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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 metabolites and TNFα in CSF from rabbit pups following IVH. OxyHb (A), metHb (B), and TNFα (D) were quantified in intraventricular CSF at 24 (n = 6), 48 (n = 6), and 72 (n = 10) hours, as described in the Methods section, and the ratio of oxyHb/metHb was calculated (C). Horizontal lines depict median values. Median values of metHb and TNFα were increased at 72 hours as compared to corresponding values at 24 and 48 hours (P <0.01, Mann–Whitney U). The correlation between TNFα and metHb (E) at 72 hours was determined by linear regression analysis (r2 = 0.896, P <0.001). CSF, cerebrospinal fluid; Hb, hemoglobin; IVH, intraventricular hemorrhage; metHb, methemoglobin; oxyHb, oxyhemoglobin.
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Figure 2: Hb metabolites and TNFα in CSF from rabbit pups following IVH. OxyHb (A), metHb (B), and TNFα (D) were quantified in intraventricular CSF at 24 (n = 6), 48 (n = 6), and 72 (n = 10) hours, as described in the Methods section, and the ratio of oxyHb/metHb was calculated (C). Horizontal lines depict median values. Median values of metHb and TNFα were increased at 72 hours as compared to corresponding values at 24 and 48 hours (P <0.01, Mann–Whitney U). The correlation between TNFα and metHb (E) at 72 hours was determined by linear regression analysis (r2 = 0.896, P <0.001). CSF, cerebrospinal fluid; Hb, hemoglobin; IVH, intraventricular hemorrhage; metHb, methemoglobin; oxyHb, oxyhemoglobin.

Mentions: Intraventricular CSF concentrations of extracellular oxyHb, metHb, and TNFα in rabbit pups with IVH were assessed at 24, 48, and 72 hours of age (Figure 2). The median concentration of oxyHb did not change significantly over time (Figure 2A) whereas that of metHb was significantly increased at 72 hours compared to values at 24 and 48 hours (Figure 2B), illustrated by the increasing ratio of metHb/oxyHb over time (Figure 2C). Similarly to metHb, the concentration of TNFα increased significantly over time and was highest at 72 hours as compared to 24 and 48 hours (Figure 2D). Furthermore, concentrations of metHb and TNFα exhibited a strong positive correlation at 72 hours (r2 = 0.896, P <0.001, Figure 2E) whereas no correlation was observed between oxyHb and TNFα (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)

Hb metabolites and TNFα in CSF from rabbit pups following IVH. OxyHb (A), metHb (B), and TNFα (D) were quantified in intraventricular CSF at 24 (n = 6), 48 (n = 6), and 72 (n = 10) hours, as described in the Methods section, and the ratio of oxyHb/metHb was calculated (C). Horizontal lines depict median values. Median values of metHb and TNFα were increased at 72 hours as compared to corresponding values at 24 and 48 hours (P <0.01, Mann–Whitney U). The correlation between TNFα and metHb (E) at 72 hours was determined by linear regression analysis (r2 = 0.896, P <0.001). CSF, cerebrospinal fluid; Hb, hemoglobin; IVH, intraventricular hemorrhage; metHb, methemoglobin; oxyHb, oxyhemoglobin.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Hb metabolites and TNFα in CSF from rabbit pups following IVH. OxyHb (A), metHb (B), and TNFα (D) were quantified in intraventricular CSF at 24 (n = 6), 48 (n = 6), and 72 (n = 10) hours, as described in the Methods section, and the ratio of oxyHb/metHb was calculated (C). Horizontal lines depict median values. Median values of metHb and TNFα were increased at 72 hours as compared to corresponding values at 24 and 48 hours (P <0.01, Mann–Whitney U). The correlation between TNFα and metHb (E) at 72 hours was determined by linear regression analysis (r2 = 0.896, P <0.001). CSF, cerebrospinal fluid; Hb, hemoglobin; IVH, intraventricular hemorrhage; metHb, methemoglobin; oxyHb, oxyhemoglobin.
Mentions: Intraventricular CSF concentrations of extracellular oxyHb, metHb, and TNFα in rabbit pups with IVH were assessed at 24, 48, and 72 hours of age (Figure 2). The median concentration of oxyHb did not change significantly over time (Figure 2A) whereas that of metHb was significantly increased at 72 hours compared to values at 24 and 48 hours (Figure 2B), illustrated by the increasing ratio of metHb/oxyHb over time (Figure 2C). Similarly to metHb, the concentration of TNFα increased significantly over time and was highest at 72 hours as compared to 24 and 48 hours (Figure 2D). Furthermore, concentrations of metHb and TNFα exhibited a strong positive correlation at 72 hours (r2 = 0.896, P <0.001, Figure 2E) whereas no correlation was observed between oxyHb and TNFα (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