Abstract Body: Background: Real-time assessment of leukocyte subsets in atherosclerosis is limited by gating and motion artifacts in large arteries. We describe a femoral artery ligation–induced model of accelerated atherosclerosis that enables ungated intravital multiphoton microscopy (MPM) of immune cell trafficking in free-breathing mice. Using this, we tracked plaque CD11b+ cells in vivo and evaluated CD11b+ subsets based on uptake of low–molecular weight FITC–dextran (LMW-FD), a feature of M2-slanted pinocytosis-capable macrophages.
Aims: To (i) develop an accelerated femoral atherosclerosis model capable of motion-compensated free-breathing MPM (ii) track CD11b+ subsets based on LMW-FD uptake in atheroma in vivo.
Methods: Male ApoE-/- mice (N=104) were used to develop a femoral artery double ligation model of accelerated atherosclerosis (Figure, A.) After establishing consistent lesions in 84 mice, MPM cell tracking was performed in 20 mice. On day 14, the femoral artery was surgically exposed, and MPM imaging was performed for 1 hr following intravenous injection of a CD11b–647 ab and 3 kDa LMW-FD. After MPM, femoral plaques were harvested for histological analyses. iNOS immunofluorescence assessed colocalization between CD11b+ cells retaining LMW-FD (CD11b+ LMW-FD+) and iNOS+ cells. MPM analyses focused on CD11b and LMW-FD uptake in intimal plaque regions, with colocalization quantified by Manders’ correlation coefficient (MCC).
Results: Day 14 post-ligation plaques harvested from the femoral artery exhibited key features of atherosclerosis, including increased inflammation (CD68+) and lipid deposition (Sudan Black+). The femoral model enabled motion-compensated MPM with stable single-cell tracking, capturing CD11b+ leukocyte deceleration during rolling and firm adhesion, and retention of LMW-FD in a subset of CD11b+ cells. Most plaque CD11b+ cells were also LMW-FD+ in vivo (MCC= 67±22%, n=13) and on ex vivo frozen sections (MCC= 72±10%, n=18). CD11b+LMW-FD+ cells colocalized less often with the inflammatory marker iNOS (MCC= 9.8%±3.7%) compared with CD11b+LMW-FD- cells (MCC= 33.1%±16.4%) (Figure).
Conclusion: An accelerated femoral atherosclerosis model enables simplified, free-breathing, non-cardiac gated MPM tracking of CD11b+ leukocytes in vivo. MPM revealed a subset of CD11b+ cells that can retain 3kDa LMW-FD. These cells show reduced iNOS colocalization, indicating the potential to further investigate this M2-slanted macrophage subset in atheroma progression.