Folic acid deficiency enhanced microglial immune response via the Notch1/nuclear factor kappa B p65 pathway in hippocampus following rat brain I/R injury and BV2 cells

Recent studies revealed that folic acid deficiency (FD) increased the likelihood of stroke and aggravated brain injury after focal cerebral ischaemia. The microglia‐mediated inflammatory response plays a crucial role in the complicated pathologies that lead to ischaemic brain injury. However, whether FD is involved in the activation of microglia and the neuroinflammation after experimental stroke and the underlying mechanism is still unclear. The aim of the present study was to assess whether FD modulates the Notch1/nuclear factor kappa B (NF‐κB) pathway and enhances microglial immune response in a rat middle cerebral artery occlusion‐reperfusion (MCAO) model and oxygen‐glucose deprivation (OGD)‐treated BV‐2 cells. Our results exhibited that FD worsened neuronal cell death and exaggerated microglia activation in the hippocampal CA1, CA3 and Dentate gyrus (DG) subregions after cerebral ischaemia/reperfusion. The hippocampal CA1 region was more sensitive to ischaemic injury and FD treatment. The protein expressions of proinflammatory cytokines such as tumour necrosis factor‐α, interleukin‐1β and interleukin‐6 were also augmented by FD treatment in microglial cells of the post‐ischaemic hippocampus and in vitro OGD‐stressed microglia model. Moreover, FD not only dramatically enhanced the protein expression levels of Notch1 and NF‐κB p65 but also promoted the phosphorylation of pIkBα and the nuclear translocation of NF‐κB p65. Blocking of Notch1 with N‐[N‐(3, 5‐difluorophenacetyl)‐l‐alanyl]‐S‐phenylglycine t‐butyl ester partly attenuated the nuclear translocation of NF‐κB p65 and the protein expression of neuroinflammatory cytokines in FD‐treated hypoxic BV‐2 microglia. These results suggested that Notch1/NF‐κB p65 pathway‐mediated microglial immune response may be a molecular mechanism underlying cerebral ischaemia‐reperfusion injury worsened by FD treatment.     

Suppression of Brain Mast Cells Degranulation Inhibits Microglial Activation and Central Nervous System Inflammation

Brain inflammation has a critical role in the pathophysiology of brain diseases. Microglia, the resident immune cells in the brain, play an important role in brain inflammation, while brain mast cells are the “first responder” in the injury rather than microglia. Functional aspects of mast cell-microglia interactions remain poorly understood. Our results demonstrated that site-directed injection of the “mast cell degranulator” compound 48/80 (C48/80) in the hypothalamus induced mast cell degranulation, microglial activation, and inflammatory factor production, which initiated the acute brain inflammatory response. “Mast cell stabilizer” disodium cromoglycate (cromolyn) inhibited this effect, including decrease of inflammatory cytokines, reduced microglial activation, inhibition of MAPK and AKT pathways, and repression of protein expression of histamine receptor 1 (H₁R), histamine receptor 4 (H₄R), protease-activated receptor 2 (PAR2), and toll-like receptor 4 (TLR4) in microglia. We also demonstrated that C48/80 had no effect on microglial activation in mast cell-deficient Kit^W-sh/W-sh mice. These results implicate that activated brain mast cells trigger microglial activation and stabilization of mast cell inhibits microglial activation-induced central nervous system (CNS) inflammation. Interactions between mast cells and microglia could constitute a new and unique therapeutic target for CNS immune inflammation-related diseases.     

TLR4 signaling is involved in the protective effect of propofol in BV2 microglia against OGD/reoxygenation

Propofol exhibits neuroprotective effects against hypoxic–ischemic brain injury, but the underlying mechanisms are still not clear. Toll-like receptor 4 (TLR4) plays a considerable role in the induction of innate immune and inflammatory responses. The purposes of this study are to investigate the effect of propofol on the oxygen and glucose deprivation (OGD)/reoxygenation (OGD/R) BV2 microglia and to explore the role of TLR4/myeloid differentiation protein 88 (MyD88)/nuclear factor-kappa B (NF-κB) pathway in the neuroprotective effects of propofol. BV2 microglia were placed into an airtight chamber and in glucose-free medium for OGD/reoxygenation. Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide assay. TLR4 and its downstream signaling molecules, MyD88 and NF-κB expressions were detected by Western blotting. Level of tumor necrosis factor alpha (TNF-α) in culture medium was determined with enzyme-linked immunosorbent assay. BV2 microglia apoptosis was determined by flow cytometry. We found that pretreatment with propofol significantly alleviated the hypoxic injury in BV2 microglia. Propofol inhibited upregulation of TLR4, MyD88, and NF-κB expressions in BV2 microglia exposed to OGD/reoxygenation. Propofol pretreatment also significantly reduced the production of TNF-α and apoptosis in OGD/reoxygenation BV2 microglia. The results indicated that TLR4 and its downstream MyD88-dependent signaling pathway contributed to neuroprotection of propofol to microglia exposed to OGD/reoxygenation.     

Astrocytes in injury states rapidly produce anti-inflammatory factors and attenuate microglial inflammatory responses

Microglia are known to be a primary inflammatory cell type in the brain. However, microglial inflammatory responses are attenuated in the injured brain compared to those in cultured pure microglia. In the present study, we found that astrocytes challenged by oxygen-glucose deprivation (OGD) or H(2) O(2) released soluble factor(s) and attenuated microglial inflammatory responses. Conditioned medium prepared from astrocytes treated with OGD (OGD-ACM) or H(2) O(2) (H(2) O(2) -ACM) significantly reduced the levels of interferon-γ (IFN-γ)-induced microglial inflammatory mediators, including inducible nitric oxide synthase, at both the mRNA and protein levels. The anti-inflammatory effect of astrocytes appeared very rapidly (within 5min), but was not closely correlated with the extent of astrocyte damage. Both OGD-ACM and H(2) O(2) -ACM inhibited STAT nuclear signaling, as evidenced by a reduction in both STAT-1/3 binding to the IFN-γ-activated site and IFN-γ-activated site promoter activity. However, both phosphorylation and nuclear translocation of STAT-1/3 was unchanged in IFN-γ-treated microglia. The active component(s) in OGD-ACM were smaller than 3kDa, and displayed anti-inflammatory effects independent of protein synthesis. These results suggest that, in the injured brain, astrocytes may act as a controller to rapidly suppress microglial activation.     

Prion protein expression differences in microglia and astroglia influence scrapie-induced neurodegeneration in the retina and brain of transgenic mice

Activated microglia and astroglia are known to be involved in a variety of neurodegenerative diseases, including prion diseases. In the present experiments, we studied activation of astroglia and microglia after intraocular scrapie infection in transgenic mice expressing prion protein (PrP) in multiple cell types (tg7 mice) or in neurons only (tgNSE mice). In this model, scrapie infection and protease-resistant PrP deposition occurs in the retinas of both strains of mice, but retinal degeneration is observed only in tg7 mice. Our results showed that the retinas of tg7 and tgNSE mice both had astroglial activation with increased chemokine expression during the course of infection. However, only tg7 retinas exhibited strong microglial activation compared to tgNSE retinas, which showed little microglial activation by biochemical or morphological criteria. Therefore, microglial PrP expression might be required for scrapie-induced retinal microglial activation and damage. Furthermore, microglial activation preceded retinal neurodegeneration in tg7 mice, suggesting that activated microglia might contribute to the degenerative process, rather than being a response to the damage. Surprisingly, brain differed from retina in that an altered profile of microglial activation markers was upregulated, and the profiles in the two mouse strains were indistinguishable. Microglial activation in the brain was associated with severe brain vacuolation and neurodegeneration, leading to death. Thus, retinal and brain microglia appeared to differ in their requirements for activation, suggesting that different activation pathways occur in the two tissues.     

Notch Signaling Regulates Microglial Activation and Inflammatory Reactions in a Rat Model of Temporal Lobe Epilepsy

The inflammatory response mediated by microglia in the central nervous system is closely related to epilepsy. Notch signaling plays an important role in the microglial activation during hypoxia. This study aimed to investigate whether Notch signaling is involved in microglial activation and subsequent inflammation-related neuronal injury during the process of epileptogenesis in a rat model of temporal lobe epilepsy. By using western blotting, real-time quantitative PCR, immunohistochemistry and immunofluorescence labeling, we found that the expression of Notch signaling increased after status epilepticus and that a γ-secretase inhibitor could significantly inhibit the upregulation of Notch signaling, the activation of microglia, and the release of proinflammatory cytokines. Likewise, the neuronal apoptosis and loss in the hippocampus after SE were attenuated by the γ-secretase inhibitor. These results suggest that Notch signaling plays a key role in neuroinflammation and inflammation-related neuronal damage in epilepsy, and γ-secretase inhibitors may become a novel prospective therapeutic agent for epilepsy.     

Microglial Activation Promotes Cell Survival in Organotypic Cultures of Postnatal Mouse Retinal Explants

The role of microglia during neurodegeneration remains controversial. We investigated whether microglial cells have a neurotoxic or neuroprotective function in the retina. Retinal explants from 10-day-old mice were treated in vitro with minocycline to inhibit microglial activation, with LPS to increase microglial activation, or with liposomes loaded with clodronate (Lip-Clo) to deplete microglial cells. Flow cytometry was used to assess the viability of retinal cells in the explants and the TUNEL method to show the distribution of dead cells. The immunophenotypic and morphological features of microglia and their distribution were analyzed with flow cytometry and immunocytochemistry. Treatment of retinal explants with minocycline reduced microglial activation and simultaneously significantly decreased cell viability and increased the presence of TUNEL-labeled cell profiles. This treatment also prevented the migration of microglial cells towards the outer nuclear layer, where cell death was most abundant. The LPS treatment increased microglial activation but had no effect on cell viability or microglial distribution. Finally, partial microglial removal with Lip-Clo diminished the cell viability in the retinal explants, showing a similar effect to that of minocycline. Hence, cell viability is diminished in retinal explants cultured in vitro when microglial cells are removed or their activation is inhibited, indicating a neurotrophic role for microglia in this system.     

The Effect of Annexin A1 as a Potential New Therapeutic Target on Neuronal Damage by Activated Microglia

Brain disease is known to cause irrevocable and fatal loss of biological function once damaged. One of various causes of its development is damage to neuron cells caused by hyperactivated microglia, which function as immune cells in brain. Among the genes expressed in microglia stimulated by various antigens, annexin A1 (ANXA1) is expressed in the early phase of the inflammatory response and plays an important role in controlling the immune response. In this study, we assessed whether ANXA1 can be a therapeutic target gene for the initial reduction of the immune response induced by microglia to minimize neuronal damage. To address this, mouse-origin microglial cells were stimulated to mimic an immune response by lipopolysaccharide (LPS) treatment. The LPS treatment caused activation of ANXA1 gene and expression of inflammatory cytokines. To assess the biological function in microglia by the downregulation of ANXA1 gene, cells were treated with short hairpin RNA-ANXA1. Downregulated ANXA1 affected the function of mitochondria in the microglia and showed reduced neuronal damage when compared to the control group in the co-culture system. Taken together, our results showed that ANXA1 could be used as a potential therapeutic target for inflammation-related neurodegenerative diseases.