Retinal Degeneration--genetic/environmental animal model

Deafness &Other Communication Disorders

Investigators

Linked publications & trials

Abstract

Investigation of disease mechanisms in retinoschisis Immunohistochemistry and m-RNA localization This disease is caused by mutations in a protein called retinoschisin (RS-1) of unknown function. This study re-evaluated the role of retinoschisin in retinal development by studying prenatal and postnatal expression and localization in the normal mouse retina. RS-1 mRNA and protein expression during the retinal development were studied with in situ hybridization and immunohistochemistry. RS-1 mRNA expression in each retinal layer was confirmed by Laser Capture Microdissection (LCM) RT-PCR. The wave of RS-1 expression moves from posterior through the retina as neurons are born and layer development progresses Nearly all adult retinal neurons, with possible exception of horizontal cells, express RS-1 on basis of in situ hybridization and LCM of the outer nuclear layer (ONL), inner nuclear layer (INL), and ganglion cell layers (GCL) at adult age. Constructing a gene targeted mouse model An Rs1 targeted mouse is a useful animal model in studies on the normal biological role of the gene as well as in our novel therapeutic approaches to cure RS condition in humans. Therefore, we produced an Rs1 gene knock out mouse in collaboration with Ingenious Targeting Laboratories (ITL. Hetrozygote females are being bred to C57/black males in order to obtain the males carrying the targeted X chromosome complement.These males are expected to present a phenotype akin to that is seen in human RS patients. Photoreceptor rescue using neurotrophic factors Exogenous growth and neurotrophic factors have shown promise as protective agents against retinal photoreceptor degenerations caused by environmental stress (light damage, chemical toxicity), inherited retinal degenerations (retinitis pigmentosa) or a combination of both (age-related macular degeneration). A clone encoding a novel adhesive, growth and survival factor has been previously isolated from a cDNA library of human lens epithelial cells (LEC) and was named lens epithelium-derived growth factor (LEDGF). We cloned the rat LEDGF using RT-PCR, and the whole coding region was sequenced utilizing the Beckman CEQ? 8000 Genetic Analysis System. After comparison to human and mouse LEDGF, we fount that there is over 90% nucleotide identity among the coding regions. We produced recombinant adeno-associated viral (AAV)vector containing the LEDGF gene. This vector was injected into the eye of rats which were then exposed to light strong enough (350 lux) to damage the retinas in 24 hours. The electrophysiological response of the eyes injected AAV-LEDGF were better than eyes in the same animals injected with saline. More studies need to be done but this suggests that this growth factor can protect photoreceptors from degeneration. An encapsulated cell based delivery system for ciliary neurotrophic factor (CNTF): effects on normal rabbit ERG and retinal morphology Ciliary neurotrophic factor (CNTF) delivered intravitreally by encapsulated engineered human RPE cells (ECT) reduces photoreceptor cell death in the rcd1 dog model of retinitis pigmentosa with few, if any, side effects observed clinically or histologically. However, nothing is known about the effects of CNTF delivered by this method on retinal function. We investigated ERG and histological changes in normal rabbits with intravitreal implants of ECT devices releasing CNTF at doses found to rescue photoreceptors in the rcd1 dog. We found that CNTF did not affect the rod a- or b-wave at 25 days after implantation. These results indicate that exogenous CNTF delivered intraocularly by an ECT device at doses found to provide photoreceptor protection in a dog model of retinal degeneration does not cause acute reduction in either rod or cone function, as measured by the ERG, of normal rabbit retina. Higher dose CNTF (22 ng/day) produces small, but significant, decreases in the light-adapted b-wave that may reflect changes in cone pathway function. Changes in outer nuclear layer morphology produced by higher doses of CNTF than those previously shown to be required for maximal photoreceptor protection, are not necessarily associated with alterations of rod function over the short term. Phase I clinical trials of ECT-CNTF in retinitis pigmentosa patients This year our lab has spearheaded the development and FDA approval process, along with Neurotech, USA, of a pilot study to assess the ophthalmic and systemic safety, and to some extent efficacy, of a novel intra-ocular NT-501 implant in patients with RP and poor visual acuity in one eye. The main purpose of the study is to assess the safety of the NT-501 implant. Secondary outcomes will include the anterior chamber cell scale and vitreous haze grading to measure inflammation, which may be caused by the implant. Other secondary outcome measures related to potential product performance are visual acuity, visual fields, electroretinograms (ERG), and optical coherence tomography (OCT3) to determine retinal thickness. The first patient was to have a device implanted in September, 2003. The cone electroretinogram (ERG) in mice without functional rods We previously published an overview of the electrophysiological responses in two gene knockout mice without functional rods, the rhodopsin and NRL knockout mice. This year we studied in more detail the effects of aging and light adaptation on these all cone retinas. The rhodopsin knokout mouse has a normal number of cones and a normal cone ERG up to at least 6 weeks of age, which decline steadily to baseline by 14 weeks. The NRL knockout, on the other hand, has a cone ERG which is 2-3 times normal and all the photoreceptors in the retina have cone like morphology. However, by 8 months, NRL knockout mice have a cone ERG approximately ? that in wild type. This suggests that cones require the presence of rods to survive. The effect of dietary fatty acids docosahexeanoic acid (DHA) and docosapentaenoic acid (DPA) on the rat electroretinogram DHA is the primary long chain polyunsaturated fatty acid in photoreceptor our segment membranes. Previous evidence supports the idea that it is important for the function of neuronal membranes and, in particular the visual transduction proteins. To explore this rats were raised for 12 weeks by Dr. Norma Salem?s laboratory in NIAAA on diets geared to alter the fatty acid content of brain and retina and various behavior and biochemical tests were performed. We performed dark adapted and light adapted ERGs or 4 dietary groups of rats. The most surprising finding was diets which resulted depletion of DHA and omega 3 fatty acid and its replacement with DPA an omega 6 fatty acid in the brain had no effect on the rod or cone a- and b-waves, indicating visual transduction at the level of the retina was normal.