Spinal cord injury (SCI) is a severe traumatic condition of the central nervous system (CNS) that can cause motor, sensory, and autonomic dysfunction below the level of injury [1,2]^[1][2].

The pathophysiological process of SCI includes the primary SCI and secondary SCI. The primary SCI is mainly caused by the initial traumatic impact, resulting in the direct damage to the spinal cord tissue. It usually occurs within minutes to hours following the injury, characterized by the cell necrosis, hemorrhage, edema, etc. On the basis of the primary injury, SCI further develops within a few days to several weeks, causing a series of secondary injuries including inflammatory response, apoptosis, lipid peroxidation, and the production of a large number of free radicals. The secondary injury leads to massive neuronal and glial cell death and glial scarring, and eventually forms necrotic cysts that hinder nerve regeneration. Among them, the inflammation is considered to play an extremely important role in the SCI [6,7]^[3][4]. Inflammation after SCI is complicated and regulated by various types of cells, including microglia, astrocytes, neutrophils, monocytes, macrophages, and lymphocytes [8,9,10]^[5][6][7]. In the initial stage of SCI, various types of major inflammatory cells aggregate to the lesion, clear the damaged tissue by actively phagocytosing myelin and dead cell debris, and gather around the lesion core to limit the further expansion of the injury. However, with the progression of the SCI, the aggregation of the above-mentioned cells induces a strong immune response and secretes a large number of cytotoxic substances, such as free radicals, nitric oxide, and various inflammatory factors, including interleukin and tumor necrosis factor, etc., which further aggravates the injury of spinal cord tissue. As resident immune cells of the CNS, microglia play a major and critical role in the inflammatory response following SCI.

2. Microglia

2.1. The Origin and Physiological Role of Microglia

Microglia are resident immune cells in the CNS, accounting for approximately 5–15% of the total number of cells in the CNS [11,12]^[8][9]. They originate from myeloid progenitor cells in the yolk sac during the embryonic period and are a special type of mononuclear macrophage [13,14,15]^[10][11][12]. Under physiological conditions, microglia are in a “resting state” and characterized by small cell diameters and various morphologies, similar to neuronal structures with many tiny protrusions extending hierarchically from the cell body. While the cell body does not move much, the branches of microglia are highly dynamic, constantly stretching, moving, and therefore surveying the CNS every now and then [16]^[13]. As the first line of defense, microglia in the “resting state” were reported to directly or indirectly contact neurons, astrocytes, oligodendrocytes, etc. through their multi-level branches, and then play a dynamic surveillance role in the CNS [17,18]^[14][15]. Specifically, they are able to make a quick response to the potential pathological damage, then quickly and effectively remove invading pathogens and cell debris through phagocytosis to maintain the stability of the neural network of the CNS [19,20,21]^[16][17][18]. On the other hand, microglia function as a special kind of secretory cells, which can secrete trophic factors in the “resting state” to regulate the activity of neurons and oligodendrocytes, thereby affecting important processes such as axon growth and myelination of neurons [22]^[19]. The absence or dysregulation of such cells may directly lead to abnormal immune regulation, inflammatory storm, and neuronal death in the CNS [23,24]^[20][21].

2.2. The Role of Microglia in SCI

Microglia, as resident immune cells of the central nervous system, play a major regulatory role in the inflammatory response after SCI, but its regulatory mechanism on SCI remains unclear [27,28]^[22][23]. Following SCI, microglia are rapidly activated by various pathological factors and lead to a range of alterations in morphology and function. For cell morphology, there is an “amoeba-like” change, which is manifested by the rapid enlargement of the cell body, and the shrinkage of the elongated branch structures (they become shorter and thicker). For cell function, the proliferation and migration activities of microglia are significantly increased, and the phagocytic activity and secretion of cytokines are significantly enhanced. Although moderately activated microglia have a certain positive effect after SCI, those continuously stimulated by the injury often exhibit abnormal activation and exert toxic effects on neuronal cells, which promotes inflammatory cell aggregation and infiltration and further activates the inflammatory response in turn, thereby mediating secondary injury in the spinal cord [29,30]^[24][25].

3. The Effect of Microglia Depletion on SCI Repair

3.1. The Adverse Effects of Microglia Depletion on SCI

At present, most studies have found that the depletion of microglia before SCI significantly inhibits the repair and functional recovery in animals with SCI [49,50,51,52]^{[26][27][28][29]}. Li et al. [49]^[26] found that microglia could promote long-distance axon growth after spinal cord crush injury and removal of microglia will directly affect the process of nerve regeneration in neonatal mice with SCI. Furthermore, single-cell sequencing revealed that microglia can temporarily secrete fibronectin and its binding proteins to provide the extracellular-matrix bridge, thereby promoting spinal cord axon regeneration. Victor Bellver-Landete et al. [50]^[27] also found that microglia were an essential component of protective scars after spinal cord contusion injury, and the depletion of microglia directly led to decreased secretion of insulin-like growth factor-1, disordered scar structure, and ultimately hindered functional recovery in animals. While the methods and strategies of microglia depletion in the aforementioned studies were different, all of them found that microglia depletion would further affect the repair after SCI. Thus, microglia depletion might not improve functional recovery after SCI, but even further aggravate injury on animals in these cases.

3.2. The Beneficial Effects of Microglia Depletion on SCI

Taken together, the effects of microglia depletion on SCI are still controversial. Different microglia depleting approaches, microglia depleting periods, and SCI models directly determine the positive or negative outcomes after SCI. Notably, a growing body of evidence shows that the persistent microglia removal seemed to be not conducive to tissue repair after SCI, while selective and strategic removal of microglia might improve recovery after SCI.

4. The Strategy and Timing of Microglia Depletion

4.1. The Main Method of Microglia Depletion

The methods of microglia depletion can be mainly divided into two categories: gene manipulation and small-molecule intervention. The former mainly uses microglia-specific surface markers such as Cx3Cr1, TMEM119, Sall1, etc., to construct specific microglia transgenic animals and then achieve the conditional removal of microglia; the latter involves the design and construction of small-molecule compounds against specific microglia cell markers so as to induce apoptosis and necrosis of microglia and then deplete these cells.

4.1.1. Microglia Depletion via Gene Manipulation

Microglia depletion via gene manipulation usually requires the construction of specific transgenic mice combined with specific toxic substances, such as diphtheria toxin (DT) or herpes virus, to conditionally remove microglia. Parkhurst et al. [59]^[30] constructed Cx3cr1^CreER: Rosa26^iDTR transgenic mice and achieved conditional knockout of microglia through the CreERT system and DTR system. Specifically, they first constructed Cx3cr1^CreER mice that specifically express Cre recombinase and estrogen receptor (ER) at the site of Cx3cr1⁺ cells and obtained Cx3Cr1^CreER: Rosa26^iDTR transgenic mice by crossing Cx3Cr1^CreER mice with Rosa26^iDTR mice. Then, they induced Cre recombinase expression by tamoxifen, and then injected DT intraperitoneally to achieve Cx3cr1⁺ cells knockout. Considering that peripheral monocyte-macrophages also express Cx3cr1 protein but with more frequent cell turnover (about 7 days), the intraperitoneal injection of DT would be performed 30 days after tamoxifen induction to achieve specific microglia depletion. Similarly, CX3CR1^CreER: Rosa26^iDTA [60]^[31], Cx3cr1^Cre:Csf1r^fl mice [49]^[26], and CD11b^HSVTK transgenic mice [55]^[32] have been constructed successively to conditionally remove microglia in the CNS. They either used the CreERT system, the Cre-Flox system, or expressed suicide genes to achieve the depletion of microglia.

4.1.2. Microglia Depletion via Small-Molecule Compounds

Unlike genetic manipulation to remove microglia, microglia depletion via small-molecule compounds is more convenient and diverse. They are either designed to impress those targets important for microglial survival, such as colony-stimulating factor 1 receptor (CSF1R), or they exert direct toxic effects leading to microglial cell death. For example, CSF1 is an extremely important regulator of microglia/macrophage proliferation, differentiation, and survival, so interventions on CSF1R can achieve targeted depletion of microglia. The small-molecule compound PLX3397 [14,33,61]^[11][33][34] and its second-generation product PLX5622 [50,62,63]^[27][35][36] are currently widely used specific inhibitors of CSF1R. They are highly specific to CSF1R and able to penetrate the blood–brain barrier and competitively occupy the site of CSF1, resulting in microglial cell death with up to 99% effectiveness. At the same time, studies have shown that PLX small-molecule compounds had little effect on other types of cells and animal behavior in mice, and thus are considered very good microglia-targeted regulation drugs.

4.2. The Timing of Microglia Depletion

The optimal timing for microglia depletion remains controversial at present. The different purposes or approaches to microglia depletion also influence the timing selection of microglia depletion. Taking microglia depletion via gene manipulation as an example, some studies performed diphtheria toxin application 7 days after tamoxifen induction [72]^[37] and found that microglia were specifically reduced and lasted for a week. However, in theory, the peripheral monocytes and macrophages would also be specifically removed at this time. On the other hand, in order to obtain selective microglia depletion, many studies chose to use diphtheria toxin 3–4 weeks after tamoxifen induction [59^[30][36],63], since the peripheral macrophages would have completed the cell turnover and no longer express the diphtheria toxin receptor, whereas microglia still expressed DT receptor. However, the time frame for microglia depletion via gene manipulation is relatively short and studies have shown [29]^[24] that the number of microglia return to normal one week after the application of diphtheria toxin. As for the timing of microglia depletion via small-molecule compounds, it is more flexible and diverse. Taking the most widely used PLX3397 and PLX5622 as examples, most studies chose to apply CSF1R inhibitor three weeks or more before the injury [50,60]^[27][31] and maintain the drug to the end of the experiment. Additionally, some chose two weeks or one before the injury to remove microglia [33,49,53,73]^{[26][33][38][39]}.

5. Conclusions

Microglia play a very important role in normal physiological processes and after SCI. Following SCI, on the one hand, microglia respond quickly and play a certain role in neuroprotection and repair by removing invading pathogens and cell debris through phagocytosis. On the other hand, continuous activated microglia often exert adverse effects on neuronal cells, promote inflammatory cell aggregation and infiltration, and further activate the inflammatory response in turn, thereby mediating secondary injury in the spinal cord. The effects of microglia depletion on SCI are still controversial, and different microglia depletion strategies greatly affect the outcome of tissue repair after SCI. Current studies have found that continuous microglia depletion is not conducive to tissue repair after SCI, and selective short-term depletion of microglia could effectively improve tissue repair and promote functional recovery after injury. Regarding the removal method and removal timing of microglia, there is still no uniform paradigm. Different strategies or purposes of microglia depletion determine the actual intervention timing and ultimately affect the outcome of animal SCI.