Thei, LJ; (2014) The Role of MAP Kinase Cascade in Neonatal Brain Response to Hypoxia-Ischemic Insult. Doctoral thesis , UCL (University College London).

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Abstract

Babies that are born more than 8 weeks premature or those deprived of Oxygen during the perinatal period are susceptible to brain injury, particularly in conjunction with maternal or fetal infection, leading to neurological deficits later in life. Multiple studies have shown that even brief exposure to hypoxic conditions will cause rapid and selective increase in specific mitogen-activated protein kinases including extracellular signal - related kinase 1 and 2 (ERK1/2) and C-Jun N-terminus Kinases 1 to 3 (JNK1-3) as well as the JNK downstream effector: C-Jun. To gain insight into the in vivo function of some of these intracellular pathways that contribute to neuronal damage in ischemic postnatal brains, we examined the effects of global or cell type specific as single or combined deletions of ERK 1 and 2 and both cell type specific or functional deletions of C-Jun. In addition, ERK phosphorylation was prevented by the administration of the selective MEK inhibitor SL327. This was observed with both pre- and post-injury application. Lastly, to explore the effect of endotoxin as a sensitising agent prior to neonatal hypoxia ischemia: lipopolysaccharide given to neonatal mutant mice, with sensitising effects noted at a dosage of 0.6mg/kg of LPS and a time interval between endotoxin administration and hypoxia-ischemia of 12hr. The Rice-Vannucci mouse model is a well-established experimental paradigm for hypoxic ischemic injury, providing insights into molecular signals that determine both white and grey matter tissue loss. Normal pERK is detectable in periventricular white matter axons (15-45min post HI), followed by white and grey matter glia and cortical neurons (1-4h post HI), returning to normal by 8hr. Mice with double mutation of global ERK1 and neuronal ERK2 deletion showed a lack of pERK expression through the forebrain following HI. In LPS-sensitised HI, we observed a strong decrease in infarct size, histological brain injury and microglial activation in cortex, striatum, and thalamus. A more discreet effect was seen in subcortical white matter and hippocampus. ERK1 deletion attenuated the effect of neuronal ERK2 deletion. These results were reproduced following severe HI insult alone. Astrocytic ERK mutation exhibited a polar response with a 3-4fold increase in microglia activation and the number of dying cells within grey matter regions. Global inactivation of ERK, through pharmacological ERK inhibitor SL327 significantly reduced cell death, and associated microglial activation in both grey and white matter at 48h following HI insult. Application of SL327 even as late as 1hr post insult significantly reduced brain damage induced by mild HI exposure. Systemic administration 1hr after severe HI dramatically increased the survival rate of pups at 48hr post insult by 80% compared to sham-treated controls. Deletion of C-Jun in all neural-epithelial lineage cells resulted in a strong increase in injury following severe and LPS-sensitised insult. In contrast, neuron-specific deletion of C-Jun resulted in neuroprotection. Tunel positive cell death was significantly reduced compared to control groups, in white matter, cortex and thalamus. Microglial activation, and infarct volume loss was more discreetly decreased with a notable effect in cortex. C-Jun expression in astrocytes is not a major contributor to ischemic damage response, with very mild reduction in markers for cell death and microglia recruitment following severe hypoxia, and no change observed between mutants and controls after endotoxin-sensitised HI. Replacement of the four JNK-dependent C-Jun phosphorylation sites (jun4A) resulted in a mild decrease in cell death and microglial activation compared to littermate controls, which did not reach the level of statistical significance. Overall, the data points to an important role of neuronal ERK and C-Jun in both a cellular and biochemical response to HI in the neonatal cerebral brain, but also argues against the involvement of JNK-dependent C-Jun phosphorylation in mediating neural damage. In addition, inhibition of ERK via pharmaceutical agents shows promise in decreasing morbidity and mortality caused by mild to moderate HI exposure. Lastly, neuroepithelial C-Jun expression appears to be a critical factor in normal cortical development and employment of endogenous neuroprotective mechanisms against postnatal insult.

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