Figure 1.
Schematic of the imaging protocol used to define the time window of the VEGF-induced increase in BBB permeability.
The number of animals per subgroup is shown above.
Figure 2.
Changes in BBB permeability as revealed by MRI.
Mice were pretreated with saline (Group A), 1.6 µg VEGF (Group B), or 3.0 µg VEGF (Group C) by venous injection 8 hours prior to administration of the contrast agent (Gd-DTPA). Mice from the three treatment groups were scanned before (Figure 2.1 A1, B1 and C1) and immediately after Gd-DTPA injection (Figure 2.1 A2, B2 and C2). Regions of interest (ROIs) was manually defined in both cerebral hemispheres and basal ganglia. ROI1 and ROI2 are located over regions of cerebral cortex and ROI3 and ROI4 over the basal ganglia. ROI5 is over the water tube. Compared to the saline-treated control group (Figure 2.1 A2), signal intensity enhancement was observed after treatment with 1.6 µg VEGF, particularly around cerebral ventricles (Figure 2.1 B2, arrow). Pretreatment with 3.0 µg VEGF also resulted in an obvious signal intensity enhancement in both cerebral cortex and basal ganglia (Figure 2.1 C2, arrows) comparable with saline treatment (Figure 2.1 A2). Arrow head indicates the water tube. (2.2, 2.3) Statistical analysis of signal intensity changes from the cerebrum and basal ganglia. Signal intensity values of each animal were calculated as follows: ROIa = (ROI1/ROI5+ROI2/ROI5)/2, ROIb = (ROI3/ROI5+ROI4/ROI5)/2. ROIa and ROIb were used for the statistical analysis of the three groups. ROIa, signal intensity from the cerebral hemisphere. ROIb, signal intensity from the basal ganglia.
Figure 3.
Permeability of blood vessels as revealed by Evans blue staining (2%, 3 ml/kg).
Evans blue was injected 30 minutes before sacrifice. (A1) In the control group, red fluorescence was restricted to CNS blood vessels. (A2) By contrast, 8 hours after a single low dose of VEGF (1.6 µg), diffuse red fluorescence intensity was observed in the brain parenchyma as well as vessels. (A3) Pretreatment with 3.0 µg VEGF 8 hours prior to Evan blue injection resulted in more intense red fluorescence from the brain parenchyma, while the signal from blood vessels was reduced. Neural cells are DAPI-stained and images are merged. The white box in the top right corner of the MRI image indicates the location of the tissue used for histological study. Scale bar = 200 µm.
Figure 4.
MRI and histological study of brain edema.
(4.1) ADC maps for all three treatment groups. No obvious signs of edema were observed in cerebrum and basal ganglia. Arrow indicates water tube. (4.2, 4.3) Statistic analysis of ADC values from the cerebrum and basal ganglia. ADC values for each animal were calculated as follows: ROIc = (ROI1+ROI2)/2, ROId = (ROI3+ROI4)/2. ROIc and ROId were used for the statistic analysis. ROIc, ADC values of the cerebral hemisphere. ROId, ADC values of the basal ganglia. (4.4) Histological staining using HE. No obvious differences were detected among the three groups. Small voids were detected between the parenchyma and outer vessel wall (arrows). Scale barz = 100 µm.