Longitudinal Intravital Imaging of the Retina Reveals Long-term Dynamics of Immune Infiltration and Its Effects on the Glial Network in Experimental Autoimmune Uveoretinitis, without Evident Signs of Neuronal Dysfunction in the Ganglion Cell Layer

Abstract

A hallmark of autoimmune retinal inflammation is the infiltration of the retina with cells of the innate and adaptive immune system, leading to detachment of the retinal layers and even to complete loss of the retinal photoreceptor layer. As the only optical system in the organism, the eye enables non-invasive longitudinal imaging studies of these local autoimmune processes and of their effects on the target tissue. Moreover, as a window to the central nervous system (CNS), the eye also reflects general neuroinflammatory processes taking place at various sites within the CNS. Histological studies in murine neuroinflammatory models, such as experimental autoimmune uveoretinitis (EAU) and experimental autoimmune encephalomyelitis, indicate that immune infiltration is initialized by effector CD4+ T cells, with the innate compartment (neutrophils, macrophages, and monocytes) contributing crucially to tissue degeneration that occurs at later phases of the disease. However, how the immune attack is orchestrated by various immune cell subsets in the retina and how the latter interact with the target tissue under in vivo conditions is still poorly understood. Our study addresses this gap with a novel approach for intravital two-photon microscopy, which enabled us to repeatedly track CD4+ T cells and LysM phagocytes during the entire course of EAU and to identify a specific radial infiltration pattern of these cells within the inflamed retina, starting from the optic nerve head. In contrast, highly motile CX3CR1+ cells display an opposite radial motility pattern, toward the optic nerve head. These inflammatory processes induce modifications of the microglial network toward an activated morphology, especially around the optic nerve head and main retinal blood vessels, but do not affect the neurons within the ganglion cell layer. Thanks to the new technology, non-invasive correlation of clinical scores of CNS-related pathologies with immune infiltrate behavior and subsequent tissue dysfunction is now possible. Hence, the new approach paves the way for deeper insights into the pathology of neuroinflammatory processes on a cellular basis, over the entire disease course.

Coregistered Spectral Optical Coherence Tomography and Two‐Photon Microscopy for Multimodal Near‐Instantaneous Deep‐Tissue Imaging

“…Whereas the OCT signal highlighted several retinal layers, 2PM allowed us to retrieve the localization of CX 3 CR1 + cells, presumably tissue‐resident microglia based on their GFP fluorescence. These cells play a crucial role in the disease development in the chronically inflamed central nervous system , in general, and chronically inflamed retina , in particular, as shown by us and by others.…”

“…Due to the nonlinear excitation of femtosecond pulsed lasers, the illumination in 2PM affects only a thin layer of tissue located at the focal plane. By controlling the laser power in the range of few tens of milliwatts, photodamage is typically reduced to a negligible level for mammalian tissues . Based on elastic reflection and interference, OCT typically employs low coherence light sources at extremely low powers (in the range of microwatts) .…”

Intravenous indocyanine green dye is insufficient for robust immune cell labelling in the human retina

“…No rapid alterations were seen during imaging sessions, potential cell labelling could only be identified in post-processing and the expected speed of migration within retinal tissue remains unknown. Evidence from 2-photon imaging of the inflamed mouse eye however demonstrates myeloid cell movement may be much slower than expected, even compared to other tissues, at around 1 µm per minute (20). This could explain the larger time intervals required to observe signal position shifts that could represent labelled immune cells.…”

Molecular mobility and activity in an intravital imaging setting – implications for cancer progression and targeting

“…For example, IVM demonstrated that fluorescently labelled bisphosphonates, which have been shown to possess anti-tumour activity, were taken up by tumour-associated macrophages (TAMs), potentially corrupting their tumourpromoting functions (Junankar et al, 2015). Here IVM identified Imaging of fibrillar collagen in tissues including muscle, skin and tumours (Brown et al, 2003;Campagnola et al, 2002;Koenig and Riemann, 2003;Kular et al, 2015;Plotnikov et al, 2006;Zipfel et al, 2003) Imaging of fibrillar collagen in human breast tissue in relation to mammographic density (Huo et al, 2015), during cancer cell invasion (Andrlová et al, 2017) or anti-cancer drug targeting of the stromal ECM (Chang et al, 2017;Miller et al, 2015;Vennin et al, 2017) Third harmonic generation (THG) Excitation primarily occurs at water-lipid and water-protein interfaces, to provide structural information (e.g. cell morphology and surrounding ECM).…”

“…For example, live cell imaging in the retina was recently performed to observe dynamic radial infiltration patterns of immune cells in a mouse model of autoimmune retinal inflammation (Bremer et al, 2016). The auricular skin of the ear provides another easily accessible site to extract valuable in vivo cell biology without the need for surgical intervention.…”