Mouse retinal cell behaviour in space and time using light sheet fluorescence microscopy

Abstract

As the general population ages, more people are affected by eye diseases, such as retinopathies. It is therefore critical to improve imaging of eye disease mouse models. Here, we demonstrate that 1) rapid, quantitative 3D and 4D (time lapse) imaging of cellular and subcellular processes in the mouse eye is feasible, with and without tissue clearing, using light-sheet fluorescent microscopy (LSFM); 2) flat-mounting retinas for confocal microscopy significantly distorts tissue morphology, confirmed by quantitative correlative LSFM-Confocal imaging of vessels; 3) LSFM readily reveals new features of even well-studied eye disease mouse models, such as the oxygen-induced retinopathy (OIR) model, including a previously unappreciated ‘knotted’ morphology to pathological vascular tufts, abnormal cell motility and altered filopodia dynamics when live-imaged. We conclude that quantitative 3D/4D LSFM imaging and analysis has the potential to advance our understanding of the eye, in particular pathological, neurovascular, degenerative processes.

Remodeling of the Microvasculature: May the Blood Flow Be With You

“…Likewise, confocal microscopy of vessel-stained tissues such as whole-mount inflamed intestine combined with 3D-image rendering has turned into an approach that provides sufficient resolution to identify and quantify capillary holes/pillars, loops, and duplications ( Figure 3B ) ( Karthik et al, 2018 ; Esteban et al, 2020 ), all hallmarks of capillary splitting, circumventing the limitations of previously used corrosion cast techniques and electron microscopy ( Nowak-Sliwinska et al, 2018 ). Light-sheet fluorescent microscopy (LSFM) has recently been applied to the quantitative 3D and 4D analysis of endothelial cell motility and filopodia dynamics in blood flow-free ex vivo postnatal mouse retina ( Prahst et al, 2020 ). Advanced microscopy techniques will undoubtedly improve our knowledge about capillary remodeling in more complex vascular beds what combined with the use of fluorescent reporter animal lines may provide unprecedented visualization and information about these events.…”

“…Notably, the use of the Raichu Rac1 FRET sensor established the role for the activity of this small GTPase in regulating directed endothelial cell migration during capillary pruning in the brain vasculature of zebrafish (Chen et al, 2012). LifeAct reporter has recently been used for visualization of endothelial cell filopodia and dynamics in the postnatal mouse retina ex vivo (Riedl et al, 2008;Prahst et al, 2020) and it could be a useful tool to analyze cytoskeletal rearrangements of endothelial cells during capillary remodeling. A better knowledge of the molecular actors involved in each step of segment pruning or splitting could help designing new fluorescent reporter animal lines.…”

Blood Flow Forces in Shaping the Vascular System: A Focus on Endothelial Cell Behavior

“…In addition, the contribution of different flow-derived forces (e.g., shear stress and circumferential stretch) to specific EC behaviors involved in vascular morphogenesis still needs to be clarified. These open questions are difficult and will benefit from a number of new tools: optogenetics (Chow and Vermot, 2017;Krueger et al, 2019), functionalized protein binders (Bieli et al, 2016), three dimensional live-imaging and processing (Campinho et al, 2020;Prahst et al, 2020) as well as tissue engineering approaches where forces are trackable and accurately controlled (Duchemin et al, 2019b;Vianello and Lutolf, 2019).…”

Three-dimensional Imaging Coupled with Topological Quantification Uncovers Retinal Vascular Plexuses Undergoing Obliteration

“…Advances in optical clearing techniques have rendered the tissue or organ systems (including retinas) from different animal models transparent for LSFM imaging 22 , 23 , 25 , 26 , 33 , 41 . These techniques remove lipids from tissues to minimize photon scattering while stabilizing the 3-D structural conformation 42 - 44 to allow for deep tissue penetration for large samples 42 , 45 - 48 .…”

“…The current gold standard to image and quantify changes in the murine retinal vascular network typically utilizes whole-mount samples with 2-D analysis 17 - 21 . Previous groups have utilized 3-D imaging to demonstrate vaso-obliteration in the rat model 22 and report the local 'knotted' morphology and vascular tufts during neovascularization in murine OIR 23 . To quantify the early abnormalities and progression of OIR in 3-D for the entire retinal vasculature, we hereby coupled selective plane illumination microscopy also known as light-sheet fluorescence microscopy (LSFM), with quantitative 3-D topological analyses to interrogate the entire retinal vasculature in response to hyperoxia-induced microvascular obliteration 24 - 28 .…”