Dipyridamole potentiated the trypanocidal effect of nifurtimox and improved the cardiac function in NMRI mice with acute chagasic myocarditis

Figures

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  Fig. 1. : effect of nifurtimox (Nfx) and dipyridamole (DPY) in epimastigote axenic culture. After epimastigote growth in liver infusion tryptose (LIT) medium at 28ºC under continuous rotational shaking until they achieved logarithmic growth with a density of 4 x 106 parasites/mL, moment Nfx (1 µM to 1 mM) and/or DPY (10 µM to 1 mM) was added and then incubated for 48 h. (A) Normalised dose response curves for Nfx (left) and DPY (right); observe that Nfx had 10 times more potency than DPY on the inhibition of epimastigote proliferation. (B) DPY (20 µM) was assayed at 5, 10 and 15 µM doses of Nfx; observe that DPY potentiated the effect of Nfx.

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  Fig. 2. : parasitaemia and survival in infected mice treated with nifurtimox (Nfx) and/or dipyridamole (DPY). Parasitaemia was quantified using Newbauer’s camera seeded with 5 µL of blood diluted 1:10 in NaCl 0.9%; mortality was registered until 50% of mice for the determined group died. (A) Shows parasitaemia obtained at the end of the experiment; observe that groups treated with Nfx alone or combined with DPY had parasitaemia close to 0, while parasitaemia persisted in control Chagas mice and DPY-treated mice. However, parasitaemia with DPY was significantly lower than control Chagas mice. (B) Shows parasitaemia evolution as a function of time in mice treated with Nfx 40 mg/kg (black filled squares) and Nfx 40 mg/kg plus DPY 30 mg/kg (open diamond); parasitaemia achieved values close to 0 from the 25th day, and the DPY-Nfx group tended to increase parasitaemia in the first week. (C) Shows survival curves for control Chagas (open circle), DPY 30 mg/kg (open diamond), Nfx 10 mg/kg (black filled square), Nfx 40 mg/kg (hexagon total black filled), Nfx 10 mg/kg plus DPY (square half black filled) and Nfx 40 mg/kg plus dipyridamole (hexagon half black filled) groups. The control Chagas and…

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  Fig. 4. : typical electrocardiographic (ECG) traces and qualitative ECG disorders observed in healthy, untreated and treated chagasic mice. The mean ± standard error (SEM) of the qualitative ECG disorders (rhythm, conduction and repolarisation disorders) observed in mice of the different experimental groups are shown in the upper panel on the left. Healthy mice have practically no disorders, whereas the group of chagasic mice treated with nifurtimox (Nfx) had a significantly greater increase in these disorders compared with the remaining groups. In panel A1, we show a typical record of a healthy mouse. Traces of mice with chagasic myocarditis are shown in panels B1 and B2; in B1, we can observe low-voltage QRS complexes with a Rr’ wave suggestive of His intranodal block and multiple rhythmical atrial ectopics that are magnified in the inserted box suggestive of atrial flutter; in B2, we can perceive a complex rhythm disorder with ventricular extrasystoles, a very low voltage QRS complex, a deep S wave and a negative T wave. Traces of mice with chagasic myocarditis treated with Nfx at doses of 10 mg/kg of body weight are shown in panels C1 and C2 where there is a worsening in the…

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  Fig. 5. : histopathological findings in haematoxylin-eosin stained heart tissue. (A) Shows a heart tissue sample from healthy mice; (B) from Trypanosoma cruzi-infected mice where amastigote nests (arrows), mononuclear infiltrate and myocitolisis can be observed; (C) 40 mg/kg Nfx-treated mice lacked amastigotes present, but fibrosis areas were present; and (D) 40 mg/kg Nfx plus 30 mg/kg-treated mice lacked evident lesions or parasite forms.

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  Fig. 6. : histopathological findings in heart tissue stained by Masson’s trichrome technique. (A) Shows a sample from Trypanosoma cruzi-infected mice and (B) from 40 mg/kg nifurtimox (Nfx)-treated mice; note that there is extensive lax fibrosis with persistent inflammatory infiltrate and an absence of cardiomyocytes in chagasic heart sections, while in the heart sections of mice treated with Nfx, the fibrosis is less extensive, compact and interfibrilar.

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  Fig. 3. : assessment of nifurtimox (Nfx) side effects by behavioural tests in healthy mice. (A) Shows results obtained from the hot plate test, which assesses cutaneous pain sensitivity, and no significant difference was observed between healthy mice and healthy mice treated with 40 mg/kg Nfx for 30 days. (B) Shows results obtained from the open field test, which assesses horizontal and vertical motility; Nfx-treated healthy mice displayed significantly less motility compared to healthy untreated mice. (C) Shows results obtained from the formalin test, which assesses neural and inflammatory pain sensitivity; licking was only significantly higher at 5 and 45 min in Nfx-treated healthy mice; however, in the other time windows, no differences were observed.