Article Figures & Data
Figures
- Figure 1. Reversible brain swelling in experimental cerebral malaria.
(A, B, C) Subtraction images of T1-weighted images are displayed to illustrate the degree and spatial distribution of blood–brain barrier disruption (BBBD) in surviving and non-surviving mice. (D) Survival curves of wild-type infected mice and mice after single vaccination (A n = 15, B n = 5; C n = 13). (E) The difference in signal intensity (ΔSI) as a measure of BBBD is present in the olfactory bulb, cortex and brainstem in all groups. Most pronounced signal alterations are seen in the olfactory bulb. In cortex and brainstem less BBBD is seen in survivors. After edema resolves BBB, normalizes and returns to baseline values. (F) The degree of brain swelling is shown at baseline, in non-survivors and survivors at the acute stage and after edema has reversed in surviving mice. Peak edema occurred at day 8.1 ± 0.6 d post infection in non-immunized non-survivors, at day 8.8 ± 0.4 d post infection in non-immunized survivors and at 9.4 ± 0.8 d post infection. Edema lasted 1–3 d. Day 11 after infection was the last day edema was observed. Day 14 after infection was the imaging time point for reversed edema in all mice. Significance levels were tested with one-way ANOVA.
- Figure S1. Edema duration.
Exemplary T2w sagittal images of three surviving mice are displayed. Turquoise squares surround images with edema. Edema (white arrows), visible as increased (bright) T2w signal was evident for 3 d, if it had reached the dorsal migratory stream, or lasted for 1–2 d if mainly the olfactory bulb and rostral migratory stream were affected.
- Figure S2. Parasitemia course.
Parasitemia increases slower in surviving mice and is significantly different to non-survivors from day 7 to day 9. * indicates significance levels as calculated by the unpaired, two tailed t test.
- Figure 2. Reversible edema is induced by transcellular blood brain barrier disruption.
(A) Tomographic slices of example vessels are displayed with 3D models projected on the slice. Black squares are magnified below to demonstrate intact tight junctions (red) and adjacent adherence junctions (blue). White squares are magnified below and illustrate the increased number of vesicles in the endothelium (yellow), occurring during transcellular blood–brain barrier disruption. Scale bar first row 1 μm, scale bar second and third rows 200 nm. (B) DNP-Gold labelled Tokuyasu sections of mice injected with DNP-albumin. In a control mouse without edema, DNP-albumin remains intraluminal (first images). In an experimental cerebral malaria mouse with edema, DNP-albumin reaches the parenchymal side via vesicles (second image). L = lumen, E = endothelium, P = parenchyma. Scale bar 100 nm.
- Figure S3. Basal lamina and tight junctions.
(A) Dissolvement of the basal lamina in an example vessel from a non-surviving mouse is shown (arrowhead). (B) Abnormal tight junction with gaps (arrows) are seen in non-survivors and rarely in mice after edema had reversed, but not in surviving mice at peak edema. Scale bar 200 nm.
- Figure 3. Reperfusion injury on the microscopic and ultrastructural level.
(A) Exemplary T2*w images are displayed. At baseline, no microhemorrhages are apparent (arrows). In severe disease a high microhemorrhage load is seen, mainly in the olfactory bulb (OB). In survivors microhemorrhages increase after edema has resolved. (B) Graphs illustrate the increase of microhemorrhage volume, whereas brain volume reverses. (C) Segmented vessels (gold) on R2* datasets are displayed. The segmentation delineates vessel lumen and vessel wall. In non-survivors vessel volume is highest. (D) R2* vessel volume throughout the groups is presented. (n = 5–13). (E) Ultrastructural endothelial changes on TEM images are illustrated by one example of a vessel from each group. In healthy mice, the endothelium is thin with an open lumen. Astrocyte end feet cover the endothelium. In non-survivors, the endothelium is thicker, the lumen collapsed, and surrounded by swollen astrocyte end feet. Survivors in the acute stage of disease show a slight increase in endothelial thickness, but also swollen astrocyte end feet. Astrocyte end feet swelling decreases after edema has reversed, but endothelial thickness slightly increases. (F) Average endothelial thickness in all groups is displayed (n = 3–5); Significance levels were tested with one-way ANOVA.
- Figure 4. Correlation and time course of reperfusion injury and behavioral changes.
(A) Microhemorrhage volume correlates with the degree of brain swelling at the acute stage of the disease. (B) Behavioral changes were assessed with the Rapid Murine Coma and Behavioral Scale (RMCBS) score. At baseline all mice displayed a healthy score of 20. Survivors showed altered behavior during the acute stage of disease but returned back to normal in most mice. (C) The ratio of inner area of the endothelium versus the outer area of the endothelium is displayed as a measure of collapse of lumen. Significance levels were tested with one-way ANOVA. Spearman’s analyzes were used for correlation analyses.
- Figure 5. Brain swelling and occurrence of microhemorrhages in pediatric and adult cerebral malaria (CM).
(A) Normalized brain volume in children was significantly increased compared with age-matched controls and more pronounced in children compared with adults. In adults, no significant increase compared with healthy volunteers was noted, despite a spread in brain volume in non-fatal CM and fatal CM. Normalized brain volumes could only be calculated in datasets without motion artefacts. Red circles label data points of patients with successful brain volume calculation and presence of microhemorrhages. In this cross-sectional dataset, brain volumes of patients with microhemorrhages show a wide range. Significance levels were tested with one-way ANOVA. (B) Exemplary susceptibility-weighted images are shown. In controls no microhemorrhages are visible. In non-fatal and fatal disease red arrows point to exemplary microhemorrhages at the subcortical white matter.
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