Abstract Body: Background: Intravital imaging is a valuable tool for monitoring real-time leukocyte behavior, providing cellular-resolution insights into disease progression and therapies. Nevertheless, the pulsatile motion and large size of vessels during real-time imaging of atherosclerotic arteries pose challenges. Atherosclerotic femoral arteries of smaller size and low pulsatility may avoid these limitations.
Aim: We aimed to develop an atherosclerosis model in mouse femoral arteries and use advanced intravital imaging to gain insights into monocyte extravasation, a key proatherogenic event driving plaque formation and progression.
Methods: Male ApoE -/- mice (n=4 per batch, 60 total) consumed a high-cholesterol diet for two weeks. A week later, bilateral partial femoral artery ligation was performed to induce disturbed blood flow and accelerate plaque formation. Later atherosclerotic areas in femoral artery were imaged using both epifluorescence and two-photon microscopy with retro-orbital injections of CD11b antibody and FITC dextran. CD11b antibody (PE/Cyanine5) was used to observe monocyte behavior, while FITC dextran (3kD) was used to assess blood flow. Histological analysis examined plaque morphology.
Results: Atherosclerotic plaque formation varied, occurring between days 7 and 21 in the mouse femoral artery. Epifluorescence microscopy showed focal enhancement of atherosclerotic plaques between the ligations, as evidenced by an increased CD11b signal (Figure 1). Two-photon microscopy further revealed monocyte migration into the intima and enabled visualization of second harmonic generation of vascular collagen. Histological analysis showed plaques in the early stages of atherosclerosis, as evidenced by increased CD68⁺ macrophages and lipid accumulation, but lacked characteristics associated with advanced plaques, such as necrotic core formation, fibrosis, fibrous cap thinning, or smooth muscle proliferation. Efforts to quantify and track monocyte migration into the intima are ongoing. Imaging additional inflammation markers, such as neutrophil markers, is also being explored.
Conclusion: Our results establish the use of femoral atherosclerosis for intravital imaging (epifluorescence and 2-multiphoton microscopies) of vascular inflammation and permeability. CD11b is a reliable marker for intravital imaging of monocytes, but further characterization of CD11b⁺ cells is essential to determine the myeloid cell subsets involved in plaque formation and progression.