Limits...
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.

Show MeSH

Related in: MedlinePlus

TNFα and GFAP immunofluorescence following IVH. Immunofluorescence of rabbit pup brain sections at 72 hours following IVH was performed as described in the Methods section. Sections were stained against TNFα (green, AF-488) and GFAP (red, Cy3) and displayed a co-staining for TNFα in GFAP-positive astrocytes. Scale bar, 20 μm. V, ventricle. GFAP, glial fibrillary acidic protein; IVH, intraventricular hemorrhage.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3750409&req=5

Figure 5: TNFα and GFAP immunofluorescence following IVH. Immunofluorescence of rabbit pup brain sections at 72 hours following IVH was performed as described in the Methods section. Sections were stained against TNFα (green, AF-488) and GFAP (red, Cy3) and displayed a co-staining for TNFα in GFAP-positive astrocytes. Scale bar, 20 μm. V, ventricle. GFAP, glial fibrillary acidic protein; IVH, intraventricular hemorrhage.

Mentions: The median levels of mRNA expression for TNFα, IL-1β, TLR-4 and HO-1 were increased in periventricular brain tissue in rabbit pups with IVH as compared to control pups (Figure 3). The increases in TNFα, IL-1β and TLR-4 mRNA were significant (P <0.05, TNFα; P <0.01, IL-1β; P <0.001, TLR-4) at 72 hours but not at 24 hours. Correspondingly, the median level of TNFα protein was increased at 72 hours in IVH pups (67.9 pg/mg total protein) as compared to control pups (15.4 pg/mg total protein; P = 0.02) but not at 24 hours (Figure 4). In addition, immunohistochemistry showed positive TNFα staining in periventricular brain tissue in rabbit pups with IVH at 72 hours (Figure 5). Counter staining for astrocyte activation (GFAP-positive staining) revealed the presence of TNFα in GFAP-positive astrocytes but also in non–GFAP-positive cells.


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)

TNFα and GFAP immunofluorescence following IVH. Immunofluorescence of rabbit pup brain sections at 72 hours following IVH was performed as described in the Methods section. Sections were stained against TNFα (green, AF-488) and GFAP (red, Cy3) and displayed a co-staining for TNFα in GFAP-positive astrocytes. Scale bar, 20 μm. V, ventricle. GFAP, glial fibrillary acidic protein; IVH, intraventricular hemorrhage.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: TNFα and GFAP immunofluorescence following IVH. Immunofluorescence of rabbit pup brain sections at 72 hours following IVH was performed as described in the Methods section. Sections were stained against TNFα (green, AF-488) and GFAP (red, Cy3) and displayed a co-staining for TNFα in GFAP-positive astrocytes. Scale bar, 20 μm. V, ventricle. GFAP, glial fibrillary acidic protein; IVH, intraventricular hemorrhage.
Mentions: The median levels of mRNA expression for TNFα, IL-1β, TLR-4 and HO-1 were increased in periventricular brain tissue in rabbit pups with IVH as compared to control pups (Figure 3). The increases in TNFα, IL-1β and TLR-4 mRNA were significant (P <0.05, TNFα; P <0.01, IL-1β; P <0.001, TLR-4) at 72 hours but not at 24 hours. Correspondingly, the median level of TNFα protein was increased at 72 hours in IVH pups (67.9 pg/mg total protein) as compared to control pups (15.4 pg/mg total protein; P = 0.02) but not at 24 hours (Figure 4). In addition, immunohistochemistry showed positive TNFα staining in periventricular brain tissue in rabbit pups with IVH at 72 hours (Figure 5). Counter staining for astrocyte activation (GFAP-positive staining) revealed the presence of TNFα in GFAP-positive astrocytes but also in non–GFAP-positive cells.

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