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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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Hb metabolite–induced TNFα protein secretion in astrocyte cell cultures. Determination of TNFα protein concentration in culture medium of primary rabbit astrocyte cell cultures, exposed for one to four hours to oxyHb, cyan-Hb, metHb and hemin at 1, 5 and 15 μM, respectively, using ELISA, as described in the Methods section. Exposure to oxyHb (filled circles) and cyan-Hb (open circles) is illustrated in panel A and metHb (filled squares) and hemin (open squares) in panel B. Continuous line = 15 μM; dotted line = 5 μM; hatched line = 1 μM. Results are from triplicate experiments and are presented as mean ± SEM. MetHb at 1, 5 and 15 μM versus control, all P <0.01 (ANOVA for repeated measures). ANOVA, analysis of variance; Hb, hemoglobin; metHb, methemoglobin; oxyHb, oxyhemoglobin; SEM, standard error of the mean.
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Figure 7: Hb metabolite–induced TNFα protein secretion in astrocyte cell cultures. Determination of TNFα protein concentration in culture medium of primary rabbit astrocyte cell cultures, exposed for one to four hours to oxyHb, cyan-Hb, metHb and hemin at 1, 5 and 15 μM, respectively, using ELISA, as described in the Methods section. Exposure to oxyHb (filled circles) and cyan-Hb (open circles) is illustrated in panel A and metHb (filled squares) and hemin (open squares) in panel B. Continuous line = 15 μM; dotted line = 5 μM; hatched line = 1 μM. Results are from triplicate experiments and are presented as mean ± SEM. MetHb at 1, 5 and 15 μM versus control, all P <0.01 (ANOVA for repeated measures). ANOVA, analysis of variance; Hb, hemoglobin; metHb, methemoglobin; oxyHb, oxyhemoglobin; SEM, standard error of the mean.

Mentions: The strong correlation observed between extracellular metHb and TNFα in intraventricular CSF suggested that metHb or metabolites of Hb may be causal initiators of inflammation following IVH. These relationships were evaluated in primary rabbit astrocyte cell cultures. Exposing the astrocytes to oxyHb, cyan-Hb, metHb or hemin at concentrations of 1 to 15 μM led to dose-dependent HO-1 mRNA expression with each reagent as compared to controls (Figure 6C). TNFα mRNA expression increased dose-dependently only after exposure to metHb but to not oxyHb, cyan-Hb or hemin (Figure 6A) whereas no reagent affected IL-1β mRNA expression as compared to control cultures (Figure 6B). Furthermore, exposure to metHb caused a significant, dose-dependent increase in TNFα protein concentrations in culture medium at one, two, and four hours of exposure, with the highest levels noted after two hours (Figure 7B). Exposure to oxyHb at corresponding concentrations resulted in a small but significant increase in levels of TNFα protein (Figure 7A). This increase was not observed following cyan-Hb exposure, strongly indicating that conversion of oxyHb to metHb is necessary for TNFα induction. Additionally, exposure to free hemin did not cause a measurable increase in TNFα protein in culture medium (Figure 7B). In order to assure that the results obtained with metHb were not caused by endotoxin contamination in the purchased metHb derivative, confirming experiments were performed. Endotoxin-free oxyHb (in-house purified as described in the Methods section) was oxidized to metHb under sterile conditions (as described in the Methods section), and astrocytes were exposed to oxyHb and metHb as described above. Analysis of TNFα mRNA from astrocytes [see Additional file 1: Figure S1A] and of TNFα protein [see Additional file 1: Figure S1B] in culture medium displayed very similar results as those obtained with purchased and purified metHb.


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)

Hb metabolite–induced TNFα protein secretion in astrocyte cell cultures. Determination of TNFα protein concentration in culture medium of primary rabbit astrocyte cell cultures, exposed for one to four hours to oxyHb, cyan-Hb, metHb and hemin at 1, 5 and 15 μM, respectively, using ELISA, as described in the Methods section. Exposure to oxyHb (filled circles) and cyan-Hb (open circles) is illustrated in panel A and metHb (filled squares) and hemin (open squares) in panel B. Continuous line = 15 μM; dotted line = 5 μM; hatched line = 1 μM. Results are from triplicate experiments and are presented as mean ± SEM. MetHb at 1, 5 and 15 μM versus control, all P <0.01 (ANOVA for repeated measures). ANOVA, analysis of variance; Hb, hemoglobin; metHb, methemoglobin; oxyHb, oxyhemoglobin; SEM, standard error of the mean.
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Figure 7: Hb metabolite–induced TNFα protein secretion in astrocyte cell cultures. Determination of TNFα protein concentration in culture medium of primary rabbit astrocyte cell cultures, exposed for one to four hours to oxyHb, cyan-Hb, metHb and hemin at 1, 5 and 15 μM, respectively, using ELISA, as described in the Methods section. Exposure to oxyHb (filled circles) and cyan-Hb (open circles) is illustrated in panel A and metHb (filled squares) and hemin (open squares) in panel B. Continuous line = 15 μM; dotted line = 5 μM; hatched line = 1 μM. Results are from triplicate experiments and are presented as mean ± SEM. MetHb at 1, 5 and 15 μM versus control, all P <0.01 (ANOVA for repeated measures). ANOVA, analysis of variance; Hb, hemoglobin; metHb, methemoglobin; oxyHb, oxyhemoglobin; SEM, standard error of the mean.
Mentions: The strong correlation observed between extracellular metHb and TNFα in intraventricular CSF suggested that metHb or metabolites of Hb may be causal initiators of inflammation following IVH. These relationships were evaluated in primary rabbit astrocyte cell cultures. Exposing the astrocytes to oxyHb, cyan-Hb, metHb or hemin at concentrations of 1 to 15 μM led to dose-dependent HO-1 mRNA expression with each reagent as compared to controls (Figure 6C). TNFα mRNA expression increased dose-dependently only after exposure to metHb but to not oxyHb, cyan-Hb or hemin (Figure 6A) whereas no reagent affected IL-1β mRNA expression as compared to control cultures (Figure 6B). Furthermore, exposure to metHb caused a significant, dose-dependent increase in TNFα protein concentrations in culture medium at one, two, and four hours of exposure, with the highest levels noted after two hours (Figure 7B). Exposure to oxyHb at corresponding concentrations resulted in a small but significant increase in levels of TNFα protein (Figure 7A). This increase was not observed following cyan-Hb exposure, strongly indicating that conversion of oxyHb to metHb is necessary for TNFα induction. Additionally, exposure to free hemin did not cause a measurable increase in TNFα protein in culture medium (Figure 7B). In order to assure that the results obtained with metHb were not caused by endotoxin contamination in the purchased metHb derivative, confirming experiments were performed. Endotoxin-free oxyHb (in-house purified as described in the Methods section) was oxidized to metHb under sterile conditions (as described in the Methods section), and astrocytes were exposed to oxyHb and metHb as described above. Analysis of TNFα mRNA from astrocytes [see Additional file 1: Figure S1A] and of TNFα protein [see Additional file 1: Figure S1B] in culture medium displayed very similar results as those obtained with purchased and purified metHb.

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