Efficacy of microbubble-enhanced sonothrombolysis on platelet-rich thrombi and erythrocyte-rich thrombi of different ages / 中国医学影像技术

Objective To investigate the efficacy of microbubble-enhanced sonothrombolysis on platelet-rich thrombi (PRT) and erythrocyte-rich thrombi (ERT) in different ages.Methods PRT and ERT in different ages were prepared both in vitro and in vivo of common carotid artery in rats.All the participants were divided into 8 groups with 4 in vitro and another 4 in vivo experiment,including PRT 3 h,PRT 24 h,ERT 3 h,ERT 24h in vitro groups and PRT 3 h,PRT 24 h,ERT 3 h,ERT 24 h in vivo groups.Microbubble-enhanced sonothrombolysis was carried out in both in vitro and in vivo experiments,and the ultrasonic images were collected.The components of PRT and ERT were identified by histopathological examination.The percentage increase of luminal cross sectional area and lytic ratio in vitro,and the recanalization rate and mean blood flow velocity of common carotid artery in vivo were mainly analyzed.Results After sonothrombolysis,both in vitro and in vivo experiment showed there was no statistically significant difference of the percentage increase of luminal cross sectional area ([121.12 ± 13.21]％ vs [130.09 ± 15.34]％),lytic ratio ([39.83± 7.09]％ vs [42.14±5.17]％),recanalization rate (83.33％ vs 91.67％) and blood flow velocity of common carotid artery ([0.21±0.02]m/s vs [0.22±0.01]m/s) between PRT 3 h group and ERT 3 h group (both P＞0.05).PRT 24 h group compared with EPR 24 h group,PRT 24 h group compared with PRT 3 h group,as well as ERT 24 h group compared with ERT 3 h group,the percent increase of luminal cross sectional area,lytic ratio,recanalization rate and blood flow velocity of common carotid artery reduced (all P＜0.05).Conclusion The efficacy of microbubble-enhanced sonothrombolysis on PRT and ERT in vitro and in vivo of of rat common carotid artery model decrease with the increase of thrombus age,especially for the PRT.

Preparation and characterization of microbubbles loaded with hydrogen sulfide / 南方医科大学学报

<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>

Temporal variation of serum interleukin-37 and its relation with C-reactive protein in patients with acute ST-segment elevation myocardial infarction / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the temporal changes of serum interleukin-37 (IL-37) concentration following acute ST-segment elevation myocardial infarction (ASTEMI) and the relationship between IL-37 and C-reactive protein (CRP) in patients with ASTEMI.</p><p><b>METHODS</b>This analysis was conducted in a cohort of 20 patients with an established diagnosis of ASTEMI and 26 patients admitted for chest pain but with normal findings in coronary angiography (control) between June 2012 and December 2013. Venous blood was collected at days 1, 3, 5, and 7 after myocardial infarction for measurement of serum IL-37 and CRP levels using enzyme-linked immunosorbent assay (ELISA).</p><p><b>RESULTS</b>Compared with the control group, the patients in ASTEMI group showed a significant acute elevation of IL-37 level on day 1 following myocardial infarction; IL-37 level reached the peak on day 3 and began to decrease on day 5, followed by a significant decrease on day 7. The time course of post-infarction CRP changes was consistent with that of IL-37 variations and showed a positive correlation the latter (r=0.63, P<0.05).</p><p><b>CONCLUSION</b>IL-37 may participate in the inflammatory responses in ASTEMI.</p>

Ultrasound molecular imaging of endothelial vascular endothelial growth factor recptor-2 expression during hind-limb ischemia-mediated angiogensis / 中华超声影像学杂志

Objective To explore the feasibility of evaluation of mice hind limb ischemia-mediated angiogenesis with ultrasound molecular imaging using molecular probes targeted to angiogenesis endothelial marker VEGFR-2.Methods A mice model of unilateral hind-limb ischemia was induced by femoral artery excision in 12 experimental mice.Ultrasound molecular imaging of the ischemia and contralateral non-ischemia hind-limbs was performed in all mice on day 7 after surgery at 8 minutes after intravenous injection of either VEGFR-2 targeting microbubbles or isotype control microbubbles in random with 30 min interval,and the video intensity (VI) was measured.Following ultrasound imaging,the hind-limb was harvested for immunohistochemical analysis.Results As expected,VI in the ischemia hind-limb was significantly higher (P ＜0.05) for MBvEGFR-2 [(25.6 ± 4.3)U] as compared with MBIso[(6.7 ± 1.6)U].However,the ultrasound signal in the non-ischemia hind-limb was low for both MBvEGFR-2 [4.4 ± 1.5)U] and MBIso [(4.6 ± 1.6)U].A marked endothelial VEGFR-2 expression in ischemia hind-limb was confirmed by immunohistochemistry.Conclusions Ultrasound molecular imaging using molecular probes targeted to angiogenesis endothelial VEGFR-2 can effectively evaluate ischemia-mediated angiogenesis.

Construction and fluorescence intensity of lipid ultrasound microbubbles with monoclonal antibody targeted to leukaemia inhibitory factor receptor / 中华超声影像学杂志

Objective To investigate fluorescence intensity of lipid ultrasound microbubbles constructed in vitro and targeted to leukaemia inhibitory factor receptor (LIFR) with a monoclonal antibody.MethodsThe LIFR-targeted ultrasound mierobubbles (MB-BSB-LIFR-AB) were constructed using a technology of biotin-avidin bridge.FITC labeled Avidin was incubated with lipid ultrasound microbubbles (MB) and biotinylated lipid microbubbles (MB-B).Two dilutions (1:4 and 1:16) of DTAF second antibody were incubated with four types of ultrasound microbubbles,including MB,MB-B,biotinavidin-MB (MB-BS),MB-BSB-LIFR-AB.The fluorescence intensity of microhubbles were graded as 0,1,2to 3.ResultsAfter incubating with FITC-avidin,MB-B displayed bright green fluorescence ( grade 3),but MB had no fluorescence ( grade 0).After incubating with two dilutions of DTAF second antibody (1:4 and 1:16),MB-BSB-LIFR-AB displayed brightest green fluorescence (grade 3) in both concentration,while MB-BS and MB-B only displayed dim green fluorescence (grade 1 ) at the dilution of 1:4,with MB displaying no fluorescence at either dilution (grade 0).Conclusions LIFR monoclonal antibody can be effectively conjugated to MB-B with biotin-avidin bridge.Fluorescence detection is a simple method for investigating the conjugation reliability of targeted lipid ultrasound microbubbles.

Adhesive behavior of dual-targeted microbubbles carrying both Sialyl Lewisx and anti-ICAM-1 monoclonal antibody in vitro / 中华超声影像学杂志

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.

Binding capability of microbubbles targeted to VCAM-1 under pulsatile high-shear flow conditions / 中华超声影像学杂志

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.

Visually assessment of matrigel angiogenesis with ultrasound molecular imaging using microbubbles targeted to endothelial αv-integrins / 中华超声影像学杂志

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.

Preparation of biotin-avidin mediated KDR-targeted liposome ultrasound contrast agent and targeted experiment in vitro / 中华超声影像学杂志

Objective To prepare a new kind of targeted liposome ultrasound contrast agent with small peptide K237 as the ligand which can combine specifically with KDR which is the main receptor of VEGF.and to test its capability in vitro. Methods Targeted bubbles(P-Bio-Av-Bio-Mbs) were formed through "biotin-avidin" bridge grafting, then they were incubated respectively with LOVO, HUVECs and LS174T which were KDR positive or negative expressed in various cells,meanwhile incubated LOVO cells with FITC- P-Bio-Av-Bio-Mbs,FITC-P-Mbs and FITC-Mbs respectively. After that, the rosette formation rate and fluorescence intensity of the combination between microbubbles and cells were observed with microscope and fluorescence microscope. After being incubated with small peptide K237 of 10 μg and 50 μg, LOVO cells were incubated with P-Bio-Av-Bio-Mbs for observing the distribution of microbubbles. Results In KDR sharply positive expressed LOVO cells, the surrounding rosette formation rate was as high as 90. 52% with the fluorescence intensity of grade 3, and it was 53. 46% with grade 2 fluorescence intensity rate in KDR positive expressed HUVECs cells, while in KDR negative expressed LS174T cells, there were few microbubbles surrounded with rosette formation rate of 5. 57% and fluorescence intensity rate of grade 0-1, therefore there were significant statistic differences in rosette formation rate among groups ( P < 0.05). After LOVO cells combined with FITC-P-Bio-Av-Bio-Mbs, FITC-P-Mbs and FITC-Mbs respectively,there were significant differences in their rosette formation rate, namely 89.62%, 7. 56% , 0 with the fluorescence rate of 3,0 - 1 and 0 respectively. Targeted cells pretreated with 10 pg K237 showed significant decreased rosette formation,and there was no formation in 50 ?g pretreated group. Conclusions KDR-Targeted liposome contrast agent with small peptide K237 liganded has been successfully prepared through biotin-avidin mediation and could combine specifically and high efficiently with targeted cells in vitro. The KDR-targeted molecular imaging of tumor neovascularizaiton may provide a new approach for early diagnosis of carcinoma.

Preparation and evaluation of nanometer-scale bubbles with surfaces of N-palmitoyl chitosan / 中华超声影像学杂志

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.

Evaluation of left ventricular diastolic and systolic function of dogs on different status of acute myocardial ischemia with velocity vector imaging / 中国医学影像技术

Objective To evaluate left ventricular diastolic and systolic function of the dogs during different status of acute myocardial ischemia with velocity vector imaging (VVI). Methods Six healthy mongrel dogs were enrolled. Home-made artery constrictor was placed in the proximal coronary artery after opening the chest of dogs to produced different grades of coronary artery stenosis. Mean of peak diastolic velocity (Em), peak strain rate of diastole (Esr), mean of peak systolic velocity (Sm), peak strain rate of systole (Ssr), ejection fraction (EF) and left ventricular pressure were measured. Results There was good linear correlation between Em and Esr by VVI and left ventricular end diastolic pressure (LVEDP) (r=-0.834, P＜0.001 and r=-0.703, P＜0.001, respectively). Em and LVEDP had better correlation than that between Esr and LVEDP (P=0.032). Sm, Ssr or EF had significant correlation with +dp/dt_(max) (r=0.883, P＜0.001, r=0.772, P＜0.001 and r=0.647, P＜0.001, respectively). Significant difference was observed for correlation coefficient between Em and Esr and EF (P＜0.001). Conclusion Em and Sm are sensitive echocardiography indexes to evaluate left ventricular diastolic and systolic function, and are better than EF, Esr and Ssr.

Velocity vector imaging combined with contrast echocardiography in evaluation of relationship between myocardial perfusion and diastolic function in dog models of coronary artery stenosis / 中国医学影像技术

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.

Construction of targeted ultrasound contrast agent with anti-IgG monoclone antibody and the evaluation on the effectiveness of its targeted adhesion in vitro / 中华超声影像学杂志

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.