Figure 4.

Histopathological alterations that indicate neurovirulence were reduced in Nos2^−/− mice infected with DENV. Mice were i.c. inoculated with 4 × 10² pfu of DENV-3 (MG20). The brains of 4 animals per group were collected at day 5 or 8 post-infection (d.p.i.) and routinely processed for histopathological analysis. Mice inoculated with uninfected C6/36 cell supernatants were used as controls. Collected mice brains: A and B-Uninfected; C and D-DENV-3-infected WT at 5th d.p.i.; E and F- DENV-3-infected WT at 8th d.p.i.; G and H-DENV-3-infected Nos2^−/− at 5th d.p.i.; I and J-DENV-3-infected Nos^−/− at 8th d.p.i. Histopathological alterations were not observed in the brains of uninfected WT mice (A and B). Note the endothelial response (thick arrows) and perivascular leukocyte cell migration (thin arrows) as well as the intense vacuolization of the brain tissue (arrowhead), suggesting neuronal degeneration, necrosis, and apoptosis (*) (E and F). The images were captured at different magnifications, bar = 10 micrometers. (K) The area of the lesion was expressed as the inflammatory area per 100 μm² of brain tissue. The results are representative of two similar and independent experiments. Data from the different groups were compared using a one-way analysis of variance (ANOVA) (GraphPad Prism software, San Diego, USA). *P < 0.05 when comparing the 8th d.p.i. in the WT mice with the other bars. N.I. = uninfected.