Fetal stress-mediated hypomethylation increases the brain susceptibility to hypoxic–ischemic injury in neonatal rats

https://doi.org/10.1016/j.expneurol.2015.10.007Get rights and content

Highlights

  • Fetal hypoxia induces global DNA hypomethylation in offspring brain.

  • DNA hypomethylation increases HIF-1α expression in the developing brain.

  • DNA hypomethylation sensitizes brain to HI injury in a HIF-1α-dependent manner.

  • Hypomethylation contributes to fetal hypoxia enhanced susceptibility to HIE.

Abstract

Background and purpose

Fetal hypoxia increases brain susceptibility to hypoxic–ischemic (HI) injury in neonatal rats. Yet mechanisms remain elusive. The present study tested the hypothesis that DNA hypomethylation plays a role in fetal stress-induced increase in neonatal HI brain injury.

Methods

Pregnant rats were exposed to hypoxia (10.5% O2) from days 15 to 21 of gestation and DNA methylation was determined in the developing brain. In addition, 5-aza-2′-deoxycytidine (5-Aza) was administered in day 7 pups brains and the HI treatment was conducted in day 10 pups. Brain injury was determined by in vivo MRI 48 h after the HI treatment and neurobehavioral function was evaluated 6 weeks after the HI treatment.

Results

Fetal hypoxia resulted in DNA hypomethylation in the developing brain, which persisted into 30-day old animals after birth. The treatment of neonatal brains with 5-Aza induced similar hypomethylation patterns. Of importance, the 5-Aza treatment significantly increased HI-induced brain injury and worsened neurobehavioral function recovery six weeks after the HI-treatment. In addition, 5-Aza significantly increased HIF-1α mRNA and protein abundance as well as matrix metalloproteinase (MMP)-2 and MMP-9, but decreased MMP-13 protein abundance in neonatal brains. Consistent with the 5-Aza treatment, hypoxia resulted in significantly increased expression of HIF-1α in both fetal and neonatal brains. Inhibition of HIF-1α blocked 5-Aza-mediated changes in MMPs and abrogated 5-Aza-induced increase in HI-mediated brain injury.

Conclusion

The results suggest that fetal stress-mediated DNA hypomethylation in the developing brain causes programming of hypoxic–ischemic sensitive phenotype in the brain and increases the susceptibility of neonatal brain to hypoxic–ischemic injury in a HIF-1α-dependent manner.

Introduction

Increasing evidence suggests that epigenetic mechanisms are of critical importance in regulating gene expression patterns, profoundly impacting normal brain development and programming of adaptive/maladaptive phenotypes in response to various environmental cues (Choi and Friso, 2010, Dauncey, 2012, Li et al., 2012a, Mehler, 2008). DNA methylation is the most characterized epigenetic mechanism, once considered as an inherently stable mark incapable of rapid change (Godfrey et al., 2007, Moore et al., 2013). Recently, emerging evidence has demonstrated that DNA undergoes rapid methylation and demethylation in the brain by means of distinct mechanisms in an activity-dependent fashion, which is critical for various types of brain function and physiological activity (Borrelli et al., 2008, Guo et al., 2011a, Guo et al., 2011b). However, pathological DNA methylation or demethylation profiles may result in aberrant gene expression and thus contribute to multiple brain pathologies in various neuropsychiatric conditions (Gräff et al., 2011, Hwang et al., 2013, Ikegame et al., 2013, Levenson and Sweatt, 2005).

Neonatal hypoxic–ischemic encephalopathy (HIE) is one of major causes of acute brain damage and mortality as well as chronic neurological disability in newborns (Chen et al., 2009b, Vannucci, 2000). Due to the poor understanding of the basic pathogenesis, few universally accepted therapy is available for neonatal HIE except that some studies implied the possible therapeutic effects of moderate hypothermia intervention (Perlman, 2006, Rees et al., 2011). Various candidate mechanisms have been proposed to elucidate the underlying pathogenesis in HIE, of which several molecules were considered as players and promising therapeutic targets, including the glucocorticoid receptor, angiotensin II type 2 receptor, hypoxia-inducible factor 1α (HIF-1α) and matrix metalloproteinase (MMP), etc. (Chen et al., 2008, Chen et al., 2009a, Gonzalez-Rodriguez et al., 2014a, Gonzalez-Rodriguez et al., 2014b, Li et al., 2012b, Li et al., 2013). Of great interest, recent studies revealed that adverse intrauterine environment may contribute to aberrant brain development and program a sensitive brain phenotype to neonatal HI insult (Gonzalez-Rodriguez et al., 2014b, Li et al., 2012b, Ma and Zhang, 2015).

Gestational hypoxia is a common stress to the fetal development and increases the risk of neonatal morbidity and mortality (Ma et al., 2014, Ma and Zhang, 2015). Our previous study revealed that chronic fetal hypoxia resulted in increased brain susceptibility to HI injury in neonatal rats (Gonzalez-Rodriguez et al., 2014b), whilst the underlying mechanisms are not fully elucidated. Herein, we presented a novel finding that gestational hypoxia induced a significant decrease in global DNA methylation and a sustained increase in HIF-1α in the developing brain. Of importance, we demonstrated that DNA hypomethylation in the brain significantly increased HI-induced brain injury in neonatal rats in a HIF-1α-dependent manner and worsened long-term neurobehavioral deficits, which may underlie fetal stress-induced programming of HI sensitive phenotype in the developing brain.

Section snippets

Experimental animals

Pregnant Sprague Dawley rats were purchased from Charles River Laboratories (Portage, MI). For the hypoxic treatment, pregnant animals were randomly divided into 2 groups: normoxic control and hypoxic treatment (10.5% O2, days 15 to 21 of gestation), as described previously (Patterson et al., 2010). On day 21 of pregnancy, some animals were killed and brains were isolated from fetuses (E21). Other animals were allowed to give birth, and brains collected from 12-day old (P12) pups and P30

Fetal hypoxia and 5-Aza induced global hypomethylation in the developing brain

Fig. 1A showed a developmental regulation of DNA methylation levels in the brain. Compared with the fetal brain, there was a significant increase in global methylation in the brain of P30 animals. Of importance, gestational hypoxia resulted in a significant decrease in methylation levels in the fetal brain, which persisted in the postnatal development, inhibiting the increase of methylation during the brain development (Fig. 1A). We investigated the role of hypomethylation in fetal

Discussion

The present study confers several novel findings. Firstly, we showed that gestational hypoxia altered normal developmental patterns of DNA methylation, resulting in a persistent global hypomethylation status in the developing brain. We then demonstrated that DNA hypomethylation in the brain resulted in a significant increase in neonatal brain vulnerability to HI-induced injury and worsened subsequent neurobehavioral dysfunction. Furthermore, we revealed that DNA methylation was an important

Sources of funding

This work was supported by National Institutes of Health grants HL82779 (L.Z.), HL83966 (L.Z.), and HL118861 (L.Z.).

Disclosures

The authors declare no competing financial interests.

Acknowledgments

A portion of this research used the Loma Linda University School of Medicine Advanced Imaging and Microscopy Core, a facility supported in part by the National Science Foundation through the Major Research Instrumentation program of the Division of Biological Infrastructure Grant No. 0923559 and the Loma Linda University School of Medicine.

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