In vitro drug assays

With regard to the drug mixture preparations, tropisetron monohydrochloride (CAS no.: 105,826–92-4, Product no.: T104; Sigma-Aldrich, St. Louis, MO, USA) and granisetron hydrochloride (CAS no.: 107,007–99-8, Product no.: G3796; Sigma-Aldrich China, Shanghai, China), which are soluble in water, were diluted directly in the culture medium. However, CsA (CAS no.: 59865-13-3, Product no.: 30024, Sigma-Aldrich USA) and ricobendazole (ABZSO) (CAS no: 54029-12-8, Product no: 19953; Sigma-Aldrich China) were first dissolved in dimethyl sulfoxide (DMSO)(CAS no.: D8779, Product no: 67-68-5, Sigma-Aldrich USA) at an amount of < 0.3%, which has no deleterious effect on the parasite [26], and then diluted in the culture medium. Treatment of the PSC was initiated 1 day after initiation of in vitro culture. For all drugs, three final concentrations were prepared:50, 150 and 250 µM. Additionally, two cell culture flasks containing non-treated PSC were used as control cultures; one with culture medium only, and the other supplemented with DMSO. All treated PSC and the two controls were dispersed in the flasks at approximately equal numbers (n = 25,000), with medium change on day 5 of the initiation of the drug assessment. Protoscolex viability assessment following initiation of drug exposure was performed at 1-h inspections during the first 12 h and then at 24-h intervals up to the tenth day. The viability of the PSC was evaluated after gentle mixing of the solutions using an eosin 0.1% exclusion test [23] with microscopic observation of change in the color of the PSC. Viability was calculated as the proportion of viable PSC among the total number of PSC in ten microscopic fields at ×10 magnification. Each run of drugs tests was carried out in three replicates per treatment condition and repeated three times. After 24 h of drug exposure, some samples of treated PSC were harvested from the preparations containing 250 µM of the respective drug for SEM, and from those containing 50 and 250 μM for RNA extraction and comparison with the two controls.