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1.
Figure 8

Figure 8. Trypanosomes enter the brain parenchyma within hours.. From: Early Invasion of Brain Parenchyma by African Trypanosomes.

Selected frames from intravital movies show motile BSF in the brain parenchyma 5 h after infection with 106 BSF. Note the absence of arrested leukocytes and leakage of the vascular marker into the surrounding tissue. Upper panel: A motile Tbr-T BSF (red) is located in the cerebral parenchyma adjacent to a PCV. Lower panel: A motile Tbb GVR/35-PKH67 BSF (green) can be seen next to a PCV. Intravascular parasites (arrow) are rare. The vascular lumen is red, nuclei are blue. Bars = 20 µm. Movie S10 and S11.

Ute Frevert, et al. PLoS One. 2012;7(8):e43913.
2.
Figure 3

Figure 3. Trypanosomes travel with the flagellar tip leading.. From: Early Invasion of Brain Parenchyma by African Trypanosomes.

Temporary leukocyte-mediated interruption of the blood flow allows a parasite to swim freely in a capillary. Note that the negatively stained blood cells (dark) in the vascular lumen become distorted to streaks with increasing velocity. Two days post infection with 106 Tbb-O BSF. Bars = 20 µm. Movie S3.

Ute Frevert, et al. PLoS One. 2012;7(8):e43913.
3.
Figure 2

Figure 2. Trypanosomes divide within the brain parenchyma.. From: Early Invasion of Brain Parenchyma by African Trypanosomes.

DNA staining reveals two nuclei (large Hoechst-stained organelles) and two kinetoplasts (small Hoechst-stained organelles) within a motile extravascular BSF (red). The figure shows individual frames from an intravital movie recorded 24 h post infection with 106 Tbb-O. The vascular lumen is green, nuclei are blue. The panels on the right show the two nuclei and two kinetoplasts of the dividing parasite in the blue channel. Bars = 20 µm. Movie S2.

Ute Frevert, et al. PLoS One. 2012;7(8):e43913.
4.
Figure 7

Figure 7. Leukocyte recruitment to PCVs.. From: Early Invasion of Brain Parenchyma by African Trypanosomes.

The number of arrested mononuclear leukocytes increases between 24 h and 48 h post infection with 105 Tbb-O. In contrast, arrested cells are absent from the cortical microvasculature of an uninfected control mouse. Whereas intravascular Tbb-O are rare at 24 h, multiple red streaks in the vascular lumen indicate the increase in parasitemia at 48 h post infection. Bars = 20 µm. Movie S8 and S9.

Ute Frevert, et al. PLoS One. 2012;7(8):e43913.
5.
Figure 1

Figure 1. Trypanosomes enter the brain parenchyma.. From: Early Invasion of Brain Parenchyma by African Trypanosomes.

One day post infection with 106 Tbb-O, mice were subjected to craniotomy and examined by confocal microscopy. The vascular lumen was visualized by intravenous inoculation of Alexa 647-conjugated BSA (shown in green). A motile Tbb-O BSF (red) is located adjacent to a PCV. RBCs in the lumen of the PCV exclude the vascular maker and appear as negatively stained (dark) streaks. Nuclei were stained with Hoechst (blue). Bar = 20 µm. Movie S1.

Ute Frevert, et al. PLoS One. 2012;7(8):e43913.
6.
Figure 6

Figure 6. Leukocyte recruitment and vascular occlusion.. From: Early Invasion of Brain Parenchyma by African Trypanosomes.

Upper panel: Two days after infection with 105 Tbb-O, a motile BSF (red) is located in the brain parenchyma near a PCV. While numerous other trypanosomes travel at bloodstream velocity in the PCV, the flow in a neighboring capillary (top) is impaired by arrested leukocytes. Lower panel: An arrested mononuclear leukocyte occludes a capillary (arrowhead) and diverts the blood flow into a collateral microvessel (arrow). Some Tbb-O (red) appear distorted due to their high velocity. Bars = 20 µm. Movie S6 and S7.

Ute Frevert, et al. PLoS One. 2012;7(8):e43913.
7.
Figure 5

Figure 5. Leukocyte recruitment and vascular leakage.. From: Early Invasion of Brain Parenchyma by African Trypanosomes.

Two days post infection with 106 Tbb-O BSF, the number of both intra- and extravascular trypanosomes has increased. A) Motile BSF are located in the PVS (arrowhead) surrounding a PCV and in the parenchyma (arrows). B) A cluster of extravascular BSF can be seen in a PVS (arrowhead), while numerous others have entered the parenchyma (arrows). The vascular marker BSA-Alexa 647 (shown in green) has leaked into the PVS. The PCVs contain arrested leukocytes (★). The narrow dark streaks in the PCV represent fast-moving blood cells and demonstrate unimpaired blood flow. Bars = 20 µm. Movie S4 and S5.

Ute Frevert, et al. PLoS One. 2012;7(8):e43913.
8.
Figure 4

Figure 4. Brain sections reveal extravascular trypanosomes after low-dose infection.. From: Early Invasion of Brain Parenchyma by African Trypanosomes.

Screening of large areas of brain tissue was required to detect extravascular (A, arrow) and intravascular (B, arrow) Tbb-O (red) 24 h after infection with 105 BSF. Infected mice were injected with BSA-Alexa 647 (green) and the nuclear stain Hoechst (blue) prior to removal of the brain. Immediately after vibratome sectioning, 100 µm slices of live brain tissue were subjected to ex vivo imaging. Traces of the vascular marker remain visible after sectioning and outline the microvascular lumen. Bars = 20 µm.

Ute Frevert, et al. PLoS One. 2012;7(8):e43913.

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