Investigating Pharmacological Inhibition of the NLRP3 Inflammasome Acutely and Chronically Post-Hypoxia in a Moderate Mouse Model of Hypoxic-Ischaemic Encephalopathy.

Abstract

Neonatal Encephalopathy (NE) describes syndromes of "disturbed neurological function" during the neonatal period induced by a range of aetiologies and is the leading cause of death and disability in neonates worldwide. The most common aetiology is hypoxic-ischaemic encephalopathy (HIE), which accounts for 29% of NE cases and has an incidence of 1 to 3 in 1000 live births in developed countries. HIE is the most common cause of neonatal seizures, and the co-occurrence of HIE and neonatal seizures is strongly associated with poorer neurological outcomes. Diagnostic modalities and treatment strategies are suboptimal at best and rely upon an incredibly narrow therapeutic window of 6 hours post-injury to have any protective effects, highlighting a significant unmet clinical need. The involvement of neuroinflammation and microglia in HIE pathology has been demonstrated in preclinical and human studies, which is significant given their intrinsic role in neurodevelopment. Even in moderate models of HIE, microglia populations in the hippocampus are significantly elevated. Thanks to these advances, anti-inflammatory treatment strategies have garnered promising results in attenuating acute injury and managing chronic neuroinflammation. One pathway which has emerged is the NLRP3 inflammasome, responsible for IL-1β release by microglia, which is elevated post-HIE, and targeting activating receptors, such as P2X7R, has been neuroprotective. Therefore, this thesis aims to characterise microglia in the hippocampus using phenotypes and activation markers, followed by NOD-like Receptor Family Pyrin Domain containing 3 (NLRP3) inflammasome inhibition studies using MCC950 to understand how neuroinflammation in the hippocampus is impacted by inflammasome inhibition. To do this, global hypoxia was induced at Postnatal Day 7 and microglia in the hippocampus were analysed at 72 hours and 5 weeks post-hypoxia. This found that microglia populations were elevated at both timepoints and expressed higher proportions of pro-inflammatory microglia and fewer homeostatic microglia. Furthermore, neonatal reflex development was negatively impacted by hypoxia, as were hippocampal behaviours later in life such as anxiety and spatial memory. To understand the role of inflammasome signalling in these deficits, mice were treated with MCC950 immediately post-hypoxia. Proinflammatory microglia were significantly reduced acutely but not chronically, and homeostatic microglia were increased at the chronic stage. Neonatal reflexes were only partially attenuated, 5 weeks later, spatial memory and anxiety were significantly improved. Accompanying gene and protein expression studies were performed, which attenuated expression of pro-inflammatory markers, while inducing persistent upregulation of the classical complement pathway acutely and chronically. Next, a different approach was adopted whereby MCC950 was administered twice daily from PND21 to PND26 to see whether an anti-inflammatory approach is still neuroprotective far past the traditional treatment window. Proinflammatory microglia were significantly reduced at 5 weeks post-hypoxia, although proportions of homeostatic microglia were practically unchanged. MCC950 attenuated behavioural deficits, having a particularly beneficial effect on anxiety compared to spatial memory. Overall, the results of this thesis provide compelling evidence that a significant and chronic immune and microglial response is induced by a moderate model of global hypoxia. Inhibiting the NLRP3 inflammasome after and 2 weeks post-hypoxia provides promising neuroprotective effects, opening potential avenues for future research into anti-inflammatory treatment approaches for HIE.