Imaging immune cell type and behavior in the living retina using adaptive optics

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Abstract: Immune cell response to ocular inflammation is involved in various prevalent retinal diseases that lead to blindness such as glaucoma, age related macular degeneration, uveitis and diabetic retinopathy. However, the microscopic and translucent nature of circulating and resident immune cells has made study of such cells challenging in the living eye. Further complicating matters, immune cells move at fast speeds (centimeters/second) when circulating in big vessels and glacial pace at micrometers/minute when in tissue. This project will combine a number of innovations that overcome many of these challenges to study single immune cells in the living eye in their response to inflammation. The use of adaptive optics scanning light ophthalmoscope (AOSLO) to image mouse retina will provide a noninvasive in vivo imaging method that can resolve single immune cells and track their behavior through the entire course of inflammation. With our AOSLO, we have recently demonstrated that phase contrast combined with both fast and time-lapse videography now enables us to visualize these microscopic immune cells using near infrared light alone and without fluorescence contrast. Therefore, this project will seek to study immune cell dynamics in two models of inflammation in the living mouse eye and track the cellular responses to initiation, escalation and resolution. And while the approach is non-invasive and does not require use of fluorescence, we will combine fluorescence confirmation of specific leukocyte populations that contribute to the inflammatory response. Our project tackles three synergistic aims to track the dynamic nature of inflammation using two established models: Model 1) An endotoxin induced uveitis model using lipopolysaccharide (LPS) injection in vitreous of the eye. This results in an acute, short-term form of inflammation. We will track the initial escalation of inflammation in the retina by monitoring behavior of tissue resident microglia and systemic early responder neutrophils. And Model 2) An autoimmune uveitis condition that is induced in a healthy host mouse by injecting a subpopulation of fluorescent CD4+ T cells that are reactive to interphotoreceptor retinoid binding protein from a different donor mouse. Here, we will characterize the behavior of these foreign reactive T cells in healthy retina, and their interaction with the host immune system. In both inflammatory models, we will characterize the initiation, escalation, infiltration and resolution of immune cells in the retina by longitudinally tracking their behavior from hours to days to months. Finally, in a third aim, we will also study the changes in blood flow in response to these inflammatory models. For both endotoxin mediated and autoimmune inflammatory models described above, we will monitor structural and functional changes in retinal vasculature from the largest arteries and veins to single file flow capillaries to understand the interplay between blood flow and immune cell arrival into the retina. Already, we have observed that the vascular response is not homogeneous, and arterioles, venules and capillaries respond in different ways to the inflammatory insult and likely mediate the location, arrival and type of immune cell passage. Together, the longitudinal monitoring of inflammation and blood flow will provide new insights to the retinal immune response using innovations in high-resolution ophthalmoscopy.