Recent studies in the field of SCIRI pathology, in addition to many other neurological diseases, point to the dual role of microglial cells and macrophages in neuronal injury and recovery [23, 24]. In support of the hypothesis for a protective role for microglia and macrophages, it has been reported that microglial depletion by using the colony-stimulating factor-1 receptor (CSF-1R) inhibitor PLX5622 resulted in increased vascular leaking in both white and gray matter in the spinal cord when hypoxic . However, mounting evidence has revealed that robust activation of microglial cells and macrophages contribute to a deterioration of neurological outcomes [26, 27]. Activated microglia/macrophages have been reported to exacerbate damage by interacting with multiple pathological processes other than inflammation during injury, including crosstalk with astrocytes that affects proliferation , oligodendrocyte apoptosis, and demyelination . However, the infiltration of MDMs and the precise inflammatory profile of MDMs and microglia during SCIRI remain largely unresolved. Therefore, a more clear understanding of the dynamic alterations of M1 and M2 phenotypes of microglia and macrophages will advance our understanding of SCIRI and inform future treatment options.
The relative secretion levels of pro- and anti-inflammatory factors provide an indication of the activation state of microglia and macrophages. Thus, we studied the expression of inflammatory markers at the transcriptional level. Firstly, similar with traumatic brain injury , a sharp upregulation of M1-type genes, such as iNOS, TNF-α, CD86, and CD16, was observed in the present study, which appeared mostly at an early stage (almost within the 1st week). M1-type cells are believed to release devastating levels of pro-inflammatory mediators (e.g. TNF-α, IL-1β, and IL-6) while oxidative metabolites impair axonal regrowth . In contrast, M2 biomarkers, including CD204 and CD206, became elevated at later stages, mainly after the 1st week. The recruitment of M2 microglia and macrophages may represent an endogenous process aimed at restricting ischemic damage by releasing protective and neurotrophic factors, scavenging cell debris, and resolving local inflammation . Secondly, the pattern of M1 to M2 conversion occurred 7 days post-injury, coincident with improved BMS scores, alleviation of morphological damage, and loss of motor neurons in the spinal cord at a later stage. Finally, we also found that the expression of some M2 biomarkers (such as Arg-1 and IL-4) altered in an uncoordinated manner, as reported in other studies [31–33], suggesting that inflammatory microenvironments may favor a particular biomarker but not others. Taken together, these data suggest a distinct pattern of microglial and macrophage phenotypic change from M2 to M1 in spinal cord injury, indicating that different pathological mechanisms underlie the migration of the two cell types to the spinal cord.
Peripheral monocytes are known to enter the CNS following injury and contribute to traumatic injury in mouse models of multiple sclerosis [34, 35]. Monocyte infiltration occurs 1 and 3 d after the onset of status epilepticus, whereas in the majority of CNS disorders, such as stroke and experimental autoimmune encephalomyelitis (EAE), a significant influx of MDMs is initiated into the injured area on day 3 . In the present study, we also found that monocyte infiltration was not immediately evident after injury until day 3. The underlying mechanisms that limit monocyte transmigration into acutely re-perfused spinal cord tissue are yet to be fully understood. As reported in previous studies , the migration of leukocytes into the CNS is highly regulated, requiring coordinated activation of the leukocytes and endothelium, and the presence of appropriate chemoattractant gradients between the blood and brain. Furthermore, we have demonstrated that invading MDMs decrease from day 7 after SCIRI. Such changes were similar to the infiltration of MDMs in mice in which spinal cord injuries occurred [10, 13, 16], suggesting that the ablation of MDMs from the second week onward may have no effect on functional recovery. Taken together, these two observations indicate that the role of MDMs is essentially transiently restricted to the first week after SCIRI, and probably only between days 3 and 7 post-injury.
Importantly, the present study highlights the fact that infiltrating MDMs and resident microglia differ in phenotype following SCIRI. Although indistinguishable by standard immunohistochemical techniques, these two macrophage populations are commonly viewed as functionally homogenous . Previous SCIRI-related studies have generally suppressed or eliminated these cells at the site of the lesion. However, multiple other studies have reported distinct developmental origins of MDMs in other CNS diseases, suggesting that they may exert functions different from those of microglia in pathological processes . For example, previous studies have established that invading monocytes induce axonal damage by initiating demyelination, whereas microglia clear debris . Furthermore, the effect of monocyte infiltration is dependent on disease context. In EAE mice, infiltrating MDMs are highly inflammatory compared with microglia, and their depletion results in enhanced recovery . Conversely, MDMs provide neuroprotection and promote recovery following spinal cord injury . The results of the present study, however, attribute a pro-inflammatory function to MDMs, primarily at an early stage following SCIRI. In addition, resident microglia play a pro-inflammatory role in the early response to SCIRI, followed by an increased proportion of M2 phenotype that promotes endogenous repair at a later stage. These findings indicate that damage and repair during SCIRI are elegantly modulated via endogenous mechanisms involving coordinated phenotype modulation of microglia and MDMs that are dependent on timing.
Together, the study brings new insight into the long-standing unsolved problems regarding the differential contributions of MDMs and microglia to recovery from SCIRI. We have revealed a pro-inflammatory effect of infiltrating MDMs restricted probably to between day 3 and 7 post-injury, while later resident microglia increased their conversion to an M2 phenotype that enhanced endogenous repair. Importantly, in a previous study  we reported an increased ratio of M1/M2-like monocytes in the peripheral circulation in patients with Stanford type-A aortic dissection (AAD), indicating that once AAD patients are subjected to the process of deep hypothermic circulatory arrest, increased numbers of M1 polarized macrophages migrate to the re-perfused spinal cord, aggravating the severity of inflammation in the micro-environment of the site of the lesion. Thus, a better understanding of the differential phenotypes of activated resident microglia and infiltrating MDMs following CNS might enable the development of novel approaches to attenuate SCIRI, for example, by timely targeting infiltrating monocytes through regulation of monocyte migration and intracellular signaling may help for the recovery.
Several limitations of this study merit comment. First, the mice only underwent moderate paresis after clamping across the descending aorta for 9 min. In this regard, the number of invading MDMs in spinal cord may differ according to the degree of paresis. More severe paresis should be also observed. However, most mice would die within several days after clamping more than 9 min to cause severe paresis, which did not fulfil the requirement of a survival time as long as 21 days to explore the dynamic polarization of macrophage/microglia. Indeed, most clinical patients undergoing moderate paresis instead of severe paresis, which makes this model closer to clinical setting. Second, although the polarization of MDMs was determined in the present study, further studies to intervene these cells are needed.