Snjolaug Sveinsdottir

Hemoglobin induces inflammation after preterm intraventricular hemorrhage by methemoglobin formation

BackgroundCerebral 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.MethodsUsing 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.ResultsFollowing 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.ConclusionsFollowing 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.

Altered Expression of Aquaporin 1 and 5 in the Choroid Plexus following Preterm Intraventricular Hemorrhage.

Intraventricular hemorrhage (IVH) with posthemorrhagic ventricular dilatation (PHVD) is a common cause of hydrocephalus in infants. Dysregulation of cerebrospinal fluid (CSF) production by the choroid plexus may contribute to the development of PHVD. The aquaporins (AQPs), transmural water transporting proteins, are believed to contribute to CSF production. The aim of the study was to characterize the expression and localization of AQP1, 4 and 5 in the choroid plexus following preterm IVH. Using a preterm rabbit pup model, the mRNA expression, protein level and localization of AQP1, 4 and 5 were investigated in the choroid plexus at 24 and 72 h following IVH with PHVD. Further, AQP1, 4 and 5 expression were characterized in primary human plexus epithelial cells exposed to CSF from preterm human infants with IVH and to hemoglobin metabolites. IVH with PHVD in the immature brain caused a downregulation of AQP1 mRNA, the key AQP in CSF production, but an upregulation of AQP1 protein level with apical epithelial cell localization. Notably, AQP5 was expressed in the choroid plexus with upregulated mRNA expression and protein levels during PHVD with apical epithelial cell localization. Analysis of human choroid plexus epithelial cells in vitro, following exposure to posthemorrhagic CSF and to hemin, displayed results concordant with those observed in vivo, i.e. downregulation of AQP1 mRNA and upregulation of AQP5 mRNA expression. AQP4 was neither detectable in vivo nor in vitro. The changes observed in AQP1 and AQP5 expression in the choroid plexus suggest an adaptive response following IVH with possible functional implications for the development of PHVD.

High presence of extracellular hemoglobin in the periventricular white matter following preterm intraventricular hemorrhage

Severe cerebral intraventricular hemorrhage (IVH) in preterm infants continues to be a major clinical problem, occurring in about 15–20% of very preterm infants. In contrast to other brain lesions the incidence of IVH has not been reduced over the last decade, but actually slightly increased. Currently over 50% of surviving infants develop post-hemorrhagic ventricular dilatation and about 35% develop severe neurological impairment, mainly cerebral palsy and intellectual disability. To date there is no therapy available to prevent infants from developing either hydrocephalus or serious neurological disability. It is known that blood rapidly accumulates within the ventricles following IVH and this leads to disruption of normal anatomy and increased local pressure. However, the molecular mechanisms causing brain injury following IVH are incompletely understood. We propose that extracellular hemoglobin is central in the pathophysiology of periventricular white matter damage following IVH. Using a preterm rabbit pup model of IVH the distribution of extracellular hemoglobin was characterized at 72 h following hemorrhage. Evaluation of histology, histochemistry, hemoglobin immunolabeling and scanning electron microscopy revealed presence of extensive amounts of extracellular hemoglobin, i.e., not retained within erythrocytes, in the periventricular white matter, widely distributed throughout the brain. Furthermore, double immunolabeling together with the migration and differentiation markers polysialic acid neural cell adhesion molecule (PSA-NCAM) demonstrates that a significant proportion of the extracellular hemoglobin is distributed in areas of the periventricular white matter with high extracellular plasticity. In conclusion, these findings support that extracellular hemoglobin may contribute to the pathophysiological processes that cause irreversible damage to the immature brain following IVH.

Impaired Cerebellar Maturation, Growth Restriction, and Circulating Insulin-Like Growth Factor 1 in Preterm Rabbit Pups

Cerebellar growth is impeded following very preterm birth in human infants and the observed reduction in cerebellar volume is associated with neurodevelopmental impairment. Decreased levels of circulating insulin-like growth factor 1 (IGF-1) are associated with decreased cerebellar volume. The relationship between preterm birth, circulating IGF-1, and key cell populations supporting cerebellar proliferation is unknown. The aim of this study was to evaluate the effect of preterm birth on postnatal growth, circulating IGF-1, and cerebellar maturation in a preterm rabbit pup model. Preterm rabbit pups (PT) were delivered by cesarean section at day 29 of gestation, cared for in closed incubators with humidified air, and gavage fed with formula. Control term pups (T) delivered by spontaneous vaginal delivery at day 32 of gestation were housed and fed by their lactating doe. In vivo perfusion-fixation for immunohistochemical evaluation of cerebellar proliferation, cell maturation, and apoptosis was performed at repeated time points in PT and T pups. Results show that the mean weight of the pups and circulating IGF-1 protein levels were lower in the PT group at all time points (p < 0.05) than in the T group. Postnatal weight development correlated with circulating IGF-1 (r2 = 0.89) independently of gestational age at birth and postnatal age. The proliferative (Ki-67-positive) portion of the external granular layer (EGL) was decreased in the PT group at postnatal day 2 (P2) compared to in the T group (p = 0.01). Purkinje cells exhibited decreased calbindin staining at P0 (p = 0.003), P2 (p = 0.004), and P5 (p = 0.04) in the PT group compared to in the T group. Staining for sonic hedgehog was positive in neuronal EGL progenitors and Purkinje cells at early time points but was restricted to a well-defined Purkinje cell monolayer at later time points. Preterm birth in rabbit pups is associated with lower circulating levels of IGF-1, decreased postnatal growth, and decreased cerebellar EGL proliferation and Purkinje cell maturation. The preterm rabbit pup model exhibits important characteristics of human preterm birth, and may thus be suitable for the evaluation of interventions aiming to modify growth and cerebellar development in the preterm population.

PO-0395 Scavenging Of Extracellular Haemoglobin Modifies The Monocyte-macrophage Recruitment And Differentiation In The Intraventricular Space Following Ivh

Introduction Severe cerebral intraventricular haemorrhage (IVH) in preterm infants continues to be a major clinical problem. To date, no available therapy prevents infants from neurologic sequel following IVH. Recruitment of monocytes-macrophages and periventricular infiltration is a key step in the inflammatory response leading to brain damage. The sequence of the recruitment and profiling of monocytes-macrophages following IVH is not well characterised. We have previously shown that extracellular haemoglobin induces chemotactic cytokines following IVH. Haptoglobin is a haemoglobin scavenger and could potentially protect the immature brain from the detrimental effects of haemoglobin. Objective To characterise the recruitment and differentiation of monocytes-macrophages in the intraventricular space following IVH and to investigate if haemoglobin scavenging with haptoglobin alters the recruitment and differentiation. Methods Using a preterm rabbit pup model of IVH we characterised the immune cell recruitment and differentiation in intraventricular cerebrospinal fluid (CSF) at 24 to 72 h following haemorrhage. Using flow cytometry, immunohistochemistry and mRNA and protein analysis we characterised the systemic and CSF infiltrating macrophages in animals with IVH, sham controls and animals treated with intraventricular injections of haptoglobin. Results Following IVH, there is an infiltration of M1 macrophages into the intraventricular CSF. Intraventricular introduction of the haemoglobin-scavenger haptoglobin modifies them into alternative M2 macrophages, expressing CD163. This causes a subsequent in vivo clearance of the accumulated haemoglobin. Conclusion Following IVH, intraventricular haptoglobin treatment modifies macrophage differentiation, initiating clearance of extracellular haemoglobin. Treatment of haptoglobin might be a feasible approach to protect the immature brain following IVH.

Arterial diameter change measurements in premature rabbit pups using B-Mode images

To further promote cardiovascular research there is a growing need for arterial characterization on small animals. Arterial wall movement measurements form the basis for most stiffness related parameters. As far as we know, previous reports on lumen diameter change measurements in small animals have required ECG triggering and cross-correlation techniques on the RF-signal using extremely high frame rate. The first aim of this study was to evaluate a novel method, ARTerIal Characterization (ARTIC), for robust diameter measurements in premature rabbit pups using conventional scanning technique (B-mode, 230 Hz). Although blood pressure is important for arterial stiffness measurements, it is often impossible to obtain without affecting the animal setup. However, arterial stiffness can also be assessed by measuring the pressure independent transition strain as recently proposed by Danpinid et al. (Ultrasonics 50, 2010:654-665). The second aim was to investigate the feasibility of measuring transition strain in premature rabbit pups using the obtained diameter curve. The diameter was 874/82 m (mean/SD), CV 2.9%. The distension was 24±3 m, CV 7.3%. The transition strain was 1.55±0.46%, CV 12.8%. In summary, ARTIC can measure arterial diameter and distension in small animals using B-Mode images at regular frame rates. ARTIC also has a low computation time suitable for real-time implementation. Measurements of transition strain on premature rabbit pups are feasible using B-Mode images at regular frame rate.

310 Methemoglobin Determines Degree of Pro-Inflammation and Choroid Plexus Apoptosis Following Intraventricular Hemorrhage in Preterm Rabbit Pups

Background and Aims Inflammation following intraventricular hemorrhage (IVH) has been shown to cause periventricular brain damage. Released free hemoglobin may be an early up-stream initiator of inflammation in the intraventricular space and cause damage to the choroid plexus. We aimed to evaluate release of free hemoglobin, the relationship to inflammation in the intraventricular space and effects on choroid plexus epithelium following IVH. Methods IVH was induced in preterm rabbit pups. Intraventricular samples of cerebrospinal fluid (CSF) were obtained under guidance of high-frequency ultrasound at 24, 48 and 72h following IVH for measurement of free oxyhemoglobin (OxyHb), methemoglobin (MetHb) and TNFa. Analysis of gene expression (RT-PCR) for TNFa, IL-1b and heme-oxygenase-1 (HO-1) and immunostaining with electronmicroscopy (EM) was performed in choroid plexus obtained at 72h. Results Intraventricular CSF concentrations of free OxyHb remained stable whereas those of MetHb and TNFa increased from 24 to 72 h. There was a highly positive correlation between concentrations of MetHb and TNFa in CSF at 72h (r= 0.98, p<0.0001). Levels of mRNA for HO-1, IL-1 b and TNF-a were up-regulated in choroid plexus following IVH as compared to controls (all p<0.01). EM showed choroid plexus epithelium with fragmented microvilli, apoptotic bodies, swollen mitochondria and increased epithelial expression of TNFa following IVH (fig. 1). Abstract 310 Figure 1 Conclusions Amount of released free Hb, subsequently auto-oxidised to MetHb determines degree of pro-inflammation in intraventricular CSF following IVH. This is associated with severe apoptosis in choroid plexus epithelium.