FV 1091. C1-esterase Inhibitor Treatment Prevents Blood–Brain Barrier Dysfunction in the Neonatal Mouse Brain after Acute Systemic Hypoxia

Background: Hypoxic-ischemic (HI) complications represent the main cause of acquired perinatal brain injury of term and near-term newborns. Neuroprotective effects of therapeutic hypothermia (TH) in terms with HI encephalopathy are promising; however, long-term morbidity still remains > 50%. Regarding adjuvant treatment approaches to TH, inflammatory and excitotoxic pathways involved in blood–brain barrier (BBB) vulnerability and hypoxic brain edema seem to provide putative therapeutic targets. Here, we investigated impacts of C1-esterase inhibitor (C1-INH) treatment on hypoxia-induced changes of structural and functional BBB properties in the neonatal mouse brain.

Methods: A 7-day-old wild-type mice (P7; C57BL6/NCrl) were exposed to normoxia or hypoxia (8% O₂) for 6 hours (INVIVO₂ 400 hypoxia workstation; Baker Ruskinn) and treated with C1-INH (Berinert, CSL Behring; 7.5–30 IU/kg, i. p.) at the end of hypoxia exposure. Brains were dissected after a regeneration period of 24 hours. Cerebral expression of proapoptotic genes (Bnip3, Dusp1), matrix metalloproteinases (MMP), MMP inhibitors, and tight junctions (ZO-1, occludin, claudin-1, −5) was analyzed by qRT-PCR. Degree of apoptotic cell death was quantified by cleaved caspase 3 (CC3) and TUNEL staining. Protein levels of S100b and albumin in plasma and brain lysates were analyzed by ELISA.

Results: Acute hypoxia significantly enhanced mRNA expression of Bnip3 and Dusp1 and increased the number of CC3- and TUNEL-positive cells in the parietal cortex, hippocampus, and subventricular zone compared with normoxic controls. In addition, systemic hypoxia led to significant increase of S100b protein levels in brain tissue and plasma (2- and 32-fold, respectively) associated with elevated cerebral albumin concentrations. MMP-2, −9, and −14, TIMP-2, as well as ZO-1, occludin, claudin-1, and −5 mRNA levels were unchanged.

In response to C1-INH treatment, cerebral Bnip3 (0.69 ± 0.06 vs. 1.10 ± 0.07, p = 0.0090) and Dusp1 (0.69 ± 0.05 vs. 1.16 ± 0.10, p = 0.0116) mRNA levels as well as hypoxia-induced elevated numbers of CC3- and TUNEL-positive cells significantly decreased compared with controls. Interestingly, C1-INH decreased S100b protein levels in both brain (4.34 ± 0.51 ng/mg vs. 9.77 ± 1.72 ng/mL) and plasma (7.00 ± 6.86 vs. 222.74 ± 40.76 pg/mg) in the hypoxic brains compared with controls. Additionally, passage of albumin from blood to the hypoxic brain parenchyma was abolished compared with controls. Concerning effects of C1-INH on tight junction expression, a dose-dependent increase of occludin mRNA levels (2.49 ± 0.25 vs. 5.15 ± 0.99, p = 0.0002) was observed.

Conclusion: Present neonatal mouse model is appropriate to mimic functional disruption of the immature BBB due to acute systemic hypoxia suggesting S100b as early marker of BBB dysfunction. Our data demonstrate for the first time that C1-INH treatment significantly augmented mRNA expression of specific tight junction proteins, abolished escalating plasma levels of S100b, and decreased proapoptotic mechanisms in the hypoxic neonatal mouse brain indicating prevention of hypoxia-induced BBB damage. The significance of C1-INH treatment as a promising neuroprotective therapeutic option remains to be elucidated.

No conflict of interest has been declared by the author(s).