Acute CD increases the cerebral leukocyte-endothelium interaction, the percentage of microvessels presenting platelet-leukocyte aggregates and oxidative stress.

<p>An increased number of rolling cells was found in the cerebral venular segment of infected mice (A). The NI group presented only 3±0.5 cells/min. At 8 dpi, the animals presented 6.3±0.8 cells/min, and at 15 dpi, the number of rolling leukocytes increased to 16±0.6 cells/min. The leukocyte adhesion analysis (B) showed that 1±0.2 cells/min/100 µm adhered to the venular segment in the NI group. This number was 0.4±0.2 cells/min/100 µm at 8 dpi and 8.6±1.7 cells/min/100 µm at 15 dpi. At 15 dpi, a high percentage of venules showed microvascular platelet-leukocyte aggregates (PLAs) (C). The malondialdehyde (MDA) levels in the brain of the non-infected and <i>T. cruzi</i>-infected mice showed an increase in oxidative stress only at 8 dpi (D). A–C and H–K, bar = 100 µm. One-way ANOVA test; <i>p</i><0.05^*, <i>p</i><0.01^**and <i>p</i><0.001^***, comparing the infected animals at 15 dpi with the NI group; <i>p</i><0.001^### and <i>p</i><0.01^##, comparing the infected animals at 15 and 8 dpi. Values are the means ± SEM (n = 4–8 animals/group); dpi: days post-infection; NI: non-infected.</p

Swiss Webster mice infected with the Y strain of <i>T. cruzi</i>-developed acute CD.

<p>Mice were infected with 10⁴ blood trypomastigote forms, and the following parameters were evaluated in a kinetic study: (A) parasitemia, (B) weight and (C) survival rate. The parasitemia peak occurred at 8 dpi (A). <i>T. cruzi</i> infection induced a significant body weight decrease in a time-dependent manner, starting at 12 dpi. At 22 dpi, the average weight of the NI mice was 31.4±1.9 g, while that of the <i>T. cruzi</i>-infected group was 23.8±2.5 g (B). At 22 dpi, only 20% of the infected animals survived (C). Quantitative data are expressed as the means ± SEM (n = 20). One-way ANOVA test, <i>p</i><0.05^* and <i>p</i><0.001^***, comparing the infected group at 15 dpi with the NI group; dpi: days post-infection; NI: non-infected.</p

Acute CD causes cerebral functional capillary rarefaction.

<p>Perfused cerebral arterioles and capillaries (arrows) can be observed by the fluorescence of FITC-dextran in the non-infected (A) and <i>T. cruzi</i>-infected animals at 8 (B) and 15 (C) dpi. A collapse in the microcirculation can be observed at 15 dpi (C). In (D), the graph shows a significant reduction in the number of perfused blood vessels (capillary density) in the infected animals at 15 dpi (405±31.4 capillaries/mm²) compared with the non-infected controls (514±1 capillaries/mm²) and with the <i>T. cruzi</i>-infected mice at 8 dpi (535±31.2 capillaries/mm²). Quantitative data are expressed as means ± SEM (n = 5–8/group). One-way ANOVA test; bar = 100 µm; dpi: days post-infection; <i>p</i><0.05: * comparing the infected animals at 15 dpi with NI group; # comparing 15 to 8 dpi. dpi: days post infection. NI: non-infected.</p

Rhodamine-labeled leukocytes in cerebral venules in acute CD.

<p>The images show venules of the non-infected (A) and <i>T. cruzi</i>-infected (B) mice at 8 and 15 dpi (C and D). The leukocyte-endothelium interaction (arrows) in the venules of the infected animals can be observed. Note the microvascular platelet-leukocyte aggregates (arrowhead) at 15 dpi in the infected animals (C and D). dpi: days post-infection; NI: non-infected.</p

Structural investigation of C6/36 and Vero cell cultures infected with a Brazilian Zika virus - Fig 6

<p><b>C6/36 cells infected with ZIKV analyzed by transmission electron microscopy (TEM) at different time points post-infection (A-B: 48 hr p.i., C-D: 72 hr p.i.).</b> Several large viroplasm-like perinuclear compartments (V) (A-B) and ZIKV particles (*) measuring approximately 40–50 nm in diameter in the endoplasmic reticulum cisternae (RER) (A, B) and in lysosomes (L) (C-D) were observed. Nucleocapsids were observed inside the rER (D). Thickening of the nuclear membrane and rough endoplasmic reticulum cisternae (rER) (black head arrow) (C), numerous lysosomes (L) (C, D) and vesicular compartments associated with rER (arrow) (C) measuring approximately 100 nm in diameter were observed. Nucleus (N).</p

Detection of ZIKV RNA and viral load quantification from C6/36 and Vero cell supernatants at 24, 48 and 72 hr p.i. by quantitative real-time RT-PCR.

<p>Detection of ZIKV RNA and viral load quantification from C6/36 and Vero cell supernatants at 24, 48 and 72 hr p.i. by quantitative real-time RT-PCR.</p

Uninfected C6/36 cells (negative controls) at different time points in culture.

<p><b>No cellular ultrastructural alterations were observed.</b> (A–B) 24 hr of culture; (C) 48 hr of culture; (D) 72 hr of culture. Rough endoplasmic reticulum cisternae (rER), nucleus (N).</p

Immunolocalization of ZIKV E protein (4G2, green) [arrow] in the cytoplasm of Vero cells infected with ZIKV at different time points post-infection.

<p>(A) Uninfected cells; (B) 24 hr p.i; (C) 48 hr p.i.; (D) 72 hr p.i. The nuclei were counterstained with DAPI (blue). (E) The graph represents the mean ± standard deviation of the infection percentage (%). A higher number of infected cells was observed at 24 hr p.i.</p

Uninfected Vero cells (negative controls) at different culture time points.

<p>No cellular ultrastructural alterations were observed. (A) 24 hr of culture; (B) 48 hr of cultures; (C): 72 hr of culture. Nucleus (N), mitochondria (M).</p

Immunolocalization of ZIKV E protein (4G2, green) [arrow] inside cytoplasm of C6/36 cells infected with ZIKV at different time points post infection.

<p>(A) Uninfected cells; (B) 24 hr p.i.;(C/D) 48 hr p. i.; (E) 72 hr p.i. The nuclei (N) were counterstained with DAPI (blue). (F) The graph represents the mean ± standard deviation of the infection percentage (%). The highest number of infected cells was observed at 72 hr p.i.</p