Cell Death and Microglia in the Developing Brain

Abstract

Introduction: Sudden unexpected death in infancy (SUDI) is the leading cause of post-neonatal death in the developed world. SUDI has been sub-categorised into explained causes, such as motor vehicle accidents, drowning and known illness (eSUDI); or unexplained causes, which includes sudden infant death syndrome (SIDS) and undetermined causes of death. SIDS by definition is a diagnosis of exclusion, with the pathogenesis currently elusive with no clear pathological defining features. It has been shown that SIDS infants have multiple abnormalities within brainstem regions crucial to cardio-respiratory control with this proposed to contribute to their premature death, however the linking pathway(s) are yet to be identified. Thus, investigation of the neuropathological changes remains a critical pillar of SIDS research. Notably, increased cell death, beyond that required in normal physiological development, has been implicated in SIDS, with amassing evidence in the literature of apoptotic cell death in regions of the SIDS brain, most notably in the brainstem and hippocampus. To build upon this, this thesis aimed to extend the scope of brain regions examined in the context of cell death in eSUDI and SIDS cases. Furthermore, this thesis also focused on a new cell type of interest in SUDI, being microglia. The role of microglia in the context of central nervous system insult and disease is a rapidly evolving field of neuropathological research, however, to date, literature in the infant brain is limited (reviewed section 2.4.4). Overall, this thesis aimed to: firstly, examine the methods of classifying unexplained SUDI cases to standardise our dataset. Secondly, to investigate cell death marker expression, cell stress and apoptotic pathways, and microglia populations in the developing human brain, specifically to (i) identify variability amongst brain regions, (ii) variability between SUDI sub-groups, and (iii) the effects of intrinsic and extrinsic stimuli. Finally, to investigate the effects of intermittent hypercapnic hypoxia and nicotine exposure (two leading risk factor models of SIDS) in the developing piglet brain.

Methods: Both human (Chapters 3-6) and piglet (Chapter 7) brain tissue samples were utilised for this thesis. The primary method used was immunohistochemistry, with the distribution of cell death (active caspase-9, active caspase-3, TUNEL) and microglia (ionised calcium binding adaptor molecule-1 (Iba1), cluster of differentiation factor 68 (CD68) and human leukocyte antigen clone DR-DP-DQ (HLA)) marker expression explored amongst a broad range of brain regions. Human brain tissue was also investigated using a commercially available Cell Stress and Apoptosis Signalling Antibody Array to investigate 18 promoter, executioner, inhibitor and regulator proteins of the cell stress and apoptosis cascade.

Results: Of the SUDI cases collected 2008-2012, there was a high level of variation in the diagnostic classification of SIDS and undetermined, thus necessitating the convening of an expert panel to apply a standardised classification of unexplained SUDI, SIDS and its subgroups (Chapter 3). On examining the neurological tissue, regional heterogeneity in all cell death and microglia markers was observed within the developing infant brain. During the first year of life, less than 20% of all neurons are undergoing physiological apoptosis (Chapter 4), and microglia occupy less than 5% of the total area (Chapter 6) in any given brain region. Changes in SIDS brain tissue were region dependent, with the brainstem and amygdala identified as regions of interest in the context of cell death (Chapter 4); the temporal cortex in the context of promoters of cell stress and apoptosis (Chapter 5); and the hippocampus in regards to changes in microglial populations (Chapter 6). Changes predominated in SIDS II suggesting the observations are influenced by confounding risk factors. In the piglet brain, continuous nicotine exposure was not associated with any changes in microglia, however acute/sub-acute IHH mediated region-dependent changes, particularly in the hippocampus (Chapter 7).

Conclusion: The neuropathological findings from this work highlight that cell death and microglia markers are heterogeneously expressed in regions of the postnatal developing brain. Region-specific changes in the SIDS brain also have potential links to extrinsic stimuli, based on the findings in the piglet model. The brainstem, amygdala and broader temporal cortex are regions of interest in the context of SIDS and cell death, while the hippocampus is a region of interest in the context of microglia. This thesis provides a comprehensive analysis of microglial and cell death distribution and markers in the SIDS brain, contributing to our understanding of region-unique vulnerabilities. The results suggest new brain regions, and new markers of cell stress and apoptotic pathways, that can be targeted in future studies of the pathogenesis of SIDS.