RESUMO
Microglia and astrocytes are the main components of the central nervous system (CNS). Upon activation, microglia is able to phagocyte cell debris, pathogens, and toxins; astrocytes support neuronal functions, blood-brain barrier (BBB) homeostasis, and neurotransmitter uptake and metabolism. Furthermore, both cell types can produce cytokines and chemokines. Aging impacts microglia and astrocytes by promoting the production of pro-inflammatory cytokines, impairing microglial phagocytosis and motility and astrocyte glutamate uptake. During neurodegenerative and neuroinflammatory diseases, the aging process may be accelerated contributing to the alteration of CNS glial cells functions. Multiple sclerosis (MS) is an autoimmune, demyelinating disease in which immunosenescence can promote the conversion from relapsing-remitting form to progressive disease. The murine model of experimental autoimmune encephalomyelitis (EAE) allows to investigate MS pathogenesis. Furthermore, EAE can be developed as acute or progressive, mimicking different forms of human MS. Microglia and astrocytes report morphological and functional changes during neuroinflammation that can be investigated in different ways. We here present a protocol for the study of glial cell activation in the spinal cord tissue of EAE mice.
Assuntos
Astrócitos , Encefalomielite Autoimune Experimental , Gliose , Microglia , Medula Espinal , Animais , Microglia/metabolismo , Microglia/patologia , Camundongos , Medula Espinal/patologia , Medula Espinal/metabolismo , Encefalomielite Autoimune Experimental/patologia , Encefalomielite Autoimune Experimental/metabolismo , Astrócitos/metabolismo , Astrócitos/patologia , Gliose/patologia , Gliose/metabolismo , Imunofluorescência/métodos , Modelos Animais de Doenças , Esclerose Múltipla/patologia , Esclerose Múltipla/metabolismoRESUMO
Unlike mammals, some nonmammalian species recruit Müller glia for retinal regeneration after injury. Identifying the underlying mechanisms may help to foresee regenerative medicine strategies. Using a Xenopus model of retinitis pigmentosa, we found that Müller cells actively proliferate upon photoreceptor degeneration in old tadpoles but not in younger ones. Differences in the inflammatory microenvironment emerged as an explanation for such stage dependency. Functional analyses revealed that enhancing neuroinflammation is sufficient to trigger Müller cell proliferation, not only in young tadpoles but also in mice. In addition, we showed that microglia are absolutely required for the response of mouse Müller cells to mitogenic factors while negatively affecting their neurogenic potential. However, both cell cycle reentry and neurogenic gene expression are allowed when applying sequential pro- and anti-inflammatory treatments. This reveals that inflammation benefits Müller glia proliferation in both regenerative and nonregenerative vertebrates and highlights the importance of sequential inflammatory modulation to create a regenerative permissive microenvironment.
Assuntos
Proliferação de Células , Células Ependimogliais , Regeneração , Retina , Animais , Células Ependimogliais/metabolismo , Células Ependimogliais/patologia , Camundongos , Retina/patologia , Retina/metabolismo , Doenças Neuroinflamatórias/metabolismo , Doenças Neuroinflamatórias/patologia , Doenças Neuroinflamatórias/etiologia , Microglia/metabolismo , Microglia/patologia , Modelos Animais de Doenças , Retinose Pigmentar/patologia , Retinose Pigmentar/metabolismo , Inflamação/patologia , Inflamação/metabolismo , LarvaRESUMO
Traumatic brain injury (TBI) is an acquired insult to the brain caused by an external mechanical force, potentially resulting in temporary or permanent impairment. Microglia, the resident immune cells of the central nervous system, are activated in response to TBI, participating in tissue repair process. However, the underlying epigenetic mechanisms in microglia during TBI remain poorly understood. ARID1A (AT-Rich Interaction Domain 1 A), a pivotal subunit of the multi-protein SWI/SNF chromatin remodeling complex, has received little attention in microglia, especially in the context of brain injury. In this study, we generated a Arid1a cKO mouse line to investigate the potential roles of ARID1A in microglia in response to TBI. We found that glial scar formation was exacerbated due to increased microglial migration and a heightened inflammatory response in Arid1a cKO mice following TBI. Mechanistically, loss of ARID1A led to an up-regulation of the chemokine CCL5 in microglia upon the injury, while the CCL5-neutralizing antibody reduced migration and inflammatory response of LPS-stimulated Arid1a cKO microglia. Importantly, administration of auraptene (AUR), an inhibitor of CCL5, repressed the microglial migration and inflammatory response, as well as the glial scar formation after TBI. These findings suggest that ARID1A is critical for microglial response to injury and that AUR has a therapeutic potential for the treatment of TBI.
Assuntos
Lesões Encefálicas Traumáticas , Quimiocina CCL5 , Proteínas de Ligação a DNA , Camundongos Knockout , Microglia , Fatores de Transcrição , Animais , Lesões Encefálicas Traumáticas/patologia , Lesões Encefálicas Traumáticas/metabolismo , Lesões Encefálicas Traumáticas/genética , Microglia/metabolismo , Microglia/patologia , Quimiocina CCL5/metabolismo , Quimiocina CCL5/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Camundongos , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Movimento Celular , Cicatriz/patologia , Cicatriz/metabolismo , Camundongos Endogâmicos C57BL , MasculinoRESUMO
BACKGROUND: Tobacco smoking is the leading cause of preventable death and disease worldwide, with over 8 million annual deaths attributed to cigarette smoking. This study investigates the impact of cigarette smoke and heated tobacco products (HTPs) on microglial function, focusing on toxicological profiles, inflammatory responses, and oxidative stress using ISO standard and clinically relevant conditions of exposure. METHODS: We assessed cell viability, reactive oxygen species (ROS) production, lipid peroxidation, mitochondrial function, unfolded protein response, and inflammation in human microglial cells (HMC3) exposed to cigarette smoke, HTP aerosol or nicotine. RESULTS: Our findings show that cigarette smoke significantly reduces microglial viability, increases ROS formation, induces lipid peroxidation, and reduces intracellular glutathione levels. Cigarette smoke also alters the expression of genes involved in mitochondrial dynamics and biogenesis, leading to mitochondrial dysfunction. Additionally, cigarette smoke impairs the unfolded protein response, activates the NF-κB pathway, and induces a pro-inflammatory state characterized by increased TNF and IL-18 expression. Furthermore, cigarette smoke causes DNA damage and decreases the expression of the aging marker Klotho ß. In contrast, HTP, exhibited a lesser degree of microglial toxicity, with reduced ROS production, lipid peroxidation, and mitochondrial dysfunction compared to conventional cigarettes. CONCLUSION: These results highlight the differential toxicological profile of cigarette smoke and HTP on microglial cells, suggesting a potential harm reduction strategy for neurodegenerative disease for smokers unwilling or unable to quit.
Assuntos
Sobrevivência Celular , Inflamação , Peroxidação de Lipídeos , Microglia , Mitocôndrias , Estresse Oxidativo , Espécies Reativas de Oxigênio , Fumaça , Produtos do Tabaco , Resposta a Proteínas não Dobradas , Estresse Oxidativo/efeitos dos fármacos , Humanos , Espécies Reativas de Oxigênio/metabolismo , Inflamação/patologia , Microglia/efeitos dos fármacos , Microglia/metabolismo , Microglia/patologia , Produtos do Tabaco/efeitos adversos , Fumaça/efeitos adversos , Mitocôndrias/metabolismo , Mitocôndrias/efeitos dos fármacos , Peroxidação de Lipídeos/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Linhagem Celular , Temperatura Alta , NF-kappa B/metabolismo , Nicotiana/efeitos adversos , Dano ao DNARESUMO
Aging is the greatest known risk factor for most neurodegenerative diseases. Myelin degeneration is an early pathological indicator of these diseases and a normal part of aging; albeit, to a lesser extent. Despite this, little is known about the contribution of age-related myelin degeneration on neurodegenerative disease. Microglia participate in modulating white matter events from demyelination to remyelination, including regulation of (de)myelination by the microglial innate immune receptor triggering receptor expressed on myeloid cells 2 (TREM2). Here, we demonstrate Trem2-deficiency aggravates and accelerates age-related myelin degeneration in the striatum. We show TREM2 is necessary for remyelination by recruiting reparative glia and mediating signaling that promotes OPC differentiation/maturation. In response to demyelination, TREM2 is required for phagocytosis of large volumes of myelin debris. In addition to lysosomal regulation, we show TREM2 can modify the ER stress response, even prior to overt myelin debris, that prevents lipid accumulation and microglial dysfunction. These data support a role for Trem2-dependent interactions in age-related myelin degeneration and suggest a basis for how early dysfunctional microglia could contribute to disease pathology through insufficent repair, defective phagocytosis, and the ER stress response.
Assuntos
Envelhecimento , Glicoproteínas de Membrana , Microglia , Bainha de Mielina , Receptores Imunológicos , Animais , Camundongos , Envelhecimento/patologia , Envelhecimento/metabolismo , Doenças Desmielinizantes/patologia , Doenças Desmielinizantes/metabolismo , Doenças Desmielinizantes/genética , Estresse do Retículo Endoplasmático/fisiologia , Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/deficiência , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microglia/metabolismo , Microglia/patologia , Bainha de Mielina/patologia , Bainha de Mielina/metabolismo , Fagocitose/genética , Receptores Imunológicos/metabolismo , Receptores Imunológicos/genética , Receptores Imunológicos/deficiência , Remielinização/fisiologia , Masculino , FemininoRESUMO
BACKGROUND: Glaucoma is a leading cause of blindness, affecting retinal ganglion cells (RGCs) and their axons. By 2040, it is likely to affect 110 million people. Neuroinflammation, specifically through the release of proinflammatory cytokines by M1 microglial cells, plays a crucial role in glaucoma progression. Indeed, in post-mortem human studies, pre-clinical models, and ex-vivo models, RGC degeneration has been consistently shown to be linked to inflammation in response to cell death and tissue damage. Recently, Rho kinase inhibitors (ROCKis) have emerged as potential therapies for neuroinflammatory and neurodegenerative diseases. This study aimed to investigate the potential effects of three ROCKis (Y-27632, Y-33075, and H-1152) on retinal ganglion cell (RGC) loss and retinal neuroinflammation using an ex-vivo retinal explant model. METHODS: Rat retinal explants underwent optic nerve axotomy and were treated with Y-27632, Y-33075, or H-1152. The neuroprotective effects on RGCs were evaluated using immunofluorescence and Brn3a-specific markers. Reactive glia and microglial activation were studied by GFAP, CD68, and Iba1 staining. Flow cytometry was used to quantify day ex-vivo 4 (DEV 4) microglial proliferation and M1 activation by measuring the number of CD11b+, CD68+, and CD11b+/CD68+ cells after treatment with control solvent or Y-33075. The modulation of gene expression was measured by RNA-seq analysis on control and Y-33075-treated explants and glial and pro-inflammatory cytokine gene expression was validated by RT-qPCR. RESULTS: Y-27632 and H-1152 did not significantly protect RGCs. By contrast, at DEV 4, 50 µM Y-33075 significantly increased RGC survival. Immunohistology showed a reduced number of Iba1+/CD68+ cells and limited astrogliosis with Y-33075 treatment. Flow cytometry confirmed lower CD11b+, CD68+, and CD11b+/CD68+ cell numbers in the Y-33075 group. RNA-seq showed Y-33075 inhibited the expression of M1 microglial markers (Tnfα, Il-1ß, Nos2) and glial markers (Gfap, Itgam, Cd68) and to reduce apoptosis, ferroptosis, inflammasome formation, complement activation, TLR pathway activation, and P2rx7 and Gpr84 gene expression. Conversely, Y-33075 upregulated RGC-specific markers, neurofilament formation, and neurotransmitter regulator expression, consistent with its neuroprotective effects. CONCLUSION: Y-33075 demonstrates marked neuroprotective and anti-inflammatory effects, surpassing the other tested ROCKis (Y-27632 and H-1152) in preventing RGC death and reducing microglial inflammatory responses. These findings highlight its potential as a therapeutic option for glaucoma.
Assuntos
Fármacos Neuroprotetores , Piridinas , Células Ganglionares da Retina , Quinases Associadas a rho , Animais , Piridinas/farmacologia , Quinases Associadas a rho/metabolismo , Quinases Associadas a rho/antagonistas & inibidores , Fármacos Neuroprotetores/farmacologia , Ratos , Células Ganglionares da Retina/efeitos dos fármacos , Células Ganglionares da Retina/patologia , Células Ganglionares da Retina/metabolismo , Doenças Neuroinflamatórias/tratamento farmacológico , Doenças Neuroinflamatórias/metabolismo , Retina/efeitos dos fármacos , Retina/patologia , Retina/metabolismo , Amidas/farmacologia , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Ratos Sprague-Dawley , Neuroproteção/efeitos dos fármacos , Neuroproteção/fisiologia , Microglia/efeitos dos fármacos , Microglia/metabolismo , Microglia/patologia , Inibidores de Proteínas Quinases/farmacologia , Masculino , Traumatismos do Nervo Óptico/tratamento farmacológico , Traumatismos do Nervo Óptico/patologia , Traumatismos do Nervo Óptico/metabolismo , Isoquinolinas , SulfonamidasRESUMO
Progressive supranuclear palsy (PSP) is an incurable neurodegenerative disease characterized by 4-repeat (0N/4R)-Tau protein accumulation in CNS neurons. We generated transgenic zebrafish expressing human 0N/4R-Tau to investigate PSP pathophysiology. Tau zebrafish replicated multiple features of PSP, including: decreased survival; hypokinesia; impaired optokinetic responses; neurodegeneration; neuroinflammation; synapse loss; and Tau hyperphosphorylation, misfolding, mislocalization, insolubility, truncation, and oligomerization. Using automated assays, we screened 147 small molecules for activity in rescuing neurological deficits in Tau zebrafish. (+)JQ1, a bromodomain inhibitor, improved hypokinesia, survival, microgliosis, and brain synapse elimination. A heterozygous brd4+/- mutant reducing expression of the bromodomain protein Brd4 similarly rescued these phenotypes. Microglial phagocytosis of synaptic material was decreased by (+)JQ1 in both Tau zebrafish and rat primary cortical cultures. Microglia in human PSP brains expressed Brd4. Our findings implicate Brd4 as a regulator of microglial synaptic elimination in tauopathy and provide an unbiased approach for identifying mechanisms and therapeutic targets in PSP.
Assuntos
Animais Geneticamente Modificados , Modelos Animais de Doenças , Microglia , Paralisia Supranuclear Progressiva , Sinapses , Fatores de Transcrição , Peixe-Zebra , Proteínas tau , Animais , Humanos , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Proteínas tau/metabolismo , Proteínas tau/genética , Microglia/metabolismo , Microglia/patologia , Sinapses/metabolismo , Paralisia Supranuclear Progressiva/metabolismo , Paralisia Supranuclear Progressiva/genética , Paralisia Supranuclear Progressiva/patologia , Azepinas/farmacologia , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Triazóis/farmacologia , Ratos , Proteínas de Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Encéfalo/metabolismo , Encéfalo/patologia , Fagocitose , Neurônios/metabolismo , Proteínas que Contêm Bromodomínio , Proteínas de Ciclo CelularRESUMO
Astrocytes and microglia can adopt two distinct phenotypes in various pathological processes: neurotoxic A1/M1 and neuroprotective A2/M2. Recent evidence suggests that these cells play a significant role in epileptogenesis. The objective of this study was to characterize the phenotype of astrocytes and microglial cells in the hippocampus and temporal cortex of young male Wistar rats at 3 h, 1, 3, and 7 days after pentylenetetrazole-induced seizures. RT-qPCR was employed to examine the expression of glial genes (Gfap, Aif1, Slc1a1, Slc1a2, Slc1a3, Itpr2, Gdnf, Bdnf, Fgf2, Tgfb, Il1b, Tnf, Il1rn, Lcn2, S100a10, Nlrp3, Arg1). The most notable alterations in the expression of glial genes were observed on the first day following seizures in the temporal cortex. An increase in the expression of the Gfap, Slc1a2, Slc1a1, Il1b, Tnfa, Bdnf, and Fgf2 genes, and the A2 astrocyte condition marker S100a10, was observed. An increase in the expression of the Gfap and Slc1a2 genes was observed in the hippocampus on the first day after seizures. However, in contrast to the changes observed in the cortex, the changes in the hippocampus were opposite for the Il1rn, Bdnf, Tgfb, and Arg1 genes. Nevertheless, the alterations in GFAP and EAAT2 protein levels were not corroborated by Western blot analysis. Conversely, a more comprehensive immunohistochemical analysis confirmed an augmentation in the number of GFAP-positive cells in the hippocampus 1 day after seizures. Based on the presented evidence, we can conclude that a single convulsive seizure episode in 3-week-old rats results in transient astroglial activation and polarization to a neuroprotective phenotype (A2).
Assuntos
Astrócitos , Hipocampo , Microglia , Pentilenotetrazol , Ratos Wistar , Convulsões , Lobo Temporal , Animais , Masculino , Hipocampo/metabolismo , Hipocampo/patologia , Astrócitos/metabolismo , Astrócitos/patologia , Convulsões/induzido quimicamente , Convulsões/metabolismo , Convulsões/patologia , Ratos , Pentilenotetrazol/toxicidade , Microglia/metabolismo , Microglia/patologia , Lobo Temporal/metabolismo , Lobo Temporal/patologia , FenótipoRESUMO
Traumatic brain injury (TBI) affects millions globally, with a majority of TBI cases being classified as mild, in which diffuse pathologies prevail. Two of the pathological hallmarks of TBI are diffuse axonal injury (DAI) and microglial activation. While progress has been made investigating the breadth of TBI-induced axonal injury and microglial changes in rodents, the neuroinflammatory progression and interaction between microglia and injured axons in humans is less well understood. Our group previously investigated microglial process convergence (MPC), in which processes of non-phagocytic microglia directly contact injured proximal axonal swellings, in rats and micropigs acutely following TBI. These studies demonstrated that MPC occurred on injured axons in the micropig, but not in the rat, following diffuse TBI. While it has been shown that microglia co-exist and interact with injured axons in humans post-TBI, the occurrence of MPC has not been quantitatively measured in the human brain. Therefore, in the current study we sought to validate our pig findings in human postmortem tissue. We investigated MPC onto injured axonal swellings and intact myelinated fibers in cases from individuals with confirmed DAI and control human brain tissue using multiplex immunofluorescent histochemistry. We found an increase in MPC onto injured axonal swellings, consistent with our previous findings in micropigs, indicating that MPC is a clinically relevant phenomenon that warrants further investigation.
Assuntos
Axônios , Lesão Axonal Difusa , Microglia , Humanos , Microglia/patologia , Microglia/metabolismo , Axônios/patologia , Axônios/metabolismo , Animais , Masculino , Suínos , Lesão Axonal Difusa/patologia , Lesão Axonal Difusa/metabolismo , Feminino , Encéfalo/patologia , Encéfalo/metabolismo , Lesões Encefálicas Traumáticas/patologia , Lesões Encefálicas Traumáticas/metabolismo , Pessoa de Meia-Idade , Autopsia , Adulto , Idoso , RatosRESUMO
A recent study has introduced a recombinant fusion protein, consisting of the extracellular domain (ECD) of p75 and the Fc fragment of human immunoglobulin IgG1 (p75ECD-Fc), as a multifaceted agent within the nervous system. This research aimed to assess the effects of p75ECD-Fc on neuronal growth and the restoration of neurological functions in rats afflicted with neonatal hypoxic-ischemic encephalopathy (NHIE). In vitro analyses revealed that 1⯵M p75ECD-Fc treatment markedly increased cell viability and facilitated neurite outgrowth in neurons exposed to oxygen-glucose deprivation (OGD). Subsequent in vivo studies determined that a dose of 78.6⯵g/3⯵l of p75ECD-Fc significantly mitigated brain damage and both acute and long-term neurological impairments, outperforming the therapeutic efficacy of hypothermia, as evidenced through behavioral assessments. Additionally, in vivo immunostaining showed that p75ECD-Fc administration enhanced neuronal survival and regeneration, and reduced astrocytosis and microglia activation in the cortex and hippocampus of NHIE rats. A noteworthy shift from A1 to A2 astrocyte phenotypes and from M1 to M2 microglia phenotypes was observed after p75ECD-Fc treatment. Furthermore, a co-expression of the p75 neurotrophin receptor (p75NTR) and Nestin was identified, with an overexpression of Nestin alleviating the neurological dysfunction induced by NHIE. Mechanistically, the neuroprotective effects of p75ECD-Fc, particularly its inhibition of neuronal apoptosis post-OGD, may be attributed to Nestin. Taken together, these results highlight the neuroprotective and anti-inflammatory effects of p75ECD-Fc treatment through the modulation of glial cell phenotypes and the Nestin-mediated inhibition of neuronal apoptosis, positioning it as a viable therapeutic approach for NHIE.
Assuntos
Animais Recém-Nascidos , Apoptose , Hipóxia-Isquemia Encefálica , Fragmentos Fc das Imunoglobulinas , Nestina , Ratos Sprague-Dawley , Animais , Hipóxia-Isquemia Encefálica/tratamento farmacológico , Hipóxia-Isquemia Encefálica/patologia , Hipóxia-Isquemia Encefálica/metabolismo , Apoptose/efeitos dos fármacos , Nestina/metabolismo , Fragmentos Fc das Imunoglobulinas/farmacologia , Ratos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Fármacos Neuroprotetores/farmacologia , Proteínas Recombinantes de Fusão/farmacologia , Masculino , Sobrevivência Celular/efeitos dos fármacos , Microglia/efeitos dos fármacos , Microglia/patologia , Microglia/metabolismo , Humanos , Receptores de Fator de Crescimento Neural/metabolismo , Modelos Animais de DoençasRESUMO
Microglia-driven neuroinflammation plays an important role in the development of Alzheimer's disease. Microglia activation is accompanied by the formation and chronic expression of TLR4 inflammarafts, defined as enlarged and cholesterol-rich lipid rafts serving as an assembly platform for TLR4 dimers and complexes of other inflammatory receptors. The secreted apoA-I binding protein (APOA1BP or AIBP) binds TLR4 and selectively targets cholesterol depletion machinery to TLR4 inflammaraft-expressing inflammatory, but not homeostatic microglia. Here we demonstrated that amyloid-beta (Aß) induced formation of TLR4 inflammarafts in microglia in vitro and in the brain of APP/PS1 mice. Mitochondria in Apoa1bp-/- APP/PS1 microglia were hyperbranched and cupped, which was accompanied by increased reactive oxygen species and the dilated endoplasmic reticulum. The size and number of Aß plaques and neuronal cell death were significantly increased, and the animal survival was decreased in Apoa1bp-/-APP/PS1 compared to APP/PS1 female mice. These results suggest that AIBP exerts control of TLR4 inflammarafts and mitochondrial dynamics in microglia and plays a protective role in Alzheimer's disease associated oxidative stress and neurodegeneration.
Assuntos
Doença de Alzheimer , Modelos Animais de Doenças , Camundongos Transgênicos , Mitocôndrias , Receptor 4 Toll-Like , Animais , Camundongos , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Doença de Alzheimer/genética , Receptor 4 Toll-Like/metabolismo , Receptor 4 Toll-Like/genética , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Microglia/metabolismo , Microglia/patologia , Feminino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , HumanosRESUMO
Spinal cord injury (SCI) triggers microglial/monocytes activation with distinct pro-inflammatory or inflammation-resolving phenotypes, which potentiate tissue damage or facilitate functional repair, respectively. The major integrin Mac-1 (CD11b/CD18), a heterodimer consisting of CD11b and CD18 chains, is expressed in multiple immune cells of the myeloid lineage. Here, we examined the effects of CD11b gene ablation in neuroinflammation and functional outcomes after SCI. qPCR analysis of C57BL/6 female mice showed upregulation of CD11b mRNA starting from 1 d after injury, which persisted up to 28 d. CD11b knockout (KO) mice and their wildtype littermates were subjected to moderate SCI. At 1 d post-injury, qPCR showed increased expression of genes involved with inflammation-resolving processes in CD11b KO mice. Flow cytometry analysis of CD45intLy6C-CX3CR1+ microglia, CD45hiLy6C+Ly6G- monocytes, and CD45hiLy6C+Ly6G+ neutrophils revealed significantly reduced cell counts as well as reactive oxygen species (ROS) production in CD11b KO mice at d3 post-injury. Further examination with NanoString and RNA-seq showed upregulation of pro-inflammatory genes, but downregulation of the ROS pathway. Importantly, CD11b KO mice exhibited significantly improved locomotor function, reduced cutaneous mechanical/thermal hypersensitivity, and limited tissue damage at 8 weeks post-injury. Collectively, our data suggest an important role for CD11b in regulating tissue inflammation and functional outcome following SCI.
Assuntos
Antígeno CD11b , Recuperação de Função Fisiológica , Traumatismos da Medula Espinal , Animais , Feminino , Camundongos , Antígeno CD11b/metabolismo , Modelos Animais de Doenças , Inflamação/patologia , Antígeno de Macrófago 1/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microglia/metabolismo , Microglia/patologia , Traumatismos da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/patologia , Traumatismos da Medula Espinal/genéticaRESUMO
Ischaemic stroke is a common condition that can lead to cerebral ischaemia-reperfusion injury. Phillygenin (PHI), a natural bioactive compound derived from Forsythia suspensa, has been shown to play a crucial role in regulating inflammation across various diseases. However, its specific regulatory effects in ischaemic stroke progression remain unclear. In this study, we established a middle cerebral artery occlusion (MCAO) rat model. Treatment with PHI (50 or 100 mg/kg) significantly reduced cerebral infarction in MCAO rats. PHI treatment also mitigated the increased inflammatory response observed in these rats. Additionally, PHI suppressed microglial activation by reducing iNOS expression, a marker of M1-type polarization of microglia, and attenuated increased brain tissue apoptosis in MCAO rats. Furthermore, PHI's anti-inflammatory effects in MCAO rats were abrogated upon co-administration with GW9662, a peroxisome proliferator-activated receptor γ (PPARγ) inhibitor. In summary, PHI attenuated microglial activation and apoptosis in cerebral ischaemia-reperfusion injury through PPARγ activation, suggesting its potential as a therapeutic agent for mitigating cerebral ischaemia-reperfusion injury.
Assuntos
Apoptose , Infarto da Artéria Cerebral Média , Microglia , PPAR gama , Ratos Sprague-Dawley , Traumatismo por Reperfusão , Animais , PPAR gama/metabolismo , Apoptose/efeitos dos fármacos , Traumatismo por Reperfusão/tratamento farmacológico , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Microglia/efeitos dos fármacos , Microglia/metabolismo , Microglia/patologia , Ratos , Masculino , Infarto da Artéria Cerebral Média/tratamento farmacológico , Infarto da Artéria Cerebral Média/patologia , Infarto da Artéria Cerebral Média/metabolismo , Isquemia Encefálica/tratamento farmacológico , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patologia , LignanasRESUMO
Progression of acute traumatic brain injury (TBI) into chronic neurodegeneration is a major health problem with no protective treatments. Here, we report that acutely elevated mitochondrial fission after TBI in mice triggers chronic neurodegeneration persisting 17 months later, equivalent to many human decades. We show that increased mitochondrial fission after mouse TBI is related to increased brain levels of mitochondrial fission 1 protein (Fis1) and that brain Fis1 is also elevated in human TBI. Pharmacologically preventing Fis1 from binding its mitochondrial partner, dynamin-related protein 1 (Drp1), for 2 weeks after TBI normalizes the balance of mitochondrial fission/fusion and prevents chronically impaired mitochondrial bioenergetics, oxidative damage, microglial activation and lipid droplet formation, blood-brain barrier deterioration, neurodegeneration, and cognitive impairment. Delaying treatment until 8 months after TBI offers no protection. Thus, time-sensitive inhibition of acutely elevated mitochondrial fission may represent a strategy to protect human TBI patients from chronic neurodegeneration.
Assuntos
Lesões Encefálicas Traumáticas , Dinaminas , Mitocôndrias , Dinâmica Mitocondrial , Proteínas Mitocondriais , Lesões Encefálicas Traumáticas/metabolismo , Lesões Encefálicas Traumáticas/patologia , Animais , Dinaminas/metabolismo , Dinaminas/genética , Proteínas Mitocondriais/metabolismo , Proteínas Mitocondriais/genética , Humanos , Camundongos , Mitocôndrias/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/patologia , Estresse Oxidativo , Encéfalo/patologia , Encéfalo/metabolismo , Microglia/metabolismo , Microglia/patologia , Doença Crônica , Modelos Animais de Doenças , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologiaRESUMO
Diabetic retinopathy (DR) is a common complication of diabetes characterized by vascular pathology and neuroinflammation. Pentraxin 3 (PTX3) is a soluble pattern recognition molecule that functions at the crossroads between innate immunity, inflammation, and tissue remodeling. DR is known to involve inflammatory pathways, although the potential relevance of PTX3 has not been explored. We found that PTX3 protein levels increased in the retina of diabetic mice. Similarly, evaluation of a publicly available transcriptomic human dataset revealed increased PTX3 expression in DR with diabetic macular edema and proliferative retinopathy, when compared to nondiabetic retinas or diabetic retinas without complications. To further understand the role of PTX3 within DR, we employed the streptozotocin-induced diabetes model in PTX3 knockout mice (PTX3KO), which were followed up for 9 mo to evaluate hallmarks of disease progression. In diabetic PTX3KO mice, we observed decreased reactive gliosis, diminished microglia activation, and reduced vasodegeneration, when compared to diabetic PTX3 wild-type littermates. The decrease in DR-associated pathological features in PTX3KO retinas translated into preserved visual function, as evidenced by improved optokinetic response, restored b-wave amplitude in electroretinograms, and attenuated neurodegeneration. We showed that PTX3 induced an inflammatory phenotype in human retinal macroglia, characterized by GFAP upregulation and increased secretion of IL6 and PAI-1. We confirmed that PTX3 was required for TNF-α-induced reactive gliosis, as PTX3KO retinal explants did not up-regulate GFAP in response to TNF-α. This study reveals a unique role for PTX3 as an enhancer of sterile inflammation in DR, which drives pathogenesis and ultimately visual impairment.
Assuntos
Proteína C-Reativa , Diabetes Mellitus Experimental , Retinopatia Diabética , Camundongos Knockout , Retina , Animais , Retinopatia Diabética/metabolismo , Retinopatia Diabética/patologia , Retinopatia Diabética/genética , Retinopatia Diabética/fisiopatologia , Camundongos , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/fisiopatologia , Diabetes Mellitus Experimental/complicações , Retina/metabolismo , Retina/patologia , Proteína C-Reativa/metabolismo , Proteína C-Reativa/genética , Humanos , Componente Amiloide P Sérico/metabolismo , Componente Amiloide P Sérico/genética , Masculino , Camundongos Endogâmicos C57BL , Inflamação/metabolismo , Inflamação/patologia , Microglia/metabolismo , Microglia/patologia , Edema Macular/metabolismo , Edema Macular/patologia , Edema Macular/genética , Proteínas do Tecido NervosoAssuntos
Doença de Alzheimer , Apolipoproteína E4 , Microglia , Neutrófilos , Tauopatias , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Humanos , Microglia/metabolismo , Microglia/patologia , Feminino , Masculino , Apolipoproteína E4/genética , Apolipoproteína E4/metabolismo , Neutrófilos/metabolismo , Tauopatias/genética , Tauopatias/patologia , Tauopatias/metabolismo , Transdução de Sinais/genética , AnimaisRESUMO
Abnormal glial activation promotes neurodegeneration in Alzheimer's disease (AD), the most common cause of dementia. Stimulation of the cGAS-STING pathway induces microglial dysfunction and sterile inflammation, which exacerbates AD. We showed that inhibiting STING activation can control microglia and ameliorate a wide spectrum of AD symptoms. The cGAS-STING pathway is required for the detection of ectopic DNA and the subsequent immune response. Amyloid-ß (Aß) and tau induce mitochondrial stress, which causes DNA to be released into the cytoplasm of microglia. cGAS and STING are highly expressed in Aß plaque-associated microglia, and neuronal STING is upregulated in the brains of AD model animals. The presence of the APOE ε4 allele, an AD risk factor, also upregulated both proteins. STING activation was necessary for microglial NLRP3 activation, proinflammatory responses, and type-I-interferon responses. Pharmacological STING inhibition reduced a wide range of AD pathogenic features in AppNL-G-F/hTau double-knock-in mice. An unanticipated transcriptome shift in microglia reduced gliosis and cerebral inflammation. Significant reductions in the Aß load, tau phosphorylation, and microglial synapse engulfment prevented memory loss. To summarize, our study describes the pathogenic mechanism of STING activation as well as its potential as a therapeutic target in AD.
Assuntos
Doença de Alzheimer , Modelos Animais de Doenças , Proteínas de Membrana , Microglia , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Doença de Alzheimer/genética , Doença de Alzheimer/etiologia , Animais , Microglia/metabolismo , Microglia/patologia , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Camundongos , Humanos , Peptídeos beta-Amiloides/metabolismo , Camundongos Transgênicos , Nucleotidiltransferases/metabolismo , Nucleotidiltransferases/genética , Transdução de SinaisRESUMO
Traumatic brain injury (TBI) is an incurable and overwhelming disease accompanied with serve disability and huge financial burden, where the overproduced reactive oxygen species (ROS) can exacerbate the secondary injury, leading to massive apoptosis of neurons. In this study, ß-cyclodextrin (CD)-capped hyperbranched polymers containing selenium element (HSE-CD) were crosslinked with CD-modified hyaluronic acid (HA-CD) and amantadine-modified hyaluronic acid (HA-AD) to obtain a ROS-responsive ointment (R-O). The structures of synthesized polymers were characterized with 1H nuclear magnetic resonance, and the properties of ointment were investigated with rheology and antioxidation. Compared to non-ROS-responsive ointment (N-O), the R-O ointment had stronger efficiency in decreasing the ROS level in BV2 cells in vitro. In a controlled rat cortical impact (CCI) model, the R-O ointment could relieve the DNA damage and decrease apoptosis in injured area via reducing the ROS level. Besides, after the R-O treatment, the rats showed significantly less activated astrocytes and microglia, a lower level of pro-inflammatory cytokines and a higher ratio of M2/M1 macrophage and microglia. Moreover, compared to the TBI group the R-O ointment promoted the doublecortin (DCX) expression and tissue structure integrity around the cavity, and promoted the recovery of nerve function post TBI. STATEMENT OF SIGNIFICANCE: Traumatic brain injury (TBI) is an incurable and overwhelming disease, leading to severe disability and huge social burden, where reactive oxygen species (ROS) are considered as one of the most significant factors in the secondary injury of TBI. A ROS responsive supramolecular ointment containing di-selenide bonds was injected in rats with controlled cortical impact. It relieved the DNA damage and decreased apoptosis in the injured area via reducing the ROS levels, downregulated neuroinflammation, and improved neurological recovery of TBI in vivo. This designed self-adaptive biomaterial effectively regulated the pathological microenvironment in injured tissue, and achieved better therapeutic effect.
Assuntos
Lesões Encefálicas Traumáticas , Pomadas , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio , Selênio , Animais , Selênio/química , Selênio/farmacologia , Lesões Encefálicas Traumáticas/tratamento farmacológico , Lesões Encefálicas Traumáticas/patologia , Masculino , Espécies Reativas de Oxigênio/metabolismo , Ratos , Apoptose/efeitos dos fármacos , Proteína Duplacortina , Linhagem Celular , Camundongos , Microglia/efeitos dos fármacos , Microglia/patologia , Microglia/metabolismoRESUMO
Uveitis is a vision-threatening disease primarily driven by a dysregulated immune response, with retinal microglia playing a pivotal role in its progression. Although the transcription factor EGR2 is known to be closely associated with uveitis, including Vogt-Koyanagi-Harada disease and Behcet's disease, and is essential for maintaining the dynamic homeostasis of autoimmunity, its exact role in uveitis remains unclear. In this study, diminished EGR2 expression was observed in both retinal microglia from experimental autoimmune uveitis (EAU) mice and inflammation-induced human microglia cell line (HMC3). We constructed a mice model with conditional knockout of EGR2 in microglia and found that EGR2 deficiency resulted in increased intraocular inflammation. Meanwhile, EGR2 overexpression downregulated the expression of inflammatory cytokines as well as cell migration and proliferation in HMC3 cells. Next, RNA sequencing and ChIP-PCR results indicated that EGR2 directly bound to its downstream target growth differentiation factor 15 (GDF15) and further regulated GDF15 transcription. Furthermore, intravitreal injection of GDF15 recombinant protein was shown to ameliorate EAU progression in vivo. Meanwhile, knockdown of GDF15 reversed the phenotype of EGR2 overexpression-induced microglial inflammation in vitro. In summary, this study highlighted the protective role of the transcription factor EGR2 in AU by modulating the microglial phenotype. GFD15 was identified as a downstream target of EGR2, providing a unique target for uveitis treatment.
Assuntos
Doenças Autoimunes , Proteína 2 de Resposta de Crescimento Precoce , Fator 15 de Diferenciação de Crescimento , Microglia , Uveíte , Animais , Humanos , Camundongos , Doenças Autoimunes/imunologia , Doenças Autoimunes/genética , Doenças Autoimunes/patologia , Doenças Autoimunes/metabolismo , Linhagem Celular , Modelos Animais de Doenças , Proteína 2 de Resposta de Crescimento Precoce/metabolismo , Proteína 2 de Resposta de Crescimento Precoce/genética , Fator 15 de Diferenciação de Crescimento/metabolismo , Fator 15 de Diferenciação de Crescimento/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microglia/metabolismo , Microglia/patologia , Fenótipo , Retina/metabolismo , Retina/patologia , Uveíte/imunologia , Uveíte/metabolismo , Uveíte/patologia , Uveíte/genéticaRESUMO
Neonatal hypoxic-ischemic encephalopathy (HIE) occurs in 1.5 per 1000 live births, leaving affected children with long-term motor and cognitive deficits. Few animal models of HIE incorporate maternal immune activation (MIA) despite the significant risk MIA poses to HIE incidence and diagnosis. Our non-invasive model of HIE pairs late gestation MIA with postnatal hypoxia. HIE pups exhibited a trend toward smaller overall brain size and delays in the ontogeny of several developmental milestones. In adulthood, HIE animals had reduced strength and gait deficits, but no difference in speed. Surprisingly, HIE animals performed better on the rotarod, an assessment of motor coordination. There was significant upregulation of inflammatory genes in microglia 24 h after hypoxia. Single-cell RNA sequencing (scRNAseq) revealed two microglia subclusters of interest following HIE. Pseudobulk analysis revealed increased microglia motility gene expression and upregulation of epigenetic machinery and neurodevelopmental genes in macrophages following HIE. No sex differences were found in any measures. These results support a two-hit noninvasive model pairing MIA and hypoxia as a model for HIE in humans. This model results in a milder phenotype compared to established HIE models; however, HIE is a clinically heterogeneous injury resulting in a variety of outcomes in humans. The pathways identified in our model of HIE may reveal novel targets for therapy for neonates with HIE.