Study On The Effect Of High Intensity Focused Ultrasound On Hydatid Cyst Of Echinococcus Granulosus

Background and purpose:Cystic echinococcosis (also called hydatidosis) is a health-threatening zoonosis caused by the larval stage of Echinococcus granulosus. But there is currently no agreement about the ideal therapy for hydatidosis. The accepted surgical approaches for hydatidosis are all invasive and pose a risk of recurrence and postoperative complications due to rupture of cysts and/or spillage of the contents. Albendazole is the drug of choice for chemotherapy. However, its concentrations in the target organ are not optimal, and are much lower than in plasma because of thick cyst wall. Increases in dose may increase side-effects such as diarrhea, nausea, vomiting, aminotransferase elevation, leucopenia and may even result in death. High Intensity Focused Ultrasound (HIFU) is a non-invasive technique which can kill histiocytes and induce necrosis in the targeted area. The aim of this work was to observe the effect of HIFU on echinococcus and to explore whether this new and non-invasive therapy could be more effective and with fewer complications for cystic echinococcosis. Materials and Methods:1. Germinal cells of Echinococcus granulosus from naturally infected sheep were primarily cultured and identified and the optimal parameters relevant to the effects of HIFU on the proliferation of germinal cells selected by MTT colorometric assay; cells being treated with 10% lethal dosages of HIFU, electron microscopy, AnnexinV/PI labeling method, fluorescence microscopy combined with flow cytometry were applied to observe apoptosis in cells and analyze cell cycle as well as to identify the activity of Caspase-3; western blot used to detect the apoptosis-related protein expressions of P53,BcL-2/Bax and Fas etc.2. HIFU dosage-effect was obtained from protoscolices isolation. Irradiated with HIFU of maximal tolerable dosage, parasite morphology and mortality was observed by fluorescence microscopy and the inhibitory effect of HIFU on protoscolices was investigated by trypan-blue exclusion assay; the infection effect was learned from the mean weight of hydatid cysts and hydatid morphological changes through infection experiments in vivo; meanwhile, the activity of ACP,G-6-P and SDH of protoscolices were detected by enzymatic histochemistry.3. The animal model of cystic echinococcosis was established using protoscolices from naturally infected sheep. Irritated with HIFU of curve scanning mode around cysts, the morphology of hydatid walls was observed in comparison with the wet weights of hydatid and the apoptotic induction of the wall cells detected by TUNEL assay to evaluate the effects of HIFU on the cysts; the level of IgG, IFN-γ, IL-2, IL-4 and IL-10 in serum was detected by EL1SA, the counts and subsets of lymphocyte in peripheral blood detected by flow cytometry, mRNA expressions of lymphocytokines detected by RT-PCR to learn the immunoreactive change of the mice treated with HIFU.Results:1. The results of MTT demonstrate that HIFU does have the effects of growth inhibition on germinal cells in vitro. Cell apoptosis was similar under fluorescence microscopy EM and FCM, with 10% lethal dosages of HIFU (20W×20s,40W×10s and160W×5s), while the cell apoptosis and the activity of Caspase-3 increased, but the expression of P53,B cL-2/Bax and Fas were not be detected.2. HIFU damage on protoscolices and the protoscolicidal effect was dose-dependent. HIFU at 200Ws (or above) could easily cause immediate death of protoscolices even with instant irradiation; HIFU at 25 W×20 s and 50 W×10 s could be used as maximal tolerable dosages. Under the effect of HIFU of maximal tolerable dosages, the growth of protoscolices that survived the exposure to HIFU was obviously suppressed in vitro and the mean weight of hydatid cysts resulting from such protoscolices was less than that in control group. The positive reactive product of G-6-P and SDH decreased markedly in HIFU treated protoscolices, and there was no difference between HIFU treated protoscolices and control group in ACP which were detected by enzymatic histochemistry.3. The mean weight of hydatid cysts decreased obviously, and the structure of the germinal membrane of irradiated cysts was not as normal or intact as those non-irradiated ones, and morphological changes related to degeneration were observed after being irritated with HIFU of curve scanning mode around cysts. The level of IgG almot remained no difference as previous in serum by ELISA. The content of CD4+ increased and as well as the value of CD4+/CD8+ in lymphocyte. The results of RT-PCR demonstrate that the mRNA expression of IFN-γwas up-regulated while IL-4 down-regulated, in accordance with the results that the contents of IFN-γand IL-2 increasing while IL-4 and IL-10 decreasing.Conclusions:1. HIFU demonstrated an immediate damaging effect on both germinal cells and protoscolices of Echinococcus granulosu, and inhibited the proliferations as well in vitro.2. HIFU was capable of inhibiting the growth of cysts in animal mode in vivo, and could be a possible therapeutic option for cystic echinococcosis.3. The mechanism maybe related to directly damaging, promiting apoptosis, suppressing the enzymatic activities and re-motivating the efficient immune responses of the host on the parasite as well.