Elsevier

Neuroscience Letters

Volume 738, 1 November 2020, 135389
Neuroscience Letters

Research article
Oxygen exposure in early life activates NLRP3 inflammasome in mouse brain

https://doi.org/10.1016/j.neulet.2020.135389Get rights and content

Highlights

  • High levels of oxygen exposure in early life results in NLRP3 inflammasome formation in preterm infants.

  • NLRP3 inflammasome activation is accompained by neuronal damage and cell death in preterm brain.

Abstract

Despite widely known detrimental effects on the developing brain, supplemental oxygen is still irreplaceable in the management of newborn infants with respiratory distress. Identifying downstream mechanisms underlying oxygen toxicity is a key step for development of new neuroprotective strategies. Main purpose of this study is to investigate whether NLRP3 inflammasome activation has a role in the pathogenesis of hyperoxia-induced preterm brain injury. C57BL6 pups were randomly divided into either a hyperoxia group (exposed to 90 % oxygen from birth until postnatal day 7) or control group (maintained in room air; 21 % O2). At postnatal day 7, all animals were sacrificed. Immunohistochemical examination revealed that hyperoxic exposure for seven days resulted in a global increase in NLRP3 and IL-1β immunopositive cells in neonatal mouse brain (p ≤ 0.001). There was a significant rise in Caspase-1 positive cell count in prefrontal and parietal area in the hyperoxia group when compared with controls (p ≤ 0.001). Western blot analysis of brain tissues showed elevated NLRP3, IL-1β and Caspase-1 protein levels in the hyperoxia group when compared with controls (p ≤ 0.001). To the best of our knowledge, this is the first study that investigates an association between hyperoxia and establishment of NLRP3 inflammasome in preterm brain.

Introduction

Supplemental oxygen plays a critical role in the management of both term and preterm infants with respiratory distress [1,2]. However, with our increased understanding regarding adverse effects of this molecule, there has been a renewed interest for better oxygen management in neonatal intensive care units. During fetal life, brain is primed to maintain its development in an environment with relative low levels of oxygen. Mean PaO2 in the intrauterine life is 3.2 kPa, that is equivalent to arterial oxygen saturations of about 70 % [3]. Following birth, even in the absence of supplemental oxygen, preterm infants are subjected to relative high levels of oxygen that results in generation of reactive oxygen species (ROS). Free radical attack is one of the principal downstream mechanisms leading to dysmaturational events in preterm brain by triggering maturational arrest of premyelinating oligodendrocytes, neuronal loss and subsequent gliosis [4].

As being one of the most powerful and commonly used drug in neonatal medicine, supplemental oxygen should be carefully titrated in order to prevent hyperoxia induced brain injury [3]. Although several well designed randomized controlled trials for target oxygen saturations of preterm infants were published in past years [5], in current practice, we still have scarce evidence regarding the optimum dose of oxygen [1]. Moreover, those infants who require oxygen support are usually the ones with more limited availability of antioxidant defense systems for proper handling the burden of oxygen (e.g., infants born preterm or undergone perinatal asphyxia) [1]. Brain injury and permanent neurological deficits continue to impair the well-being of many children, and protective strategies are particularly required in cases requiring oxygen support [6]. Gaining a clear understanding of the mechanisms of oxygen toxicity is essential to develop new therapeutic avenues for neuroprotection of babies on supplemental oxygen [7].

NLRP3 (NOD-, LRR- and pyrin domain-containing protein 3) is an intracellular multimeric protein complex resulting in Caspase 1-dependent release of the pro-inflammatory cytokines and finally cell death upon activation [8]. NLRP3 is shown to be involved in the pathogenesis of brain injury in mouse model of hypoxic ischemic encephalopathy [9,10] however the role in hyperoxia- induced-brain injury remains to be investigated. Assembly of the NLRP3 inflammasome can occur by diverse cellular insults one of which is increased production of ROS [11]. Oxidative stress can promote various inflammatory cascades in the preterm brain, such as upregulation of certain cytokines and increased expression of redox sensitive transcription factors (i.e., NF-κB) [12,13] which may trigger the two critical steps in the cellular process of NLRP3 inflammasome establishment: priming and activation [14]. Priming process is associated with inflammatory stimuli that promote NF-κB-mediated NLRP3 and pro-IL-1β expression and the activation process results in inflammasome assembly with Caspase-1-mediated IL-1β and IL-18 secretion. Moreover, modulation of ROS generation has been reported to interfere with NLRP3 inflammasome activation [14].

Main purpose of this study is to determine the role of NLRP3 activation in a mouse model of hyperoxia-induced preterm brain injury and if any, investigate the downstream mechanisms associated.

Section snippets

Animals

This study was performed in accordance with the guidelines provided by the Experimental Animal Laboratory and approved by the Animal Care and Use Committee of the Dokuz Eylül University School of Medicine. C57BL6 mice were maintained on standard 12 h light-dark cycles, at 21 °C temperature with 45 % humidity and with ad libitum access to standard food and water. All experiments were performed during the light phase of the 12 h day/night cycle.

Neonatal hyperoxia model

After giving birth, dams and their newborns were

Hyperoxia leads brain and body weight loss

Brain and body weights were measured at PN7, and both brain and body weights were found to be lower in the hyperoxia group compared to control (p = 0.04, p = 0.004 respectively) (Table 2).

Hyperoxia increases NLRP3, Caspase-1 and IL1β protein expression

In order to understand the relation between the hyperoxia-induced preterm brain injury and inflammasome activation, NLRP3, Caspase-1 and IL1β levels in brain tissue were determined by immunohistochemical assay. There were a global increase in NLRP3 immunopositive cells in brain tissues of hyperoxia group when

Discussion

In the present study, we succesfully demonstrated that seven days of exposure to hyperoxic environment leads to NLRP3 inflammasome activation in the brain tissue of neonatal mice. To the best of our knowledge, this is the first study to investigate the association between hyperoxia and establishment of NLRP3 inflammasome in brain.

Inflammasomes are known to be involved in the pathogenesis of acute hyperoxic lung injury in adults for several years [23,24]. The inflammasome protein complex is

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sector

CRediT authorship contribution statement

Serap Cilaker Micili: Conceptualization, Methodology, Writing - review & editing. Defne Engür: Conceptualization, Methodology, Writing - review & editing. Sermin Genc: Project administration. Ilkcan Ercan: Methodology, Resources. Sıla Soy: Methodology, Resources. Bora Baysal: Investigation. Abdullah Kumral: Supervision, Project administration.

Declaration of Competing Interest

None.

References (37)

  • W.A. Carlo et al.

    Oxygen therapy for preterm infants

    Clin. Perinatol.

    (2019)
  • Q. Liu et al.

    The role of mitochondria in NLRP3 inflammasome activation

    Mol. Immunol.

    (2018)
  • O.D. Saugstad et al.

    Oxygen therapy of the newborn from molecular understanding to clinical practice

    Pediatr. Res.

    (2019)
  • O.D. Saugstad

    Hyperoxia in the term newborn: more evidence is still needed for optimal oxygen therapy

    Acta Paediatr.

    (2012)
  • W. Tin et al.

    Optimum oxygen therapy in preterm babies

    Arch. Dis. Child. Fetal Neonatal Ed.

    (2007)
  • L.M. Askie et al.

    Neonatal oxygenation prospective meta-analysis (NeOProM) collaboration

    JAMA

    (2018)
  • B. Reich et al.

    Hyperoxia and the Immature Brain

    Dev. Neurosci.

    (2016)
  • K.V. Swanson et al.

    The NLRP3 inflammasome: molecular activation and regulation to therapeutics

    Nat. Rev. Immunol.

    (2019)
  • Cited by (0)

    1

    These author contributed equally to this work.

    View full text