Limits...
Extracellular hemoglobin - mediator of inflammation and cell death in the choroid plexus following preterm intraventricular hemorrhage.

Gram M, Sveinsdottir S, Cinthio M, Sveinsdottir K, Hansson SR, Mörgelin M, Åkerström B, Ley D - J Neuroinflammation (2014)

Bottom Line: Following IVH + PHVD, an up-regulation of mRNA for the receptor-related genes TLR-4, IL1R1, FAS, the transcription factor NF-Κβ and for the pro-inflammatory and chemotactic effector molecules, IL-1β, TNFα, MCP-1, IL-8, and IL-6 was observed in the choroid plexus at 24 and 72 hours.This was associated with structural disintegration, caspase activation and cell death in the choroid plexus epithelium.In vitro characterization of choroid plexus epithelial cells, following exposure to hemorrhagic CSF and to the Hb-metabolites metHb and heme, displayed apoptotic and necrotic cell death and an up-regulation of receptor-related and inflammatory effector molecules similar to that observed in vivo following IVH + PHVD.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Lund University, Lund, S-221 84, Sweden. magnus.gram@med.lu.se.

ABSTRACT

Background: Intraventricular hemorrhage (IVH) with post-hemorrhagic ventricular dilatation (PHVD) is a major cause of neurodevelopmental impairment and mortality in preterm infants. The mechanisms leading to PHVD and brain damage remain largely unknown. The choroid plexus and the ependyma, which constitute an essential part of the blood-brain barrier (BBB), are the first structures to encounter the damaging effects of extravasated blood. The breakdown of the BBB is a critical upstream event leading to brain damage following IVH. In this study we investigated the impact of hemorrhage and hemoglobin (Hb) metabolites on the choroid plexus epithelium.

Methods: Using a preterm rabbit pup model of IVH, the structural and functional integrity, cellular, inflammatory and oxidative response of the choroid plexus, at 24 and 72 hours following IVH + PHVD, were investigated. In order to further characterize cellular and molecular mechanisms, primary human choroid plexus epithelial cells were exposed to cerebrospinal fluid (CSF) from preterm infants with IVH as well as to Hb-metabolites. Finally, the blocking effects of the Hb-scavenger haptoglobin (Hp) were investigated both in vivo and in vitro.

Results: Following IVH + PHVD, an up-regulation of mRNA for the receptor-related genes TLR-4, IL1R1, FAS, the transcription factor NF-Κβ and for the pro-inflammatory and chemotactic effector molecules, IL-1β, TNFα, MCP-1, IL-8, and IL-6 was observed in the choroid plexus at 24 and 72 hours. This was associated with structural disintegration, caspase activation and cell death in the choroid plexus epithelium. In vitro characterization of choroid plexus epithelial cells, following exposure to hemorrhagic CSF and to the Hb-metabolites metHb and heme, displayed apoptotic and necrotic cell death and an up-regulation of receptor-related and inflammatory effector molecules similar to that observed in vivo following IVH + PHVD. Intraventricular injection of the Hb-scavenger Hp in vivo and co-incubation with Hp in vitro reversed or reduced the cellular activation, inflammatory response, structural damage and cell death.

Conclusion: Hb-metabolites are important causal initiators of cell death following IVH and removal or scavenging of Hb-metabolites may present an efficient means to reduce the damage to the immature brain following IVH.

Show MeSH

Related in: MedlinePlus

Analysis of structural damage and cell death in the choroid plexus. Rabbit pups with IVH + PHVD (B, D, F) or sham controls (A, C, E) were euthanized at 72 hours of age and the brains were removed from the skulls, sectioned at the level of the midseptal nucleus and placed in formaldehyde for subsequent histochemical analysis with H&E (A and B) and IHC analysis against cleaved caspase-3 (E and F) as described in Materials and Methods. For EM-IHC (C and D), the choroid plexus from pups with IVH + PHVD and sham controls at 72 hours was fixed and prepared as described in the Materials and Methods and observed in a Jeol JEM 1230 electron microscope. Recessed picture display mitochondrial structure. P = periventricular tissue, CP = choroid plexus, V = ventricle, Vi = villi, M = mitochondria. Arrow illustrates cleaved caspase-3 positive staining. Scale bar in (A) indicate 50 μm (is applicable for panels (A) and (B)), in (C) indicate 500 nm (is applicable for (C) and (D)) and (E) indicate 20 μm (is applicable for (E) and (F)). IHC, immunohistochemistry; IVH, intraventricular hemorrhage; PHVD, post-hemorrhagic ventricular dilatation.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4269927&req=5

Fig1: Analysis of structural damage and cell death in the choroid plexus. Rabbit pups with IVH + PHVD (B, D, F) or sham controls (A, C, E) were euthanized at 72 hours of age and the brains were removed from the skulls, sectioned at the level of the midseptal nucleus and placed in formaldehyde for subsequent histochemical analysis with H&E (A and B) and IHC analysis against cleaved caspase-3 (E and F) as described in Materials and Methods. For EM-IHC (C and D), the choroid plexus from pups with IVH + PHVD and sham controls at 72 hours was fixed and prepared as described in the Materials and Methods and observed in a Jeol JEM 1230 electron microscope. Recessed picture display mitochondrial structure. P = periventricular tissue, CP = choroid plexus, V = ventricle, Vi = villi, M = mitochondria. Arrow illustrates cleaved caspase-3 positive staining. Scale bar in (A) indicate 50 μm (is applicable for panels (A) and (B)), in (C) indicate 500 nm (is applicable for (C) and (D)) and (E) indicate 20 μm (is applicable for (E) and (F)). IHC, immunohistochemistry; IVH, intraventricular hemorrhage; PHVD, post-hemorrhagic ventricular dilatation.

Mentions: Structural and cellular damage to the choroid plexus was investigated following IVH in preterm rabbit pups. Histopathological analysis with H&E staining displayed extensive structural damage to the epithelial cells at 72 hours after IVH (Figure 1A and B). Substantial ultrastructural damage to the choroid plexus was seen as determined by EM; for example, disintegration of normal epithelial structure and swollen mitochondria (Figure 1C and D). IHC analysis of apoptosis, using antibodies towards cleaved caspase-3, displayed a strong staining at 24 hours following IVH (Figure 1F), which was not observed in the control animals (Figure 1E).Figure 1


Extracellular hemoglobin - mediator of inflammation and cell death in the choroid plexus following preterm intraventricular hemorrhage.

Gram M, Sveinsdottir S, Cinthio M, Sveinsdottir K, Hansson SR, Mörgelin M, Åkerström B, Ley D - J Neuroinflammation (2014)

Analysis of structural damage and cell death in the choroid plexus. Rabbit pups with IVH + PHVD (B, D, F) or sham controls (A, C, E) were euthanized at 72 hours of age and the brains were removed from the skulls, sectioned at the level of the midseptal nucleus and placed in formaldehyde for subsequent histochemical analysis with H&E (A and B) and IHC analysis against cleaved caspase-3 (E and F) as described in Materials and Methods. For EM-IHC (C and D), the choroid plexus from pups with IVH + PHVD and sham controls at 72 hours was fixed and prepared as described in the Materials and Methods and observed in a Jeol JEM 1230 electron microscope. Recessed picture display mitochondrial structure. P = periventricular tissue, CP = choroid plexus, V = ventricle, Vi = villi, M = mitochondria. Arrow illustrates cleaved caspase-3 positive staining. Scale bar in (A) indicate 50 μm (is applicable for panels (A) and (B)), in (C) indicate 500 nm (is applicable for (C) and (D)) and (E) indicate 20 μm (is applicable for (E) and (F)). IHC, immunohistochemistry; IVH, intraventricular hemorrhage; PHVD, post-hemorrhagic ventricular dilatation.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4269927&req=5

Fig1: Analysis of structural damage and cell death in the choroid plexus. Rabbit pups with IVH + PHVD (B, D, F) or sham controls (A, C, E) were euthanized at 72 hours of age and the brains were removed from the skulls, sectioned at the level of the midseptal nucleus and placed in formaldehyde for subsequent histochemical analysis with H&E (A and B) and IHC analysis against cleaved caspase-3 (E and F) as described in Materials and Methods. For EM-IHC (C and D), the choroid plexus from pups with IVH + PHVD and sham controls at 72 hours was fixed and prepared as described in the Materials and Methods and observed in a Jeol JEM 1230 electron microscope. Recessed picture display mitochondrial structure. P = periventricular tissue, CP = choroid plexus, V = ventricle, Vi = villi, M = mitochondria. Arrow illustrates cleaved caspase-3 positive staining. Scale bar in (A) indicate 50 μm (is applicable for panels (A) and (B)), in (C) indicate 500 nm (is applicable for (C) and (D)) and (E) indicate 20 μm (is applicable for (E) and (F)). IHC, immunohistochemistry; IVH, intraventricular hemorrhage; PHVD, post-hemorrhagic ventricular dilatation.
Mentions: Structural and cellular damage to the choroid plexus was investigated following IVH in preterm rabbit pups. Histopathological analysis with H&E staining displayed extensive structural damage to the epithelial cells at 72 hours after IVH (Figure 1A and B). Substantial ultrastructural damage to the choroid plexus was seen as determined by EM; for example, disintegration of normal epithelial structure and swollen mitochondria (Figure 1C and D). IHC analysis of apoptosis, using antibodies towards cleaved caspase-3, displayed a strong staining at 24 hours following IVH (Figure 1F), which was not observed in the control animals (Figure 1E).Figure 1

Bottom Line: Following IVH + PHVD, an up-regulation of mRNA for the receptor-related genes TLR-4, IL1R1, FAS, the transcription factor NF-Κβ and for the pro-inflammatory and chemotactic effector molecules, IL-1β, TNFα, MCP-1, IL-8, and IL-6 was observed in the choroid plexus at 24 and 72 hours.This was associated with structural disintegration, caspase activation and cell death in the choroid plexus epithelium.In vitro characterization of choroid plexus epithelial cells, following exposure to hemorrhagic CSF and to the Hb-metabolites metHb and heme, displayed apoptotic and necrotic cell death and an up-regulation of receptor-related and inflammatory effector molecules similar to that observed in vivo following IVH + PHVD.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Lund University, Lund, S-221 84, Sweden. magnus.gram@med.lu.se.

ABSTRACT

Background: Intraventricular hemorrhage (IVH) with post-hemorrhagic ventricular dilatation (PHVD) is a major cause of neurodevelopmental impairment and mortality in preterm infants. The mechanisms leading to PHVD and brain damage remain largely unknown. The choroid plexus and the ependyma, which constitute an essential part of the blood-brain barrier (BBB), are the first structures to encounter the damaging effects of extravasated blood. The breakdown of the BBB is a critical upstream event leading to brain damage following IVH. In this study we investigated the impact of hemorrhage and hemoglobin (Hb) metabolites on the choroid plexus epithelium.

Methods: Using a preterm rabbit pup model of IVH, the structural and functional integrity, cellular, inflammatory and oxidative response of the choroid plexus, at 24 and 72 hours following IVH + PHVD, were investigated. In order to further characterize cellular and molecular mechanisms, primary human choroid plexus epithelial cells were exposed to cerebrospinal fluid (CSF) from preterm infants with IVH as well as to Hb-metabolites. Finally, the blocking effects of the Hb-scavenger haptoglobin (Hp) were investigated both in vivo and in vitro.

Results: Following IVH + PHVD, an up-regulation of mRNA for the receptor-related genes TLR-4, IL1R1, FAS, the transcription factor NF-Κβ and for the pro-inflammatory and chemotactic effector molecules, IL-1β, TNFα, MCP-1, IL-8, and IL-6 was observed in the choroid plexus at 24 and 72 hours. This was associated with structural disintegration, caspase activation and cell death in the choroid plexus epithelium. In vitro characterization of choroid plexus epithelial cells, following exposure to hemorrhagic CSF and to the Hb-metabolites metHb and heme, displayed apoptotic and necrotic cell death and an up-regulation of receptor-related and inflammatory effector molecules similar to that observed in vivo following IVH + PHVD. Intraventricular injection of the Hb-scavenger Hp in vivo and co-incubation with Hp in vitro reversed or reduced the cellular activation, inflammatory response, structural damage and cell death.

Conclusion: Hb-metabolites are important causal initiators of cell death following IVH and removal or scavenging of Hb-metabolites may present an efficient means to reduce the damage to the immature brain following IVH.

Show MeSH
Related in: MedlinePlus