Two-photon microscopy on vital carotid arteries: imaging the relationship between collagen and inflammatory cells in atherosclerotic plaques

We used two-photon laser scanning microscopy TPLSM to demonstrate for the first time its potential in studying relational details at the cellular level of atherogenesis in intact, viable mouse carotid arteries. Isolated and mounted arteries of ApoE-/-mice, aged 15 or 21 weeks 7 and 13 weeks on western diet, were imaged after label- ing with specific fluorescent markers for cell nuclei, inflammatory cells, collagen, and lipids. Data were compared with C57BL6/J mice fed a chow diet. Control vessels had intact endothelium without ad- hering blood cells or significant intimal collagen labeling. In ApoE-/- mice already at 15 weeks, inflammatory cells adhered to the endot- helium and increased labeling of collagen was observed in tunica intima at both lesion-prone and non-lesion-prone sites, indicating en- dothelium activation. In plaques, internalized inflammatory cell den- sity increased with age and plaque progression in tunicae adventitia and intima, but not media. In the whole plaque, aging or plaque progression did not alter the direct relationship between inflammatory cells and collagen. However, within the fibrous caps specifically, di- rect contact between inflammatory cells and collagen increased with age. This study demonstrates the potential of TPLSM in determining detailed information regarding the complex relationship between in- flammatory cells and collagen during atherogenesis. © 2008 Society of