Eveline Strackx

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.

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.

Fetal asphyxia leads to a decrease in dorsal raphe serotonergic neurons.

The aim of the present study was to determine the effects of fetal asphyxia (FA) on anxiety and serotonergic neurons in young adult and middle-aged rats. FA was induced at embryonic day 17 by clamping the uterine circulation for 75 min. Anxiety-related behavior was tested in an open field, and design-based stereology was used for counting serotonergic (5-hydroxytryptamine/serotonin, 5-HT) neurons in the dorsal raphe nucleus (DRN). The open field revealed increased anxiety in the 19-month-old FA rats in comparison to control animals. No significant differences were found in DRN 5-HT neuron numbers at 6 months. At 19 months, however, FA significantly lowered the mean density and volume of 5-HT neurons in the DRN as compared to controls. Further, an age-related reduction was found in the total number, the mean density and the mean volume of 5-HT neurons within the FA group. In conclusion, FA is associated with increased anxiety and age-related changes in 5-HT immunohistochemistry within the DRN. These results support the notion that insults caused by asphyxiation during critical periods of brain development could create a predisposition to serotonergic abnormalities and anxiety deficits in adulthood.

Lipopolysaccharide-Induced Chorioamnionitis Causes Acute Inflammatory Changes in the Ovine Central Nervous System

OBJECTIVE To better understand the inflammatory response in the central nervous system (CNS) after lipopolysaccharide (LPS)-induced chorioamnionitis. STUDY DESIGN Fetal sheep were exposed to intra-amniotic LPS 2 or 14 days before preterm delivery at 125 days of gestation. mRNA levels of cytokines, TLRs and anti-oxidants were determined in different CNS regions. RESULTS Interleukin 1β levels increased in hippocampus, cortex and cerebellum 2 days after LPS exposure, while Interleukin 8 levels increased in the periventricular white matter as well. Levels returned back to control levels after 14 days. Tumor necrosis factor-α levels increased in hippocampus and cortex after 2 days. Toll like receptor 4 levels was upregulated in all grey matter regions 2 and 14 days after exposure. Glutathione s-transferase mRNA levels were lower after 2 and 14 days in all grey matter regions. CONCLUSION Intra-amniotic LPS exposure causes acute and region-specific changes in inflammatory markers in the fetal brain, with grey matter being more affected than white matter. CONDENSATION Intra-amniotic LPS exposure causes acute and region-specific changes in cytokines, TLR and anti-oxidants levels, with grey matter being more affected than white matter.

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.

Preconditioning by oxygen-glucose deprivation preserves cell proliferation and reduces cytotoxicity in primary astrocyte cultures.

Hypoxic-ischemic preconditioning (HIPC) has a neuroprotective effect against a subsequent, more severe perinatal hypoxic-ischemic episode. The protective processes of preconditioning (PC) in the immature brain remain undefined but are most likely related to the immune cells of the central nervous system. To determine the role of astrocytes in HIPC, we initially exposed primary rat astrocytes to oxygen-glucose deprivation (OGD) for 30 minutes as a PC stimulus. A subsequent more severe insult was induced 24 hours later by exposing the astrocytes to OGD for 3 hours. These experiments revealed that OGD for 3 hours induced increased cytotoxicity as measured by lactate dehydrogenase in primary astrocytes, which was diminished in astrocytes earlier subjected to PC. Moreover, decreased cell proliferation, as measured by Ki67, and lower cytokine expression (IL-1β, TNF-α, IL-6, IL-10) were observed in astrocytes subjected to OGD for 3 hours, while these levels remained unchanged in PC+OGD cells. Therefore, we speculate that PC by OGD may affect the survival, proliferation and function of primary cultured astrocytes which may partly explain the neuroprotective properties of HIPC seen in HI rat models.

The effects of fetal and perinatal asphyxia on neuronal cytokine levels and ceramide metabolism in adulthood.

In a rat model of global fetal and perinatal asphyxia, we investigated if asphyxia and long-lasting brain tolerance to asphyxia (preconditioning) are mediated by modifications in inflammatory cytokines and ceramide metabolism genes in prefrontal cortex, hippocampus and caudate-putamen at the age of 8months. Most significant changes were found in prefrontal cortex, with reduced LAG1 homolog ceramide synthase 1 expression after both types of asphyxia. Additionally, sphingosine kinase 1 was upregulated in those animals that experienced the combination of fetal and perinatal asphyxia (preconditioning), suggesting increased cell proliferation. While cytokine levels are normal, levels of ceramide genes were modulated both after fetal and perinatal asphyxia in the adult prefrontal cortex. Moreover, the combination of two subsequent asphyctic insults provides long-lasting neuroprotection in the prefrontal cortex probably by maintaining normal apoptosis and promoting cell proliferation. Better understanding of the effects of asphyxia on ceramide metabolism will help to understand the changes leading to brain tolerance and will open opportunities for the development of new neuroprotective therapies.

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 asphyxia leads to the loss of striatal presynaptic boutons in adult rats

Fetal asphyxic insults in the brain are known to be associated with developmental and neurological problems like neuromotor disorders and cognitive deficits. Little is known, however, about the long‐term consequences of fetal asphyxia contributing to the development of different neurological diseases common in the adult or the aging brain. For that reason the present study aimed to investigate the long‐term effects of fetal asphyxia on synaptic organization within the adult rat brain. Fetal asphyxia was induced at embryonic day 17 by 75‐min clamping of the uterine and ovarian arteries. Presynaptic bouton densities and numbers were analyzed in the striatum and prefrontal cortex at the age of 19 months. A substantial decrease in presynaptic bouton density and number was observed in the striatum of fetal asphyxia rats compared to control rats, while an increase was found in the fifth layer of the prefrontal cortex. These results suggest that fetal asphyxia can have long‐lasting effects on synaptic organization that might contribute to a developmental etiology of different neurological disorders and aging.

Age-Related Changes and Effects of Mild Hypothermia on Carotid Artery Reactivity in Newborn Rats

Therapeutic hypothermia has become a standard neuroprotective treatment in term newborn infants following perinatal asphyxia. Hypothermia-induced changes in the reactivity of the vessels supplying the brain might play a role in its therapeutic or side effects. We investigated the putative age-related changes and the effect of clinically relevant cooling (33°C) on the reactivity of the newborn rat carotid artery. Carotid artery rings from 2-3 days old and 9-10 days old rats were mounted in myographs and studied at 33°C and 37°C. Hypothermia did not significantly affect the contractions induced by KCl and U46619, nor the relaxations induced by acetylcholine (ACh), the nitric oxide (NO) donor sodium nitroprusside (SNP), the NO-independent stimulator of soluble guanylate cyclase (sGC) BAY 41-2272, the β -adrenoceptor agonist isoproterenol, the adenylate cyclase activator forskolin, and acute hypoxia (PO2 3 kPa). The relaxations induced by ACh, isoproterenol, the β 2-adrenoceptor agonist salbutamol, the β 3-adrenoceptor agonist CL-316243 and acute hypoxia increased with postnatal age and were impaired by endothelium removal or by inhibition of NO synthase (L-NAME) or sGC (ODQ). In contrast, the relaxations induced by SNP, BAY 41-2272 and forskolin were endothelium-independent and did not change with age. In conclusion, mild hypothermia (33°C) does not affect the reactivity of neonatal rat carotid arteries. Our data suggest a reduced NO bioavailability in the carotid artery during the first days of life. This transient reduction in endothelium-dependent relaxation might play a role in the adaptation of the circulatory system to birth and in the neonatal vascular response to insults such as hypoxia.