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.

The TRPM group of cation channels plays diverse roles ranging from sensory signaling to Mg²⁺ homeostasis. In most metazoan organisms the TRPM subfamily is comprised of multiple members, including eight in humans. However, the Drosophila TRPM subfamily is unusual in that it consists of a single member. Currently, the functional requirements for this channel have not been reported. Here, we found that the Drosophila TRPM protein was expressed in the fly counterpart of mammalian kidneys, the Malpighian tubules, which function in the removal of electrolytes and toxic components from the hemolymph. We generated mutations in trpm and found that this resulted in shortening of the Malpighian tubules. In contrast to all other Drosophila trp mutations, loss of trpm was essential for viability, as trpm mutations resulted in pupal lethality. Supplementation of the diet with a high concentration of Mg²⁺ exacerbated the phenotype, resulting in growth arrest during the larval period. Feeding high Mg²⁺ also resulted in elevated Mg²⁺ in the hemolymph, but had relatively little effect on cellular Mg²⁺. We conclude that loss of Drosophila trpm leads to hypermagnesemia due to a defect in removal of Mg²⁺ from the hemolymph. These data provide the first evidence for a role for a Drosophila TRP channel in Mg²⁺ homeostasis, and underscore a broad and evolutionarily conserved role for TRPM channels in Mg²⁺ homeostasis.

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.