Corrigendum: High Presence of Extracellular Hemoglobin in the Periventricular White Matter Following Preterm Intraventricular Hemorrhage.

[This corrects the article DOI: 10.3389/fphys.2016.00330.].

The heme and radical scavenger α1-microglobulin (A1M) confers early protection of the immature brain following preterm intraventricular hemorrhage.

BACKGROUND: Germinal matrix intraventricular hemorrhage (GM-IVH) is associated with cerebro-cerebellar damage in very preterm infants, leading to neurodevelopmental impairment. Penetration, from the intraventricular space, of extravasated red blood cells and extracellular hemoglobin (Hb), to the periventricular parenchyma and the cerebellum has been shown to be causal in the development of brain injury following GM-IVH. Furthermore, the damage has been described to be associated with the cytotoxic nature of extracellular Hb-metabolites. To date, there is no therapy available to prevent infants from developing either hydrocephalus or serious neurological disability. Mechanisms previously described to cause brain damage following GM-IVH, i.e., oxidative stress and Hb-metabolite toxicity, suggest that the free radical and heme scavenger α1-microglobulin (A1M) may constitute a potential neuroprotective intervention. METHODS: Using a preterm rabbit pup model of IVH, where IVH was induced shortly after birth in pups delivered by cesarean section at E29 (3 days prior to term), we investigated the brain distribution of recombinant A1M (rA1M) following intracerebroventricular (i.c.v.) administration at 24 h post-IVH induction. Further, short-term functional protection of i.c.v.-administered human A1M (hA1M) following IVH in the preterm rabbit pup model was evaluated. RESULTS: Following i.c.v. administration, rA1M was distributed in periventricular white matter regions, throughout the fore- and midbrain and extending to the cerebellum. The regional distribution of rA1M was accompanied by a high co-existence of positive staining for extracellular Hb. Administration of i.c.v.-injected hA1M was associated with decreased structural tissue and mitochondrial damage and with reduced mRNA expression for proinflammatory and inflammatory signaling-related genes induced by IVH in periventricular brain tissue. CONCLUSIONS: The results of this study indicate that rA1M/hA1M is a potential candidate for neuroprotective treatment following preterm 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.

Cerebellar Exposure to Cell-Free Hemoglobin Following Preterm Intraventricular Hemorrhage: Causal in Cerebellar Damage?

Decreased cerebellar volume is associated with intraventricular hemorrhage (IVH) in very preterm infants and may be a principal component in neurodevelopmental impairment. Cerebellar deposition of blood products from the subarachnoid space has been suggested as a causal mechanism in cerebellar underdevelopment following IVH. Using the preterm rabbit pup IVH model, we evaluated the effects of IVH induced at E29 (3 days prior to term) on cerebellar development at term-equivalent postnatal day 0 (P0), term-equivalent postnatal day 2 (P2), and term-equivalent postnatal day 5 (P5). Furthermore, the presence of cell-free hemoglobin (Hb) in cerebellar tissue was characterized, and cell-free Hb was evaluated as a causal factor in the development of cerebellar damage following preterm IVH. IVH was associated with a decreased proliferative (Ki67-positive) portion of the external granular layer (EGL), delayed Purkinje cell maturation, and activated microglia in the cerebellar white matter. In pups with IVH, immunolabeling of the cerebellum at P0 demonstrated a widespread presence of cell-free Hb, primarily distributed in the white matter and the molecular layer. Intraventricular injection of the Hb scavenger haptoglobin (Hp) resulted in a corresponding distribution of immunolabeled Hp in the cerebellum and a partial reversal of the damaging effects observed following IVH. The results suggest that cell-free Hb is causally involved in cerebellar damage following IVH and that blocking cell-free Hb may have protective effects.

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.

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

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-Κß and for the pro-inflammatory and chemotactic effector molecules, IL-1ß, 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.

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.

Automatic measurements of diameter, distension and intima media thickness of the aorta in premature rabbit pups using B-mode images.

To improve cardiovascular research, there is a growing need for arterial characterization in small animals. We developed a method, ARTIC (arterial characterization) for measuring lumen diameter, distension and intima media thickness (IMT). In this study ARTIC was used to automatically characterize the aorta of premature rabbit pups. Automatic measurements were compared with manual measurements, both performed by three observers. Diameter was 769 ± 140 µm (manual) and 766 ± 142 µm (automatic), distension was 35 ± 15 µm (manual) and 40 ± 12 µm (automatic) and IMT was 84 ± 11 µm (manual) and 88 ± 8 µm (automatic) (mean ± standard deviation). The variation in the measured diameter, distension and IMT ranged from 1.1% to 26.0% (manual) and from 1.0% to 9.0% (automatic). The intra-class correlation coefficient ranged from 33.0% to 99.3% (manual) and from 76.9% to 99.6% (automatic). The evaluation revealed that it is feasible to use ARTIC on B-mode images of arteries with small dimensions, which makes it a useful tool for arterial characterization in small animals.

Hemoglobin induces inflammation after preterm intraventricular hemorrhage by methemoglobin formation.

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-1ß, 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.

High-frequency ultrasound in the evaluation of cerebral intraventricular haemorrhage in preterm rabbit pups.

Cerebral intraventricular haemorrhage (IVH) is the most common cause of severe neurologic impairment following preterm birth in human infants. Ideally, an animal model for cerebral IVH should allow for reliable noninvasive evaluation of haemorrhagic extension and of subsequent development of posthaemorrhagic ventricular dilatation (PHVD). The aim of this study was to evaluate the use of high-frequency ultrasound (HFU) in premature rabbit pups with cerebral IVH induced by IP glycerol injection. Serial examinations using HFU enabled an accurate description of haemorrhagic extension and measurement of progressive PHVD over 72 h. The coefficient of variation for inter- and intraobserver variability in two measurements of ventricular size was less than 8.8% and 9.3%, respectively. Repeated ultrasound-guided intraventricular injection and sampling could be performed in vivo excluding requirement of stereotactic procedures and sedation. Application of HFU is a powerful tool for the evaluation of mechanisms involved in cerebral IVH and PHVD in the preterm rabbit pup model.