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Title: Characterisation of neuroserpin protein expression throughout development and investigation of putative neuroprotective properties in rodent models of neonatal hypoxia ischaemia
Author: Millar, Lancelot
ISNI:       0000 0004 7971 6094
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2018
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Neonatal hypoxia ischaemia remains the most common cause of infant mortality globally, accounting for persistent motor and cognitive disorders in a substantial proportion of survivors. Currently, there is only one licensed treatment which aims to reduce brain damage and improve cognitive function: hypothermia. Increased understanding of the infant brain's response to hypoxic ischaemic injury and novel treatment design are of utmost importance. This thesis characterises expression of neuronally- secreted serine protease inhibitor neuroserpin, previously established as a protective factor in rodent models of adult stroke, throughout development and in rodent models of mild-to-moderate neonatal hypoxic ischaemic injury. The expression profile of neuroserpin protein was examined in the developing mouse and human brain using immunohistochemistry. Results closely corresponded with published mRNA expression patterns, demonstrating an enrichment of neuroserpin-immunoreactive neurons in Layer V and the transitory deep layer known as subplate in murine cerebral cortex. Within the subplate, neuroserpin- immunoreactive neurons formed an early-born subpopulation, partially overlapping conserved subplate molecular markers. Expression peaked at the murine brain development stage equivalent to birth in human, suggesting a potential developmental function for neuroserpin. Investigations in human foetal brain tissue confirmed neuroserpin-immunoreactivity within the deep layers of the cortical plate. Neuroserpin expression patterns were also quantified in rodent models of neonatal hypoxia ischaemia, adapted from the Rice-Vannucci method. In a preterm rat model and a neonatal mouse model, the number of neuroserpin-immunoreactive neurons within the subplate underwent a statistically significant increase in the hypoxic-ischaemic hemisphere compared to the control hemisphere and sham-operated brains, with no significant change in any other cortical layer. However, semi- quantitative Western blot detected no significant difference in total cortical neuroserpin protein between hypoxic-ischaemic and control hemispheres in neonatal mouse brains. It is possible that neuroserpin contributes to a highly anatomically-localised or time-point specific endogenous response to neonatal hypoxia ischaemia. The proposed neuroprotective properties of neuroserpin were investigated in two models of neonatal hypoxia ischaemia, yielding inconclusive results. Oxygen glucose deprivation experiments in cultured Neuro-2-A neuroblastoma cells and primary cortical neurons in vitro demonstrated a modest but statistically significant increase in staining for metabolic viability marker 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide (MTT) when treated with exogenous neuroserpin. The functional significance of this effect remains to be evaluated. The Serpini1 knock-out mouse, which does not manufacture functional neuroserpin protein, was used to assess the vulnerability of neurons to hypoxic-ischaemic brain damage in the absence of neuroserpin in vivo. Serpini1 knock-out mouse pups and wild-type littermates underwent adapted Rice-Vannucci neonatal hypoxia ischaemia, before being perfused with metabolic activity marker 2,3,5- triphenyltetrazolium chloride (TTC) 48 hours after injury. No significant difference in staining intensity was detected between genotypes, although methodological refinement is required. Immunohistochemistry for glial inflammatory markers also found mixed results, with only one of three markers investigated demonstrating a significant difference in immunoreactivity between genotypes. This thesis examines putative neuroprotective protein neuroserpin expression and reports a neuron- specific expression enrichment around term. Any endogenous upregulation of neuroserpin expression following rodent neonatal hypoxia ischaemia is modest, and evidence for substantial variability in injury severity in the absence of neuroserpin from these initial investigations is weak.
Supervisor: Molnar, Zoltan Sponsor: Academy of Medical Sciences ; Medical Research Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Neurosciences ; Developmental neurobiology ; Developmental Neuroanatomy