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. 2022 Nov 28;220(2):e20211390. doi: 10.1084/jem.20211390

Figure 1.

Figure 1.

Expression of Notch4*tetEC from birth led to brain AVMs by P16. (A–C) Whole-mount frontal cortex from P16 brain with FITC-lectin+ ECs to highlight blood vessels. Arrowheads indicate AV connections with capillary diameter in P16 control brains (A and C) and enlargement in Notch4*tetEC brains (B and C). a, artery; v, vein. N = 4 Notch4*tetEC mice (113 connections; 20.26 ± 3.28 μm); N = 3 controls (88 connections; 5.90 ± 0.08 μm). P = 0.0220. Two independently repeated experiments. (D–F) Microsphere passage assay. FITC-microspheres (15 μm diameter, too large to pass capillaries) were injected into the left common carotid artery to circulate. Microspheres lodged in capillaries in control P16 brains (D and D′); microspheres passed through AV shunts in Notch4*tetEC P16 brains and lodged in lungs (E and E′). N = 3 Notch4*tetEC mice (92.76 ± 1.64%); N = 3 controls (24.62 ± 22.47%). P = 0.0346. Three independently repeated experiments. (G and H) MICROFIL casting of P16 brain vasculature. Cerebellum is shown, as cartooned in (I). Vessels were enlarged and tortuous in Notch4*tetEC cerebellum (N = 3), as compared to the control (N = 3). Three independently repeated experiments. (J and K) Saline-perfused whole brain showed evidence for hemorrhage in Notch4*tetEC brain (arrowhead) and not in control. Three independently repeated experiments. (L) Brain regions depicted in cartoon. A, anterior; P, posterior. N = 3 Notch4*tetEC mice; N = 3 controls. (M) H&E staining showed normal histology in the sagittal section through P16 control cerebellum (N = 3). (N) Minor tissue lesions (arrows) and disrupted Purkinje neurons (asterisks) were seen in P16 Notch4*tetEC cerebellum (N = 4). ml, molecular layer; gl, internal granule layer. Scale bar, 200 μm. Two independently repeated experiments. (O) Schematic indicates the cerebellar region of the sagittal brain section shown in M and N, P and Q. (P and Q) Hypoxyprobe immunostaining showed regions of hypoxic cells (arrowheads) in Notch4*tetEC cerebellum but not in controls. Quantified in (R). Lectin-positive staining indicated perfused vessels. N = 3 Notch4*tetEC mice (10 fields; 6.12 ± 0.28%); N = 3 controls (12 fields; 0.13 ± 0.03%). P = 0.0007. Insets show DAPI+ nuclei. Three independently repeated experiments. Scale bars: 100 μm in A, B, P, and Q; 5 mm in D–E′, G, H, J, and K; 200 μm in M and N. *P<0.05; ***P<0.001.