ORIGINAL ARTICLE Gestational age at initial exposure to in utero inflammation influences the extent of diaphragm dysfunction in preterm lambs KANAKESWARY KARISNAN, 1 ANTHONY J. BAKKER, 1,2 YONG SONG, 1 PETER B. NOBLE, 1 J. JANE PILLOW 1,2 AND GAVIN JON PINNIGER 1 1 School of Anatomy, Physiology and Human Biology and 2 Centre for Neonatal Research and Education, School of Paediatrics and Child Health, University of Western Australia, Perth, Western Australia, Australia ABSTRACT Background and objective: In utero infection may critically influence diaphragm development and pre- dispose preterm infants to postnatal respiratory failure.We aimed to determine how frequency and ges- tational age (GA) at time of intra-amniotic (IA) lipopolysaccharide (LPS) exposure affects preterm dia- phragm function. Methods: Pregnant ewes received IA injections of saline or 10-mg LPS at 7 days or 21 days or weekly injec- tions 21, 14 and 7 days before delivery at 121-day GA. Foetal lambs were killed with pentobarbitone (150 mg/ kg; intravenous). Diaphragm contractile function was measured in vitro. Muscle fibre type, activation of protein synthesis and degradation pathways, pro- inflammatory signalling and oxidative stress were evaluated using immunofluorescence staining, RT-qPCR, ELISA, Western blotting and biochemical assay. Results: In utero LPS exposure significantly impaired diaphragm contractile function. LPS exposure 7 days before delivery caused maximum specific twitch and tetanic forces 30% lower than controls.When the initial LPS exposure occurred 21 days before delivery maximum specific forces were 40% lower than con- trols. Earlier LPS exposure also prolonged twitch con- traction time, increased fatigue resistance and elevated protein carbonyl content. Despite increased white blood cell counts and interleukin-6 mRNA expression following weekly LPS exposure, there were no signifi- cant differences in contractile properties between exposure 21 days before delivery and repeated LPS groups suggesting that frequency of inflammatory exposure does not influence the severity of contractile dysfunction. Conclusions: GA at time of initial LPS exposure, rather than frequency of exposure, determines the extent of inflammation-induced diaphragm dysfunction. Key words: chorioamnionitis, contractile dysfunction, inflam- mation, preterm diaphragm. Abbreviations: 1/2 RT, half relaxation times; CSA, cross- sectional area; ELISA, enzyme-linked immunosorbent assay; FI, fatigue index; GA, gestational age; IA, intra-amniotic; IL, interleu- kin; LPS, lipopolysaccharide; MHC, myosin heavy chain; OCT, optical cutting temperature; RT-qPCR, reverse transcription quan- titative polymerase chain reaction; TTP, time to peak. INTRODUCTION Impaired diaphragm function is considered infre- quently as a major factor contributing to develop- ment of postnatal respiratory failure, despite the vital role of the diaphragm to self-sufficient breathing. However, like the lung, preterm diaphragm is struc- turally and functionally immature at birth 1,2 and less able to cope with an increased work of breathing compared with term diaphragm. Furthermore, many extremely preterm infants are exposed to an inflam- matory environment in utero that affects skeletal muscle function. Therefore, the immature diaphragm is vulnerable to adverse in utero exposures that may contribute to inefficient spontaneous breathing and development of respiratory failure requiring mechanical ventilator support. In utero exposure to inflammation, as occurs in chorioamnionitis, is associated with adverse respira- tory outcomes after birth. 3 Subclinical/histological chorioamnionitis is present in up to 70% of extremely Correspondence: Gavin Jon Pinniger, School of Anatomy, Physiology and Human Biology, University of Western Australia, 35 Stirling Highway, M309, Perth, WA 6009, Australia. Email: gavin.pinniger@uwa.edu.au Received 13 November 2014; invited to revise 1 February, 27 April and 19 May 2015; revised 28 March, 14 May and 11 June 2015; accepted 28 June 2015 (Associate Editor: Yuanlin Song). Article first published online: 26 August 2015 SUMMARY AT A GLANCE We investigated timing and frequency effects of in utero lipopolysaccharide (LPS) exposure on dia- phragm function in preterm lambs. LPS exposure earlier in gestation caused extensive alterations to diaphragm function regardless of frequency. Inflammatory exposure during development causes diaphragm dysfunction, which may con- tribute to respiratory failure in preterm infants. © 2015 Asian Pacific Society of Respirology Respirology (2015) 20, 1255–1262 doi: 10.1111/resp.12615