Research articleOxygen exposure in early life activates NLRP3 inflammasome in mouse 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.
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These author contributed equally to this work.