Near-infrared fluorescence imaging of carotid plaques with Feraheme-Alexa750 in an atherosclerotic murine model

Objectives: Early detection and intervention of vulnerable plaques can improve coronary artery disease morbidities by preventing serious and often fatal symptoms. Feraheme, an iron oxide nanoparticle, is recognized by proinflammatory and anti-inflammatory macrophages via scavenger receptor type AI/II, and it has been used as an MRI contrast agent for various applications including carotid atherosclerosis imaging. We hypothesize that Feraheme-conjugated NIR fluorophore (Alexa750) can allow us to locate inflamed plaques with near-infrared fluorescence (NIRF) imaging.

Methods: 8-week-old male FVB/NJ mice (n=4) were fed a 0.2% high-cholesterol diet for eight weeks after initial acclimation. Streptozotocin (STZ, 40 mg/kg) was injected peritoneally for 8 consecutive days at week 4 to induce diabetes for accelerating plaque progression. Two weeks after inducing diabetes, the left carotid artery (LCA) was surgically ligated to mimic stenosis. The intact right carotid artery (RCA) served as a negative control. At week 8, mice were fasted for 6 hours before Feraheme-Alexa750 (5 mg/kg) was injected retro-orbitally. In vivo Imaging was performed with an in-house multispectral NIRF imaging system (Ex: 760 nm, Em: 785 nm LP) at various time points (baseline, 1 h, 7 h, and 24 h after injection of contrast agent). Animals were euthanized at the final imaging time point and organs were harvested for ex vivo imaging and future histology analysis.

Results: Ex vivo imaging showed significantly higher fluorescent signal in the LCA compared to the control RCA for all mice even at a short 100 ms exposure time (mean signal-to-background ratio [SBR] of 1.70 vs. 1.02, p=0.0262). Imaging of harvested organs showed predominant uptake in the liver (signal-to-background ratio, average SBR=7.6±2.6). In vivo imaging over time showed high fluorescent signal over the chest region and localized at cervical lymph nodes.

Conclusions: NIRF imaging with Feraheme-Alexa750 shows promise in the detection of atherosclerotic plaques, particularly for intravascular imaging applications. Continued investigation will involve larger sample sizes with histological analyses in mouse models and in larger animals using intravascular NIRF imaging. Acknowledgements: The authors would like to thank the MGH Center for Comparative Medicine animal facility staff. Support for this work partially came from the National Heart, Lung, and Blood Institute and the National Institute of Biomedical Imaging and Bioengineering of the National Institutes of Health under award numbers R00HL127180 and T32EB013180.