scholarly journals The MAO Inhibitor Tranylcypromine Alters LPS- and Aβ-Mediated Neuroinflammatory Responses in Wild-type Mice and a Mouse Model of AD

Cells ◽  
2020 ◽  
Vol 9 (9) ◽  
pp. 1982
Author(s):  
HyunHee Park ◽  
Kyung-Min Han ◽  
Hyongjun Jeon ◽  
Ji-Soo Lee ◽  
Hyunju Lee ◽  
...  
Keyword(s):  
Mouse Model ◽  
Proinflammatory Cytokine ◽  
Microglial Activation ◽  
Microglial Cells ◽  
Wild Type ◽  
Mao Inhibitor ◽  
Cytokine Levels ◽  
Bv2 Microglial Cells

Monoamine oxidase (MAO) has been implicated in neuroinflammation, and therapies targeting MAO are of interest for neurodegenerative diseases. The small-molecule drug tranylcypromine, an inhibitor of MAO, is currently used as an antidepressant and in the treatment of cancer. However, whether tranylcypromine can regulate LPS- and/or Aβ-induced neuroinflammation in the brain has not been well-studied. In the present study, we found that tranylcypromine selectively altered LPS-induced proinflammatory cytokine levels in BV2 microglial cells but not primary astrocytes. In addition, tranylcypromine modulated LPS-mediated TLR4/ERK/STAT3 signaling to alter neuroinflammatory responses in BV2 microglial cells. Importantly, tranylcypromine significantly reduced microglial activation as well as proinflammatory cytokine levels in LPS-injected wild-type mice. Moreover, injection of tranylcypromine in 5xFAD mice (a mouse model of AD) significantly decreased microglial activation but had smaller effects on astrocyte activation. Taken together, our results suggest that tranylcypromine can suppress LPS- and Aβ-induced neuroinflammatory responses in vitro and in vivo.

scholarly journals Dasatinib regulates LPS-induced microglial and astrocytic neuroinflammatory responses by inhibiting AKT/STAT3 signaling

2019 ◽  
Vol 16 (1) ◽  
Author(s):  
Ka-Young Ryu ◽  
Hyun-ju Lee ◽  
Hanwoong Woo ◽  
Ri-Jin Kang ◽  
Kyung-Min Han ◽  
...  
Keyword(s):  
Proinflammatory Cytokine ◽  
Tween 80 ◽  
Subcellular Fractionation ◽  
Microglial Cells ◽  
Wild Type ◽  
Stat3 Signaling ◽  
Tnf Α ◽  
Cytokine Levels ◽  
Primary Astrocytes ◽  
Bv2 Microglial Cells

Abstract Background The FDA-approved small-molecule drug dasatinib is currently used as a treatment for chronic myeloid leukemia (CML). However, the effects of dasatinib on microglial and/or astrocytic neuroinflammatory responses and its mechanism of action have not been studied in detail. Methods BV2 microglial cells, primary astrocytes, or primary microglial cells were treated with dasatinib (100 or 250 nM) or vehicle (1% DMSO) for 30 min or 2 h followed by lipopolysaccharide (LPS; 200 ng/ml or 1 μg/ml) or PBS for 5.5 h. RT-PCR, real-time PCR; immunocytochemistry; subcellular fractionation; and immunohistochemistry were subsequently conducted to determine the effects of dasatinib on LPS-induced neuroinflammation. In addition, wild-type mice were injected with dasatinib (20 mg/kg, intraperitoneally (i.p.) daily for 4 days or 20 mg/kg, orally administered (p.o.) daily for 4 days or 2 weeks) or vehicle (4% DMSO + 30% polyethylene glycol (PEG) + 5% Tween 80), followed by injection with LPS (10 mg/kg, i.p.) or PBS. Then, immunohistochemistry was performed, and plasma IL-6, IL-1β, and TNF-α levels were analyzed by ELISA. Results Dasatinib regulates LPS-induced proinflammatory cytokine and anti-inflammatory cytokine levels in BV2 microglial cells, primary microglial cells, and primary astrocytes. In BV2 microglial cells, dasatinib regulates LPS-induced proinflammatory cytokine levels by regulating TLR4/AKT and/or TLR4/ERK signaling. In addition, intraperitoneal injection and oral administration of dasatinib suppress LPS-induced microglial/astrocyte activation, proinflammatory cytokine levels (including brain and plasma levels), and neutrophil rolling in the brains of wild-type mice. Conclusions Our results suggest that dasatinib modulates LPS-induced microglial and astrocytic activation, proinflammatory cytokine levels, and neutrophil rolling in the brain.

scholarly journals Astrocyte-Derived CCL2 is Associated with M1 Activation and Recruitment of Cultured Microglial Cells

2016 ◽  
Vol 38 (3) ◽  
pp. 859-870 ◽  
Author(s):  
Mingfeng He ◽  
Hongquan Dong ◽  
Yahui Huang ◽  
Shunmei Lu ◽  
Shu Zhang ◽  
...  
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Microglial Activation ◽  
Culture Media ◽  
Microglial Cells ◽  
Migration Ability ◽  
Migration Assay ◽  
Tnf Α

Background/Aims: Microglia are an essential player in central nervous system inflammation. Recent studies have demonstrated that the astrocytic chemokine, CCL2, is associated with microglial activation in vivo. However, CCL2-induced microglial activation has not yet been studied in vitro. The purpose of the current study was to understand the role of astrocyte-derived CCL2 in microglial activation and to elucidate the underlying mechanism(s). Methods: Primary astrocytes were pre-treated with CCL2 siRNA and stimulated with TNF-α. The culture medium (CM) was collected and added to cultures of microglia, which were incubated with and without CCR2 inhibitor. Microglial cells were analyzed by quantitative RT-PCR to determine whether they polarized to the M1 or M2 state. Microglial migratory ability was assessed by transwell migration assay. Results: TNF-α stimulated the release of CCL2 from astrocytes, even if the culture media containing TNF-α was replaced with fresh media after 3 h. CM from TNF-α-stimulated astrocytes successfully induced microglial activation, which was ascertained by increased activation of M1 and enhanced migration ability. In contrast, CM from astrocytes pretreated with CCL2 siRNA showed no effect on microglial activation, compared to controls. Additionally, microglia pre-treated with RS102895, a CCR2 inhibitor, were resistant to activation by CM from TNF-α-stimulated astrocytes. Conclusion: This study demonstrates that the CCL2/CCR2 pathway of astrocyte-induced microglial activation is associated with M1 polarization and enhanced migration ability, indicating that this pathway could be a useful target to ameliorate inflammation in the central nervous system.

scholarly journals Murine monoclonal antibodies against RBD of SARS-CoV-2 neutralize authentic wild type SARS-CoV-2 as well as B.1.1.7 and B.1.351 viruses and protect in vivo in a mouse model in a neutralization dependent manner

2021 ◽  
Author(s):  
Fatima Amanat ◽  
Shirin Strohmeier ◽  
Wen-Hsin Lee ◽  
Sandhya Bangaru ◽  
Andrew B Ward ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Severe Acute Respiratory Syndrome ◽  
Mouse Model ◽  
Neutralizing Antibodies ◽  
Dependent Manner ◽  
Receptor Binding Domain ◽  
Wild Type ◽  
Murine Monoclonal Antibodies

After first emerging in December 2019 in China, severe acute respiratory syndrome 2 (SARS-CoV-2) has since caused a pandemic leading to millions of infections and deaths worldwide. Vaccines have been developed and authorized but supply of these vaccines is currently limited. With new variants of the virus now emerging and spreading globally, it is essential to develop therapeutics that are broadly protective and bind conserved epitopes in the receptor binding domain (RBD) or the whole spike of SARS-CoV-2. In this study, we have generated mouse monoclonal antibodies (mAbs) against different epitopes on the RBD and assessed binding and neutralization against authentic SARS-CoV-2. We have demonstrated that antibodies with neutralizing activity, but not non-neutralizing antibodies, lower viral titers in the lungs when administered in a prophylactic setting in vivo in a mouse challenge model. In addition, most of the mAbs cross-neutralize the B.1.351 as well as the B.1.1.7 variants in vitro.

scholarly journals Marinobufagenin Inhibits Neutrophil Migration and Proinflammatory Cytokines

2019 ◽  
Vol 2019 ◽  
pp. 1-11 ◽  
Author(s):  
Deyse C. M. Carvalho ◽  
Luiz Henrique Agra Cavalcante-Silva ◽  
Éssia de A. Lima ◽  
José G. F. M. Galvão ◽  
Anne K. de A. Alves ◽  
...  
Keyword(s):  
Cell Migration ◽  
Proinflammatory Cytokines ◽  
Proinflammatory Cytokine ◽  
Biological Activities ◽  
Neutrophil Migration ◽  
Peritoneal Macrophages ◽  
Cytokine Levels ◽  
Anti Inflammatory

Cardiotonic steroids, such as ouabain and digoxin, are known to bind to Na+/K+-ATPase and to promote several biological activities, including anti-inflammatory activity. However, there are still no reports in the literature about inflammation and marinobufagenin, a cardiotonic steroid from the bufadienolide family endogenously found in mammals. Therefore, the aim of this work was to analyze, in vivo and in vitro, the role of marinobufagenin in acute inflammation. Swiss mice were treated with 0.56 mg/kg of marinobufagenin intraperitoneally (i.p.) and zymosan (2 mg/mL, i.p.) was used to induce peritoneal inflammation. Peritoneal fluid was collected and used for counting cells by optical microscopy and proinflammatory cytokine quantification (IL-1β, IL-6, and TNF-α) by immunoenzymatic assay (ELISA). Zymosan stimulation, as expected, induced increased cell migration and proinflammatory cytokine levels in the peritoneum. Marinobufagenin treatment reduced polymorphonuclear cell migration and IL-1β and IL-6 levels in the peritoneal cavity, without interfering in TNF-α levels. In addition, the effect of marinobufagenin was evaluated using peritoneal macrophages stimulated by zymosan (0.2 mg/mL) in vitro. Marinobufagenin treatment at different concentrations (10, 100, 1000, and 10000 nM) showed no cytotoxic effect on peritoneal macrophages. Interestingly, the lowest concentration, which did not inhibit Na+/K+-ATPase activity, attenuated proinflammatory cytokines IL-1β, IL-6, and TNF-α levels. To investigate the putative mechanism of action of marinobufagenin, the expression of surface molecules (TLR2 and CD69) and P-p38 MAPK were also evaluated, but no significant effect was observed. Thus, our results suggest that marinobufagenin has an anti-inflammatory role in vivo and in vitro and reveals a novel possible endogenous function of this steroid in mammals.

scholarly journals Modulation of Glial Responses by Furanocembranolides: Leptolide Diminishes Microglial Inflammation in Vitro and Ameliorates Gliosis In Vivo in a Mouse Model of Obesity and Insulin Resistance

Marine Drugs ◽  
2020 ◽  
Vol 18 (8) ◽  
pp. 378
Author(s):  
Miriam Corraliza-Gómez ◽  
Amalia B. Gallardo ◽  
Ana R. Díaz-Marrero ◽  
José M. de la Rosa ◽  
Luis D’Croz ◽  
...  
Keyword(s):  
Mouse Model ◽  
Microglial Cells ◽  
Obese Mouse ◽  
Neurodegenerative Process ◽  
Age Related ◽  
Bv2 Cells ◽  
Anti Inflammatory ◽  
Inflammatory Molecules

Neurodegenerative diseases are age-related disorders caused by progressive neuronal death in different regions of the nervous system. Neuroinflammation, modulated by glial cells, is a crucial event during the neurodegenerative process; consequently, there is an urgency to find new therapeutic products with anti-glioinflammatory properties. Five new furanocembranolides (1−5), along with leptolide, were isolated from two different extracts of Leptogorgia sp., and compound 6 was obtained from chemical transformation of leptolide. Their structures were determined based on spectroscopic evidence. These seven furanocembranolides were screened in vitro by measuring their ability to modulate interleukin-1β (IL-1β) production by microglial BV2 cells after LPS (lipopolysaccharide) stimulation. Leptolide and compounds 3, 4 and 6 exhibited clear anti-inflammatory effects on microglial cells, while compound 2 presented a pro-inflammatory outcome. The in vitro results prompted us to assess anti-glioinflammatory effects of leptolide in vivo in a high-fat diet-induced obese mouse model. Interestingly, leptolide treatment ameliorated both microgliosis and astrogliosis in this animal model. Taken together, our results reveal a promising direct biological effect of furanocembranolides on microglial cells as bioactive anti-inflammatory molecules. Among them, leptolide provides us a feasible therapeutic approach to treat neuroinflammation concomitant with metabolic impairment.

scholarly journals Efficacy of BB-83698, a Novel Peptide Deformylase Inhibitor, in a Mouse Model of Pneumococcal Pneumonia

2004 ◽  
Vol 48 (1) ◽  
pp. 80-85 ◽  
Author(s):  
E. Azoulay-Dupuis ◽  
J. Mohler ◽  
J. P. Bédos
Keyword(s):  
Mouse Model ◽  
Lung Tissue ◽  
Resistant Strain ◽  
Peptide Deformylase ◽  
In Vitro Activity ◽  
Wild Type ◽  
Virulent Strains ◽  
Resistant Strains

ABSTRACT The efficacy of BB-83698, a novel potent peptide deformylase inhibitor, was evaluated in a mouse model of acute pneumonia. The Streptococcus pneumoniae isolates tested included four virulent strains (one penicillin-susceptible wild-type strain, one macrolide-resistant strain, and two quinolone-resistant mutants [a mutant carrying mutations in ParC and GyrA and an efflux mutant] isogenic to the wild type) and two poorly virulent penicillin-resistant strains. Pneumonia was induced by intratracheal inoculation of 105 CFU (virulent strains) into immunocompetent mice or 107 CFU (less virulent strains) into leukopenic mice. Animals received three or six subcutaneous injections of antibiotics at 12- or 24-h intervals, with antibiotic treatment initiated at 3, 6, 12, or 18 h postinfection (p.i.). BB-83698 showed potent in vitro activity against all strains (MICs, 0.06 to 0.25 μg/ml). In the in vivo model, all control animals died within 2 to 5 days of infection. BB-83698 (80 mg/kg of body weight twice daily or 160 mg/kg once daily) protected 70 to 100% of the animals, as measured 10 days p.i., regardless of the preexisting resistance mechanisms. In contrast, the survival rates for animals treated with the comparator antibiotics were 30% for animals treated with erythromycin (100 mg/kg) and infected with the macrolide-resistant strain, 34% for animals treated with amoxicillin (200 mg/kg every 8 h) and infected with the penicillin-resistant strain, and 0 and 78% for animals treated with ciprofloxacin (250 mg/kg) and infected with the ParC and GyrA mutant and the efflux mutant, respectively. At 80 mg/kg, BB-83698 generated a peak concentration in lung tissue of 61.9 μg/ml within 1 h and areas under the concentration-times curves of 57.4 and 229.4 μg · h/ml for plasma and lung tissue, respectively. The emergence of S. pneumoniae isolates with reduced susceptibilities to BB-83698 was not observed following treatment with a suboptimal dosing regimen. In conclusion, the potent in vitro activity of BB-83698 against S. pneumoniae, including resistant strains, translates into good in vivo efficacy in a mouse pneumonia model.

CHIR258, a Novel Multi-Targeted Tyrosine Kinase Inhibitor, for the Treatment of t(4;14) Multiple Myeloma.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 641-641 ◽  
Author(s):  
Suzanne Trudel ◽  
Zhi Hua Li ◽  
Ellen Wei ◽  
Marion Wiesmann ◽  
Katherine Rendahl ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Mouse Model ◽  
Tyrosine Kinase ◽  
Cell Lines ◽  
Small Molecule ◽  
Kinase Inhibitor ◽  
Wild Type ◽  
Myeloma Cells

Abstract The t(4;14) translocation that occurs uniquely in a subset (15%) of multiple myeloma (MM) patients results in the ectopic expression of the receptor tyrosine kinase, Fibroblast Growth Factor Receptor3 (FGFR3). Wild-type FGFR3 induces proliferative signals in myeloma cells and appears to be weakly transforming in a hematopoeitic mouse model. The subsequent acquisition of FGFR3 activating mutations in some MM is associated with disease progression and is strongly transforming in several experimental models. The clinical impact of t(4;14) translocations has been demonstrated in several retrospective studies each reporting a marked reduction in overall survival. We have previously shown that inhibition of activated FGFR3 causes morphologic differentiation followed by apoptosis of FGFR3 expressing MM cell lines, validating activated FGFR3 as a therapeutic target in t(4;14) MM and encouraging the clinical development of FGFR3 inhibitors for the treatment of these poor-prognosis patients. CHIR258 is a small molecule kinase inhibitor that targets Class III–V RTKs and inhibits FGFR3 with an IC50 of 5 nM in an in vitro kinase assay. Potent anti-tumor and anti-angiogenic activity has been demonstrated in vitro and in vivo. We employed the IL-6 dependent cell line, B9 that has been engineered to express wild-type FGFR3 or active mutants of FGFR3 (Y373C, K650E, G384D and 807C), to screen CHIR258 for activity against FGFR3. CHIR258 differentially inhibited FGF-mediated growth of B9 expressing wild-type and mutant receptors found in MM, with an IC50 of 25 nM and 80 nM respectively as determined by MTT proliferation assay. Growth of these cells could be rescued by IL-6 demonstrating selectivity of CHIR258 for FGFR3. We then confirmed the activity of CHIR258 against FGFR3 expressing myeloma cells. CHIR258 inhibited the viability of FGFR3 expressing KMS11 (Y373C), KMS18 (G384D) and OPM-2 (K650E) cell lines with an IC50 of 100 nM, 250 nM and 80 nM, respectively. Importantly, inhibition with CHIR258 was still observed in the presence of IL-6, a potent growth factors for MM cells. U266 cells, which lack FGFR3 expression, displayed minimal growth inhibition demonstrating that at effective concentrations, CHIR258 exhibits minimal nonspecific cytotoxicity on MM cells. Further characterization of this finding demonstrated that inhibition of cell growth corresponded to G0/G1 cell cycle arrest and dose-dependent inhibition of downstream ERK phosphorylation. In responsive cell lines, CHIR258 induced apoptosis via caspase 3. In vitro combination analysis of CHIR258 and dexamethasone applied simultaneously to KMS11 cells indicated a synergistic interaction. In vivo studies demonstrated that CHIR258 induced tumor regression and inhibited growth of FGFR3 tumors in a plasmacytoma xenograft mouse model. Finally, CHIR258 produced cytotoxic responses in 4/5 primary myeloma samples derived from patients harboring a t(4;14) translocation. These data indicate that the small molecule inhibitor, CHIR258 potently inhibits FGFR3 and has activity against human MM cells setting the stage for a Phase I clinical trial of this compound in t(4;14) myeloma.

scholarly journals Mutation of the Maturase Lipoprotein Attenuates the Virulence of Streptococcus equi to a Greater Extent than Does Loss of General Lipoprotein Lipidation

2006 ◽  
Vol 74 (12) ◽  
pp. 6907-6919 ◽  
Author(s):  
Andrea Hamilton ◽  
Carl Robinson ◽  
Iain C. Sutcliffe ◽  
Josh Slater ◽  
Duncan J. Maskell ◽  
...  
Keyword(s):  
Mouse Model ◽  
Mutant Strain ◽  
Natural Host ◽  
Wild Type ◽  
Animal Suffering ◽  
Organ Cultures ◽  
Mucus Production ◽  
Streptococcus Equi

ABSTRACT Streptococcus equi is the causative agent of strangles, a prevalent and highly contagious disease of horses. Despite the animal suffering and economic burden associated with strangles, little is known about the molecular basis of S. equi virulence. Here we have investigated the contributions of a specific lipoprotein and the general lipoprotein processing pathway to the abilities of S. equi to colonize equine epithelial tissues in vitro and to cause disease in both a mouse model and the natural host in vivo. Colonization of air interface organ cultures after they were inoculated with a mutant strain deficient in the maturase lipoprotein (ΔprtM 138 - 213, with a deletion of nucleotides 138 to 213) was significantly less than that for cultures infected with wild-type S. equi strain 4047 or a mutant strain that was unable to lipidate preprolipoproteins (Δlgt 190 - 685). Moreover, mucus production was significantly greater in both wild-type-infected and Δlgt 190 - 685-infected organ cultures. Both mutants were significantly attenuated compared with the wild-type strain in a mouse model of strangles, although 2 of 30 mice infected with the Δlgt 190 - 685 mutant did still exhibit signs of disease. In contrast, only the ΔprtM 138 - 213 mutant was significantly attenuated in a pony infection study, with 0 of 5 infected ponies exhibiting pathological signs of strangles compared with 4 of 4 infected with the wild-type and 3 of 5 infected with the Δlgt 190 - 685 mutant. We believe that this is the first study to evaluate the contribution of lipoproteins to the virulence of a gram-positive pathogen in its natural host. These data suggest that the PrtM lipoprotein is a potential vaccine candidate, and further investigation of its activity and its substrate(s) are warranted.

scholarly journals 15-Deoxy-γ12,14-prostaglandin J2 Reduces Liver Impairment in a Model of ConA-Induced Acute Hepatic Inflammation by Activation of PPARγand Reduction in NF-κB Activity

PPAR Research ◽  
2014 ◽  
Vol 2014 ◽  
pp. 1-10 ◽  
Author(s):  
Kan Chen ◽  
Jingjing Li ◽  
Junshan Wang ◽  
Yujing Xia ◽  
Weiqi Dai ◽  
...  
Keyword(s):  
Autoimmune Hepatitis ◽  
Proinflammatory Cytokine ◽  
Histological Grade ◽  
Hepatic Inflammation ◽  
Protection Mechanism ◽  
Liver Impairment ◽  
Cytokine Levels ◽  
Prostaglandin J2

Objective. 15-Deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) reduces inflammation and has been identified as an anti-inflammatory prostaglandin in numerous animal models. In this study, we investigated both effects of 15d-PGJ2 and its protection mechanism in concanavalin A- (ConA-) induced autoimmune hepatitis in mice.Materials and Methods. In vivo, Balb/C mice were injected with ConA (25 mg/kg) to induce acute autoimmune hepatitis, and 15d-PGJ2 (10 μg or 25 μg) was administered 1 h before the ConA injection. The histological grade, proinflammatory cytokine levels, and NF-κB and PPARγactivity were determined 6, 12, and 24 h after the ConA injection. In vitro, LO2 cells and RAW264.7 cells were pretreated with 15d-PGJ2 (2 μM) 1 h before the stimulation with ConA (30 μg/mL). The NF-κB and PPARγactivity were determined 30 min after the ConA administration.Results. Pretreatment with 15d-PGJ2 reduced the pathological effects of ConA-induced autoimmune hepatitis and significantly reduced the levels of cytokines after injection. 15d-PGJ2 activated PPARγ, blocked the degradation of IκBα, and inhibited the translocation of NF-κB into the nucleus.Conclusion. These results indicate that 15d-PGJ2 protects against ConA-induced autoimmune hepatitis by reducing proinflammatory cytokines. This reduction in inflammation may correlate with the activation of PPARγand the reduction in NF-κB activity.

scholarly journals Rapid Morphologic and Molecular Activation of Microglial Cells by Stimulation of the P2X7 Receptor Correlates with Neuron Loss

2021 ◽  
Author(s):  
Keith E Campagno ◽  
Wennan Lu ◽  
Assraa Hassan Jassim ◽  
Farraj Albalawi ◽  
Aurora Cenaj ◽  
...  
Keyword(s):  
Cell Body ◽  
Microglial Activation ◽  
Morphological Changes ◽  
Mechanical Strain ◽  
Atp Release ◽  
Microglial Cells ◽  
Microglial Migration ◽  
Process Retraction

Abstract Background: The endogenous signals leading to microglial activation represent central components of neuroinflammatory cascades. Given ATP release accompanies mechanical strain to neural tissue, and the P2X7R for ATP is expressed on microglial cells, we examined the morphological and molecular consequences of P2X7R stimulation in vivo and in vitro in detail to enhance understanding of the response. Methods: IL-1β release was determined with ELISA. Expression of mRNA used qPCR. ATP release was determined with the luciferin/luciferase assay while fura-2 indicated cytoplasmic calcium. Microglial migration used Boyden chambers. Morphological changes were quantified from Iba1-immunostained cells. Results: Sholl analysis of Iba1-stained cells showed retraction of microglial ramifications one day after injection of P2X7R agonist BzATP into mouse retinae. Mean branch length also decreased, while cell body size and expression of Nos2, Tnfa, Arg1, Chil3 increased. BzATP induced similar morphological changes in ex vivo tissue isolated from Cx3CR1-GFP mice, suggesting cell recruitment was unnecessary. Primary microglial cultures were developed to investigate the autonomous nature of the response. Isolated microglial cells expressed P2X7R, while increased intracellular Ca 2+ triggered by BzATP and blocked by antagonist A839977 confirmed functional expression. BzATP induced process retraction and cell body enlargement within minutes in isolated microglial cells, and increased expression of Nos2 and Arg1 . BzATP both increased expression of IL-1β, and triggered a substantial release, suggesting P2X7R both primes and activates the NLRP3 inflammasome. ATP increased microglial migration, but this required P2Y12R, not P2X7R involvement. As ATP release often accompanies mechanical strain, responses to intraocular pressure elevation were determined. Transient elevation increased ATP release and led to microglial process retraction, cell body enlargement and gene upregulation resembling the responses to BzATP injection. These pressure-dependent changes to microglia were reduced in P2X7R -/- mice. Critically, the loss of retinal ganglion cell neurons accompanying increased pressure was correlated with microglial activation in C57Bl/6J, but not P2X7R -/- mice.Conclusions: P2X7R stimulation induced morphological and molecular markers of activation in retinal microglial cells in vivo and in vitro , affecting IL-1β release and rapid process retraction but not cell migration. Parallel responses accompanied transient pressure elevation, suggesting ATP release and P2X7R stimulation contribute to the microglial response to rising pressure.

Sign in / Sign up

Export Citation Format

Share Document