| Background and objectiveEchinococcus granulosus, the etiologic agent of cystic echinococcosis (CE) in humans and other animal hosts, is distributed worldwide. Echino-coccosis is an increasing public health and socioeconomic concern.. 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 ofthe 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. HIFU, in which the ultrasound is focused to a focal zone, can deposit ultrasound mechanical energy and produce clinically relevant bio-effects in the targeted tissue, and has been widely applied to treat some tumors as a non-invasive therapeutic option. In HIFU therapy, absorption of ultrasonic energy at the focus leads to local heating that can result in apoptosis and thermal necrosis when the local temperature rises above60℃. However, success of this therapeutic modality depends critically on the ability to focus HIFU energy accurately at the diseased site. Our group has previously found that HIFU could damage protoscolices in vitro, but the death rate was limited by cyst fluid with characters of less focus HIFU energy and fast loss of heat. In this study, we prospectively suggest that a kind of special particle with superabsorbent ability was added into the cyst fluid of Echinococcus granulosus and would strongly focus HIFU energy. Starch based superabsorbent polymer (SAR) of poly (sodium acry late) was prepared by reverse phase suspension polymerization. They are solid, granular or powder cross linked polymers that rapidly absorb and retain large volumes of aqueous solutions. The absorptive properties of SAR are ideally suited for the absorption and solidification of various types of liquids like water, sludge, blood etc. which has caused an increased use of this substance in the production of hygiene products and agriculture.In this study, we prospectively suggest that a kind of special particle with superabsorbent ability was added into the cyst fluid of Echinococcus granulosus and would strongly focus HIFU energy. By the present work evaluated whether or not a superabsorbent polymer (SAR) could enhance the damage efficacy of high intensity focused ultrasound (HIFU) on the viability of E. granulosus protoscolices in vitro.Materials and Methods1. A super absorbent resin was synthesized with grafting acrylic acid and corn starch as elementary materials by solution polymerization in the presence of N, N-Methylene bisacrylamide as cross-linker and potassium per-sulfate as initiator. The products synthesizing were observed through infrared spectrum and light microscope.2. The instantaneous water absorption ratios of this resin were measured when it, of which are different particle sizes, mixed with the different liquids such as demineralized water, physiological saline, anhydrous alcohol and hydatid cyst fluid at different temperatures of20℃,25℃, and37℃respectively. The water preserving capabilities of the resin of several different particle sizes were measured.3.The protoscolices were cultivated in the physiological saline with superabsorbent resin at37℃. and the major chemical constituent in the hydatid fluid with superabsorbent resin were measured.4. HIFU of100W acoustic power, and some dose of superabsorbent resin, were used to treat5000protoscolices in2milliliter protoscolices suspension. The temperature of protoscolices suspension was investigated at different HIFU exposure time (5,10,20,30,40,50,60seconds) respectively, the structures of protoscolices were observed by light microscopy. To better understand the biological mechanisms responsible for the regulation of protoscolices death, we examined the activity of succinate dehydrogenase (SDH) and Glucose-6-phosphatase of the protoscolices treated with HIFU.5. HIFU of200W acoustic power, and some dose of superabsorbent resin, were used to treat hydatid of E. granulosus. All the experimental hydatid of E. granulosus were randomly assigned into four groups by intervention study as follows:A)with no exposure to HIFU was considered as the blank group; B)with exposure to HIFU was considered as the test control group (200power); C)with exposure to HIFU combined with lg SAR in the hydatid liquid and D) with exposure to HIFU combined with2g SAR werconsidered as the test group (the parameter same as the forward). The changes of structure of procolices and cyst ball of hydatid were by light microscope, scanning and transmission electric microscope, and detected the apoptosis of protolices by Hochest assay. The temperature of monitoring points in the hydatid liver were measured by temperature sensitive thermometer.Results1. A transparent graticule ultrastructures was observed in the water-absorbing resin under light microscope and through infrared spectrum.2. The absorption ratio of the resin to deionized water, and hydatid cyst fluid, physiological saline were the maximum, being of700g/g and200-300g/g respectively. It’s ratio to physiological saline, however, was only25times of its mass, and less absorptive to anhydrous alcohol. Of which160-180mesh size particles, to deionized water could reach to700times, and a peak of water absorption was observed within4to8minutes. Although a same maximum absorption ratio was observed in the resin with a particle size of80-120mesh, but it took16minutes for the water absorption peak. At room temperature of25℃, the water-preserving rate of the resin with a particle size of160-180mesh was55%in2days, but it was58%in5days for the resin of which size was0.5×0.5×0.5mm. The water-absorbing rate was almost same for a resin with a particular size mixed the above mentioned liquids at different temperature of20℃,25℃, and37℃respectively.3. The lively ability of protoscolices in the physiological saline at37℃had not been reflected by Super Absorbent Resin (SAR) within the8minutes. The major chemical constituent had not preonunced changes by Super Absorbent Resin (SAR).4. The rising effect of temperature of protoscolices suspension treated with HIFU was slow, and the death rate of protoscolices was73.7%in the group of HIFU treatment time of40seconds; In the group of HIFU treatment time of40seconds, the rising effect was quick, and the death rate of protoscolices was100%in the group of HIFU combined with SAR. The best protoscolicidal effect (100%) of HIFU of100W acoustic power combined with SAR was obtained after40,50, and60seconds respectively. The live protoscolices in the blank control group showed distinct inner structure. The membrane of dead protoscolices were stained to blue exposed by the100W acoustic power of HIFU, showed shrunken and black calcareous corpuscles, disorder and decreasing hooks though of intact membrane; some protoscolices lost hooks and teared open on membrane were observed. In the group of HIFU combined with SAR, it was found that the superabsorbent polymer around the spoiled protoscolices and the destruction of much stronger than in the group of HIFU. The dead protoscolices exhibited the reduction or absence of SDH and G-6-P staining intensity in the parenchymal cell and calcareous corpuscles undergoing HIFU irradiation, where a large number of apoptotic, necrotic cell were evident; but was markedly preserved in the blank group.5. The average death rates of procolices in the Ⅰ、Ⅱ、Ⅲ、Ⅳgroup were22.94%、55.28%、63.4%、72.4%。The dead pritoscolices such as lost-alively and crimple and teard into piece were observed by light microscope, the blue and reflected light particles of the proscolices observed by fluorescence microscope exposured by the HIFU combined with SAR were more than the simple HIFU group. The decreasing glycogenosome and appeared to black body and karyorrhexis of the proscolices were observed by transmission electron microscope, it showed germinal layer growed thinner and interruption, separated from the cuticulae layer, wherein appeared to vacuoles and concentric circles. The protein concentration of major component in the hydatid fluid ofⅠ、Ⅱ、 Ⅲgroup were lg/100ml、2g/100ml、2g/100ml。The average temperatures of hydatid fluid were21.3℃、31.1℃、38.9℃、44.6℃;were21.3℃、38.9℃、49℃、55℃in the cystwall;21.3℃、32.28℃、34.5℃、37.8℃in the which was5mm between liver and hydatid cyst。Conclusions1. Super Absorbent Resin (SAR) is very unique water-absorbing and water-holding materials. They can absorb up to700times of their own weight of pure water and even under pressure resist release of the absorbed water. The temperature change has little effect on the rate of water-absorbing. Solution properties, especially the water content of solution greatly impact the water-absorbing. Therefore, the water absorbency of the particles is the highest in the deionized water, followed by hydatid cyst fluid, normal saline, anhydrous alcohol for almost no water absorption.2. The lively ability of protoscolices in the physiological saline had not been reflected by Super Absorbent Resin (SAR) within the8minutes.3. These results suggest that HIFU induced damage and loss of viability of protoscolices and hydatid, and Super Absorbent Resin (SAR) enhanced the HIFU energy and caused more severe destruction efficacy of HIFU irradiation on protoscolices and hydatid, and that mitochondrial energetic function is involved in the regulation of cell death pathways in the pathogenesis of protoscolices. |
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