Journal: PLOS Pathogens

Article Title: Benznidazole treatment leads to DNA damage in Trypanosoma cruzi and the persistence of rare widely dispersed non-replicative amastigotes in mice

doi: 10.1371/journal.ppat.1011627

Figure Lengend Snippet: (a) Experimental outline. MA104 cells were infected with T . cruzi CL Luc::mNeon in 24-well plates. They were treated with either benznidazole (BZ) (200 μM) for 24 hours, or tert-butyl hydroperoxide (TBHP) (50 μM) for 3 days. Post-fixation, as a control group, untreated cells were treated with DNase. TUNEL assays were then performed and cells imaged using a Nikon Ti-2 E inverted microscope (Materials and Methods). (b) Representative images showing infected cells following each of the treatments. Parasites (green fluorescence), DNA (blue, DAPI), TUNEL (red). The enlarged inset (left) highlights replicating kinetoplast DNA (kDNA). The white arrows in the non-treated images show the location of a highly infected cell in which all the parasites have differentiated into TUNEL-negative non-replicating trypomastigotes. White scale bars = 10 μm.

Article Snippet: The coverslips were washed (x3) in PBS, mounted on slides with VECTASHIELD with DAPI (Vector Laboratories, Inc.), and then examined using Zeiss LSM880 confocal or inverted Nikon Ti-2 E inverted microscopes.

Techniques: Infection, TUNEL Assay, Inverted Microscopy, Fluorescence

Journal: PLOS Pathogens

Article Title: Benznidazole treatment leads to DNA damage in Trypanosoma cruzi and the persistence of rare widely dispersed non-replicative amastigotes in mice

doi: 10.1371/journal.ppat.1011627

Figure Lengend Snippet: CB17 SCID mice were infected with T . cruzi and 10 days post-infection they were treated once daily with 25 mg/kg benznidazole for 5 days. EdU labelling was then carried out as described previously (Materials and Methods). Mice were euthanised (16 dpi), and cardiac sections prepared and imaged using a Nikon Ti-2 E inverted microscope (9 mice per group, 3 randomly selected sections from each mouse) (a) Infection burden per infected cardiac cell (nest), with the number of EdU+ve/-ve parasites indicated (see also ). (b) Images showing an infected cardiomyocyte from a non-treated mouse that contains both replicating and non-replicating amastigotes. The inset shows an example of a single serial cross section derived by 3-dimensional confocal laser scanning microscopy (z-stacking), which was used to determine the precise number of amastigotes in the infected cell (white arrow indicates intensely stained kinetoplast DNA) (see for 3-dimensional image of this infected cell). DNA (blue, DAPI); EdU+ve amastigotes (red); parasites (green fluorescence). White scale bar = 10 μm. (c) Images of infected cardiomyocytes from benznidazole-treated mice. None of the amastigotes detected were EdU+ve. A purple arrow indicates two host cells that were in S-phase during the period of EdU exposure (upper image). White scale bars = 20 μM. The insets (right) show enlarged images of DAPI stained host cell nuclei and single infecting amastigotes (green). Full 3-dimensional images of parasites that persist after benznidazole treatment are shown in and Videos.

Article Snippet: The coverslips were washed (x3) in PBS, mounted on slides with VECTASHIELD with DAPI (Vector Laboratories, Inc.), and then examined using Zeiss LSM880 confocal or inverted Nikon Ti-2 E inverted microscopes.

Techniques: Infection, Inverted Microscopy, Derivative Assay, Confocal Laser Scanning Microscopy, Staining, Fluorescence