Live cell multiphoton microscopy of atherosclerotic plaques in mouse aortas

Atherosclerosis is a chronic inflammatory disease with both innate and adaptive immune components. Various static methods have been applied to investigate mechanisms of atherosclerosis development. However, they did not allow for monitoring dynamic changes in leukocyte behavior in normal and atherosclerotic aortas. Live cell imaging is necessary to study dynamic or transient leukocyte functions relevant to disease pathology, such as antigen presentation, cell migration, and cell-cell interaction. We developed a protocol for ex vivo multiphoton microscopy of atherosclerotic aortas and used it to demonstrate that antigen presentation may occur within the arterial wall. Aortas are harvested from transgenic reporter mice with fluorescent myeloid cells and then incubated with labeled T cells. The cells are imaged with a multiphoton microscope, while a superfusion system maintains the explant in physiologic condition. Cells are tracked from the videos, and their motion is quantified. This system was used to demonstrate antigen presentation in the arterial wall in the context of atherosclerosis.