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.].

Relation of Retinopathy of Prematurity to Brain Volumes at Term Equivalent Age and Developmental Outcome at 2 Years of Corrected Age in Very Preterm Infants.

BACKGROUND: Retinopathy of prematurity (ROP) is a major complication of preterm birth and has been associated with later visual and nonvisual impairments. OBJECTIVES: To evaluate relationships between any stage of ROP, brain volumes, and developmental outcomes. METHODS: This study included 52 very preterm infants (gestational age [mean ± SD]: 26.4 ± 1.9 weeks). Total brain, gray matter, unmyelinated white matter (UWMV), and cerebellar volumes were estimated in 51 out of 52 infants by magnetic resonance imaging at term-equivalent age. Bayley Scales of Infant Development were used to assess developmental outcomes in 49 out of 52 infants at a mean corrected age of 24.6 months. RESULTS: Nineteen out of 52 infants developed any stage of ROP. Infants with ROP had a lower median (IQR) UWMV (173 [156-181] vs. 204 [186-216] mL, p < 0.001) and cerebellar volume (18.3 [16.5-20] vs. 22.3 [20.3-24.7] mL, p < 0.001) than infants without ROP. They also had a lower median (IQR) mental developmental index (72 [56-83] vs. 100 [88-104], p < 0.001) and a lower psychomotor developmental index (80 [60-85] vs. 92 [81-103], p = 0.002). Brain volumes and developmental outcomes did not differ among infants with different stages of ROP. CONCLUSION: Any stage of ROP in preterm infants was associated with a reduced brain volume and an impaired developmental outcome. These results suggest that common pathways may lead to impaired neural and neurovascular development in the brain and retina and that all stages of ROP may be considered in future studies on ROP and development.

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.