Abstract:
Hypoxic-ischaemic brain injury occurring in utero or during labour and delivery has devastating consequences on the neurological outcome of premature infants. While the aetiology of brain injury is likely to be multifactorial, inflammation appears to play a significant role in the pathogenesis of hypoxic-ischaemic injury in the developing brain. Emerging evidence suggests that Toll-like receptors (TLRs) are key immunomodulators, and can both increase and reduce injury. Previously our research group identified a candidate pathway for protection involving upregulation of cerebral TLR7 expression associated with reduced preterm brain injury. Since such evidence pointed to a potential neuroprotective role for TLR7 activation, the broad objectives of my PhD studies were to assess the therapeutic potential of the synthetic TLR7 agonist, Gardiquimod (GDQ), in a clinically relevant preterm fetal sheep model of asphyxia induced by umbilical cord occlusion. The first set of studies investigated the impact of central administration of GDQ on short-term neural outcome and neurophysiological and cardiovascular adaptation following asphyxia. Results demonstrated that GDQ significantly improved survival of oligodendrocytes within the white matter and subcortical neurons after 3 days recovery. This provided the first evidence that therapeutic manipulation via the TLR7 agonist may offer the potential to protect the immature brain against hypoxia-ischaemic brain injury. In addition to neuroprotective effects, my next study illustrated that post-asphyxial administration of GDQ was associated with prolonged fetal hypertension, accompanied by early induction of seizures and greater seizure burden. Whether the observed physiological changes had an effect on neurological outcome remain unclear. However, these findings support the pervading controversial relationship between potential modulators of brain injury, including seizures, and observed histopathological changes in the setting of asphyxia. The second set of studies investigated the long-term effects of central administration of GDQ on neural outcome and physiological recovery following asphyxia. This is important as mounting evidence suggests that hypoxic-ischaemic brain injury can evolve over days and even weeks following the initial insult. The first study revealed that GDQ did not significantly change the number of oligodendrocytes within white matter regions at 7 days recovery. Whilst GDQ exhibited protective effects within a specific region of the hippocampus, it was without effect in other subcortical regions, but exacerbated neuronal injury within the caudate nucleus. These findings appeared not supportive of a long-term neuroprotective action. Additionally, electroencephalography measures showed that GDQ induced greater intensity of epileptiform discharge activity during the early tertiary phase of recovery. Whether the delayed epileptiform activity drives cell injury within the caudate nucleus and potentially underlies reduced longterm efficacy of GDQ remains unknown. Nevertheless, a potential relationship was demonstrated by the significant correlation between epileptiform activity and reduced number of neurons for both the caudate nucleus and CA1/2 region of the hippocampus. Furthermore, this study also demonstrated a discordant neurophysiological response to post-asphyxial administration of GDQ, compared to the 3 day cohort, exhibiting profound suppression of overt seizures. Overall, the findings presented in this thesis showed that although GDQ provided neuroprotection after 3 days post-asphyxial recovery it did not appear to offer longer-term neuroprotection, at least using the acute dosing regimen and dose levels adopted in our present studies. Our current data also demonstrate that as part of preclinical investigations of potential neuroprotective therapies, including the use of immunomodulators, it is important to examine both short- and long-term effects on physiological parameters in order to fully understand their impact. Given the early and profound neuroprotective effects, the TLR7 agonist, GDQ, as well as its downstream immunomodulatory pathways merit further evaluation for the treatment of preterm hypoxic-ischaemic brain injury.