Disruption of Bmal1 Impairs Blood–Brain Barrier Integrity via Pericyte Dysfunction

BBB hyperpermeability in Bmal1_nestin^−/− mice. A, Wet/dry weight ratios of control and Bmal1_nestin^−/− mice (control, n = 6; Bmal1_nestin^−/−, n = 5). B, Confocal images of the cerebral cortex of mice injected with Evans Blue after 24 h of circulation. C, Confocal images of the cerebral cortex in mice injected with biotin after 24 h of circulation. Sections were stained with Alexa Fluor 488–streptavidin (1:400; Invitrogen) and anti-laminin/Alexa Fluor 546-conjugated goat anti-rabbit IgG. The leakage area of biotin was quantified (control, n = 3; Bmal1_nestin^−/−, n = 3). D, Three-dimensional reconstruction of confocal image z-stacks of cerebral cortex vasculatures depicted by CD31 (endothelial cells), Desmin (pericytes), CD13 (pericytes), and PDGFRβ (pericytes) in Bmal1_nestin^−/− mice at age 8–12 weeks. Vascular density, pericyte coverage, and the number of PDGFRβ⁺ cells were calculated (control, n = 3; Bmal1_nestin^−/−, n = 3). E, Confocal images of Aqp4 (astrocyte end-feet) and CD31 in the cerebral cortex of Bmal1_nestin^−/− mice. The Aqp4⁺ area was calculated (control, n = 3; Bmal1_nestin^−/−, n = 3). *p < 0.05; **p < 0.01 when compared with littermate controls.

Cerebral astroglial activation and the loss of pericyte marker expression in Bmal1_nestin^−/− mice are age dependent. A, Confocal images of GFAP, Desmin, CD13, and PDGFRβ in the cerebral cortex of Bmal1_nestin^−/− mice at 1, 2, and 4 weeks of age. B, Quantification of GFAP⁺ and PDGFRβ⁺ cells in the cerebral cortex of control and Bmal1_nestin^−/− mice at various time points. The percentage areas of Desmin⁺ or CD13⁺ pericytes vs CD31⁺ vessels were calculated (control, n = 3; Bmal1_nestin^−/−, n = 3). *p < 0.05 compared with littermate controls.

Neuron-specific (Bmal1_synapsinI^−/−) and astrocyte-specific (Bmal1_s100β^−/−) Bmal1 knock-out mice do not exhibit the same abnormal phenotypes as Bmal1_nestin^−/− mice. A, Open-field test to measure novelty-induced locomotor activity in Bmal1_synapsinI^−/− (control, n = 3; Bmal1_synapsinI^−/−, n = 3) and Bmal1_100β^−/− mice (control, n = 10; Bmal1_s100β^−/−, n = 6) at 8–12 weeks of age. B, Immunohistochemical analysis of GFAP in the cerebral cortex of Bmal1_synapsinI^−/− and Bmal1_s100β^−/− mice at age 8–12 weeks. The number of positive cells per square millimeter was calculated. C, Confocal image of cerebral cortex vasculatures depicted by CD31, Desmin, CD13, and PDGFRβ staining in Bmal1_synapsinI^−/− and Bmal1_s100β^−/− mice at age 8–12 weeks. The percentage area of Desmin⁺ or CD13⁺pericytes vs CD31⁺ vessels and the number of PDGFRβ⁺ cells were calculated.

Pericytes in the brain are Nestin-GFP⁺. A, B, Confocal image of GFP (green) and Hoechst 33342 DNA staining (blue) in the cerebral cortex (A) or spinal cord (B) of Nestin-GFP transgenic mice at age 8–12 weeks. The area surrounded by a white dashed line denotes the dorsal horn of the spinal cord. C, Immunohistochemical analysis of Bmal1 in the cerebral cortex of Nestin-GFP⁺ mice at 8–12 weeks of age. D, Immunohistochemical analysis of NeuN, GFAP, CD11b, CD31, αSMA, Desmin, CD13, and PDGFRβ in the cerebral cortex of Nestin-GFP⁺ mice at age 8–12 weeks. The area surrounded by a white dashed line denotes the αSMA⁺ area. Arrows indicate the PDGFRβ⁺ cells. E, The percentage of each marker-positive cell type among Nestin-GFP⁺ cells in a confocal image from the immunohistochemical analysis presented in D (n = 3; 2–3 slices per independent mouse). Figure 5-1. Additional data for Nestin-GFP⁺ cells in the brain. A, Confocal image of GFP (green) and Hoechst 33342 DNA staining (blue) in the hippocampal dentate gyrus and CA1 and CA3 regions of Nestin-GFP transgenic mice at 8–12 weeks of age. B, Low-magnification images of immunohistochemical staining for CD31, Desmin, CD13, and PDGFRβ in the cerebral cortex of Nestin-GFP⁺ mice. C, Representative confocal laser-scanning orthoimage of z-stacks of CD31, Desmin, and CD13 in the cerebral cortex of Nestin-GFP⁺ mice.