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Background: Preterm birth implies an array of respiratory diseases including apnea of prematurity and bronchopulmonary dysplasia (BPD). Caffeine has been introduced to treat apneas but also appears to reduce rates of BPD. Oxygen is essential when treating preterm infants with respiratory problems but high oxygen exposure aggravates BPD. This experimental study is aimed at investigating the action of caffeine on inflammatory response and cell death in pulmonary tissue in a hyperoxia-based model of BPD in the newborn rat. Material/Methods . Lung injury was induced by hyperoxic exposure with 80% oxygen for three (P3) or five (P5) postnatal days with or without recovery in ambient air until postnatal day 15 (P15). Newborn Wistar rats were treated with PBS or caffeine (10 mg/kg) every two days beginning at the day of birth. The effects of caffeine on hyperoxic-induced pulmonary inflammatory response were examined at P3 and P5 immediately after oxygen exposure or after recovery in ambient air (P15) by immunohistological staining and analysis of lung homogenates by ELISA and qPCR., Results: Treatment with caffeine significantly attenuated changes in hyperoxia-induced cell death and apoptosis-associated factors. There was a significant decrease in proinflammatory mediators and redox-sensitive transcription factor NF κ B in the hyperoxia-exposed lung tissue of the caffeine-treated group compared to the nontreated group. Moreover, treatment with caffeine under hyperoxia modulated the transcription of the adenosine receptor (Adora)1. Caffeine induced pulmonary chemokine and cytokine transcription followed by immune cell infiltration of alveolar macrophages as well as increased adenosine receptor (Adora1, 2a, and 2b) expression., Conclusions: The present study investigating the impact of caffeine on the inflammatory response, pulmonary cell degeneration and modulation of adenosine receptor expression, provides further evidence that caffeine acts as an antioxidative and anti-inflammatory drug for experimental oxygen-mediated lung injury. Experimental studies may broaden the understanding of therapeutic use of caffeine in modulating detrimental mechanisms involved in BPD development., Competing Interests: The authors declare that there is no conflict of interest., (Copyright © 2020 Stefanie Endesfelder et al.)

Background: While additional oxygen supply is often required for the survival of very premature infants in intensive care, this also brings an increasing risk of progressive lung diseases and poor long-term lung outcomes. Caffeine is administered to neonates in neonatal intensive care for the prevention and treatment of apneas and has been shown to reduce BPD incidence and the need for mechanical ventilation, although it is still unclear whether this is due to a direct pulmonary action via antagonism of adenosine receptors and/or an indirect action. This experimental study aims to investigate the action of caffeine on the oxidative stress response in pulmonary tissue in a hyperoxia-based model of bronchopulmonary dysplasia in newborn rats., Methods: Newborn Wistar rats were exposed to 21% or 80% oxygen for 3 (P3) or 5 (P5) postnatal days with or without recovery on room air until postnatal day 15 (P15) and treated with vehicle or caffeine (10 mg/kg) every 48 h beginning on the day of birth. The lung tissue of the rat pups was examined for oxidative stress response at P3 and P5 immediately after oxygen exposure or after recovery in ambient air (P15) by immunohistological staining and analysis of lung homogenates by ELISA and qPCR., Results: Lungs of newborn rats, corresponding to the saccular stage of lung development and to the human lung developmental stage of preterms, showed increased rates of total glutathione and hydrogen peroxide, oxidative damage to DNA and lipids, and induction of second-phase mediators of antioxidative stress response (superoxide dismutase, heme oxygenase-1, and the Nrf2/Keap1 system) in response to hyperoxia. Caffeine reduced oxidative DNA damage and had a protective interference with the oxidative stress response., Conclusion: In addition to the pharmacological antagonism of adenosine receptors, caffeine appears to be a potent antioxidant and modulates the hyperoxia-induced pulmonary oxidative stress response and thus protective properties in the BPD-associated animal model. Free-radical-induced damage caused by oxidative stress seems to be a biological mechanism progress of newborn diseases. New aspects of antioxidative therapeutic strategies to passivate oxidative stress-related injury should be in focus of further investigations.

In preterm infants, phenobarbital is the first-line antiepileptic drug for neonatal seizures while caffeine is used for the treatment of apnea. Data from experimental animals suggest that phenobarbital and other anticonvulsants are toxic for the developing brain, while neuroprotective effects have been reported for caffeine both in newborn rodents and preterm human infants. To characterize the interaction of phenobarbital and caffeine in the hippocampus of the developing rodent brain, we examined the effects of both drugs given separately or together on postnatal neurogenesis after administration to neonatal rats throughout postnatal day (P) 4 to P6. Phenobarbital treatment (50 mg/kg) resulted in a significant decrease of proliferative capacity in the dentate gyrus. Phenobarbital also reduced expression of neuronal markers (doublecortin (DCX), calretinin, NeuN), neuronal transcription factors (Pax6, Sox2, Tbr1/2, Prox1), and neurotrophins (NGF, BDNF, NT-3) up to 24 h after the last administration. The phenobarbital-mediated impairment of neurogenesis was largely ameliorated by preconditioning with caffeine (10 mg/kg). In contrast, caffeine alone reduced proliferative capacity and expression of the neuronal markers DCX and NeuN at 6 h, but increased expression of neurotrophins and neuronal transcription factors at 6 and 12 h. These results indicate that administration of phenobarbital during the vulnerable phase of brain development negatively interferes with neuronal development, which can be prevented in part by co-administration of caffeine.

Phenobarbital is the most commonly used drug for the treatment of neonatal seizures but may induce neurodegeneration in the developing brain. Methylxanthine caffeine is used for the treatment of apnea in newborn infants and appears to be neuroprotective, as shown by antiapoptotic and anti-inflammatory effects in oxidative stress models in newborn rodents and reduced rates of cerebral palsy in human infants treated with caffeine. We hypothesized that caffeine may counteract the proapoptotic effects of phenobarbital in newborn rats. Postnatal day 4 (P4) rats received phenobarbital (50 mg/kg) +/- caffeine (10 mg/kg) for three consecutive days. Brains examined at 6, 12, and 24 h after last injection of phenobarbital showed a drastic increase of apoptotic cell death (TUNEL+), which was attenuated by co-treatment with caffeine at 6 and 24 h but not at 12 h. Phenobarbital also increased protein levels of apoptosis inducing factor (AIF) and cleaved caspase-3, which was reduced by caffeine co-administration at all time points investigated. RNA expression of the pro-inflammatory cytokines TNFα, IFNγ, and IL-1β, but not IL-18, was upregulated by phenobarbital. Co-treatment with caffeine significantly decreased these upregulations at all time points investigated, while caffeine without phenobarbital resulted in increased expression of TNFα, IL-1β, and IL-18, but not IFNγ at 6 h. Downregulation of the adenosine A1 and A2a receptors, both of which bind caffeine, by 24 h of phenobarbital exposure was partly antagonized by caffeine. These results raise the possibility that the phenobarbital-induced adverse effects could be reduced by a co-treatment with caffeine.

Sequelae of prematurity triggered by oxidative stress and free radical-mediated tissue damage have coined the term "oxygen radical disease of prematurity". Caffeine, a potent free radical scavenger and adenosine receptor antagonist, reduces rates of brain damage in preterm infants. In the present study, we investigated the effects of caffeine on oxidative stress markers, anti-oxidative response, inflammation, redox-sensitive transcription factors, apoptosis, and extracellular matrix following the induction of hyperoxia in neonatal rats. The brain of a rat pups at postnatal Day 6 (P6) corresponds to that of a human fetal brain at 28-32 weeks gestation and the neonatal rat is an ideal model in which to investigate effects of oxidative stress and neuroprotection of caffeine on the developing brain. Six-day-old Wistar rats were pre-treated with caffeine and exposed to 80% oxygen for 24 and 48 h. Caffeine reduced oxidative stress marker (heme oxygenase-1, lipid peroxidation, hydrogen peroxide, and glutamate-cysteine ligase catalytic subunit (GCLC)), promoted anti-oxidative response (superoxide dismutase, peroxiredoxin 1, and sulfiredoxin 1), down-regulated pro-inflammatory cytokines, modulated redox-sensitive transcription factor expression (Nrf2/Keap1, and NFκB), reduced pro-apoptotic effectors (poly (ADP-ribose) polymerase-1 (PARP-1), apoptosis inducing factor (AIF), and caspase-3), and diminished extracellular matrix degeneration (matrix metalloproteinases (MMP) 2, and inhibitor of metalloproteinase (TIMP) 1/2). Our study affirms that caffeine is a pleiotropic neuroprotective drug in the developing brain due to its anti-oxidant, anti-inflammatory, and anti-apoptotic properties., Competing Interests: The authors declare no conflict of interest.

Caffeine administered to preterm infants has been shown to reduce rates of cerebral palsy and cognitive delay, compared to placebo. We investigated the neuroprotective potential of caffeine for the developing brain in a neonatal rat model featuring transient systemic hyperoxia. Using 6-day-old rat pups, we found that after 24 and 48h of 80% oxygen exposure, apoptotic (TUNEL(+)) cell numbers increased in the cortex, hippocampus, and central gray matter, but not in the hippocampus or dentate gyrus. In the dentate gyrus, high oxygen exposure led to a decrease in the number of proliferating (Ki67(+)) cells and the number of Ki67(+) cells double staining for nestin (immature neurons), doublecortin (progenitors), and NeuN (mature neurons). Absolute numbers of nestin(+), doublecortin(+), and NeuN(+) cells also decreased after hyperoxia. This was mirrored in a decline of transcription factors expressed in immature neurons (Pax6, Sox2), progenitors (Tbr2), and mature neurons (Prox1, Tbr1). Administration of a single dose of caffeine (10mg/kg) before high oxygen exposure almost completely prevented these effects. Our findings suggest that caffeine exerts protection for neonatal neurons exposed to high oxygen, possibly via its antioxidant capacity., (© 2013 Elsevier Inc. All rights reserved.)

In preterm human infants, briefly elevated concentrations of oxygen are associated with a prolonged increase in blood chemokine concentrations and the development of bronchopulmonary dysplasia (BPD). Caffeine given to preterm infants for the prevention or treatment of apnoea has been shown to reduce the rate of BPD. We tested the hypotheses that infant rats exposed to a combination of caffeine and hyperoxia would be less susceptible to lung injury than those exposed to hyperoxia alone and that caffeine decreases the pulmonary tissue expression of chemokines and leukocyte influx following hyperoxia. Using 6-day-old rat pups, we demonstrated that 24 h of 80% oxygen exposure caused pulmonary recruitment of neutrophils and macrophages. High levels of oxygen upregulated the expression of: the CXC chemokines, cytokine-induced neutrophil chemoattractant-1 and macrophage inflammatory protein-2; the CC-chemokine monocyte chemoattractant protein-1; the pro-inflammatory cytokines tumour necrosis factor-α and interleukin-6, as measured by realtime PCR after the administration of caffeine (10 mg · kg(-1) body weight); and attenuated chemokine and cytokine upregulation, as well as the influx of CD11b(+), ED-1(+) and myeloperoxidase(+) leukocytes. These experiments suggest that protective effects of caffeine in the neonatal lung are mediated, at least in part, by reduction of pulmonary inflammation.