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Hemoglobin induces inflammation after preterm intraventricular hemorrhage by methemoglobin formation.

Gram M, Sveinsdottir S, Ruscher K, Hansson SR, Cinthio M, Akerström B, Ley D - J Neuroinflammation (2013)

Bottom Line: Also, the mRNA expression of TNFα, IL-1β, and Toll-like receptor-4 and TNFα protein levels were significantly increased in periventricular tissue at 72 hours, which was accompanied by extensive astrocyte activation (that is, glial fibrillary acidic protein (GFAP)staining).Thus, the formation of metHb might be a crucial initial event in the development of brain damage following preterm IVH.Accordingly, removal, scavenging, or neutralization of Hb could present a therapeutic opportunity and plausible approach to decreasing the damage in the immature brain following preterm IVH.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Infection Medicine, Lund University, S-221 84 Lund, Sweden. magnus.gram@med.lu.se

ABSTRACT

Background: Cerebral intraventricular hemorrhage (IVH) is a major cause of severe neurodevelopmental impairment in preterm infants. To date, no therapy is available that prevents infants from developing serious neurological disability following IVH. Thus, to develop treatment strategies for IVH, it is essential to characterize the initial sequence of molecular events that leads to brain damage. In this study, we investigated extracellular hemoglobin (Hb) as a causal initiator of inflammation in preterm IVH.

Methods: Using a preterm rabbit pup model, we investigated the molecular mechanisms and events following IVH. We also characterized the concentrations of cell-free Hb metabolites and pro-inflammatory mediators in the cerebrospinal fluid (CSF) of preterm human infants and rabbit pups. Finally, Hb metabolites were evaluated as causal initiators of inflammation in primary rabbit astrocyte cell cultures.

Results: Following IVH in preterm rabbit pups, the intraventricular CSF concentration of cell-free methemoglobin (metHb) increased from 24 to 72 hours and was strongly correlated with the concentration of TNFα at 72 hours (r2 = 0.896, P <0.001). Also, the mRNA expression of TNFα, IL-1β, and Toll-like receptor-4 and TNFα protein levels were significantly increased in periventricular tissue at 72 hours, which was accompanied by extensive astrocyte activation (that is, glial fibrillary acidic protein (GFAP)staining). Furthermore, exposure of primary rabbit astrocyte cell cultures to metHb caused a dose-dependent increase in TNFα mRNA and protein levels, which was not observed following exposure to oxyhemoglobin (oxyHb) or hemin. Finally, a positive correlation (r2 = 0.237, P <0.03) between metHb and TNFα concentrations was observed in the CSF of preterm human infants following IVH.

Conclusions: Following preterm IVH, increased metHb formation in the intraventricular space induces expression of pro-inflammatory cytokines. Thus, the formation of metHb might be a crucial initial event in the development of brain damage following preterm IVH. Accordingly, removal, scavenging, or neutralization of Hb could present a therapeutic opportunity and plausible approach to decreasing the damage in the immature brain following preterm IVH.

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Fenton reaction–induced TLR-4, TNFα, IL-1β and HO-1 mRNA expression in astrocyte cell cultures. Primary rabbit astrocyte cell cultures were exposed to a mixture of (NH4)Fe(SO4)2, hydrogen peroxide and ascorbate (the Fenton reaction) for four hours at concentrations of 10 μM (NH4)Fe(SO4)2 + 100 μM ascorbate + 20 μM H2O2 (white bars) or 50 μM (NH4)Fe(SO4)2, 500 μM ascorbate + 100 μM H2O2 (shaded bars). mRNA expression of TLR-4 (A), TNFα (B), IL-1β (C) and HO-1 (D) was determined using real-time PCR, as described in the Methods section. The mRNA expression of TLR-4, TNFα, IL-1β and HO-1 was normalized against GAPDH and is given as fold change. The fold-change values were calculated by normalizing against control samples from untreated cells. Results are from triplicate experiments and presented as mean ± SEM. Differences between the respective exposures and control conditions were analyzed using Mann–Whitney U. *** P <0.001. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HO-1, heme oxygenase; SEM, standard error of the mean; TLR-4, Toll-like receptor-4.
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Figure 8: Fenton reaction–induced TLR-4, TNFα, IL-1β and HO-1 mRNA expression in astrocyte cell cultures. Primary rabbit astrocyte cell cultures were exposed to a mixture of (NH4)Fe(SO4)2, hydrogen peroxide and ascorbate (the Fenton reaction) for four hours at concentrations of 10 μM (NH4)Fe(SO4)2 + 100 μM ascorbate + 20 μM H2O2 (white bars) or 50 μM (NH4)Fe(SO4)2, 500 μM ascorbate + 100 μM H2O2 (shaded bars). mRNA expression of TLR-4 (A), TNFα (B), IL-1β (C) and HO-1 (D) was determined using real-time PCR, as described in the Methods section. The mRNA expression of TLR-4, TNFα, IL-1β and HO-1 was normalized against GAPDH and is given as fold change. The fold-change values were calculated by normalizing against control samples from untreated cells. Results are from triplicate experiments and presented as mean ± SEM. Differences between the respective exposures and control conditions were analyzed using Mann–Whitney U. *** P <0.001. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HO-1, heme oxygenase; SEM, standard error of the mean; TLR-4, Toll-like receptor-4.

Mentions: Exposure to the Fenton reaction, (that is, a mixture of Fe3+, ascorbate and hydrogen peroxide) was performed to evaluate the pro-inflammatory effect of the hydroxyl radical. Exposure to the Fenton reaction–generated hydroxyl radicals caused a highly significant upregulation of TLR-4 and HO-1 mRNA expression, but not of TNFα or IL-1β (Figure 8). Furthermore, no increase in TNFα protein concentrations was observed in culture medium of cells exposed to the Fenton-reaction mixture, as compared to control cultures (not shown).


Hemoglobin induces inflammation after preterm intraventricular hemorrhage by methemoglobin formation.

Gram M, Sveinsdottir S, Ruscher K, Hansson SR, Cinthio M, Akerström B, Ley D - J Neuroinflammation (2013)

Fenton reaction–induced TLR-4, TNFα, IL-1β and HO-1 mRNA expression in astrocyte cell cultures. Primary rabbit astrocyte cell cultures were exposed to a mixture of (NH4)Fe(SO4)2, hydrogen peroxide and ascorbate (the Fenton reaction) for four hours at concentrations of 10 μM (NH4)Fe(SO4)2 + 100 μM ascorbate + 20 μM H2O2 (white bars) or 50 μM (NH4)Fe(SO4)2, 500 μM ascorbate + 100 μM H2O2 (shaded bars). mRNA expression of TLR-4 (A), TNFα (B), IL-1β (C) and HO-1 (D) was determined using real-time PCR, as described in the Methods section. The mRNA expression of TLR-4, TNFα, IL-1β and HO-1 was normalized against GAPDH and is given as fold change. The fold-change values were calculated by normalizing against control samples from untreated cells. Results are from triplicate experiments and presented as mean ± SEM. Differences between the respective exposures and control conditions were analyzed using Mann–Whitney U. *** P <0.001. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HO-1, heme oxygenase; SEM, standard error of the mean; TLR-4, Toll-like receptor-4.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3750409&req=5

Figure 8: Fenton reaction–induced TLR-4, TNFα, IL-1β and HO-1 mRNA expression in astrocyte cell cultures. Primary rabbit astrocyte cell cultures were exposed to a mixture of (NH4)Fe(SO4)2, hydrogen peroxide and ascorbate (the Fenton reaction) for four hours at concentrations of 10 μM (NH4)Fe(SO4)2 + 100 μM ascorbate + 20 μM H2O2 (white bars) or 50 μM (NH4)Fe(SO4)2, 500 μM ascorbate + 100 μM H2O2 (shaded bars). mRNA expression of TLR-4 (A), TNFα (B), IL-1β (C) and HO-1 (D) was determined using real-time PCR, as described in the Methods section. The mRNA expression of TLR-4, TNFα, IL-1β and HO-1 was normalized against GAPDH and is given as fold change. The fold-change values were calculated by normalizing against control samples from untreated cells. Results are from triplicate experiments and presented as mean ± SEM. Differences between the respective exposures and control conditions were analyzed using Mann–Whitney U. *** P <0.001. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HO-1, heme oxygenase; SEM, standard error of the mean; TLR-4, Toll-like receptor-4.
Mentions: Exposure to the Fenton reaction, (that is, a mixture of Fe3+, ascorbate and hydrogen peroxide) was performed to evaluate the pro-inflammatory effect of the hydroxyl radical. Exposure to the Fenton reaction–generated hydroxyl radicals caused a highly significant upregulation of TLR-4 and HO-1 mRNA expression, but not of TNFα or IL-1β (Figure 8). Furthermore, no increase in TNFα protein concentrations was observed in culture medium of cells exposed to the Fenton-reaction mixture, as compared to control cultures (not shown).

Bottom Line: Also, the mRNA expression of TNFα, IL-1β, and Toll-like receptor-4 and TNFα protein levels were significantly increased in periventricular tissue at 72 hours, which was accompanied by extensive astrocyte activation (that is, glial fibrillary acidic protein (GFAP)staining).Thus, the formation of metHb might be a crucial initial event in the development of brain damage following preterm IVH.Accordingly, removal, scavenging, or neutralization of Hb could present a therapeutic opportunity and plausible approach to decreasing the damage in the immature brain following preterm IVH.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Infection Medicine, Lund University, S-221 84 Lund, Sweden. magnus.gram@med.lu.se

ABSTRACT

Background: Cerebral intraventricular hemorrhage (IVH) is a major cause of severe neurodevelopmental impairment in preterm infants. To date, no therapy is available that prevents infants from developing serious neurological disability following IVH. Thus, to develop treatment strategies for IVH, it is essential to characterize the initial sequence of molecular events that leads to brain damage. In this study, we investigated extracellular hemoglobin (Hb) as a causal initiator of inflammation in preterm IVH.

Methods: Using a preterm rabbit pup model, we investigated the molecular mechanisms and events following IVH. We also characterized the concentrations of cell-free Hb metabolites and pro-inflammatory mediators in the cerebrospinal fluid (CSF) of preterm human infants and rabbit pups. Finally, Hb metabolites were evaluated as causal initiators of inflammation in primary rabbit astrocyte cell cultures.

Results: Following IVH in preterm rabbit pups, the intraventricular CSF concentration of cell-free methemoglobin (metHb) increased from 24 to 72 hours and was strongly correlated with the concentration of TNFα at 72 hours (r2 = 0.896, P <0.001). Also, the mRNA expression of TNFα, IL-1β, and Toll-like receptor-4 and TNFα protein levels were significantly increased in periventricular tissue at 72 hours, which was accompanied by extensive astrocyte activation (that is, glial fibrillary acidic protein (GFAP)staining). Furthermore, exposure of primary rabbit astrocyte cell cultures to metHb caused a dose-dependent increase in TNFα mRNA and protein levels, which was not observed following exposure to oxyhemoglobin (oxyHb) or hemin. Finally, a positive correlation (r2 = 0.237, P <0.03) between metHb and TNFα concentrations was observed in the CSF of preterm human infants following IVH.

Conclusions: Following preterm IVH, increased metHb formation in the intraventricular space induces expression of pro-inflammatory cytokines. Thus, the formation of metHb might be a crucial initial event in the development of brain damage following preterm IVH. Accordingly, removal, scavenging, or neutralization of Hb could present a therapeutic opportunity and plausible approach to decreasing the damage in the immature brain following preterm IVH.

Show MeSH
Related in: MedlinePlus