The PHIFU Irradiation Perfusion Isolated Tissue Preliminary Experimental Study

Background and objectiveHigh Intensity focused ultrasound technology is a recently developedclinical non-invasive therapy strategy which interacts with tissue by thermaleffect, mechanical effect and cavitation effect. It achieves therapeuticpurpose by thermal ablation due to constant US energy deposition in targettissue and has been used for a wide range of tumor therapy. Continuous highintensity focused ultrasound is currently used for one trial thermal ablationof tumor. It primarily causes high thermal effect whereas enduringtreatments with high power would result in some complications, such as skinburns, gastrointestinal perforation and etc. Organ perfusion in vitro canpromise an approximation with that in vivo, and avoid the interference fromcomplicated beam path、respiration、steady blood, hence the object of thisstudy. Pulsed high intensity focused ultrasound,(pHIFU) different from thepast continuous wave HIFU irradiation method, due to the biological effectdepends on its sound power, duty cycle (duty cycle), the pulse repetitionfrequency, exposure time and depthmultiple parameters. pHIFU useintermittent energy delivery method, to avoid the continuous HIFUirradiation for a long time, or to improve the the HIFU treatment efficiency, reduce the occurrence of complications. In addition, the isolated organperfusion simulated organ function in vivo, but not subject to the living bodysoft tissue the complex acoustic channel and respiratory interference,therefore chosen as the object of the present study in vitro liver perfusion.based on liver perfusion in vitro model, first,process pHIFU irradiation ofreperfusion group and non-reperfusion group to explore pyrogenicdeposition of the liver perfusion pHIFU irradiation.Then, process pHIFUirradiation with different duty cycle and the irradiation time to study thedifferences between it and the cHIFU from tissue damage results, thesonographic appearance of the temperature rise and cavitation signal.Provide experimental basis for pHIFU research and provide new ideas andbasis for clinical HIFU applications.Material and methods1.Perfusion in vitro recycling liver modelingPerfusion in vitro recycling liver modeling[1-2]: Fetch fresh and integralpig liver(keep intact hepatic artery、portal vein、hepatic vein、gallbladder、cystic duct). Mix heparin with normal saline by1:100for40ml, thencompound the mixture with2000ml sodium lactate ringer’s injection. Usethis hybrid to clean the portal vein、hepatic artery to make them clear withoutblock and then immersed in sodium lactate ringer with a constanttemperature of4℃. Deliver the prepared liver to the lab within2hours.Ligature the cystic duct, drain the common bile duct, intubate the hepatic artery and portal vein and connect them with cycling pump, turn on theoxygenator and keep the oxygen flow at5L/min, adjust vascellum flow ofthe cycling pump to build a circulation of hepatic artery with18ml/min/kg、portal vein with40ml/min/kg and hepatic vein. Observe liver morphology,ultrasound imaging performance, record hemodynamic, biochemicalparameters and structure changes before and after perfusion.2. pHIFU irradiation perfusion in vitro recycling liver2.1Effect of liver perfusion on thermal deposition caused by pHIFUirradiationThe perfusion recycling standards will be divided into isolated liverreperfusion and non-reperfusion group, the the fixed irradiation power of80W, pulse repetition frequency of100Hz, irradiation depth of20mm. Andselect the duty cycle of30%,20%,10%, the irradiation time20s, sentinelirradiation. Duty cycle-the time parameters in the two groups experimentwas repeated20times, a total of120irradiation. The thermocouple is used tomeasure the temperature in target region during the irradiation and the dataare recorded by reading program. B-Ultra sonogram are immediatelycollected after irradiation and compared with the sonogram of the same spotbefore irradiation. After the irradiation, cut the liver into slices withthicknesses of1-2mm, find the layer with the utmost damage and use Jupiterto measure the actual necrosis volume of the liver after digitalphotographing. 2.2pHIFU and cHIFU contrast study under perfusion conditionOn the basis of in vitro liver recycling model, perform fixed-pointirradiation with an irradiation power of80W, a depth of20mm, pulserepeating frequency of100Hz, and different duty cycles(100%、30%、20%、10%respectively). It becomes cHIFU when the duty cycle is100%and theirradiation period is4s and(4、8、12、16、20)s for other duty cycle conditions.Each duty cycle-time group were the irradiation20points of320irradiated.Collect the two-dimensional sonogram before the irradiation and at everytime point after the HIFU irradiation, then reprocess the image to measurethe gray enhancement value. Collect the positive cavitation signal of each ofthe duty cycle-time group above. Thermolcouple probe measures thetemperature of the focal region. After the irradiation, cut the liver into1-2mm slices, digitally photograph the slice with the maximum damage and useJupiter to measure the actual necrosis area of the liver and to display theutmost section, measure the utmost damage radial line and the biologicalfocal region area, and fetch the border area tissue between the normal andnecrosis tissue for pathological examination.Results:1、12cases liver after4h perfusion, the liver surface color is uniformrosy, soft texture; stable hemodynamics and liver function: the portal veinand hepatic artery flow rate of13.9±1.7cm/s,38.9±2.2cm/s; averagepressureis were9.05±1.4mmHg and112.6±10.6mmHg; the bile and ALT enzyme secretion stability; the liver weight gain than of beforeperfusion perfusion lobular structure of the liver tissue integrity in the lightmicroscope, with time extension, cell swelling and sinusoidal widened.2、Duty cycle–time parameters for the10%-20s,20%-20s,30%-20s,in the perfusion, Perfusion group and non-reperfusion group each irradiationpoints have coagulation necrosis and hyperechoic.Target area of necrosiswere19.23±1.33mm~2,28.39±1.28mm~2,35.87±1.40mm~2; B ultrasoundecho enhancement area were13.82±1.22mm2,21.48±1.04mm2,27.41±1.42mm2；Gray value added was49.3±11.78,82.3±17.14,103.6±15.17;the highest temperature at the focal region of irradiation were46.54±0.43℃,56.68±1.16℃,61.21±1.50℃. in the non perfusion, target area ofnecrosis were23.61±1.31mm2,35.76±1.18mm~2,41.45±1.32mm2; Bultrasound echo enhancement area were35.98±1.77mm2,27.40±1.71mm~2,19.28±1.42mm2, Gray value added was87.08±15.10different,90.04±14.84,106.44±18.57; the highest temperature at the focal region ofirradiation were50.38±0.49℃,66.23±1.58℃,71.54±1.61℃.3、Under the pulse condition：duty ratio30%, the irradiation time8s;duty ratio20%, irradiation time12s; the target damage areas of duty ratio10%and irradiation time20s were greater than or equal to4seconds ofcontinuous wave irradiation; Pulse required irradiation dose was less thancontinuous wave irradiation dose required dose.4、in the same time,and a certain range, the pulse of HIFU with the increase of duty ratio, caused by the target area of necrosis area alsoincreased; in the duty ratio of30%,20%, the target area of necrosis increasedwith the time, but the parameters of10%-8s and10%-12s caused the targetarea of necrosis were10.31±1.19mm~2,14.38±1.57mm~2, the differenceswere not obvious.5、Duty cycle–time Parameters were30%-4s、20%-4s、10%-4s，thetarget echo enhancement gray values were59.15±18.99,47.77±14.12,30±14.73, there were significant difference (p<0.05); The gray value ofechogenic at irradiation time of8s is significantly greater than gray value ofechogenic at irradiation time of4s. Irradiation time≥8s, In the same dutycycle,caused echogenic gray value increases with the duty cycle withoutsignificant increases,irradiation time is larger than4S, the same duty causedecho enhancement gray values were not significantly different, the dutyratio between20%and30%also had no difference of gray values, but10%and the other two groups with significant difference.6、In the duty ratio of30%,20%,10%,the target areas produced bystrong echo increased with the extension of time, but the parameters for10%-8s,10%-12s,10%-16s, there were no significant differences betweenthe strong echo areas.7、Pulse HIFU with the duty ratio (10%,20%,30%), the highesttemperature were46.54±0.43℃,56.68±1.16℃,61.21±1.50℃, ContinuousHIFU irradiation4s caused the temperature of target tissues were 63.36±1.22℃.8、 At the duty cycle-time parameters100%-4s,10%-20s,20%-20s,30%-20s, HIFU irradiation, the maximum RMS values were1.52±0.69mv,1.49±0.8mv,1.46±0.76mv,1.50±0.58mv, the difference was notstatisticallysignificant (p>0.05).Conclusions：1、 Isolated perfused liver morphology, ultrasound imaging andhistology showed no significant abnormal, stable liver function andhemodynamic, are capable of simulating living donor liver bloodcirculation, that meet the high intensity focused ultrasound experimentalstudies in vitro liverperfusion model was successfully established;2、 Under the same duty cycle-time parameters, due to thenon-reperfusion group necrosis of the target area, echogenic area and theaverage maximum temperature is greater than the reperfusion group, thedifference was significant; no significant difference was in gray-scalevalue-added;3、under the same injury caused by the cHIFU and pHIFU irradiationreperfusion live, the case of the former than the latter required a larger dose;average maximum temperature due to pHIFU is less than that due to cHIFU,and the temperature rise is slower than of cHIFU.3、The same parameters of HIFU irradiation，echo enhancement areasin recirculating liver were less than those in non-recirculating liver, the differences were significant, Gray value added were no significantdifferences.4、In each duty cycle, pHIFU irradiation reperfusion due to livernecrosis target area, echogenic area of the average maximum temperatureincreases as time increases. In the same time, the target area of necrosis,the echogenic size of the area, with an average maximum temperatureincreases with the duty cycle.5、Irradiation time of4s, gray value of echogenic increases with theduty cycle, the difference was significant (p <0.05). Irradiation time≥8s,difference of gray value of echogenic was not significant between dutycycle of20%group and30%group. gray values were significantlydifferent in10%group and the other two group. In each duty cycle, theirradiation time is in less than8s, gray value of echogenic tended toincrease. After8s, gray values of echogenicwere not significantly increased,the approximate saturation.6、At80w sound power, either continuous wave or the pulse wave ofduty cycle of10%,20%,30%irradiation perfusion liver,there was nosignificant cavitation occuring.