Abstract
Purpose :
To study retinal mouse metabolic states ex-vivo using novel tissue preparation and fluorescent lifetime imaging microscopy (FLIM).
Methods :
Wild type C57BL/6J mice were included in the study. Following euthanasia and enucleation, corneas were removed, and eyecups were fixed overnight in a 4% Paraformaldehyde (PFA) solution. The next day, eyecups were embedded in polyacrylamide, and 200 μm thick retinal sections were made using a vibratome. Images were taken using the Leica SP8 DIVE FALCON for multiphoton FLIM measurements of metabolic states and tissue structure, using the phasor approach to FLIM analysis. Tissue excitation was performed with 740 nm light from our ultrafast tunable laser at ~500 μW power. FLIM was collected simultaneously on two hybrid detectors from ~425-475 nm for NADH and ~530-650 nm for FAD, retinoids and hemoglobin.
Results :
The percentage of bound NADH was ~50% in the retinal pigment epithelium, ~55% in the photoreceptor layer and 60-70% in the inner retina, indicating higher glycolysis in the outer retina and higher oxidative phosphorylation in the inner retina. This finding in fixed tissues, recapitulated previous findings of live-tissue metabolic FLIM NADH measurements. Additionally, our study showed that the hemoglobin signal in the vasculature has a distinct FLIM profile allowing visibility of retinal capillaries on 3-dimensional images as they penetrate the retina and form capillary plexus at different layers. This novel tissue preparation, imaging, and analysis pipeline enable the reconstruction of metabolic states of tissue layers, cells, stroma, and vasculature in 3 dimensions, without the need for staining techniques.
Conclusions :
FLIM of retinal sections in mice showed a predominance of glycolysis in the outer retina and oxidative phosphorylation in the inner retina. With additional autofluorescence excitation and emission strategies, FLIM could be used to define the structure and metabolism of healthy versus diseased retinas.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.