Abstract
Purpose :
Early vascular changes in response to tissue inflammation includes the selective rolling and crawling of circulating immune cells along the venular endothelium. Recently, we have imaged single immune cells in vivo using label-free adaptive optics scanning light ophthalmoscopy (AOSLO) with phase contrast. Here, we image and measure the rolling/crawling speeds of immune cells that precede cell transmigration into the retina.
Methods :
A custom AOSLO with phase contrast capabilities (796 nm, 200-500 µW, 800 µm pinhole, 30 airy disc diameter offset) was used. Image locations were within 10° of the optic disc, focusing on arterioles and venules adjacent to the ganglion cell layer. C57BL/6J male mice were anesthetized using ketamine (100 mg/kg)-xylazine (10 mg/kg) and isoflurane (1% v/v). Baseline videos were captured before mice were injected intravitreally with 0.5 ng of lipopolysaccharide in 1 µL PBS. The same mice were reimaged within five epochs: 0.5, 4, 6, 24 and 72 hours post injection. Rolling cell speed was quantified at 6 hours (n=4 venules in 3 mice, 53 cells tracked) by manually tracking cell position over time using ImageJ Manual Tracking plugin (Fig 1).
Results :
By morphology, we observed two types of adherent cells; those with a circular appearance rolling along the endothelium and a more adherent class of cells showing tank-treading behaviors (Fig 2). Rolling and crawling immune cells were found in venules; arterioles showed few endothelial interactions. Rolling and crawling was predominantly observed at the 4-6 hour time epoch; few rolling cells were observed in 24 and 72 hours epochs. Average rolling speed was 10.9±9.9 µm/s. Speeds were variable across the same cells (0-103.9 µm/s) indicating patches of greater adhesion. Two cells were nearly stationary (0.2±0.9µm/s), indicating firmly adherent cells that may later extravasate. We observed one cell actively moving against the direction of blood flow in a venule shown as negative velocity -0.3±0.5 µm/s (Fig.2).
Conclusions :
Here we reveal some of the earliest features of inflammation at the single cell level in vivo. Phase contrast AOSLO enables label-free noninvasive measurements, allowing an unperturbed assessment of retinal inflammatory response. This work provides a foundation for detailed study of immune cell adhesion in vivo, a key target for new drugs that seek to modulate such behavior.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.