Impaired blood‐brain barrier function represents an important component of hypoxic‐ischemic brain injury in the perinatal period. Proinflammatory cytokines could contribute to ischemia‐related blood‐brain barrier dysfunction. IL‐6 increases vascular endothelial cell monolayer permeability in vitro. However, contributions of IL‐6 to blood‐brain barrier abnormalities have not been examined in the immature brain in vivo. We generated pharmacologic quantities of ovine‐specific neutralizing anti‐IL‐6 mAbs and systemically infused mAbs into fetal sheep at 126 days of gestation after exposure to brain ischemia. Anti‐IL‐6 mAbs were measured by ELISA in fetal plasma, cerebral cortex, and cerebrospinal fluid, blood‐brain barrier permeability was quantified using the blood‐to‐brain transfer constant in brain regions, and IL‐6, tight junction proteins, and plasmalemma vesicle protein (PLVAP) were detected by Western immunoblot. Anti‐IL‐6 mAb infusions resulted in increases in mAb ( P < 0.05) in plasma, brain parenchyma, and cerebrospinal fluid and decreases in brain IL‐6 protein. Twenty‐four hours after ischemia, anti‐IL‐6 mAb infusions attenuated ischemia‐related increases in blood‐brain barrier permeability and modulated tight junction and PLVAP protein expression in fetal brain. We conclude that inhibiting the effects of IL‐6 protein with systemic infusions of neutralizing antibodies attenuates ischemia‐related increases in blood‐brain barrier permeability by inhibiting IL‐6 and modulates tight junction proteins after ischemia.—Zhang, J., Sadowska, G. B., Chen, X., Park, S. Y., Kim, J.‐E., Bodge, C. A., Cummings, E., Lim, Y.‐P., Makeyev, O., Besio, W. G., Gaitanis, J., Banks, W. A., Stonestreet, B. S. Anti‐IL‐6 neutralizing antibody modulates blood‐brain barrier function in the ovine fetus. FASEB J. 29, 1739‐1753 (2015). www.fasebj.org

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.

Objectives: The blood-brain barrier is a selective diffusion barrier between brain parenchyma and the intravascular compartment. Tight junctions are integral components of the blood-brain barrier. Pro-inflammatory cytokines are important in the pathogenesis of brain injury and could modify the protein constituents of tight junctions. We hypothesized that interleukin-6 (IL-6) downregulates key protein constituents of endothelial tight junctions (e.g. occludin and claudin-5). Methods: We examined the effects of IL-6 on tight junction protein expression using an in vitro blood-brain barrier model. We isolated microvessels from yearling and adult ovine cerebral cortex and placed them into culture with IL-6 concentrations of 0 (control, phosphate-buffered saline), 1, 10, and 100 ng/ml for 24 h. Cerebral microvessels were harvested, Western immunoblot performed for occludin and claudin-5, densitometry performed, and results expressed as a ratio to control values. Results: Western immunoblot analysis showed that treatment with 100 ng/ml of IL-6, but not the lower concentrations, reduced (p < 0.05) occludin expression in microvessels from yearling and adult sheep and claudin-5 in microvessels from adult sheep. However, treatment with 10 ng/ml of IL-6 increased claudin-5 in microvessels from yearling sheep. The percent of lactate dehydrogenase released from the microvessels into the surrounding media was not increased by IL-6 treatment, suggesting that the reductions in tight junction proteins did not result from cell death. Treatment of adult cerebral cortical microvessels with IL-6 preincubated with anti-IL-6 monoclonal antibodies partially attenuated the reduction in claudin-5. Conclusion: We conclude that IL-6 modulates tight junction protein expression in cerebral cortical microvessels from yearling and adult sheep.

Perinatal hypoxic‐ischemic (HI)‐related brain injury is an important cause of morbidity and long‐standing disability in newborns. The only currently approved therapeutic strategy available to reduce brain injury in the newborn is hypothermia. Therapeutic hypothermia can only be used to treat HI encephalopathy in full‐term infants and survivors remain at high risk for a wide spectrum of neurodevelopmental abnormalities as a result of residual brain injury. Therefore, there is an urgent need for adjunctive therapeutic strategies. Inflammation and neurovascular damage are important factors that contribute to the pathophysiology of HI‐related brain injury and represent exciting potential targets for therapeutic intervention. In this review, we address the role of each component of the neurovascular unit (NVU) in the pathophysiology of HI‐related injury in the neonatal brain. Disruption of the blood–brain barrier (BBB) observed in the early hours after an HI‐related event is associated with a response at the basal lamina level, which comprises astrocytes, pericytes, and immune cells, all of which could affect BBB function to further exacerbate parenchymal injury. Future research is required to determine potential drugs that could prevent or attenuate neurovascular damage and/or augment repair. However, some studies have reported beneficial effects of hypothermia, erythropoietin, stem cell therapy, anti‐cytokine therapy and metformin in ameliorating several different facets of damage to the NVU after HI‐related brain injury in the perinatal period.

Objectives: The cytokines interleukin (IL)-1β and IL-6 are modulators of the neuroimmune axis and have been implicated in neuronal cell death cascades after ischemia or infection. Previous work has shown that some cross-species conservation exists between human and rodent blood-brain barrier (BBB) transport systems. To further assess cross-species conservation of cytokine transport across the BBB, the current studies investigated permeability and inhibition of ovine IL-1β and IL-6 in the mouse. Methods: IL-1β or IL-6 was radioactively labeled with ¹³¹I and injected into the jugular vein at time zero. A subset of mice received 1 or 3 µg/mouse of an unlabeled ovine or murine cytokine (IL-1β or IL-6) to assess self- and/or cross-inhibition of transport. Permeability was assessed using multiple-regression analysis. Results: There was a significant linear relationship for both ovine ¹³¹I-IL-1β and ¹³¹I-IL-6 between brain/serum ratios and exposure time, indicating BBB permeability. Inclusion of 3 µg/mouse unlabeled ovine IL-1β or IL-6 significantly reduced the transport of ovine ¹³¹I-IL-1β or ¹³¹I-IL-6, respectively, across the BBB. Transport of both ovine ¹³¹I-IL-1β and ¹³¹I-IL-6 was significantly inhibited by 1 µg/mouse of murine IL-1β or IL-6, respectively. In contrast, 1 µg/mouse of unlabeled ovine IL-1β or IL-6 did not inhibit the transport of murine ¹³¹I-IL-1β or ¹³¹I-IL-6. Conclusions: Ovine IL-1β and IL-6 cross the mouse BBB by saturable transport. Inhibition of transport by murine homologs indicates that both species use the same transport mechanisms. Conversely, an inability of ovine cytokines to significantly inhibit the transport of murine cytokines indicates that mouse BBB has a lower affinity for ovine than murine cytokines. Knowledge of species-conserved BBB transport mechanisms may facilitate the development of novel animal models of central nervous system pathogenesis.

Pro-inflammatory cytokines contribute to hypoxic–ischemic brain injury. Blood–brain barrier (BBB) dysfunction represents an important component of hypoxic–ischemic brain injury in the fetus. Hypoxic–ischemic injury could accentuate systemic cytokine transfer across the fetal BBB. There has been considerable conjecture suggesting that systemic cytokines could cross the BBB during the perinatal period. Nonetheless, evidence to support this contention is sparse. We hypothesized that ischemia–reperfusion increases the transfer of systemic interleukin-1β (IL-1β) across the BBB in the fetus. Ovine fetuses at 127 days of gestation were studied 4 hours after 30 minutes of bilateral carotid artery occlusion and compared with a nonischemic group. Recombinant ovine IL-1β protein was expressed from an IL-1β pGEX-2 T vector in E. coli BL-21 cells and purified. The BBB function was quantified in 12 brain regions using a blood-to-brain transfer constant with intravenous¹²⁵I-radiolabeled IL-1β (¹²⁵I-IL-1β). Interleukin-1β crossed the intact BBB in nonischemic fetuses. Blood-to-brain transport of¹²⁵I-IL-1β was higher ( P < 0.05) across brain regions in fetuses exposed to ischemia–reperfusion than nonischemic fetuses. We conclude that systemic IL-1β crosses the intact fetal BBB, and that ischemia–reperfusion increases transfer of this cytokine across the fetal BBB. Therefore, altered BBB function after hypoxia–ischemia facilitates entry of systemic cytokines into the brain of the fetus.