Physical interactions between activated microglia and injured axons: do all contacts lead to phagocytosis?

Axonal injury is a pathological hallmark of both head injury and inflammatory-mediated neurological disorders, including multiple sclerosis (Schirmer et al., 2013). Such axonal disruptions and/or disconnections typically result in proximal axonal segments that remain in continuity with the neuronal soma while losing contact with their distal targets. These disconnected axonal segments contribute to loss of signal transduction and overall circuit disruption, via subsequent deafferentation. Due to the disruption of anterograde transport in this cohort of axons, immunolabeling of the normally transported protein, amyloid precursor protein (APP), in post-mortem brain tissue is the most commonly used method for the visualization of the swollen end of proximal axonal segments. While proximal axonal segments remain connected to the neuronal cell body, axonal segments distal to the point of injury progress to anterograde Wallerian degeneration, forming myelin and axonal debris that negatively affect the surrounding tissue (Vargas and Barres, 2007; Mietto et al., 2015). Wallerian debris signals the resident microglia within the central nervous system (CNS) to activate and become phagocytic. Phagocytosis of this Wallerian debris by microglia, and later by peripheral monocytes, in instances of blood-brain barrier breakdown, has been well characterized and is regarded as one of the main beneficial effects of acute neuroinflammation in both the healthy and injured brain. Specifically, mutations of the Mecp2 or Trem2 genes in microglia, resulting in reduced or absent phagocytic function, are correlated to the negative outcomes of Rett syndrome or Alzheimer’s respectively (Chen and Trapp, 2015; Mietto et al., 2015). Microglia, however, have multiple roles, apart from phagocytosis. Within the healthy brain surveying microglia have highly ramified morphologies and are dynamic cells that, as the name suggests, survey the immediate environment and form brief, but frequent contacts with axonal segments (Kumar and Loane, 2012; Eyo and Wu, 2013). Virtually nothing is known regarding the molecular mechanisms mediating microglial process interactions with axons, however, microglial signaling molecules, such as fractalkine, DAP12 and complement proteins, are vital for the proper formation and maintenance of neuronal circuits. Further, the absence of microglia in the normal brain results in a host of negative effects, including extensive developmental defects and loss of neuronal electrophysiological adaptation to inflammatory signals (Eyo and Wu, 2013). Upon activation, microglia undergo distinct morphological changes, transforming from highly ramified phenotypes to microglia, with truncated processes, larger cell bodies, and less complex process networks or amoeboid morphologies. As noted previously, phagocytosis by morphologically simple or amoeboid phagocytic microglia is vital for clearance of debris from degenerating neurons and distal axonal segments (Chen and Trapp, 2015; Mietto et al., 2015). These phagocytic activities have been the primary focus of axon/microglial associations following injury. In contrast, knowledge regarding interactions between activated microglia and the disconnected swellings of proximal axonal segments has been extremely limited. Accordingly, our recent study began to explore this interaction (Lafrenaye et al., 2015). Using an adapted central fluid percussion injury model of mild traumatic brain injury we evaluated the extent of axonal injury and microglial activation in the micro pig 6 hours following injury. This mild central fluid percussion injury model has been well characterized and is routinely used to generate diffuse axonal injury in rodents; however, to our knowledge this was the first recorded use of this mild injury paradigm within the adult micro pig. Due to the similarity in neuroanatomy and systemic immune response between pigs and humans, this could constitute a highly clinically relevant model system for the study of axonal injury in an experimental setting. Systemic physiological readings of blood pressure, heart rate, temperature, hemoglobin oxygen saturation and blood gasses were rigorously monitored and maintained within normal ranges to ensure that brain pathology was not complicated by systemic physiological abnormality (Lafrenaye et al., 2015). Mild diffuse central fluid percussion brain injury did not produce gross pathology, such as contusion or hematoma formation. Injured axons, however, were found diffusely scattered throughout the thalamic domain (Lafrenaye et al., 2015). These same thalamic sites also demonstrated robust microglial activation, determined by both expression of ionized calcium-binding adapter molecule 1 (Iba1) and morphological characteristics consistent with microglial activation (Kumar and Loane, 2012).