Characterisation of neuroserpin protein expression throughout development and investigation of putative neuroprotective properties in rodent models of neonatal hypoxia ischaemia

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

Perinatal and Neonatal Hypoxia Ischaemia: The Unique Challenges of Treating the Infant Brain

Hypoxic ischaemic injury can damage the brain at any age. However, the infant brain displays a unique profile of sensitivity and resistance compared to adult ischaemic stroke patients. Both pathology and response to treatment are uniquely affected by the molecular landscape of the neonatal brain. With new revelations in the biology of brain injury in perinates and neonates being discovered, as global mortality and morbidity increases research funding into infant brain injury, it is important to raise awareness of the unparalleled challenge of treating these young patients. This chapter will review currently known differences between the infant and adult brain response to hypoxia, and address existing treatments alongside proposed treatments not yet evaluated by clinical trial

Newcastle Disease Virus in Madagascar: Identification of an Original Genotype Possibly Deriving from a Died Out Ancestor of Genotype IV

In Madagascar, Newcastle disease (ND) has become enzootic after the first documented epizootics in 1946, with recurrent annual outbreaks causing mortality up to 40%. Four ND viruses recently isolated in Madagascar were genotypically and pathotypically characterised. By phylogenetic inference based on the F and HN genes, and also full-genome sequence analyses, the NDV Malagasy isolates form a cluster distant enough to constitute a new genotype hereby proposed as genotype XI. This new genotype is presumably deriving from an ancestor close to genotype IV introduced in the island probably more than 50 years ago. Our data show also that all the previously described neutralising epitopes are conserved between Malagasy and vaccine strains. However, the potential implication in vaccination failures of specific amino acid substitutions predominantly found on surface-exposed epitopes of F and HN proteins is discussed

Characterisation of neuroserpin protein expression throughout development and investigation of putative neuroprotective properties in rodent models of neonatal hypoxia ischaemia

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.</p

Neonatal Hypoxia Ischaemia: Mechanisms, Models, and Therapeutic Challenges

Neonatal hypoxia-ischaemia (HI) is the most common cause of death and disability in human neonates, and is often associated with persistent motor, sensory, and cognitive impairment. Improved intensive care technology has increased survival without preventing neurological disorder, increasing morbidity throughout the adult population. Early preventative or neuroprotective interventions have the potential to rescue brain development in neonates, yet only one therapeutic intervention is currently licensed for use in developed countries. Recent investigations of the transient cortical layer known as subplate, especially regarding subplate's secretory role, opens up a novel set of potential molecular modulators of neonatal HI injury. This review examines the biological mechanisms of human neonatal HI, discusses evidence for the relevance of subplate-secreted molecules to this condition, and evaluates available animal models. Neuroserpin, a neuronally released neuroprotective factor, is discussed as a case study for developing new potential pharmacological interventions for use post-ischaemic injury

The Duration, Dynamics, and Determinants of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Antibody Responses in Individual Healthcare Workers

Abstract Background Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immunoglobulin G (IgG) antibody measurements can be used to estimate the proportion of a population exposed or infected and may be informative about the risk of future infection. Previous estimates of the duration of antibody responses vary. Methods We present 6 months of data from a longitudinal seroprevalence study of 3276 UK healthcare workers (HCWs). Serial measurements of SARS-CoV-2 anti-nucleocapsid and anti-spike IgG were obtained. Interval censored survival analysis was used to investigate the duration of detectable responses. Additionally, Bayesian mixed linear models were used to investigate anti-nucleocapsid waning. Results Anti-spike IgG levels remained stably detected after a positive result, for example, in 94% (95% credibility interval [CrI] 91–96%) of HCWs at 180 days. Anti-nucleocapsid IgG levels rose to a peak at 24 (95% CrI 19–31) days post first polymerase chain reaction (PCR)-positive test, before beginning to fall. Considering 452 anti-nucleocapsid seropositive HCWs over a median of 121 days from their maximum positive IgG titer, the mean estimated antibody half-life was 85 (95% CrI 81–90) days. Higher maximum observed anti-nucleocapsid titers were associated with longer estimated antibody half-lives. Increasing age, Asian ethnicity, and prior self-reported symptoms were independently associated with higher maximum anti-nucleocapsid levels and increasing age and a positive PCR test undertaken for symptoms with longer anti-nucleocapsid half-lives. Conclusions SARS-CoV-2 anti-nucleocapsid antibodies wane within months and fall faster in younger adults and those without symptoms. However, anti-spike IgG remains stably detected. Ongoing longitudinal studies are required to track the long-term duration of antibody levels and their association with immunity to SARS-CoV-2 reinfection. </jats:sec