Xiaodi Chen

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

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.

Neutralizing anti-interleukin-1β antibodies modulate fetal blood-brain barrier function after ischemia.

We have previously shown that increases in blood-brain barrier permeability represent an important component of ischemia-reperfusion related brain injury in the fetus. Pro-inflammatory cytokines could contribute to these abnormalities in blood-brain barrier function. We have generated pharmacological quantities of mouse anti-ovine interleukin-1β monoclonal antibody and shown that this antibody has very high sensitivity and specificity for interleukin-1β protein. This antibody also neutralizes the effects of interleukin-1β protein in vitro. In the current study, we hypothesized that the neutralizing anti-interleukin-1β monoclonal antibody attenuates ischemia-reperfusion related fetal blood-brain barrier dysfunction. Instrumented ovine fetuses at 127 days of gestation were studied after 30 min of carotid occlusion and 24h of reperfusion. Groups were sham operated placebo-control- (n=5), ischemia-placebo- (n=6), ischemia-anti-IL-1β antibody- (n=7), and sham-control antibody- (n=2) treated animals. Systemic infusions of placebo (0.154M NaCl) or anti-interleukin-1β monoclonal antibody (5.1±0.6 mg/kg) were given intravenously to the same sham or ischemic group of fetuses at 15 min and 4h after ischemia. Concentrations of interleukin-1β protein and anti-interleukin-1β monoclonal antibody were measured by ELISA in fetal plasma, cerebrospinal fluid, and parietal cerebral cortex. Blood-brain barrier permeability was quantified using the blood-to-brain transfer constant (Ki) with α-aminoisobutyric acid in multiple brain regions. Interleukin-1β protein was also measured in parietal cerebral cortices and tight junction proteins in multiple brain regions by Western immunoblot. Cerebral cortical interleukin-1β protein increased (P<0.001) after ischemia-reperfusion. After anti-interleukin-1β monoclonal antibody infusions, plasma anti-interleukin-1β monoclonal antibody was elevated (P<0.001), brain anti-interleukin-1β monoclonal antibody levels were higher (P<0.03), and interleukin-1β protein concentrations (P<0.03) and protein expressions (P<0.001) were lower in the monoclonal antibody-treated group than in placebo-treated-ischemia-reperfusion group. Monoclonal antibody infusions attenuated ischemia-reperfusion-related increases in Ki across the brain regions (P<0.04), and Ki showed an inverse linear correlation (r= -0.65, P<0.02) with anti-interleukin-1β monoclonal antibody concentrations in the parietal cortex, but had little effect on tight junction protein expression. We conclude that systemic anti-interleukin-1β monoclonal antibody infusions after ischemia result in brain anti-interleukin-1β antibody uptake, and attenuate ischemia-reperfusion-related interleukin-1β protein up-regulation and increases in blood-brain barrier permeability across brain regions in the fetus. The pro-inflammatory cytokine, interleukin-1β, contributes to impaired blood-brain barrier function after ischemia in the fetus.

Effects of Interleukin-6 on the Expression of Tight Junction Proteins in Isolated Cerebral Microvessels from Yearling and Adult Sheep

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.

Anti–IL-6 neutralizing antibody modulates blood-brain barrier function in the ovine fetus

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

Interleukin-1β Transfer across the Blood–Brain Barrier in the Ovine Fetus

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 intravenous 125I-radiolabeled IL-1β (125I-IL-1β). Interleukin-1β crossed the intact BBB in nonischemic fetuses. Blood-to-brain transport of 125I-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.

Expression and localization of Inter-alpha Inhibitors in rodent brain.

Inter-alpha Inhibitor Proteins (IAIPs) are a family of related serine protease inhibitors. IAIPs are important components of the systemic innate immune system. We have identified endogenous IAIPs in the central nervous system (CNS) of sheep during development and shown that treatment with IAIPs reduces neuronal cell death and improves behavioral outcomes in neonatal rats after hypoxic-ischemic brain injury. The presence of IAIPs in CNS along with their exogenous neuroprotective properties suggests that endogenous IAIPs could be part of the innate immune system in CNS. The purpose of this study was to characterize expression and localization of IAIPs in CNS. We examined cellular expressions of IAIPs in vitro in cultured cortical mouse neurons, in cultured rat neurons, microglia, and astrocytes, and in vivo on brain sections by immunohistochemistry from embryonic (E) day 18 mice and postnatal (P) day 10 rats. Cultured cortical mouse neurons expressed the light chain gene Ambp and heavy chain genes Itih-1, 2, 3, 4, and 5 mRNA transcripts and IAIP proteins. IAIP proteins were detected by immunohistochemistry in cultured cells as well as brain sections from E18 mice and P10 rats. Immunoreactivity was found in neurons, microglia, astrocytes and oligodendroglia in multiple brain regions including cortex and hippocampus, as well as within both the ependyma and choroid plexus. Our findings suggest that IAIPs are endogenous proteins expressed in a wide variety of cell types and regions both in vitro and in vivo in rodent CNS. We speculate that endogenous IAIPs may represent endogenous neuroprotective immunomodulatory proteins within the CNS.

Neutralizing anti-interleukin-1β antibodies reduce ischemia-related interleukin-1β transport across the blood–brain barrier in fetal sheep

Hypoxic ischemic insults predispose to perinatal brain injury. Pro-inflammatory cytokines are important in the evolution of this injury. Interleukin-1β (IL-1β) is a key mediator of inflammatory responses and elevated IL-1β levels in brain correlate with adverse neurodevelopmental outcomes after brain injury. Impaired blood-brain barrier (BBB) function represents an important component of hypoxic-ischemic brain injury in the fetus. In addition, ischemia-reperfusion increases cytokine transport across the BBB of the ovine fetus. Reducing pro-inflammatory cytokine entry into brain could represent a novel approach to attenuate ischemia-related brain injury. We hypothesized that infusions of neutralizing IL-1β monoclonal antibody (mAb) reduce IL-1β transport across the BBB after ischemia in the fetus. Fetal sheep were studied 24-h after 30-min of carotid artery occlusion. Fetuses were treated with placebo- or anti-IL-1β mAb intravenously 15-min and 4-h after ischemia. Ovine IL-1β protein expressed from IL-1β pGEX-2T vectors in Escherichia coli (E. coli) BL-21 cells was produced, purified, and radiolabeled with 125I. BBB permeability was quantified using the blood-to-brain transfer constant (Ki) with 125I-radiolabeled-IL-1β. Increases in anti-IL-1β mAb were observed in the brain of the mAb-treated group (P<0.001). Blood-to-brain transport of 125I-IL-1β was lower (P<0.04) across brain regions in the anti-IL-1β mAb-treated than placebo-treated ischemic fetuses. Plasma 125I-IL-1β counts were higher (P<0.001) in the anti-IL-1β mAb- than placebo-treated ischemic fetuses. Systemic infusions of anti-IL-1β mAb reduce IL-1β transport across the BBB after ischemia in the ovine fetus. Our findings suggest that conditions associated with increases in systemic pro-inflammatory cytokines and neurodevelopmental impairment could benefit from an anti-cytokine therapeutic strategy.

Ontogeny of tight junction protein expression in the ovine cerebral cortex during development

Tight junctions of the blood-brain barrier are composed of transmembrane and associated cytoplasmic proteins. The transmembrane claudin proteins form the primary seal between endothelial cells and junctional adhesion molecules (JAMs) regulate tight junction formation. We have previously shown that claudin-1, claudin-5, zonula occludens (ZO)-1, and ZO-2 exhibit differential developmental regulation from 60% of gestation up to maturity in adult sheep. The purpose of the current study was to examine developmental changes in claudin-3, -12, and JAM-A protein expression in cerebral cortices of fetuses at 60%, 80%, and 90% gestation, and in newborn and adult sheep. We also examined correlations between changes in endogenous cortisol levels and tight junction protein expression in cerebral cortices of the fetuses. Claudin-3, -12 and JAM-A expressions were determined by Western immunoblot. Claudin-3 and -12 were lower (P<0.01) at 60%, 80%, 90% and in newborns than in adults, and JAM-A was lower in adults than in fetuses at 80% and 90% gestation. Claudin-3 expression demonstrated a direct correlation with increasing plasma cortisol levels (r=0.60, n=15, P<0.02) in the fetuses. We conclude that: claudin-3, -12 and JAM-A are expressed as early as 60% of gestation in ovine cerebral cortices, exhibit differential developmental regulation, and that increasing endogenous glucocorticoids modulate claudin-3 expression in the fetus.

Systemic infusions of anti-interleukin-1β neutralizing antibodies reduce short-term brain injury after cerebral ischemia in the ovine fetus

Perinatal hypoxic-ischemic reperfusion (I/R)-related brain injury is a leading cause of neurologic morbidity and life-long disability in children. Infants exposed to I/R brain injury develop long-term cognitive and behavioral deficits, placing a large burden on parents and society. Therapeutic strategies are currently not available for infants with I/R brain damage, except for hypothermia, which can only be used in full term infants with hypoxic-ischemic encephalopathy (HIE). Moreover, hypothermia is only partially protective. Pro-inflammatory cytokines are key contributors to the pathogenesis of perinatal I/R brain injury. Interleukin-1β (IL-1β) is a critical pro-inflammatory cytokine, which has been shown to predict the severity of HIE in infants. We have previously shown that systemic infusions of mouse anti-ovine IL-1β monoclonal antibody (mAb) into fetal sheep resulted in anti-IL-1β mAb penetration into brain, reduced I/R-related increases in IL-1β expression and blood-brain barrier (BBB) dysfunction in fetal brain. The purpose of the current study was to examine the effects of systemic infusions of anti-IL-1β mAb on short-term I/R-related parenchymal brain injury in the fetus by examining: 1) histopathological changes, 2) apoptosis and caspase-3 activity, 3) neuronal degeneration 4) reactive gliosis and 5) myelin basic protein (MBP) immunohistochemical staining. The study groups included non-ischemic controls, placebo-treated ischemic, and anti-IL-1β mAb treated ischemic fetal sheep at 127days of gestation. The systemic intravenous infusions of anti-IL-1β mAb were administered at fifteen minutes and four hours after in utero brain ischemia. The duration of each infusion was two hours. Parenchymal brain injury was evaluated by determining pathological injury scores, ApopTag® positive cells/mm2, caspase-3 activity, Fluoro-Jade B positive cells/mm2, glial fibrillary acidic protein (GFAP) and MBP staining in the brains of fetal sheep 24h after 30min of ischemia. Treatment with anti-IL-1β mAb reduced (P<0.05) the global pathological injury scores, number of apoptotic positive cells/mm2, and caspase-3 activity after ischemia in fetal sheep. The regional pathological scores and Fluoro-Jade B positive cells/mm2 did not differ between the placebo- and anti-IL-1β mAb treated ischemic fetal sheep. The percent of the cortical area stained for GFAP was lower (P<0.05) in the placebo ischemic treated than in the non-ischemic group, but did not differ between the placebo- and anti-IL-1β mAb treated ischemic groups. MBP immunohistochemical expression did not differ among the groups. In conclusion, infusions of anti-IL-1β mAb attenuate short-term I/R-related histopathological tissue injury, apoptosis, and reduce I/R-related increases in caspase-3 activity in ovine fetal brain. Therefore, systemic infusions of anti-IL-1β mAb attenuate short-term I/R-related parenchymal brain injury in the fetus.

Ischemia Reduces Inter‐Alpha Inhibitor Proteins in the Brain of the Ovine Fetus

Hypoxic‐ischemic (HI) brain injury is a major cause of neurological abnormalities in the perinatal period. Inflammation contributes to the evolution of HI brain injury. Inter‐alpha inhibitor proteins (IAIPs) are a family of proteins that are part of the innate immune system. We have reported that endogenous IAIPs exhibit developmental changes in ovine brain and that exogenous IAIP treatment reduces neuronal death in HI neonatal rats. However, the effects of HI on endogenous IAIPs in brain have not been previously examined. In this study, we examined the effects of ischemia‐reperfusion on endogenous IAIPs levels in fetal sheep brain. Cerebral cortex, cerebellum, cervical spinal cord, choroid plexus, and CSF were snap frozen from sham control fetuses at 127 days gestation and after 30‐min of carotid occlusion and 4‐, 24‐, and 48‐h of reperfusion. IAIP levels were determined by Western immunoblot. IAIP expressions of the 250 kDa Inter‐alpha inhibitor (IaI) and 125 kDa Pre‐alpha inhibitor (PaI) in cerebral cortex and PaI in cerebellum were reduced (p < 0.05) 4‐h after ischemia compared with controls and returned toward control levels 24‐ and 48‐h after ischemia. CSF PaI and IaI were reduced 48 h after ischemia. We conclude that IAIPs in cerebral cortex and cerebellum are reduced by brain ischemia, and return toward control levels between 24 and 48 h after ischemia. However, changes in CSF IAIPs were delayed, exhibiting decreases 48 h after ischemia. We speculate that the decreases in endogenous IAIPs reflect increased utilization, potentially suggesting that they have endogenous neuroprotective properties.