Ischemia-reperfusion impairs blood-brain barrier function and alters tight junction protein expression in the ovine fetus

Abstract

The blood-brain barrier is a restrictive interface between the brain parenchyma and the intravascular compartment. Tight junctions contribute to the integrity of the blood-brain barrier. Hypoxic-ischemic damage to the blood-brain barrier could be an important component of fetal brain injury. We hypothesized that increases in blood-brain barrier permeability after ischemia depend upon the duration of reperfusion and that decreases in tight junction proteins are associated with the ischemia-related impairment in blood-brain barrier function in the fetus. Blood-brain barrier function was quantified with the blood-to-brain transfer constant (K(i)) and tight junction proteins by Western immunoblot in fetal sheep at 127 days of gestation without ischemia, and 4, 24, or 48 h after ischemia. The largest increase in K(i) (P<0.05) was 4 h after ischemia. Occludin and claudin-5 expressions decreased at 4 h, but returned toward control levels 24 and 48 h after ischemia. Zonula occludens-1 and -2 decreased after ischemia. Inverse correlations between K(i) and tight junction proteins suggest that the decreases in tight junction proteins contribute to impaired blood-brain barrier function after ischemia. We conclude that impaired blood-brain barrier function is an important component of hypoxic-ischemic brain injury in the fetus, and that increases in quantitatively measured barrier permeability (K(i)) change as a function of the duration of reperfusion after ischemia. The largest increase in permeability occurs 4 h after ischemia and blood-brain barrier function improves early after injury because the blood-brain barrier is less permeable 24 and 48 than 4 h after ischemia. Changes in the tight junction molecular composition are associated with increases in blood-brain barrier permeability after ischemia.

Figure 1

ECoG (A) and carotid blood flow (B) plotted for the non-ischemic (n=4 up to 24-h and n=2 up to 48-h because the ECoG in one animal at 24-h was not adequate, and n=5 up to 24-h and n=2 up to 48-h for ECoG and carotid blood flow, respectively), I/R group includes I/R-4, n=5, I/R-24 n=5, and I/R-48, n=5 plotted against study time in hours, before ischemia at baseline, and during ischemia and reperfusion. ECoG (A) Y-axis is the normalized ECoG power as difference from the individually averaged baseline ECoG values. Values are median ± SD and mean ± SD for ECoG and carotid arterial blood flow, respectively. When n=2, values plotted as separate symbols rather than means. (A) *P<0.05 ischemia vs. baseline within I/R group, but not non-ischemic group and (B) *P<0.05 ischemia vs. baseline within I/R group.

Figure 2

Blood-to-brain transfer constants (K_i) in the non-ischemic (n=5), I/R-4 (n=5), I/R-24 (n=5), and I/R-48 (n=5) groups plotted for 11 brain regions. Values are mean ± SD. *P<0.05 vs. nonischemic group, + P<0.05 vs. I/R-4 group.

Figure 3

Blood-to-brain transfer constants (K_i) in the non-ischemic (n=5), I/R-4 (n=5), I/R-24 (n=5), and I/R-48 (n=5) groups plotted for the frontal, parietal and occipital cerebral cortices. Values are mean±SD. *P<0.05 vs. non-ischemic group.

Figure 4

Tight junction protein expression in the cerebral cortex, hippocampus, and cerebellum. Group legends and numbers as for figure 2. A. Occludin, B. Claudin-1, and C. Claudin-5. Representative Western immunoblot shown for each tight junction protein; IC indicates the internal control standard protein derived from the same adult cerebral cortex. The extracts from the cerebral cortex, hippocampus, and cerebellum from the different treatment groups were placed on different immunoblots. Therefore, for the purpose of illustration, the immunoblot that most closely represented the mean value of the bar graph for each protein and brain region of each group was selected for illustration above the bar graph. GAPDH is also shown as a loading control. Values are expressed as mean ± SD. *P <0.05 vs. non-ischemic group, + P <0.05 vs. I/R-4 group.

Figure 5

Tight junction protein expression in the cerebral cortex, hippocampus, and cerebellum. Group legends and numbers as for figure 2. A. ZO-1, B. ZO-2 in the non-ischemic, I/R-4, I/R-24, and I/R-48 groups. Representative Western immunoblot shown for each tight junction protein; IC indicates the internal control standard protein derived from the same adult cerebral cortex. Values are expressed as mean ± SD. *P <0.05 vs. non-ischemic group.

Figure 6

K_i values measured with AIB in the non-ischemic (n=4), I/R-4-treated (n=5), I/R-24-treated (n=4), and I/R-48-treated (n=5) fetuses plotted against claudin-5 protein expression in the cerebral cortex. Stepwise multiple regression analysis using the Dummy coding variables, r = −0.54, n=18, P<0.05.