Supplementary MaterialsSupplementary figures
Supplementary MaterialsSupplementary figures. The plaque necrotic center/fiber cap (NC/FC) ratio and vulnerability index were calculated to evaluate plaque vulnerability. Twenty-four hours after TUS treatment at 3.0 MPa, the MVD in the plaque was substantially decreased by 84% (p < 0.05), while there was almost no change in MVD and neovessel density (NVD) in normal tissues, including skeletal muscle, mesentery and skin. Additionally, a marked reduction in the number of immature vessels was observed in the plaques (reduced by 90%, p < 0.05), whereas the number of mature Broxyquinoline vessels was not significantly decreased. Furthermore, TUS treatment at 3.0 MPa significantly improved plaque stability, as reflected by the NC/FC ratio and vulnerability index, which may be due to the selective destruction of intraplaque neovascularization by TUS treatment, thereby decreasing the extravasation of erythrocytes and leading to vascular inflammation alleviation and thin-cap fibroatheroma reduction. Conclusions: TUS treatment at 3.0 MPa selectively depleted plaque neovessels and improved the stability of vulnerable plaques through a reduction in erythrocyte extravasation and inflammatory mediator influx, with no significant effect on normal tissue. experiment, the endothelial cells were arranged in a single layer at the bottom of a 30-mm diameter petri dish filled with DMEM. Then, as described previously 26, the petri dish was inverted into a 100 mm petri dish and then filled with DMEM, sterile saline made up of 1.5 108 microbubbles was injected into the medium through a syringe and homemade pillow. The transducer was disinfected with 70% alcohol and then placed vertically against the dish with the aid of coupling gel. The transducer was operated at a frequency of 1 1 MHz with a pulse repetition frequency of 10 Hz and a duty cycle of 0.19%. The acoustic pressure output could be switched between 1.0 MPa and 5.0 MPa. The treatment was performed with an intermittent Broxyquinoline mode of 2 seconds on and 8 Broxyquinoline seconds off for 30 seconds. The cavitation generated by MBs is usually represented by two-dimensional imaging in the water in Fig. S1A, B. MB preparation and characterization The MBs were generated as previously described 16. The concentration size and distribution of MBs were analyzed using Rabbit Polyclonal to CBR1 a coulter counter (Multisizer III, Beckman Coulter, FL, USA), which was presented in Fig. S1C, D. The structure of MBs was visualized by a microscope (BX51; Olympus, Tokyo, Japan). The cavitation of MBs was determined by the therapeutic US system described above. B-mode images were acquired before and after treatment by microbubble-enhanced ultrasound (MEUS). Histology and immunohistology After the mice were sacrificed, tissues of the abdominal aorta were fixed in 4% paraformaldehyde and embedded in paraffin. Serial 3-m thick paraffin sections were cut and stained with H&E, Masson’s trichrome staining and immunostaining Broxyquinoline for the observation of histological changes as described previously 27. To evaluate plaque vulnerability, the plaque necrotic center/fiber cap (NC/FC) ratio and vulnerability index were calculated as described previously. H&E and Masson’s trichrome (MST-8003; Matxin Labs Pvt., Ltd., Bangalore, India) were used to measure the area of lipid deposition and collagen fiber content. Sections were incubated using a rabbit polyclonal against mouse -easy muscle actin (-SMA) and cluster of differentiation (CD) 68 (all from Abcam, Cambridge, MA, USA) to stain for easy muscle cells (SMCs) and macrophages, respectively. The positive staining areas of SMCs, macrophages, lipid deposition and collagen were quantified using Image-Pro Plus (IPP, Media Cybernetics, Rockville, MD, USA) by two individuals who were blinded to the experimental design and are expressed as the percentage of positive-to-total plaque area. The NC/FC ratio was measured as the ratio of lipid deposition to collagen fiber, and the plaque vulnerability index was calculated using the following formula: vulnerability index = (macrophages %+ lipids %) / (SMCs %+ collagen %). To evaluate the microvessel density (MVD), sections were also subjected to immunostaining with an anti-CD31 antibody Broxyquinoline (Abcam, Cambridge, MA, USA) to label endothelial cells. Microvessels were identified as channels surrounded by a layer of endothelial cells. Density counts of microvessels were calculated as CD31-positive endothelial cells with or without a lumen in 5 randomly selected high-power (400) fields from 6 individual sections of each sample. The MVD was quantified by two individuals who were blinded to the experimental design and is expressed as the average number of microvessels per field. To explore the mechanism of the US-MB treatment-induced reversal of plaque instability, sections were stained with an.