Limits...
Differential effects of domoic acid and E. coli lipopolysaccharide on tumor necrosis factor-alpha, transforming growth factor-beta1 and matrix metalloproteinase-9 release by rat neonatal microglia: evaluation of the direct activation hypothesis.

Mayer AM, Guzman M, Peksa R, Hall M, Fay MJ, Jacobson PB, Romanic AM, Gunasekera SP - Mar Drugs (2007)

Bottom Line: LPS [3 ng/mL] but not domoic acid [1 mM] stimulated a statistically significant increase in TNF-alpha mRNA and protein generation.Furthermore, both LPS and domoic acid did not significantly affect TGF-beta1 gene expression and protein release.However, in contrast, no statistically significant increase in MMP-9 expression and release was observed after domoic acid treatment.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Chicago College of Osteopathic Medicine, Midwestern University, 555 31st Street, Downers Grove, Illinois 60515, USA. amayer@midwestern.edu

ABSTRACT
The excitatory amino acid domoic acid is the causative agent of amnesic shellfish poisoning in humans. The in vitro effects of domoic acid on rat neonatal brain microglia were compared with E. coli lipopolysaccharide (LPS), a known activator of microglia mediator release over a 4 to 24 hour observation period. LPS [3 ng/mL] but not domoic acid [1 mM] stimulated a statistically significant increase in TNF-alpha mRNA and protein generation. Furthermore, both LPS and domoic acid did not significantly affect TGF-beta1 gene expression and protein release. Finally, an in vitro exposure of microglia to LPS resulted in statistically significant MMP-9 expression and release, thus extending and confirming our previous observations. However, in contrast, no statistically significant increase in MMP-9 expression and release was observed after domoic acid treatment. Taken together our observations do not support the hypothesis that a short term (4 to 24 hours) in vitro exposure to domoic acid, at a concentration toxic to neuronal cells, activates rat neonatal microglia and the concomitant release of the pro-inflammatory mediators tumor necrosis factor-alpha (TNF-alpha) and matrix metalloproteinases-9 (MMP-9), as well as the anti-inflammatory cytokine transforming growth factor beta1 (TGF-beta1).

No MeSH data available.


Related in: MedlinePlus

RT-PCR analysis of TGF-β1 gene expression in rat neonatal microglia after in vitro treatment with LPS or DOM. Rat neonatal microglia (2.8–5.0 x 106 cells/culture dish) were treated with (A) LPS [3 ng/mL] or (B) DOM [1mM] for 4 to 24 hours as described in Experimental. Amplification of TGF-β1 and S12 mRNAs showed the predicted cDNA size after separation on a 1.5 % agarose gel and visualization by SYBRR Gold nucleic acid staining. The S12 ribosomal RNA gene product was amplified for 25 and 30 cycles and demonstrated equal loading of the gels. Quantification of the TGF-β1 product was done after 20 cycles shown to be in the linear range. The figure depicts one of two similar experiments.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2367328&req=5

f5-md503113: RT-PCR analysis of TGF-β1 gene expression in rat neonatal microglia after in vitro treatment with LPS or DOM. Rat neonatal microglia (2.8–5.0 x 106 cells/culture dish) were treated with (A) LPS [3 ng/mL] or (B) DOM [1mM] for 4 to 24 hours as described in Experimental. Amplification of TGF-β1 and S12 mRNAs showed the predicted cDNA size after separation on a 1.5 % agarose gel and visualization by SYBRR Gold nucleic acid staining. The S12 ribosomal RNA gene product was amplified for 25 and 30 cycles and demonstrated equal loading of the gels. Quantification of the TGF-β1 product was done after 20 cycles shown to be in the linear range. The figure depicts one of two similar experiments.

Mentions: Activation of microglia has been shown to cause the release of the anti-inflammatory cytokine TGF-β1 [7,8]. As shown in Figure 5A, untreated (control) microglia showed constitutive TGF-β1 mRNA expression. Interestingly, stimulation of microglia with LPS [3 ng/ml] resulted in a time-dependent decrease in TGF-β1 mRNA expression relative to controls as determined by semi-quantitative RT-PCR within the linear range of amplification (25 cycles). In contrast, as shown in Figure 5B, domoic acid [1mM] did not appear to affect basal TGF-β1 mRNA expression as determined by semi-quantitative RT-PCR over the linear range of amplification (25 cycles).


Differential effects of domoic acid and E. coli lipopolysaccharide on tumor necrosis factor-alpha, transforming growth factor-beta1 and matrix metalloproteinase-9 release by rat neonatal microglia: evaluation of the direct activation hypothesis.

Mayer AM, Guzman M, Peksa R, Hall M, Fay MJ, Jacobson PB, Romanic AM, Gunasekera SP - Mar Drugs (2007)

RT-PCR analysis of TGF-β1 gene expression in rat neonatal microglia after in vitro treatment with LPS or DOM. Rat neonatal microglia (2.8–5.0 x 106 cells/culture dish) were treated with (A) LPS [3 ng/mL] or (B) DOM [1mM] for 4 to 24 hours as described in Experimental. Amplification of TGF-β1 and S12 mRNAs showed the predicted cDNA size after separation on a 1.5 % agarose gel and visualization by SYBRR Gold nucleic acid staining. The S12 ribosomal RNA gene product was amplified for 25 and 30 cycles and demonstrated equal loading of the gels. Quantification of the TGF-β1 product was done after 20 cycles shown to be in the linear range. The figure depicts one of two similar experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2367328&req=5

f5-md503113: RT-PCR analysis of TGF-β1 gene expression in rat neonatal microglia after in vitro treatment with LPS or DOM. Rat neonatal microglia (2.8–5.0 x 106 cells/culture dish) were treated with (A) LPS [3 ng/mL] or (B) DOM [1mM] for 4 to 24 hours as described in Experimental. Amplification of TGF-β1 and S12 mRNAs showed the predicted cDNA size after separation on a 1.5 % agarose gel and visualization by SYBRR Gold nucleic acid staining. The S12 ribosomal RNA gene product was amplified for 25 and 30 cycles and demonstrated equal loading of the gels. Quantification of the TGF-β1 product was done after 20 cycles shown to be in the linear range. The figure depicts one of two similar experiments.
Mentions: Activation of microglia has been shown to cause the release of the anti-inflammatory cytokine TGF-β1 [7,8]. As shown in Figure 5A, untreated (control) microglia showed constitutive TGF-β1 mRNA expression. Interestingly, stimulation of microglia with LPS [3 ng/ml] resulted in a time-dependent decrease in TGF-β1 mRNA expression relative to controls as determined by semi-quantitative RT-PCR within the linear range of amplification (25 cycles). In contrast, as shown in Figure 5B, domoic acid [1mM] did not appear to affect basal TGF-β1 mRNA expression as determined by semi-quantitative RT-PCR over the linear range of amplification (25 cycles).

Bottom Line: LPS [3 ng/mL] but not domoic acid [1 mM] stimulated a statistically significant increase in TNF-alpha mRNA and protein generation.Furthermore, both LPS and domoic acid did not significantly affect TGF-beta1 gene expression and protein release.However, in contrast, no statistically significant increase in MMP-9 expression and release was observed after domoic acid treatment.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Chicago College of Osteopathic Medicine, Midwestern University, 555 31st Street, Downers Grove, Illinois 60515, USA. amayer@midwestern.edu

ABSTRACT
The excitatory amino acid domoic acid is the causative agent of amnesic shellfish poisoning in humans. The in vitro effects of domoic acid on rat neonatal brain microglia were compared with E. coli lipopolysaccharide (LPS), a known activator of microglia mediator release over a 4 to 24 hour observation period. LPS [3 ng/mL] but not domoic acid [1 mM] stimulated a statistically significant increase in TNF-alpha mRNA and protein generation. Furthermore, both LPS and domoic acid did not significantly affect TGF-beta1 gene expression and protein release. Finally, an in vitro exposure of microglia to LPS resulted in statistically significant MMP-9 expression and release, thus extending and confirming our previous observations. However, in contrast, no statistically significant increase in MMP-9 expression and release was observed after domoic acid treatment. Taken together our observations do not support the hypothesis that a short term (4 to 24 hours) in vitro exposure to domoic acid, at a concentration toxic to neuronal cells, activates rat neonatal microglia and the concomitant release of the pro-inflammatory mediators tumor necrosis factor-alpha (TNF-alpha) and matrix metalloproteinases-9 (MMP-9), as well as the anti-inflammatory cytokine transforming growth factor beta1 (TGF-beta1).

No MeSH data available.


Related in: MedlinePlus