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Objective:To evaluate the effect of reactivated ultrasound contrast agent on prevention of ischemia-reperfusion (IR) injury in rat hindlimb ischemia model.Methods:Microbubbles were compressed into nanodroplets (NDs) and the particle size range was determined.In vitro experiments were carried out to observe the acoustic phase transition of nanodroplets in the ultrasound field and the change of dissolved oxygen (DO) concentration in the surrounding solution. Forty-one male Sprague Dawley rats were divided into 5 groups: acoustic nanodroplet vaporization treatment group (NDs+ US group, n=9), saline+ ultrasound treatment group (Saline+ US group, n=8), nanodroplet without ultrasound treatment group (NDs group, n=8), ischemia/reperfusion injury group (IRI group, n=8) and sham operation group (Sham group, n=8). Ultrasound imaging was performed before operation and 12 hours after reperfusion to evaluate the improvement of hemodynamics of criminal artery under different treatments. Tissue injury were evaluated by analyzing immunohistochemistry staining results. Results:The formed NDs ranged in size from approximately 68.0-295.4 nm and the highest concentration in the 100 nm range. In vitro studies, a decrease in DO was measured during the phase transition.In the animal experiment, after ischemia/reperfusion, NDs+ US, Saline+ US, NDs and IRI groups demonstrated a significantly higher resistance index (RI) and pualsatility index (PI) of the right common iliac artery compared with before operation (NDs+ US group: PI 1.79±0.17 vs 1.57±0.23, P=0.014; RI 0.80±0.02 vs 0.75±0.04, P=0.002. Saline+ US group: PI 2.29±0.16 vs 1.57±0.16, P<0.001; RI 0.90±0.06 vs 0.74±0.03, P<0.001. NDs group: PI 2.17±0.14 vs 1.53±0.15, P<0.001; RI 0.91±0.04 vs 0.75±0.04, P<0.001. IRI group: PI 2.12±0.22 vs 1.58±0.20, P<0.001; RI 0.88±0.04 vs 0.75±0.04, P<0.001). The increases of PI and RI (ΔPI, ΔRI) in NDs+ US group were higher than those in sham group (all P<0.05), but significantly lower than those in saline+ US group, NDS group and IRI group (all P<0.05). Immunohistochemical results indicated the percentages malondialdehyde (MDA) positive cells in Saline+ US, NDs and IRI groups were higher than those in NDs+ US and sham groups (all P<0.05). Conclusions:The acoustic phase transition of nanodroplets in the ultrasound field can reduce ischemia/reperfusion injury and improve hemodynamics abnormality after reperfusion.
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<p><b>OBJECTIVE</b>To prepare a phospholipid-coated microbubble loaded with hydrogen sulfide (HSMB) and evaluate its physicochemical and acoustic properties.</p><p><b>METHODS</b>Hydrogen sulfide and perfluoropropane were mixed at the ratios of 4:0, 3:1, 2:2, 1:3, and 0:4 to prepare hydrogen sulfide-loaded microbubbles (termed HSMB4:0, HSMB3:1, HSMB2:2, HSMB1:3, and HSMB0:4, respectively). The microbubble concentration and diameter were investigated and their stability were evaluated. The optimal ratio of hydrogen sulfide and perfluoropropane was determined according to the changes of microbubble concentration. The changes of dissolved hydrogen sulfide and concentration of the microbubbles were investigated after exposure to ultrasound, and their acoustic enhancement effects in the myocardium and kidney were observed after intravenous injection in rats.</p><p><b>RESULTS</b>HSMBs were milky in color and spherical in shape without aggregations. The concentrations of HSMB4:0 and HSMB3:1 were lower than that of HSMB2:2 and decreased with time. HSMB2:2, HSMB1:3 and HSMB0:4 showed comparable concentrations and were stable within 72 h. After exposure to ultrasound, the concentration of HSMB2:2 decreased while the dissolved hydrogen sulfide increased significantly. Intravenous injection of HSMB2:2 produced a satisfactory contrast-enhancing effect in the myocardium and kidney of rats.</p><p><b>CONCLUSION</b>HSMB prepared with the hydrogen sulfide to perfluoropropane ratio of 2:2 has excellent contrast-enhancing effect and is capable of carrying and releasing hydrogen sulfide upon ultrasound exposure to potentially allow visual site-specific delivery of hydrogen sulfide.</p>
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Animals , Rats , Contrast Media , Chemistry , Fluorocarbons , Chemistry , Heart , Hydrogen Sulfide , Chemistry , Kidney , Microbubbles , Phospholipids , Chemistry , UltrasonicsABSTRACT
Objective To assess the adhesive behavior of dual-targeted microbubbles carrying both Sialyl Lewisx and anti-ICAM-1 monoclonal antibodies in vitro. Methods Selectin-targeted (with Sialyl Lewisx) microbubbles (MB-S),ICAM-1-targeted (with anti-ICAM-1 monoclonal antibodies) microbubbles (MB-Ⅰ),and dual-targeted (with both ligands) microbubbles(MB-D) were prepared by attaching the ligands to the biotinylated lipid-microbubbles via multi-step avidin biotin bridging chemistry. A parallel plate flow chamber combined with a novel automated tracking algorithm,were used to analyze the transient velocities,rolling and firmly adherent numbers of microbubbles at various shear stress (0. 6,2.0 and 4.0 dyn/cm2)over 6 min. Microbubbles detachments were tested by ramping up the shear stress at 30 s intervals. Results At 0.6 dyn/cm2 shear stress, the rolling numbers of MB-S and MB-D were remarkably more than that of MB-I( P＜0.05), while at 2.0 and 4.0 dyn/cm2 MB-S performed higher rolling efficiency as compared with either MB-I and MB-D ( P＜ 0.05). In all flow conditions, the adhesive numbers of MB-D to the targets were obviously greater than those of MB-S and MB-I ( P＜ 0.05). Half-maximal detachment decreased gradually in MB-I, MB-D and MB-S by turns ( P＜ 0.05). Conclusions MB-I, MB-S and MB-D have different adhesive behaviors. MB-I exhibites primarily firm adhesion with low rolling efficiency, while MB-S reveales unstable or transient adhesion with high rolling efficiency,and MB-D exhibites firm adhesion with high rolling efficiency. MB-D may be suitable for molecular imaging in high-flow vessels.
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Objective To develop nanometer-scale bubbles with surfaces of N-palmitoyl chitosan(PLCS) as ultrasound contrast agent and evaluate its characteristics and acoustic effects in vivo. Methods The PLCS nanobubbles were prepared using a cutting technique at differential high-frequency of shear speed. Both optical and transmission electron micrography were performed to determine the nanobubble size and morphology. Concentration, size-distribution and zeta potential of the PLCS nanobubbles were measured by cell counting chamber, Malvern lazer particle analyzer and zeta-sizer at 1-day, 45-day and 90-day. The acoustic effects of the PLCS nanobubbles on myocardium and renal tissue in 6 normal rats were observed using bolus infusion of the nanobubbles intravenously. The maximum video intensity(VI) was measured.Results The PLCS nanobubbles with nice round-shape and uniform site-distribution were demonstrated.The mean diameter,concentration and zeta potential of the PLCS nanobubbles were (617 ± 12) nm, (7.2 ±0.6) × 109/ml and (52.9 ± 1.3)mV at the 1-day,and all of parameters did not change significantly in 45-day and 90-day ( P ＞ 0. 05). A significant contrast-enhancement was noted on myocardium and renal tissue during infusion of the nanobubbles. VI on both tissues was (15.6 ± 1.1)GU and (27.3 ± 2.5)GU,respectively. The visual contrast-enhancement last up to (10 ± 2)min. Conclusions The PLCS nanometerscale bubbles have excellent physical-features and contrast-enhanced ultrasound effects in vivo. It may develop as a novel contrast ultrasound agent which could cross endothelial cell membrances.
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Objective To assess the binding capability of microbubbles targeted to VCAM-1 using the parallel plate flow chamber mimic the pulsatile high-shear flow conditions of artery. Methods Targeted microbubbles were designed by conjugating monoclonal antibodies against mouse VCAM-1 to the lipid shell of the microbubbles via an "avidin-biotin" bridge. The binding and retention of targeted microbubbles to VCAM-1 (MBv) immobilized on a culture dish were assessed in a flow chamber at variable shear stress (0.5～ 16.0 dynes/cm2 ). The pulsatile flow conditions were generated and compared to the continuous flow conditions. The retentive ability of MBv was evaluated by the detachment test. Results The marked binding of MBv were seen in pulsatile and continuous flow conditions at low-shear flow conditions of 0.5 ～ 2dyn/cm2 ,but the binding rate in the pulsatile flow group was higher ( P ＜0. 05) than that in the continuous flow conditions. Furthermore,the marked binding of MBv was still noted at the highest shear rates (4～8dyn/cm2) under pulsatile flow conditions, while it was not observed under continuous flow conditions. The half detachment rate of MBv was high up to (20.7 ± 3. 1)dyn/cm2. Conclusions The targeted microbubbles binding to VCAM-1 specific and effective at high-shear stress under pulsatile flow conditions. The molecular ultrasound imaging can be potentially used in the high-shear conditions artery system.
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Objective To explore the feasibility of visually assessment of angiogenesis in a murine model of subcutaneous matrigel plugs with ultrasound molecular imaging(UMI) using microbubbles(MB)targeted to endothelial αv-integrins. Methods Matrigel angiogenesis was created by subcutaneous implantation of FGF-2 enriched matrigel in 10 mice. On day 10, UMI of the matrigel was performed in all mice at 6 minutes after intravenous injection of either αv-integrin targeting microbubbles(MBα) or isotype control microbubbles(MBc) in random with 30 min interval,and the video intensity(Ⅵ) was measured. To further test the specificity of the signal coming from MBα,antibody against αv-integrin was injected 10 min before microbubbles injection. Following UMI,all matrigels were harvested for histological analysis. Results As expected,VI of the matrigel was significantly higher ( P ＜0.05) for MBα (20. 5 ± 3.3)U as compared with MBc (4. 8 ± 1.5)U. After blocking with antibody against αv-integrin,a great decrease was observed in the MBα group [VI (4.6 ± 1.2) U, P ＜0.05] while no significant difference was noted for MBc [VI (4. 9 ±1.5)U, P ＞ 0.05 ]. Neovessels within matrigel was positive for αv-integrin. Conclusions UMI with microbubbles targeted to αv-integrins can be effective and specific in evaluating the angiogenesis in a murine model of subcutaneous matrigel plugs.
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Objective To evaluate the relationship between myocardial perfusion and diastolic function with velocity vector imaging (VVI) combined with myocardial contrast echocardiography (MCE) in dog models of coronary artery stenosis at rest and stress. Methods Different stenoses in anterior descending branch were made in 8 dogs. Before and after coronary artery stenosis, VVI evaluation was made on short axis image, then MCE were performed in the left ventricular mastoid muscle section at rest and in the peak dose of dobutamine. The myocardial blood flow A·β value and peak diastolic strain rate (SR_(dia)) on the direction of the circumference of the short view were measured, and the relationship between them was analyzed. Results At rest, no significant difference of A·β value nor SR_(dia) was found between the stenotic bed and normal bed when coronary stenosis was mild or moderate. However, A·β value and SR_(dia) of the stenotic bed were smaller than those in the normal bed when coronary stenosis was severe (P<0.05). At dobutamine stress, A·β value and SR_(dia) of the stenotic bed were already less than those in the normal bed when coronary stenosis was mild or moderate. A·β values and SR_(dia) of the stenotic bed decreased further compared to the normal bed (P<0.05) when coronary artery was severe. At both rest and stress, the standard A·β value was strongly correlated with SR_(dia) (r_(rest)=0.57,r_(stress)=0.72,P<0.01). Conclusion VVI can not only evaluate the diastolic function of myocardial segments on the short axis view, but also reflect changes of myocardial perfusion to a certain extent.
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Objective To construct targeted ultrasound contrast agent carried goat anti-mouse IgG antibody (UCA-IgG) and evaluate the effectiveness of its targeted adhesion using parallel plate flow chamber. Methods The ultrasound contrast agent targeted to mouse IgG was designed by conjugating monoclonal antibodies against mouse lgG to the lipid monolayer shell of the agent using biotin-streptavidin. The binding of IgG antibodies to the ultrasound contrast agent were identified by fluorescence in vitro. The attachment and detachment of UCA-IgG to mouse IgG immobilized on a culture dish were assessed in a parallel-plate flow chamber. While the plate lacked mouse IgG,or blocked with large number of goat anti-mouse IgG were served as two control groups. Results UCA-IgG issued a bright green fluorescence, while the contral lipid ultrasound contrast agent didn't show fluorescence. The number of UCA-IgG bound to mouse IgG of experimental group was greater than two control groups,increased with increasing coverslips surface antibody concentrations (P<0. 05),and there was significant positive correlation between the number of UCA-IgG bound to mouse IgG and time of combination (P<0.05). The adhesion rate of experimental group increased with shear stress before 0. 5×10-5 N/cm2 (P<0.05) and then decreased (P<0. 05). There was limited adherence of control groups to the UCA-IgG. The stess of half-maximal detachment was increased with increasing coverslips surface antibody concentrations (P<0.05). Conclusions UCA-IgG could adhere to mouse IgG in the physical conditions. It may provide strong supports for studying other targeted ultrasound contrast agent preliminary and fatherly in vitro.