Overweight worsens apoptosis, neuroinflammation and blood-brain barrier damage after hypoxic ischemia in neonatal brain through JNK hyperactivation

Abstract

BACKGROUND: Apoptosis, neuroinflammation and blood-brain barrier (BBB) damage affect the susceptibility of the developing brain to hypoxic-ischemic (HI) insults. c-Jun N-terminal kinase (JNK) is an important mediator of insulin resistance in obesity. We hypothesized that neonatal overweight aggravates HI brain damage through JNK hyperactivation-mediated upregulation of neuronal apoptosis, neuroinflammation and BBB leakage in rat pups. METHODS: Overweight (OF) pups were established by reducing the litter size to 6, and control (NF) pups by keeping the litter size at 12 from postnatal (P) day 1 before HI on P7. Immunohistochemistry and immunoblotting were used to determine the TUNEL-(+) cells and BBB damage, cleaved caspase-3 and poly (ADP-ribose) polymerase (PARP), and phospho-JNK and phospho-BimEL levels. Immunofluorescence was performed to determine the cellular distribution of phospho-JNK. RESULTS: Compared with NF pups, OF pups had a significantly heavier body-weight and greater fat deposition on P7. Compared with the NF-HI group, the OF-HI group showed significant increases of TUNEL-(+) cells, cleaved levels of caspase-3 and PARP, and ED1-(+) activated microglia and BBB damage in the cortex 24 hours post-HI. Immunofluorescence of the OF-HI pups showed that activated-caspase 3 expression was found mainly in NeuN-(+) neurons and RECA1-(+) vascular endothelial cells 24 hours post-HI. The OF-HI group also had prolonged escape latency in the Morris water maze test and greater brain-volume loss compared with the NF-HI group when assessed at adulthood. Phospho-JNK and phospho-BimEL levels were higher in OF-HI pups than in NF-HI pups immediately post-HI. JNK activation in OF-HI pups was mainly expressed in neurons, microglia and vascular endothelial cells. Inhibiting JNK activity by AS601245 caused more attenuation of cleaved caspase-3 and PARP, a greater reduction of microglial activation and BBB damage post-HI, and significantly reduced brain damage in OF-HI than in NF-HI pups. CONCLUSIONS: Neonatal overweight increased HI-induced neuronal apoptosis, microglial activation and BBB damage, and aggravated HI brain damage in rat pups through JNK hyperactivation.