Acute exposure to zinc oxide nanoparticles critically disrupts operation of the respiratory neural network in neonatal rat

Highlights

• Exposure to zinc oxide nanoparticles disrupts the neonatal respiratory network rhythm.

• Zinc oxide nanoparticles (ZnO NPs) depolarize pre-Bötzinger inspiratory neurons.

• ZnO NPs affect bioelectrical properties by reducing action potential amplitude.

• ZnO NPs depress inspiratory drive and have deleterious effects on respiratory centers.

Abstract

Due to their extremely small size that gives them unique physicochemical properties, nanoparticles (NPs) are used in the production of everyday materials. However, NPs can accumulate in body organs and could cause various diseases. Moreover, NPs that cross biological membranes such as the blood-brain barrier can aggregate in the brain and potentially produce neuronal damage. Although studies have reported the effects of diverse NPs on the bioelectrical properties of individual neurons, their potential influences on the operation of whole neuronal networks have not been documented. Here, we aimed to evaluate the effects of an acute exposure to zinc oxide (ZnO) NPs on the central neural networks responsible for mammalian respiratory rhythm generation. Using an isolated ex vivo brainstem-spinal cord preparation from neonatal rat in which the circuitry for the central respiratory command remained intact, we show that ZnO NPs accelerate, then profoundly disrupt respiratory-related activity produced by the pre-Bötzinger complex (preBötC) responsible for inspiratory rhythm generation. Consequently, a sudden and definitive cessation of respiratory-related activity occurs in ZnO NPs-exposed preparations. Part of these effects is related to zinc ions released from NPs. Using brainstem slice preparations containing the preBötC network, whole-cell patch-clamp recordings revealed that ZnO NPs depolarize preBötC inspiratory neurons and affect their bioelectrical properties by reducing the amplitude of action potentials, thereby leading to a depression of intra-network activity and the ultimate termination of respiratory rhythmogenesis. These findings support the conclusion that ZnO NPs may have deleterious effects on the central respiratory centers of newborn mammals.

Introduction

Research in the field of nanotechnology has been developing almost exponentially during the last decades. The unique properties of nanoparticles (NPs) has enabled the development of products with specialized features, allowing significant innovations in a variety of human activities such as computer technology, electronics, energy, aerospace, the food processing industry, cosmetics and medicine (Buzea et al., 2007). However, due to their extremely small size (<100 nm), NPs can easily enter the human body through different pathways (Kao et al., 2012; Oberdorster et al., 2004) and can cross most biological membranes including the blood-brain barrier (Sarkar et al., 2017). Subsequently, NPs can accumulate in the brain (Simko and Mattsson, 2010) and could be at the origin of cerebral dysfunctions and diseases (Feng et al., 2015; Leite et al., 2015; Migliore et al., 2015; Struzynska and Skalska, 2018).

Among many types of nanomaterials, NPs of zinc oxide (ZnO) are one of the most abundantly used. Due to their electrical properties (She et al., 2008), ZnO nanowires are used for nanotechnology in the field of nanoelectronics, sensors, light-emitting diodes and nanopiezotronics (Wang, 2008). Studies have also reported that ZnO NPs show selective toxicity to different bacterial systems (Reddy et al., 2007) and could therefore be used for food packaging material to prevent bacterial contamination (Tankhiwale and Bajpai, 2012). In the field of medicine, ZnO NPs exert a selective cytotoxic action on rapidly proliferating benign or malignant cells (Akhtar et al., 2012), and exhibit a strong preferential ability to kill cancerous T cells (Hanley et al., 2008). Furthermore, they may be combined with biodegradable chitosan for tumor-targeted drug delivery (Yuan et al., 2010). They also have promising applications as a nerve guidance channel substrate to promote nervous tissue regeneration (Seil and Webster, 2008). Additionally, ZnO NPs are widely used in cosmetics (Vaseem et al., 2010) and are present in sunscreens as a protective ingredient to reflect ultraviolet light radiation (McSweeney, 2016). Consequently, these ubiquitous applications lead to increasing occupational and consumer exposure to ZnO NPs, although the adverse consequences for human health in general, and for the functioning of the central nervous system (CNS) in particular, remain poorly documented.

Recently, a number of studies have reported that ZnO NPs can cross the blood-brain barrier (Shim et al., 2014b) and have the potential ability to reach the CNS where neurotoxic effects could develop. Exposure of human neuronal (Valdiglesias et al., 2013) and astrocyte-like (Lai et al., 2008) cells to ZnO NPs has been found to induce a dose-dependent decrease in cell survival through genotoxic effects and apoptosis. Similarly, when mouse neural stem cells were treated with ZnO NPs, an increasing number of apoptotic and necrosis cells has been observed (Deng et al., 2009). After a chronic exposure to ZnO NPs, an increased brain oxidative stress has been detected in mice, leading to neurotoxic manifestations (Shrivastava et al., 2014). It is also noteworthy that ZnO NPs may interact with cerebral proteins and alter their biological functions (Shim et al., 2014a). Studies conducted on hippocampal neurons have demonstrated that ZnO NPs alter the activity of various voltage-gated ion channels (for review see Yang et al., 2010), contributing to significant alterations in cell excitability (Zhao et al., 2009). Consequently, an alteration in synaptic plasticity occurs and participates in the attenuation of spatial learning and memory capability (Han et al., 2011). However, there is still a lack of information concerning the effects of these NPs on the activity of an entire neural network engaged in a vital motor function such as respiration.

The aim of the present study was to determine the effects of an acute exposure to ZnO NPs on the bioelectrical activity of neurons belonging to the respiratory centers that control rhythmic diaphragm muscle contraction in mammals. Using an ex vivo isolated brainstem–spinal cord and brainstem slice preparations from neonatal rat, we report for the first time deleterious and neurotoxic influences of ZnO NPs on the operation of the central respiratory network, leading to an abrupt and definitive arrest of respiratory-related activity.