| PrefaceAtherosclerosis ( AS) is one of the most common systemic cardiovascular disease, which related to kinds of risk factors and involved in many vessels with serious damage. Early diagnosis and treatment are important. Recently, many scholars explored the methods to make early diagnosis and improve the accuracy by ultrasonic technology. Rabbits often used as AS models for its similar vascular style to human and sensitive to cholesterol food. But the ultrasonic study is a bit restricted because abdominal aorta wall is thin and the location is deep.With the apparence of new kinds acoustic contrast agent and development of contrast technology, contrast ultrasonography has become a focus in research nowadays, which can significantly enhance the scatter signal, improve the sensitivity of ultrasonic diagnosis and has a promising aspect. At present, more studies had been applied in liver and heart, while,less in vessel disease.The purpose of this study is to observe grey scale, pulse and color Doppler imaging of normal rabbit common carotid artery (CCA) and abdominal aorta (ABAO) using conventional imaging mode by intravenously injected sonicated perfluorocarbon albumin contrast agent, to analysis the time - intensity curve (TIC) changes using different injective means. Rabbit atherosclerosis models were established, the grey intensity and ultrasound gradeing scale on vessel wall lesions before and after contrast were observed. Acoustic density changes of abdominal aorta wall were detected by acoustic densitometry. Compared with the same period pathology and histology study, changes of plaque detectable rate before and after contrast were retrospectively analyzed, the value of contrast agent on atherosclerosis and the feasibility of acoustic densitometry on enhanced effectswere explored and thus provide assist for further atherosclerosis study.Methods1. Contrast enhanced ultrasonography on CCA and ABAO in normal rabbits and changes of time - intensity curve by different injection methodsAnimal preparation: Ten healthy Japanese white rabbits, male or female, weighing 2. 0 -2. 2 kg, were anesthetized with pentobarbital, secured in supine position and abdomen was shaved. Routine surface electrocardiogram was monitored throughout the experiment and scalp acupuncture was placed in ear vein for contrast agent administration.Contrast agent preparation;Mixed dextrose albumin solution 5ml was sonicated with 0. 75 ml perfluoropropane using ultrasonic cell comminuting machine (Xinzhi JY92 - II type ) , at frequency of 25 KHz, diameter of acoustic probe was 6mm and the power was 500W. Ivory white microbubble agent was sealed for later use.Instruments;HP Sonos 5500 ultrasound system with 11 -3L linear phased array transducer was used, sample depth was 4cm for ABAO and 3cm for CCA. Mechanical index was 0.4 . The total gain, LGC and TGC were adjusted for ideal image, other settings were remain unchanged during the study.Contrast protocol and results analysis: ( 1 ) Conventional image mode;The transducer was placed parallelly along trachea, after optimal image of right CCA fixed , contrast agent was injected at a dose of 0. 5ml/kg by bolus tthrough ear vein, followed by a saline flush (1 ml). The spectral and color Doppler gain was set to a low level before injection to allow only weak signal can be seen. Images of gray scale and Doppler signal before and after contrast were acquired. Abdominal aorta was scanned longitudinally 15min later and previous process was repeated. Gray - intensity and Doppler signal were defined as;no enhanced signal was recorded as " - " , enhanced as " + " , and significantly enhanced as " + + ". Systolic peak blood velocity was measured before and after contrast. (2) A-coustic Densitometry (AD) and contrast process with intermitted trigger imaging mode: The trigger intermission was 1:3 cardiac cycle on T wave, contrast agentswere administered at a dose of 0. 5ml/kg by bolus injection and continuous infusion (0. lml/s) Respectively, at intervals of 15 min. Images were acquired before and after contrast administration and analyzed using internal AD analysis software, 21 x21 pixel round sample volume was applied to manually trace the relative same position of the vessel center by slow, speed. TIC was derived automatically and time - to - peak ( TP) , peak intensity ( PI) , descending slope (DS) , mean transit time (MTT) were displayed simultaneously.2. Contrast enhanced ultrasonography on abdominal aorta atherosclerosis and comparison study with pathologyAnimal preparation: Twenty - four healthy Japanese white rabbits, male or female, were averaged divided into three groups: control group ( group A ) , cholesterol groups (group B and C, raised by 2g cholesterol per day added to the common diet) , group B, group C and four rabbits in group A were checked respectively in the 8th and 16th week time point.Methods: Abdominal aorta of rabbits were longitudinally scanned by high -frequency ultrasound transducer, after getting optimal image, contrast agent was injected at a dose of 0. 5ml/kg by bolus through ear vein, followed by a saline flush(lml). Grey scale, M - mode image and integrated backscatter of ABAO were stored before and after contrast agent injection.Visual analysis: Compared with images before contrast, intimal - medial signal echo were defined as : no enhanced signal was recorded as " - " , enhanced as " + " , and significantly enhanced as " + + ". Number and location of plaques (signed by original branch of ABAO) were observed. According to Chin, the following ultrasound scale of 1 to 4 was used to denote an increasing degree of pathological changes: 1, normal apperance;2, thickened intimal - medial;3, thickened wall plus increased wall irregularity or roughness;4, discrete, well - defined lesions observed in echoes generated. Half - scores were assigned when the status of the vessel wall was deemed to fit an intermediate category.AD analysis: Rectangle sampling was selected with the size of 11 x 11 pixel, interested region including ABAO wall,lumen and adventitia along the same scanning beam were sampled using the track ball in slow velocity to avoid the interfere among each layer. Two spots were measured each time and three timesfor each spot, then averaged integrated backscatter value was get before and after contrast, including lumen (All - L) N intimal - medial ( AH - I) Aadventitia( All - A) and revised intimal - medial intensity, All equaled to the ratio of All - I and AH - L.Biochemical analyses: Serum levels of TC and LDL - C were measured u-sing 2ml blood sampling from ear middle artery at 8 weeks and 16 weeks, respectively.Specimen sampling and fixation : After ultrasonic examination, vessels were quickly separated, resected and washed by saline. Number and location of ABAO plaques were observed by naked eyes, plaques were defined as light yellow or dark yellow projections. Vessels were numbered and partly left for frost slice Sudan IH stainning, others immersed in 10% formalin, 2. 5%glutaral for HE stainning, immunohistochemistry and transmission electronic microscope checking, respectively.Light microscope checking: Routin paraffin slice was HE stained and observed in microscope, retrospective analysis was carried out on ultrasound checking before and after contrast.Electronic microscope checking;Specimen were rinsed, dehydrated, saturated, embeded in araldite and chipped to 50 ~70nm slices, the ultrastructure changes were observed.Immunohistochemical checking of bFGF^PCNA: Using Meta Morph Image System, expression of bFGF and PCNA in intimal under light microscope field of 400 times was observed, five fields were randomly selected in each- slice. Results were analyzed;bFGF was expressed in cell plasm, so cells with brown plasm were positive cells, average density of positive cells were measured. PCNA located in cell nuclear, cells with brown nuclear were positive cells, expressed ratio in intimal, namely the proporation of positive cells in total cells, was calculated.Results1. Self - made perfluorocarbon ultrasound contrast agent was ivory solutioncontaining microbubbles,the mean size was (3. 10 ±0. 81 ) jxm and concentration was 2. 8 x 10 /ml measured by HPIAS - 1000 image analysis system.Conventional image mode: Contrast effects were similar in CCA and ABAO: After 3 ~8s bolus injection, vessels were rapidly enhanced by dense bright ech-o, the duration of strong contrast effect was 12 ~28s. Pulse -wave spectral and color Doppler signal was markedly enhanced, and there were clearer or prolonged near branches of abdominal aorta presented in 6 rabbits. Then duplex gray -scale signal of vessel was gradually reduced, no enhancement can be seen till 60 - 142s, while there was still enhanced Doppler signal. The velocity of blood flow has no significant difference before and after contrast.Quantitative analysis of TIC: After bolus injection, TIC was typically presented as a single - peak curve, which steeply reached a peak value and then gradually decreased to baseline. While, by infusion injection, TIC slowly increased to a lower peak value and there exist a plateau until reduced to base level followed by cease of injection, DS can not be measured.2. Comparison study of visual scale, ultrasound grade and plaque detectable rate on ABAO lesions with pathology findings before and after contrast injection : After contrast agent injection, lumen was rapidly enhanced by dense bright echo, vessel wall image changes were observed after agents eliminated along with blood flow. Visual grades of wall grey - scale were significantly increased in group B and C. Ultrasound grade of image was clearly increased compared with that before contrast. Layer of ABAO wall was not clear enough in group A, whil? M - mode curve showed bright" double line pattern" after contrast, the structure was normal by pathology. Intimal - medial thickness of posterior wall of aorta was increased in group B, but anterior wall was not clear, after contrast, thickened intimal - medial displayed irregularly enhanced with the anterior wall signal improved. There were many foam cells in group B, which were located a-bove intima and displayed as jacinth by Sudan HI staining and can be defined as lipid deposition. Where there was no markedly thickened intimal - medial, only appeared enhanced spots on intimal,which proved to be regional lipid streak and tiny plaque. There were local moderate echo inbursted into lumen in group C, which appeared faintness before contrast and had much clear range with en-hanced surface image after contrast, filling defect in lumen when contrast agent injection also can be seen by plaque location, which was proved to be fibrin hy-perplasis plaque or early stage of atheromatous lesion.Relation between ultrasound grade and pathology: Ultrasound grade 1, aorta normal;ultrasound grade 2, foam cells deposit in intima with a few smooth muscle cells proliferation and fatty streak or plaque;ultrasound grade 3, diffused intima thicken and early stage of fibrin plaque;ultrasound grade 4, typical fibrin plaque or early atheromatous lesion..AD - IBS analysis : IBS value can not be measured in group A for the thin vessel wall. While All -1 and All value were increased in group B and C with prolonged experiment period. Compared with that before contrast, All - I, All ■-A and All value after contrast were all augmented and the difference was significant.Comparison of ABAO plaque detectable rate: No plaque was seen in group A, there were 9 local light yellow plaques projected in group B and 17 in group C, especially in arterial bifurcation. Detectable rate of plaque was increased with prolonged experiment period. Intima image was enhanced after contrast a-gent injection and caused plaque detectable rate increased, there were two and three plaques left out by conventional ultrasonic exam in group B and group C, respectively, which was found in contrast process and continued to enhance after contrast agent passed and proved to be lipid deposited and local thickening by pathology.Biochemical analyses: Serum levels of TC and LDL - C were within the normal level in group A and markedly increased in group B and group C.Electronic microscope results: Endothelium cell in group A was normal and elastic board regular, SMC in media appeared long shuttle or paralleled layers, containing abundant cell organ, chromatin was uniformity, SMC presented as contract style. In group B,endothelium cell was not contact,a great of foam cells under intima, SMC appeared between synthesized and contract style, cell organ was less and lesion extent was slight. The lesion was more serious in group C, SMC transformed to synthesized style in media, cell nuclear was big and round, plasm was abundance with lots of cell organ, which related with secretion, suchas endoplasmie reticulum and Golgi complex, actin filament reduced. Cell nuclear was shift and distortion because of swallow much lipid in plasm, there were also great deal of extracellular matrix.Immunohistochemical checking: There was no positive expression in group A. Positive cells can be seen in group B and group C, which was higher in group C compared with group B. bFGF - positive signal mainly existed in proliferated intimal and medial plasm, PCNA - positive signal mainly expressed in nuclear. Compared experiment was negative using PBS instead of bFGF and PCNA.Conclusions1. Sonicated perfluorocarbon ultrasound contrast agent injected via rabbit ear vein can achieve relatively perfect enhanced artery images, the preparation of contrast agent is simple and provide necessary condition for further artery contrast study.2. Relative concentration changes of contrast agent can be analyzed using TIC acquired by AD. After bolus contrast agent injection,TIC rapidly reached to peak intensity. While by infusion method, TP was longer,enhanced PI was reduced and MTF was prolonged, which can be helpful for lesion observation and diagnosis.3. Contrast enhanced ultrasonography can improve atherosclerotic wall image signal, ultrasound grade and plaque detectable ability, help to discover early stage of atherosclerotic lesion and accurately evaluate pathological degree.4. AD - IBS can detect vessel wall texture changes, monitor AS development and quantitatively analyze contrast enhanced acoustic intensity variation to provide further objective study measures.
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