Morphological Changes In Microglia Research Articles

Neuroprotection of Retinal Ganglion Cells Suppresses Microglia Activation in a Mouse Model of Glaucoma.

Microglial activation has been implicated in many neurodegenerative eye diseases, but the interrelationship between cell loss and microglia activation remains unclear. In glaucoma, there is no consensus yet whether microglial activation precedes or is a consequence of retinal ganglion cell (RGC) degeneration. We therefore investigated the temporal and spatial appearance of activated microglia in retina and their correspondence to RGC degeneration in glaucoma. We used an established microbead occlusion model of glaucoma in mouse whereby intraocular pressure (IOP) was elevated. Specific antibodies were used to immunolabel microglia in resting and activated states. To block retinal gap junction (GJ) communication, which has been shown previously to provide significant neuroprotection of RGCs, the GJ blocker meclofenamic acid was administered or connexin36 (Cx36) GJ subunits were ablated genetically. We then studied microglial activation at different time points after microbead injection in control and neuroprotected retinas. Histochemical analysis of flatmount retinas revealed major changes in microglia morphology, density, and immunoreactivity in microbead-injected eyes. An early stage of microglial activation followed IOP elevation, as indicated by changes in morphology and cell density, but preceded RGC death. In contrast, the later stage of microglia activation, associated with upregulation of major histocompatibility complex class II expression, corresponded temporally to the initial loss of RGCs. However, we found that protection of RGCs afforded by GJ blockade or genetic ablation largely suppressed microglial changes at all stages of activation in glaucomatous retinas. Together, our data strongly suggest that microglia activation in glaucoma is a consequence, rather than a cause, of initial RGC degeneration and death.

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.

Morphofunctional changes of microglia in adult and old Wistar rats

Alzheimer's disease (AD) is one of the most prevalent neurodegenerative diseases leading to dementia. There is no effective treatments for this disease so far, as well as a consensus concerning the mechanisms of its pathogenesis initiation. Obtaining data on them in vivo is possible only by modeling neurodegeneration in laboratory animals. Alzheimer’s disease (AD) is one of the most prevalent neurodegenerative diseases leading to dementia. There is no effective treatments for this disease so far, as well as a consensus concerning the mechanisms of its pathogenesis initiation. Obtaining data on them in vivo is possible only by modeling neurodegeneration in laboratory animals. Among the various theories of the initiation of neurodegeneration, the impact of microglia is vigorously studied recently, as well as inflammaging, which is a term for chronic age-related low-grade systemic inflammation. It manifests in the increasing number of senescent cells with senescence-associated secretory phenotype (SASP). Eventually, it leads to manifestation and progression of age-related diseases, such as AD. The aim of the study was to evaluate age-related changes in microglia, pro- and anti-inflammatory cytokines expression levels in the brain, as well as ones of microglial activation, and also subpopulations of lymphocytes in peripheral blood. We used male Wistar rats of two age groups, which were composed of old (age 24 months) and adult (age 3 months) rodents, without any additional exposure. In the hippocampus, morphological changes in microglia were assessed on preparations stained with antibodies to Iba1. In the prefrontal cortex, RT-qPCR was used to study the level of expression of pro-inflammatory IL-6 and TNFa, anti-inflammatory IL-10 and TGF-b cytokines, as well as microglial activation markers iNOS and MMP-9. In the peripheral blood, the relative numbers of the main subpopulations of lymphocytes and monocyte were measured by flow cytometry. It was shown that, compared with adult rats, old animals are characterized by significant changes in the morphology of microglia, an increase in the level of expression of pro-inflammatory and a decrease in anti-inflammatory cytokines, and an increase in microglia activation markers. With aging, a decrease in the percentage of monocytes and B cells in peripheral blood was observed. These data indicate the development of inflammaging, which displays itself in microglia activation, a shift in the balance of cytokine production towards pro-inflammatory ones, and, as a result, activation of the migration of monocytes and B lymphocytes from the blood into tissues. Thus, it is justified to study the role of inflammation in the development of AD in old animals whose physiological state corresponds to that in humans. Further research in this area will expand the understanding of the mechanisms of initiation and progression of neurodegeneration, which is necessary for the development of novel and effective therapeutic approaches to the treatment of AD.

Ovariectomy in mice primes hippocampal microglia to exacerbate behavioral sickness responses

Estrogens are a group of steroid hormones that promote the development and maintenance of the female reproductive system and secondary sex characteristics. Estrogens also modulate immune responses; estrogen loss at menopause increases the risk of inflammatory disorders. Elevated inflammatory responses in the brain can lead to affective behavioral changes, which are characteristic of menopause. Thus, here we examined whether loss of estrogens sensitizes microglia, the primary innate immune cell of the brain, leading to changes in affective behaviors. To test this question, adult C57BL/6 mice underwent an ovariectomy to remove endogenous estrogens and then received estradiol hormone replacement or vehicle. After a one-month recovery, mice received an immune challenge with lipopolysaccharide (LPS) or vehicle control treatment and underwent behavioral testing. Ovariectomized, saline-treated mice exhibited reduced social investigation compared to sham-operated mice. Furthermore, ovariectomized mice that received LPS exhibited an exacerbated decrease in sucrose preference, which was ameliorated by estradiol replacement. These results indicate that ovariectomy modulates affective behaviors at baseline and in response to an inflammatory challenge. Ovariectomy-related behavioral changes were associated with downregulation of Cx3cr1, a microglial receptor that limits activation, suggesting that estrogen loss can disinhibit microglia to immune stimuli. Indeed, estradiol treatment reduced ovariectomy-induced increases in Il1b and Il6 expression after an immune challenge. Changes in microglial reactivity following ovariectomy are likely subtle, as overt changes in microglial morphology (e.g., soma size and branching) were limited. Collectively, these results suggest that a lack of estrogens may allow microglia to confer exaggerated neuroimmune responses, thereby raising vulnerability to adverse affective- and sickness-related behavioral changes.

Microbial modulation via cross-fostering prevents the effects of pervasive environmental stressors on microglia and social behavior, but not the dopamine system.

Environmental toxicant exposure, including air pollution, is increasing worldwide. However, toxicant exposures are not equitably distributed. Rather, low-income and minority communities bear the greatest burden, along with higher levels of psychosocial stress. Both air pollution and maternal stress during pregnancy have been linked to neurodevelopmental disorders such as autism, but biological mechanisms and targets for therapeutic intervention remain poorly understood. We demonstrate that combined prenatal exposure to air pollution (diesel exhaust particles, DEP) and maternal stress (MS) in mice induces social behavior deficits only in male offspring, in line with the male bias in autism. These behavioral deficits are accompanied by changes in microglial morphology and gene expression as well as decreased dopamine receptor expression and dopaminergic fiber input in the nucleus accumbens (NAc). Importantly, the gut-brain axis has been implicated in ASD, and both microglia and the dopamine system are sensitive to the composition of the gut microbiome. In line with this, we find that the composition of the gut microbiome and the structure of the intestinal epithelium are significantly shifted in DEP/MS-exposed males. Excitingly, both the DEP/MS-induced social deficits and microglial alterations in males are prevented by shifting the gut microbiome at birth via a cross-fostering procedure. However, while social deficits in DEP/MS males can be reversed by chemogenetic activation of dopamine neurons in the ventral tegmental area, modulation of the gut microbiome does not impact dopamine endpoints. These findings demonstrate male-specific changes in the gut-brain axis following DEP/MS and suggest that the gut microbiome is an important modulator of both social behavior and microglia.

P2X7 receptor of microglia in olfactory bulb mediates the pathogenesis of olfactory dysfunction in a mouse model of allergic rhinitis.

The pathogenesis of allergic rhinitis (AR)-related olfactory dysfunction (OD) remains unknown. Inhibiting microglial response in olfactory bulb (OB) can ameliorate AR-related OD, but no precise targets have been available. In this study, we established a mouse model of ovalbumin (OVA)-induced AR and combined with the application of P2X7 receptor (P2X7R)-specific antagonists and cell culture in conditioned medium to investigate the role and mechanism of OB microglial P2X7R in AR-related OD. Serum IgE and IL-5 levels determined via ELISA and federated the number of nose-scratching to affirm the success of OVA-induced AR mouse model. Buried food pellet test was used to evaluate the olfactory function of mice. The changes of IBA1, GFAP, P2X7R, IL-1β, IL-1Ra, and CASPASE 1 were detected by quantitative polymerase chain reaction and western blotting. The levels of adenosine triphosphate (ATP) were determined by the commercialized kit. The morphological changes of microglia were assessed using immunofluorescence staining and Sholl analysis. Findings showed that AR-related OD was associated with OB microglia-mediated imbalance between IL-1β and IL-1Ra. Treatment with BBG improved the olfactory function in AR mice with restoring the balance between IL-1β and IL-1Ra. In vitro, the conditioned medium obtained after HNEpC treatment with Der p1 could activate HMC3 to arise inflammatory reaction basing on "ATP-P2X7R-Caspase 1" axis, while inhibition of its P2X7R suppressed the reaction. In brief, microglial P2X7R in OB is a direct effector molecule in AR-related OD and inhibition of it may be a new strategy for the treatment of AR-related OD.

Featured Research Images

Featured on this issue's front and back covers, we have "Mast Cell Degranulation in Response to Rho Activating Drugs" created by Juliette Eshleman and "Galaxies of Glia Interface with Technology" created by Matthew Birtle. Other featured images include "Astrocytes: the stars of the brain" by Katherine Souter, "Morphological changes in microglia upon sensing the environment" by Kira Sviderskaia, "The Solar Farming Revolution: Agrivoltaics is Changing the Game" by Camila Quiroz, "AW1, a novel Xanthomonas axonopodis pv. vasculorum bacteriophage" by Ashlyn Waters, "Golden Canola" by Sabrina Strelkov, and "Augmented Reality x Rehabilitation" by Annie Wei.

Bergapten alleviates depression-like behavior by inhibiting cyclooxygenase 2 activity and NF-κB/MAPK signaling pathway in microglia

Major depressive disorder (MDD) is a common psychiatric disorder that severely affects human life and health. However, the pathological mechanism of MDD is unclear, and effective treatment strategies are urgently needed. Microglia-mediated neuroinflammation is closely associated with the pathophysiology of depression. Bergapten (BG) is a natural pharmaceutical monomer with anti-inflammatory effects; however, its role in neuroinflammation and depression remains unclear. In this study, we employed a lipopolysaccharide (LPS) injection-induced acute depression mouse model, and found that treatment with BG significantly alleviated LPS-induced depression-like behavior in mice. BG administration largely decreased the increase in microglial numbers and rescued the microglial morphological changes induced by LPS injection. Furthermore, transcriptomic changes revealed a protective role of BG in the hippocampus of mice. Mechanistically, we found that BG directly inhibited cyclooxygenase 2 (COX2) activity, and suppressed nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways in microglia. Together, these results highlight the important role of BG in microglial activation, neuroinflammation, and depression-like behavior, thus providing a new candidate drug for depression treatment.

Degradation of NLRP3 by p62-dependent-autophagy improves cognitive function in Alzheimer's disease by maintaining the phagocytic function of microglia.

Activation of the NLRP3 inflammasome promotes microglia to secrete inflammatory cytokines and induce pyroptosis, leading to impaired phagocytic and clearance functions of microglia in Alzheimer's disease (AD). This study found that the autophagy-associated protein p62 interacts with NLRP3, which is the rate-limiting protein of the NLRP3 inflammasome. Thus, we aimed to prove that the degradation of NLRP3 occurs through the autophagy-lysosome pathway (ALP) and also demonstrate its effects on the function of microglia and pathological changes in AD. The 5XFAD/NLRP3-KO mouse model was established to study the effect of NLRP3 reduction on AD. Behavioral experiments were conducted to assess the cognitive function of the mice. In addition, immunohistochemistry was used to evaluate the deposition of Aβ plaques and morphological changes in microglia. BV2 cells treated with lipopolysaccharide (LPS) followed by Aβ1-42 oligomers were used as in vitro AD inflammation models and transfected with lentivirus to regulate the expression of the target protein. The pro-inflammatory status and function of BV2 cells were detected by flow cytometry and immunofluorescence (IF). Co-immunoprecipitation, mass spectrometry, IF, Western blot (WB), quantitative real-time PCR, and RNA-seq analysis were used to elucidate the mechanisms of molecular regulation. Cognitive function was improved in the 5XFAD/NLRP3-KO mouse model by reducing the pro-inflammatory response of microglia and maintaining the phagocytic and clearance function of microglia to the deposited Aβ plaque. The pro-inflammatory function and pyroptosis of microglia were regulated by NLRP3 expression. Ubiquitinated NLRP3 can be recognized by p62 and degraded by ALP, slowing down the proinflammatory function and pyroptosis of microglia. The expression of autophagy pathway-related proteins such as LC3B/A, p62 was increased in the AD model in vitro. P62 recognizes and binds to ubiquitin-modified NLRP3. It plays a vital role in regulating the inflammatory response by participating in ALP-associated NLRP3 protein degradation, which improves cognitive function in AD by reducing the pro-inflammatory status and pyroptosis of microglia, thus maintaining its phagocytic function.

Spatiotemporal Cofilin Signaling, Microglial Activation, Neuroinflammation, and Cognitive Impairment Following Hemorrhagic Brain Injury.

Intracerebral hemorrhage (ICH) is a significant health concern associated with high mortality. Cofilin plays a crucial role in stress conditions, but its signaling following ICH in a longitudinal study is yet to be ascertained. In the present study, we examined the cofilin expression in human ICH autopsy brains. Then, the spatiotemporal cofilin signaling, microglia activation, and neurobehavioral outcomes were investigated in a mouse model of ICH. Human autopsy brain sections from ICH patients showed increased intracellular cofilin localization within microglia in the perihematomal area, possibly associated with microglial activation and morphological changes. Various cohorts of mice were subjected to intrastriatal collagenase injection and sacrificed at time points of 1, 3, 7, 14, 21, and 28 days. Mice suffered from severe neurobehavioral deficits after ICH, lasting for 7 days, followed by a gradual improvement. Mice suffered post-stroke cognitive impairment (PSCI) both acutely and in the chronic phase. Hematoma volume increased from day 1 to 3, whereas ventricle size increased from day 21 to 28. Cofilin protein expression increased in the ipsilateral striatum on days 1 and 3 and then decreased from days 7 to 28. An increase in activated microglia was observed around the hematoma on days 1 to 7, followed by a gradual reduction up to day 28. Around the hematoma, activated microglia showed morphological changes from ramified to amoeboid. mRNA levels of inflammatory [tumor necrosis factor-α (TNF-α), interleukin 1β (IL-1β), and interleukin-6 (IL-6) and anti-inflammatory markers [interleukin-10 (IL-10), transforming growth factor-β TGF-β, and arginase I (Arg1)] increased during the acute phase and decreased in the chronic phase. Blood cofilin levels increased on day 3 and matched the increase in chemokine levels. slingshot protein phosphatase 1 (SSH1) protein, which activates cofilin, was increased from day 1 to 7. These results suggest that microglial activation might be the sequel of cofilin overactivation following ICH, leading to widespread neuroinflammation and consequent PSCI.

Dynamic changes in microglia in the mouse hippocampus during administration and withdrawal of the CSF1R inhibitor PLX3397.

Pexidartinib (PLX3397), a colony-stimulating factor-1 receptor (CSF1R) inhibitor, is currently in phase 1-3 clinical trials as a treatment for a variety of tumours. CSF1R signalling regulates the development, survival and maintenance of microglia, the resident brain innate immune cells. In this study, we examined the effects of PLX3397 in the drinking water of mice on microglia in the hippocampus using ionized calcium-binding adapter molecule 1 (Iba1, a microglial marker) immunocytochemistry. A high concentration of PLX3397 (1 mg/mL) significantly decreased the density of Iba1-immunoreactive cells after 7 days of exposure, but a low concentration of PLX3397 (0.5 mg/mL) did not. In addition, both low and high concentrations of PLX3397 significantly increased the intersection number, total length and maximum length of microglial processes in male mice. PLX3397 administered for 21 days eliminated microglia with 78% efficiency in males and 84% efficiency in females. Significant increases in microglial processes were found after both seven and 21 days of PLX3397 exposure in males, whereas decreases in microglial processes were observed after both 14 and 21 days of exposure in females. After PLX3397 withdrawal following its administration for 14 days in males, the soma size quickly returned to normal levels within a week. However, the microglial density, intersection number and total length of microglial processes after 3 days of recovery stabilized to untreated levels. In summary, these findings provide detailed insight into the dynamic changes in microglial number and morphology in the hippocampus in a dose- and time-dependent manner after PLX3397 treatment and withdrawal.

Gut-derived bacterial LPS attenuates incubation of methamphetamine craving via modulating microglia

BackgroundThe microbiota-gut-brain axis plays a critical role in the pathophysiology of neuropsychiatric disorders, and the compositions of gut microbiota are altered by addictive drugs. However, the role of gut microbiota in the incubation of methamphetamine (METH) craving remains poorly understood. Methods16S rRNA gene sequencing was performed to assess the richness and diversity of gut microbiota in METH self-administration model. Hematoxylin and eosin staining was performed to evaluate the integrity of intestinal barrier. Immunofluorescence and three-dimensional reconstruction were performed to assess the morphologic changes of microglia. Serum levels of lipopolysaccharide (LPS) were determined using the rat enzyme-linked immunosorbent assay kits. Quantitative real-time PCR was performed to assess transcript levels of dopamine receptor, glutamate ionotropic AMPA receptor 3 and brain-derived neurotrophic factor. ResultsMETH self-administration induced gut microbiota dysbiosis, intestinal barrier damage and microglia activation in the nucleus accumbens core (NAcc), which was partially recovered after prolonged withdrawal. Microbiota depletion via antibiotic treatment increased LPS levels and induced a marked change in the microglial morphology in the NAcc, as indicated by the decreases in the lengths and numbers of microglial branches. Depleting the gut microbiota also prevented the incubation of METH craving and increased the population of Klebsiella oxytoca. Furthermore, Klebsiella oxytoca treatment or exogenous administration of the gram-negative bacterial cell wall component LPS increased serum and central LPS levels, induced microglial morphological changes and reduced the dopamine receptor transcription in the NAcc. Both treatments and NAcc microinjections of gut-derived bacterial LPS significantly decreased METH craving after prolonged withdrawal. ConclusionsThese data suggest that LPS from gut gram-negative bacteria may enter circulating blood, activate microglia in the brain and consequently decrease METH craving after withdrawal, which may have important implications for novel strategies to prevent METH addiction and relapse.

Lithium Chloride Exerts Anti-Inflammatory and Neuroprotective Effects by Inhibiting Microglial Activation in LPS-Induced Retinal Injury.

To explore the anti-inflammatory and neuroprotective effects of lithium chloride (LiCl) in LPS-induced retinal injury. In vitro, primary retinal microglia were pretreated with LiCl and stimulated with lipopolysaccharide (LPS). Pro-inflammatory cytokine production, microglial morphological changes, and inflammation-associated signaling pathways were measured by real-time PCR (RT-PCR), western blotting, and immunofluorescence. Primary retinal neurons were cultured with microglial-derived conditioned medium in the absence or presence of LiCl. Neurotoxicity was evaluated by Cell Counting Kit-8 (CCK-8), terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay, and γ-H2AX detection. In vivo, an endotoxin-induced uveitis mice model was established, and each animal was given intraperitoneal injection of LiCl or vehicle. The retinal inflammatory response was measured by hematoxylin and eosin and fluorescent staining, RT-PCR, western blotting, and TUNEL assay. Retinal thickness and function were evaluated by spectral-domain optical coherence tomography and electroretinography. In vitro, LiCl exerted no obvious toxic effects on microglia and significantly decreased proinflammatory factor (inducible nitric oxide synthase, tumor necrosis factor α, interleukin 6) production, inhibited microglial activation in morphology, and suppressed nuclear factor kappa B (NF-κB), Akt, and phosphatidylinositol 3-kinase (PI3K) phosphorylation. Moreover, LiCl promoted retinal neuron survival and reduced cell apoptosis and the expression of γ-H2AX. In vivo, LiCl reduced inflammatory infiltrating cells in the vitreous cavity and decreased proinflammatory cytokine expression in retinas. LiCl suppressed LPS-induced microglial activation, proliferation, and migration. Additionally, LiCl reduced LPS-induced apoptosis of ganglion cells and retinal edema and rescued retinal functional damage. This study demonstrates that LiCl exerts anti-inflammatory and neuroprotective effects by inhibiting microglial activation via the PI3K/Akt/NF-κB pathway in LPS-induced retinal injury. LiCl provides a novel and promising option to treat retinal inflammatory diseases.

Microglial Cytokines Mediate Plasticity Induced by 10 Hz Repetitive Magnetic Stimulation.

Microglia, the resident immune cells of the CNS, sense the activity of neurons and regulate physiological brain functions. They have been implicated in the pathology of brain diseases associated with alterations in neural excitability and plasticity. However, experimental and therapeutic approaches that modulate microglia function in a brain region-specific manner have not been established. In this study, we tested for the effects of repetitive transcranial magnetic stimulation (rTMS), a clinically used noninvasive brain stimulation technique, on microglia-mediated synaptic plasticity; 10 Hz electromagnetic stimulation triggered a release of plasticity-promoting cytokines from microglia in mouse organotypic brain tissue cultures of both sexes, while no significant changes in microglial morphology or microglia dynamics were observed. Indeed, substitution of tumor necrosis factor α (TNFα) and interleukin 6 (IL6) preserved synaptic plasticity induced by 10 Hz stimulation in the absence of microglia. Consistent with these findings, in vivo depletion of microglia abolished rTMS-induced changes in neurotransmission in the mPFC of anesthetized mice of both sexes. We conclude that rTMS affects neural excitability and plasticity by modulating the release of cytokines from microglia.SIGNIFICANCE STATEMENT Repetitive transcranial magnetic stimulation (rTMS) is a noninvasive brain stimulation technique that induces cortical plasticity. Despite its wide use in neuroscience and clinical practice (e.g., depression treatment), the cellular and molecular mechanisms of rTMS-mediated plasticity remain not well understood. Herein, we report an important role of microglia and plasticity-promoting cytokines in synaptic plasticity induced by 10 Hz rTMS in organotypic slice cultures and anesthetized mice, thereby identifying microglia-mediated synaptic adaptation as a target of rTMS-based interventions.

Protective effect of Nr4a2 (Nurr1) against LPS-induced depressive-like behaviors via regulating activity of microglia and CamkII neurons in anterior cingulate cortex

Neuroinflammation is tightly associated with onset of depression. The nuclear receptor related 1 protein (Nurr1, also called Nr4a2), its roles in dopaminergic neurons is well understood, which can alleviate inflammation. Nevertheless, potential effects of Nr4a2 on neuroinflammation associated with depression still remains unclear. Chronic lipopolysaccharides (LPS) stress induced depressive-behaviors were confirmed via behavioral tests. Differentially expressed genes were detected by using RNA-sequencing. The anterior cingulate cortex (ACC) tissues were collected for biochemical experiments. The Golgi-Cox staining and virus labeling were used to evaluate the dendritic spines. We applied fluoxetine (FLX) and amodiaquine dihydrochloride (AQ, a highly selective agonist of Nr4a2) in mice. Overexpression experiments were performed by injecting with AAV-Nr4a2-EGFP into ACC. Chemogenetic activation of CamkII neurons via injecting the hM3Dq virus. Mice treated with LPS displayed depressive- and anxiety-like behaviors. The reduction of Nr4a2 and FosB induced by LPS were rescued by pretreatment with FLX or AQ. More importantly, LPS-induced behavior deficits in mice were also alleviated via fluoxetine treatment and pharmacological activation the expression of Nr4a2. Meanwhile, enhancing the level of Nr4a2 could improve dendritic spines loss of neuron and morphological changes in microglia. Overexpression of Nr4a2 in ACC reversed the depressive- and anxiety-like behaviors caused by LPS administration. Activation of CamkII neurons in ACC could robustly increase the expression of Nr4a2 and improve LPS-induced behavior deficits. Our findings demonstrate that the Nr4a2 may regulate depressive-like behaviors via alleviating the impairment of morphology and function on microglia and CamkII neurons induced by chronic neuroinflammation.

Endometriosis leads to central nervous system-wide glial activation in a mouse model of endometriosis

BackgroundChronic pelvic pain (CPP) is a common symptom of endometriosis. Women with endometriosis are also at a high risk of suffering from anxiety, depression, and other psychological disorders. Recent studies indicate that endometriosis can affect the central nervous system (CNS). Changes in the functional activity of neurons, functional magnetic resonance imaging signals, and gene expression have been reported in the brains of rat and mouse models of endometriosis. The majority of the studies thus far have focused on neuronal changes, whereas changes in the glial cells in different brain regions have not been studied.MethodsEndometriosis was induced in female mice (45-day-old; n = 6–11/timepoint) by syngeneic transfer of donor uterine tissue into the peritoneal cavity of recipient animals. Brains, spines, and endometriotic lesions were collected for analysis at 4, 8, 16, and 32 days post-induction. Sham surgery mice were used as controls (n = 6/timepoint). The pain was assessed using behavioral tests. Using immunohistochemistry for microglia marker ionized calcium-binding adapter molecule-1 (IBA1) and machine learning “Weka trainable segmentation” plugin in Fiji, we evaluated the morphological changes in microglia in different brain regions. Changes in glial fibrillary acidic protein (GFAP) for astrocytes, tumor necrosis factor (TNF), and interleukin-6 (IL6) were also evaluated.ResultsWe observed an increase in microglial soma size in the cortex, hippocampus, thalamus, and hypothalamus of mice with endometriosis compared to sham controls on days 8, 16, and 32. The percentage of IBA1 and GFAP-positive area was increased in the cortex, hippocampus, thalamus, and hypothalamus in mice with endometriosis compared to sham controls on day 16. The number of microglia and astrocytes did not differ between endometriosis and sham control groups. We observed increased TNF and IL6 expression when expression levels from all brain regions were combined. Mice with endometriosis displayed reduced burrowing behavior and hyperalgesia in the abdomen and hind-paw.ConclusionWe believe this is the first report of central nervous system-wide glial activation in a mouse model of endometriosis. These results have significant implications for understanding chronic pain associated with endometriosis and other issues such as anxiety and depression in women with endometriosis.Graphical

Dlg1 deletion in microglia ameliorates chronic restraint stress induced mice depression-like behavior.

Background: Major depression is one of the most common psychiatric disorders worldwide, inflicting suffering, significant reduction in life span, and financial burdens on families and society. Mounting evidence implicates that exposure to chronic stress can induce the dysregulation of the immune system, and the activation of brain-resident innate immune cells, microglia, leading to depression-like symptoms. However, the specific mechanisms need to be further elucidated. Method: Animal models of depression were established by chronic restraint stress (CRS), and depression-like behavior was assessed by sucrose preference test (SPT), open field test (OFT), tail suspension test (TST) and forced swimming test (FST). Microglial activation was visualized by immunofluorescent and immunohistochemical staining, and microglial morphological changes were further analyzed by skeleton analysis. The levels of inflammatory cytokines were detected by western blotting and qPCR. Result: Microglial Dlg1 knockout ameliorates CRS-induced mice depression-like behavior. In contrast to the effect of Dlg1 in the LPS-induced mouse model, Dlg1 knockout had little effect on microglial density, but significantly decreased the number of activated microglia and reversed microglia morphological changes in mice challenged with CRS. Moreover, the upregulation of inflammatory cytokines following CRS exposure was partially reversed by Dlg1 deletion. Conclusion: Our study provides the evidence that Dlg1 ablation in microglia remarkedly reverses microglial activation and depression-like behavior in mice exposed to CRS, implicating a potential target for the treatment of clinical depression.

CB2R activation ameliorates late adolescent chronic alcohol exposure-induced anxiety-like behaviors during withdrawal by preventing morphological changes and suppressing NLRP3 inflammasome activation in prefrontal cortex microglia in mice

BackgroundChronic alcohol exposure (CAE) during late adolescence increases the risk of anxiety development. Alcohol-induced prefrontal cortex (PFC) microglial activation, characterized by morphological changes and increased associations with neurons, plays a critical role in the pathogenesis of anxiety. Alcohol exposure increases NLRP3 inflammasome expression, increasing cytokine secretion by activated microglia. Cannabinoid type 2 receptor (CB2R), an essential receptor of the endocannabinoid system, regulates microglial activation and neuroinflammatory reactions. We aimed to investigate the role of CB2R activation in ameliorating late adolescent CAE-induced anxiety-like behaviors and microglial activation in C57BL/6J mice. MethodsSix-week-old C57BL/6J mice were acclimated for 7 days and then were administered alcohol by gavage (4 g/kg, 25 % w/v) for 28 days. The mice were intraperitoneally injected with the specific CB2R agonist AM1241 1 h before alcohol treatment. Anxiety-like behaviors during withdrawal were assessed by open field test and elevated plus maze test 24 h after the last alcohol administration. Microglial activation, microglia-neuron interactions, and CB2R and NLRP3 inflammasome-related molecule expression in the PFC were measured using immunofluorescence, immunohistochemical, qPCR, and Western blotting assays. Microglial morphology was evaluated by Sholl analysis and the cell body-to-total cell size index. Additionally, N9 microglia were activated by LPS in vitro, and the effects of AM1241 on NLRP3 and N9 microglial activation were investigated. ResultsAfter CAE, mice exhibited severe anxiety-like behaviors during withdrawal. CAE induced obvious microglia-neuron associations, and increased expression of microglial activation markers, CB2R, and NLRP3 inflammasome-related molecules in the PFC. Microglia also showed marked filament retraction and reduction and cell body enlargement after CAE. AM1241 treatment ameliorated anxiety-like behaviors in CAE model mice, and it prevented microglial morphological changes, reduced microglial activation marker expression, and suppressed the microglial NLRP3 inflammasome activation and proinflammatory cytokine secretion induced by CAE. AM1241 suppressed the LPS-induced increase in NLRP3 inflammasome-related molecules, IL-1β release, and M1 phenotype markers (iNOS and CD86) in N9 cell, which was reversed by CB2R antagonist treatment. ConclusionsCAE caused anxiety-like behaviors in late adolescent mice at least partly by inducing microglial activation and increasing microglia-neuron associations in the PFC. CB2R activation ameliorated these effects by preventing morphological changes and suppressing NLRP3 inflammasome activation in PFC microglia.

Novel image analysis tool for rapid screening of cell morphology in preclinical animal models of disease

The field of cell biology has seen major advances in both cellular imaging modalities and the development of automated image analysis platforms that increase rigor, reproducibility, and throughput for large imaging data sets. However, there remains a need for tools that provide accurate morphometric analysis of single cells with complex, dynamic cytoarchitecture in a high-throughput and unbiased manner. We developed a fully automated image-analysis algorithm to rapidly detect and quantify changes in cellular morphology using microglia cells, an innate immune cell within the central nervous system, as representative of cells that exhibit dynamic and complex cytoarchitectural changes. We used two preclinical animal models that exhibit robust changes in microglia morphology: (1) a rat model of acute organophosphate intoxication, which was used to generate fluorescently labeled images for algorithm development; and (2) a rat model of traumatic brain injury, which was used to validate the algorithm using cells labeled using chromogenic detection methods. All ex vivo brain sections were immunolabeled for IBA-1 using fluorescence or diaminobenzidine (DAB) labeling, images were acquired using a high content imaging system and analyzed using a custom-built algorithm. The exploratory data set revealed eight statistically significant and quantitative morphometric parameters that distinguished between phenotypically distinct groups of microglia. Manual validation of single-cell morphology was strongly correlated with the automated analysis and was further supported by a comparison with traditional stereology methods. Existing image analysis pipelines rely on high-resolution images of individual cells, which limits sample size and is subject to selection bias. However, our fully automated method integrates quantification of morphology and fluorescent/chromogenic signals in images from multiple brain regions acquired using high-content imaging. In summary, our free, customizable image analysis tool provides a high-throughput, unbiased method for accurately detecting and quantifying morphological changes in cells with complex morphologies.

The association of astrogliosis and microglial activation with aging and Alzheimer's disease pathology in the chimpanzee brain.

Aging and neurodegenerative disorders, such as Alzheimer's disease (AD), trigger an immune response known as glial activation in the brain. Recent evidence indicates species differences in inflammatory responses to AD pathology, highlighting the need for additional comparative studies to further understand human-specific neuropathologies. In the present study, we report on the occurrence of astrogliosis, microglial activation, and their relationship with age and AD-like pathology in a cohort of male and female chimpanzees (Pan troglodytes). Chimpanzees with severe astrogliosis exhibited widespread upregulation of hypertrophic astrocytes immunoreactive for glial fibrillary acidic protein (GFAP) throughout all layers of the dorsolateral prefrontal cortex and a loss of the interlaminar palisade. In addition, extreme astrogliosis was associated with increased astrocyte density in the absence of significant microglial activation and AD lesions. A shift from decreased resting to increased phagocytotic microglia occurred with aging, although proliferation was absent and no changes in astrogliosis was observed. Vascular amyloid correlated with decreased astrocyte and microglia densities, while tau lesions were associated with morphological changes in microglia and greater total glia density and glia: neuron ratio. These results further our understanding of inflammatory processes within the chimpanzee brain and provide comparative data to improve our understanding of human aging and neuropathological processes.