Interaction of Inflammation and Hyperoxia in a Rat Model of Neonatal White Matter Damage

Felix Brehmer,Christoph Bührer,Stefanie Endesfelder,Bettina Gerstner,Yohan Van De Looij,Barbara S Reinboth,Marco Sifringer,Ursula Felderhoff-Mueser,Daniela Brait,Gerald W Schlager,Carina Mallard,Sebastian Prager,Stéphane Sizonenko,Ivo Bendix,Julia Zimmermanns

doi:10.1371/journal.pone.0049023

Abstract

Intrauterine infection and inflammation are major reasons for preterm birth. The switch from placenta-mediated to lung-mediated oxygen supply during birth is associated with a sudden rise of tissue oxygen tension that amounts to relative hyperoxia in preterm infants. Both infection/inflammation and hyperoxia have been shown to be involved in brain injury of preterm infants. Hypothesizing that they might be additive or synergistic, we investigated the influence of a systemic lipopolysaccharide (LPS) application on hyperoxia-induced white matter damage (WMD) in newborn rats. Three-day-old Wistar rat pups received 0.25 mg/kg LPS i.p. and were subjected to 80% oxygen on P6 for 24 h. The extent of WMD was assessed by immunohistochemistry, western blots, and diffusion tensor (DT) magnetic resonance imaging (MRI). In addition, the effects of LPS and hyperoxia were studied in an in vitro co-culture system of primary rat oligodendrocytes and microglia cells. Both noxious stimuli, hyperoxia, and LPS caused hypomyelination as revealed by western blot, immunohistochemistry, and altered WM microstructure on DT-MRI. Even so, cellular changes resulting in hypomyelination seem to be different. While hyperoxia induces cell death, LPS induces oligodendrocyte maturity arrest without cell death as revealed by TUNEL-staining and immunohistological maturation analysis. In the two-hit scenario cell death is reduced compared with hyperoxia treated animals, nevertheless white matter alterations persist. Concordantly with these in vivo findings we demonstrate that LPS pre-incubation reduced premyelinating-oligodendrocyte susceptibility towards hyperoxia in vitro. This protective effect might be caused by upregulation of interleukin-10 and superoxide dismutase expression after LPS stimulation. Reduced expression of transcription factors controlling oligodendrocyte development and maturation further indicates oligodendrocyte maturity arrest. The knowledge about mechanisms that triggered hypomyelination contributes to a better understanding of WMD in premature born infants.

Highlights

Worldwide, about 12 to 13 million babies are born preterm each year
For evaluation of brain injury after an inflammatory stimulus followed by hyperoxia, apoptotic cell death was measured by determination of cleaved caspase-3 protein expression on P7, directly after hyperoxia
Since the final consequence of impaired oligodendroglia is the disturbance of myelination [11,32,33], we investigated the degree of myelin basic protein (MBP) expression four days after hyperoxia exposure (P11) in rat pups treated at postnatal day 3 (P3) with LPS and at P6 with hyperoxia (Fig. 2A) as well as in the time matched application (Fig. 2B)

Summary

Introduction

About 12 to 13 million babies are born preterm each year. Meaning the rate of preterm birth is about 9–10% in industrialized countries [1,2]. Intrauterine infection is a major cause for preterm birth [3,4]. Experimental and clinical data suggest that both inflammation [7,8,9] and elevated oxygen concentrations [6,10,11,12] contribute to brain injury of preterm infants. Even though this two-hit hypothesis of first infection/ inflammation followed by hyperoxia reflects the clinical conditions of many preterm infants, previous research on the combination of hyperoxia and inflammation only focused on cardiovascular diseases [13]

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Results

Conclusion