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Plaque inflammation in atherosclerotic rabbits


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Plaque inflammation in atherosclerotic rabbits

  1. 1. Plaque Inflammation in Atherosclerotic Rabbits can be Identified By SPIO; Introducing a non-invasive method for Imaging Macrophage Infiltration in active and inflamed Vulnerable Plaque Center for Vulnerable Plaque Research University of Texas-Houston and Texas Heart Institute Houston, Texas
  2. 2. Hypothesis • We proposed the use of superparamagnetic iron oxide (SPIO)-enhanced MRI to detect plaque inflammation, a key component of vulnerable plaques. SPIO nanoparticles are taken up by macrophages and alter MR relaxation properties.
  3. 3. Rupture-Prone Inflamed Plaque? Atherosclerotic plaques which are characterized by: • Active inflammation (i.e. macrophage infiltration) • Extensive angiogenesis, • Thin permeable cap • Large lipid core that are prone to rupture and cause sudden luminal clot formation and lead to heart attack and stroke.
  4. 4. Rupture-Prone Inflamed Plaque
  5. 5. Monocyte / Macrophage Recruitment into Atherosclerotic Plaques •Macrophage infiltration plays a central role in plaque vulnerability and risk of thrombotic complications. Review of Prior Studies
  6. 6. In the study by S. Patel, James T. Willerson and Edward Yeh, published in 1997, peritoneal macrophages of mouse were labeled with fluorescent latex microspheres and injected into the blood. Antibodies to ICAM-1, integrin and E-selectin were injected 6-8 hours before macrophage injection.
  7. 7. Figure: Macrophages labeled with fluorescent microspheres adhere to atherosclerotic plaques.
  8. 8. -The mean number of macrophages in the proximal 1mm of aortic root was estimated to be 143+17 per aortic root. -Antibodies against ICAM-1 and integrin significantly reduced the number of macrophage homing.
  9. 9. Steinberg et al, in 2000 published their study regarding a new method of detecting monocytes in plaque. The basic idea is the introduction into a recipient animal of leukocytes differing from those of the recipient by virtue of one easily identified and quantified genetic marker. PCR was the tool to detect the mutated leukocyte. Due to its extreme sensitivity, this test is able to detect a band in a dilution of 5 cells in 1 million unmarked cells.
  10. 10. A: Time course of the disappearance of donor monocytes purified from the blood of a wild-type donor (NAT- R) and injected intravenously into a mutant (NAT-S) recipient. B: Time course of the disappearance of donor monocytes from the blood of a mutant recipient (NAT-S) after intravenous injection of 45 ml of Whole blood from a wild type donor (NAT-R).
  11. 11. For the atherosclerotic plaque 2 different settings were Selected, Fatty streaks and more advanced lesions. They concluded that 623 per million cells in the early Fatty streaks were donor leukocytes. In more advanced stage, the aortic arch showed a maximum number of 3860 donor leukocytes per 1 million cells.(>1% of all the cells in aortic arch). The rate of leukocyte infiltration and lesion expansion will vary with time.
  12. 12. SPIOSPIO Super ParamagneticSuper Paramagnetic Iron OxideIron Oxide lBlood pool magnetic resonance (MR) imaging contrast media with a central core of iron oxide generally coated by a polysaccharide layer lShortening MR relaxation time lEngulfed by and accumulated inside cells with phagocytic activity
  13. 13. It is shown that SPIO particles after injection into the body, follow the tract of inflammation through monocyte / Macrophage system. Could it be used to detect the dynamic of macrophage recruitment in the inflammatory atherosclerotic plaque?
  14. 14. FL-labeled SPIO Incubated Macrophages 24hr
  15. 15. Atherosclerotic mice not injected with cytokines But received SPIO showing iron particles in the Monocytes in a clot Iron staining H & E Staining
  16. 16. H&E Staining Apo E-deficient mouse injected with SPIO No cytokines Iron Staining
  17. 17. Iron Staining H&E Staining Apo E-deficient mouse injected with SPIO Cytokines added
  18. 18. We chose Watanabe Hereditary Hypercholesterolemic rabbits (WHHR) and New Zealand White rabbits (NZW) for this study. We injected them with SPIO (Feridex) 1 mMol Fe/kg and obtained baseline as well as 5-day post-SPIO injection MR images of the aorta (1.5 Tesla, Signa, GE systems). Then we compared the images in hypercholesterolemic rabbits with the normal,wild type NZW rabbits. SPIO-Enhanced MRI study in Rabbits
  19. 19. Hypercholesterolemic Rabbit, Aorta, 4 days after SPIO injection Perls’ Staining H&E Staining X10 X10
  20. 20. Hypercholesterolemic Rabbit, Aorta, 4 days after SPIO injection Perls’ Staining H&E Staining X40 X40
  21. 21. Hypercholesterolemic Rabbit, Aorta, 10 days after SPIO injection Perls’ Staining H&E Staining X10 X10
  22. 22. Hypercholesterolemic Rabbit, Aorta, 10 days after SPIO injection Perls’ Staining H&E Staining X40 X40
  23. 23. Histopathologic Studies of Thoracic Aorta in Watanabe Hereditary Hypercholesterolemic Rabbit after SPIO Injection H&E staining Iron staining Macrophage staining
  24. 24. Histopathologic studies of Thoracic aorta in Watanabe Hereditary Hypercholesterolemic rabbit after SPIO injection H&E staining Macrophage staining Iron staining
  25. 25. Electron Microscopy evidence of Intracellular SPIO in the Rabbit Aorta Endothelial cell, x7500 Foamy cell, x4000
  26. 26. 0 10 20 30 40 50 60 0 10 20 30 40 50 60 70 macrophage (foam cell) density SPIOpositivecell-Iron staining Series1 Correlation between Iron positive cells in Iron staining and foam cell density in H&E staining in rabbit atherosclerotic aorta. R=0.956
  27. 27. MR Angiography 3D with Gadolinium-DTPA in Watanabe Rabbit 3D-TOF TR=59ms TE=7.0ms Flip=30 3D-TOF TR=59ms TE=7.0ms Flip=30 After SPIO injectionBefore SPIO injection Baseline Day 5
  28. 28. Rabbit ex-vivo MRI studies: After the in-vivo MR images, we sacrificed the animals and excised the aorta. Then we put the isolated aorta in a gel medium, clamped both ends and any side branches and injected gadolinium inside the lumen. We did the same procedure for all rabbits. We also used 2 more rabbits, one WHHR and one NZW that were not injected with SPIO, as control, in the ex-vivo MR study.
  29. 29. Ex-vivo MR Study of Thoracic Aorta in SPIO-injected Atherosclerotic and Normal Rabbits after Compared to Non-injected Controls. Watanabe rabbit post-SPIO Watanabe rabbit without SPIO NZW rabbit
  30. 30. Conclusion: 1) SPIO nanoparticles profoundly accumulate in some (not all) areas of atherosclerotic lesions in rabbits and mice. 2) There is a strong correlation between the areas of SPIO accumulation and macrophage density in mice and rabbit atherosclerotic plaques. 3) Non-invasive SPIO-enhanced MR imaging can identify inflamed atherosclerotic plaques.
  31. 31. Center for Vulnerable Plaque Research Houston, Texas