Investigating microglial inflammatory responses during traumatic spinal cord injury using novel cellular imaging approaches

Traumatic Spinal Cord Injury (SCI) is characterised by the loss of motor, sensory and autonomic function below the lesion level. After the initial mechanical injury, a chronic phase consisting of inflammatory cytokine signalling, axonal dieback, and astroglial scar formation persists. Microglia are resident immune cells of the CNS and play a pivotal role in SCI pathology. Understanding the role of microglia in neuroinflammation at the molecular and cellular level is essential to facilitate the development of effective therapeutic strategies. Here, we show that BV2 microglia adopt a pro-inflammatory phenotype, characterised by secretion of TNF and expression of iNOS and MHC2, when stimulated with LPS and IFNγ. In contrast, IL-13 reduces TNF secretion and MHC2 expression in BV2 microglia, thus promoting an anti-inflammatory phenotype. While these experiments inform us about the molecular nature of BV2 microglia in inflammation, they do not permit longitudinal characterisation of cell behaviour. To overcome this limitation, we have developed a novel optical imaging system capable of stable, in vitro live-cell imaging at physiological temperatures. We have analysed intracellular calcium signalling and cellular behaviour in BV2 microglia at 37°C. Finally, to further our understanding of cellular behaviour in SCI, we developed a custom 3D- printed surgical implant for use with the ‘UCLA V4’ miniature fluorescence microscope, to image the spinal cord longitudinally in awake, behaving mice. These novel imaging techniques will further advance our understanding of SCI pathology and facilitate the development of effective therapeutic strategies.