Fetal Asphyctic Preconditioning Research Articles

Fetal asphyctic preconditioning in rats results in a preserved placental inflammatory phenotype at birth

IntroductionPerinatal asphyxia (PA) is a major cause of neonatal mortality and morbidity. Research has shown that in rats fetal asphyxia (FA) can provoke neuroprotection against a subsequent more severe perinatal asphyctic insult. This is called fetal asphyctic preconditioning (PC). Our objective was to investigate alterations in the placental inflammatory phenotype associated with PC. MethodsFA was induced in the rat at embryonic day 17 by reversibly clamping the uterine circulation and PA was induced at birth by submersion of the uterine horns in a saline bath for 19 min. The effect of PC was studied by inducing FA at E17, followed by PA at E21. Placental TNF-α, IL-1β, IL-6 and IL-10 mRNA and protein levels were measured by qPCR and ELISA. ResultsIL-1β mRNA increased in the labouring FA group, but IL-1β protein decreased after both FA and PA. In the PC group, IL-1β mRNA and protein levels were similar to controls. IL-6 protein increased 6 h after FA, however decreased 24 h after FA. IL-6 mRNA was higher in the labouring PA group. IL-10 protein decreased 24 h after FA. At birth, IL-10 mRNA increased in the PA group; however, IL-10 protein decreased in both the PA and the FA group. In the PC group, IL-10 mRNA and protein were similar to control levels. DiscussionDepleted protein concentrations of IL-10 and IL-1β after one single asphyctic insult were reversed after fetal asphyctic PC. In addition, PC placentas showed less up-regulation of IL-6 mRNA compared to the PA ones. This modulated placental inflammatory phenotype might contribute to the improved neonatal outcome showed after fetal asphyctic PC.

Fetal asphyctic preconditioning protects against perinatal asphyxia- induced apoptosis and astrogliosis in neonatal brain.

Hypoxic-ischemic preconditioning is an endogenous mechanism in which exposure to a sublethal episode of hypoxia-ischemia protects against a subsequent more severe episode. Although several postnatal models of hypoxic-ischemic preconditioning have been established, hardly any perinatal models exist. Therefore, the objective of this study is to validate a new rodent model. We investigate whether mild fetal asphyxia (FA) as a preconditioning stimulus, protects against severe perinatal asphyxia (PA) when looking at neonatal brain histology. FA was induced at embryonic day 17 (E17) by temporarily clamping the uterine circulation. A caesarean section was performed at E21/22 and PA was induced by submersing the uterine horns, still containing the fetuses, in a water bath. Brains were examined for histological changes at either postnatal day 7 or 14. We used terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining to detect apoptotic cell death and a glial fibrillary acidic protein (GFAP) staining to detect reactive astrocytes. Interestingly, the preconditioned group showed significantly less perinatal mortality than non-preconditioned groups. Furthermore, preconditioned animals had significantly less TUNEL-positive cells and less GFAP-positive cells in striatum, prefrontal cortex and hippocampus compared to the non-preconditioned animals that underwent PA. Consequently, mild FA might cause neuroprotection by inducing anti-apoptotic mechanisms and attenuating astrogliosis. Considering the morphological findings in the neonatal brain from this study, together with previously reported long-term behavioral outcomes in this model, we can conclude that this is a suitable experimental model to investigate mechanisms of endogenous neuroprotection in the fetal brain. Identifying these endogenous neuroprotective mechanisms will provide novel potential targets for future pharmacological intervention in asphyctic newborns.

Fetal asphyctic preconditioning alters the transcriptional response to perinatal asphyxia.

BackgroundGenomic reprogramming is thought to be, at least in part, responsible for the protective effect of brain preconditioning. Unraveling mechanisms of this endogenous neuroprotection, activated by preconditioning, is an important step towards new clinical strategies for treating asphyctic neonates.Therefore, we investigated whole-genome transcriptional changes in the brain of rats which underwent perinatal asphyxia (PA), and rats where PA was preceded by fetal asphyctic preconditioning (FAPA). Offspring were sacrificed 6 h and 96 h after birth, and whole-genome transcription was investigated using the Affymetrix Gene1.0ST chip. Microarray data were analyzed with the Bioconductor Limma package. In addition to univariate analysis, we performed Gene Set Enrichment Analysis (GSEA) in order to derive results with maximum biological relevance.ResultsWe observed minimal, 25% or less, overlap of differentially regulated transcripts across different experimental groups which leads us to conclude that the transcriptional phenotype of these groups is largely unique. In both the PA and FAPA group we observe an upregulation of transcripts involved in cellular stress. Contrastingly, transcripts with a function in the cell nucleus were mostly downregulated in PA animals, while we see considerable upregulation in the FAPA group. Furthermore, we observed that histone deacetylases (HDACs) are exclusively regulated in FAPA animals.ConclusionsThis study is the first to investigate whole-genome transcription in the neonatal brain after PA alone, and after perinatal asphyxia preceded by preconditioning (FAPA). We describe several genes/pathways, such as ubiquitination and proteolysis, which were not previously linked to preconditioning-induced neuroprotection. Furthermore, we observed that the majority of upregulated genes in preconditioned animals have a function in the cell nucleus, including several epigenetic players such as HDACs, which suggests that epigenetic mechanisms are likely to play a role in preconditioning-induced neuroprotection.

Cerebellar cytokine expression in a rat model for fetal asphyctic preconditioning and perinatal asphyxia.

Asphyctic brain injury is a major cause of neuronal inflammation in the perinatal period. Fetal asphyctic preconditioning has been shown to modulate the cerebral inflammatory cytokine response, hereby protecting the brain against asphyctic injury at birth. This study was designated to examine the effects of perinatal asphyxia and fetal asphyctic preconditioning on the inflammatory cytokine response in the cerebellum. Fetal asphyxia was induced at embryonic day 17 by clamping the uterine vasculature for 30 min. At term birth, global perinatal asphyxia was induced by placing the uterine horns in saline for 19 min. Pro- and anti-inflammatory cytokine expression were assessed by real-time PCR and immunohistochemistry in cerebella of newborn rats. We found that tumor necrosis factor alpha and interleukin-10 mRNA were increased 12 h after fetal asphyxia, while the inflammatory cytokine response was decreased 96 h postfetal asphyxia. When applied as preconditioning stimulus, fetal asphyxia attenuates the cerebellar cytokine response. These results indicate that sublethal fetal asphyxia may protect the cerebellum from perinatal asphyxia-induced damage via inhibition of inflammation.

Acute and chronic immunomodulatory changes in rat liver after fetal and perinatal asphyxia.

Hypoxic-ischemic encephalopathy (HIE) caused by fetal and perinatal asphyxia is an important cause of mortality in the neonatal period. Not only will asphyxia affect the brain but also other organs such as the liver and kidneys. Interestingly, it has been shown that liver damage is proportional to the severity of the asphyctic insult, implying an association between liver impairment and HIE. Accordingly, we investigated in an established rat model the acute and chronic hepatic response to both fetal (FA) and perinatal asphyxia (PA). In addition, we assessed whether fetal asphyctic preconditioning (PC) would have any beneficial effect on the liver. Inflammation, ceramide signaling and hepatocellular damage were analyzed in the livers of newborn and adult rats at several short- and long-term time points after both FA and PA. We found that although FA induced an acute inflammatory response, apoptotic mRNA levels and oxidative DNA damage were decreased at 96 h post FA. Whereas increased IL-6 and IL-10 mRNA levels were observed after PA, the combination of FA and PA (PC) attenuated the inflammatory response. Moreover, 6 h after PA anti-apoptotic genes were downregulated and associated with less lipid peroxidation, while preconditioned animals were comparable to controls. In summary, asphyctic PC seems to have an acute protective effect on the liver by modulating the inflammatory, apoptotic and anti-oxidative response. More insight into the hepatic response to asphyxia is necessary, as disturbed hepatic function is associated with metabolic diseases in later life.

Fetal asphyxia induces acute and persisting changes in the ceramide metabolism in rat brain

Fetal asphyctic preconditioning, induced by a brief episode of experimental hypoxia-ischemia, offers neuroprotection to a subsequent more severe asphyctic insult at birth. Extensive cell stress and apoptosis are important contributing factors of damage in the asphyctic neonatal brain. Because ceramide acts as a second messenger for multiple apoptotic stimuli, including hypoxia/ischemia, we sought to investigate the possible involvement of the ceramide pathway in endogenous neuroprotection induced by fetal asphyctic preconditioning. Global fetal asphyxia was induced in rats by clamping both uterine and ovarian vasculature for 30 min. Fetal asphyxia resulted in acute changes in brain ceramide/sphingomyelin metabolic enzymes, ceramide synthase 1, 2, and 5, acid sphingomyelinase, sphingosine-1-phosphate phosphatase, and the ceramide transporter. This observation correlated with an increase in neuronal apoptosis and in astrocyte number. After birth, ceramide and sphingomyelin levels remained high in fetal asphyxia brains, suggesting that a long-term regulation of the ceramide pathway may be involved in the mechanism of tolerance to a subsequent, otherwise lethal, asphyctic event.

Fetal asphyctic preconditioning modulates the acute cytokine response thereby protecting against perinatal asphyxia in neonatal rats

BackgroundPerinatal asphyxia (PA) is a major cause of brain damage and neurodevelopmental impairment in infants. Recent investigations have shown that experimental sublethal fetal asphyxia (FA preconditioning) protects against a subsequent more severe asphyctic insult at birth. The molecular mechanisms of this protection have, however, not been elucidated. Evidence implicates that inflammatory cytokines play a protective role in the induction of ischemic tolerance in the adult brain. Accordingly, we hypothesize that FA preconditioning leads to changes in the fetal cytokine response, thereby protecting the newborn against a subsequent asphyctic insult.MethodsIn rats, FA preconditioning was induced at embryonic day 17 by clamping the uterine vasculature for 30 min. At term birth, global PA was induced by placing the uterine horns, containing the pups, in a saline bath for 19 min. We assessed, at different time points after FA and PA, mRNA and protein expression of several cytokines and related receptor mRNA levels in total hemispheres of fetal and neonatal brains. Additionally, we measured pSTAT3/STAT3 levels to investigate cellular responses to these cytokines.ResultsPrenatally, FA induced acute downregulation in IL-1β, TNF-α and IL-10 mRNA levels. At 96 h post FA, IL-6 mRNA and IL-10 protein expression were increased in FA brains compared with controls. Two hours after birth, all proinflammatory cytokines and pSTAT3/STAT3 levels decreased in pups that experienced FA and/or PA. Interestingly, IL-10 and IL-6 mRNA levels increased after PA. When pups were FA preconditioned, however, IL-10 and IL-6 mRNA levels were comparable to those in controls.ConclusionsFA leads to prenatal changes in the neuroinflammatory response. This modulation of the cytokine response probably results in the protective inflammatory phenotype seen when combining FA and PA and may have significant implications for preventing post-asphyctic perinatal encephalopathy.

730 Brain Genomic Response after Perinatal Asphyxia and Fetal Asphyctic Preconditioning

Background and AimsFetal asphyctic (FA) preconditioning has been shown to be effective in attenuating brain damage incurred by a subsequent perinatal asphyctic event. Unraveling mechanisms of this endogenous neuroprotection, is...

679 Inflammatory Changes and Ceramide Profiles in Rat Liver After Fetal Asphyctic Preconditioning and Perinatal Asphyxia

Background and AimsFetal (FA) and perinatal asphyxia (PA) are major causes of neonatal morbidity and death worldwide. Although most studies are focused on the brain, FA and PA are known...

Oxidative Stress in Perinatal Asphyxia and Fetal Asphyctic Preconditioning

Background: Asphyctic preconditioning is thought to attenuate the cellular stress response which reduces brain damage. Objective: We evaluated the expression of stress related genes in pre- and neonatal pups after fetal preconditioning (FA) and perinatal asphyxia (PA). Methods: FA was induced on E17 by clamping the uterine circulation for 30'. On P0 PA was induced by placing the fetuses-containing uterine horns in water for 19'. Control and FA pups were sacrificed 96h after the FA insult. Control, FA, PA and FA+PA pups were sacrificed 6h (P0) and 96h (P4) after birth. mRNA expression of iNos, nNos and Sod 1 was evaluated in the right hemisphere with qRT-PCR. Results: Sod1 was significant down-regulated (p=0.0057) on E21 for the FA group compared to controls. On P4 both the PA and the FA+PA group were down-regulated when compared to controls (p= 0.0021, p=0.0132) and compared to the FA group (p=0.0007, p=0.0297). On P4 there was a significant difference between the PA and the FA+PA groups (p=0.0005) (fig. 1). nNos expression revealed a significant increase on P0 for the PA and FA+PA groups compared to the FA group (p=0.0012, p=0.0145). iNos showed no significant differences. Conclusions: Modulation of the expression of antioxidants like Sod1 by FA could contribute to neuroprotection at birth. Additional whole-genome expression will provide a complete overview of the preconditioned phenotype.

Brain apoptosis and carotid artery reactivity in fetal asphyctic preconditioning

We aimed to develop a model of fetal hypoxia-ischemia (HI) preconditioning that reflects the pathophysiological conditions of perinatal asphyxia more closely than the existing neonatal stroke models. Fetal asphyxia (FA) was induced by clamping the uterine vasculature on embryonic day E17. At birth (P0), severe perinatal asphyxia (SPA) was induced during cesarean section. At P4, carotid arteries were studied in a wire myograph and at P8 brains were analyzed for apoptotic cell death in the prefrontal cortex and striatum. The contraction induced by K+ was significantly reduced in the carotid arteries from the SPA group and endothelium-dependent relaxation (mediated by acetylcholine) was augmented in the FA group. These changes in vascular responsiveness were not present in the animals exposed to both insults (FA + SPA). Additionally, FA+SPA animals showed lower numbers of apoptotic cells compared to SPA animals in both the prefrontal cortex and striatum. Exposure to a global fetal asphyctic insult seems to protect against the vascular alterations and the increase of apoptosis in striatum and prefrontal cortex induced by severe asphyxia at birth.

Fetal asphyctic preconditioning protects against perinatal asphyxia-induced behavioral consequences in adulthood

Perinatal asphyxia is one of the major causes of neuronal injury and impaired development in infants. We recently have shown that a brief episode of experimental fetal asphyxia (FA) can provoke an endogenous neuroprotection against subsequent severe perinatal asphyxia (SPA). The long-lasting functional consequences of FA preconditioning are not clear yet. The aim of the study was to determine if FA preconditioning can provide a long-lasting behavioral protection against SPA. FA was induced, as a preconditioning stimulus, by clamping the uterine vasculature for 30 min on E17. At birth, SPA was induced by placing the uterine horns in a water bath for 19 min. At 6 months of age, functional outcome was assessed using different behavioral tests: the open field for locomotor activity, the elevated zero maze for anxiety-related behavior, the forced swim test for depression-related behavior and the object recognition task for cognition. Data showed that FA preconditioning improved postnatal mortality after SPA. At the age of 6 months, the total distance moved in the open field and elevated zero maze was significantly less in the SPA group compared to the control groups. In addition, cognitive performance in the object recognition task was impaired in the SPA offspring compared to the control groups. Most importantly, FA preconditioning was able to preserve both locomotor activity and cognition function. In conclusion, FA preconditioning induces a long-lasting, functional protection against SPA. Therefore, this model seems to offer good opportunities for the identification and characterization of the underlying mechanisms of preconditioning.