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A direct molecular link between amyloid pathology in the brain and ocular lens has been described, with beta-amyloid pathology and unusual co-localizing equatorial supranuclear cataracts detected in lenses from patients with Alzheimer's disease but not those without disease. Amyloid accumulates in the lens as cytoplasmic aggregates within equatorial supranuclear lens fiber cells. Beta amyloid potentiates lens protein aggregation and light scattering, linking the amyloid formation regional cataractogenesis. These findings led to development of novel quantitative laser optical technology for non-invasive early detection and diagnostic assessment of amyloidisorders. Although retinal and corneal involvement in human and veterinary transmissible spongiform encephalopathy (TSE) infection are well documented, scant attention has been paid to the lens or to the possibility of using ocular or periocular tissues as a peripherally accessible diagnostic window to detect and monitor the disease process, perhaps from the earliest stages of the disease. This project is a pilot proof of principle field test of our purpose-built instruments to assess the viability of these novel diagnostic techniques for the detection and assessment of TSE.
NON-TECHNICAL SUMMARY: There are no non-invasive live animal tests for the transmissible spongiform encephalopathies of livestock. This project examines the feasibility of a non-invasive technique for detecting early accumulation of the disease associated prion protein in the ocular lens of sheep with scrapie.
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APPROACH: The proposed study will be conducted in confirmed TSE+ and control (TSE-) sheep. Sheep are diagnosed with scrapie at approximately 14 months of age through biopsy of the lymphoid tissue of the nictitating membrane and detection of PrP-d by immunohistochemistry. Sheep are naturally infected through contact with infected ewes, typically their dams, and held in quarantine at the USDA, ARS, Animal Disease Research Unit Animal Research Facility in Pullman, WA. Negative control sheep are age matched sheep with the resistant PRNP genotypes AARR and AAQR. Infected sheep typical survive through age 34-38 months (breeding ewes) and up to 48 months (neutered males). In this study, sheep will be sampled at the preclinical and very early clinical stages of disease, with scrapie confirmed at necropsy following progression to clinical disease (weight loss, ataxia, pruritis). Sheep are restrained in a chute in a holding stall at the Washington State Veterinary Teaching Hospital Large Animal facility. We will utilize complementary anterior segment laser/optical technologies of varying sensitivity and specificity (e.g., infra-red slit-lamp ophthalmoscopy, static light scattering, quasi-elastic light scattering, and fluorescence ligand scanning) for quantitative, non-invasive in vivo assessment of TSE-related PrP-Sc pathology in ocular (lens, retina) and peri-ocular tissues in TSE+ (non-symptomatic and early symptomatic) versus TSE- (control) sheep. Ex vivo imaging of eyes from sheep with late clinical disease will be examined after collection of the tissue at necropsy. The ophthalmic examinations will be conducted by research personnel using purpose-built instruments. In vivo quantitative ophthalmic analyses will be correlated with infection status, clinical staging, and ex vivo assays of PrP-Sc tissue burden/localization and organ pathology.
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PROGRESS: 2005/09 TO 2006/09<BR>
Previous findings by members of this research group discovered a direct molecular link between Alzheimer's disease (AD) beta-amyloid pathology in the brain and ocular lens. Preliminary studies had been performed on Tg2576 transgenic mice with an amyloid precursor protein mutation that results in AD pathology. The transgene was driven by the promoter for the prion protein PrP, a protein that is prodigiously expressed in the lens. The discovery of AD pathology in the lens due to this transgene expression driven by the PrP promoter suggested that prion infection might also manifest lenticular and other ocular and periocular pathology. Striking equatorial supranuclear cataracts were observed in Tg2576 mice infected with the 87V strain of scrapie. Based on these observations, the goal of this project was to assess the potential of utilizing amyloid pathology in specific subregions of the lens as an optically accessible TSE biomarker in the natural host. We investigated the possibility of related lenticular pathology or disease associated prion protein accumulations in sheep with scrapie. We collected 25-30 pairs of eyes from scrapie infected sheep and 25-30 pairs of age-matched controls. Specimens were analysed by stereophotomicroscopy, western blotting, and immunohistochemistry using a panel of PrP antibodies. Further, amyloid histofluorescence assays were performed using the amyloid-specific fluorescent stains MX-04 and X-34. The specificity of these stains in detection of both amyloid and pre-amyloid in neural and lymphoreticular sections from infected sheep was demonstrated. However, in contrast to the findings in the mouse model, the immunohistochemistry and histofluorescence results to date suggest that there are no striking infection-related differences between lens specimens obtained from scrapie positive and scrapie negative sheep.
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IMPACT: 2005/09 TO 2006/09<BR>
Accumulations of protease resistant PrP were not reproducibly observed in eyes from sheep with scrapie. However, these results do not exclude the possibility of using the eye as a disease biomarker in other species with naturally occurring TSEs, particularly disorders such as chronic wasting disease, which is characterized by amyloid accumulation. Further, the feasibility of incorporating disease-specific fluorescent markers into rapid and sensitive diagnostic tests for ruminant TSE's was demonstrated.