The application of novel high resolution imaging to interrogate leukocyte-neurovascular unit interactions in the inflamed mouse retina

The recruitment of leukocytes into the brain and retina is a principle feature of diseases as diverse as multiple sclerosis, posterior uveitis, diabetic retinopathy and stroke and yet our understanding of the biological mechanisms underpinning this process remains incomplete. One of the limitations in advancing our knowledge is that the specialised and complex vascular structure that is found in the central nervous system (CNS), the neurovascular unit (NVU), cannot be readily recapitulated in vitro. Accordingly, data from investigations that rely on cell cultures do not reveal the important and complex contribution made to the inflammatory process by all the components of the NVU, including pericytes and astroglia. Some of the most notable recent advances in our understanding of leukocyte recruitment has arisen from intravital imaging of non-CNS structures such as the cremaster muscle. This, however, tells us little about the process in the CNS where the blood-tissue barriers behave very differently. To date, live imaging of the CNS has been restricted by poor access to living brain tissue or, where this can be overcome by imaging the living retina, by inadequate resolution. In this project we propose developing and applying advanced high resolution imaging to investigate the process of leukocyte recruitment to the mouse retina. This will provide us, for the first time, with the ability to investigate leukocyte traffic at the microvascular level. The industrial partner, Cambridge Research Systems (CRS), is developing a new confocal Scanning Laser Ophthalmoscope (cSLO) for retinal imaging specifically for mice. Current commercial instruments designed for mice are simple fundus cameras with limited resolution and poor contrast. The cSLO imaging modality being developed is capable of higher resolution, much greater contrast and depth selectivity and is well suited to imaging single fluorescently labelled cells. Previously repurposed clinical SLOs have been employed to study mice with limited success and high quality mouse prototype instruments are avalable only in a few specialised research labs. The instrument under development will deliver a significant increment in mouse in-eye imaging quality and usability. It is proposed that an early stage commercial prototype will be used in this project which can be readily customised to this projects needs. Working closely with the partners engineers and the cosupervisor it is anticipated that imaging results can be fed back into the product development to enhance the instrument performance and utility throughout the period of the project. To investigate leukocyte traffic in the retina we will employ fluorescently labelled immune cells, retinal cells and cellular structures to interrogate over time the contribution of the endothelial cell, pericyte and glia in facilitating leukocyte migration. We will further investigate regions of preferential migration, the transvascular route of passage taken, and mechanisms involved in the resolution of inflammation such as immune cell intravasation. Cell deletion strategies and the use of specific inhibitors of candidate signalling pathways will be used to gain detailed insight into the role of different cells and structures of the NVU in this process. We propose that by investigating the contribution that the vascular extracellular environment makes to permissive regions will reveal novel regulating elements that are tractable to therapeutic intervention.