Fig 1.
Flow chart showing the experimental protocol with the number of animals used, death registered, and mice included in 3 (endpoint registered as “120 dpi”) or 2 (endpoint registered as “150 dpi”) independent experiments.
Fig 2.
C57BL/6 mice infected with the Colombian Trypanosoma cruzi strain control parasitemia, survive, and develop chronic phase with preserved muscle strength.
(A) Mice were infected with 100 blood trypomastigote forms, parasitemia and death were recorded weekly, and the animals were analyzed in the chronic phase of infection (120 dpi). (B) Survival curve (percentage of alive mice). (C) Parasitemia curve (Parasites x 104/mL). (D) The graph shows the results of muscle strength [gram force (gf)/body weight (g)] of T. cruzi-infected mice compared with sex- and age-matched noninfected controls (NI), at 120 dpi. Each experimental group consisted of 5 NI mice and 7–10 T. cruzi-infected mice. Each circle represents an individual mouse. Data are represented as means ± SE of three independent experiments (15 NI; 27 T. cruzi-infected mice). Data were analyzed using t-Student test.
Fig 3.
Memory loss in chronically Trypanosoma cruzi-infected C57BL/6 mice.
Mice were infected with 100 blood trypomastigote forms of the Colombian T. cruzi strain and analyzed at 120 dpi, comparing with noninfected controls (NI). (A) The graph shows the discrimination index [number of crossed line day 2 /(number of crossed line day 1 + number of crossed line day 2)] assessed by the open field test used to evaluate habituation memory. (B) The graph shows the discrimination index [time exploring the novel object/(time exploring the novel object + time exploring the familiar object)] evaluated by the novel object recognition test object applied to assess recognition memory. (C) The graph shows the latency (second; sec) measured using the aversive shock evoked test to evaluate aversive memory. (D) The graph shows the encephalon weight (mg). Each experimental group consisted of 5 NI mice and 7–10 T. cruzi-infected mice. Each circle represents an individual mouse. Data are represented as means ± SE of three independent experiments (15 NI; 27 T. cruzi-infected mice). Data were analyzed using t-Student test (A, B and D) and ANOVA-Bonferroni posttest (C). *, p<0.05, ***, p<0.001, comparing T. cruzi-infected and NI mice.
Fig 4.
Memory loss is improved by benznidazole administration to chronically Trypanosoma cruzi-infected C57BL/6 mice.
(A) Mice were infected with 100 blood trypomastigote forms of the Colombian T. cruzi strain and benznidazole (Bz, 0.1 mL gavage; 25 mg/Kg/day) or pyrogen-free water (Veh, 0.1 mL gavage) were administered for 30 consecutive days, from 120 to 150 dpi, when mice were tested. (B) The graph shows the discrimination index [number of crossed line day 2 /(number of crossed line day 1 + number of crossed line day 2)] assessed by the open field test used to evaluate habituation memory. (C) The graph shows the discrimination index [time exploring the novel object/(time exploring the novel object + time exploring the familiar object)] evaluated by the novel object recognition test. (D) The graph shows the latency (second; sec) measured using the aversive shock evoked test to evaluate aversive memory. Each experimental group consisted of 10 NI mice and 10 T. cruzi-infected mice. Each circle represents an individual mouse. Data are shown as means ± SE and represent two independent experiments. Data were analyzed using ANOVA-Bonferroni posttest. **, p<0.01 and ***, p<0.001, comparing T. cruzi-infected and NI mice; #, p<0.05 and ##, p<0.01, comparing Bz-treated and Veh-treated T. cruzi-infected.
Fig 5.
CNS tissue architecture is preserved in chronically Trypanosoma cruzi-infected C57BL/6 mice.
Mice were infected with 100 blood trypomastigote forms of the Colombian T. cruzi strain and benznidazole (Bz, 0.1 mL gavage; 25 mg/Kg/day) or pyrogen-free water (Veh, 0.1 mL gavage) were administered for 30 consecutive days, from 120 to 150 dpi, when mice were euthanized, tissue collected and fixed. Tissue sections were stained with hematoxylin and eosin. (A) Representative sections of cerebral cortex, hippocampus, and cerebellum areas of noninfected, and Veh-treated and Bz-treated T. cruzi-infected mice. Bar = 100 μm. (B) Representative sections of choroid plexus areas of noninfected, and Veh-treated and Bz-treated T. cruzi-infected mice. Bar = 100 μm. Arrows show infiltration of rare mononuclear inflammatory cells in perivascular areas.
Fig 6.
Benznidazole therapy reduces parasitemia and parasite load in the central nervous system of chronically T. cruzi-infected C57BL/6 mice.
Mice were infected with 100 blood trypomastigote forms of the Colombian T. cruzi strain and benznidazole (Bz, 0.1 mL gavage; 25 mg/Kg/day) or pyrogen-free water (Veh, 0.1 mL gavage) were administered for 30 consecutive days, from 120 to 150 dpi, when parasitemia was analyzed, mice were euthanized, tissue collected, DNA extracted and qPCR performed. (A) Parasitemia levels. Group data for qPCR detection of T. cruzi satDNA in (B) hippocampus and (C) cerebral cortex. Each experimental group consisted of 3 NI mice and 3 randomly sorted T. cruzi-infected mice per experimental group. Each circle represents an individual mouse. Data are shown as means ± SE and represent two independent experiments. Data were analyzed using ANOVA-Bonferroni posttest. ##, p<0.01 and ###, p<0.001, comparing Bz-treated and Veh-treated T. cruzi-infected.
Fig 7.
Therapeutic intervention with Bz decreases oxidative stress in cerebral cortex of chronically infected mice.
Mice were infected with 100 blood trypomastigote forms of the Colombian T. cruzi strain and benznidazole (Bz, 0.1 mL gavage; 25 mg/Kg/day) or pyrogen-free water (Veh, 0.1 mL gavage) were administered for 30 consecutive days, from 120 to 150 dpi, when mice were euthanized, tissue collected, extracts prepared and TBARS revealed the oxidative stress marker MDA in (B) hippocampus and (C) cerebral cortex. Each experimental group consisted of 3 NI mice and 3 randomly sorted T. cruzi-infected mice per experimental group. Each circle represents an individual mouse. Data are shown as means ± SE and represent two independent experiments. Data were analyzed using ANOVA-Bonferroni posttest. *, p<0.05, **, p<0.01 and ***, p<0.001, comparing T. cruzi-infected and NI mice; ##, p<0.01, comparing Bz-treated and Veh-treated T. cruzi-infected. &&, p<0.01, comparing Bz-treated and T. cruzi-infected pre-therapy (p-Th; 120 dpi).