Figure 1.
ECM correlates with microrheological alterations in postcapillary venules.
CBA/CaJ mice were infected with PbA, PyXL, or no parasites. To assess the blood flow within the cortical microvasculature, time sequences were converted to minimal projections. A) In mice with ECM, the functional postcapillary venule diameter (short arrow), i.e. the perfused portion of the vessel, is considerably reduced compared to the entire vessel diameter (long arrow). Visualization of the vascular lumen with Evans blue reveals a zone along the endothelium of postcapillary venules (A) that contains adherent leukocytes (dark circles or ovals), but is devoid of RBC (dark streaks in the center). Note that migrating leukocytes are represented multiple times in minimal projections. Leakage of Evans blue into the perivascular space and brain parenchyma is apparent on either side of the postcapillary venule (arrowheads). B) Arterioles from mice with symptomatic ECM do not exhibit any restriction in diameter. C) The small number of adherent leukocytes (dark circles) in mice with hyperparasitemia does not cause any significant restriction in the functional postcapillary venule lumen. Neither arterioles from mice with hyperparasitemia (D) nor postcapillary venules or arterioles from uninfected control mice (E and F) exhibit any microrheological alterations. Scale bars = 20 µm. See Videos S1–S5 for the corresponding dynamic data. G) Measurement of the total and functional vascular diameters and cross-sections reveals that the blood flow in postcapillary venules from mice with ECM, but not from mice with hyperparasitemia, is severely restricted. Postcapillary venules from uninfected control mice exhibit no luminal restriction.
Figure 2.
Leukocytes are recruited to cortical postcapillary venules during both ECM and hyperparasitemia.
CBA/CaJ mice were infected with PbA, PyXL, or no parasites, subjected to craniotomy at the time of neurological signs or the parasitemia exceeding 50%, and prepared for intravital microscopy. CD8+ T cells, CD4+ T cells, neutrophils, monocytes, and macrophages were labeled by intravenous inoculation of fluorochrome-conjugated mAb (see Materials and Methods for details) and appear as open green circles. The vascular lumen was visualized with Evans blue (red). Representative images from intravital microscopy movies showing arrested green CD8+ T cells (CD8a), CD4+ T cells (CD4), neutrophils (GR-1), monocytes (CD14), and macrophages (CD11b) in postcapillary venules from PbA-infected mice with ECM and PyXL-infected mice with hyperparasitemia. Note that anti-CD14 labels the endothelium (green outline) during ECM in addition to monocytes (open green circles). Scale bars = 20 µm. See Videos S6–S16 for dynamic information.
Figure 3.
Leukocyte recruitment to cortical postcapillary venules during ECM and hyperparasitemia.
A) CBA/CaJ mice were infected with PbA, PyXL, or no parasites, subjected to craniotomy at the time of neurological signs or the parasitemia exceeding 50%, and prepared for intravital microscopy. CD8+ T cells, CD4+ T cells, Gr-1+ neutrophils, CD14+ monocytes, and CD11b+ macrophages were labeled by intravenous inoculation of mAb against CD8+a, CD4+, GR-1, CD14, and CD11b, respectively. Significantly larger numbers of CD8+ T cells, neutrophils and macrophages were found in postcapillary venules from mice with ECM compared to hyperparasitemia (P<0.05). The density of CD4+ T cells and monocytes was not significantly different. Most of the arrested leukocytes are macrophages followed by neutrophils, CD8+ T cells, CD4+ T cells, and monocytes. The data represent the mean cell density/mm2 ± STD. Significance (*, P<0.05; **, P<0.001) was determined with 1-way ANOVA. See Tables S2–S6 for details. B) Compared to mice with ECM (PbA), significantly less CD8+ T cells are recruited to postcapillary venules from mice with hyperparasitemia (PyXL). Treatment with FTY720 reduces the density of CD8+ T cells to levels similar to those found in PyXL infected mice. No arrested CD8+ T cells were found in the cortical microvasculature from uninfected mice. Vascular leakage was observed in mice with ECM (day 6–8; 76 measurements from 5 mice), but not in FTY720-treated PbA-infected mice (day 8; 19 measurements from 4 mice), PyXL infected mice with hyperparasitemia (day 5; 20 measurements from 6 mice), or uninfected control mice (>10 mice). C) Leukocytes were isolated from the brains of PbA-infected (day 6–8), PbA-infected/FTY720-treated (day 9), or PyXL-infected mice (day 5) and analyzed by flow cytometry. Most of the ECM-associated leukocytes are F4/80+ macrophages followed by Ly-6C+ monocytes, Ly-6G+ neutrophils, CD8+ T cells, and CD4+ T cells. Significance (*, P<0.05; **, P<0.01) was determined with 1-way ANOVA followed by Tukey's test for multiple comparisons. See Table S7 for details. D) FTY720 treatment of PbA-infected mice reduces the number of CD45hi ( = blood-derived) macrophages significantly to a level similar to that found in PyXL-infected mice with hyperparasitemia. No significant differences were found for CD45lo ( = parenchymal) macrophages. See Table S7 for details.
Figure 4.
ECM is associated with brain recruitment of CD45+ leukocytes.
A) PbA-infected mice exhibit significantly more CD45hi and CD45lo leukocytes in the brain compared to PyXL-infected mice. FTY720 treatment of PbA-infected mice reduces the number of CD45hi and CD45lo leukocytes to levels below those observed in PyXL-infected mice. ECM is associated with larger numbers of both CD45hi and CD45lo ICAM-1+ (B), CD69+ (C), and GrB+ (D) leukocytes compared to PyXL-infected mice. FTY720 treatment reduces the number of these leukocytes significantly, but not to the level found in PyXL-infected mice. Effect of FTY720 treatment reduces the number of GrB+ CD45hi leukocytes. The data are based on groups of at least 3 mice per experimental condition. Significance (*, P<0.05; **, P<0.01) was determined with 1-way ANOVA followed by Tukey's test for multiple comparisons. See Tables S8–S11 for details.
Figure 5.
Both ECM and hyperparasitemia are associated with upregulation of endothelial ICAM-1.
CBA/CaJ mice were infected with PbA, PyXL, or no parasites (control) and inoculated with PE-conjugated anti-ICAM-1 and Evans blue. A–D) Maximum projections of representative intravital microscopy movies showing endothelial ICAM-1 expression in postcapillary venules, and less so in arterioles (Art), in mice infected with PbA and PyXL compared to uninfected control mice. Note the pronounced ICAM-1 label along the endothelial junctions. A) PbA-infected mouse with ECM (day 6), B) PyXL-infected mouse with hyperparasitemia (day 5), C) uninfected control mouse. Note that ECM, but not hyperparasitemia, is associated with the expression of ICAM-1 on the surface of arrested leukocytes (light open circles, arrows). D) Endothelial ICAM-1 is upregulated, while ICAM-1 expressing leukocytes are absent, after FTY720 treatment of PbA-infected mice (day 9, no neurological signs). Scale bar = 20 µm. E) The endothelial ICAM-1 signal is similarly increased in cortical postcapillary venules from mice infected with PbA and PyXL compared to uninfected control mice. ICAM-1 is significantly upregulated in cortical arterioles during both ECM and hyperparasitemia compared to uninfected control mice. Compared to postcapillary venules, however, the overall level of ICAM-1 expression in arterioles is significantly lower. F) ICAM-1 fluorescence emission of individual leukocytes (N = 6) is higher during ECM compared to hyperparasitemia. G) The density of ICAM-1 expressing leukocytes in postcapillary venules from mice with ECM is significantly higher compared to mice with hyperparasitemia. Significance (*, P<0.05; **, P<0.01) was determined with 1-way ANOVA followed by Tukey's test for multiple comparisons. See Table S13 for details and Videos S17–S19 for the corresponding dynamic data.
Figure 6.
ECM is associated with the recruitment of ICAM-1+ macrophages.
Leukocytes were isolated from PbA-infected, PbA-infected/FTY720-treated, or PyXL-infected mice and subjected to flow cytometric analysis. A) The vast majority of ICAM-1+ leukocytes are macrophages, which were identified with mAb F4/80. B) FTY720 treatment reduces the number of ICAM-1 macrophages to levels similar to those found in PyXL-infected mice. C) No significant difference was found for the median ICAM-1 expression levels in macrophages from PbA-infected versus PbA-infected/FTY720-treated mice. Significance (*, P<0.05; **, P<0.01) was determined with 1-way ANOVA followed by Tukey's test for multiple comparisons. See Table S14 and S15 for details.
Figure 7.
ECM correlates with the arrest of P-selectin expressing platelets in postcapillary venules.
PbA-infected mice exhibit small clusters of CD41+ platelets (blue) and patches of P-selectin (green) along the wall of postcapillary venules at the time of ECM. Platelets remained in circulation and P-selectin was not detected in postcapillary venules from PyXL-infected mice with hyperparasitemia. The vascular lumen is visualized with Evans blue (bright red). Note that Evans blue has leaked into the brain parenchyma of the PbA-infected mouse (dark red shade on either side of the postcapillary venule). Scale bars = 50 µm. See Video S20 and 22 for the corresponding dynamic data.
Figure 8.
Model for the ECM-associated venous blood flow restriction.
A) Under normal conditions, blood passes through the cerebral microvasculature without leukocytes arresting in postcapillary venules. Except for the narrow glycocalix (GC) lining the vascular endothelium, the entire vascular diameter is used for the bloodstream, the functional diameter (FD) is not restricted, and the BBB is intact. B) Infection with PbA causes endothelial junction opening at the BBB in the absence of junction protein degradation or endothelial death. During ECM, arrested leukocytes form steric obstacles that reduce the functional diameter (FD) of postcapillary venules resulting in a severe restriction of the venous blood flow. As a consequence, the paracellular leakage of plasma into the PVS (pink arrows) is exacerbated. Like uninfected RBC, iRBC travel with the bloodstream and do not arrest. C) During hyperparasitemia, significantly fewer leukocytes and no platelets adhere to the postcapillary venule endothelium compared to ECM. Consequently, the restriction in the venous blood flow is minor and there is no vascular leakage. D) Hypothetical model for pediatric P. falciparum HCM. While leukocytes, RBC, and all iRBC pass through capillaries without adhering to the endothelium, late-stage iRBC, mononuclear cells, and platelets sequester on the wall of postcapillary venules thus restricting the venous blood flow and exacerbating the leakage of plasma into the perivascular space. Because of the significant reduction in the functional diameter (FD) of the postcapillary venule, ring-stage iRBC, uninfected RBC, and most leukocytes must flow through the center of the vessel. The arterial blood flow remained unaffected under all experimental conditions. PVM = perivascular macrophage, iRBC = infected red blood cell, L = leukocyte, M = mononuclear cell, blue circles = platelets.