The data presented here provide new insight into the cellular immune response to spinal cord injury, including a detailed picture of the daily dynamics of cellular inflammation. We used a novel OptiPrep gradient cell preparation method combined with flow cytometry to assess the principal immune cell types present in the injured spinal cord. Removal of myelin debris by the OptiPrep gradient method enhanced detection of PMNs compared to enzymatic dissociation alone. Additionally, the OptiPrep method allowed detection of a graded increase in both PMNs at 1 dpi and macrophages/microglia at 7 dpi, showing a quantitative linear relationship between the cellular inflammatory response and spinal cord injury severity. The persistent multiphasic inflammatory response of PMNs, macrophages/microglia and T cells observed here by flow cytometry was supported by quantitative stereologic data at a subset of time points. Each time point in the flow cytometric timecourse was normalized to uninjured animals, providing confirmation within this study that the uninjured spinal cord has minimal immune cells, as previously reported (Popovich et al.
; Nguyen et al.
). Some temporal differences that were statistically significant in the flow cytometric data set failed to yield significant differences in the stereologic data set (). Nonetheless, the stereologic data demonstrate the chronic presence of PMNs, macrophages/microglia and T cells. Together, the data revealed a multiphasic response of cellular inflammation after spinal cord injury.
Consistent with previous reports, the first cellular inflammation phase was comprised principally of PMNs (peaking 1 dpi), macrophages/microglia (peaking 7 dpi) and T cells (peaking 9 dpi). A novel and prolonged second phase of cellular inflammation was first observed after 14 dpi and persisted throughout 180 dpi, with a dramatic peak in the macrophage/microglial response at 60 dpi that doubled the magnitude of the earlier peak. Cellular inflammation was lowest for all three cell populations 14 dpi, suggesting a clear division between the phases of inflammation.
Later peaks of cellular inflammation after 14 dpi did not coincide with changes in locomotor function, suggesting ongoing inflammation is insufficient to affect locomotor recovery at this scale of analysis. In this regard, it is possible that more subtle changes in function occur during this timeframe but require more sensitive methodology. Interestingly, the rat contusion spinal cord injury syrinx shrinks from 14 to 28 dpi (Scheff et al.
), suggesting a relationship between the second phase of the cellular inflammatory response and tissue reorganization. Accordingly, the hypothesis that this second phase of macrophage/microglial response plays a role in preventing further loss of function must be considered. In this case, one would predict that reducing cellular inflammation during this period would result in a decline in locomotor function. Accordingly, blocking sub-acute to chronic inflammation via C5aRa resulted in reduced functional recovery and myelination in the injured spinal cord. The data presented here are unique in that this is the first study to test the role of delayed inflammation, providing novel insight into the multiphasic immune response following spinal cord injury.
Data from the current and previous studies suggest that cellular inflammation has complex, time-dependent functions. Our data show that PMNs, despite a reduction in number after peaking at 1 dpi, persisted for many months, suggesting that they may have a long-term role in promoting damage and/or repair. PMNs are thought to aggravate acute spinal cord injury within several dpi and have been previously demonstrated to promote neuronal cell death in culture through PMN-to-neuron contact or release of toxic factors (Dinkel et al.
; Nguyen et al.
). However, potentially PMNs have alternative roles in the long term as they secrete growth factors and cytokines that can promote scar formation/cytogenesis after spinal cord injury (Cicco et al.
; Jablonska et al.
; Nguyen et al.
). Moreover, PMNs express complement proteins, such as C1q, which may play a complex role in the injured microenvironment (Hogasen et al.
; Nguyen et al.
). The extended presence of PMNs suggests a long-term role after spinal cord injury, which may be very different from the conventional role suggested for acute PMNs.
In parallel with these observations, the role of macrophages/microglia, detected in two distinct phases of inflammation in the present study, may also be complex and time-dependent. The first phase of macrophages/microglia, which peaked 7 dpi, exacerbated spinal cord injury (Popovich et al.
). However, the role of the second phase, which peaked 60 dpi, is not known. The data presented here suggest that early chronic inflammation from 14 to 28 dpi modulates myelination in the injured spinal cord. The successful replacement of myelin damaged by spinal cord injury is a biphasic process of debris removal followed by remyelination that may hinge on interactions between the central nervous system and the immune system. Myelin degradation after spinal cord injury results in debris around the injury site, which may inhibit oligodendrocyte progenitor cell maturation (Robinson and Miller, 1999
; Kotter et al.
). Additionally, macrophages have also been shown to modify the microenvironment, causing an upregulation of factors that promote oligodendrocyte progenitor cell proliferation (fibroblast growth factor-2 and interleukin-1β) and maturation (transforming growth factor-β1 and insulin-like growth factor-1), although factors that may inhibit successful remyelination are also upregulated (transforming growth factor-α and Scya4) (Diemel et al.
; Kotter et al.
; Setzu et al.
). The net effect of these signalling molecules probably determines how oligodendrocyte progenitor cells respond to demyelinated axons, and while there is clear evidence that inflammation aids in remyelination (Foote and Blakemore, 2005
; Setzu et al.
), the exact mechanism remains unknown.
Interestingly, infiltrated macrophages have been shown to switch from pro-inflammatory to anti-inflammatory over time (Arnold et al.
; Villalta et al.
). While the role of macrophages in spinal cord injury has been debated (Rapalino et al.
; Popovich et al.
), the data presented here suggest a functional switch over time, though we did not specifically examine macrophage phenotype. Nonetheless, a thorough understanding of the dual role of these cells in both pro- and anti-inflammatory functions is critical for a complete interpretation of the data.
Unlike PMNs and macrophages/microglia, T cells were scarcely represented in the injured spinal cord. However, the number of cells does not necessarily reflect their importance in affecting the pathophysiology of spinal cord injury. While present in lower numbers, T cells could play an important role in injury and repair by modulating the function and recruitment of both innate and adaptive immune cells after spinal cord injury (Hendrix and Nitsch, 2007
; Ankeny et al.
The issue of which factor is driving the multiphasic kinetics of cellular inflammation after spinal cord injury has not been resolved, although at least three possibilities have been identified. Normally, the spinal cord is a site of relative immunoprivilege; traumatic injury causes an opening of the blood–brain barrier by 3 dpi, which is mostly intact at 14 dpi, and open again at 28 dpi (Popovich et al.
). These periods coincide with our observations of increased immune cell presence acutely after injury, low levels at 14 dpi and increasing again beginning 21 dpi. An increased permeability of the blood–brain barrier would allow immune cells to easily enter the spinal cord from the circulation, although specific cues are also likely to be involved. One such cue could be the complement anaphylatoxin, C5a, a strong recruiter of PMNs, macrophages/microglia and T cells (Klos et al.
). Local complement activation in neurons and glia, resulting in C5a production, has been demonstrated as early as 1 dpi and persists to at least 42 dpi (Anderson et al.
). Furthermore, PMNs have been shown to express a subset of complement proteins following spinal cord injury (Nguyen et al.
), while macrophages expressed the entire complement cascade in vitro
(Johnson and Hetland, 1988
). It is unlikely that complement proteins are the only factor to recruit immune cells to the injured spinal cord. It has been suggested that a homeostatic balance of pro- and anti-inflammatory cytokines modulates the recruitment of inflammatory cells after central nervous system injury (Lenzlinger et al.
; Stoll et al.
). Pro-inflammatory cytokines are expressed mostly within days after mouse spinal cord injury, though tumour necrosis factor-α has a second phase of expression beginning 14 dpi and lasting to at least 28 dpi (Pineau and Lacroix, 2007
). The expression pattern of anti-inflammatory cytokines after spinal cord injury has not yet been thoroughly investigated, nor has any direct link between cytokine production and leucocyte recruitment.
The temporal and quantitative analysis of cellular inflammation presented here has provided new insight into the dynamics of the spinal cord injury microenvironment, and identified for the first time an extended multiphasic response of cellular inflammation. The development of a novel OptiPrep gradient cell preparation method combined with flow cytometry to assess the principal immune cell types present in the injured spinal cord allowed detection of a graded increase in both PMNs and macrophages/microglia, showing a quantitative linear relationship between the cellular inflammatory response and spinal cord injury severity. Flow cytometric quantification as conducted here also allowed accurate comparison of data across multiple time points, which was verified by quantitative stereology, identifying a time-dependent multiphasic response of cellular inflammation after spinal cord injury. Furthermore, reduction of delayed C5a-mediated macrophage/microglial infiltration after spinal cord injury resulted in decreased functional recovery, suggesting a reparative role for chronic inflammation in this model. Understanding the role of this multiphasic response in the pathophysiology of spinal cord injury is likely to be critical for the design and implementation of rational therapeutic treatment strategies, including both cell-based and pharmacological interventions.