Abstract
Purpose :
Inflammation is mediated by entry of circulating leukocytes into vascularized tissues by interaction of cell adhesion molecules expressed on the vascular wall and leukocyte surface. Until recently, these early events could not be visualized without fluorescent labels in the living retina. Here we use label-free adaptive optics scanning light ophthalmoscopy (AOSLO) to characterize leukocyte adhesion to the vessel wall by tracking leukocyte speed in response to retinal inflammation in mice.
Methods :
C57BL/6J male mice (Jax #000664) were anesthetized using ketamine (100 mg/kg)-xylazine (10 mg/kg) and 1% v/v isoflurane for 1-1.5 hours. Intravitreal injection of 0.5 ng lipopolysaccharide (LPS) in 1 µL PBS was used to induce inflammation. Mice were imaged at baseline, as well as at 6 hours post LPS injection with a custom phase-contrast AOSLO (200-500 µW of 796 nm light, 800 µm pinhole offset by 30 airy disc diameter). Rolling leukocytes in retinal venules were imaged within 10° of the optic disc. Leukocyte speed was quantified by manually marking cell position over time using ImageJ Manual Tracking plugin (350 leukocytes, 6 venules, 5 mice)
Results :
We did not observe leukocyte rolling in baseline conditions. However, many leukocytes were seen rolling at the venular walls 6 hours after LPS injection (Fig 1). Average rolling speed of 350 cells was 11.97±7.3 µm/s (mean±SD). The range of speeds was between 0-159.1 µm/s. We found specific locations along the vascular wall spanning ~25 µm where leukocytes consistently slowed down by 5.2-30.3x the average speed (Fig. 2). These zones suggest vascular areas of increased binding affinity. Conversely, there were other patches ~20 µm in length where leukocytes consistently speed up by 3.9-5x (Fig. 2), suggesting decreased adhesive molecule expression.
Conclusions :
In response to global inflammation of the eye, we find there are specific zones within vessels where leukocytes bind more strongly. Unlike ex vivo histological approaches, we can now reveal these locations using in vivo label free imaging. This provides the new potential to study local gradients in cell adhesion molecules such as selectins and integrins. This is an important step to identify the complex vascular spatial dynamics of leukocyte adhesion that precedes extravasation into the neural retina.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.