In this study, we aimed to elucidate the inflammatory cytokine profile in response to global fetal and perinatal asphyxia. Moreover, we investigated whether pro- and antiinflammatory cytokines played a role in the fetal induction of endogenous neuroprotection against PA injury. Prenatally, a time-dependent cytokine profile was observed after FA with acute downregulation of IL-1β, TNF-α and IL-10 mRNA levels. At 96 h post FA, immediately before birth, IL-6 mRNA and IL-10 protein expression were increased. Postnatally, PA rats showed higher IL-10 and IL-6 mRNA levels after PA than control rats. Moreover, in FA preconditioned rats this effect was attenuated, showing an inflammatory phenotype similar to control animals.
Cytokines have been demonstrated to play an important role in the brain response to asphyxia. Moreover, they have been implicated in playing a protective role in the induction of ischemic tolerance [13, 16]. The exact role of these cytokines and the time pattern of their expression have, however, not been elucidated yet. In human studies, IL-1β, IL-6 and TNF-α have been found to be upregulated in CSF of asphyxiated newborns compared with control neonates [9, 21]. In these studies, CSF sampling was performed at only one time point (24 h and 48 h respectively) after birth. More time points are included in experimental models of focal asphyxia. These models are mainly based on the Rice-Vannucci model , where P7 rats are exposed to unilateral carotid artery occlusion, followed by a period of systemic hypoxia. Although these models are widely used to investigate the mechanisms involved in PA, they miss the transitional physiology from intrauterine to extrauterine life. The birth process itself is a physiologically unique period, with specific hormonal changes priming the fetus to be born . This aspect was covered by the model used in our study as we awaited the vaginal delivery of the first pup so that the physiological conditions that occur during labor would be mimicked.
One aim of our study was to investigate the inflammatory mechanisms in global asphyxia. To the best of our knowledge, the role of cytokines in preconditioning during the fetal period has not been studied before. Therefore, we explored the impact of 30-min sublethal global fetal asphyxia on the levels of IL-1β, TNF-α, IL-10 and IL-6.
A time-dependent cytokine response after FA was observed prenatally. This response started 2 h after FA with decreased IL-1β mRNA levels, followed by a drop in TNF-α mRNA levels at 6 h post FA. Both IL-1β and TNF-α are key mediators of the acute inflammatory response  and have been shown to be upregulated after global cerebral hypoxia-ischemia in adult and neonatal rats [12, 25, 26]. Important differences between the addressed studies and our observations include the nature and the time point of the asphyctic insult. The FA that we induced is only a sublethal insult with no fetal losses or morbidity in adulthood . Additionally, we induced the stimulus prenatally, and it has been suggested that the fetal brain is less reactive to inflammatory reactions than a mature brain .
The antiinflammatory cytokine also showed decreased mRNA levels at 12 h after FA. IL-10 is an important antiinflammatory mediator of the immune response and has been shown to be both upregulated  and downregulated  after an hypoxic-ischemic insult. In addition, IL-10 prevents neurodegeneration by protecting neurons through activation of PI-3-kinase and STAT-3 pathways [29, 30]. Interestingly, at 96 h after FA protein expression of IL-10 was increased together with increased IL-6 mRNA levels. It is known that IL-6 is secreted together with the antiinflammatory cytokine IL-10 [21, 31], and IL-6 is a pleiotropic cytokine with both pro- and antiinflammatory effects in the injured brain [32, 33]. IL-6 has been shown to be expressed more on neurons in response to ischemia, promoting neuronal survival and development . Furthermore, a study by Yamashita and coworkers showed that endogenous IL-6 plays a critical role in preventing apoptosis of damaged neurons in the acute phase of cerebral ischemia in mice .
Taken together, these data indicate that FA induces acute changes in the cytokine response. Since IL-6 mRNA and IL-10 protein levels are increased at 96 h post FA, the time point immediately before birth, these cytokines might be involved in inducing neuroprotection.
The second aim of our study was to investigate whether the changes in cytokine response observed because of FA have any protective effect against a subsequent more severe PA insult. Our data showed decreased mRNA levels of the proinflammatory cytokines (IL-1β, TNF-α and IL-6) 2 h after birth in all experimental groups compared with control animals. Moreover, pSTAT3/STAT3 levels were decreased, indicating a downregulation in proinflammatory response. This decrease in cytokine levels is in accordance with a study from Ashdown et al., who observed decreased IL-1β and TNF-α protein and decreased IL-6 mRNA in the brain at 2 h after birth asphyxia . They claimed that the downregulation in cytokine levels was due to global birth asphyxia itself and not to the C-section. Comparison of E21 and P0 control animals revealed that IL-1β and IL-10 levels decreased because of birth. Control levels of TNF-α and IL-6 did not show decreased levels due to birth. These are remarkable findings because studies have shown that the proinflammatory response is increased during the birth process . Additionally, although IL-1β control levels decreased 2 h after birth, these levels were lower in the experimental groups than in the control groups, suggesting an interaction between the birth process and asphyxia. However, a synergic effect between birth and any asphyctic insult (FA and/or PA) was not observed for IL-1β but for the other cytokines. Hence, the drop in inflammatory cytokines 2 h after birth remains an interesting and important observation because focal asphyctic episodes have been associated with increased inflammation [6, 11, 12, 26]. Studies indicate that endogenous production of glucocorticoids may play an important role in the suppression of specific cytokine production and inflammation . As it has been established that birth is associated with higher stress hormones, such as cortisol and catecholamines , and hypoxia induces an even higher catecholamine response in the fetus , we can assume that the animals that had experienced an asphyctic insult before had higher corticosteroid levels. Accordingly, these animals would be able to downregulate the proinflammatory response as a protective mechanism.
One of the main findings of our study is that IL-10 was highly expressed in asphyxiated animals 2 h after birth. Moreover, significantly elevated mRNA levels of IL-6 were observed at 6 h and 12 h after PA. These data are comparable to the abundance of evidence indicating increased cytokine levels in the brain due to hypoxia-ischemia [6, 11, 12, 26]. Most importantly, a preconditioning effect was observed in both IL-10 and IL-6 mRNA levels. So the increase in IL-10 and IL-6 mRNA caused by PA was attenuated and comparable to control levels in the preconditioned FA-PA animals. Studies have already demonstrated that inhibition of cytokine activity reduces excitotoxic brain injury in neonatal rats [11, 40, 41]. An important role for the cytokines IL-10 and IL-6 after global asphyxia is highlighted in our study. An IL-6 response to asphyxia has been correlated with higher mortality rates . Therefore, the changes in IL-6 levels could indicate why FA preconditioned animals have a higher survival chance after PA compared with non-preconditioned animals. Although both human and experimental data show increased levels of IL-10 and IL-6 after asphyctic injury [9, 11, 12, 28, 31], the precise role of these cytokines in cerebral asphyxia has not been fully elucidated. Hence, further studies on the role of IL-10 and IL-6 in the pathophysiology of global perinatal brain injury are warranted.
It has been shown that the post-ischemic inflammatory neuronal damage is amplified by the STAT signaling pathways [42, 43]. Additionally, prevention of the post-ischemic STAT3 phosphorylation leads to significant neuroprotection and a decrease in neurological deficits . Surprisingly, we found decreased pSTAT3 expression 12 h post PA, while preconditioned animals showed baseline pSTAT3 expression. We can assume that the downregulation in pSTAT3 levels is an attempt to promote neuronal survival since we are looking at the brains of surviving offspring.