Figure 1.
Rabbit aorta cholesterol plaque formation and NP diffusion into intima in vivo.
A: Sudan IV staining (en face) of opened aorta section showing plaque (red) in 12 month diet rabbit but seldom in 3 month diet rabbit. Sections were collected as labeled for fluorescence microscopy and histology (1 cm), electron microscopy (0.5 cm), lower segments for MRI/MRS and whole mount fluorescence images (IVIS). B: Cartoon of PFC-NP structure, and 19F MR spectroscopy (top left), TEM of NP (top right); 19F and 1H MR image of test tubes containing CE core NP showing fluorine signatures (bottom) and oil core NP as control showing no signal. C: Top: En face Sudan IV staining of section with plaque (P: red) and grossly normal (N: clear) areas. Mid: Marked fluorescent nanoparticle presence in plaque (P) intima (I) of 12 mo cholesterol diet rabbit aorta (green) after 12 hours in vivo circulation. Minimal staining of adventitia (A) is noted , and none apparent in media (M) or lumen (Lu). Bottom: Fluorescent NP signals (green) in plaque intima (P), but not the adjacent grossly normal regions (N). Blue = DAPI nuclear stain.
Figure 2.
Duration of cholesterol feeding determines NP plaque penetration in vivo.
A: 6 hours circulation in vivo with Alexa fluor-488 labeled NP in 3 month cholesterol diet rabbit aorta showing modest intimal plaque thickening and little fluorescent signal (arrow). Note lack of green-yellow autofluorescence in intima and + nuclear (DAPI stain). B: In vivo 6 hours circulation with identical NP in 9 month cholesterol diet rabbit aorta indicative of marked endothelial penetration and particle trapping (arrow). C: In vivo 6 hours circulation with identical NP in 12 month cholesterol diet rabbit aorta showing extensive NP penetration into intima (green). D: Sigmoidal fitting of fluorescence scores of aortas exposed in vivo to fluorescent NP circulating for 2 hours (open) and 6 hours (solid) on rabbits fed with cholesterol for 0 to 419 days. The temporal dependence of plaque permeability on the duration of hypercholesterolemia is well approximated with a sigmoidal function.
Figure 3.
MRI imaging and quantification of NP signal in cholesterol plaques.
A: Left: 3D saggital rendering of nanoparticle signals from aorta of 12 mo cholesterol fed rabbit after 12 hours circulation time in vivo. 19F MR image registering nanoparticle fluorine cores (color) overlaid on 1H MR image (gray) of aorta show intimal (I) location of particles trapped in thickened plaque (color) atop the medial (M) layer (gray). Particle concentration per voxel is coded in nM (scale bar). Right: Close up of intimal layer. B: 1H, 19F and overlay MR transverse images of aortic rings with nanoparticles trapped in intima of thickened plaque from 9 mo cholesterol fed rabbit after 2 hour circulation in vivo. Black artifacts are small air bubbles. Note lack of 19F signal from more normal adjacent tissue sections. C: Comparison of normalized CE NP number to the endothelial surface area between 3 month diet and >7 month diet rabbit aorta samples showing 10 fold greater accumulation in older plaques.
Figure 4.
Fluorescence microscopy of rabbit aorta segments after ex vivo NP incubation for different times.
A, B: 12 month cholesterol diet rabbit incubated with Alexa Fluor 488-labeled NP (green) for 15 minutes and 6 hours, showing progressively deeper penetration into intimal plaque region. Media shows elastin autofluorescence (yellow-green) only. Blue = nuclear stain C: 3 month cholesterol diet rabbit aorta incubated for 6 hours with Alexa Fluor 488-labeled NP showing minimal plaque thickening and only minor particle accumulation at endothelial surface. D: “Fluorescence score” of ex vivo NP-treated 12-month diet rabbit aorta segments incubated for different times, confirming greater penetration with longer NP exposure times. The exponential fit to the data indicates an asymptotic increase in penetration with little additional diffusion beyond 2 hour incubations.
Figure 5.
SEM images of rabbit aorta cholesterol plaque endothelium disruption.
A: Speed vacuum dried, sputter coated SEM image of aortic endothelium of 12 month diet rabbit aorta demonstrating extensive superficial coating with crystalline structures (arrow) consistent with cholesterol that were not observed in standard ethanol dried preparations and similar to observations in human carotid endarterectomy samples made by Abela et al (see text). B: SEM images of speed vacuum dried cholesterol crystal around cavitary structure (*). C: Traditional dehydrated SEM image of cavitary structures (*) penetrating into subendothelial regions and surrounded by cholesterol crystals. D: Lower magnification SEM image with multiple endothelial erosions (arrow). E: TEM of endothelial foam cells (arrow) in cholesterol fed rabbit aorta sample. F: SEM image of normal aortic endothelium from regular diet rabbit for comparison.
Figure 6.
SEM, fluorescence microscopy and MRS of human carotid endarterectomy specimen.
A: SEM image of intimal subendothelial cholesterol crystals in carotid endarterectomy tissue. (scale bar = 50 um) B: Fluorescence microscopy of human carotid endarterectomy specimen (fresh) incubated ex vivo with Alexa Fluor-488 labeled NP (green). Lu: lumen; I: intima; M: media. (scale bar = 500 um) C: 19F MR spectroscopy of human plaque segment indicating a strong signal and the presence of trapped CE NP in plaque. (ppm: part per million). PFOB standard signal from co-registered control sample is for NP calibration.