| Background and purposeEchinococcus granulosus is a worm of cestode genus. Carnivores are the definitive hosts for the adult form of the parasite. Various species act as intermediate hosts, such as sheep and human beings, hosting the larval form of the parasite (hydatid). Human cystic Echinococcosis (also called hydatidosis) is a health-threatening infection in humans caused by the larval stage of Echinococcus granulosus,In primary Echinococcosis, larval cysts may develop in every organ,mainly in liver, lung and brain. Cystic Echinococcosis is a cosmopolitan disease. In China, it is regarded as the parasite disease that should be prevented and cured particularly, and mainly distributed in Xinjiang, Gansu, Ninxia, Qinhai, Inner Mongolia, Tibet and Sichuan as well. In China 44 % of mainland was endemic areas of Echinococcosis , in where 50 million people were at risk.There were many therapeutic options of cystic Echinococcosis, including surgical resection ,nonsurgical interventions and chemotherapy. Surgical resection was considered as the first and effective treatment, however, it risks accidental rupture and parasite dissemination during manipulation; the spillage of the antigen contained in the hydatid fluid will induce anaphylactic shock, serous case may be result in death. Nonsurgical intervention may have little damage, better recovery; but it can not damage of the germinal cell completely, hence it associated with high recurrence rate, also had risk of spillage of the hydatid fluid. The primary medical treatment was chemotherapy with benzimidazoles (Albendazole and Mebendazole), especially for cases of early stage or those unfit for surgery. However, many cases responded poorly to such chemotherapy, and the cure rate is very low. The duration of treatment was very long because of the low concentrations of Benzimidazoles in the hydatid. This may due to the laminated layer forming a barrier which renders the penetration of the drug into the cyst more difficult.High intensity focused ultrasound has characters of good penetrativity, directivity, focalization. As a new non-invasive therapeutic option, HIFU can deliver high intensity sound energy to the focal point. High temperature would be produce at the focal point, which in turn induces a coagulation necrosis of the tissue positioned at the focal point. HIFU treatment has been used clinically and experimentally for the treatment of solid tumors. However, there had few report about HIFU on cystic Echinococcosis in the literature, except our group previously found that HIFU could damage protoscoleces in the plastic test tube. Only damage the protoscoleces was low effective because new brood capsules and protoscoleces would be produced by germinal cell. Furthermore, the condition of protoscoleces in the plastic test tube was very different to it in the hydatid cyst. We want to known whether the protoscolicidal effect of HIFU treatment on the hydatid cyst is same as our previous experiment, and a new method which can completely damage the cyst wall will be explored. It is very important to know the damage effect of the HIFU irradiating directly on the cyst wall. The aim of this work was to observe the damage effect and its possible mechanisms underlying its therapeutic effects from the aspects of heat effect, the mortality rate of the protoscoleces in the hydatid fluid, activities of ALD and AKP in the hydatid fluid and pathological changes of cyst wall after HIFU irradiation. This experiment will provide helpful data to our next research that HIFU treat the hydatid cyst in the body of the experimental animal. And it may be a new alternative option for the treatment of Echinococcosis. It is prospective for application in the future.Materials and methodsPart 1: Selection of the hydatid cysts: hydatid cysts were taken from sheep infected naturally which had been slaughtered at an abatoir in Xining, Qinghai, China. The livers containing hydatid cysts were taken out just after sheep slaughtered, kept on ice and delivered immediately to the experiment site. The livers containing less hydatid cysts which can separate easily were selected. The hydatid cysts were touched carefully using hands, those with thin and elastic wall were selected for experiment. The diameter of the hydatid cysts was measured using B type ultrasound.Part 2: the grouping of the hydatid cysts:The size of the hydatid cyst should be balanced in each test group, because it was different from each other, which may affect the result of HIFU irradiation. In present study the grouping of randomized block design was taken. The detail of grouping was following: all samples were divided into different blocks based on the cyst diameter (spherical cyst based on it's diameter, irregular cyst based on it's long axis; all called diameter). The HIFU-treated groups and the control group were consisted of some cysts randomly selected from each blocks and were subjected to the following treatment: the cysts in the HIFU-treated cysts were irradiated with HIFU at sound power of 100 W, 150 W, 200 W or 250 W respectively, and the control cysts were treated with common diagnostic ultrasound. The cysts were fixed in a barrel used specifically for HIFU experiments and irradiated from the bottom of the barrelPart 3:Direct linear irradiation focused on hydatid fluid;The test groups were irradiated with HIFU at 200W sound power for 2min, 4min, 6min, 8min and 10min respectively. The temperature of the hydatid fluid, cyst wall and hepatic tissue around the cyst was detected respectively just after the HIFU irradiation. The mortality rates of protoscoleces were investigated by trypan blue exclusion. The control group was treated with common ultrasound for 2 min. Part 4: multilayer circular irradiation focused on cyst wallUsing multilayer circular irradiation, the HIFU-treated cysts were treated with 100 W, 150 W, 200 W or 250 W acoustic powers. Layers that were 5 mm thickness were irradiated along the circle once at a speed of 3 mm/s. The duration of HIFU irradiation varied from 2 min to 10 min. The control cysts were treated for 2 min with the diagnostic ultrasound located at the center of the HIFU transducer. The frequency of the diagnostic ultrasound was 3.5–5.0 MHz. The acoustic power of the diagnostic ultrasound was no more than 0.5 W. Hydatid fluid was aspirated just after irradiation, smeared onto three glass slides, stained with Trypan blue , viewed at least three fields of vision, and count more than 50 protoscoleces. Gross pathological changes in the hydatid cysts were observed when the cysts were cut open just after irradiation. Three representative tissue samples were taken from different locations in the cyst and preserved in 3% glutaral-phosphate buffer as samples for transmission electron microscopy. Another three representative tissue samples were taken, fixed in formalin for light microscopy.Results:Part 1:In the test groups, the mortality rate of protoscoleces in the hydatid fluid was elevated obviously under HIFU direct linear irradiation; the temperatures of cyst wall were higher than those in the hydatid fluid and the hepatic tissue around the cyst.Part 2: Under HIFU multilayer circular irradiation, the mortality rate of protoscoleces in the hydatid fluid was elevated obviously, the activities of LAD and AKP in the hydatid fluid were higher than those in the control group.Part 3: Under HIFU multilayer circular irradiation, it was found that the inner cyst walls of hydatids became curved, thicker, stiffer, white and less transparent, the germinal layers were detached mostly from the laminated layers under investigation of microscope; the fabric textures of laminated layer were changed obviously, the germinal cells were broken under investigation of transmission electron microscope.Conclusions:Part 1: Direct linear irradiation of HIFU focused on hydatid fluid had protoscolicidal effect, and could produce high temperature on the hydatid cyst.Part 2: Multilayer circular irradiation of HIFU focused on cyst wall induced coagulation necrosis of the cyst wall; collapse of the germinal cell, part death of the protoscoleses in the hydatid fluid, obvious elevation of the enzyme activity in the hytatid fluid. Part 3: Under the same condition,the damage effect of the cysts wall under the HIFU multilayer circular irradiation were better than it under HIFU linear irradiation; but as far as protosclicide effect and heat effect were concerned, the latter was better than former, the heat effect was poor in large cyst. |
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