BASIC SCIENCE ARTICLE Long-term neurological effects of neonatal caffeine treatment in a rabbit model of preterm birth Lennart Van der Veeken 1 , Susanne Grönlund 2 , Erik Gerdtsson 3 , Bo Holmqvist 3 , Jan Deprest 1,4 , David Ley 2 and Matteo Bruschettini 2 BACKGROUND: Neonatal caffeine treatment might affect brain development. Long-term studies show conflicting results on brain- related outcomes. Herein we aimed to investigate the long-term effects of neonatal caffeine administration in a rabbit model of preterm birth. METHODS: Preterm (born day 29) and term (day 32) pups were raised by wet nurses and allocated to treatment with saline or caffeine for 7 or 17 days. At pre-puberty, neurobehavioral tests were performed and brains were harvested for immunostaining of neurons, synapses, myelin, and astrocytes. RESULTS: Survival was lower in preterm saline pups than in controls, whereas caffeine-treated preterm pups did not differ from term control pups. Preterm saline pups covered less distance compared to controls and were more likely to stay in the peripheral zone of the open field. Corresponding differences were not seen in preterm caffeine pups. Preterm animals had lower neuron density compared to controls, which was not influenced by caffeine treatment. Synaptic density, astrocytes, and myelin were not different between groups. CONCLUSION: Caffeine appeared to be safe. All preterm rabbits had lower neuron density but anxious behavior seen in preterm saline rabbits was not seen in caffeine-treated preterm pups. Pediatric Research (2020) 87:1011–1018; https://doi.org/10.1038/s41390-019-0718-8 INTRODUCTION Premature infants are at risk for apnea of prematurity (AOP), a developmental disorder where the lack of central respiratory control leads to temporary cessation of breathing for 15–20 s leading to bradycardia and desaturation. Incidence of AOP is inversely correlated with gestational age and present in most infants born before 30 weeks. 1–4 Besides the immaturity of the respiratory system (both centrally and peripherally), there is also evidence suggesting that central nervous inflammation might play a role. 5 Methyl- xanthines are one of the most widespread drugs in neonatal medicine and have a proven efficiency for reducing the frequency of apnea and decreasing the need for mechanical ventilation. 6 Caffeine has replaced other methylxanthines, i.e., theophylline and aminophylline, due to its wider therapeutic index. Although these drugs were already used for >40 years, the CAP trial in 2006 was the first report proving their safety by demonstrating that caffeine did not harm short-term neonatal neurological outcome. 7 In further follow-up of this study, it was shown that neonates treated with caffeine had a dramatic reduction in the rate of cerebral palsy and a better neurological outcome at 18 months. 8–10 This beneficial effect was, however, less pronounced at school age. Through its action on the adenosine receptor (AR), caffeine stimulates respiration by increasing respiratory drive, enhancing diaphragmatic contractility, and increasing sensitivity to carbon dioxide. 11 AR has four different subtypes (A1AR, A2AAR, A2BAR, and A3AR), which are all antagonized by caffeine. 12,13 In the brain, mainly subtypes A1AR (hippocampus) and A2AAR (striatum) are found, for which caffeine has a high affinity. 14–16 In the central nervous system, caffeine has anti-inflammatory properties and can provide neuroprotective effects by counteracting the effects of adenosine, e.g., by antagonizing the excitatory A2A receptor. 15,17 In case of hypoxia and ischemia, levels of adenosine increase rapidly during hypoxia/ischemia and have been shown to alter normal oligodendrocyte development, necessary for myelin formation. 18 Doyle et al. demonstrated that neonates treated with caffeine had better myelin microstructure. 19 However, these findings were not seen in 11-year-old children; moreover, there were signs of a slower growth of the corpus callosum in caffeine- treated subjects. 20 Animal studies on the effects of caffeine on brain-related outcomes show conflicting results. Some studies found increased dendritic length and arborization, 21 increased myelination, 22 decreased inflammation, 23 and attenuation of deficits in spatial memory. Other studies have found no effect on developing white or gray matter, 24 and some studies found a negative impact with reduced brain weight, 25 decreased cell proliferation, 26 induced apoptosis, 27 and delayed cerebral myelin synthesis. 28 Therefore, we aimed to investigate the long-term effects of neonatal caffeine administration in a rabbit model of preterm (PT) birth. Received: 13 September 2019 Revised: 20 November 2019 Accepted: 26 November 2019 Published online: 7 December 2019 1 Department of Development and Regeneration, Cluster Woman and Child, Group Biomedical Sciences, KU Leuven University of Leuven, Leuven, Belgium; 2 Department of Clinical Sciences Lund, Paediatrics, Lund University, Skane University Hospital, Lund, Sweden; 3 ImaGene-iT AB, Medicon Village, Lund, Sweden and 4 Institute for Women’s Health, University College London, London, UK Correspondence: Matteo Bruschettini (matteo.bruschettini@med.lu.se) This paper has been presented at the 3rd Congress of joint European Neonatal Societies, Maastricht, 2019 September 17–21 www.nature.com/pr © International Pediatric Research Foundation, Inc. 2019 1234567890();,: