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Enhancement of Vascular Permeability with Low-Frequency Contrast-enhanced Ultrasound in the Chorioallantoic Membrane Model¹

¹ From the Department of Surgical and Radiological Sciences, School of Veterinary Medicine, University of California, Davis, 2112 Tupper Hall, Davis, CA 95616 (S.M.S., E.R.W.); Department of Biomedical Engineering, University of California, Davis, Calif (C.F.C., S.Q., K.W.F.); and Department of Physiology and Membrane Biology, School of Medicine, University of California, Davis, Calif (R.H.A., F.E.C.). Received January 27, 2006; revision requested March 28; revision received April 25; accepted May 31; final version accepted August 1. Supported by National Institutes of Health CA 103828. Address correspondence to S.M.S. (e-mail: smstieger{at}ucdavis.edu).

View larger version (121K): [in this window] [in a new window] [Download PPT slide]   Figure 1: A, Diagram shows experimental setup for optical imaging of the CAM. B, Image of CAM acquired at day 15. C–J, Optical images from 2.25-MHz study with PNP of 1.7 MPa. C was acquired before insonation with 2.25 MHz, and D–J were acquired 0.06, 0.12, 0.24, 1.24, 2.24, 3.24, and 4.24 seconds after insonation began, respectively. K–P, Optical images from 1.00-MHz study at PNP of 1.3 MPa. K was acquired before insonation with 1.00 MHz, and L–P were acquired at the same times as C–J. A smaller number of extravasation sites (arrows) and a more gradual, spherical extravasation is observed with 2.25-MHz insonation as compared with 1.00-MHz insonation. Scale bar = 100 µm.

View larger version (5K): [in this window] [in a new window] [Download PPT slide]   Figure 2a: (a) Schematic of parabolic blood flow in vessel. d = Opening in vessel wall, Di = inner diameter of vessel (10 µm), Do = outer diameter of vessel (12 µm), Pi = intravascular pressure (35 mm Hg), umax = maximum flow velocity (3 mm/sec). (b) Graph shows mean velocity of dextran traveling through 0.5- and 1-µm openings of vessel. * = Mean velocity could not be calculated any further because extravasation traveled beyond the computational domain. (c) Calculated images of FITC dextran, which was transported through a vessel wall opening, along with representative optical images. Inner vessel diameter is 10 µm, wall thickness is 1 µm, and vascular pressure is 35 mm Hg. Square containing each subimage indicates computational interstitial domain (400 x 400 µm2). Solid rectangle = vessel segment (10 x 80 µm2). Open rectangle = scale bar (100 µm). Inner propagating front is 90% of the dextran concentration; outer propagating front is 10% of the dextran concentration. t = Time from start of dextran extravasation.

View larger version (15K): [in this window] [in a new window] [Download PPT slide]   Figure 2b: (a) Schematic of parabolic blood flow in vessel. d = Opening in vessel wall, Di = inner diameter of vessel (10 µm), Do = outer diameter of vessel (12 µm), Pi = intravascular pressure (35 mm Hg), umax = maximum flow velocity (3 mm/sec). (b) Graph shows mean velocity of dextran traveling through 0.5- and 1-µm openings of vessel. * = Mean velocity could not be calculated any further because extravasation traveled beyond the computational domain. (c) Calculated images of FITC dextran, which was transported through a vessel wall opening, along with representative optical images. Inner vessel diameter is 10 µm, wall thickness is 1 µm, and vascular pressure is 35 mm Hg. Square containing each subimage indicates computational interstitial domain (400 x 400 µm2). Solid rectangle = vessel segment (10 x 80 µm2). Open rectangle = scale bar (100 µm). Inner propagating front is 90% of the dextran concentration; outer propagating front is 10% of the dextran concentration. t = Time from start of dextran extravasation.

View larger version (33K): [in this window] [in a new window] [Download PPT slide]   Figure 2c: (a) Schematic of parabolic blood flow in vessel. d = Opening in vessel wall, Di = inner diameter of vessel (10 µm), Do = outer diameter of vessel (12 µm), Pi = intravascular pressure (35 mm Hg), umax = maximum flow velocity (3 mm/sec). (b) Graph shows mean velocity of dextran traveling through 0.5- and 1-µm openings of vessel. * = Mean velocity could not be calculated any further because extravasation traveled beyond the computational domain. (c) Calculated images of FITC dextran, which was transported through a vessel wall opening, along with representative optical images. Inner vessel diameter is 10 µm, wall thickness is 1 µm, and vascular pressure is 35 mm Hg. Square containing each subimage indicates computational interstitial domain (400 x 400 µm2). Solid rectangle = vessel segment (10 x 80 µm2). Open rectangle = scale bar (100 µm). Inner propagating front is 90% of the dextran concentration; outer propagating front is 10% of the dextran concentration. t = Time from start of dextran extravasation.

View larger version (14K): [in this window] [in a new window] [Download PPT slide]   Figure 3a: (a) Bar graph shows occurrence of extravasation in each pressure group as a function of PNP and corresponding number of vessels affected in each field of view. (b) Bar graph shows average size of vessels affected in each field of view and area over which extravasation was observed as a percentage of each field of view. (c) Bar graph shows rate of extravasation from each vessel, calculated from the time (in seconds) required for fluid to travel 100 µm2 from the affected vessel. (d) Bar graph shows rate of fluid extravasating from openings in walls of vessels with an initial diameter between 10 and 15 µm. Dotted lines with arrows indicate low-stress group. Error bars indicate standard deviations.

View larger version (16K): [in this window] [in a new window] [Download PPT slide]   Figure 3b: (a) Bar graph shows occurrence of extravasation in each pressure group as a function of PNP and corresponding number of vessels affected in each field of view. (b) Bar graph shows average size of vessels affected in each field of view and area over which extravasation was observed as a percentage of each field of view. (c) Bar graph shows rate of extravasation from each vessel, calculated from the time (in seconds) required for fluid to travel 100 µm2 from the affected vessel. (d) Bar graph shows rate of fluid extravasating from openings in walls of vessels with an initial diameter between 10 and 15 µm. Dotted lines with arrows indicate low-stress group. Error bars indicate standard deviations.

View larger version (11K): [in this window] [in a new window] [Download PPT slide]   Figure 3c: (a) Bar graph shows occurrence of extravasation in each pressure group as a function of PNP and corresponding number of vessels affected in each field of view. (b) Bar graph shows average size of vessels affected in each field of view and area over which extravasation was observed as a percentage of each field of view. (c) Bar graph shows rate of extravasation from each vessel, calculated from the time (in seconds) required for fluid to travel 100 µm2 from the affected vessel. (d) Bar graph shows rate of fluid extravasating from openings in walls of vessels with an initial diameter between 10 and 15 µm. Dotted lines with arrows indicate low-stress group. Error bars indicate standard deviations.

View larger version (10K): [in this window] [in a new window] [Download PPT slide]   Figure 3d: (a) Bar graph shows occurrence of extravasation in each pressure group as a function of PNP and corresponding number of vessels affected in each field of view. (b) Bar graph shows average size of vessels affected in each field of view and area over which extravasation was observed as a percentage of each field of view. (c) Bar graph shows rate of extravasation from each vessel, calculated from the time (in seconds) required for fluid to travel 100 µm2 from the affected vessel. (d) Bar graph shows rate of fluid extravasating from openings in walls of vessels with an initial diameter between 10 and 15 µm. Dotted lines with arrows indicate low-stress group. Error bars indicate standard deviations.

View larger version (218K): [in this window] [in a new window] [Download PPT slide]   Figure 4: Transmission electron micrographs show range of effects of ultrasound treatment in CAM model. Slices were stained as described in Appendix E1 (http://radiology.rsnajnls.org/cgi/content/full/243/1/112/DC1). A, Image in control group with injection of dextran only shows subchorionic blood capillaries immediately next to chorionic epithelium and overlying interstitial space. B, Low-magnification view of venular vessel in control group shows chorionic epithelium and associated blood capillaries at right and allantoic epithelium at left. C, High-magnification view of wall of venular blood vessel in control group shows highly attenuated vascular endothelium. D, High-magnification view in control group shows intact junction of endothelial cells and thin basement membrane between endothelium and pericyte. E, Detailed view of F, which was obtained in high-stress group insonated at 1.00 MHz and 1.3 MPa. Gap in endothelial layer is associated with extravasation of blood (arrows). F, Overview of region near venular vessel. Extravasated erythrocytes and plasma are visible. G, Detailed view of F shows erythrocyte impacted in wall of vessel where endothelial cells are missing (arrow). H–K, Images in high-stress group insonated at 1.00 MHz and 2.3 MPa. H, Chorionic capillary. Remnants of endothelial cells remain attached to basement membrane over much of perimeter (arrows). Erythrocyte is impacted in gap in endothelial layer. I, There is a small gap in endothelial layer, though basement membrane of capillary remains intact. J, Venular microvessel shows gap in endothelial layer and separation from underlying intact pericyte (arrows). K, There are disrupted endothelial cells with plasma in space separating endothelium and pericyte, yet basement membrane on abluminal side of pericyte remains intact. One erythrocyte is impacted on basement membrane where endothelium is missing. Extravasated erythrocytes from region out of the plane of section are in interstitium. Density possibly indicating fibrin is seen adjacent to site where blood plasma is in contact with basement membrane. a = Allantoic epithelium; bm = basement membrane; C = capillary; Ch = chorionic epithelium; ec = endothelial cell; Er, er, er* = erythrocyte; ex = extravasated erythrocytes; f = fibrin; g = gap; L = lumen; N = nucleus; P = pericyte; tj = tight junction.

View larger version (25K): [in this window] [in a new window] [Download PPT slide]   Figure 5a: (a) Bar graph shows percentage of vessel wall perimeter altered by insonation. Increasing the pressure produced an effect in a larger fraction of the vessel perimeter, particularly in 25–60-µm vessels. (b) Bar graph shows length of affected perimeter normalized by vessel area to account for the higher concentration of contrast agents in larger vessels. After correction for the relative frequency of microbubbles as a function of vessel diameter, spatial extent of the effect in 7–25-µm vessels was similar to that in 25–60-µm vessels. Error bars indicate standard deviations.

View larger version (27K): [in this window] [in a new window] [Download PPT slide]   Figure 5b: (a) Bar graph shows percentage of vessel wall perimeter altered by insonation. Increasing the pressure produced an effect in a larger fraction of the vessel perimeter, particularly in 25–60-µm vessels. (b) Bar graph shows length of affected perimeter normalized by vessel area to account for the higher concentration of contrast agents in larger vessels. After correction for the relative frequency of microbubbles as a function of vessel diameter, spatial extent of the effect in 7–25-µm vessels was similar to that in 25–60-µm vessels. Error bars indicate standard deviations.