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  1. 1. Histologic evidence ofHistologic evidence of SPIO accumulation inSPIO accumulation in vulnerable plaquesvulnerable plaques Silvio Litovsky, MDSilvio Litovsky, MD June 7, 2002June 7, 2002
  2. 2. In-vitro Study of Macrophage SPIO Uptake  In a series of in-vitro studies we have tested the rate of SPIO uptake by human activated monocytes in different conditions regarding incubation time and concentration of SPIO. SPIO particles were labeled by a fluorescent dye (DCFA).
  3. 3. FL-labeled SPIO Incubated Macrophages after 24hr
  4. 4. Double DAPI Staining with Fluorescence-labeled SPIO Macrophages after 24hr Incubation
  5. 5. Macrophages labeled with fluorescent microspheres adhere to atherosclerotic plaques.
  6. 6. In-vivo distribution of SPIO in ApoE deficient and wild type mice: •For the initial study, we use the mouse model of atherosclerosis. •ApoE deficient mouse has similar atherosclerotic lesions to human and the lesions are more common in the aortic arch and thoracic aorta. • We used ApoE deficient mice and normal variant (C57BL mice) as control. •Animals were sacrificed on day 3 and 5 after injection.
  7. 7. •Tissues from different organs including liver, kidneys, lung, heart, spleen and aorta (including valve region, ascending, descending and abdominal) were obtained and stained. • We used Hematoxyline and Eosin (H&E), Iron, CD68 and Movat staining. • After Iron staining, aortic walls of the ApoE deficient mice and normal variant were compared based on the number of Iron particles.
  8. 8. ApoE k/o Mouse 5 Days AfterApoE k/o Mouse 5 Days After Injection- LiverInjection- Liver
  9. 9. Histopathologic Study of the Mice Injected with SPIO (Aortic Root) ApoE K/O mouse, Movat staining, aortic root Coronary Cross section Atherosclerotic plaque
  10. 10. Histopathologic study of ApoE K/O Mouse injected With SPIO (Aortic Root) CD68 staining (coronary plaque) Iron Staining (aortic plaque) Iron Staining (coronary section) Iron particles Iron particles
  11. 11. Histopathologic Study of Wild Type Mouse Injected with SPIO (Thoracic Aorta) H&E staining CD68 stainingIron staining
  12. 12. Comparison of the Number of the Iron Particles (per HPF) in ApoE K/O Mice Plaque vs. Normal Wall 0 5 10 15 Atherosclerotic Aorta Average number of iron particles per sample P <0.001
  13. 13. Histopathologic studies of Thoracic aorta in Watanabe Hereditary Hypercholesterolemic rabbit after SPIO injection H&E staining Macrophage staining Iron staining Iron particles
  14. 14. Hypercholesterolemic Rabbit, Aorta, 4 days after SPIO injection Perls’ Staining H&E Staining X40 X40
  15. 15. Electron Microscopy Evidence of Intracellular SPIO in the Rabbit Aorta Endothelial cell, x7500 Foamy cell, x4000
  16. 16. 0 10 20 30 40 50 60 0 10 20 30 40 50 60 70 macrophage (foam cell) density SPIOpositivecell-Iron staining Series1 R=0.956 Correlation Between Iron Positive Cells in Iron Staining and Foam Cell Density in H&E Staining in Rabbit Atherosclerotic Aorta.
  17. 17. Plaque Iron in ApoE k/o mousePlaque Iron in ApoE k/o mouse after SPIOafter SPIO H&E IRON STAIN
  18. 18. Plaque Iron in Cytokine-TreatedPlaque Iron in Cytokine-Treated Mice after SPIOMice after SPIO H&E stain Iron stain
  19. 19. Plaque Iron in Cytokine–Treated MicePlaque Iron in Cytokine–Treated Mice after SPIOafter SPIO Overlying plaques Plaque-free area
  20. 20. Conclusion: 1) SPIO nanoparticles actively accumulate in superficial areas of atherosclerotic lesions in rabbits and mice. 2) There is a strong correlation between areas of foamy cells infiltration and SPIO uptake in mice and rabbit plaques. 3) Cytokines markedly increase the SPIO uptake by atherosclerotic plaques, thus providing a method for quantifying recruitment of monocytes. 4) MRI has the potential for non-invasively assessing monocyte homing into atherosclerotic plaque.
  21. 21. Center for Vulnerable Plaque ResearchCenter for Vulnerable Plaque Research Houston, TexasHouston, Texas