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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

The time-course of MMP-2 and MMP-9 expression in rat neonatal microglia cultured in the presence of LPS or DOM. MMP-2 and MMP-9 were determined as described in Experimental. (A) SDS-PAGE zymography, (B) bargraph depicting the quantitated results. Rat neonatal microglia (2.8–5 x 106 cells/culture dish) were treated with LPS [3 ng/ml] or DOM [1 mM] for 4 to 24 hours. Data (inverse O.D. units) are expressed as mean ± SE of 2 independent experiments. *p < 0.05 or **p < 0.01 vs. vehicle control.
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f10-md503113: The time-course of MMP-2 and MMP-9 expression in rat neonatal microglia cultured in the presence of LPS or DOM. MMP-2 and MMP-9 were determined as described in Experimental. (A) SDS-PAGE zymography, (B) bargraph depicting the quantitated results. Rat neonatal microglia (2.8–5 x 106 cells/culture dish) were treated with LPS [3 ng/ml] or DOM [1 mM] for 4 to 24 hours. Data (inverse O.D. units) are expressed as mean ± SE of 2 independent experiments. *p < 0.05 or **p < 0.01 vs. vehicle control.

Mentions: We have shown that a stimulation of microglia with LPS in vitro will lead to MMP-9 release [5,27]. In order to determine whether a 4 to 24 hour stimulation of microglia with LPS [3 ng/mL] or domoic acid [1mM] leads to MMP-2 and MMP-9 secretion, cell-free media from the same cell culture dishes containing microglia used for MMP-9 mRNA analysis shown in Figures 8 and 9, were assayed for MMP-2 and MMP-9 release as described in Experimental. As shown in Figure 10, a statistically significant increase in MMP-9 release was observed 8, 16 and 24 hours after microglia were stimulated with LPS [3 ng/mL]. MMP-9 inverse optical density levels for 8, 16 and 24 hours were 0.69 ± 0.05, 0.874 ± 0.06, 0.91 ± 0.0005 (LPS-treated, n=2), respectively, vs. 0.46 ± 0.03 (untreated controls, n=2), P< 0.05 (8 hours) and P < 0.01(16 and 24 hours). In contrast to LPS, MMP-9 levels in the tissue culture media in domoic acid-treated microglia remained unchanged 4, 8, 16 and 24 hours after domoic acid treatment. Neither LPS nor domoic acid had a significant effect on MMP-2 release over the 24 hour-observation period.


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)

The time-course of MMP-2 and MMP-9 expression in rat neonatal microglia cultured in the presence of LPS or DOM. MMP-2 and MMP-9 were determined as described in Experimental. (A) SDS-PAGE zymography, (B) bargraph depicting the quantitated results. Rat neonatal microglia (2.8–5 x 106 cells/culture dish) were treated with LPS [3 ng/ml] or DOM [1 mM] for 4 to 24 hours. Data (inverse O.D. units) are expressed as mean ± SE of 2 independent experiments. *p < 0.05 or **p < 0.01 vs. vehicle control.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2367328&req=5

f10-md503113: The time-course of MMP-2 and MMP-9 expression in rat neonatal microglia cultured in the presence of LPS or DOM. MMP-2 and MMP-9 were determined as described in Experimental. (A) SDS-PAGE zymography, (B) bargraph depicting the quantitated results. Rat neonatal microglia (2.8–5 x 106 cells/culture dish) were treated with LPS [3 ng/ml] or DOM [1 mM] for 4 to 24 hours. Data (inverse O.D. units) are expressed as mean ± SE of 2 independent experiments. *p < 0.05 or **p < 0.01 vs. vehicle control.
Mentions: We have shown that a stimulation of microglia with LPS in vitro will lead to MMP-9 release [5,27]. In order to determine whether a 4 to 24 hour stimulation of microglia with LPS [3 ng/mL] or domoic acid [1mM] leads to MMP-2 and MMP-9 secretion, cell-free media from the same cell culture dishes containing microglia used for MMP-9 mRNA analysis shown in Figures 8 and 9, were assayed for MMP-2 and MMP-9 release as described in Experimental. As shown in Figure 10, a statistically significant increase in MMP-9 release was observed 8, 16 and 24 hours after microglia were stimulated with LPS [3 ng/mL]. MMP-9 inverse optical density levels for 8, 16 and 24 hours were 0.69 ± 0.05, 0.874 ± 0.06, 0.91 ± 0.0005 (LPS-treated, n=2), respectively, vs. 0.46 ± 0.03 (untreated controls, n=2), P< 0.05 (8 hours) and P < 0.01(16 and 24 hours). In contrast to LPS, MMP-9 levels in the tissue culture media in domoic acid-treated microglia remained unchanged 4, 8, 16 and 24 hours after domoic acid treatment. Neither LPS nor domoic acid had a significant effect on MMP-2 release over the 24 hour-observation period.

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