Pathological Study On Myocardial Permeability During Contrast Echocardiography In Rabbit

BackgroundAcoustic cavitation is an important mechanism for nonthermal bioeffects of ultrasound (US) and had been important in US research. With clinical application and basic research of microbubble ultrasound contrast agent (UCA), both of the bioeffects and therapeutical uses of microbubbles (MB) had been found, MB augmented cavitation had gained a wide interests. As a result, more and more attention had been paid to the bioeffects of MB.On one hand, intravenous injection of microbubbles could significantly enhance the bioeffects of US by MB mediated cavitation as cavitational nuclei. On the other hand, sonoporation and microstream generated from carrier MB disruption could be used for gene transfection or drug delivery. So, it is important to know the right way to prevent bioeffects and to carry out therapy at the same time because of the double effects of US cavitation.The side effects of the MB induced cavitation had been found in many aspects. Bioeffects like premature ventricular contractions (PVC), increased myocardial vascular permeability, petechial hemorrhages and cardiomyocyte injury were identified during contrast echocardiography under high mechanic index (MI) by Li and Miller, et al. These bioeffects tended to be closely related to the MBs dosages and the values of ultrasound MI. Cell damages indicated by Evans-blue (EB) -stained cardiomyocytes and inflammatory cell infiltration were also discovered by Miller, especially under stress test.So far, pathological studies of the microvascular bioeffects were only limited to microscopy level. The ultrastructure of related microvascular injury had never been investigated. In addition, the microvascular effects regarding stress condition needs further understanding. This study was designed to investigate mainly the pathology and microstructure ultrastructure of microvascular injury under stress or routine condition.Objectives:This study was designed to explore whether myocardial stress is an independent impact factor of myocardial bioeffects in contrast echocardiography and to investigate the related pathological changes of impacted microvascular permeability.Methods:92 healthy New Zealand rabbits with an average weight of 2.0 kg were included and divided as in Table 1. All rabbits were manipulated under proper anesthesia. Table 1. The experimental groups and the corresponding procedures Groups Microbubble(ml/kg) anesthetics imaging modalities The simple MB 0.5 xylazole and EDTA no The simple US continuous 0 xylazole and EDTA continuous diagnostic USThe simple US triggered 0 xylazole and EDTA triggered diagnostic US The routine MCE continuous 0.5 xylazole and EDTA continuous diagnostic USThe routine MCE triggered 0.5 xylazole and EDTA triggered diagnostic US The stress MCE continuous 0.5 xylazole and EDTA continuous diagnostic USThe stress MCE triggered 0.5 xylazole and EDTA triggered diagnostic USUltrasound was delivered to in vivo myocardium using a commercial diagnostic ultrasound imaging system, a GE Vivid 7 with M3S probe. The US central frequency was 1.75 MHz and mechanic index was set to 1.3.Microbubbles: Zhifuxian, a perfluoropentene filled lipid microbubbles with bubble concentration at 7×109/ml, average 2μm in diameter, and 98% of the MBs less than 8μm.Electrocardiogram was recorded during all ultrasound exposure. EB was used as a blood-pool tracer and the EB content in myocardium was harvested by formamide extrudation. EB leakage of myocardium and petechial hemorrhages of myocardium were visually quantified by subjective scores. At last, pathological exam of myocardium was performed using microscopy and transmission electron microscopy (TEM).Results:No PVC was found in the simple ultrasound and the simple MB groups, but PVCs were found in all the other experimental groups. There were no statistical PVCs differences among the experimental groups.The myocardial EB content was significantly less in the simple ultrasound and the simple MB groups than that of the experimental groups. It was particularly higher in the routine contrast triggered group and the stress group than that of the routine contrast continuous group. No EB significant differences were found between the routine contrast triggered group and the two stress group.Visual subjective scores of myocardial EB leakage were 0 in the simple ultrasound and the two simple MB groups, significantly less than that of all the other experimental groups. It was much higher in the stress triggered group than that of the routine triggered contrast group and the stress continuous.Visual scores of myocardial petechial hemorrhages were 0 in the simple ultrasound groups, the simple MB group and the routine continuous group, significantly less than that of the others. It was much higher in the stress triggered group than that of the other experimental groups.There were no obvious pathological changes in the simple MB and the simple US groups under microscopy. There were erythrocytes leakage and lymphocytes exudation from circulation into the interstitial space of myocardium in the routine continuous, and the routine triggered group, the stress continuous groups and the stress trigger group. Significant more erythrocytes leakage was found in the stress triggered group than the other two routine groups.In TEM exam, cardiomyocyte edema could only be found in the stress triggered group. Discontinuity or rupture of capillary endothelium and basement membrane, pores formation and endothelial swelling could be found in the routine continuous, the routine triggered, the stress continuous and the stress triggered groups except the simple ultrasound and the simple MB groups. The above changes tended to happen on one sidewall of the vessel. The opening of tight junction could only be found in the stress continous group.Conclusions:1. The vascular petechial hemorrhages and microvascular leakage happened much more severely in the stress mode.2. The pathological changes of high myocardial vascular permeability during contrast echocardiography may include minute pores formation on the capillary endothelium and basement membrane, and the opening of tight junction.