicated by GFAP expression in Müller cells; translocation of rhodopsin expression to the somas of rods was present in and beyond the macula. Cones adjacent to lesions were rounded and lacked outer segments; distant from the lesions, many cones prolapsed through the external limiting membrane (ELM), so that their somas lay within the subretinal space, possibly indicating breakdown of the ELM.

Anita Hendrickson, Keely Bumsted-O’Brien , Riccardo Natoli, and Jan Provis

Aims: There have been few studies of human rod development using specific molecular markers, so developmental sequences for rod fate determination, rod-specific protein express- ion and outer segment (OS) formation remain unclear. This study combines a morphological analysis with the temporal and spatial expression of rod-specific mRNA and proteins. Methods: Retinas from fetal week (Fwk) 9-40 and 1d to15mo were fixed in paraformaldehyde, and either frozen sectioned or labeled as wholemounts. Rod proteins NRL, NR2e3, Recoverin (Rec), Interphotoreceptor binding protein (IRBP), rhodopsin (Rho), peripherin (Per), arrestin (Arr) and OS sodium channel (NaCh) were tracked using immunolabeling and insitu hybridization. Results: Morphologically, rods were detected between and under the cones on the foveal edge at Fwk10.5-11; rods formed a 2-4 cell deep layer near the optic disc at Fwk12-13; and were present across peripheral retina by Fwk20-22. Rods could be identified in the outer retina weeks before the outer plexiform layer appeared. NRL, NR2e3, Rec and IRBP were expressed in rods at Fwk10.5-11 on the foveal edge, and in a few cells near the retinal edge by Fwk16. The number of labeled rods increased 4-5x between Fwk15-30, indicating rods are added over many weeks. Rho appeared in and around the fovea at Fwk15 (in situ) and Fwk15.5 (immuno). WM showed that Rho and red/green opsin were expressed simultaneously across the retina, although cones outnumbered rods during the earliest expression. Both opsins reached the retinal edge at Fwk36-38. Per, Arr and NaCh were expressed shortly after a tiny Rho+ OS was present. Rod OS elongated for at least 4 months postpartum. Conclusions: Human rods express rod fate markers NRL and NR2e3 at the same time they can be morphologically detected. Rho and rod OS structural and phototransduction proteins are expressed 4-6wk later. Although rods across the retina express all markers by birth, OS undergo significant growth into the first year.

EVIDENCE OF WIDESPREAD PHOTORECEPTOR PATHOLOGY IN AGEING AND AGE-RELATED MACULAR DEGENERATION (AMD)

Elizabeth J Shelley, Michele C Madigan, Jan M Provis

AIM: To investigate the pathology status of photoreceptors in the central retina of normal aged human retinas, and in AMD retinas. METHODS: Eyes (>70 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 out whole, flattened, and embedded in gelatin. A gelatin block (~8 x 10 mm) including the macula and adjacent region was excised, frozen and sectioned at 10µm. One in 60 sections was stained using haematoxylin and eosin for histopathology and two adjacent series were double immunolabeled, using standard methods, with antibodies to rhodopsin, long-medium wavelength sensitive (L/M) opsin and glial fibrillary acidic protein (GFAP). Selected sections were double immunolabled with antibodies against synaptic vesicle protein 2, the synaptic ribbon marker CtBP2, vesicular glutamate transporter 1 (vGluT1), cone arrestin and Müller cell markers. RESULTS: Two out of 8 eyes analysed had a history of AMD. By histopathology, 3 had changes consistent with AMD; three eyes were histologically normal; one had macular hole and one had unspecified pan-retinal photoreceptor degeneration. All retinas (8/8) showed evidence of widespread photoreceptor degeneration, particularly in the cone population. This included cone prolapse into the subretinal spcae; opsin expression in the cell membrane, axon and pedicle; axonal swelling and beading; loss of synaptic markers; low levels of arrestin; 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. In histologically normal retinas ,the foveal region seemed somewhat protected from these changes. CONCLUSION: We identified evidence of pan-retinal degeneration of photoreceptors in a small sample of aged and AMD retinas. The results suggest that in normal aging the macula may be protected from this degeneration.

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

AIM: Bright light can precondition the retina, upregulating mechanisms which increase resistance to subsequent light challenge. The present study tested whether preconditioning with another photoreceptor-lethal stimulus, hyperoxia, provides similar protection against light-induced photoreceptor death. We also tested whether conditioning with bright light is protective against hyperoxia-induced damage. METHODS: Light-sensitive BALB/c and hyperoxia-sensitive C57BL/6 strain were raised in dim (5 lux) cyclic light. At age P (postnatal day) 79-165, one group of Balb/c was placed in 75% O2 for 2 w and the other group was maintained in normoxia. Both groups were then exposed bright (1000 lux) cyclic light for 1 w. For the C57BL/6J mice, one group was exposed to bright light (1000 lux) for 1 w while the other group was maintained in dim light, before both groups were placed in 75% O2 for 2 w. Eyes were cryosectioned for TUNEL labelling and immunohistochemistry analysis. RESULTS: Light preconditioning decreased the rate of photoreceptor death induced in the C57BL/6 retina by hyperoxia, by ~50%. By contrast, hyperoxic preconditioning did not decrease 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. CONCLUSION: The mechanism by which light conditioning protects C57BL/6 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 as light stress. It is possible that the retinal mechanism for protection against bright light is more active and evolved than the protective mechanisms evoked by hyperoxia.