Figure 8: Early BBB damage leads to delayed JP degradation.

(a–d) tFCI was induced in WT and Tg-ADFm mice for 1 h followed by 24 h of reperfusion. (a) Expression of JPs occludin, VE-cadherin and ZO-1, as well as the basal lamina protein laminin in whole-cell extracts from brain microvessels. Endothelial ADFm overexpression preserved occludin, VE-cadherin and laminin from tFCI-induced degradation. (b) Double-label immunostaining for laminin and CD31 in ipsilateral cortex. Consistent with its role as a basement membrane protein, laminin is distributed in the outer layer of CD31⁺ microvessels. Square: the region enlarged in high-power images. Arrowhead: partial loss of laminin protein (green signal). The overlap coefficient of laminin and CD31 immunofluorescence along the microvessels was calculated in the ischaemic core area and inner border zone, respectively. The coefficient was reduced after tFCI due to the partial loss of laminin, which was significantly attenuated in Tg-ADFm animals. (c) Double-label immunostaining for MMP-9 and CD31 in ipsilateral cortex. MMP-9 was upregulated after tFCI, mainly in CD31⁺ microvessels (arrowhead) and in infiltrated immune cells (arrow; see Supplementary Fig. 12a). ADFm overexpression abolished the upregulation of brain MMP-9. Scale bar, 50 μm. (d) MMP-9 or MMP-2 levels were measured by gelatin zymography in brain tissues and plasma. Endothelial ADFm overexpression blocked tFCI-induced MMP-9 elevation in the brain but not in the plasma. n=5–6 mice per group. *P≤0.05, **P≤0.01 versus WT. (e,f) Blood neutrophils were extracted from MMP-9^+/+ or MMP-9^−/− mice 24 h after tFCI. HBMECs were exposed to 1 h of OGD, and immediately co-cultured with these neutrophils for 1–3 h. Expression of occludin and VE-cadherin in HBMECs was then examined. (e) OGD-challenged HBMECs became vulnerable to neutrophil-induced JP degradation. (f) The degradation of JPs was mediated by MMP-9, as MMP-9^−/− neutrophils failed to elicit protein degradation in OGD-challenged HBMECs. *P≤0.05, **P≤0.01 versus no neutrophil controls (Con). (g) HBMECs were infected with Lenti or Lenti-ADFm. After 48 h, cells were subjected to OGD, followed by co-culture with MMP-9^+/+ neutrophils for 1–2 h. ADFm expression significantly preserved occludin and VE-cadherin against neutrophil MMP-9-mediated degradation. *P≤0.05, **P≤0.01 versus OGD+Lenti. Data represent four independent experiments.