Citation

Preventing pathogenesis and developing selective therapeutic strategies are the main goals in modern biomedicine. Therefore, it is absolutely necessary to probe cellular and tissue functions in the living organism. Animal models can be used to study dierent aspects of chronic inammatory diseases taking place in the central nervous system (CNS). Imaging the brain stem, spinal cord or cortex needs invasive and often terminal surgery. Thus, longitudinal measurements in one and the same animal at several time points during the whole time course of disease are not possible. The retina is the only part of the CNS that can be imaged non-invasively by optical methods. Changes in the retina may reect pathogenesis from other parts of the CNS as earlier studies have shown e.g. in patients with multiple sclerosis in which thinning of the retinal nerve ber layer is one of the rst symptoms. In the here presented work a new method has been developed to observe the retina from living mice longitudinally. The method is based on the two-photon-absorption, thereby near infrared radiation is used to excite uorescent proteins or dyes. This allows imaging of cellular inltration, migration and function of uorescently labelled leukocytes in vivo. The new approach was tested in the animal model for experimental autoimmune uveoretinitis (EAU) where T cells overcome the blood-retinal-barrier to inict retinal inammation followed by recruitment of peripheral leukocytes that induce tissue damage. The process of CD4 + T cell inltration starts from the optic nerve head towards the retinal periphery followed by LysM + phagocyte inltration in a radial manner. The CX3CR1 + microglia show a change from a more probing state towards an activated phenotype and accumulate mainly around disrupted blood vessels. A small population of activated and highly motile CX3CR1 + cells could only be observed in the perivasculature. Interestingly, most of this motile cells are moving towards the optic nerve head in mice aected by EAU compared to healthy controls. Unexpectedly, functional calcium measurements during the course of EAU up to 28 days after immunisation showed no signicant increase in intracellular calcium levels as an indicator for cellular apoptosis in the ganglion cell layer. The newly developed approach allows longitudinal retinal imaging of cellular inltration, migration and function over time repeatedly over the whole course of chronic inammatory diseases. This method is not only limited to ocular diseases but also can be used to observe indirectly pathogenic processes of the CNS.