Preclinical imaging of the rodent eye with multi-functional optical coherence tomography

Major causes of blindness are still not entirely understood which complicates early diagnosis and hinders the development of proper treatment. In order to understand the pathogenesis of vision threatening diseases such as age-related macular degeneration (AMD) or glaucoma, a variety of small animal models have been developed. The gold standard for such preclinical investigations is histology which provides microscopic images of anatomic details and tissue microstructure. However, animals have to be killed in order to perform histology. Hence, there is a great demand for non-invasive imaging methods in order to enable the measurement of anatomical and physiological parameters in vivo and in situ, to reduce the number of animals required, and to enable longitudinal studies in the same animal. It is the aim of this project to develop multi-functional optical coherence tomography (OCT) as a non-invasive alternative to histology in preclinical research in rodent models. OCT is an emerging optical technology which performs high-resolution, cross-sectional and three-dimensional (3D) imaging of tissue morphology in situ and in real time. OCT has become a clinical standard in human ophthalmology and was recently demonstrated as a feasible tool for imaging the rodent retina. Functional OCT methods such as polarization sensitive (PS) OCT and Doppler OCT provide additional information about tissue structure (polarization properties) and function (blood flow) that is inaccessible with standard OCT technology images based solely on the intensity of backscattered light. The additional contrast provided by functional OCT methods extends and improves image contrast and enables quantitative measurements. In this project, a novel multi-functional OCT system will be developed and optimized for both PS- and Doppler OCT imaging in a single system in the mouse and rat eye. Using this system, the following properties and parameters of healthy rodent eyes will be investigated in order to set the base for investigations in disease models: Quantitative mapping of melanin pigmentation based on depolarization of light Measurement of retinal nerve fiber layer birefringence Measurement of scleral fiber orientation and birefringence Mapping retinal vasculature and blood flow measurement For validation, the results will be correlated with state-of-the-art confocal, two photon and electron microscopy of histological sections of the same structures. Finally, relevant rodent models in AMD and glaucoma research will be investigated with the multi-functional OCT system in order to study the pathogeneses and to identify early disease markers. Quantitative functional imaging will provide access to a set of anatomical and physiological parameters that cannot be accessed using any other previously reported techniques. Furthermore, since future biomedical research based on OCT supports the principle of the "Three R`s," to Replace, Reduce and Refine the use of animals for scientific purposes.

In this project, a novel prototype for optical imaging in rodent eyes was developed and employed in several preclinical studies of small animal models for ocular diseases. The prototype provides non-invasive in-vivo micrometer-scale imaging of the rodent retina at rapid acquisition rates and with multi-functional contrast. In addition to morphological imaging based on the reflectivity of different tissues, our prototype simultaneously also reveals tissue polarization properties and 3D angiography of microvasculature. This threefold contrast was exploited to perform quantitative imaging including retinal layer thickness mapping, localized measurements of melanin pigmentation density and perfusion mapping. After specification measurements and imaging experiments in healthy rodents, we employed the prototype for several short-term and longitudinal studies in disease models of ocular pathology. We showed that both structural and functional changes can be monitored and measured in vivo when the intraocular pressure is changed. Important aspects of age-related macular degeneration (AMD) such as the development of abnormal retinal vessels and subtle changes of outer retinal structures were detected and quantitatively assessed in longitudinal studies of two mouse models. The results from the high-resolution imaging studies were correlated with gold standard histological work-up. However, unlike histology, which requires that animals be killed, our optical imaging approach can be performed repeatedly at the exact same eye location as animals age and therefore enables in-vivo and in-situ investigations of lesion development over time. Therefore, the methodology developed in this project not only supports the Three Rs to replace, reduce and refine but also represents a powerful technology for longitudinal studies of rodent models focusing on improved diagnostics and treatment of major causes of blindness affecting millions of humans worldwide.