An Experimental Study Of Ultrasound Imaging With The Self-made Intravenous Acoustic Contrast Agent

ObjectiveRecently, with the improvement of the equipment and the development of the contrast agent, it is a hot topic of ultrasound and make rapid progress. Because it is a noninvasive method, making intravenous myocardial contrast agent is more important. The purpose of this study is to evaluate the safety, steady and repetitively of intravenous self - made contrast agent in normal open chest dogs, and evaluate the myocardium, liver and kidney contrast effect of imaging. Combined second harmonic imaging (SHI) , trigger imaging (TI) and power doppler imaging (PDI) technplogy quantitative assessment of canine normal and graded coronary artery stenosis with myocardial contrast echocardiography(MCE) ,asse-ment of myocardial viability, evaluate coronary flow reserve.MethodsStudy of ultrasound imaging effects and safety with self - made acoustic contrast agentPreparation of self - made sonicated contrast agent: Selected 10 ml aliquots of 5% human albumin and 5% Dextranum 40 were mixed in 20ml upright syringe which connect to one of the three path catheters. A sonicator is used to created microbubbles. The experiment is sonicating 150minutes with levels 100 output. During the early time of the sonicating, 1ml perfluorocarbon gases were injected to the bottom of the syringe from another end of three path catheters. U-sing the 0.5ml lowest liquids, the left is sealed for preparation. Three samples were made for every condition. The size and concentration of microbubbles weremeasured under the light microscopy by the improved Neubauer method.Animal preparation;Eight mongrel dogs (weight, 15kg ~25kg) were used for the experiment. They were anesthetized with 30mg/kg of 30% sodium pento-barbital, intubated, and ventilated by respirator pump. Two catheters were introduced into double femoral veins to infuse contrast agent and fluids. HR,MAP and ECG were monitored during the experiment. A medial thoracotomy was performed , and the heart was suspended in a pericardial cradle.MCE protocol;Philips SONOS 5500 system was used for MCE, with S4 probe and L7540 probe. Intermittent harmonic imaging was performed in the pa-rasternal short - axis plain at mid papillary muscle level. In abdomen, fixed in the right liver lobe, the long axis of kidney. The transducer was placed in a saline bath over the heart which served as an acoustic interface, with the mechanical index set to maximum. At the beginning of the experiment, the gain, the depth and the focus were adjusted at the baseline , the myocardium , liver and kidney was almost black and only endocardial bounders should be visible. The parameters of the equipment (the transmit power, focus, overall gain, depth, et al) were held throughout the experiment. The baseline 2D image was acquired. Self - made contrast agent was intravenous injected by a gently agitate infusion pump at a rate of 0. 5ml/min, 1. Oml/min, 2.0ml/min, 3. Oml/min. End - systolic images were acquired at pulsing intervals ranging from 3 to maximal cycles. The double frames were used. The second image was acquired 15ms after the first one and used as a background image to avoid artifacts. MCE were repeated six times in every mongeral dog. All images were recorded on the MO disk and tape. The ECG and hemodynamic parameters were monitored during the experiment.Tissue slice and pathologic examinationAfter the experiment, the dogs were killed by injection 10% KCL;the hearts were removed, and cut into 0. 5cm thick slices. Pick up three piece of tissue from different region with lung, liver, kidney respectively , The slices were immersed in a solution of 10% formalin and stained by the routine HE method.Quantitative Assessment of canine myocardial blood flow by self -made intravenous myocardial contrast agentAnimal preparation: Fifteen mongrel dogs were used. They were prepared to MCE as the same with previous experiment. A median thoracotomy was performed, and the heart was suspended in a pericardial cradle. The proximal of the left arterial descending coronary artery ( LAD) was dissected freely from surrounding tissue and was made graded coronary stenosis by the bloker, placed a electromagnetic blood flowmeter in the approximately of LAD. radiolabeled mi-crospheres were injected into the left artium after MCE for comparision .15 dogs were underwent progressive stenosis of LAD, graded coronay stenosis was divided with mild( <25% ) , moderate ( <50% ) and serious ( >75% ) graded coronary stenosis. Contrast were used in 12 open chest dogs at baseline, during hyperemia with adenosine( 140|xg/kg/min,intravenous for 6 minutes)and in different severity of LAD stenosis. LAD was occlusion for 3 - 4 hours, followed by 2 hours reperfusion.MCE protocol: PHILIPS SONOS 5500 echocardiographic system and S4 probe was used. Contrast, Second Harmonic Imaging and Angio were introduced. The parameters of the equipment were held throughout the experiment. The baseline 2D image was acquired. Self - made contrast agent was intravenous injected by a gently agitate infusion pump at a rate of 1. Oml/min. The double frames were used. The second image was acquired 15ms after the first one and used as a background image to avoid artifacts. End - systolic images were obtained at pulsing intervals ranging from 1 cycles step by step to steady imaging.Date analysisQualitative analysis: visual observation, The contrast was graded qualitatively as 0 = no contrast, 1 = some visual contrast, 2 = moderate, 3 = marked visual contrast.Quantitative analysis: Analysis of video intensity: Power images were transferred into digital image using Photoshop 7.01 software. The video intensity was divided by 0 ~255. The unit was grey unit. The samples were obtained with LV myocardium, livers and kidneys respectively. The left ventricle was imaged on short - axis view at mid - papillary muscle level, and assigned to 6 segments: anterior, lateral, posterior, inferior, posteroseptal, and anteroseptal. Averagepixel intensity was measured in regions of interest drawn over the LAD and LCx beds in aligned frames selected from different pulsing intervals. The videointensi-ty measurements were plotted against pulsing interval and fit to a previously described exponential function: y=A(l -e~pt), where y is equal to videointensi-ty measured at varying pulsing intervals, and t, A, and are parameters derived from curve fits as described below. (3 is mean velocity, A is a peak plateau videointensity, MBF(A ? (3 value) was calculated by multiplying the rate of replenishment and the plateau of myocardial video intensity.MBF measurement by radiolabeled microspheres: Approximately 2x106 30 - 50|jjn radiolabeled 5 mci mTc - MAA microspheres suspended in 4 ml 0. 9% saline solution were injected into the LA at last stage. Reference samples were withdrawn from the femoral artery over 180 seconds with a 5 ml/min constant -rate withdrawal pump. At the end of the experiment, the short - axis slice of the left ventricle corresponding to the MCE image was cut into 16 wedge - shaped pieces. All samples were counted in a well counter with a multichannel analyzer. Corrections were made for activity spilling from one window to the next with a custom - designed computer program.Flow to each myocardial sample was calculated from the-equation Qm = (Cm xQr)/Cr, whereQm is blood flow to the myocardial segment( ml/min) , Cro is tissue counts, Qr is rate of arterial sample withdrawal (ml/min) , and Cr is counts in the arterial reference sample. Transmural blood flow (ml ? min"1 ? g"1) was derived by dividing the sum of flows to the individual segments by their combined weight.Tissue slice and pathologic examination0. 5% Evan's Blue 10ml were injected in left artium. After the experiment, the dogs were euthanized;the hearts were removed, and cut into 0. 5cm thick slices. The slices were immersed in a solution of 2% 2,3,5 -tripheylterrazoli-um chloride at 37X, for 20 minutes, and the infracted myocardium was identified as the region that failed to demonstrate brick red staining followed by fixation in 10% formalin after they were took photos by a Sony digital camera. The myocardium from different regions according to the TTC dye were cut and stained by the routine HE method.Statistical analysis-. The data are expressed as mean SD. The data was compared with t test. The correlation was evaluated using the linear regression a-nalysis technique. The statistical analysis was performed using version 11.0 of SPSS and Microsoft Excel 2000 software.ResultsStudy of ultrasound imaging effects , reproducibility and safety with self -made acoustic contrast agent The mean size of the microbubbles of the contrast agent was about 3. 20 ±0.75 u,m, and the concentration was 3.7 x lOVml. The size of 95% of the microbubbles were < 6. 0|xm. The steady of microbubbles was 96% in normal temperature. Microbubbles were made and injected repeatly in different time,the results were no significantly difference.Heart rate, mean aortic pressure, arterial blood gas and ECG were monitored during the experiment, there weren' t showed any distinctive changes (P > 0.05). The results showed no significantly change in biochemical indexs of e-lectrolyte, serum myocardial enzymogram, liver and kidney functions, and so on. Microbubles produced no hemodynamic changes and no significant side effects. Multi - orangic pathological examination were normal.Contrast effect:Qualitative analysis;Satisfactory opacification is acquired after contrast a-gent injection in the normal open chest dogs. Progressively greater opacification were notified in the normal myocardium, hepatic parenchyma and renal cortex.Myocardium opacification was obvious, the success rate of MCE is 100%. MCE with four kinds injection velocity all produced good myocardium opacification, and velocity of 1. Oml/min, 2. Oml/min both produce satisfactory opacification.After contrasting, 2D grey scale of liver and kidney were obviously enhanced. Echo of vessels in hepatic was enhanced, the pass -way was clearly to be seen. Doppler showed blood flow signs strengthen. Opacification of liver was a litter better than that of kidney with same amount of contrast agent.Quantitative analysis:Analysis of video intensity:Before and after MCE, the contrast VI differerces in the normal myocardium, hepatic parenchyma and renal cortex were significant (P <0.05).PDI showed that there were red and yellow in myocardium uniform.In rest, MCE data derived from MCE time - VI curve in normal and mild, morderate coronary stenosis are not significent differerces. But MCE data are signidicent changes between normal and serve coronary stenosis.A ? 3 ratio with rest and adenosine are different. There are more than three times in normal coronary, but only little increase in differerce coronary stenosis. We can assess the graded coronary stenoses by normalizaed A ? p.Study the accurate of measurement regional myocardial blood flow with MCE, the normalized A ? ￡} in ischemic regions /non ischemic regions coparee with the normalized MBF measurement by radiolabeled microspheres, there was a good correlation between the normalized A ? 3 and the normalized MBF ( y = 1. 002x -0.041 ,r=0. 844,P<0.01).In resting and adenosine stress, MCE parameters were compared with different coronary artery stenosis, the changes were used to evaluate coronary flow reserve, and evaluate coronary artery stenosis degree.The ratio of the perfusion defected areas measured with left ventricular area by energy mode were compared with the ratio of infarct area of TTC histochemic stain with left ventricular area, y = 1.481x +3. 761 ,r =0. 816,P <0.01.Large perfusion defected area could be observed immediately after coronary occlusion, the size of perfusion defected area decreased following the occlusion maintain 3 ~4 hours. After reperfusion, infarct area can be assessed accurately with adenosine stress MCE. MCE parameters of normal region, ischemic region and infarct were changed significantly.ConclusionThe mean size of the microbubbles of the contrast agent was about 3.20 ± 0.75 jxm, and the concentration was 3.7 x lOVml. The size of 95% of the microbubbles were <6.0jn,m. The microbubbles were made homogeneous, steadyand reproducibility.Heart rate, mean aortic pressure, arterial blood gas, ECG , electrolyte, serum myocardial enzymogram, liver and kidney functions were monitored during the experiment, Microbubles produced no hemodynamic changes and no significant side effects.Satisfactory opacification was acquired after injection self - made contrast a-gent in the myocardium, hepatic parenchyma and renal cortex.MCE images were acquired with SHI,TT,PDI by intravenous injection self - made contrast agent, time - intensity curves can be obtained. A ? (3 value of MCE were well correlated with MBF measurement by radiolabeled microspheres, MCE can access the regional myocardial perfusion quantitatively.In resting and adenosine stress, MCE parameters were be compared with different coronary artery stenosis, the changes were used to evaluate coronary flow reserve, and evaluate coronary artery stenosis degree.After reperfusion, the ulmost infarct area could be assessed accurately with Adenosine stress MCE, and detect the savage myocardium of the risk area.