Figure 7.
CCM lesions are surrounded by hypoxia; more and larger lesions result in a hypoxic parenchyma. (A) Representative image of wild-type (upper panel) and Ccm3-iECKO (lower panel) P8 cerebellum sections: vessels are stained with CD31 (green), a marker of hypoxia (Hypoxyprobe, red), and DAPI (blue). Merged images (right panel) show that hypoxia appears in the periphery of the lesions. (B) Quantification of the hypoxic area in the cerebellum (cb) of wild-type and Ccm3-iECKO mice (P = .0173). A Mann-Whitney U test was used to compare the 2 groups. Each point represents 1 biological replicate (n = 5-6 mice per group), the bar indicates the mean of each group, and the error bars represent the standard deviation. (C) Representative images of CLARITY-treated cerebellums of wild-type (upper) and Ccm3-iECKO (lower) mice stained with a marker of hypoxia (Hypoxyprobe, red), and collagen IV (white). (D) A magnified region of the dashed green square marked in panel C, illustrating how hypoxia and lesion distance were measured. The left panel shows hypoxia surrounding a lesion in 1 cerebellar lobe. Collagen IV is used to mark the vasculature. The right panel shows how lesion area, hypoxic area, and hypoxic distance were measured and the graph below shows the fluorescence intensity of hypoxia (gray value) after the hypoxic distance in micrometers was measured across the lesion. (E) Hypoxia intensity increases with distance from the lesions. Hypoxia intensity is shown as AU, which were determined by dividing the fluorescence intensity surrounding the lesions by the fluorescence intensity at the perimeter of the same lesions. Mean hypoxia intensity and lesion distance were determined for a total of 262 lesions from 3 Ccm3-iECKO mice. (F) The lesions were grouped by size (i-iv). The number of analyzed lesions in each group is indicated in parenthesis on the upper right corner of each graph. Data are shown as mean with 95% confidence intervals. (G) A total of 37 parts of the cerebellum (∼750 × 750 μm), such as in the lower image of panel C, were randomly selected from 5 sections of 3 Ccm3-iECKO mice and were evaluated for lesion size and hypoxic area. A correlation analysis was done between hypoxic area and lesion area. The Spearman correlation coefficient (r = 0.5339) and the corresponding P value (P = .0007) are indicated on the plot.

CCM lesions are surrounded by hypoxia; more and larger lesions result in a hypoxic parenchyma. (A) Representative image of wild-type (upper panel) and Ccm3-iECKO (lower panel) P8 cerebellum sections: vessels are stained with CD31 (green), a marker of hypoxia (Hypoxyprobe, red), and DAPI (blue). Merged images (right panel) show that hypoxia appears in the periphery of the lesions. (B) Quantification of the hypoxic area in the cerebellum (cb) of wild-type and Ccm3-iECKO mice (P = .0173). A Mann-Whitney U test was used to compare the 2 groups. Each point represents 1 biological replicate (n = 5-6 mice per group), the bar indicates the mean of each group, and the error bars represent the standard deviation. (C) Representative images of CLARITY-treated cerebellums of wild-type (upper) and Ccm3-iECKO (lower) mice stained with a marker of hypoxia (Hypoxyprobe, red), and collagen IV (white). (D) A magnified region of the dashed green square marked in panel C, illustrating how hypoxia and lesion distance were measured. The left panel shows hypoxia surrounding a lesion in 1 cerebellar lobe. Collagen IV is used to mark the vasculature. The right panel shows how lesion area, hypoxic area, and hypoxic distance were measured and the graph below shows the fluorescence intensity of hypoxia (gray value) after the hypoxic distance in micrometers was measured across the lesion. (E) Hypoxia intensity increases with distance from the lesions. Hypoxia intensity is shown as AU, which were determined by dividing the fluorescence intensity surrounding the lesions by the fluorescence intensity at the perimeter of the same lesions. Mean hypoxia intensity and lesion distance were determined for a total of 262 lesions from 3 Ccm3-iECKO mice. (F) The lesions were grouped by size (i-iv). The number of analyzed lesions in each group is indicated in parenthesis on the upper right corner of each graph. Data are shown as mean with 95% confidence intervals. (G) A total of 37 parts of the cerebellum (∼750 × 750 μm), such as in the lower image of panel C, were randomly selected from 5 sections of 3 Ccm3-iECKO mice and were evaluated for lesion size and hypoxic area. A correlation analysis was done between hypoxic area and lesion area. The Spearman correlation coefficient (r = 0.5339) and the corresponding P value (P = .0007) are indicated on the plot.

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