Dataset Information

Sudden cessation of fluoxetine before alcohol drinking reinstatement alters microglial morphology and TLR4/inflammatory neuroadaptation in the rat brain.

ABSTRACT: Preclinical studies on the effects of abrupt cessation of selective serotonin reuptake inhibitors (SSRIs), a medication often prescribed in alcohol use disorder (AUD) patients with depression, results in alcohol consumption escalation after resuming drinking. However, a potential neuroinflammatory component on this escalation remains unexplored despite the immunomodulatory role of serotonin. Here, we utilized a rat model of 14-daily administration of the SSRI fluoxetine (10 mg/kg/day) along alcohol self-administration deprivation to study the effects of fluoxetine cessation on neuroinflammation after resuming alcohol drinking. Microglial morphology and inflammatory gene expression were analyzed in prelimbic cortex, striatum, basolateral amygdala and dorsal hippocampus. Results indicated that alcohol drinking reinstatement increased microglial IBA1 immunoreactivity and altered morphometric features of activated microglia (fractal dimension, lacunarity, density, roughness, and cell area, perimeter and circularity). Despite alcohol reinstatement, fluoxetine cessation modified microglial morphology in a brain region-specific manner, resulting in hyper-ramified (spatial complexity of branching), reactive (lower heterogeneity and circularity)-like microglia. We also found that microglial cell area correlated with changes in mRNA expression of chemokines (Cx3cl1/fractalkine, Cxcl12/SDF1α, Ccl2/MCP1), cytokines (IL1β, IL6, IL10) and the innate immune toll-like receptor 4 (TLR4) in dorsal hippocampus. Specifically, TLR4 correlated with microglial spatial complexity assessed by fractal dimension in striatum, suggesting a role in process branching. These findings suggest that alcohol drinking reinstatement after fluoxetine treatment cessation disturbs microglial morphology and reactive phenotype associated with a TLR4/inflammatory response to alcohol in a brain region-specific manner, facts that might contribute to alcohol-induced damage through the promotion of escalation of alcohol drinking behavior.

SUBMITTER: Aranda J

PROVIDER: S-EPMC8354990 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description:BACKGROUND:Neuraminidase (NA) is a sialidase present, among various locations, in the envelope/membrane of some bacteria/viruses (e.g., influenza virus), and is involved in infectiveness and/or dispersion. The administration of NA within the brain lateral ventricle represents a model of acute sterile inflammation. The relevance of the Toll-like receptors TLR2 and TLR4 (particularly those in microglial cells) in such process was investigated. METHODS:Mouse strains deficient in either TLR2 (TLR2-/-) or TLR4 (TLR4-/-) were used. NA was injected in the lateral ventricle, and the inflammatory reaction was studied by immunohistochemistry (IBA1 and IL-1?) and qPCR (cytokine response). Also, microglia was isolated from those strains and in vitro stimulated with NA, or with TLR2/TLR4 agonists as positive controls (P3C and LPS respectively). The relevance of the sialidase activity of NA was investigated by stimulating microglia with heat-inactivated NA, or with native NA in the presence of sialidase inhibitors (oseltamivir phosphate and N-acetyl-2,3-dehydro-2-deoxyneuraminic acid). RESULTS:In septofimbria and hypothalamus, IBA1-positive and IL-1?-positive cell counts increased after NA injection in wild type (WT) mice. In TLR4-/- mice, such increases were largely abolished, while were only slightly diminished in TLR2-/- mice. Similarly, the NA-induced expression of IL-1?, TNF?, and IL-6 was completely blocked in TLR4-/- mice, and only partially reduced in TLR2-/- mice. In isolated cultured microglia, NA induced a cytokine response (IL-1?, TNF?, and IL-6) in WT microglia, but was unable to do so in TLR4-/- microglia; TLR2 deficiency partially affected the NA-induced microglial response. When WT microglia was exposed in vitro to heat-inactivated NA or to native NA along with sialidase inhibitors, the NA-induced microglia activation was almost completely abrogated. CONCLUSIONS:NA is able to directly activate microglial cells, and it does so mostly acting through the TLR4 receptor, while TLR2 has a secondary role. Accordingly, the inflammatory reaction induced by NA in vivo is partially dependent on TLR2, while TLR4 plays a crucial role. Also, the sialidase activity of NA is critical for microglial activation. These results highlight the relevance of microbial NA in the neuroinflammation provoked by NA-bearing pathogens and the possibility of targeting its sialidase activity to ameliorate its impact.

Project description:Resident macrophages in the CNS microglia become activated and produce proinflammatory molecules upon encountering bacteria or viruses. TLRs are a phylogenetically conserved diverse family of sensors that drive innate immune responses following interactions with PAMPs. TLR3 and TLR4 recognize viral dsRNA Poly (I:C) and bacterial endotoxin LPS, respectively. Importantly, these receptors differ in their downstream adaptor molecules. Thus far, only a few studies have investigated the effects of TLR3 and TLR4 in macrophages. However, a genome-wide search for the effects of these TLRs has not been performed in microglia using RNA-seq. Gene expression patterns were determined for the BV-2 microglial cell line when stimulated with viral dsRNA Poly (I:C) or bacterial endotoxin LPS to identify novel transcribed genes, as well as investigate how differences in downstream signaling could influence gene expression in innate immunity.Sequencing assessment and quality evaluation revealed that common and unique patterns of proinflammatory genes were significantly up-regulated in response to TLR3 and TLR4 stimulation. However, the IFN/viral response gene showed a stronger response to TLR3 stimulation than to TLR4 stimulation. Unexpectedly, TLR3 and TLR4 stimulation did not activate IFN-ß and IRF3 in BV-2 microglia. Most importantly, we observed that previously unidentified transcription factors (TFs) (i.e., IRF1, IRF7, and IRF9) and the epigenetic regulators KDM4A and DNMT3L were significantly up-regulated in both TLR3- and TLR4-stimulated microglia. We also identified 29 previously unidentified genes that are important in immune regulation. In addition, we confirmed the expressions of key inflammatory genes as well as pro-inflammatory mediators in the supernatants were significantly induced in TLR3-and TLR4-stimulated primary microglial cells. Moreover, transcriptional start sites (TSSs) and isoforms, as well as differential promoter usage, revealed a complex pattern of transcriptional and post-transcriptional gene regulation upon infection with TLR3 and TLR4. Furthermore, TF motif analysis (-950 to +50 bp of the 5' upstream promoters) revealed that the DNA sequences for NF-?B, IRF1, and STAT1 were significantly enriched in TLR3- and TLR4-stimulated microglia.These unprecedented findings not only permit a comparison of TLR3-and TLR4-stimulated genes but also identify new genes that have not been previously implicated in innate immunity.

Project description:BACKGROUND:Microglial cells are tissue-resident macrophages of the central nervous system. They are extremely dynamic, sensitive to their microenvironment and present a characteristic complex and heterogeneous morphology and distribution within the brain tissue. Many experimental clues highlight a strong link between their morphology and their function in response to aggression. However, due to their complex "dendritic-like" aspect that constitutes the major pool of murine microglial cells and their dense network, precise and powerful morphological studies are not easy to realize and complicate correlation with molecular or clinical parameters. METHODS:Using the knock-in mouse model CX3CR1(GFP/+), we developed a 3D automated confocal tissue imaging system coupled with morphological modelling of many thousands of microglial cells revealing precise and quantitative assessment of major cell features: cell density, cell body area, cytoplasm area and number of primary, secondary and tertiary processes. We determined two morphological criteria that are the complexity index (CI) and the covered environment area (CEA) allowing an innovative approach lying in (i) an accurate and objective study of morphological changes in healthy or pathological condition, (ii) an in situ mapping of the microglial distribution in different neuroanatomical regions and (iii) a study of the clustering of numerous cells, allowing us to discriminate different sub-populations. RESULTS:Our results on more than 20,000 cells by condition confirm at baseline a regional heterogeneity of the microglial distribution and phenotype that persists after induction of neuroinflammation by systemic injection of lipopolysaccharide (LPS). Using clustering analysis, we highlight that, at resting state, microglial cells are distributed in four microglial sub-populations defined by their CI and CEA with a regional pattern and a specific behaviour after challenge. CONCLUSIONS:Our results counteract the classical view of a homogenous regional resting state of the microglial cells within the brain. Microglial cells are distributed in different defined sub-populations that present specific behaviour after pathological challenge, allowing postulating for a cellular and functional specialization. Moreover, this new experimental approach will provide a support not only to neuropathological diagnosis but also to study microglial function in various disease models while reducing the number of animals needed to approach the international ethical statements.

Project description:Rationale: Ischemic white matter damage frequently results in myelin loss, accompanied with microglial activation. We previously found that directing microglia towards an anti-inflammatory phenotype provided a beneficial microenvironment and helped maintain white matter integrity during chronic cerebral hypoperfusion. However, the molecular mechanisms underlying microglial polarization remain elusive. Methods: Hypoperfusion induced white matter damage mice model and lipopolysaccharide (LPS) induced primary cultured microglia were established. Autophagy activation in microglia was detected both in vivo and in vitro by immunofluorescence, Western blot and electron microscopy. Autophagy inhibitors/agonist were administrated to investigate the role of autophagic process in modulating microglial phenotypes. Quantitative real time-polymerase chain reaction and Western blot were carried out to investigate the possible pathway. Results: We identified rapid accumulation of autophagosomes in primary cultured microglia exposed to LPS and within activated microglia during white matter ischemic damage. Autophagy inhibitors switched microglial function from pro-inflammatory to anti-inflammatory phenotype. Furthermore, we found TLR4, one of the major receptors binding LPS, was most highly expressed on microglia in corpus callosum during white matter ischemic damage, and TLR4 deficiency could mimic the phenomenon in microglial functional transformation, and exhibit a protective activity in chronic cerebral hypoperfusion. Whereas, the anti-inflammatory phenotype of microglia in TLR4 deficiency group was largely abolished by the activation of autophagic process. Finally, our transcriptional analysis confirmed that the up-regulation of STAT1 and down-regulation of STAT6 in microglia exposure to LPS could be reversed by autophagy inhibition. Conclusion: These results indicated that TLR4-dependent autophagy regulates microglial polarization and induces ischemic white matter damage via STAT1/6 pathway.

Dataset Information

Sudden cessation of fluoxetine before alcohol drinking reinstatement alters microglial morphology and TLR4/inflammatory neuroadaptation in the rat brain.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets