Number Of Microglia Research Articles

Hydroxytyrosol-Rich Olive Mill Wastewater, a Potential Protector Against Dyslipidemia, Diabetes, and Diabetic Retinopathy in Psammomys obesus.

Olive mill wastewater (OMWW), a byproduct of olive oil extraction, constitutes a natural resource of phenolic compounds. Hydroxytyrosol (HT), the predominant compound, was reported to have antioxidant, anti-inflammatory, and neuroprotective effects. This research aims to evaluate the effect of OMWW bioproduct rich in HT on retinal glial function, glutamate metabolism, and synaptic transmission alterations mediated by hyperglycemia and dyslipidemia in high-calorie diet (HCD)-induced diabetic retinopathy (DR) in Psammomys obesus. Animals were divided into four groups. Two diabetic animal groups (D) received an HCD, one untreated (D) and another receiving HT-OMWW treatment (20mg/kg body weight: bw) (D+); the two other groups were used as controls (C and C+). During 7 months, food and water intake, body weight, glycemia, hematocrit, and serum lipid parameters were assessed. At 3, 5, and advanced 7 months of DR, immunohistochemical studies were performed to identify key proteins implicated in the protection of DR. HT-OMWW has anti-obesity, hypoglycemic, and hypolipidemic effects. Its long-term administration attenuates retinal glial reactivity, microglia number, changes in glutamate homeostasis, and synaptic function in diabetic animals with retinopathy. These results suggest that HT-OMWW extract seems to have promising in vivo anti-diabetic, anti-dyslipidemic, and neuroprotective effects in P. obesus, a model of DR-like humans.

S100A9 protein activates microglia and stimulates phagocytosis, resulting in synaptic and neuronal loss.

S100 calcium-binding protein A9 (S100A9, also known as calgranulin B) is expressed and secreted by myeloid cells under inflammatory conditions, and S100A9 can amplify inflammation. There is a large increase in S100A9 expression in the brains of patients with neurodegenerative diseases, such as Alzheimer's disease, and S100A9 has been suggested to contribute to neurodegeneration, but the mechanisms are unclear. Here we investigated the effects of extracellular recombinant S100A9 protein on microglia, neurons and synapses in primary rat brain neuronal-glial cell cultures. Incubation of cell cultures with 250-500 nM S100A9 caused neuronal loss without signs of apoptosis or necrosis, but accompanied by exposure of the "eat-me" signal - phosphatidylserine on neurons. S100A9 caused activation of microglial inflammation as evidenced by an increase in the microglial number, morphological changes, release of pro-inflammatory cytokines, and increased phagocytic activity. At lower concentrations, 10-100 nM S100A9 induced synaptic loss in the cultures. Depletion of microglia from the cultures prevented S100A9-induced neuronal and synaptic loss, indicating that neuronal and synaptic loss was mediated by microglia. These results suggest that extracellular S100A9 may contribute to neurodegeneration by activating microglial inflammation and phagocytosis, resulting in loss of synapses and neurons. This further suggests the possibility that neurodegeneration may be reduced by targeting S100A9 or microglia.

Texture analysis of optical coherence tomography retinal images: Its potential use for the early diagnosis of diabetic retinopathy

Aims/Purpose: Diabetic retinopathy (DR) is usually diagnosed many years after diabetes onset. The early diagnosis of DR remains challenging, and identifying novel biomarkers is crucial. We aim to identify novel early biomarkers for DR, based on texture analysis of optical coherence tomography (OCT) retinal images.Methods: A type 1 diabetes (T1D) rat (Wistar) model (streptozotocin‐induced) was used. Volume OCT scans (N = 40‐44) and electroretinograms (N = 25) were acquired before and after 1, 2 and 4 weeks of diabetes. Automated OCT image segmentation was performed, followed by retinal thickness and texture analysis. Changes in blood‐retinal barrier (tight junctions and permeability), glial reactivity and nitrosative stress were assessed.Results: Early T1D induced significant changes in retinal texture. Several texture metrics, autocorrelation, correlation, homogeneity, information measure of correlation II (IMCII), inverse difference moment normalized (IDMN), inverse difference normalized (IDN), and sum average, decreased in all retinal layers at week 4. Correlation, homogeneity, IMCII, IDMN, and IDN decreased at week 2. T1D induced a progressive weekly reduction in the texture metrics. T1D also induced significant thinning of the inner plexiform and inner nuclear layers, inner/outer photoreceptor segments, and total retina, at weeks 1, 2 and 4. Impaired retinal function was also observed (increased latencies in a‐wave and oscillatory potentials at weeks 1, 2 and 4), as well as decreased tight junction proteins immunoreactivity and increased microglial cell number and nitrosative stress, but vascular permeability was unchanged.Conclusions: Early T1D impacts retinal texture, concomitant with molecular, cellular and physiological retinal changes, thus unlocking the potential of texture analysis for DR early diagnosis, but this needs further proof in clinical studies.Support: FCT, Portugal: 2020.07432.BD, PEst UIDB/04539/Base/2020 and UIDP/04539/Programatico/2020, and PEst UIDB/04950/Base/2020 and UIDP/04950/Programatico/2020.

Microglia stimulation produces antidepressant-like effects in a mouse depression model induced by adolescent chronic unpredictable stress.

Depression triggered by harmful stress in adolescents is a common phenomenon that can lead to serious social problems. Its treatment is still frustrating in the clinic. We reported the phenomenon that a 12-day chronic unpredictable stress (CUS), starting on postnatal day 28, led to a significant decrease in the number of microglia in the dentate gyrus of the hippocampus in adult mice. Reversal of this decline by a single injection of low-dose lipopolysaccharide (LPS), a classical immunostimulant, could rapidly reverse the depression-like behaviors induced by 12 days of CUS stimulation during adolescence. In the dose-dependent experiment, a single injection of LPS at doses of 75 and 100 μg/kg, but not at doses of 25 and 50 μg/kg, produced an antidepressant effect in mice exposed to 12-day CUS during adolescence. The time-dependent experiment showed that the antidepressant effect of the single LPS injection (100 μg/kg) occurred at time points 5 and 8 hours, but not 3 hours after LPS injection. The antidepressant effect of the single LPS injection (100 μg/kg) lasted for at least 7 days, and 14 days after the single LPS injection, a repeated injection could still induce the depressed mice to develop an antidepressant phenotype. Furthermore, inhibition of microglia by minocycline or depletion of microglia by PLX3397 was found to prevent the antidepressant effect of the single LPS injection. These results suggest that reversing the decline of microglia in the dentate gyrus may be a potential strategy for the treatment of depression induced by harmful stress in adolescents.

Intrauterine Growth Restriction Alters Postnatal Hippocampal Dentate Gyrus Neuron and Microglia Morphology and Cytokine/Chemokine Milieu in Mice.

Infants born with intrauterine growth restriction (IUGR) have up to a five-fold higher risk of learning and memory impairment than those with normal growth. Using a mouse model of hypertensive diseases of pregnancy (HDP) to replicate uteroplacental insufficiency (UPI), we have previously shown that UPI causes premature embryonic hippocampal dentate gyrus (DG) neurogenesis in IUGR offspring. The DG is a brain region that receives the first cortical information for memory formation. In the current study, we examined the postnatal DG neuron morphology one month after delivery (P28) using recombinant adeno-associated viral labeling of neurons. We also examined DG microglia's morphology using immunofluorescent histochemistry and defined the hippocampal cytokine/chemokine milieu using Luminex xMAP technology. We found that IUGR preserved the principal dendrite lengths but decreased the dendritic branching and volume of DG neurons. IUGR augmented DG microglial number and cell size. Lastly, IUGR altered the hippocampal cytokine/chemokine profile in a sex-specific manner. We conclude that the prematurely-generated neuronal progenitors develop abnormal morphologies postnatally in a cell-autonomous manner. Microglia appear to modulate neuronal morphology by interacting with dendrites amidst a complex cytokine/chemokine environment that could ultimately lead to adult learning and memory deficits in our mouse model.

The effects of developmental sub-chronic low-level lead exposure on microglia and a test of possible mitigation by apigenin in C57BL/6/J young mice

Developmental chronic low-level lead (Pb) exposure disrupts central nervous system function and diminishes neurocognition. Microglial cell activation might contribute to these deficits. The present study evaluated the effects of developmental sub-chronic low-level lead exposure on microglial cells and the possible effectiveness of a natural anti-inflammatory intervention with apigenin to mitigate these effects. From PND 0 to 28, 87 C57BL/6 J mice were exposed to one of six treatment conditions: 0 ppm Pb; 30 ppm Pb; 430 ppm Pb; 30 ppm Pb + 400 ppm apigenin; 430 ppm Pb + 400 ppm apigenin; or 400 ppm apigenin, via dam's drinking water. Following sacrifice, brain tissue was harvested and microglial cells were labeled via immunohistochemistry and counted within the dentate gyrus (DG) using unbiased stereology methods. It was hypothesized that developmental sub-chronic low-level lead exposure would increase microglial cell numbers within the DG and that apigenin treatment may mitigate these effects. A significant effect of treatment group was found and post-hoc analyses revealed that Pb-exposure generated an increased number of microglial cells as compared to controls. Interestingly, the 30 ppm Pb with apigenin treatment group did not generate microglial cell numbers different from the control group unexposed to Pb. These results suggested that developmental sub-chronic low-level lead exposure increased microglial cell activation within the DG and that, at low-levels of Pb exposure, apigenin treatment may mitigate these effects. These results provided the groundwork for studies that could identify an effective intervention to alleviate the effects of developmental chronic low-level lead exposure in child neurocognition.

IL-17A Mediates Depressive-Like Symptoms by Inducing Microglia Activation in Psoriasiform Dermatitis Mice.

Psoriasis is recognized as a systemic disease for its accompanying comorbidities, among which psychological disorders present a high incidence rate and affect patients' life quality. Interleukin (IL)-17A is the central pathological factor in the pathogenesis and development of psoriasis. To clarify if psoriasis-induced systemic IL-17A increase can mediate the neuronal inflammation and result in depressive-like symptoms. Psoriasiform dermatitis model was established by imiquimod (IMQ) application on male BALB/c mice and IL-17A intervention was performed by lateral ventricular catheterization. Skin structural, histopathological characteristics, and behavioral tests were assessed. Serum IL-17A levels were detected by Enzyme-linked immunosorbent assay. mRNA expression of pro-inflammatory factors IL-1β, IL-6, and tumor necrosis factor-α (TNF-α) as well as anti-inflammatory factors IL-4 and IL-10 in the hippocampus and cortex were measured by RT-qPCR. The number of microglia and hippocampal neurons was quantified by immunofluorescent assay. IMQ treatment resulted in significant skin structural and histopathological characters of psoriasiform dermatitis with elevated serum IL-17A levels, obvious depressive-like behaviors, microglia activation with increased IL-1β, IL-6, and TNF-α expression levels in the hippocampus and cortex, and notable inhibition of hippocampal neurogenesis. While, IL-17A neutralization by intracerebroventricular injection of anti-IL-17A antibody can remarkably inhibit microglia activation and decrease the abnormally increased expression levels of IL-1β, IL-6, and TNF-α in the hippocampus and cortex of psoriasiform dermatitis mice, promote hippocampal neurogenesis, thus alleviate the depressive-like behaviors. In the pathological condition of psoriasis, systemic IL-17A elevation can trigger microglia activation, provoke pro-inflammation mediators to release, evoke neuroinflammation, subsequently inhibit hippocampal neurogenesis, and result in depression. IL-17A, as an important pathogenic factor in psoriasis, contributes to its critical role in mediating systemic inflammation and depression comorbidity.

Dorsal root ganglia CSF1+ neuronal subtypes have different impact on macrophages and microglia after spared nerve injury.

Colony-stimulating factor 1 (CSF1) is a growth factor secreted by dorsal root ganglia (DRG) neurons important for DRG macrophages and spinal cord (SC) microglia injury-induced proliferation and activation, specifically released after spared nerve injury (SNI). In this study, we investigated if SNI-induced CSF1 expression and perineuronal rings of macrophages around mouse DRG neurons vary between L3-L5 DRG and with the neuronal type, and if the CSF1+ neuronal projections at the SC dorsal horns were associated with an increased microglial number in the corresponding laminae. Seven days after surgery, L3-L5 DRG as well as their corresponding segments at the SC level were collected, frozen, and cut. DRG sections were double-immunostained using antibodies against CSF1 and NF200, CGRP or IB4, while SC sections were immunostained using a fluorescent Nissl Stain and analyzed for CX3CR1-GFP microglia number and distribution by an in-house ImageJ Plug-in. Our results showed that SNI-induced CSF1 expression was common for all subtypes of mouse DRG neurons, being responsible for attracting more resident macrophages around them in a DRG-dependent manner, with L4 showing the stronger response and CSF1+/NF200+ neurons showing the highest incidence. Even though the total number of microglia in the SC ipsilateral dorsal horns increased after SNI, the increase at their specific laminar projection sites did not mirror the incidence of DRG neuronal subtypes among CSF1+ neurons. Taken together, these results contribute to a more comprehensive understanding of the connection between CSF1 and macrophage/microglia response after SNI and emphasize the importance of considering L3-L5 DRG individually when investigating SNI-neuropathic pain pathogenesis in mice.

Lipopolysaccharide induces neuroinflammation in a valproic acid male model of autism

BackgroundAutism spectrum disorders (ASD) are characterized by social skill deficits and behavior impairments. Exposure to valproic acid (VPA) has been linked to ASD in humans and ASD-like behaviors in rodents. Clinical evidence suggests that immunological damage can worsen ASD symptoms in humans. ObjectiveThis study aimed to investigate the potential of lipopolysaccharide (LPS) to induce neuroinflammation in a VPA-induced autism male model. Materialsand methods: Pregnant Sprague Dawley rats were injected with 500mg/kg of VPA on gestational day 12.5 to create an ASD rat model in their offspring. Male offspring from VPA-injected group received 10mg/kg of LPS on postnatal day 20. Immunohistochemistry, western blotting, and immunofluorescence were used to assess the expression of NF-κB signaling pathway-related proteins and microglia in the prefrontal cortex and hippocampus. Gene Ontology and pathway enrichment analyses were conducted to predict the function of key synaptic proteins, which were further validated through real-time polymerase chain reaction analysis. ResultsThe results showed that VPA exposure led to increased locomotor activity, social impairment, and repetitive behaviors in male rats. NF-κB signaling pathway-related proteins were upregulated, and microglial numbers were elevated in the VPA-induced group. Furthermore, synaptic dysfunction was observed in the brains of offspring exposed to VPA. Importantly, LPS administration exacerbated autism-related behaviors in VPA-exposed male rats by promoting NF-κB signaling pathway activation, increasing microglial numbers, and downregulating key synaptic proteins. ConclusionsThis study not only contributed to understanding the importance of the NF-κB signaling pathway, microglia, and synaptic proteins in the progression of ASD, but also identified that LPS induces neuroinflammation in a valproic acid-induced male model of autism.

Transient CSF1R inhibition ameliorates behavioral deficits in Cntnap2 knockout and valproic acid-exposed mouse models of autism

Microglial abnormality and heterogeneity are observed in autism spectrum disorder (ASD) patients and animal models of ASD. Microglial depletion by colony stimulating factor 1-receptor (CSF1R) inhibition has been proved to improve autism-like behaviors in maternal immune activation mouse offspring. However, it is unclear whether CSF1R inhibition has extensive effectiveness and pharmacological heterogeneity in treating autism models caused by genetic and environmental risk factors. Here, we report pharmacological functions and cellular mechanisms of PLX5622, a small-molecule CSF1R inhibitor, in treating Cntnap2 knockout and valproic acid (VPA)-exposed autism model mice. For the Cntnap2 knockout mice, PLX5622 can improve their social ability and reciprocal social behavior, slow down their hyperactivity in open field and repetitive grooming behavior, and enhance their nesting ability. For the VPA model mice, PLX5622 can enhance their social ability and social novelty, and alleviate their anxiety behavior, repetitive and stereotyped autism-like behaviors such as grooming and marble burying. At the cellular level, PLX5622 restores the morphology and/or number of microglia in the somatosensory cortex, striatum, and hippocampal CA1 regions of the two models. Specially, PLX5622 corrects neurophysiological abnormalities in the striatum of the Cntnap2 knockout mice, and in the somatosensory cortex, striatum, and hippocampal CA1 regions of the VPA model mice. Incidentally, microglial dynamic changes in the VPA model mice are also reported. Our study demonstrates that microglial depletion and repopulation by transient CSF1R inhibition is effective, and however, has differential pharmacological functions and cellular mechanisms in rescuing behavioral deficits in the two autism models.

Glial Cell Responses and Gene Expression Dynamics in Retinas of Treated and Untreated RPE65 Mutant Dogs.

The long-term evaluation of RPE65 gene augmentation initiated in middle-aged RPE65 mutant dogs previously uncovered notable inter-animal and intra-retinal variations in treatment efficacy. The study aims to gain deeper insights into the status of mutant retinas and assess the treatment impact. Immunohistochemistry utilizing cell-specific markers and reverse transcription-quantitative PCR (RT-qPCR) analysis were conducted on archival retinal sections from normal and RPE65 mutant dogs. Untreated middle-aged mutant retinas exhibited marked downregulation in the majority of 20 examined genes associated with key retinal pathways. These changes were accompanied by a moderate increase in microglia numbers, altered expression patterns of glial-neuronal transmitter recycling proteins, and gliotic responses in Müller glia. Analysis of advanced-aged mutant dogs revealed mild outer nuclear layer loss in the treated eye compared to moderate loss in the corresponding retinal regions of the untreated control eye. However, persistent Müller glial stress response along with photoreceptor synapse loss were evident in both treated and untreated eyes. Photoreceptor synaptic remodeling, infrequent in treated regions, was observed in all untreated advanced-aged retinas, accompanied by a progressive increase in microglial cells indicative of ongoing inflammation. Interestingly, about half of the examined genes showed similar expression levels between treated and untreated advanced-aged mutant retinas, with some reaching normal levels. Gene expression data suggest a shift from pro-degenerative mechanisms in middle-aged mutant retinas to more compensatory mechanisms in preserved retinal regions at advanced stages, despite ongoing degeneration. Such shift, potentially attributed to a number of surviving resilient cells, may influence disease patterns and treatment outcomes.

Locomotor and gait changes in the LPS model of neuroinflammation are correlated with inflammatory cytokines in blood and brain.

Lipopolysaccharide (LPS) challenge in mice has been used to identify the mechanisms and therapeutics for neuroinflammation. In this study, we aimed to comprehensively evaluate the behavioral changes including locomotion, exploration, and memory, correlating them with a panel of thirteen inflammatory cytokines in both blood and brain.We found that acute LPS administration (0.83 mg/Kg i.p.) reduced body weight, food intake, and glucose levels compared to the saline-injected mice, concomitant with decreased activity in home cage monitoring. Locomotion was significantly reduced in Open Field, Introduced Object, and Y-Maze tests. Decreased exploratory behavior in the Y-Maze and Introduced Object tests was noticed, by measuring the number of arms explored and object interaction time, respectively. Additionally, in rotarod, LPS administration led to a significant decrease in the distance achieved, while in the MouseWalker, LPS led to a reduction in average velocity.LPS induced a decrease in microglia ramification index in the motor cortex and the striatum, while surprisingly a reduction in microglia number was observed in the motor cortex.The concentrations of thirteen cytokines in the blood were significantly altered, while only CXCL1, CCL22, CCL17, G-CSF, and IL-12p40 were changed in the brain. Correlations between cytokine levels in blood and brain were found, most notably for CCL17 and CCL22. TGFβ was the only one with negative correlations to other cytokines. Correlations between cytokines and behavior changes were also disclosed, especially for CCL17, CCL22, G-CSF, and IL-6 and negatively for TGFβ and IL-10.In summary, our study employing acute LPS challenge in mice has revealed a comprehensive profile of behavioral alterations alongside significant changes in inflammatory cytokine levels, both in peripheral blood and brain tissue. These findings contribute to a deeper understanding of the interplay between inflammation and behavior, with possible implications for identifying prognostics and therapeutic targets for neuroinflammatory conditions.

Tracking changes in functionality and morphology of repopulated microglia in young and old mice

BackgroundMicroglia (MG) are myeloid cells of the central nervous system that support homeostasis and instigate neuroinflammation in pathologies. Single-cell RNA sequencing (scRNA-seq) revealed the functional heterogeneity of MG in mouse brains. Microglia are self-renewing cells and inhibition of colony-stimulating factor 1 receptor (CSF1R) signaling depletes microglia which rapidly repopulate. The functions of repopulated microglia are poorly known.MethodsWe combined scRNA-seq, bulk RNA-seq, immunofluorescence, and confocal imaging to study the functionalities and morphology of repopulated microglia.ResultsA CSRF1R inhibitor (BLZ-945) depleted microglia within 21 days and a number of microglia was fully restored within 7 days, as confirmed by TMEM119 staining and flow cytometry. ScRNA-seq and computational analyses demonstrate that repopulated microglia originated from preexisting progenitors and reconstituted functional clusters but upregulated inflammatory genes. Percentages of proliferating, immature microglia displaying inflammatory gene expression increased in aging mice. Morphometric analysis of MG cell body and branching revealed a distinct morphology of repopulated MG, particularly in brains of old mice. We demonstrate that with aging some repopulated MG fail to reach the homeostatic phenotype. These differences may contribute to the deterioration of MG protective functions with age.

Running exercise decreases microglial activation in the medial prefrontal cortex in an animal model of depression

BackgroundRunning exercise effectively ameliorates depressive symptoms in humans and depression-like behaviors in animals, but the underlying mechanisms remain unclear. Microglia-mediated neuroinflammation plays a major role in the development of depression. The medial prefrontal cortex (mPFC) is a key brain region involved in depression and is sensitive to physical activity. Whether the antidepressant effect of running exercise involves changes in mPFC microglia is not understood. MethodsThe animals were subjected to chronic unpredictable stress (CUS) intervention followed by treadmill running. The sucrose preference test and elevated plus maze test or tail suspension test were used for behavioral assessment of the animals. The number of microglia in the mPFC was quantified by immunohistochemistry and stereology. The density and morphology of microglia were analyzed via immunofluorescence staining combined with three-dimensional laser scanning techniques. The mRNA expressions of inflammatory cytokines in the mPFC were examined via quantitative real-time PCR. ResultsRunning exercise effectively alleviated depressive-like behaviors in depression model animals. Running exercise reversed the increase in the number of microglia and the density of activated microglia in the mPFC of CUS animals. Running exercise effectively reversed the changes in microglia (reduced cell body area, total branch length and branch complexity) in the mPFC of CUS animals. Furthermore, running exercise regulated the gene expressions of pro−/antiinflammatory cytokines in the mPFC of CUS animals. ConclusionsOur results suggested that the antidepressant effects of running exercise may involve decreasing the number of activated microglia, reversing morphological changes in microglia in the mPFC, and reducing inflammatory responses.

Phloretin alleviates sleep deprivation-induced cognitive impairment by reducing inflammation through PPARγ/NF-κB signaling pathway

Sleep loss leads to significant pathophysiological consequences, including cognitive impairment. The neuroinflammation are pivotal factors in the pathogenesis of cognitive impairment induced by sleep loss. The phloretin (PHL), derived from peel of juicy fruits, has demonstrated potent anti-inflammatory properties. However, the precise influence of PHL on the cognitive impairment triggered by sleep loss and its underlying mechanism remain uncertain. In the present study, mice were subjected to sleep deprivation (SD) paradigm. Cognitive impairment induced by SD were significantly relieved by administration of PHL in a dose-dependent manner. Furthermore, PHL not only mitigated the synaptic losses but also enhanced dendritic spine density and neuronal activity within mice hippocampus following exposure to SD. Moreover, PHL treatment decreased the microglial numbers and altered microglial morphology in the hippocampus to restore the M1/M2 balances; these effects were accompanied by regulation of pro−/anti-inflammatory cytokine production and secretion in SD-exposed mice. Additionally, in vivo and in vitro studies showed PHL might attenuate the inflammation through the PPARγ/NF-κB pathway. Our findings suggest that PHL exerts inhibitory effects on microglia-mediated neuroinflammation, thereby providing protection against cognitive impairment induced by SD through a PPAR-γ dependent mechanism. The results indicate PHL is expected to provide a valuable candidate for new drug development for SD-induced cognitive impairment in the future.

Pregnancy ameliorates neuropathic pain through suppression of microglia and upregulation of the δ-opioid receptor in the anterior cingulate cortex in late-pregnant mice.

Pregnancy-induced analgesia develops in late pregnancy, but its mechanisms are unclear. The anterior cingulate cortex (ACC) plays a key role in the pathogenesis of neuropathic pain. The authors hypothesized that pregnancy-induced analgesia ameliorates neuropathic pain by suppressing activation of microglia and the expression of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, and by upregulating opioid receptors in the ACC in late-pregnant mice. Neuropathic pain was induced in non-pregnant (NP) or pregnant (P) C57BL/6JJmsSlc female mice by partial sciatic nerve ligation (PSNL). The nociceptive response was evaluated by mechanical allodynia and activation of microglia in the ACC was evaluated by immunohistochemistry. The expressions of phosphorylated AMPA receptors and opioid receptors in the ACC were evaluated by immunoblotting. In von Frey reflex tests, NP-PSNL-treated mice showed a lower 50% paw-withdrawal threshold than NP-Naïve mice on experimental day 9. No difference in 50% paw-withdrawal threshold was found among the NP-Naïve, NP-Sham, P-Sham, and P-PSNL-treated mice. The number of microglia in the ACC was significantly increased in NP-PSNL-treated mice compared to NP-Sham mice. Immunoblotting showed significantly increased expression of phosphorylated AMPA receptor subunit GluR1 at Ser831 in NP-PSNL-treated mice compared to NP-Sham mice. Immunoblotting also showed significantly increased δ-opioid receptor in the ACC in P-Sham and P-PSNL-treated mice compared to NP-Sham mice. Pregnancy-induced analgesia ameliorated neuropathic pain by suppressing activation of microglia and the expression of phosphorylated AMPA receptor subunit GluR1 at Ser831, and by upregulation of the δ-opioid receptor in the ACC in late-pregnant mice.

Early brain neuroinflammatory and metabolic changes identified by dual tracer microPET imaging in mice with acute liver injury.

Acute liver injury (ALI) that progresses into acute liver failure (ALF) is a life-threatening condition with an increasing incidence and associated costs. Acetaminophen (N-acetyl-p-aminophenol, APAP) overdosing is among the leading causes of ALI and ALF in the Northern Hemisphere. Brain dysfunction defined as hepatic encephalopathy is one of the main diagnostic criteria for ALF. While neuroinflammation and brain metabolic alterations significantly contribute to hepatic encephalopathy, their evaluation at early stages of ALI remained challenging. To provide insights, we utilized post-mortem analysis and non-invasive brain micro positron emission tomography (microPET) imaging of mice with APAP-induced ALI. Male C57BL/6 mice were treated with vehicle or APAP (600 mg/kg, i.p.). Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), liver damage (using H&E staining), hepatic and serum IL-6 levels, and hippocampal IBA1 (using immunolabeling) were evaluated at 24h and 48h. Vehicle and APAP treated animals also underwent microPET imaging utilizing a dual tracer approach, including [11C]-peripheral benzodiazepine receptor ([11C]PBR28) to assess microglia/astrocyte activation and [18F]-fluoro-2-deoxy-2-D-glucose ([18F]FDG) to assess energy metabolism. Brain images were pre-processed and evaluated using conjunction and individual tracer uptake analysis. APAP-induced ALI and hepatic and systemic inflammation were detected at 24h and 48h by significantly elevated serum ALT and AST levels, hepatocellular damage, and increased hepatic and serum IL-6 levels. In parallel, increased microglial numbers, indicative for neuroinflammation were observed in the hippocampus of APAP-treated mice. MicroPET imaging revealed overlapping increases in [11C]PBR28 and [18F]FDG uptake in the hippocampus, thalamus, and habenular nucleus indicating microglial/astroglial activation and increased energy metabolism in APAP-treated mice (vs. vehicle-treated mice) at 24h. Similar significant increases were also found in the hypothalamus, thalamus, and cerebellum at 48h. The individual tracer uptake analyses (APAP vs vehicle) at 24h and 48h confirmed increases in these brain areas and indicated additional tracer- and region-specific effects including hippocampal alterations. Peripheral manifestations of APAP-induced ALI in mice are associated with brain neuroinflammatory and metabolic alterations at relatively early stages of disease progression, which can be non-invasively evaluated using microPET imaging and conjunction analysis. These findings support further PET-based investigations of brain function in ALI/ALF that may inform timely therapeutic interventions.

Photoreceptor regeneration occurs normally in microglia-deficient irf8 mutant zebrafish following acute retinal damage

Microglia are resident immune cells in the central nervous system, including the retina that surveil the environment for damage and infection. Following retinal damage, microglia undergo morphological changes, migrate to the site of damage, and express and secrete pro-inflammatory signals. In the zebrafish retina, inflammation induces the reprogramming and proliferation of Müller glia and the regeneration of neurons following damage or injury. Immunosuppression or pharmacological ablation of microglia reduce or abolish Müller glia proliferation. We evaluated the retinal architecture and retinal regeneration in adult zebrafish irf8 mutants, which have significantly depleted numbers of microglia. We show that irf8 mutants have normal retinal structure at 3 months post fertilization (mpf) and 6 mpf but fewer cone photoreceptors by 10 mpf. Surprisingly, light-induced photoreceptor ablation induced Müller glia proliferation in irf8 mutants and cone and rod photoreceptor regeneration. Light-damaged retinas from both wild-type and irf8 mutants show upregulated expression of mmp-9, il8, and tnfβ pro-inflammatory cytokines. Our data demonstrate that adult zebrafish irf8 mutants can regenerate normally following acute retinal injury. These findings suggest that microglia may not be essential for retinal regeneration in zebrafish and that other mechanisms can compensate for the reduction in microglia numbers.

Regulatory T cells administration reduces anxiety-like behavior in mice submitted to chronic restraint stress.

Major depression disorder (MDD) and anxiety are common mental disorders that significantly affect the quality of life of those who suffer from them, altering the person's normal functioning. From the biological perspective, the most classical hypothesis explaining their occurrence relies on neurotransmission and hippocampal excitability alterations. However, around 30% of MDD patients do not respond to medication targeting these processes. Over the last decade, the involvement of inflammatory responses in depression and anxiety pathogenesis has been strongly acknowledged, opening the possibility of tackling these disorders from an immunological point of view. In this context, regulatory T cells (Treg cells), which naturally maintain immune homeostasis by suppressing inflammation could be promising candidates for their therapeutic use in mental disorders. To test this hypothesis, C57BL/6 adult male mice were submitted to classical stress protocols to induce depressive and anxiety-like behavior; chronic restriction stress (CRS), and chronic unpredictable stress (CUS). Some of the stressed mice received a single adoptive transfer of Treg cells during stress protocols. Mouse behavior was analyzed through the open field (OFT) and forced swim test (FST). Blood and spleen samples were collected for T cell analysis using cell cytometry, while brains were collected to study changes in microglia by immunohistochemistry. Mice submitted to CRS and CUS develop anxiety and depressive-like behavior, and only CRS mice exhibit lower frequencies of circulating Treg cells. Adoptive transfer of Treg cells decreased anxiety-like behavior in the OFT only in CRS model, but not depressive behavior in FST in neither of the two models. In CRS mice, Treg cells administration lowered the number of microglia in the hippocampus, which increased due this stress paradigm, and restored its arborization. However, in CUS mice, Treg cells administration increased microglia number with no significant effect on their arborization. Our results for effector CD4+ T cells in the spleen and microglia number and morphology in the hippocampus add new evidence in favor of the participation of inflammatory responses in the development of depressive and anxiety-like behavior and suggest that the modulation of key immune cells such as Treg cells, could have beneficial effects on these disorders.

Chronic activation of a negative engram induces behavioral and cellular abnormalities.

Negative memories engage a brain and body-wide stress response in humans that can alter cognition and behavior. Prolonged stress responses induce maladaptive cellular, circuit, and systems-level changes that can lead to pathological brain states and corresponding disorders in which mood and memory are affected. However, it is unclear if repeated activation of cells processing negative memories induces similar phenotypes in mice. In this study, we used an activity-dependent tagging method to access neuronal ensembles and assess their molecular characteristics. Sequencing memory engrams in mice revealed that positive (male-to-female exposure) and negative (foot shock) cells upregulated genes linked to anti- and pro-inflammatory responses, respectively. To investigate the impact of persistent activation of negative engrams, we chemogenetically activated them in the ventral hippocampus over 3 months and conducted anxiety and memory-related tests. Negative engram activation increased anxiety behaviors in both 6- and 14-month-old mice, reduced spatial working memory in older mice, impaired fear extinction in younger mice, and heightened fear generalization in both age groups. Immunohistochemistry revealed changes in microglial and astrocytic structure and number in the hippocampus. In summary, repeated activation of negative memories induces lasting cellular and behavioral abnormalities in mice, offering insights into the negative effects of chronic negative thinking-like behaviors on human health.