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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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High-frequency ultrasound of normal brain and cerebral IVH. Coronal images obtained by high-frequency ultrasound displaying a normal brain with no IVH and cerebral IVH at six hours of age in preterm rabbit pups. Vertical ruler indicates 10 mm. IVH, intraventricular hemorrhage.
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Figure 1: High-frequency ultrasound of normal brain and cerebral IVH. Coronal images obtained by high-frequency ultrasound displaying a normal brain with no IVH and cerebral IVH at six hours of age in preterm rabbit pups. Vertical ruler indicates 10 mm. IVH, intraventricular hemorrhage.

Mentions: The animal protocols were approved by the Swedish Animal Ethics Committee in Lund. The experiments were performed using rabbit pups from a half-breed between New Zealand White and Lop, delivered at gestational day 29 (full gestational age 32 days). The pups were delivered by cesarean section after the does were anesthetized with i.v. propofol (5 mg/kg) and by local infiltration of the abdominal wall using lidocaine with adrenaline (10 mg/ml + 5 μl/ml, 20 to 30 ml). After delivery, the pups were dried vigorously, weighed, and placed in an infant incubator with a constant temperature of 36°C and 60% ambient humidity. At two to three hours of age, the pups were fed 1 ml of cat milk formula (KMR; PetAg Inc., Hampshire, IL, USA), and subsequently every 12 hours, increasing each meal by 0.5 ml. At two hours of age, 165 pups (from 25 litters) received an i.p. injection of 50% glycerol (6.5 g/kg, endotoxin-free as analyzed below) to induce IVH [10,19]. Using HFU imaging of the brain (VisualSonics Vevo 2100, VisualSonics Inc., Toronto, Canada, with a MS-550D 40MHz transducer) enabled accurate distinction of hemorrhagic extension [22] and was performed at 6, 24, 48, and 72 hours of age. At six hours of age, 110 pups (67%) displayed severe IVH (distended lateral ventricles filled by high-echogenic content with no parenchymal extension); 15 pups (9%) displayed a small/minor IVH (high-echogenic content within the lateral ventricles with no ventricular distention); and 40 pups (24%) displayed no signs of IVH. Images of pups with severe IVH or without signs of hemorrhage, as determined by HFU at six hours of age, are given in Figure 1. In this study, only pups with severe IVH (here referred to as the IVH group) and pups with no signs of cerebral hemorrhage (sham control group) were included (as determined by HFU at six hours of age). All control animals used in this study had received an i.p. injection of glycerol but did not exhibit any sign of IVH on cerebral ultrasound, thus ensuring that differences seen in IVH animals and control animals were not due to glycerol toxicity. In 22 rabbit pups with severe IVH, ultrasound-guided CSF sampling was performed at 24 (n = 6), 48 (n = 6) and 72 (n = 10) hours of age, as described previously [22]. Following CSF sampling, which was only performed once in each pup, the pups were euthanized and not further included in the study, that is, no further tissue or CSF sampling was performed on these pups. Accurate intraventricular CSF sampling from control pups was not possible due to the small, slit-like size of the ventricles in pups without hemorrhage. Immediately after sampling, the CSF samples were centrifuged (2,000 × g, 20°C, 10 minutes) to remove cells, and the supernatant was stored at -80°C until further analysis, as described below.


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)

High-frequency ultrasound of normal brain and cerebral IVH. Coronal images obtained by high-frequency ultrasound displaying a normal brain with no IVH and cerebral IVH at six hours of age in preterm rabbit pups. Vertical ruler indicates 10 mm. IVH, intraventricular hemorrhage.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: High-frequency ultrasound of normal brain and cerebral IVH. Coronal images obtained by high-frequency ultrasound displaying a normal brain with no IVH and cerebral IVH at six hours of age in preterm rabbit pups. Vertical ruler indicates 10 mm. IVH, intraventricular hemorrhage.
Mentions: The animal protocols were approved by the Swedish Animal Ethics Committee in Lund. The experiments were performed using rabbit pups from a half-breed between New Zealand White and Lop, delivered at gestational day 29 (full gestational age 32 days). The pups were delivered by cesarean section after the does were anesthetized with i.v. propofol (5 mg/kg) and by local infiltration of the abdominal wall using lidocaine with adrenaline (10 mg/ml + 5 μl/ml, 20 to 30 ml). After delivery, the pups were dried vigorously, weighed, and placed in an infant incubator with a constant temperature of 36°C and 60% ambient humidity. At two to three hours of age, the pups were fed 1 ml of cat milk formula (KMR; PetAg Inc., Hampshire, IL, USA), and subsequently every 12 hours, increasing each meal by 0.5 ml. At two hours of age, 165 pups (from 25 litters) received an i.p. injection of 50% glycerol (6.5 g/kg, endotoxin-free as analyzed below) to induce IVH [10,19]. Using HFU imaging of the brain (VisualSonics Vevo 2100, VisualSonics Inc., Toronto, Canada, with a MS-550D 40MHz transducer) enabled accurate distinction of hemorrhagic extension [22] and was performed at 6, 24, 48, and 72 hours of age. At six hours of age, 110 pups (67%) displayed severe IVH (distended lateral ventricles filled by high-echogenic content with no parenchymal extension); 15 pups (9%) displayed a small/minor IVH (high-echogenic content within the lateral ventricles with no ventricular distention); and 40 pups (24%) displayed no signs of IVH. Images of pups with severe IVH or without signs of hemorrhage, as determined by HFU at six hours of age, are given in Figure 1. In this study, only pups with severe IVH (here referred to as the IVH group) and pups with no signs of cerebral hemorrhage (sham control group) were included (as determined by HFU at six hours of age). All control animals used in this study had received an i.p. injection of glycerol but did not exhibit any sign of IVH on cerebral ultrasound, thus ensuring that differences seen in IVH animals and control animals were not due to glycerol toxicity. In 22 rabbit pups with severe IVH, ultrasound-guided CSF sampling was performed at 24 (n = 6), 48 (n = 6) and 72 (n = 10) hours of age, as described previously [22]. Following CSF sampling, which was only performed once in each pup, the pups were euthanized and not further included in the study, that is, no further tissue or CSF sampling was performed on these pups. Accurate intraventricular CSF sampling from control pups was not possible due to the small, slit-like size of the ventricles in pups without hemorrhage. Immediately after sampling, the CSF samples were centrifuged (2,000 × g, 20°C, 10 minutes) to remove cells, and the supernatant was stored at -80°C until further analysis, as described below.

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