Regulation Of Glial Activation Research Articles

PKM2 Nuclear Translocation Promotes Glial Cell Activation and Aggravates the Brain Injury of Intracerebral Hemorrhage.

The purpose of this study was to investigate the potential involvement of pyruvate kinase M2 (PKM2), an enzyme acting as a rate-limiting enzyme in the final phase of glycolysis, in the regulation of glial activation and brain damage of intracerebral hemorrhage (ICH). Western blotting and immunofluorescence were performed to investigate PKM2 expression, terminal deoxynucleotidyl transferase deoxyurinary triphosphate (dUTP) nick end labeling staining, hematoxylin and eosin staining, and behavioral tests were employed to evaluate the brain damage of ICH mice, and RNA-seq and bioinformatic analyses were performed to detect gene expression changes in ICH mice treated with TEPP-46. Increased PKM2 levels in perihematomal brain tissue were found starting from 3 days following ICH and peaked at 5 and 7 days post ICH. The increased expression of PKM2 was mainly co-localized with glial fibrillary acidic protein (GFAP)+ astrocytes and ionized calcium binding adaptor molecule-1 (IBA-1)+ microglia. Furthermore, we observed a notable increase in the nuclear translocation of PKM2 in glial cells following ICH. TEPP-46 treatment significantly reduced PKM2 nuclear translocation, and effectively attenuated glial activation and brain injury, and improved functional recovery of mice with ICH. RNA-seq data indicated that 91.1% (205/225) of differentially expressed genes (DEGs) were down-regulated in the TEPP-46 treated groups compared with the vehicle-treated groups in ICH brains. Furthermore, bioinformatic analyses revealed that these down-regulated DEGs were involved in a variety of biological processes, including autophagy and metabolic processes. In addition, the majority of these downregulated DEGs had a primary high expression in neurons, with subsequent expression seen in endothelial cells, microglia, and astrocytes. These results indicate that increased PKM2 nuclear translocation promotes the activation of glial cells after ICH, hence aggravating ICH-induced brain damage, and aggravates the brain injury induced by ICH. This highlights a potential therapeutic target for inhibiting glial activation to attenuate brain injury after ICH.

BFGF-like Activity Supported Tissue Regeneration, Modulated Neuroinflammation, and Rebalanced Ca2+ Homeostasis following Spinal Cord Injury.

A spinal cord injury (SCI) is a well-defined debilitating traumatic event to the spinal cord that usually triggers permanent changes in motor, sensory, and autonomic functions. Injured tissue becomes susceptible to secondary mechanisms caused by SCIs, which include pro-inflammatory cytokine release, the activation of astrocytes and microglia, and increased neuronal sensibility. As a consequence, the production of factors such as GFAP, IBA-1, TNF-α, IL-1β, IFN-γ, and S100-β slow down or inhibit central nervous system (CNS) regeneration. In this regard, a thorough understanding of the mechanisms regulating the CNS, and specifically SCI, is essential for the development of new therapeutic strategies. It has been demonstrated that basic fibroblast growth factor (bFGF) was successful in the modulation of neurotrophic activity, also promoting neurite survival and tissue repair, thus resulting in the valuable care of CNS disorders. However, bFGF therapeutic use is limited due to the undesirable effects developed following its administration. Therefore, the synthetic compound mimetic of bFGF, SUN11602 (with chemical name 4-[[4-[[2-[(4-Amino-2,3,5,6-tetramethylphenyl)amino]acetyl]methylamino]-1-piperidinyl]methyl]benzamide), has been reported to show neuroprotective activities similar to those of bFGF, also demonstrating a good pharmacokinetic profile. Here, we aimed to investigate the neuroprotective activity of this bFGF-like compound in modulating tissue regeneration, neuroinflammation, and Ca2+ overload by using a subacute mouse model of SCI. SUN11602 (1, 2.5, and 5 mg/kg) was administered orally to mice for 72 h daily following the in vivo model of SCI, which was generated by the extradural compression of the spinal cord. The data obtained demonstrated that SUN11602 treatment considerably decreased motor alteration and diminished the neuroinflammatory state through the regulation of glial activation, the NF-κB pathway, and kinases. Additionally, by controlling Ca2+-binding proteins and restoring neurotrophin expression, we showed that SUN11602 therapy restored the equilibrium of the neuronal circuit. Because of these findings, bFGF-like compounds may be an effective tool for reducing inflammation in SCI patients while enhancing their quality of life.

Mulberry vinegar attenuates lipopolysaccharide and interferon gamma-induced inflammatory responses in C6 glial cells.

The present study evaluated the effect of mulberry vinegar (MV) on the regulation of the inflammatory responses using C6 glial cells. Treatment with lipopolysaccharide (LPS) and interferon-γ (IFN-γ) induced the nitric oxide and reactive oxygen species generation, while pre-incubation with MV inhibited these formations in a concentration-dependent manner. MV treatment also decreased the production of pro-inflammatory cytokines, such as interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-α in C6 glial cells stimulated by LPS/IFN-γ. Compared to the LPS/IFN-γ-treated control group, the MV-treated group exerts downregulation in the protein expressions of inducible nitric oxide synthase and cyclooxygenase-2, through inhibition of nuclear factor-κB activation. Protein expressions of glial fibrillary acidic protein and ionized calcium-binding adapter molecule 1 were also decreased in the MV-treated group. These findings suggest that MV prevents neuroinflammation by regulating the NF-κB signaling pathway and glial activation. PRACTICAL APPLICATION: Mulberry fruits (Morus alba L.) have been commonly consumed as juice or jam. It is a rich source of anthocyanins that might be associated with beneficial effects on human health, including the anti-oxidant, anti-inflammatory, anti-obesity, and anti-diabetic effects. Mulberry vinegar was produced by alcohol and acetic fermentation of mulberry juice, and they possessed a protective effect against LPS/IFN-γ-stimulated inflammatory responses in glial cells via regulation of glial activation and NF-κB signaling pathway (i.e., downregulation of iNOS, COX-2, TLR4, p-IκB, and NF-κB p65 protein expressions). Although further research especially animal and clinical trials are still necessary, the present study will be helpful to scale-up the production of functional vinegar with neuroprotective and anti-inflammatory properties using mulberry.

Exosomes derived from bone-marrow mesenchymal stem cells alleviate cognitive decline in AD-like mice by improving BDNF-related neuropathology

BackgroundAlzheimer's disease (AD) is a neurodegenerative disease characterized by a progressive decline in cognitive ability. Exosomes derived from bone-marrow mesenchymal stem cells (BMSC-exos) are extracellular vesicles that can execute the function of bone-marrow mesenchymal stem cells (BMSCs). Given the versatile therapeutic potential of BMSC and BMSC-exos, especially their neuroprotective effect, the aim of this study was to investigate the potential effect of BMSC-exos on AD-like behavioral dysfunction in mice and explore the possible molecular mechanism.MethodsBMSC-exos were extracted from the supernatant of cultured mouse BMSCs, which were isolated from the femur and tibia of adult C57BL/6 mice, purified and sorted via flow cytometry, and cultured in vitro. BMSC-exos were identified via transmission electron microscopy, and typical marker proteins of exosomes were also detected via Western blot. A sporadic AD mouse model was established by intracerebroventricular injection of streptozotocin (STZ). Six weeks later, BMSC-exos were administered via lateral ventricle injection or caudal vein injection lasting five consecutive days, and the control mice were intracerebroventricularly administered an equal volume of solvent. Behavioral performance was observed via the open field test (OFT), elevated plus maze test (EPM), novel object recognition test (NOR), Y maze test (Y-maze), and tail suspension test (TST). The mRNA and protein expression levels of IL-1β, IL-6, and TNF-α in the hippocampus were measured via quantitative polymerase chain reaction (qPCR) and Western blot, respectively. Moreover, the protein expression of Aβ1-42, BACE, IL-1β, IL-6, TNF-α, GFAP, p-Tau (Ser396), Tau5, synaptotagmin-1 (Syt-1), synapsin-1, and brain-derived neurotrophic factor (BDNF) in the hippocampus was detected using Western blot, and the expression of GFAP, IBA1, Aβ1−42 and DCX in the hippocampus was measured via immunofluorescence staining.ResultsLateral ventricle administration, but not caudal vein injection of BMSC-exos improved AD-like behaviors in the STZ-injected mouse model, as indicated by the increased number of rearing, increased frequency to the central area, and increased duration and distance traveled in the central area in the OFT, and improved preference index of the novel object in the NOR. Moreover, the hyperactivation of microglia and astrocytes in the hippocampus of the model mice was inhibited after treatment with BMSC-exos via lateral ventricle administration, accompanied by the reduced expression of IL-1β, IL-6, TNF-α, Aβ1-42, and p-Tau and upregulated protein expression of synapse-related proteins and BDNF. Furthermore, the results of the Pearson test showed that the preference index of the novel object in the NOR was positively correlated with the hippocampal expression of BDNF, but negatively correlated with the expression of GFAP, IBA1, and IL-1β. Apart from a positive correlation between the hippocampal expression of BDNF and Syt-1, BDNF abundance was found to be negatively correlated with markers of glial activation and the expression of the inflammatory cytokines, Aβ1-42, and p-Tau, which are characteristic neuropathological features of AD.ConclusionsLateral ventricle administration, but not caudal vein injection of BMSC-exos, can improve AD-like behavioral performance in STZ-injected mice, the mechanism of which might be involved in the regulation of glial activation and its associated neuroinflammation and BDNF-related neuropathological changes in the hippocampus.

Histamine in the Crosstalk Between Innate Immune Cells and Neurons: Relevance for Brain Homeostasis and Disease.

Histamine is a biogenic amine playing a central role in allergy and peripheral inflammatory reactions and acts as a neurotransmitter and neuromodulator in the brain. In the adult, histamine is produced mainly by mast cells and hypothalamic neurons, which project their axons throughout the brain. Thus, histamine exerts a range of functions, including wakefulness control, learning and memory, neurogenesis, and regulation of glial activity. Histamine is also known to modulate innate immune responses induced by brain-resident microglia cells and peripheral circulating monocytes, and monocyte-derived cells (macrophages and dendritic cells). In physiological conditions, histamine per se causes mainly a pro-inflammatory phenotype while counteracting lipopolysaccharide-induced inflammation both in microglia, monocytes, and monocyte-derived cells. In turn, the activation of the innate immune system can profoundly affect neuronal survival and function, which plays a critical role in the onset and development of brain disorders. Therefore, the dual role of histamine/antihistamines in microglia and monocytes/macrophages is relevant for identifying novel putative therapeutic strategies for brain diseases. This review focuses on the effects of histamine in innate immune responses and the impact on neuronal survival, function, and differentiation/maturation, both in physiological and acute (ischemic stroke) and chronic neurodegenerative conditions (Parkinson's disease).

Anti‑inflammatory role of Prunus persica L. Batsch methanol extract on lipopolysaccharide‑stimulated glial cells.

Glial cells are the resident immune cells of the central nervous system. Reactive glial cells release inflammatory mediators that induce neurotoxicity or aggravate neurodegeneration. Regulation of glial activation is crucial for the initiation and progression of neuropathological conditions. Constituents of the peach tree (Prunus persica L. Batsch), which has a global distribution, have been found to exert therapeutic effects in pathological conditions, such as rashes, eczema and allergies. However, the therapeutic potential of its aerial parts (leaves, fruits and twigs) remains to be elucidated. The present study aimed to evaluate the anti-inflammatory role of P. persica methanol extract (PPB) on lipopolysaccharide (LPS)-stimulated glial cells. High-performance liquid chromatography coupled with tandem mass spectrometry analysis showed that PPB contained chlorogenic acid and catechin, which have antioxidant properties. Western blot and reverse transcription polymerase chain reaction results indicated that PPB reduced the transcription of various proinflammatory enzymes (nitric oxide synthase and cyclooxygenase-2) and cytokines [tumor necrosis factor-α, interleukin (IL)-1β and IL-6] in LPS-stimulated BV2 cells. In addition, PPB inhibited the activation of NF-κB and various mitogen-activated protein kinases required for proinflammatory mediator transcription. Finally, nitrite measurement and immunocytochemistry results indicated that PPB also suppressed nitrite production and NF-κB translocation in LPS-stimulated primary astrocytes. Thus, PPB may be used as a potential therapeutic agent for neurodegenerative diseases and neurotoxicity via the suppression of glial cell activation.

The effect of exercise frequency on neuropathic pain and pain-related cellular reactions in the spinal cord and midbrain in a rat sciatic nerve injury model

BackgroundExercise regimens are established methods that can relieve neuropathic pain. However, the relationship between frequency and intensity of exercise and multiple cellular responses of exercise-induced alleviation of neuropathic pain is still unclear. We examined the influence of exercise frequency on neuropathic pain and the intracellular responses in a sciatic nerve chronic constriction injury (CCI) model.Materials and methodsRats were assigned to four groups as follows: CCI and high-frequency exercise (HFE group), CCI and low-frequency exercise (LFE group), CCI and no exercise (No-Ex group), and naive animals (control group). Rats ran on a treadmill, at a speed of 20 m/min, for 30 min, for 5 (HFE) or 3 (LFE) days a week, for a total of 5 weeks. The 50% withdrawal threshold was evaluated for mechanical sensitivity. The activation of glial cells (microglia and astrocytes), expression of brain-derived neurotrophic factor (BDNF) and μ-opioid receptor in the spinal dorsal horn and endogenous opioid in the midbrain were examined using immunohistochemistry. Opioid receptor antagonists (naloxone) were administered using intraperitoneal injection.ResultsThe development of neuropathic pain was related to the activation of glial cells, increased BDNF expression, and downregulation of the μ-opioid receptor in the ipsilateral spinal dorsal horn. In the No-Ex group, neuropathic pain showed the highest level of mechanical hypersensitivity at 2 weeks, which improved slightly until 5 weeks after CCI. In both exercise groups, the alleviation of neuropathic pain was accelerated through the regulation of glial activation, BDNF expression, and the endogenous opioid system. The expression of BDNF and endogenous opioid in relation to exercise-induced alleviation of neuropathic pain differed in the HFE and LFE groups. The effects of exercise-induced alleviation of mechanical hypersensitivity were reversed by the administration of naloxone.ConclusionThe LFE and HFE program reduced neuropathic pain. Our findings indicated that aerobic exercise-induced alleviated neuropathic pain through the regulation of glial cell activation, expression of BDNF in the ipsilateral spinal dorsal horn, and the endogenous opioid system.

G5G2.5 core-shell tecto-dendrimer specifically targets reactive glia in brain ischemia.

Secondary neuronal death is a serious stroke complication. This process is facilitated by the conversion of glial cells to the reactive pro-inflammatory phenotype that induces neurodegeneration. Therefore, regulation of glial activation is a compelling strategy to reduce brain damage after stroke. However, drugs have difficulties to access the CNS, and to specifically target glial cells. In the present work, we explored the use core-shell polyamidoamine tecto-dendrimer (G5G2.5 PAMAM) and studied its ability to target distinct populations of stroke-activated glial cells. We found that G5G2.5 tecto-dendrimer is actively engulfed by primary glial cells in a time- and dose-dependent manner showing high cellular selectivity and lysosomal localization. In addition, oxygen-glucose deprivation or lipopolysaccharides exposure invitro and brain ischemia invivo increase glial G5G2.5 uptake; not being incorporated by neurons or other cell types. We conclude that G5G2.5 tecto-dendrimer is a highly suitable carrier for targeted drug delivery to reactive glial cells invitro and invivo after brain ischemia.

Enhanced microglial activity in FAAH(-/-) animals.

Several lines of evidence suggest that the endocannabinoid system is involved in the regulation of glial activity. Enhanced levels of the endocannabinoid N-arachidonoyl ethanolamine (AEA, also referred to as anandamide) as well as non-cannabinoid lipids like palmitoylethanolamine (PEA) due to genetic deletion or pharmacologic blockade of its degrading enzyme fatty acid amide hydrolase (FAAH) reduced neuroinflammatory changes in models of neurodegeneration. Now we addressed the question if genetic deletion of FAAH also influences age-related neuroinflammation. To answer this question we compared the number and size of microglia in young and old wild-type and FAAH(-/-) mice and analysed the distribution of microglia sizes in the four groups. Additionally, we analysed IL-6 and IL-1β levels with ELISA and astrocyte activities as ratio of GFAP-positive areas in the hippocampus. Ageing was associated with an increased number and activity of microglia, elevated IL-6 and IL-1β levels and enhanced area covered by astrocytes in wild-type animals. Unexpectedly, in FAAH(-/-) animals the number of microglia and the ratio of activated microglia and IL-1β level were already higher in young animals than in age-matched wild-type controls. There was no further age-related increase in these inflammation markers in the knockout line. Our results suggest that AEA is involved in the regulation of microglia activity. Life-long elevation of AEA levels disturbs microglial regulation and leads to pro-inflammatory changes.

Role of CB1 cannabinoid receptors on GABAergic neurons in brain aging

Brain aging is associated with cognitive decline that is accompanied by progressive neuroinflammatory changes. The endocannabinoid system (ECS) is involved in the regulation of glial activity and influences the progression of age-related learning and memory deficits. Mice lacking the Cnr1 gene (Cnr1(-/-)), which encodes the cannabinoid receptor 1 (CB1), showed an accelerated age-dependent deficit in spatial learning accompanied by a loss of principal neurons in the hippocampus. The age-dependent decrease in neuronal numbers in Cnr1(-/-) mice was not related to decreased neurogenesis or to epileptic seizures. However, enhanced neuroinflammation characterized by an increased density of astrocytes and activated microglia as well as an enhanced expression of the inflammatory cytokine IL-6 during aging was present in the hippocampus of Cnr1(-/-) mice. The ongoing process of pyramidal cell degeneration and neuroinflammation can exacerbate each other and both contribute to the cognitive deficits. Deletion of CB1 receptors from the forebrain GABAergic, but not from the glutamatergic neurons, led to a similar neuronal loss and increased neuroinflammation in the hippocampus as observed in animals lacking CB1 receptors in all cells. Our results suggest that CB1 receptor activity on hippocampal GABAergic neurons protects against age-dependent cognitive decline by reducing pyramidal cell degeneration and neuroinflammation.

Focal cerebral ischemia upregulates SHP-1 in reactive astrocytes in juvenile mice

The role of the tyrosine phosphatase SHP-1 in the hematopoietic system has been well studied; however, its role in the central nervous system (CNS) response to injury is not well understood. Previous studies in our laboratory have demonstrated increased immunoreactivity for SHP-1 in a subset of reactive astrocytes that do not appear to enter the cell cycle following deafferentation of the chicken auditory brainstem. In order to determine whether mammalian astrocytes also upregulate SHP-1 immunoreactivity following CNS injury, a mouse model of focal cerebral ischemia was utilized to study SHP-1 expression. The brains of 3-week-old mice were analyzed at four time points following permanent middle cerebral artery occlusion (MCAO): 1, 3, 7, and 14 days. Our results demonstrate consistent infarct volumes within surgical groups, and infarct volumes decrease as a function of time from 1 day (maximum infarct volume) to 14 days (minimum infarct volume) post-MCAO. In addition, SHP-1 protein levels are upregulated following cerebral ischemia and this increase peaks at 7 days post-MCAO. Analysis of confocal images further reveals that immunoreactivity for SHP-1 occurs predominantly in GFAP+ reactive astrocytes, although a small percentage of F4-80+ microglia are also double labeled for SHP-1 at early times post-MCAO. These SHP-1+ reactive astrocytes do not appear to enter the cell cycle (as defined by PCNA immunoreactivity), confirming our previous studies in the avian auditory brainstem. These results suggest that SHP-1 plays an important role in the regulation of glial activation and proliferation in the ischemic CNS.

Neurodegeneration and glial activation patterns after mechanical nerve injury are differentially regulated by non-MHC genes in congenic inbred rat strains

Ventral root avulsion in the rat leads to a retrograde response, with activation of glia and up-regulation of immunologic cell surface molecules such as major histocompatibility complex (MHC) antigens, and the subsequent degeneration of a large proportion of the lesioned motoneurons. Herein, we examined several inbred congenic rat strains previously known to react differently to experimentally induced autoimmune diseases and demonstrate a substantial genetic diversity in the regulation of glial activation and neuron death in this injury model. The panel of examined inbred rat strains included DA(RT1AV1), PVG.1AV1, LEW.1AV1, LEW.1N, BN(RT1N) and E3(RT1U), and the following parameters were determined: (1) MHC class II expression on glia; (2) expression of glial fibrillary acidic protein, C3 complement, and microglial response factor-1 mRNAs in glia; (3) levels of the tumor necrosis factor-alpha and interleukin-1beta cytokine mRNAs; (4) degree of motoneuron loss. The findings of considerable strain-dependent differences in all parameters studied demonstrate important polymorphisms in the genetic regulation of these events. Furthermore, some of the studied features segregated from each other, suggesting independent regulatory mechanisms. Genes outside of the MHC complex are mainly implicated as being of importance for the phenotypic differences, as significant differences were recorded between the MHC congenic strains differing in the non-MHC genes but not vice versa. These results contribute new important insights into the genetic regulation of glial reactivity and neuron death after mechanical nerve injuries. In addition, the finding of conspicuous strain-dependent differences makes it necessary to consider the genetic background when designing and interpreting animal experiments involving noxious insults to the central nervous system resulting in glial activation and nerve cell loss.

Transforming growth factor beta expression in reactive spinal cord microglia and meningeal inflammatory cells during experimental allergic neuritis.

Experimental allergic neuritis (EAN), an inflammatory demyelinating disorder of the peripheral nervous system, is preceded and accompanied by a massive microglial reaction in the spinal cord which occurs in the absence of inflammatory cells infiltrating the cord parenchyma. Since transforming growth factor beta (TGF-beta) has been shown to play a beneficial role in experimental autoimmune disease and might be involved in the regulation of glial activity, we have investigated the expression of TGF-beta in EAN spinal cord and nerve root tissue. Adoptive transfer EAN was induced by the injection of neurotogenic T-cells specific for the P2 myelin protein. In normal spinal cord tissue, both TGF-beta 1 and TGF-beta 3 mRNA were constitutively expressed at low levels. Already 3 days following injection of P2-specific T-cells, TGF-beta 1 mRNA levels began to increase, peaked at day 6 at levels about tenfold above normal, and thereafter declined. TGF-beta 3 was induced even earlier with a sharp rise at day 3 and a peak fourfold above normal at day 4. In situ hybridization for TGF-beta 1 performed on spinal cord sections 6 days after injection of cells localized TGF-beta 1 mRNA to many nonneuronal cells with the typical morphology of microglia. In addition, TGF-beta 1 mRNA was observed in the meninges, and massive accumulation of signal was seen over inflammatory cells infiltrating the nerve roots. Our data indicate that TGF-beta 1 and -beta 3 are involved in regulating the glial response in EAN and that activated microglial cells might control their own activity state by expressing TGF-beta 1.(ABSTRACT TRUNCATED AT 250 WORDS)