| Objective: The study analyzeds the effectiveness and safety of a novel ablation methodality-nanosecond pulsed electric field electroporation on echinococcosis granulosis by experimental treatment in vitro and in vivo.The feasibility of treatment parameter,the dose effect were investigated for the clinical application.The mice model of cystic echinococcus with natural infection route was established by injecting echinococcus granulosis protoscolex through portal vein.By adjusting the number of protoscolex injected to the portal vein,the model was optimized,and the analogue clinical hepatic cystic echinococosis model was achieved.By selecting appropriate pulse number and the electric field strength on cystic echinococcus with fixed pulse duration and frequency,the effective treatment parameters were screened.The design and placement of electrode was also investigated.Methods:(1)The protoscolices were collect from patients with confirmed CE1 hepatic cystic echinococosis in their radical surgety.The protoscolex was digested by 1% pepsase and adjusted to different injection concentrations: 2000/100 ul,200/100 ul and 100/100 ul.After injecting them in the portal vein of C57BL/6 mice,the animal survival,the hepatic hydatid cyst formation,the cyst growth were followed up ultrasomic;(2)The in vivo effecacy of ns PEF on CE was observed on CE1 bearing C57BL/6 mice.The effective ns PEF treatment parameters were screened the size,shape,structure of cys 1w、4w、8w post ns PEF treatment.The parameters were optimized by comparing field strength at 21、14、7、0k V/cm of fixed pulse duration 350 nm,frequency 1Hz/s,50 impulse;(3)The scolicidal effect of ns PEF was evaluated on protoscolex in vitro.The shape,activity and integrity of protoscolex were observed on the 1st,3rd and 7th day post ns PEF treatment.The dose effect was investigated.Different field strengths(29、21、14、7、0k V/cm)and pulse number(50,100 pulses)were comparedof fixed pulse duration 350 nm,frequency 1Hz/s.Morphological changes in the structure of protoscolex was recored by HE stain,scanning electron microscopy.The same treatment condition was also applied on the hypatic cysts ex vivo from CE2.The ns PEF parameters were repeated ex vivo at 29、21、14、7、0k V/cm and 50,100 pulses of fixed pulse duration 350 nm,frequency 1Hz/s.The changes in cyst wall and germinal layer were examed by HE stain.Results:(1)After the mice have been injected 2000/100 ul,200/100 ul and 100/100 ul protoscolex suspension on the main portal vein respectively,the infection rate of the mice were 90%(9/10)、100%(10/10)、63.6%(7/11)respectively,the encapsulated ratio were 0.7% ~ 4.1%,0.6% ~ 1.9%,0.6% ~ 3.9% respectively.Pathological section indicated the size of majority of the protoscolex had shrunk,inflammatory zone had formed on the 7th day.The survived protoscolex have developed vesica on the 21 st day.The cystic echinococosis lesion formed on the 42 nd day;(2)The highest electric field strength at 21 k V/cm showed the most significant growth inhibition compared with the control group at the same time post ns PEF ablation(P<0.05),showing a decreasing trend in cyst diameter.At the 1,4,8w in the ns PE-treated group,the average cyst diameters decreased 1.4mm,1.2mm,1.7mm respectively.Visible structure changes were found in the ns PEF treated groups.The cyst wall became thickened,and the transparency decreased.As a comparison,the control group without ns PEF showed the smooth,transparent,thin and flexible syst wall.The pathological section suggests at the 8th week,the external fibrous tissue thickened and inflammatory reaction zone extended in the 21 k V/cm ns PEF group.The average cyst diameter in 14 k V/cm group also showed growth inhibit,significantely different from control group(P<0.05)by increased-0.4mm,0.4mm,-0.1mm at the 1,4,8w respectively.The diameter of mice hepatic cystic echinococosis lesion that has received 7k V/cm ns PEF increased-0.2mm,0.9mm,and 4.2mm at the 1,4,8w respectively,showing no siginificant difference with control group(P>0.5);(3)On the 1st and the 3rd day after ns PEF,the scolicidal effect of the protoscolex in 29、21k V/cm group were significantly higher than that of control group(P<0.05).With 50 pulses,the scolicidal effect of the protoscolex from 29、21k V/cm pulses group were 16.5%,12.9% respectively on the 1st day,while the scolicidal rates of the protoscolex from 29、21k V/cm groups were 19%,12.6% on the 3rd day.Seven days after ns PEF,the scolicidal rates of the protoscolex from 29,21 and 14 k V/cm groups were different from that of the protoscolex from the control group(P<0.05).With 50 pulses,the scolicidal rates of the protoscolex from 29,21 and 14 k V/cm impulse group is 71.7%,64.3%,and 48.3% respectively on the 7th day.With 100 pulses,the scolicidal rates from29 and 21 k V/cm groups were 32.3%,15.6% respectively on the 1st day,and 32.3%,16.7% on the third day.With 100 pulses,the scolicidal rates from 29,21 and 14 k V/cm groups were 79.2%,68.7%,and 49.2% respectively on the 7th day.Inlayer slide down occurred one day after ns PEF,which was significantly different from the control group(P<0.05).The higher the field strengthwas,the more likely for the collapse to happen.With fixed 50 impulse,the collapse rates of the 1st and the 3rd day were 75% and 100% in 29 k V/cm group,the collapse rates of the 1st,the 3rd and the 5th day were 50%,75%,100% in 21 k V/cm group,and the collapse rates of the 1st,the 3rd,the 5th and the 7th day were 25%,62.5%,75%,and 75% respectively in 14 k V/cm group.With fixed impulse 100,the collapse rates of the 1st,the 3rd,the 5th and the 7th day were 62.5%,87.5%,87.5%,and 100% respectively in 29 k V/cm group,compared with those in 21 k V/cm group(50%,75%,75% and 100% respectively),and 14 k V/cm group(75%,87.5%,87.5% and 100%).Conclusion:(1)The hepatic cystic echinococosis infection model of mice can be successfully established by injecting protoscolex on the portal vein of the mouse.The optimal injecting number of 200 protoscolices in 100 ul achieved the higher infection rate and more vesica than the other two concentrations(2000/100 ul and 100/100 ul.).1 to maximum 4 hydatid cysts formed in liver,which imitating the natural injection route and course of hepatic cystic echinococosis in human;(2)21k V/cm field strength showed the most siginificant inhibiting effect on ablating mice hepatic cystic echinococosis lesion in vivo in terms of cyst-wall thickening and transparency decrease.14 k V/cm can also inhibit the growth of mice hepatic cystic echinococosis lesion while 7k V/cm field strengthshowed no effect.When applied appropriate parameter,ns PEF inhibit growth of hepatic cystic echinococosis lesion in vivo;(3)With fixed pulse duration 350 nm,frequency 1Hz/s,the 29 and 21 k V/cm field strength showed the most effective scolicidal effect on protoscolex in vitro.With 29,21 and 24 k V/cm field strength,the germinal layer collapsed on the first day after impulse.ns PEF showed the dose effect on CE ablation.With higherfield strength and more pulse numbers,germinal layer was more likely to collapse. |
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