Hemostatic Effects Of Ultrasound In A Liver Avulsion Injury Model

BackgroundThe liver is a very fragile structure, which supply with a plentiful blood. It issusceptible to traumatic injury because of its large size and fixed location relatively.There are severals of emergency treatment approaches to liver ruptures currently,including electric coagulation, argon hemostasis, simple suture, gauze packing, partialhepatectomy, hepatic artery embolization or ligation and so on. Surgical methods canresult in a operative damage although they are effective. Moreover, surgical treatmentoften demands a certain extent of device and technological supports, and some patientsmight suffer hemorrhagic shock, DIC and death on the way to the hospital before gettinga proper clinical treatment. The less invasiveness processes which were guided bycontrast-enhanced ultrasound（CEUS）to prevent bleeding include hemostatic agentsinjection, microwave coagulation, percutaneous radiofrequency coagulation and highintensity focused ultrasound (HIFU). These processes can achieve good hemostatic effectwith minimally invasiveness and recover rapidily. However,such processes have somelimitations. For example, the efficacy of hemostatic agents which injected in the woundsof the large hemorrhage because of serious injury have not been satisfied and they canresult in some discomfort. The mechanism of other three methods (microwavecoagulation, percutaneous radiofrequency coagulation and high intensity focusedultrasound) to cause hemostasis is resulting in coagulation necrosis by heating the targettissue above70oC. However, the temperatures which access of70oC can lead todehydration of target tissue, and result in deformation of other adjacent structures. And itmight permit a prolonged treatment time because of the heat sink effect. Morever, suchabove hemostasis processes can only obtain contact hemostasis directily on some limitedsurface region or a point. Some studies showed that cavitation through HIFU could promote the formation of microthrombosis and homogenates to go over the wound areas,which can stop the wound from bleeding and enhance the thermal hemostasis.Ultrasound combined with microbubbles (MB) not only can guide the progress ofthe above invasiveness hemostasis methods, but it also damage the tiny blood vessels tostop bleeding of livers or spleens in the laceration models when cavitation has happened,this method’s name is angiotripsy. However, the wounds made in these trauma modelswere corresponding to injury scale I.These models were neat cuts and the hemorrhagewas unstable and small. Thus, It can not exclude some factors such as contactcompression or activation of clotting which could cause a observed hemostasis. Thepurpose of our study was to explore feasibility and pathological characteristics ofmicrobubble-enhanced ultrasound (MEUS) hemostasis though optimizing the setting ofthe devices of pulsed non-focused ultrasound of the model of liver avulsion injury (scaleII), the another form of liver trauma.ObjectiveMEUS could interrupt the circulation in the normal organs such as liver,spleen, andinsonated on the lacerations of that organs, the hemorrhage of the wound decreasedsignificantly. However, factors such as activation of clotting and contact compressioncausing observed hemostasis could not be excluded. The aim of this study was toevaluate the hemostatic effect of microbubble-enhanced pulsed, low-intensity ultrasoundin a New Zealand White rabbit model of avulsion trauma of the liver and to optimizeacoustic parameters so as to further explore the feasibility, scope and histologicalmechanisms of MEUS for a useful new treatment.MethodsFourteen healthy male New Zealand White rabbits for the experiment. They wererandomly assigned to three groups: microbubble-enhanced ultrasound (MEUS)(n=6),therapeutic ultrasound alone without microbubbles (TUS)(n=4), and microbubbles only with sham ultrasound exposure (MB)(n=4).Established model of liver avulsion injury: Anesthetized animals placed in a supineposition, and the left hepatic lobe was stretched out carefully with saline gauze, then fixedin situ. A model of a liver avulsion wound was created from the edge of the left liver lobeusing a scissor.Methods of the groups:(1) MEUS group: The wound bed was insonated directly for5min with intravenous injection of microbubbles in the first4min. The treatmenttransducer moved at a uniform speed without any pressure to insonate throughout thewound.(2) TUS group: The same method was used to insonate the wound, but salinesolution instead of microbubbles to be injected intravenously.(3) MB group: The samedose of microbubbles was injected intravenously, and to insonate the wound in the sameway without pulses were transmitted.Methods of bleeding score:(1) Visual scores of bleeding: Observed the bleedingwound by naked-eye and make a score for the wound.(2) The blood from wound wascollected with absorbent paper for30s both before and after insonation, and weighed onthe precision scale. The difference of bleeding wounds before and after irradiation is thenet weight of each group.Condition of contrast enhanced ultrasonography: Observed the perfusion of contrastenhanced ultrasonography for each liver both before and after radiation and made thegrades, then analysed the acoustic density (PI, AUC).Histological Examinations: After the experiment, we selected three rabbits from theMEUS group randomly, and all the rabbits from the control groups were euthanized bypentobarbital. The abdominal wall of the remaining three rabbits were closed after the fedfor48h, and were given penicillin and gentamicin daily to prevent infection. Theremainder of the animals were euthanized and prepared for histological examination in thesame way.Data analysis:Data on bleeding rate, acoustic density analysis (PI, AUC) of each group before andafter insonation were expressed as mean±SD, and the data of bleeding visual scores of each group before and after insonation were expressed as medians (Q25, Q75). Thedifferences in the bleeding rate, PI, AUC of each group before and after insonation wereanalyzed by paired-samples Student’s t-test, and the data from each group before and afterinsonation were compared by using one-way ANOVA and LSD test for analyzing thedifferences between two groups when the total difference was significant. The visualbleeding scores of each group before and after insonation were compared by the Wilcoxontest and data of each corresponding group was compared by the Kruskal-Wallis H andMann-Whitney U tests. We used Pearson’s correlation to analyze the correlations betweenbleeding rate with PI, bleeding rate with AUC, and PI with AUC, and Spearman’scorrelation was used to analyze the correlation between bleeding rate with visual score. AP value <0.05was considered to be statistically significant. All the data were analyzed byusing SPSS13.0software.ResultsThe bleeding visual score of the MEUS group after the insonation, compared withthat of pre-insonation situation, decreased from grade4(3,4) to1(0.75,1), and thedifference was statistically significant (P <0.05); Differences between the MEUS groupand the control group after insonation were statistically significant (P <0.05)The bleeding rates of livers were similar before insonation, ranging from0.012±0.007g/s to0.019±0.014g/s, without significant differences between any two groups(P>0.05). After insonation, the bleeding rate of the MEUS group decreased afterinsonation (P <0.05). Differences between the MEUS group and the control groups afterinsonation were significantly different (P <0.05).Contrast agent was well perfused to all the corresponding regions of target liversbefore treatment, the insonation regions of TUS group and MB group. Microbubbleswere delivered to the liver tissue rapidly, and enhanced the tissue homogeneouslywithout any defects. The scoring grade was0. In the MEUS Group，it was discoveredthat the microbubbles were delivered to liver tissue slowly and unevenly. The livertissues were transformed into irregular non-enhanced areas of varying sizes with the edges of the defects being poorly perfused. The score was grade Ⅱ.Analysis of acoustic density: Before insonation, the differences in PI and AUCamong three groups were not significant (P>0.05). After insonation, the PI of the MEUSGroup decreased from22.000±6.090%to9.517±1.380%, and AUC decreased from2173.983±839.190%s to872.583±196.409%s. The differences were statisticallysignificant (P <0.05). The comparison between the MEUS Group and the control groupsafter insonation were significantly different (P <0.05).Correlation analysis: there was excellent positive correlation in bleeding rate, PI andAUC, respectively after insonation (P <0.05). The correlation of PI and AUC betweenvisual score and bleeding rate was positive (P <0.05).In the control groups, HE sections showed that the hepatic cord and plate structureremained intact after insonation. There were small numbers of erythrocytes scattered inthe sinusoids which were of normal size. There were four kinds of lesions in the MEUSgroup after insonation:1. hepatocytes were swollen and cloudy, compressing thesinusoids and perisinusoidal space at the wound surface;2. a large number oferythrocytes that accumulated in the sinusoids, perisinusoidal and small veins, distantfrom the wound surface.3. significant hemorrhage into periportal parenchyma.4.scattered necrosis with infiltration of inflammatory cells appeared within the liver tissuein the insonation area. After48h, the surfaces of the wounds were found to be gray andsmooth and without any bleeding (bleed visual scores were grade0). The HE sectionsshowed that the area of the lesion was about1.5cm away from the wound surface, andthe necrosis in the lesion area had expanded. There was some degeneration surroundingthe peripheral zone of the necrotic cells. Infiltration of a few inflammatory cells was seenin the areas of degenerated cells. There was a clear boundary between the lesions andnormal tissues, and a small number of erythrocytes were found to be scattered in liversinusoids near the lesions.Conclusions1、This experiment almost completely blocked the circulation of hepatic blood by ultrasound combined microbubbles.2、After the insonation of the Ultrasound combined microbubbles,the total amountof bleeding of the liver avulsion injury reduced by at least ten-fold. MEUS is anoninvasive method for Hemostasis, and potential to become a useful new method forthe clinical treatment of liver avulsion injury.3、We think that hemostatic effect of the MEUS was achieved as hepatocytes andcapillary walls were subjected to various degrees of damage. Sinusoids andperisinusoidal spaces became compressed, and small vessels became congested in theportal areas of the bleeding site.4、The devices of pulsed non-focused ultrasound and the ultrasound contrast agentmicrobubbles were light-weight and portable, and had the potential to be a new methodfor clinical to use in the accident field conducivily.