Current Knowledge of Microglia in Traumatic Spinal Cord Injury

Microglia are the resident immune cells in the central nervous system (CNS). After traumatic spinal cord injury (SCI), microglia undergo activation, proliferation, and changes in gene and protein expression and morphology, with detrimental and beneficial effects. Activated microglia cause secondary neuronal injury via the production of proinflammatory cytokines, reactive oxygen species, and proteases. However, activated microglia also promote neuronal repair through the secretion of anti-inflammatory growth factors and cytokines. Proinflammatory cytokines increase endothelial permeability, promote A1 astrocyte activation and axonal demyelination, and reduce neural stem/progenitor cells (NSPCs), leading to the exacerbation of neuronal injury. In contrast, anti-inflammatory factors facilitate angiogenesis, reduce reactive astrocytes, and promote axonal remyelination and the propagation of NSPCs, contributing to tissue repair and locomotor recovery. Due to its limited regenerative capacity, the CNS requires beneficial microglia for continuous protection against injury. Understanding and regulating microglial activation status are beneficial to reducing detrimental effects and promoting repair behaviors and to obtain more information on efficient therapies for traumatic SCI. This review discusses microglial activation and the differences between microglia and similar immune cells, microglial interactions with other cells in the spinal cord, and the progress in the development of therapies targeting microglia in SCI.

Melatonin Modulates Microglia Activation in Neuro-inflammation by Regulating the ER Stress/PPARd/SIRT1 Signaling Pathway

Background: Activated microglia-mediated neuro-inflammation plays a vital aspect in regulating the micromilieu of the central nervous system. Neuro-inflammation involves distinct alterations of microglial phenotypes, containing nocuous pro-inflammatory (M1) phenotype and neuroprotective anti-inflammatory (M2) phenotype. Currently, there is no effective treatment for modulating such alterations. Little evidence shows that melatonin prevents the detrimental cascade of activated microglia-mediated neuro-inflammation. Methods: The expression levels of M1/M2 marker of primary microglia influenced by Melatonin were detected via qPCR. Functional activities were explored by western blotting, luciferase activity, EMSA, and ChIP assay. Structure interaction was assessed by molecular docking and LIGPLOT analysis. ER stress detection was examined by ultrastructure TEM, calapin activity, and ERSE assay. The neurobehavioral evaluations and immunofluorescence staining in animals were used for investigation of Melatonin on the neuroinflammation in vivo. Results: Melatonin had targeted on Peroxisome Proliferator Activated Receptor Delta (PPARd) activity, boosted LPS-stimulated alterations in polarization from the M1 to the M2 phenotype, and thereby inhibited NFkB–IKKb activation in primary microglia. The PPARd agonist L-165041 or over-expression of PPARd plasmid (ov-PPARd) showed similar results. Molecular docking screening, dynamic simulation approaches, and biological studies of melatonin showed that the activated site was located at PPARd (phospho-Thr256-PPARd). Furthermore, we found that activated microglia had lowered PPARd activity as well as the downstream SIRT1 formation via enhancing ER stress. Melatonin, PPARd agonist and ov-PPARd all effectively reversed the above-mentioned effects. Melatonin blocked ER stress by regulating calapin activity and expression in LPS-activated microglia. Additionally, melatonin or L-165041 ameliorated the neurobehavioral deficits in LPS-aggravated neuroinflammatory mice through blocking microglia activities, and also promoted phenotype changes to M2-predominant microglia. Conclusions Melatonin suppressed neuro-inflammation in vitro and in vivo by tuning microglial activation through the ER stress-dependent PPARd/SIRT1 signaling cascade. We proposed that this treatment strategy is an encouraging pharmacological approach for the remedy of neuro-inflammation associated disorders.

Microglia Polarization from M1 toward M2 Phenotype Is Promoted by Astragalus Polysaccharides Mediated through Inhibition of miR-155 in Experimental Autoimmune Encephalomyelitis

Activated microglia is considered to be major mediators of the neuroinflammatory environment in demyelinating diseases of the central nervous system (CNS). Activated microglia are mainly polarized into M1 type, which plays a role in promoting inflammation and demyelinating. However, the proportion of microglia polarized into M2 type is relatively low, which cannot fully play the role of anti-inflammatory and resistance to demyelinating. Our previous study found that Astragalus polysaccharides (APS) has an immunomodulatory effect and can inhibit neuroinflammation and demyelination in experimental autoimmune encephalomyelitis (EAE), which is a classic animal model of CNS demyelinating disease. In this study, we found that APS was effective in treating EAE mice. It restored microglia balance by inhibiting the polarization of microglia to M1-like phenotype and promoting the polarization of microglia to M2-like phenotype in vivo and in vitro. miR-155 is a key factor in regulating microglia polarization. We found that APS could inhibit the expression level of miR-155 in vivo and in vitro. Furthermore, we performed transfection overexpression and blocking experiments. The results showed that miR-155 mediated the polarization of microglia M1/M2 phenotype, while the selective inhibitor of miR-155 attenuated the inhibition of APS on microglia M1 phenotype and eliminated the promotion of APS on microglia M2 phenotype. Microglia can secrete IL-1α, TNF-α, and C1q after polarizing into M1 type and induce the activation of A1 neurotoxic astrocytes, further aggravating neuroinflammation and demyelination. APS reduced the secretion of IL-1α, TNF-α, and C1q by activated microglia, thus inhibited the formation of A1 neurotoxic astrocytes. In summary, our study suggests that APS regulates the polarization of microglia from M1 to M2 phenotype by inhibiting the miR-155, reduces the secretion of inflammatory factors, and inhibits the activation of neurotoxic astrocytes, thus effectively treating EAE.

Neuroinflammation, body temperature and behavioural changes in CD1 male mice undergoing acute restraint stress: An exploratory study

Background Animal models used to study pathologies requiring rehabilitation therapy, such as cardiovascular and neurologic disorders or oncologic disease, must be as refined and translationally relevant as possible. Sometimes, however, experimental procedures such as those involving restraint may generate undesired effects which may act as a source of bias. However, the extent to which potentially confounding effects derive from such routine procedures is currently unknown. Our study was therefore aimed at exploring possible undesirable effects of acute restraint stress, whereby animals were exposed to a brightly lit enclosed chamber (R&L) similar to those that are commonly used for substance injection. We hypothesised that this would induce a range of unwanted physiological alterations [such as neuroinflammatory response and changes in body weight and in brown adipose tissue (BAT)] and behavioural modification, and that these might be mitigated via the use of non-aversive handling methods: Tunnel Handling (NAH-T) and Mechanoceptive Handling (NAH-M)) as compared to standard Tail Handling (TH). Methods Two indicators of physiological alterations and three potentially stress sensitive behavioural parameters were assessed. Physiological alterations were recorded via body weight changes and assessing the temperature of Brown Adipose Tissue (BAT) using infra-red thermography (IRT), and at the end of the experiment we determined the concentration of cytokines CXCL12 and CCL2 in bone marrow (BM) and activated microglia in the brain. Nest complexity scoring, automated home-cage behaviour analysis (HCS) and Elevated Plus Maze testing (EPM) were used to detect any behavioural alterations. Recordings were made before and after a 15-minute period of R&L in groups of mice handled via TH, NAH-T or NAH-M. Results BAT temperature significantly decreased in all handling groups following R&L regardless of handling method. There was a difference, at the limit of significance (p = 0.06), in CXCL12 BM content among groups. CXCL12 content in BM of NAH-T animals was similar to that found in Sentinels, the less stressed group of animals. After R&L, mice undergoing NAH-T and NAH-M showed improved body-weight maintenance compared to those exposed to TH. Mice handled via NAH-M spent a significantly longer time on the open arms of the EPM. The HCS results showed that in all mice, regardless of handling method, R&L resulted in a significant reduction in walking and rearing, but not in total distance travelled. All mice also groomed more. No difference among the groups was found in Nest Score, in CCL2 BM content or in brain activated microglia. Conclusions Stress induced by a common restraint procedure caused metabolic and behavioural changes that might increase the risk of unexpected bias. In particular, the significant decrease in BAT temperature could affect the important metabolic pathways controlled by this tissue. R&L lowered the normal frequency of walking and rearing, increased grooming and probably carried a risk of low-grade neuro-inflammation. Some of the observed alterations can be mitigated by Non-aversive handlings.

Ca2+ activity is required for injury-induced migration of microglia

Objectives: Microglia are the resident immune cells in the brain. Brain injury can activate the microglia and induce its directional migration towards injury sites for exerting immune functions. While extracellular ATP released from the injury site mediates the directionality of activated microglia's migration, what endows activated microglia with migration capability remains largely unexplored. Methods: In the present study, we used the larval zebrafish as an in vivo model to visualize the dynamics of both morphology and Ca2+ activity of microglia during its migration evoked by local brain injury. Results: We found that, in response to local injury, activated microglia exhibited an immediate Ca2+ transient and later elevated Ca2+ bursts frequency during its migration towards the local injury site (P < 0.01). Furthermore, suppression of Ca2+ activities significantly retarded microglial migration (P < 0.05). Conclusion: Thus, our study suggests that intracellular Ca2+ activity is required for activated microglia's migration.

Cycloastragenol, a Triterpenoid Saponin, Regulates Oxidative Stress, Neurotrophic Dysfunctions, Neuroinflammation and Apoptotic Cell Death in Neurodegenerative Conditions

Here, we have unveiled the effects of cycloastragenol against Aβ (Amyloid-beta)-induced oxidative stress, neurogenic dysfunction, activated mitogen-activated protein (MAP) kinases, and mitochondrial apoptosis in an Aβ-induced mouse model of Alzheimer’s disease (AD). The Aβ-induced mouse model was developed by the stereotaxic injection of amyloid-beta (5 μg/mouse/intracerebroventricular), and cycloastragenol was given at a dose of 20 mg/kg/day/p.o for 6 weeks daily. For the biochemical analysis, we used immunofluorescence and Western blotting. Our findings showed that the injection of Aβ elevated oxidative stress and reduced the expression of neurogenic markers, as shown by the reduced expression of brain-derived neurotrophic factor (BDNF) and the phosphorylation of its specific receptor tropomyosin receptor kinase B (p-TrKB). In addition, there was a marked reduction in the expression of NeuN (neuronal nuclear protein) in the Aβ-injected mice brains (cortex and hippocampus). Interestingly, the expression of Nrf2 (nuclear factor erythroid 2–related factor 2), HO-1 (heme oxygenase-1), p-TrKB, BDNF, and NeuN was markedly enhanced in the Aβ + Cycloastragenol co-treated mice brains. We have also evaluated the expressions of MAP kinases such as phospho c-Jun-N-terminal kinase (p-JNK), p-38, and phospho-extracellular signal-related kinase (ERK1/2) in the experimental groups, which suggested that the expression of p-JNK, p-P-38, and p-Erk were significantly upregulated in the Aβ-injected mice brains; interestingly, these markers were downregulated in the Aβ + Cycloastragenol co-treated mice brains. We also checked the expression of activated microglia and inflammatory cytokines, which showed that cycloastragenol reduced the activated microglia and inflammatory cytokines. Moreover, we evaluated the effects of cycloastragenol against mitochondrial apoptosis and memory dysfunctions in the experimental groups. The findings showed significant regulatory effects against apoptosis and memory dysfunction as revealed by the Morris water maze (MWM) test. Collectively, the findings suggested that cycloastragenol regulates oxidative stress, neurotrophic processes, neuroinflammation, apoptotic cell death, and memory impairment in the mouse model of AD.

Absence of Apolipoprotein E is associated with exacerbation of prion pathology and promotes microglial neurodegenerative phenotype

Prion diseases or prionoses are a group of rapidly progressing and invariably fatal neurodegenerative diseases. The pathogenesis of prionoses is associated with self-replication and connectomal spread of PrPSc, a disease specific conformer of the prion protein. Microglia undergo activation early in the course of prion pathogenesis and exert opposing roles in PrPSc mediated neurodegeneration. While clearance of PrPSc and apoptotic neurons have disease-limiting effect, microglia-driven neuroinflammation bears deleterious consequences to neuronal networks. Apolipoprotein (apo) E is a lipid transporting protein with pleiotropic functions, which include controlling of the phagocytic and inflammatory characteristics of activated microglia in neurodegenerative diseases. Despite the significance of microglia in prion pathogenesis, the role of apoE in prionoses has not been established. We showed here that infection of wild type mice with 22L mouse adapted scrapie strain is associated with significant increase in the total brain apoE protein and mRNA levels and also with a conspicuous cell-type shift in the apoE expression. There is reduced expression of apoE in activated astrocytes and marked upregulation of apoE expression by activated microglia. We also showed apoE ablation exaggerates PrPSc mediated neurodegeneration. Apoe−/− mice have shorter disease incubation period, increased load of spongiform lesion, pronounced neuronal loss, and exaggerated astro and microgliosis. Astrocytes of Apoe−/− mice display salient upregulation of transcriptomic markers defining A1 neurotoxic astrocytes while microglia show upregulation of transcriptomic markers characteristic for microglial neurodegenerative phenotype. There is impaired clearance of PrPSc and dying neurons by microglia in Apoe−/− mice along with increased level of proinflammatory cytokines. Our work indicates that apoE absence renders clearance of PrPSc and dying neurons by microglia inefficient, while the excess of neuronal debris promotes microglial neurodegenerative phenotype aggravating the vicious cycle of neuronal death and neuroinflammation.

The secretome of M1 and M2 microglia differently regulate proliferation, differentiation and survival of adult neural stem/progenitor cell

Microglia has been reported to be able to regulate the proliferation, differentiation and survival of adult Neural stem/progenitor cells (NSPCs) by modulating the microenvironment, which results in different consequences of adult neurogenesis. However, whether the microglial activation is beneficial or harmful to NSPCs is still controversial because of the complexity and variability of microglial activation phenotypes. In this study, we detected the expression levels of M1 marker and M2 marker in IFN-γ- and IL-4-induced microglia at different time, respectively. The phenotypic markers of M1 and M2 microglia were stable for 24 h after removal of IFN-γ and IL-4 intervention, but exhibited different change patterns during the next 24 h. Then, the adult NSPCs were treated by the conditioned medium from IFN-γ- and IL-4-activated microglia. The conditioned medium from IFN-γ-activated microglia promoted apoptosis and astroglial differentiation of NSPCs, while suppressed proliferation and neuronal differentiation of NSPCs. However, the conditioned medium from IL-4-activated microglia exhibited opposite effects on these physiological processes. In addition, the direct treatment of IFN-γ or IL-4 alone did not significantly affect the proliferation, differentiation and survival of NSPCs. These results suggest that the secretome of pro-inflammatory (M1) and anti-inflammatory (M2) microglia differently regulated the proliferation, differentiation and survival of adult NSPCs. These findings will help further study the biological mechanism of microglia regulating neurogenesis, and provide a therapeutic strategy for neurological diseases by regulating microglial phenotypes to affect neurogenesis.