Figure 1.
Expression of ZO-1 and Cl-5 in cortical microvessels of the neurovascular unit in vitro model.
(A, B) CD31 stainings demonstrating preservation of microvascular morphology in the COSC of the coculture. (C), Endfeet of Astrocytes (arrowheads in C) contacting CD31 positive microvessels. D-F, Microvessels express the tight-junction proteins ZO-1 (panel D) and Claudin-5 (panel E), which show a co-localization (panel F). Scale bars in C and F: 20 µm; in A, B and D, E: 20 µm.
Figure 2.
Application of Z-DEVD-fmk preserves TEER and TJ monolayer integrity under OGD.
(A-F) Continuous expression of Cl-5 (panel A) and ZO-1 (panel D) under normoxic conditions. After 30 min of OGD Cl-5 and ZO-1 are prominently disrupted (arrowheads and insets in B and E) and display a ragged shape. Analogous to the protective effect of Z-DEVD-fmk on TEER (panel I), Z-DEVD-fmk also has a protective effect on ZO-1 and Cl-5 expression, but does not completely prevent TJ disruption (arrowheads in C and F). (G) Immunohistochemical co-staining with propidium iodide (red) and caspase-3 (green) shows high amounts of cleaved caspase-3 after OGD which are lowered by pre-treatment with Z-DEVD-fmk (H). I, Pre-treatment with 50 µmol/l Z-DEVD-fmk for 2 hours preserves TEER values significantly after hypoxia (no inhibitor 266.1±7.7 OHM×cm2 vs. Z-DEVD-fmk: 317.3±15.5 OHM×cm2, n = 6 coculture preparations, P<0.05).
Figure 3.
Z-DEVD-fmk preserves TJ expression in brain endothelial cell membranes under OGD.
Fluorescence intensity of ZO-1 and Cl-5 in cell membranes related to the fluorescence intensity of the cytoplasm: Cell membranes are represented by values in V1 and V3. The fluorescence intensity of the cytoplasm is the average intensity of the distance V1-V3: V2. (A-E) Intensity histograms of ZO-1 in cells that were not pre-treated with Z-DEVD-fmk before OGD (A and B) have lower peak values at cell membranes (B) related to the intensity of the cytoplasm of the corresponding cell. (C and D) In contrast, high peak values in relation to the intensity of the cytoplasm were observed in cells that were pre-treated with Z-DEVD-fmk. (E) Z-DEVD-fmk significantly preserved ZO-1 intensity at cell membranes under OGD (no inhibitor: 1.24±0.1 vs. Z-DEVD-fmk: 1.84±0.1, n = 12 cells from 3 coculture preparations, P<0.001). (F–J) Quantification of Cl-5 at cell membranes demonstrates that Z-DEVD-fmk has a protective effect on Cl-5 alignment at cell membranes. (F) BEC with impaired Cl-5 expression at the cell membrane as shown in the corresponding linescan histogram (G). (H) Preserved Cl-5 localization at the cell membrane in a BEC pre-treated with Z-DEVD-fmk before OGD with sustained peak values in the linescan histogram (I). (J) Quantification of linescan histograms shows a significant protective effect of Z-DEVD-fmk for Cl-5 expression at cell membranes (no inhibitor: 1.9±0.21 vs. Z-DEVD-fmk: 3.19±0.41, n = 12 cells from 3 coculture preparations, P<0.05).
Figure 4.
OGD induced microvascular TJ disruption is prevented by Z-DEVD-fmk.
(A-D), Depletion of oxygen and glucose results in disruptions of ZO-1 (arrowheads in A, C) and Cl-5 (arrowheads in B, D, I) in cortical microvessels in the NVU model. (E and F) Z-DEVD-fmk significantly reduced gap formation in Cl-5 (15.33±1.32% no inhibitor vs. 4.69±0.84% +Z-DEVD-fmk, P<0.001, n = 28–46 microvessels, 6–7 coculture preparations) and ZO-1 (23.87±2.15% no inhibitor vs. 12.59±2.15% +Z-DEVD-fmk, P<0.001, n = 26–42 microvessels, 6–7 coculture preparations) after 30 min of OGD. (J-O) Here, Z-DEVD-fmk prevents disruptions of ZO-1 and Cl-5.
Figure 5.
ZO-1 disruption and MAP-2 damage in the guinea pig isolated whole brain after MCAO.
(A-C) Cerebral microvessels have continuous ZO-1 alignment in the control hemisphere, with only slight discontinuities in ZO-1 staining (arrowhead in C). (D) MAP-2 staining demonstrates early hypoxic tissue damage after 30 min of MCAO without reperfusion in the occluded MCA-territory of the guinea pig whole brain. The control hemisphere shows no hypoxic damage. (E-H) Numerous disruptions of ZO-1 in cortical microvessels were found after 30 min of MCAO without reperfusion (arrowheads in E-H).
Figure 6.
OGD induced caspase-3 expression is only partially associated with DNA-fragmentation.
(A, B) Positive TUNEL-labeling demonstrates DNA-fragmentation after 30 min of OGD in the bEnd.3 cells (arrowheads in A and B). (C-F) Elevated levels of active caspase-3 (C and D: red stained immunoreactive cleaved caspase-3) are only partially expressed in TUNEL positive cells (insets in E and F, note the fragmentation of the nucleus in F). (G), Caspase-3 immunoreactivity is significantly higher (arrowheads H) after 30 min of OGD as compared to normoxic (arrowheads I) conditions in the brain endothelial monolayer area of the neurovascular unit model (normoxia: 16.85%±2.78% caspase-3 positive quadrants per cell vs. OGD: 31.31%±5.71% caspase-3 positive quadrants per cell, n = 3 coculture preparations: 9 RFV per group, P<0.05).