Chronic hypoxia alters cardiac mitochondrial complex protein expression and activity in fetal guinea pigs in a sex-selective manner.

We hypothesize that intrauterine hypoxia (HPX) alters the mitochondrial phenotype in fetal hearts contributing to developmental programming. Pregnant guinea pigs were exposed to normoxia (NMX) or hypoxia (HPX, 10.5% O2), starting at early [25 days (25d), 39d duration] or late gestation (50d, 14d duration). Near-term (64d) male and female fetuses were delivered by hysterotomy from anesthetized sows, and body/organ weights were measured. Left ventricles of fetal hearts were excised and frozen for measurement of expression of complex (I-V) subunits, fusion (Mfn2/OPA1) and fission (DRP1/Fis1) proteins, and enzymatic rates of I and IV from isolated mitochondrial proteins. Chronic HPX decreased fetal body weight and increased relative placenta weight regardless of timing. Early-onset HPX increased I, III, and V subunit levels, increased complex I but decreased IV activities in males but not females (all P < 0.05). Late-onset HPX decreased (P < 0.05) I, III, and V levels in both sexes but increased I and decreased IV activities in males only. Both HPX conditions decreased cardiac mitochondrial DNA content in males only. Neither early- nor late-onset HPX had any effect on Mfn2 levels but increased OPA1 in both sexes. Both HPX treatments increased DRP1/Fis1 levels in males. In females, early-onset HPX increased DRP1 with no effect on Fis1, whereas late-onset HPX increased Fis1 with no effect on DRP1. We conclude that both early- and late-onset HPX disrupts the expression/activities of select complexes that could reduce respiratory efficiency and shifts dynamics toward fission in fetal hearts. Thus, intrauterine HPX disrupts the mitochondrial phenotype predominantly in male fetal hearts, potentially altering cardiac metabolism and predisposing the offspring to heart dysfunction.

Effect of chronic perinatal hypoxia on the role of rho-kinase in pulmonary artery contraction in newborn lambs.

Exposure to chronic hypoxia during gestation predisposes infants to neonatal pulmonary hypertension, but the underlying mechanisms remain unclear. Here, we test the hypothesis that moderate continuous hypoxia during gestation causes changes in the rho-kinase pathway that persist in the newborn period, altering vessel tone and responsiveness. Lambs kept at 3,801 m above sea level during gestation and the first 2 wk of life were compared with those with gestation at low altitude. In vitro studies of isolated pulmonary arterial rings found a more forceful contraction in response to KCl and 5-HT in high-altitude compared with low-altitude lambs. There was no difference between the effects of blockers of various pathways of extracellular Ca(2+) entry in low- and high-altitude arteries. In contrast, inhibition of rho-kinase resulted in significantly greater attenuation of 5-HT constriction in high-altitude compared with low-altitude arteries. High-altitude lambs had higher baseline pulmonary artery pressures and greater elevations in pulmonary artery pressure during 15 min of acute hypoxia compared with low-altitude lambs. Despite evidence for an increased role for rho-kinase in high-altitude arteries, in vivo studies found no significant difference between the effects of rho-kinase inhibition on hypoxic pulmonary vasoconstriction in intact high-altitude and low-altitude lambs. We conclude that chronic hypoxia in utero results in increased vasopressor response to both acute hypoxia and serotonin, but that rho-kinase is involved only in the increased response to serotonin.

Chronic prenatal hypoxia induces epigenetic programming of PKC{epsilon} gene repression in rat hearts.

RATIONALE: Epidemiological studies demonstrate a clear association of adverse intrauterine environment with an increased risk of ischemic heart disease in adulthood. Hypoxia is a common stress to the fetus and results in decreased protein kinase C epsilon (PKCepsilon) expression in the heart and increased cardiac vulnerability to ischemia and reperfusion injury in adult offspring in rats. OBJECTIVES: The present study tested the hypothesis that fetal hypoxia-induced methylation of cytosine-phosphate-guanine dinucleotides at the PKCepsilon promoter is repressive and contributes to PKCepsilon gene repression in the heart of adult offspring. METHODS AND RESULTS: Hypoxic treatment of pregnant rats from days 15 to 21 of gestation resulted in significant decreases in PKCepsilon protein and mRNA in fetal hearts. Similar results were obtained in ex vivo hypoxic treatment of isolated fetal hearts and rat embryonic ventricular myocyte cell line H9c2. Increased methylation of PKCepsilon promoter at SP1 binding sites, -346 and -268, were demonstrated in both fetal hearts of maternal hypoxia and H9c2 cells treated with 1% O(2) for 24 hours. Whereas hypoxia had no significant effect on the binding affinity of SP1 to the unmethylated sites in H9c2 cells, hearts of fetuses and adult offspring, methylation of both SP1 sites reduced SP1 binding. The addition of 5-aza-2'-deoxycytidine blocked the hypoxia-induced increase in methylation of both SP1 binding sites and restored PKCepsilon mRNA and protein to the control levels. In hearts of both fetuses and adult offspring, hypoxia-induced methylation of SP1 sites was significantly greater in males than in females, and decreased PKCepsilon mRNA was seen only in males. In fetal hearts, there was significantly higher abundance of estrogen receptor alpha and beta isoforms in females than in males. Both estrogen receptor alpha and beta interacted with the SP1 binding sites in the fetal heart, which may explain the sex differences in SP1 methylation in the fetal heart. Additionally, selective activation of PKCepsilon restored the hypoxia-induced cardiac vulnerability to ischemic injury in offspring. CONCLUSIONS: The findings demonstrate a direct effect of hypoxia on epigenetic modification of DNA methylation and programming of cardiac PKCepsilon gene repression in a sex-dependent manner, linking fetal hypoxia and pathophysiological consequences in the hearts of adult offspring.

Chronic fetal hypoxia produces selective brain injury associated with altered nitric oxide synthases.

OBJECTIVE: The purpose of this study was to investigate the impact of chronic hypoxia on the nitric oxide synthase isoenzymes in specific brain structures. STUDY DESIGN: Time-mated pregnant guinea pigs were exposed to 10.5% molecular oxygen for 14 days (animals with chronic fetal hypoxia; HPX) or room air (control animals; NMX); L-N6-(1-iminoethyl)-lysine (L-NIL; an inducible nitric oxide synthase inhibitor, 1 mg/kg/d) was administered to HPX group for 14 days (L-NIL + HPX). Fetal brains were harvested at term. Multilabeled immunofluorescence was used to generate a brain injury map. Laser capture microdissection and quantitative polymerase chain reaction were applied; cell injury markers, apoptosis activation, neuron loss, total nitric oxide, and the levels of individual nitric oxide synthase isoenzymes were quantified. RESULTS: Chronic hypoxia causes selective fetal brain injury rather than global. Injury is associated with differentially affected nitric oxide synthases in both neurons and glial cells, with inducible macrophage-type nitric oxide synthase up-regulated at all injury sites. L-NIL attenuated the injury, despite continued hypoxia. CONCLUSION: These studies demonstrate that chronic hypoxia selectively injures the fetal brain in part by the differential regulation of nitric oxide synthase isoenzymes in an anatomic- and cell-specific manner.

Results of and further prevention of hypoxic fetal brain damage by inhibition of xanthine oxidase enzyme with allopurinol.

Several experimental models on adult and newborn animals showed that in cerebral hypoxic-ischemic conditions similar to clinical states the main source of the excessive production of free oxygen radicals is the highly activated xanthine oxidase (XO) enzyme reaction. Long before this data were available, it became known that the main role of allopurinol (AP) is the inhibition of XO. On the basis of these results, many therapeutic trials with AP were performed both in experimental and clinical studies of ischemia and reperfusion. However, it has been shown that only preventive administration of AP has favorable effects. The explanation for the poor results of AP treatment in human fetal brain damage (FBD) cases is that the drug was applied postnatally. The clinical studies performed in healthy laboring mothers whose deliveries were complicated with FBD showed that placental transfer after prenatal administration of AP may be effective in protecting newborns at increased risk of hypoxic-ischemic cerebral damage. Further controlled trials are required to determine if the prophylactic use of the drug might prevent hypoxic-ischemic injuries when the drug is administered immediately prior to impending fetal hypoxia, or even in deliveries at risk of developing hypoxia.

Maturation and long-term hypoxia-induced acclimatization responses in PKC-mediated signaling pathways in ovine cerebral arterial contractility.

In the developing fetus, cerebral arteries (CA) show striking differences in signal transduction mechanisms compared with the adult, and these differences are magnified in response to high-altitude long-term hypoxia (LTH). In addition, in the mature organism, cerebrovascular acclimatization to LTH may be associated with several clinical problems, the mechanisms of which are unknown. Because PKC plays a key role in regulating CA contractility, in fetal and adult cerebral arteries, we tested the hypothesis that LTH differentially regulates the PKC-mediated Ca(2+) sensitization pathways and contractility. In four groups of sheep [fetal normoxic (FN), fetal hypoxic (FH), adult normoxic (AN), and adult hypoxic (AH)], we examined, simultaneously, responses of CA tension and intracellular Ca(2+) concentration and measured CA levels of PKC, ERK1/2, RhoA, 20-kDa myosin light chain, and the 17-kDa PKC-potentiated myosin phosphatase inhibitor CPI-17. The PKC activator phorbol 12,13-dibutyrate (PDBu) produced robust contractions in all four groups. However, PDBu-induced contractions were significantly greater in AH CA than in the other groups. In all CA groups except AH, in the presence of MEK inhibitor (U-0126), the PDBu-induced contractions were increased a further 20-30%. Furthermore, in adult CA, PDBu led to increased phosphorylation of ERK1, but not ERK2; in fetal CA, the reverse was the case. PDBu-stimulated ERK2 phosphorylation also was significantly greater in FH than FN CA. Also, although RhoA/Rho kinase played a significant role in PDBu-mediated contractions of FN CA, this was not the case in FH or either adult group. Also, whereas CPI-17 had a significant role in adult CA contractility, this was not the case for the fetus. Overall, in ovine CA, the present study demonstrates several important maturational and LTH acclimatization changes in PKC-induced contractile responses and downstream pathways. The latter may play a key role in the pathophysiologic disorders associated with acclimatization to high altitude.

Prenatal hypoxia causes a sex-dependent increase in heart susceptibility to ischemia and reperfusion injury in adult male offspring: role of protein kinase C epsilon.

The present study tested the hypothesis that protein kinase C (PKC) epsilon plays a key role in the sex dichotomy of heart susceptibility to ischemia and reperfusion injury in adult offspring resulting from prenatal hypoxic exposure. Time-dated pregnant rats were divided between normoxic and hypoxic (10.5% O(2) on days 15-21 of gestation) groups. Hearts of 3-month-old progeny were subjected to ischemia and reperfusion (I/R) injury in a Langendorff preparation. Preischemic values of left ventricle (LV) function were the same between control and hypoxic animals. Prenatal hypoxia significantly decreased postischemic recovery of LV function and increased cardiac enzyme release and infarct size in adult male, but not female, rats. This was associated with significant decreases in PKC(epsilon) and phospho-PKC(epsilon) levels in the LV of the male, but not female, rats. The PKC(epsilon) translocation inhibitor peptide (PKC(epsilon)-TIP) significantly decreased phospho-PKC(epsilon) in control male rats to the levels found in the hypoxic animals and abolished the difference in I/R injury observed between the control and hypoxic rats. In females, PKC(epsilon)-TIP inhibited PKC(epsilon) phosphorylation and decreased postischemic recovery of LV function equally well in both control and hypoxic animals. PKC(epsilon)-TIP had no effect on PKCdelta activation in either male or female hearts. The results demonstrated that prenatal hypoxia caused an increase in heart susceptibility to ischemia and reperfusion injury in offspring in a sex-dependent manner, which was due to fetal programming of PKC(epsilon) gene repression resulting in a down-regulation of PKC(epsilon) function in the heart of adult male offspring.

Chronic hypoxia increases inducible NOS-derived nitric oxide in fetal guinea pig hearts.

Intrauterine hypoxia impacts fetal growth and organ function. Inducible nitric oxide synthase (iNOS) and neuronal NOS (nNOS) expression was measured to assess the response of fetal hearts to hypoxic (HPX) stress. Pregnant guinea pigs were housed in a hypoxic chamber (10.5% O2 for 14 d, n = 17) or room air [normoxic (NMX), n = 17]. Hearts of anesthetized near-term fetuses were removed. mRNA [hypoxia-inducible factor, (HIF)-1alpha, 1beta, 2alpha, 3alpha, iNOS, and nNOS] and protein levels (HIF-1alpha, iNOS, and nNOS) of fetal cardiac left ventricles were quantified by real time polymerase chain reaction (PCR) and Western analysis, respectively. Cardiac nitrite/nitrate levels were measured in the presence/absence of L-N6-(1-iminoethyl)-lysine (L-NIL), an iNOS inhibitor, administered to pregnant sows. Hypoxia significantly increased fetal cardiac HIF-1alpha and -2alpha mRNA, HIF-1alpha protein but not HIF-3alpha or -1beta mRNA levels. Hypoxia increased both iNOS mRNA (by 5x) and protein (by 23%) levels but had no effect on nNOS levels. Nitrite/nitrate levels were increased in HPX hearts by 2.5x and decreased with L-NIL by 67 +/- 14%. Thus, up-regulation of iNOS-derived nitric oxide (NO) generation is an important mechanism by which fetal hearts respond to chronic hypoxic stress.

Prenatal hypoxia induces increased cardiac contractility on a background of decreased capillary density.

BACKGROUND: Chronic hypoxia in utero (CHU) is one of the most common insults to fetal development and may be associated with poor cardiac recovery from ischaemia-reperfusion injury, yet the effects on normal cardiac mechanical performance are poorly understood. METHODS: Pregnant female wistar rats were exposed to hypoxia (12% oxygen, balance nitrogen) for days 10-20 of pregnancy. Pups were born into normal room air and weaned normally. At 10 weeks of age, hearts were excised under anaesthesia and underwent retrograde 'Langendorff' perfusion. Mechanical performance was measured at constant filling pressure (100 cm H2O) with intraventricular balloon. Left ventricular free wall was dissected away and capillary density estimated following alkaline phosphatase staining. Expression of SERCA2a and Nitric Oxide Synthases (NOS) proteins were estimated by immunoblotting. RESULTS: CHU significantly increased body mass (P < 0.001) compared with age-matched control rats but was without effect on relative cardiac mass. For incremental increases in left ventricular balloon volume, diastolic pressure was preserved. However, systolic pressure was significantly greater following CHU for balloon volume = 50 microl (P < 0.01) and up to 200 microl (P < 0.05). For higher balloon volumes systolic pressure was not significantly different from control. Developed pressures were correspondingly increased relative to controls for balloon volumes up to 250 microl (P < 0.05). Left ventricular free wall capillary density was significantly decreased in both epicardium (18%; P < 0.05) and endocardium (11%; P < 0.05) despite preserved coronary flow. Western blot analysis revealed no change to the expression of SERCA2a or nNOS but immuno-detectable eNOS protein was significantly decreased (P < 0.001) in cardiac tissue following chronic hypoxia in utero. CONCLUSION: These data offer potential mechanisms for poor recovery following ischaemia, including decreased coronary flow reserve and impaired angiogenesis with subsequent detrimental effects of post-natal cardiac performance.

Neurosteroids in the fetus and neonate: potential protective role in compromised pregnancies.

Complications during pregnancy and birth asphyxia lead to brain injury, with devastating consequences for the neonate. In this paper we present evidence that the steroid environment during pregnancy and at birth aids in protecting the fetus and neonate from asphyxia-induced injury. Earlier studies show that the placental progesterone production has a role in the synthesis and release of neuroactive steroids or their precursors into the fetal circulation. Placental precursor support leads to remarkably high concentrations of allopregnanolone in the fetal brain and to a dramatic decline with the loss of the placenta at birth. These elevated concentrations influence the distinct behavioral states displayed by the late gestation fetus and exert a suppressive effect that maintains sleep-like behavioral states that are present for much of fetal life. This suppression reduces CNS excitability and suppresses excitotoxicity. With the availability of adequate precursors, mechanisms within the fetal brain ultimately control neurosteroid levels. These mechanisms respond to episodes of acute hypoxia by increasing expression of 5alpha-reductase and P450scc enzymes and allopregnanolone synthesis in the brain. This allopregnanolone response, and potentially that of other neurosteroids including 5alpha-tetrahydrodeoxycorticosterone (TH-DOC), reduces hippocampal cell death following acute asphyxia and suggests that stimulation of neurosteroid production may protect the fetal brain. Importantly, inhibition of neurosteroid synthesis in the fetal brain increases the basal cell death suggesting a role in controlling developmental processes late in gestation. Synthesis of neurosteroid precursors in the fetal adrenal such as deoxycorticosterone (DOC), and their conversion to active neurosteroids in the fetal brain may also have a role in neuroprotection. This suggests that the adrenal glands provide precursor DOC for neurosteroid synthesis after birth and this may lead to a switch from allopregnanolone alone to neuroprotection mediated by allopregnanolone and TH-DOC.

Programmed cell death and differential JNK, p38 and ERK response in a prenatal acute hypoxic hypoxia model.

We previously found that prenatal hypoxia induces a significant increase in the levels of active Caspase 3 at 60 min post-hypoxia (p-h) and in the number of TUNEL-positive pyknotic cells, which peaks at 6h p-h. The aim of this work was to study alterations in MAPKs pathways and the effect of specific inhibitors of the JNK (SP600125) and p38 (SB203580) pathways following acute hypoxia in chick optic lobe at embryonic day (ED) 12. To this end, JNK, p38 and ERK1-2 protein kinase expression levels were determined by Western blot in both their active and inactive forms, evaluated at successive p-h times. At 10 and 30 min p-h the P-JNK/JNK ratio was 1.912+/-0.341 and 1.920+/-0.304, respectively. Concomitantly, at 0 min p-h the P-p38/p38 ratio was 1.657+/-0.203. Lastly, the P-ERK/ERK ratio proving non-significant throughout. When inhibitors for JNK and p38 were used, we observed a decrease in the values of active Caspase 3 at 60 min p-h, which correlated with the control values in the parameters of TUNEL-positive cells at 6h p-h. Analysis for P-ATF-2 demonstrated an increase in hypoxic embryos compared to control ones which was reverted in a dose-dependent manner with the use of both inhibitors. All these results indicate that at ED 12, acute hypoxia might be differentially activating JNK and p38 pathways, without affecting the ERK pathway, which in turn would be activating Caspase 3, thus leading to cell death by apoptosis. Furthermore, JNK and p38 activation precede in time the programmed cell death induced by hypoxia.

Neuroprotection of brain-derived neurotrophic factor against hypoxic injury in vitro requires activation of extracellular signal-regulated kinase and phosphatidylinositol 3-kinase.

Intrauterine asphyxia is one of the major contributors for perinatal death, mental and physical disorders of surviving children. Brain-derived neurotrophic factor (BDNF) provides a promising solution to hypoxic injury due to its survival-promoting effects. In an attempt to identify possible molecular mechanisms underlying the neuroprotective role of BDNF, we studied extracellular signal-regulated kinase (ERK), phosphatidylinositol 3-kinase (PI-3-K) and p38 mitogen-activated protein kinase (MAPK) pathways. We demonstrated that BDNF protected cortical neurons against hypoxic injury in vitro via activation of both the ERK and PI-3-K pathways but not the p38 MAPK pathway. We also showed that both hypoxic stimuli and exogenous BDNF treatment phosphorylated the cyclic AMP response element-binding protein (CREB) and that CREB phosphorylation induced by BDNF was mediated via the ERK pathway in cultured cortical neurons.

Chronic hypoxia differentially increases glutathione content and gamma-glutamyl cysteine synthetase expression in fetal guinea pig organs.

OBJECTIVE: Glutathione is a natural antioxidant in the fetus and adult. We sought to determine whether maternal hypoxia alters glutathione levels in fetal organs as an adaptive response to the reduced oxygenation. STUDY DESIGN: Timed pregnant guinea pigs were housed in either a Plexiglas chamber containing 10.5% O(2) from 46 to 60 days gestation (HPX, n=6) or in room air, as the normoxic control (NMX, n=5). Pregnant guinea pigs were anesthetized at near term ( approximately 60 days, term=65 days) and liver, lungand kidney were excised from anesthetized fetuses and stored frozen (-80 degrees C) prior to sample processing. Using the hypoxia marker, pimonidazole, we measured a hypoxia-induced increase in stained cells of fetal liver compared to no change in either the lung or kidney. To measure the effect of hypoxia among different organs, total glutathione (GSH) content and protein levels of gamma-glutamyl cysteine synthetase (gamma-GCS) were measured from the same organs. RESULTS: Maternal hypoxia increased (P<0.05) total glutathione levels by 121% in the fetal liver but had no effect in either fetal lung or kidney. Chronic hypoxia increased (P<0.05) gamma-GCS protein levels in all three fetal organs studied. CONCLUSION: These results demonstrate that the fetal response to maternal hypoxia may be organ specific. The increase in fetal liver glutathione via upregulation of gamma-GCS may be an important adaptive response to prolonged hypoxic stress.

Hypoxia blocks 11beta-hydroxysteroid dehydrogenase type 2 induction in human trophoblast cells during differentiation by a time-dependent mechanism that involves both translation and transcription.

The present study was undertaken to determine (1) if hypoxia-induced down-regulation of placental 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2; encoded by HSD11B2 gene) activity and protein in human trophoblast cells during in vitro differentiation was mediated at the level of HSD11B2 gene transcription; and (2) whether the reduced placental 11beta-HSD2 in pregnancies complicated with fetal growth restriction (FGR) was a consequence of intrinsic abnormalities in trophoblast cells. Trophoblast cells were isolated from uncomplicated pregnancies and those complicated with FGR at term, and cultured for up to 72 h under normoxic (20% oxygen) or hypoxic (1% oxygen) conditions. Under normoxia, 11beta-HSD2 activity and protein increased progressively over the 72 h culture period, which was accompanied by a corresponding rise in 11beta-HSD2 mRNA. As demonstrated previously, hypoxia blocked the increase in levels of both 11beta-HSD2 activity and protein within the first 24h. In contrast, although hypoxia also prevented the rise in 11beta-HSD2 mRNA, it did not do so until 48 h. This time-dependent effect of hypoxia on placental 11beta-HSD2 activity/protein and mRNA suggests a dual mechanism of action whereby hypoxia may induce a rapid down-regulation of 11beta-HSD2 protein synthesis, which occurs initially at the level of translation, and later extends to the level of transcription. Indeed, transient transfection studies demonstrated that hypoxia diminished HSD11B2 promoter activity. When trophoblast cells isolated from FGR placentas were cultured and allowed to differentiate under the same conditions, they not only exhibited a similar pattern of 11beta-HSD2 activity and mRNA expression but also responded to hypoxia similarly to those from normal placentas. This suggests that the reduced placental 11beta-HSD2 in FGR is not due to intrinsic abnormalities in trophoblast cells, but likely a result of extrinsic factors associated with FGR.

Cellular levels of TrkB and MAPK in the neuroprotective role of BDNF for embryonic rat cortical neurons against hypoxia in vitro.

Intrauterine asphyxia often results in neonatal loss or mental retardation. Brain-derived neurotrophic factor (BDNF) has been shown to be a protective agent against hypoxic damage to neurons. To understand the signaling mechanism underling the neuroprotective function of BDNF and to find therapeutic interventions for intrauterine asphyxia, we utilized an immunofluorescent technique to measure the intracellular levels of tyrosine kinase B (TrkB), phosphorylated TrkB, and the mitogen-activated protein kinase (MAPK) in the rat embryonic cortical neurons cultured in hypoxic conditions with and without BDNF pretreatment. The results showed that the fluorescent intensity of TrkB and phosphorylated TrkB in the cytoplasm and the fluorescent intensity of MARK in both cytoplasma and nucleus of the neurons were significantly increased in the presence of BDNF. The results indicate that the neuroprotective function of BDNF against hypoxia-induced neurotoxicity requires the participation of TrkB and is transduced via the Ras-MAPK signaling pathway.

Adrenocorticotropic Hormone and PI3K/Akt Inhibition Reduce eNOS Phosphorylation and Increase Cortisol Biosynthesis in Long-Term Hypoxic Ovine Fetal Adrenal Cortical Cells.

This study was designed to determine the role of the MEK/ERK1/2 and PI3K/Akt pathways in cortisol production and endothelial nitric oxide synthase (eNOS) phosphorylation (peNOS) in the ovine fetal adrenal in response to long-term hypoxia (LTH). Pregnant ewes were maintained at high altitude (3820 m) for the last 100 days of gestation (dGa). At 138 to 142 dGa, fetal adrenal cortical cells (FACs) were collected from LTH and age-matched normoxic fetuses. Cortisol production and peNOS were measured in response to pretreatment with the MEK/ERK1/2 pathway inhibitor UO126 (UO) and adrenocorticotropic hormone (ACTH) stimulation. UO126 reduced ACTH-stimulated cortisol in both normoxic and LTH FACs. UO126 alone or in combination with ACTH reduced peNOS in the normoxic group, while ACTH alone or ACTH + UO inhibited peNOS in LTH FACs. Additionally, cortisol was measured in response to pretreatment with UO and treatment with 22R-hydroxycholesterol (22R-OHC) or water-soluble cholesterol (WSC) with and without ACTH stimulation. UO126 had no effect on 22R-OHC-treated cells, but reduced cortisol in cells treated with WSC and/or ACTH. Cortisol and peNOS were also measured in response to pretreatment with PI3K/Akt pathway inhibitor Wortmannin (WT) and ACTH stimulation. Wortmannin further increased cortisol under ACTH-stimulated conditions and, like ACTH, reduced peNOS in LTH but not normoxic FACs. Together, these data suggest that in LTH FACs MEK/ERK1/2 does not regulate peNOS but that UO acts downstream from eNOS, possibly at cholesterol transport, to affect cortisol production in LTH FACs, while the PI3K/Akt pathway, along with ACTH, regulates peNOS and plays a role in the fetal adaptation to LTH in FACs.

Developmental changes of gene expression in heme metabolic enzymes in rat placenta.

Transcription levels of the non-specific delta-aminolevulinate synthase (ALAS-N) and heme oxygenase-1 (HO-1) in the placenta at the terminal stage of pregnancy were comparable to those in the female adult liver and in the spleen, respectively. Immunohistochemical studies demonstrated that both enzymes were exclusively expressed in the trophoblast. During gestation, transcript of ALAS-N slightly increased, while HO-1 mRNA significantly decreased. Induced acute fetal hypoxia resulted in an increase in ALAS-N mRNA and in a decrease in HO-1 mRNA. These findings indicate that placental heme metabolism is influenced by the oxygen supply.

Chronic hypoxemia: effects on developing nitrergic and dopaminergic amacrine cells.

PURPOSE: Very low birth weight and growth-restricted children have visual impairments including reduced contrast sensitivity, a parameter mediated in part by dopaminergic amacrine cells. The origin of these deficits is uncertain. In experimental fetal growth restriction, induced by placental insufficiency, the morphology and number of dopaminergic amacrine cells as identified by tyrosine hydroxylase staining were examined. In addition, the subclass of nitrergic amacrine cells was examined because nitric oxide released from nitric oxide synthase- containing neurons is proposed as a mediator of neurotoxicity and might contribute to the injury of dopaminergic amacrine cells in this situation. METHODS: Fetal sheep were subjected to 20 or 30 days of placental embolization leading to fetal hypoxemia, hypoglycemia, and growth restriction during the last third of gestation (term, approximately 147 days). Retinal tissue was prepared as wholemounts or cryostat sections and analyzed for retinal area, total number, density, somal size and cell process length of amacrine cells immunoreactive for tyrosine hydroxylase or nitric oxide synthase, and widths of retinal layers. Retinas from fetal sheep at 72, 96, 113, and 140 days' gestation and adults were collected for an ontogenetic study of tyrosine hydroxylase-immunoreactive neurons. RESULTS: In growth-restricted fetuses, the number of tyrosine hydroxylase-immunoreactive neurons and the total length of stained processes per cell were significantly reduced compared to control fetuses. The total number of neuronal nitric oxide synthase- containing neurons was not different between growth-restricted and control fetuses. The thickness of the inner retinal layers was reduced in hypoxemia. CONCLUSIONS: There is damage to tyrosine hydroxylase-immunoreactive amacrine cells during fetal chronic placental insufficiency. This damage might be involved in the altered retinal dopaminergic system observed in very low birth weight infants, some of whom are growth-restricted. Furthermore, a differential susceptibility of tyrosine hydroxylase-immunoreactive and neuronal nitric oxide synthase-containing amacrine cells to hypoxemic injury has been demonstrated. These observations add to the current hypothesis that neuronal nitric oxide synthase- containing neurons are resistant to hypoxemic injury and may be involved in mediating some of the neuronal damage that results from hypoxemic insults.

Hemodynamic and metabolic responses to moderate asphyxia in brain and skeletal muscle of late-gestation fetal sheep.

The purpose of this study was to investigate metabolic and hemodynamic responses in two fetal tissues, hindlimb muscle and brain, to an episode of acute moderate asphyxia. Near-infrared spectroscopy was used to measure changes in total hemoglobin concentration ([tHb]) and the redox state of cytochrome oxidase (COX) simultaneously in the brain and hindlimb of near-term unanesthetized fetal sheep in utero. Oxygen delivery (DO(2)) to, and consumption (VO(2)) by, each tissue was derived from the arteriovenous difference in oxygen content and blood flow, measured by implanted flow probes. One hour of moderate asphyxia (n = 11), caused by occlusion of the maternal common internal iliac artery, led to a significant fall in DO(2) to both tissues and to a significant drop in VO(2) by the head. This was associated with an initial fall in redox state COX in the leg but an increase in the brain. [tHb], and therefore blood volume, fell in the leg and increased in the brain. These data suggest the presence of a fetal metabolic response to hypoxia, which, in the brain, occurs rapidly and could be neuroprotective.

Ornithine decarboxylase activity in fetal and newborn rat brain: responses to hypoxic and carbon monoxide hypoxia.

In response to acute maternal hypoxia, ornithine decarboxylase (ODC) activity increased significantly in fetal rat brain, peaking at 4 h. This was associated with increased ODC mRNA and elevated polyamine concentrations. To correlate this response with development, we measured ODC activity in the rat from gestational day E 17 to postnatal day P 10. We also examined to what extent hypoxia induces increased ODC activity in adult rat brains and whether the response to chronic hypoxia differed from that to acute hypoxia. To test the hypothesis that this increased activity is due to hypoxic hypoxia per se, we subjected pregnant dams to inspired carbon monoxide concentrations ranging from 150 to 1000 ppm and assayed ODC activity in the fetal brain 4 h later. In the fetus, ODC activity was elevated on E 17 in the cerebrum and cerebellum. It declined gradually to about one-tenth E 17 levels by E 21 and remained low thereafter except for a postnatal elevation in the cerebellum on P 3. In response to 10.5% O2, in the 3-day-old rat, ODC activity peaked between 2 and 3 h of hypoxia, increasing 3-fold in the hippocampus and 2-fold in cerebellum. Similar increases were seen in the hypoxic adult rat brain. In inspired oxygen dose-response studies, exposure of P 3 rat pups to 13.25% O2 for 2.5 h produced a 1.5-fold increase in ODC activity; 10.5% O2 produced a 2-3-fold increase while in response to 9% O2, ODC activity remained at baseline levels. With maternal CO-hypoxia, ODC activity increased in the fetal brain at 4 h, as seen with hypoxic-hypoxia. For example, in hippocampus, ODC activity doubled at 500 ppm and tripled at 600 ppm. We conclude: (1) apparently, the ability to respond thus is not lost as the animal ages and may represent an important cellular response to acute hypoxia; (2) the increase in hypoxic-induced ODC activity is relative to the already elevated activity seen from E 17 to E 20; a vast reserve for the induction of fetal ODC activity probably exists and may indicate the importance of this enzyme during this time frame for differentiation and growth promotion; and (3) the CO-hypoxia studies suggest that some aspects of the cellular responses to CO- and hypoxic-hypoxia are similar.