High-resolution intravital imaging reveals that blood-derived macrophages but not resident microglia facilitate secondary axonal dieback in traumatic spinal cord injury

Abstract

After traumatic spinal cord injury, functional deficits increase as axons die back from the center of the lesion and the glial scar forms. Axonal die back occurs in two phases: an initial axon intrinsic stage that occurs over the first several hours and a secondary phase which takes place over the first few weeks after injury. Here, we examine the secondary phase, which is marked by infiltration of macrophages. Using powerful time lapse multi-photon imaging, we captured images of interactions between Cx3cr1+/GFP macrophages and microglia and Thy-1YFP axons in a mouse dorsal column crush spinal cord injury model. Over the first few weeks after injury, axonal retraction bulbs within the lesion are static except when axonal fragments are lost by a blebbing mechanism in response to physical contact followed by phagocytosis by mobile Cx3Cr1+/GFP cells. Utilizing a radiation chimera model to distinguish marrow-derived cells from radio-resistant CNS resident microglia, we determined that the vast majority of accumulated cells in the lesion are derived from the blood and only these are associated with axonal damage. Interestingly, CNS-resident Cx3Cr1+/GFP microglia did not increasingly accumulate nor participate in neuronal destruction in the lesion during this time period. Additionally, we found that the blood-derived cells consisted mainly of singly labeled Ccr2+/RFP macrophages, singly labeled Cx3Cr1+/GFP macrophages and a small population of double-labeled cells. Since all axon destructive events were seen in contact with a Cx3Cr1+/GFP cell, we infer that the CCR2 single positive subset is likely not robustly involved in axonal dieback. Finally, in our model, deletion of CCR2, a chemokine receptor, did not alter the position of axons after dieback. Understanding the in vivo cellular interactions involved in secondary axonal injury may lead to clinical treatment candidates involving modulation of destructive infiltrating blood monocytes.

Beneficial effects of IL-37 after spinal cord injury in mice

“…Here we show that hIL-37tg mice exhibit enhanced locomotor function associated with attenuated tissue damage after SCI. Blood-borne monocytes infiltrating into the injured spinal cord mediate axonal retraction (19,20), and this deleterious effect is due to products released by macrophages or by integral macrophage membrane proteins, which inhibit the growth and guidance of axons (19,20). Other inhibitory molecules include chondroitin sulfate proteoglycans, Nogo, ephrins, and semaphorins, which are expressed at the lesion site by astrocytes, oligodendrocytes, and some precursor cells (7).…”

“…Because infiltrating macrophages inhibit axonal outgrowth by releasing soluble factors and by cell-cell interaction (19,20), we sought to evaluate whether there was enhanced axonal regeneration in hIL-37tg mice after complete spinal cord transection. Both the hIL-37tg and WT mice presented complete hindlimb paralysis after the injury and lacked functional improvement at 10 wk postinjury (BMS score 0).…”

Interleukin 33 Initiates CNS Inflammation Following Traumatic Injury

“…Work by Jerry Silver and colleagues have discovered direct contacts between macrophages and neurons, both in vivo and in vitro, which appear to precede axon retraction (Evans et al, 2014;Horn et al, 2008). The nature of these interactions, its molecular mediators or purposes, are unclear.…”

“…Macrophages are robustly recruited along the entire nerve following PNS injury, where they play the beneficial role of clearing myelin and apoptotic debris (Brosius and Barres, 2014). The role of macrophages following CNS injury , however, is more controversial, with works showing them to be either beneficial (Figure 2.3) (Batchelor et al, 1999;Kotter et al, 2001;London et al, 2011;Prewitt et al, 1997;Shechter et al, 2009Shechter et al, , 2013Yin et al, 2006) or harmful (Evans et al, 2014;Horn et al, 2008;McPhail et al, 2004;Popovich et al, 1999).…”

“…Axon retraction in vivo is sharply reduced by clodronate liposome treatment, liposomes that kill engulfing phagocytes (mainly macrophages and microglia) (Horn et al, 2008). Recently they discerned the major culprit to be monocyte derived macrophages rather than microglia using a GFP bone-marrow chimera (Evans et al, 2014).…”