Study Of An In Vivo Fluorescent Imaging Mouse Model Of Aveolar Echinococcus

Background: Alveolar echinococcosis(AE), which is considered to be one of the most lethal helminthic infections of humans, poses a great threat to millions of humans in wide areas of the Northern hemisphere. The current chemotherapy, such as albendazole, has been shown to exert a parasitostatic rather than a parasiticidal effect. A further disadvantage of the present treatment is that it has, in certain cases, proven to be ineffective, and the recurrence rate is rather high once chemotherapy is stopped. Thus, the development of new treatments of AE is anticipated.The conventional method for monitoring infection in the drug sensitivity test model is based on the estimation of parasite loads in target organs by anatomopathological observations,such as microscope examination of lesions. However these techniques are cumbersome, laborious and render the longitudinal monitoring of an infection in the same animal exceedingly difficult. Over the past decade, noninvasive small animal imaging has gained increasing importance in research of tumor and infectious disease because it enables to study disease development with time in the same animal and often reduces the overall number of animals needed. The feasibility of utilizing in vivo imaging to monitor the infected murine models is becoming increasingly recognized.Objects: We aimed to generate a kind of fluorescent protoscolices in vivo imaging model and thus meet the demand of real-time monitoring of AE mouse models.Methods: Metacestodes were isolated from the peritoneal cavity of AE infected Mongolian gerbils(Meriones unguiculatus). Metacestodes were then cut into pieces which been filtered and ground to get the protoscolices. Viability experiment was performed using protoscolices treated with different concentration of Met and ABZSO. Three groups of protoscolices were incubated with JC-1 dye. Images were taken using a confocal microscope and the intensity rate of red and green fluorescence was analyzed. Three groups of mice were injected with Met-treated protoscolices, Met combined with ABZSO-treated protoscolices and control protoscolices respectively. The inoculation was beneath the liver capsule and images were photographed by IVIS Lumina in vivo imaging device. Intensity rate of red and green fluorescence were analyzed and compared with the in vitro result. Finally, to explore the fluorescence changes over time, we monitored the in vivo fluorescence changing process in mice inoculated with untreated protoscolices.Results: According to Methylene blue exclusion viability test, the viability rate of 10 mM Met group is 27.43%(51/186) which is significantly lower than Con group(96.27%, 136/141),1m M Met group(90.01%, 158/175) and 5m M Met group(73.48%, 113/153)(P<0.05). The viability rate of 15μM ABZSO combined with 10 m M Met group is 4.63%(5/107), which is lower than its combination with 5m M(46.30%, 82/177), 1m M(69.73%, 103/147) and single treatment(82.35%, 198/240)(P<0.05).With the confocal microscope, the intensity rate of red and green fluorescence of the group of Met combined with ABZSO showed to be 0.29, which is lower than Met group(1.06) and Con group(4.53)(P<0.05). This indicated that Met treatment induced destruction of protoscolices mitochondrial function, which was in consistence with the in vitro study. Next, we tracked and monitored red and green fluorescence respectively. Red fluorescence of JC-1 in Met combined with ABZSO-treated protoscolices declined more rapidly as compared with that in Met treated and control protoscolices. After 48 h, red fluorescence of three groups showed no statistical difference, which indicated that attenuation at this time affected the accuracy of monitoring. Green fluorescence of Met treated group was more evident than that of Con group at 24 h, 36 h and 48h(P<0.01). Combinaton treated group had more intensive green fluorescence than Met treated group at 24 h and 36 h.Mice were injected with three groups of protoscolices beneath the liver capsule and fluorescent images were photographed in vivo. The intensity rate of red and green fluorescence of the Met group is significantly lower than that of Con group(P<0.01). Met combined with ABZSO group had the lowest one(P<0.01), which was in consistence with the in vitro confocal microscope results. These data demonstrated that AE infections can be imaged properly in vivo by fluorescence using JC-1. Besides, the locality and viability of protoscolices in living mice could be assessed by measuring the JC-1 fluorescent in- tensity ratio. Finally, to explore the fluorescence changes over time, we monitored the in vivo fluorescence changing process in mice inoculated with untreated protoscolices. Red fluorescence decayed faster than green fluorescence. This may because the anti-photobleaching ability of monomeric form of JC-1 is stronger than that of aggregate. Conclusion: JC-1 can be used to establish in vivo fluorescent model and thus monitor the location and viability of AE infection. The proper spectral unmixing method eliminated the background signal due to residue fur and the efficient monitoring time is 36 h. Fluorescent imaging has the potential to become a new monitoring measure in AE mouse models. It could prove to be of significant value in future in vivo drug trials against E. multilocularis when some long term fluorescent markers are acquired.