Elsevier

Experimental Neurology

Volume 261, November 2014, Pages 386-395
Experimental Neurology

Regular Article
Secretions from placenta, after hypoxia/reoxygenation, can damage developing neurones of brain under experimental conditions

https://doi.org/10.1016/j.expneurol.2014.05.003Get rights and content

Highlights

  • Altered oxygen stimulates human placenta to secrete factors that cause neurone damage.

  • Embryonic cortical neurones respond in vitro to the secretions with increased Ca2+ and ROS.

  • Neurones exposed to secretions show decreased dendritic lengths, spine density and synaptic activity.

  • One active factor in the secretions is identified as glutamate.

  • The secretions have the potential to alter brain development in vivo.

Abstract

Some psychiatric diseases in children and young adults are thought to originate from adverse exposures during foetal life, including hypoxia and hypoxia/reoxygenation. The mechanism is not understood. Several authors have emphasised that the placenta is likely to play an important role as the key interface between mother and foetus. Here we have explored whether a first trimester human placenta or model barrier of primary human cytotrophoblasts might secrete factors, in response to hypoxia or hypoxia/reoxygenation, that could damage neurones. We find that the secretions in conditioned media caused an increase of [Ca2 +]i and mitochondrial free radicals and a decrease of dendritic lengths, branching complexity, spine density and synaptic activity in dissociated neurones from embryonic rat cerebral cortex. There was altered staining of glutamate and GABA receptors. We identify glutamate as an active factor within the conditioned media and demonstrate a specific release of glutamate from the placenta/cytotrophoblast barriers invitro after hypoxia or hypoxia/reoxygenation. Injection of conditioned media into developing brains of P4 rats reduced the numerical density of parvalbumin-containing neurones in cortex, hippocampus and reticular nucleus, reduced immunostaining of glutamate receptors and altered cellular turnover. These results show that the placenta is able to release factors, in response to altered oxygen, that can damage developing neurones under experimental conditions.

Introduction

Some human diseases originate from events during foetal life (Barker, 2004). A variety of prenatal insults, including hypoxia, hypoxia/reoxygenation, and infection, as early as late first trimester, are associated with an increased risk of neurodevelopmental disorders including schizophrenia, attention deficit/hyperactivity disorder and autism (Fatemi and Folsom, 2009). In animal models even relatively brief periods of foetal hypoxia may lead to the death of susceptible neuronal populations (Rees etal., 2011). The principal pathways for this are initiated by energy depletion followed by an increased neuronal release and a reduced glialuptake of glutamate, an accumulation of cytosolic calcium and a generation of reactive oxygen species (Rees etal., 2011). The death of neurones is thought to occur in three stages. There is an initial period of cell dysfunction and oxidative stress. 8 to 48 h later there is a secondary phase of injury, which results in a neuroinflammatory response, mitochondrial permeabilisation, reperfusion and a loss of cerebral autoregulation. Weeks or months later there may be tertiary injuries as a result of a sensitization to inflammation, persistent gliosis and epigenetic changes (Baburamani etal., 2012, Chicha etal., 2014).

However the precise mechanisms of how transient gestational challenges can lead to neurodevelopmental diseases in later life are largely unknown, and it is thought that the placenta is likely to play a key role (Hsiao and Patterson, 2012, Rapoport etal., 2012). As Hsiao and Patterson emphasise, disruptions to the maternal or intrauterine environment are necessarily conveyed to the developing embryo viathe placenta. Where gestational challenges are confined to the uteroplacental compartment, in models of intrauterine infection and intrauterine growth restriction, primary insults to placenta can manifest in perinatal brain damage in the offspring (Hsiao and Patterson, 2012, Mikaelsson etal., 2013).

The past 5 years have seen particular interest in the contribution of placental pathology to neurodevelopmental disorders (Rapoport etal., 2012). Furthermore the placenta actively secretes molecules that are important for infant brain development and which might be affected by gestational challenges (Bonnin etal., 2011, McKay, 2011).

Previously we showed that the placenta or a model placental barrier responds to toxins or altered oxygen by secreting factors that cause genetic damage in fibroblasts or human embryonic stem cells (Bhabra etal., 2009, Sood etal., 2011). Therefore we explored here whether it would also respond to altered oxygen by secreting factors that could damage developing neurones under experimental conditions.

We show that the placenta responds invitro to hypoxia or hypoxia/reoxygenation by secreting factors that increase calcium and mitochondrial free radicals in embryonic cortical neurones invitro and reduce synaptic activity, dendritic length, branching complexity and spine density. Exposure of a developing brain to media conditioned by placenta under hypoxia and hypoxia reoxygenation results in decreased density of parvalbumin containing neurones in cortex hippocampus and reticular nucleus. We identify glutamate as an active factor mediating these changes.

Section snippets

Preparation of barriers/explants

Bilayered BeWo cell barriers were prepared on transwell inserts at 2% or 21% oxygen according to our previous protocols (Sood etal., 2011). Human placenta from 1st and 3rd trimester was obtained with ethical approval and patient consent from patients with normal pregnancies at voluntary termination of pregnancy or elective caesarean section.

Primary villous cytotrophoblasts, were extracted from 3rd trimester placenta according to our previous protocols (Tannetta etal., 2008). They were used to

Ca2 +, ROS

The media conditioned by placenta after hypoxia/reoxygenation caused a large rapid increase in intracellular [Ca2 +]i in embryonic cortical neurones grown for 12 days before exposure (Fig. 2A) in comparison to media conditioned by placenta at 21% oxygen without change. There were increased mitochondrial reactive oxygen species (Supplementary Figs. 1A–E) without oxidative damage of the inner mitochondrial membrane or apoptosis (Supplementary Figs. 1F–I). Media conditioned by a BeWo cell barrier, a

Discussion

The results show that the placenta responds invitro to hypoxia or hypoxia/reoxygenation by secreting factors which cause dendrite shortening, increased mitochondrial free radicals, increased calcium and altered immunostaining of both glutamate and GABA receptors in dissociated neurones from embryonic cortex invitro. We identify glutamate as a key active factor that is secreted from the placenta and show that this can be released from human cytotrophoblasts, which are known to face the foetal

Acknowledgments

The financial support of the Perivoli Trust is gratefully acknowledged. MG acknowledges financial support from the EC-FP7 (Marie Curie Network “NAMASEN” grant n. 264872), the Research Foundation Flanders (grant n. G.0888.12N) and the Interuniversity Attraction Poles Program (IUAP), initiated by the Belgian Science Policy Office.

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