Microglial Phenotype Research Articles

ATP-P2X7R-mediated microglia senescence aggravates retinal ganglion cell injury in chronic ocular hypertension

BackgroundDysfunction of microglia during aging affects normal neuronal function and results in the occurrence of neurodegenerative diseases. Retinal microglial senescence attributes to retinal ganglion cell (RGC) death in glaucoma. This study aims to examine the role of ATP-P2X7R in the mediation of microglia senescence and glaucoma progression.MethodsForty-eight participants were enrolled, including 24 patients with primary open-angle glaucoma (POAG) and age-related cataract (ARC) and 24 patients with ARC only. We used ARC as the inclusion criteria because of the availability of aqueous humor (AH) before phacoemulsification. AH was collected and the adenosine triphosphate (ATP) concentration was measured by ATP Assay Kit. The chronic ocular hypertension (COH) mouse model was established by microbead occlusion. Microglia were ablated by feeding PLX5622 orally. Mouse bone marrow cells (BMCs) were prepared and infused into mice through the tail vein for the restoration of microglia function. Western blotting, qPCR and ELISA were performed to analyze protein and mRNA expression in the ocular tissue, respectively. Microglial phenotype and RGC survival were assessed by immunofluorescence. The mitochondrial membrane potential was measured using a JC-1 assay kit by flow cytometry.ResultsATP concentrations in the AH were increased in older adults and patients with POAG. The expression of P2X7R was upregulated in the retinal tissues of mice with glaucoma, and functional enrichment analysis showed that P2X7R was closely related to cell aging. Through in vivo and in vitro approaches, we showed that pathological activation of ATP-P2X7R induced accelerated microglial senescence through impairing PTEN-induced kinase 1 (PINK1)-mediated mitophagy, which led to RGC damage. Additionally, we found that replacement of senescent microglia in COH model of old mice with BMCs from young mice reversed RGC damage.ConclusionATP-P2X7R induces microglia senescence by inhibiting PINK1-mediated mitophagy pathway. Specific inhibition of ATP-P2X7R may be a fundamental approach for targeted therapy of RGC injury in microglial aging-related glaucoma.

Microglial crosstalk with astrocytes and immune cells in amyotrophic lateral sclerosis.

In recent years, biomedical research efforts aimed to unravel the mechanisms involved in motor neuron death that occurs in amyotrophic lateral sclerosis (ALS). While the main causes of disease progression were first sought in the motor neurons, more recent studies highlight the gliocentric theory demonstrating the pivotal role of microglia and astrocyte, but also of infiltrating immune cells, in the pathological processes that take place in the central nervous system microenvironment. From this point of view, microglia-astrocytes-lymphocytes crosstalk is fundamental to shape the microenvironment toward a pro-inflammatory one, enhancing neuronal damage. In this review, we dissect the current state-of-the-art knowledge of the microglial dialogue with other cell populations as one of the principal hallmarks of ALS progression. Particularly, we deeply investigate the microglia crosstalk with astrocytes and immune cells reporting in vitro and in vivo studies related to ALS mouse models and human patients. At last, we highlight the current experimental therapeutic approaches that aim to modulate microglial phenotype to revert the microenvironment, thus counteracting ALS progression.

The GPR84 molecule is a mediator of a subpopulation of retinal microglia that promote TNF/IL-1α expression via the rho-ROCK pathway after optic nerve injury.

Resident microglia are important to maintain homeostasis in the central nervous system, which includes the retina. The retinal microglia become activated in numerous pathological conditions, but the molecular signatures of these changes are poorly understood. Here, using an approach based on FACS and RNA-seq, we show that microglial gene expression patterns gradually change during RGC degeneration induced by optic nerve injury. Most importantly, we found that the microglial cells strongly expressed Tnf and Il1α, both of which are known to induce neurotoxic reactive astrocytes, and were characterized by Gpr84high -expressing cells in a particular subpopulation. Moreover, ripasudil, a Rho kinase inhibitor, significantly blunted Gpr84 expression and cytokine induction in vitro and in vivo. Finally, GPR84-deficient mice prevented RGC loss in optic nerve-injured retina. These results reveal that Rho kinase-mediated GPR84 alteration strongly contribute to microglial activation and promote neurotoxicity, suggesting that Rho-ROCK and GPR84 signaling may be potential therapeutic targets to prevent the neurotoxic microglial phenotype induced by optic nerve damage, such as occurs in traumatic optic neuropathy and glaucoma.

18β-glycyrrhetinic acid ameliorates MPTP-induced neurotoxicity in mice through activation of microglial anti-inflammatory phenotype.

18β-glycyrrhetinic acid (18β-GA) has been reported to have anti-inflammatory and neuroprotective effects. However, the therapeutic effect of 18β-GA in Parkinson's disease (PD) has not been defined. The current study aimed to evaluate the potential therapeutic effects of 18β-GA in treating PD by mitigating 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neurotoxicity. The study showed that 18β-GA has anti-inflammatory effects by upregulating TREM2 expression in BV2 cells, which correlates with the presence of NF-E2-related factor-2 (Nrf2). 18β-GA reduced inflammation in BV2 cells treated with 1-methyl-4- phenylpyridinium (MPP+) by enhancing TREM2 expression, which promotes an anti-inflammatory microglial phenotype. Repeated administration of 18β-GA in MPTP-treated mice led to therapeutic effects by enhancing TREM2 expression, resulting in the activation of anti-inflammatory microglia. Moreover, 18β-GA attenuated the decrease in brain-derived neurotrophic factor (BDNF) levels in both MPP+-induced BV2 cells and MPTP-intoxicated mice, indicating the involvement of BDNF in the beneficial effects of 18β-GA. It is probable that activating microglial anti-inflammatory response through TREM2 expression might serve as a novel therapeutic strategy for PD. Additionally, 18β-GA seems to hold potential as a new therapeutic agent for PD.

Persistence of Hyper-Ramified Microglia in Porcine Cortical Gray Matter after Mild Traumatic Brain Injury.

Traumatic brain injury (TBI) is a major contributor to morbidity and mortality in the United States as several million people visit the emergency department every year due to TBI exposures. Unfortunately, there is still no consensus on the pathology underlying mild TBI, the most common severity sub-type of TBI. Previous preclinical and post-mortem human studies have detailed the presence of diffuse axonal injury following TBI, suggesting that white matter pathology is the predominant pathology of diffuse brain injury. However, the inertial loading produced by TBI results in strain fields in both gray and white matter. In order to further characterize gray matter pathology in mild TBI, our lab used a pig model (n = 25) of closed-head rotational acceleration-induced TBI to evaluate blood-brain barrier disruptions, neurodegeneration, astrogliosis, and microglial reactivity in the cerebral cortex out to 1 year post-injury. Immunohistochemical staining revealed the presence of a hyper-ramified microglial phenotype-more branches, junctions, endpoints, and longer summed process length-at 30 days post injury (DPI) out to 1 year post injury in the cingulate gyrus (p < 0.05), and at acute and subacute timepoints in the inferior temporal gyrus (p < 0.05). Interestingly, we did not find neuronal loss or astroglial reactivity paired with these chronic microglia changes. However, we observed an increase in fibrinogen reactivity-a measure of blood-brain barrier disruption-predominately in the gray matter at 3 DPI (p = 0.0003) which resolved to sham levels by 7 DPI out to chronic timepoints. Future studies should employ gene expression assays, neuroimaging, and behavioral assays to elucidate the effects of these hyper-ramified microglia, particularly related to neuroplasticity and responses to potential subsequent insults. Further understanding of the brain's inflammatory activity after mild TBI will hopefully provide understanding of pathophysiology that translates to clinical treatment for TBI.

Gut microbiota regulates blood‐cerebrospinal fluid barrier function and Aβ pathology

Accumulating evidence indicates that gut microbiota dysbiosis is associated with increased blood–brain barrier (BBB) permeability and contributes to Alzheimer's disease (AD) pathogenesis. In contrast, the influence of gut microbiota on the blood‐cerebrospinal fluid (CSF) barrier has not yet been studied. Here, we report that mice lacking gut microbiota display increased blood‐CSF barrier permeability associated with disorganized tight junctions (TJs), which can be rescued by recolonization with gut microbiota or supplementation with short‐chain fatty acids (SCFAs). Our data reveal that gut microbiota is important not only for the establishment but also for the maintenance of a tight barrier. Also, we report that the vagus nerve plays an important role in this process and that SCFAs can independently tighten the barrier. Administration of SCFAs in AppNL‐G‐F mice improved the subcellular localization of TJs at the blood‐CSF barrier, reduced the β‐amyloid (Aβ) burden, and affected microglial phenotype. Altogether, our results suggest that modulating the microbiota and administering SCFAs might have therapeutic potential in AD via blood‐CSF barrier tightening and maintaining microglial activity and Aβ clearance.

The gut microbiome regulates astrocyte reaction to Aβ amyloidosis through microglial dependent and independent mechanisms

BackgroundPrevious studies show that antibiotic-mediated (abx) alteration of the gut microbiome (GMB) results in a reduction of amyloid beta (Aβ) plaques and proinflammatory microglial phenotype in male APPPS1-21 mice. However, the effect of GMB perturbation on astrocyte phenotypes and microglial-astrocyte communication in the context of amyloidosis has not been examined.MethodsTo study whether the GMB modulates astrocyte phenotype in the context of amyloidosis, APPPS1-21 male and female mice were treated with broad-spectrum abx leading to GMB perturbation. GFAP + astrocytes, plaque-associated astrocytes (PAA), PAA morphological parameters, and astrocyte complement component C3 levels were quantified using a combination of immunohistochemistry, immunoblotting, widefield microscopy, and confocal microscopy. Furthermore, these same astrocyte phenotypes were assessed in abx-treated APPPS1-21 male mice that received either fecal matter transplant (FMT) from untreated APPPS1-21 male donors to restore their microbiome or vehicle control. To assess complete absence of the GMB on astrocyte phenotypes, the same astrocyte phenotypes were quantified in APPPS1-21 male mice raised in germ-free (GF) or specific-pathogen free conditions (SPF). Lastly, we assessed whether microglia are necessary for abx-induced astrocyte phenotypes by depleting microglia in APPPS1-21 male mice via treatment with a colony-stimulating factor 1 receptor (CSF1R) inhibitor (PLX5622) and vehicle control or PLX5622 and abx.ResultsHerein, we demonstrate that postnatal treatment of male APPPS1-21 mice with broad-spectrum abx leading to GMB perturbation reduces GFAP + reactive astrocytes and PAAs, suggesting that the GMB plays a role in regulating reactive astrocyte induction and recruitment to Aβ plaques. Additionally, we show that compared to controls, PAAs in abx-treated male APPPS1-21 mice exhibit an altered morphology with increased number and length of processes and reduced astrocytic complement C3, consistent with a homeostatic phenotype. GFAP + astrocyte reduction, PAA reduction, astrocyte morphological changes, and C3 levels are restored when abx-treated mice are subject to FMT from untreated APPPS1-21 male donor mice. Next, we found that APPPS1-21 male mice raised in GF conditions have similar astrocyte phenotypes as abx-treated male APPPS1-21 male mice. Correlational analysis revealed that pathogenic bacteria depleted by abx correlate with GFAP + astrocytosis, PAAs, and astrocyte morphological changes. Finally, we determined that abx-mediated reduction in GFAP + astrocytosis, PAAs, and astrocytic C3 expression is independent of microglia. However, abx-induced astrocyte morphological alterations are dependent on the presence of microglia, suggesting that there is both microglial independent and dependent GMB control of reactive astrocyte phenotypes.ConclusionsWe show for the first time, in the context of amyloidosis, that the GMB plays an important role in controlling reactive astrocyte induction, morphology, and astrocyte recruitment to Aβ plaques. GMB regulation of these astrocytic phenotypes is both independent and dependent on microglia.

Complement C1q drives microglia-dependent synaptic loss and cognitive impairments in a mouse model of lipopolysaccharide-induced neuroinflammation

Activated microglia and subsequent release of pro-inflammatory cytokines result in neuroinflammatory status which further damage neurological function including cognitive impairments in various neurological conditions. However, the underlying molecular mechanisms during these pathological processing remain unknown. In the current study, mice received intraperitoneal administrations of LPS (0.5 mg/kg, daily, Escherichia coli O55:B5) for seven consecutive days and their different cohorts were used for behavioral assessment with open field, Y maze, and novel object recognition test or for electrophysiology recordings of mEPSC, LFP or LTP in in vivo or ex vivo preparation. The hippocampus from some cohorts were harvested for immunostaining or Western blotting of c1q, Iba-1, CD68, PSD95 and dendritic spine density or for transcriptome and proteomics analysis. Repeated LPS injections induced an up-regulation of complement system protein c1q and distinct microglial phenotype with an enrichment of the complement–phagosome pathway. Microglial synaptic engulfment and profound synaptic loss were found. These pathological changes were accompanied with the significantly decreased excitatory synaptic transmission, disturbed theta oscillations, impaired hippocampal long-term potentiation, and cognitive impairments. Notably, neutralization of c1q signaling robustly prevented these changes. Collectively, our data provide evidence that activated microglia and complement cascade c1q signaling in the hippocampus may account for synaptic loss and cognitive impairments in a mouse model of neuroinflammation induced by repeated LPS injections. Our work implicates that complement system may be a therapeutic target for developing therapies to prevent or treat cognitive disorders related to neuroinflammation or other disease conditions including neurodegenerative disease per se.

Prolonged Early Exposure to a High-Fat Diet Augments the Adverse Effects on Neurobehavior and Hippocampal Neuroplasticity: Involvement of Microglial Insulin Signaling

High-fat diet (HFD) consumption may contribute to the high prevalence of cognitive-emotional issues in modern society. Our group previously demonstrated that mice fed a HFD for a prolonged period developed more severe neurobehavioral disturbances when first exposed to a HFD in the juvenile period than in adulthood, suggesting an initial age-related difference in the detrimental effects of long-term HFD feeding; however, the mechanism underlying this difference remains unclear. Here, male C57BL/6J mice initially aged 4 (IA4W) or 8 (IA8W) weeks were fed a control diet (CD) or HFD for 6 months and then subjected to metabolic, neurobehavioral, and histomorphological examinations. Although the detrimental effects of long-term HFD feeding on metabolism and neurobehavior were observed in mice of both ages, IA4W-HFD mice showed significant cognitive inflexibility accompanied by significantly greater levels of anxiety-like behavior than age-matched controls. Hippocampal neuroplasticity and microglial phenotype were altered by HFD feeding, while significant morphological alterations were more frequently observed in IA4W-HFD mice than IA8W-HFD mice. Additionally, significantly increased hippocampal microglial engulfment of postsynaptic proteins and elevated phospho-insulin-receptor levels were observed in IA4W-HFD but not IA8W-HFD mice. These findings suggest that aberrant microglia-related histomorphological changes in the hippocampus underlie the exacerbated detrimental neurobehavioral effects of prolonged early HFD exposure and propose that enhanced insulin signaling might drive microglial dysfunction after prolonged early HFD exposure.

Innate immune activation and aberrant function in the R6/2 mouse model and Huntington's disease iPSC-derived microglia.

Huntington's disease (HD) is an inherited autosomal dominant neurodegenerative disease caused by CAG repeats in exon 1 of the HTT gene. A hallmark of HD along with other psychiatric and neurodegenerative diseases is alteration in the neuronal circuitry and synaptic loss. Microglia and peripheral innate immune activation have been reported in pre-symptomatic HD patients; however, what "activation" signifies for microglial and immune function in HD and how it impacts synaptic health remains unclear. In this study we sought to fill these gaps by capturing immune phenotypes and functional activation states of microglia and peripheral immunity in the R6/2 model of HD at pre-symptomatic, symptomatic and end stages of disease. These included characterizations of microglial phenotypes at single cell resolution, morphology, aberrant functions such as surveillance and phagocytosis and their impact on synaptic loss in vitro and ex vivo in R6/2 mouse brain tissue slices. To further understand how relevant the observed aberrant microglial behaviors are to human disease, transcriptomic analysis was performed using HD patient nuclear sequencing data and functional assessments were conducted using induced pluripotent stem cell (iPSC)-derived microglia. Our results show temporal changes in brain infiltration of peripheral lymphoid and myeloid cells, increases in microglial activation markers and phagocytic functions at the pre-symptomatic stages of disease. Increases in microglial surveillance and synaptic uptake parallel significant reduction of spine density in R6/2 mice. These findings were mirrored by an upregulation of gene signatures in the endocytic and migratory pathways in disease-associated microglial subsets in human HD brains, as well as increased phagocytic and migratory functions of iPSC-derived HD microglia. These results collectively suggest that targeting key and specific microglial functions related to synaptic surveillance and pruning may be therapeutically beneficial in attenuating cognitive decline and psychiatric aspects of HD.

Controlled Decompression Alleviates Motor Dysfunction by Regulating Microglial Polarization via the HIF-1α Signaling Pathway in Intracranial Hypertension.

Decompressive craniectomy (DC) is a major form of surgery that is used to reduce intracranial hypertension (IH), the most frequent cause of death and disability following severe traumatic brain injury (sTBI) and stroke. Our previous research showed that controlled decompression (CDC) was more effective than rapid decompression (RDC) with regard to reducing the incidence of complications and improving outcomes after sTBI; however, the specific mechanisms involved have yet to be elucidated. In the present study, we investigated the effects of CDC in regulating inflammation after IH and attempted to identify the mechanisms involved. Analysis showed that CDC was more effective than RDC in alleviating motor dysfunction and neuronal death in a rat model of traumatic intracranial hypertension (TIH) created by epidural balloon pressurization. Moreover, RDC induced M1 microglia polarization and the release of pro-inflammatory cytokines. However, CDC treatment resulted in microglia primarily polarizing into the M2 phenotype and induced the significant release of anti-inflammatory cytokines. Mechanistically, the establishment of the TIH model led to the increased expression of hypoxia-inducible factor-1α (HIF-1α); CDC ameliorated cerebral hypoxia and reduced the expression of HIF-1α. In addition, 2-methoxyestradiol (2-ME2), a specific inhibitor of HIF-1α, significantly attenuated RDC-induced inflammation and improved motor function by promoting M1 to M2 phenotype transformation in microglial and enhancing the release of anti-inflammatory cytokines. However, dimethyloxaloylglycine (DMOG), an agonist of HIF-1α, abrogated the protective effects of CDC treatment by suppressing M2 microglia polarization and the release of anti-inflammatory cytokines. Collectively, our results indicated that CDC effectively alleviated IH-induced inflammation, neuronal death, and motor dysfunction by regulating HIF-1α-mediated microglial phenotype polarization. Our findings provide a better understanding of the mechanisms that underlie the protective effects of CDC and promote clinical translational research for HIF-1α in IH.

Microglial Caspase-3 is essential for modulating hippocampal neurogenesis

Adult hippocampal neurogenesis (AHN) is a process involved in numerous neurodegenerative diseases. Many researchers have described microglia as a key component in regulating the formation and migration of new neurons along the rostral migratory stream. Caspase-3 is a cysteine-aspartate-protease classically considered as one of the main effector caspases in the cell death program process. In addition to this classical function, we have identified the role of this protein as a modulator of microglial function; however, its action on neurogenic processes is unknown. The aim of the present study is to identify the role of Caspase-3 in neurogenesis-related microglial functions. To address this study, Caspase-3 conditional knockout mice in the microglia cell line were used. Using this tool, we wanted to elucidate the role of this protein in microglial function in the hippocampus, the main region in which adult neurogenesis takes place. After the reduction of Caspase-3 in microglia, mutant mice showed a reduction of microglia in the hippocampus, especially in the dentate gyrus region, a region inherently associated to neurogenesis. In addition, we found a reduction in doublecortin-positive neurons in conditional Caspase-3 knockout mice, which corresponds to a reduction in neurogenic neurons. Furthermore, using high-resolution image analysis, we also observed a reduction in the phagocytic capacity of microglia lacking Caspase-3. Behavioral analysis using object recognition and Y-maze tests showed altered memory and learning in the absence of Caspase-3. Finally, we identified specific microglia located specifically in neurogenic niche positive for Galectin 3 which colocalized with Cleaved-Caspase-3 in control mice. Taken together, these results showed the essential role of Caspase-3 in microglial function and highlight the relevant role of this specific microglial phenotype in the maintenance of AHN in the hippocampus.

Netrin-1 controls inflammation in response to ischemic stroke through altering microglia phenotype.

The current approaches that are used to treat ischemic stroke suffer from poor targeting, lack of effectiveness, and potential off-target effects, necessitating the development of new therapeutic strategies to enhance neuronal cell survival and regeneration. This study aimed to investigate the role of microglial Netrin-1 in ischemic stroke, a topic that has not been fully understood. Netrin-1 levels and its primary receptor expressions were investigated in cerebral microglia from acute ischemic stroke patients and age-matched control subjects. A public database (GEO148350), which supplied RNAseq results for rat cerebral microglia in a middle cerebral artery occlusion (MCAO) model, was analyzed to assess the expression of Netrin-1, its major receptors, and genes related to macrophage function. A microglia-specific gene targeting approach and a delivery system allowing for crossing the blood-brain barrier were applied in a mouse model for ischemic stroke to investigate the role of microglial Netrin-1. Netrin-1 receptor signaling in microglia was observed and the effects on microglial phenotype, apoptosis, and migration were analyzed. Across human patients, rat and mouse models, activation of Netrin-1 receptor signaling was mainly conducted via its receptor UNC5a in microglia, which resulted in a shift in microglial phenotype towards an anti-inflammatory or M2-like state, leading to a reduction in apoptosis and migration of microglia. Netrin-1-induced phenotypic change in microglia exerted protective effects on neuronal cells in vivo during ischemic stroke. Our study highlights the potential of targeting Netrin-1 and its receptors as a promising therapeutic strategy for promoting post-ischemic survival and functional recovery.

Microglial cell response to experimental periodontal disease

ObjectivesMicroglial activation is critical for modulating the neuroinflammatory process and the pathological progression of neurodegenerative diseases, such as Alzheimer's disease (AD). Microglia are involved in forming barriers around extracellular neuritic plaques and the phagocytosis of β-amyloid peptide (Aβ). In this study, we tested the hypothesis that periodontal disease (PD) as a source of infection alters inflammatory activation and Aβ phagocytosis by the microglial cells.MethodsExperimental PD was induced using ligatures in C57BL/6 mice for 1, 10, 20, and 30 days to assess the progression of PD. Animals without ligatures were used as controls. Maxillary bone loss and local periodontal tissue inflammation associated with the development of PD were confirmed by morphometric bone analysis and cytokine expression, respectively. The frequency and the total number of activated microglia (CD45+ CD11b+ MHCII+) in the brain were analyzed by flow cytometry. Mouse microglial cells (1 × 105) were incubated with heat-inactivated bacterial biofilm isolated from the ligatures retrieved from the teeth or with Klebsiella variicola, a relevant PD-associated bacteria in mice. Expression of pro-inflammatory cytokines, toll-like receptors (TLR), and receptors for phagocytosis was measured by quantitative PCR. The phagocytic capacity of microglia to uptake β-amyloid was analyzed by flow cytometry.ResultsLigature placement caused progressive periodontal disease and bone resorption that was already significant on day 1 post-ligation (p < 0.05) and continued to increase until day 30 (p < 0.0001). The severity of periodontal disease increased the frequency of activated microglia in the brains on day 30 by 36%. In parallel, heat-inactivated PD-associated total bacteria and Klebsiella variicola increased the expression of TNFα, IL-1β, IL-6, TLR2, and TLR9 in microglial cells (1.6-, 83-, 3.2-, 1.5-, 1.5-fold, respectively p < 0.01). Incubation of microglia with Klebsiella variicola increased the Aβ-phagocytosis by 394% and the expression of the phagocytic receptor MSR1 by 33-fold compared to the non-activated cells (p < 0.0001).ConclusionsWe showed that inducing PD in mice results in microglia activation in vivo and that PD-associated bacteria directly promote a pro-inflammatory and phagocytic phenotype in microglia. These results support a direct role of PD-associated pathogens in neuroinflammation.

#6315 CHARACTERIZATION OF BRAIN DAMAGE AFTER ACUTE KIDNEY INJURY

Abstract Background and Aims Acute kidney injury (AKI) is a very common condition in inpatients. Severe AKI is frequently associated with by neurological disorders such as confusion, the mechanisms of which are poorly understood. We have shown in several animal models that chronic kidney disease is associated with impaired cognitive performance such as memory and increased permeability of the blood-brain barrier (BBB), related to the accumulation of uremic toxin indoxyl sulfate. The goal of these experiments was to highlight the influence of AKI on cerebral impairment in mice, particularly the BBB permeability, and to describe the mechanisms involved. Method We performed an AKI in C57/Bl6 WT mice, by unilateral nephrectomy and transient ischemia-reperfusion of the remaining kidney for 20 minutes (RIRI20 group) and compared them to two other groups: control mice and mice with unilateral ischemia-reperfusion without nephrectomy (IR20). Modified neurological severity score (mNSS) was performed daily. BBB permeability was assessed by quantification of the Evans Blue leakage in the brain and by cerebral 68Ga-DTPA TEP/CT imaging. Cerebral immuno-histochemistry was performed to evaluate the neuro-inflammatory processes. Results Glomerular filtration rate at Day 1 was lower in AKI group compared to the control group 0.21 ± 0.26 vs. 0.94 ± 0.13 mL/min/100g b.w (p&amp;lt;0.0001) and IR group: 0.75 ± 0.17 mL/min/100g b.w (p = 0.0007). Mice with AKI displayed a neurological impairment at day 1 and day 2 post-AKI, compared to control mice and IR20 mice, with mainly an impairment of the balance. Mean mNSS score at Day 1 was 2.6 ± 4.5 in AKI group, and 0.0 ±-0.0 in both the control and IR groups (p = 0.002). BBB permeability at Day 2 was higher in the AKI group than in the control group (p = 0.10) and the IR group (p = 0.0006) by Evans blue staining. Cerebral TEP imaging found similar results with increased BBB permeability in AKI mice to compared to control mice at D1 and D2 (p = 0.004 and p = 0.009, respectively). On immunohistochemistry, brains of mice after AKI displayed more astrocytosis and microglial activation compared to brains of control mice. Conclusion AKI in mice is specifically associated with early neurological damage, unlike renal ischemia-reperfusion alone, related to a BBB disruption in the first two days after AKI. AKI appears to induce astrocytosis and altered microglial phenotype, potentially due to increased neuroinflammatory processes following BBB disruption.

Synaptamide Ameliorates Hippocampal Neurodegeneration and Glial Activation in Mice with Traumatic Brain Injury.

Traumatic brain injury (TBI) is a major concern for public health worldwide, affecting 55 million people and being the leading cause of death and disability. To improve the outcomes and effectiveness of treatment for these patients, we conducted a study on the potential therapeutic use of N-docosahexaenoylethanolamine (synaptamide) in mice using the weight-drop injury (WDI) TBI model. Our study focused on exploring synaptamide's effects on neurodegeneration processes and changes in neuronal and glial plasticity. Our findings showed that synaptamide could prevent TBI-associated working memory decline and neurodegenerative changes in the hippocampus, and it could alleviate decreased adult hippocampal neurogenesis. Furthermore, synaptamide regulated the production of astro- and microglial markers during TBI, promoting the anti-inflammatory transformation of the microglial phenotype. Additional effects of synaptamide in TBI include stimulating antioxidant and antiapoptotic defense, leading to the downregulation of the Bad pro-apoptotic marker. Our data suggest that synaptamide has promising potential as a therapeutic agent to prevent the long-term neurodegenerative consequences of TBI and improve the quality of life.

Microglia in the context of multiple sclerosis.

Multiple sclerosis (MS) is an inflammatory and neurodegenerative disease that commonly results in nontraumatic disability in young adults. The characteristic pathological hallmark of MS is damage to myelin, oligodendrocytes, and axons. Microglia provide continuous surveillance in the CNS microenvironment and initiate defensive mechanisms to protect CNS tissue. Additionally, microglia participate in neurogenesis, synaptic refinement, and myelin pruning through the expression and release of different signaling factors. Continuous activation of microglia has been implicated in neurodegenerative disorders. We first review the lifetime of microglia, including the origin, differentiation, development, and function of microglia. We then discuss microglia participate in the whole processes of remyelination and demyelination, microglial phenotypes in MS, and the NF-κB/PI3K-AKT signaling pathway in microglia. The damage to regulatory signaling pathways may change the homeostasis of microglia, which would accelerate the progression of MS.

Oleoylethanolamide Treatment Modulates Both Neuroinflammation and Microgliosis, and Prevents Massive Leukocyte Infiltration to the Cerebellum in a Mouse Model of Neuronal Degeneration.

Neurodegenerative diseases involve an exacerbated neuroinflammatory response led by microglia that triggers cytokine storm and leukocyte infiltration into the brain. PPARα agonists partially dampen this neuroinflammation in some models of brain insult, but neuronal loss was not the triggering cause in any of them. This study examines the anti-inflammatory and immunomodulatory properties of the PPARα agonist oleoylethanolamide (OEA) in the Purkinje Cell Degeneration (PCD) mouse, which exhibits striking neuroinflammation caused by aggressive loss of cerebellar Purkinje neurons. Using real-time quantitative polymerase chain reaction and immunostaining, we quantified changes in pro- and anti-inflammatory markers, microglial density and marker-based phenotype, and overall leukocyte recruitment at different time points after OEA administration. OEA was found to modulate cerebellar neuroinflammation by increasing the gene expression of proinflammatory mediators at the onset of neurodegeneration and decreasing it over time. OEA also enhanced the expression of anti-inflammatory and neuroprotective factors and the Pparα gene. Regarding microgliosis, OEA reduced microglial density-especially in regions where it is preferentially located in PCD mice-and shifted the microglial phenotype towards an anti-inflammatory state. Finally, OEA prevented massive leukocyte infiltration into the cerebellum. Overall, our findings suggest that OEA may change the environment to protect neurons from degeneration caused by exacerbated inflammation.

Dendrimer-enabled targeted delivery attenuates glutamate excitotoxicity and improves motor function in a rabbit model of cerebral palsy.

Glutamate carboxypeptidase II (GCPII), localized on the surface of astrocytes and activated microglia, regulates extracellular glutamate concentration in the central nervous system (CNS). We have previously shown that GCPII is upregulated in activated microglia in the presence of inflammation. Inhibition of GCPII activity could reduce glutamate excitotoxicity, which may decrease inflammation and promote a 'normal' microglial phenotype. 2-(3-Mercaptopropyl) pentanedioic acid (2-MPPA) is the first GCPII inhibitor that underwent clinical trials. Unfortunately, immunological toxicities have hindered 2-MPPA clinical translation. Targeted delivery of 2-MPPA specifically to activated microglia and astrocytes that over-express GCPII has the potential to mitigate glutamate excitotoxicity and attenuate neuroinflammation. In this study, we demonstrate that 2-MPPA when conjugated to generation-4, hydroxyl-terminated polyamidoamine (PAMAM) dendrimers (D-2MPPA) localize specifically in activated microglia and astrocytes only in newborn rabbits with cerebral palsy (CP), not in controls. D-2MPPA treatment led to higher 2-MPPA levels in the injured brain regions compared to 2-MPPA treatment, and the extent of D-2MPPA uptake correlated with the injury severity. D-2MPPA was more efficacious than 2-MPPA in decreasing extracellular glutamate level in ex vivo brain slices of CP kits, and in increasing transforming growth factor beta 1 (TGF-β1) level in primary mixed glial cell cultures. A single systemic intravenous dose of D-2MPPA on postnatal day 1 (PND1) decreased microglial activation and resulted in a change in microglial morphology to a more ramified form along with amelioration of motor deficits by PND5. These results indicate that targeted dendrimer-based delivery specifically to activated microglia and astrocytes can improve the efficacy of 2-MPPA by attenuating glutamate excitotoxicity and microglial activation.

Microglia-derived TNF-α contributes to RVLM neuronal mitochondrial dysfunction via blocking the AMPK–Sirt3 pathway in stress-induced hypertension

BackgroundNeuroinflammation in the rostral ventrolateral medulla (RVLM) has been associated with the pathogenesis of stress-induced hypertension (SIH). Neuronal mitochondrial dysfunction is involved in many pathological and physiological processes. However, the impact of neuroinflammation on neuronal mitochondrial homeostasis and the involved signaling pathway in the RVLM during SIH are largely unknown.MethodsThe morphology and phenotype of microglia and the neuronal mitochondrial injury in vivo were analyzed by immunofluorescence, Western blot, RT-qPCR, transmission electron microscopy, and kit detection. The underlying mechanisms of microglia-derived tumor necrosis factor‐α (TNF-α) on neuronal mitochondrial function were investigated through in vitro and in vivo experiments such as immunofluorescence and Western blot. The effect of TNF-α on blood pressure (BP) regulation was determined in vivo via intra-RVLM microinjection of TNF-α receptor antagonist R7050.ResultsThe results demonstrated that BP, heart rate (HR), renal sympathetic nerve activity (RSNA), plasma norepinephrine (NE), and electroencephalogram (EEG) power increased in SIH rats. Furthermore, the branching complexity of microglia in the RVLM of SIH rats decreased and polarized into M1 phenotype, accompanied by upregulation of TNF‐α. Increased neuronal mitochondria injury was observed in the RVLM of SIH rats. Mechanistically, Sirtuin 3 (Sirt3) and p-AMPK expression were markedly downregulated in both SIH rats and TNF-α–treated N2a cells. AMPK activator A769662 upregulated AMPK–Sirt3 signaling pathway and consequently reversed TNF-α–induced mitochondrial dysfunction. Microinjection of TNF-α receptor antagonist R7050 into the RVLM of SIH rats significantly inhibited the biological activities of TNF-α, increased p‐AMPK and Sirt3 levels, and alleviated neuronal mitochondrial injury, thereby reducing c-FOS expression, RSNA, plasma NE, and BP.ConclusionsThis study revealed that microglia-derived TNF-α in the RVLM impairs neuronal mitochondrial function in SIH possibly through inhibiting the AMPK–Sirt3 pathway. Therefore, microglia-derived TNF-α in the RVLM may be a possible therapeutic target for the intervention of SIH.