Near-term Ovine Fetus Research Articles

Changes in Cellular Localization of Inter-Alpha Inhibitor Proteins after Cerebral Ischemia in the Near-Term Ovine Fetus.

Inter-alpha Inhibitor Proteins (IAIPs) are key immunomodulatory molecules. Endogenous IAIPs are present in human, rodent, and sheep brains, and are variably localized to the cytoplasm and nuclei at multiple developmental stages. We have previously reported that ischemia-reperfusion (I/R) reduces IAIP concentrations in the fetal sheep brain. In this study, we examined the effect of I/R on total, cytoplasmic, and nuclear expression of IAIPs in neurons (NeuN+), microglia (Iba1+), oligodendrocytes (Olig2+) and proliferating cells (Ki67+), and their co-localization with histones and the endoplasmic reticulum in fetal brain cells. At 128 days of gestation, fetal sheep were exposed to Sham (n = 6) or I/R induced by cerebral ischemia for 30 min with reperfusion for 7 days (n = 5). Although I/R did not change the total number of IAIP+ cells in the cerebral cortex or white matter, cells with IAIP+ cytoplasm decreased, whereas cells with IAIP+ nuclei increased in the cortex. I/R reduced total neuronal number but did not change the IAIP+ neuronal number. The proportion of cytoplasmic IAIP+ neurons was reduced, but there was no change in the number of nuclear IAIP+ neurons. I/R increased the number of microglia and decreased the total numbers of IAIP+ microglia and nuclear IAIP+ microglia, but not the number of cytoplasmic IAIP+ microglia. I/R was associated with reduced numbers of oligodendrocytes and increased proliferating cells, without changes in the subcellular IAIP localization. IAIPs co-localized with the endoplasmic reticulum and histones. In conclusion, I/R alters the subcellular localization of IAIPs in cortical neurons and microglia but not in oligodendrocytes or proliferating cells. Taken together with the known neuroprotective effects of exogenous IAIPs, we speculate that endogenous IAIPs may play a role during recovery from I/R.

Distance to Healthy Metabolic and Cardiovascular Dynamics From Fetal Heart Rate Scale-Dependent Features in Pregnant Sheep Model of Human Labor Predicts the Evolution of Acidemia and Cardiovascular Decompensation.

The overarching goal of the present work is to contribute to the understanding of the relations between fetal heart rate (FHR) temporal dynamics and the well-being of the fetus, notably in terms of predicting the evolution of lactate, pH and cardiovascular decompensation (CVD). It makes uses of an established animal model of human labor, where 14 near-term ovine fetuses subjected to umbilical cord occlusions (UCO) were instrumented to permit regular intermittent measurements of metabolites lactate and base excess, pH, and continuous recording of electrocardiogram (ECG) and systemic arterial blood pressure (to identify CVD) during UCO. ECG-derived FHR was digitized at the sampling rate of 1,000 Hz and resampled to 4 Hz, as used in clinical routine. We focused on four FHR variability features which are tunable to temporal scales of FHR dynamics, robustly computable from FHR sampled at 4 Hz and within short-time sliding windows, hence permitting a time-dependent, or local, analysis of FHR which helps dealing with signal noise. Results show the sensitivity of the proposed features for early detection of CVD, correlation to metabolites and pH, useful for early acidosis detection and the importance of coarse time scales (2.5–8 s) which are not disturbed by the low FHR sampling rate. Further, we introduce the performance of an individualized self-referencing metric of the distance to healthy state, based on a combination of the four features. We demonstrate that this novel metric, applied to clinically available FHR temporal dynamics alone, accurately predicts the time occurrence of CVD which heralds a clinically significant degradation of the fetal health reserve to tolerate the trial of labor.

In utero hypoxia altered Ang II-induced contraction via PKCβ in fetal cerebral arteries.

Cerebral circulation is important in fetal brain development, and angiotensin II (Ang II) plays vital roles in regulation of adult cerebral circulation. However, functions of Ang II in fetal cerebral vasculature and influences of in utero hypoxia on Ang II-mediated fetal cerebral vascular responses are largely unknown. This study investigated the effects and mechanisms of in utero hypoxia on fetal middle cerebral arteries (MCA) via Ang II. Near-term ovine fetuses were exposed to in utero hypoxia, and fetal MCA responses to Ang II were tested for vascular tension, calcium transient, and molecular analysis. Ang II caused significant dose-dependent contraction in control fetal MCA. Ang II-induced MCA constriction was decreased significantly in hypoxic fetuses. Neither losartan (AT1R antagonist, 10-5 mol/L) nor PD123,319 (AT2R antagonist, 10-5 mol/L) altered Ang II-mediated contraction in fetal MCA. Phenylephrine-mediated constriction was also significantly weaker in hypoxic fetuses. Bay K8644 caused similar contractions between the two groups. Protein expression of L-type voltage-dependent calcium channels was unchanged. There were no differences in caffeine-mediated vascular tension or calcium transients. Contraction induced by PDBu (PKC agonist) was obviously weaker in hypoxic MCA. Protein expression of PKCβ was reduced in the hypoxic compared with the control, along with no differences in phosphorylation levels. The results showed that fetal MCA was functionally responsive to Ang II near term. Intrauterine hypoxia reduced the vascular agonist-mediated contraction in fetal MCA, probably via decreasing PKCβ and its phosphorylation, which might play protective effects on fetal cerebral circulation against transient hypoxia.

Vagal contributions to fetal heart rate variability: an omics approach

Objective: Fetal heart rate variability (fHRV) is an important indicator of health and disease, yet its physiological origins, neural contributions, in particular, are not well understood. We aimed to develop novel experimental and data analytical approaches to identify fHRV measures reflecting the vagus nerve contributions to fHRV. Approach: In near-term ovine fetuses, a comprehensive set of 46 fHRV measures was computed from fetal pre-cordial electrocardiogram recorded during surgery and 72 h later without (n = 24) and with intra-surgical bilateral cervical vagotomy (n = 15). Main results: The fetal heart rate did not change due to vagotomy. We identify fHRV measures specific to the vagal modulation of fHRV: multiscale time irreversibility asymmetry index (AsymI), detrended fluctuation analysis (DFA) α1, Kullback–Leibler permutation entropy (KLPE) and scale-dependent Lyapunov exponent slope (SDLE α). Significance: We provide a systematic delineation of vagal contributions to fHRV across signal-analytical domains which should be relevant for the emerging field of bioelectronic medicine and the deciphering of the ‘vagus code’. Our findings also have clinical significance for in utero monitoring of fetal health during surgery.Key points•Fetal surgery causes a complex pattern of changes in heart rate variability measures with an overall reduction of complexity or variability.•At 72 h after surgery, many of the HRV measures recover and this recovery is delayed by an intrasurgical cervical bilateral vagotomy.•We identify HRV pattern representing complete vagal withdrawal that can be understood as part of ‘HRV code’, rather than any single HRV measure.•We identify HRV biomarkers of recovery from fetal surgery and discuss the effect of anticholinergic medication on this recovery.

RNAseq profiling of primary microglia and astrocyte cultures in near-term ovine fetus: A glial in vivo-in vitro multi-hit paradigm in large mammalian brain

BackgroundThe chronically instrumented fetal sheep is a widely used animal model to study fetal brain development in health and disease, but no methods exist yet to interrogate dedicated brain cell populations to identify their molecular and genomic phenotype. For example, the molecular mechanisms whereby microglia or astrocytes contribute to inflammation in the brain remain incompletely understood. New methodHere we present a protocol to derive primary pure microglial or astrocyte cultures from near-term fetal sheep brain, after the animals have been chronically instrumented and studied in vivo. Next, we present the implementation of whole transcriptome sequencing (RNAseq) pipeline to deeper elucidate the phenotype of such primary sheep brain glial cultures. ResultsWe validate the new primary cultures method for cell purity and test the function of the glial cells on protein (IL-1β) and transcriptome (RNAseq) levels in response to a lipopolysaccharide (LPS) challenge in vitro. Comparison with existing methodsThis method represents the first implementation of pure microglial or astrocytes cultures in fetal sheep brain. ConclusionsThe presented approach opens new possibilities for testing not only supernatant protein levels in response to an in vitro challenge, but also to evaluate changes in the transcriptome of glial cells derived from a large mammalian brain bearing high resemblance to the human brain. Moreover, the presented approach lends itself to modeling the complex multi-hit paradigms of antenatal and perinatal cerebral insults in vivo and in vitro.

Brain Injury and Inflammatory Response to Umbilical Cord Occlusions Is Limited With Worsening Acidosis in the Near-Term Ovine Fetus.

We hypothesized that repetitive umbilical cord occlusions (UCOs) with worsening fetal acidemia will lead to an inflammatory response within the brain and thereby brain injury which will be exacerbated by chronic hypoxemia and low-grade infection. Chronically instrumented fetal sheep served as controls (N = 10) or underwent repeated UCOs for up to 4 hours or until arterial pH was <7.00. Normoxic-UCO (N = 9) and hypoxic-UCO (N = 5) fetuses had arterial O2 saturation pre-UCOs of >55% and <55%, respectively, whereas lipopolysaccharide (LPS) UCO fetuses (N = 6) received LPS intra-amniotic (2 mg/h) starting 1 hour pre-UCOs. Animals were euthanized at 48 hours of recovery with fetal brains processed for assessment of inflammation (microglia and mast cell counts) and injury (necrosis-hematoxylin and eosin-and apoptosis-cleaved caspase-3 and terminal deoxynucleotidyl transferase dUTP nick end labeling [TUNEL]). Repetitive UCOs resulted in severe acidemia in most animals with pH approaching 7.00 for all 3 UCO groups. However, there was no significant effect on measures of brain inflammation or injury, except in the LPS-UCO animals where TUNEL-positive cells were increased in the hippocampus, although small animal numbers in the hypoxic-UCO group may have limited the ability to detect significance in their TUNEL cell findings. We were therefore unable to confirm our working hypothesis since the near-term ovine fetal brain showed remarkable tolerance for these cord occlusion insults and likely involving protective metabolic mechanisms, despite the severe acidemia noted.

Decreased neuroinflammation correlates to higher vagus nerve activity fluctuations in near-term ovine fetuses: a case for the afferent cholinergic anti-inflammatory pathway?

BackgroundNeuroinflammation in utero may contribute to brain injury resulting in life-long neurological disabilities. The pivotal role of the efferent cholinergic anti-inflammatory pathway (CAP) in controlling inflammation, e.g., by inhibiting the HMGB1 release, via the macrophages’ α7 nicotinic acetylcholine receptor (α7nAChR) has been described in adults, but its importance in the fetus is unknown. Moreover, it is unknown whether CAP may also exert anti-inflammatory effects on the brain via the anatomically predominant afferent component of the vagus nerve.MethodsWe measured microglial activation in the ovine fetal brain near term 24 h after the umbilical cord occlusions mimicking human labor versus controls (no occlusions) by quantifying HMGB1 nucleus-to-cytosol translocation in the Iba1+ and α7nAChR+ microglia. Based on multiple clinical studies in adults and our own work in fetal autonomic nervous system, we gauged the degree of CAP activity in vivo using heart rate variability measure RMSSD that reflects fluctuations in vagus nerve activity.ResultsRMSSD correlated to corresponding plasma IL-1β levels at R = 0.57 (p = 0.02, n = 17) and to white matter microglia cell counts at R = −0.89 (p = 0.03). The insult increased the HMGB1 translocation in α7nAChR+ microglia in a brain region-dependent manner (p < 0.001). In parallel, RMSSD at 1 h post insult correlated with cytosolic HMGB1 of thalamic microglia (R = −0.94, p = 0.005), and RMSSD at pH nadir correlated with microglial α7nAChR in the white matter (R = 0.83, p = 0.04). Overall, higher RMSSD values correlated with lower HMGB1 translocation and higher α7nAChR intensity per area in a brain region-specific manner.ConclusionsAfferent fetal CAP may translate increased vagal cholinergic signaling into suppression of cerebral inflammation in response to near-term hypoxic acidemia as might occur during labor. Our findings suggest a new control mechanism of fetal neuroinflammation via the vagus nerve, providing novel possibilities for its non-invasive monitoring in utero and for targeted treatment.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-016-0567-x) contains supplementary material, which is available to authorized users.

Adaptive shut-down of EEG activity predicts critical acidemia in the near-term ovine fetus.

In fetal sheep, the electrocorticogram (ECOG) recorded directly from the cortex during repetitive heart rate (FHR) decelerations induced by umbilical cord occlusions (UCO) predictably correlates with worsening hypoxic-acidemia. In human fetal monitoring during labor, the equivalent electroencephalogram (EEG) can be recorded noninvasively from the scalp. We tested the hypothesis that combined fetal EEG – FHR monitoring allows for early detection of worsening hypoxic-acidemia similar to that shown for ECOG-FHR monitoring. Near-term fetal sheep (n = 9) were chronically instrumented with arterial and venous catheters, ECG, ECOG, and EEG electrodes and umbilical cord occluder, followed by 4 days of recovery. Repetitive UCOs of 1 min duration and increasing strength (with regard to the degree of reduction in umbilical blood flow) were induced each 2.5 min until pH dropped to <7.00. Repetitive UCOs led to marked acidosis (arterial pH 7.35 ± 0.01 to 7.00 ± 0.03). At pH of 7.22 ± 0.03 (range 7.32–7.07), and 45 ± 9 min (range 1 h 33 min–20 min) prior to attaining pH < 7.00, both ECOG and EEG amplitudes began to decrease ∼fourfold during each FHR deceleration in a synchronized manner. Confirming our hypothesis, these findings support fetal EEG as a useful adjunct to FHR monitoring during human labor for early detection of incipient fetal acidemia.

Antenatal Maternal Long-Term Hypoxia: Acclimatization Responses with Altered Gene Expression in Ovine Fetal Carotid Arteries

In humans and other species, long-term hypoxia (LTH) during pregnancy can lead to intrauterine growth restriction with reduced body/brain weight, dysregulation of cerebral blood flow (CBF), and other problems. To identify the signal transduction pathways and critical molecules, which may be involved in acclimatization to high altitude LTH, we conducted microarray with advanced bioinformatic analysis on carotid arteries (CA) from the normoxic near-term ovine fetus at sea-level and those acclimatized to high altitude for 110+ days during gestation. In response to LTH acclimatization, in fetal CA we identified mRNA from 38 genes upregulated >2 fold (P<0.05) and 9 genes downregulated >2-fold (P<0.05). The major genes with upregulated mRNA were SLC1A3, Insulin-like growth factor (IGF) binding protein 3, IGF type 2 receptor, transforming growth factor (TGF) Beta-3, and genes involved in the AKT and BCL2 signal transduction networks. Most genes with upregulated mRNA have a common motif for Pbx/Knotted homeobox in the promoter region, and Sox family binding sites in the 3′ un translated region (UTR). Genes with downregulated mRNA included those involved in the P53 pathway and 5-lipoxygenase activating proteins. The promoter region of all genes with downregulated mRNA, had a common 49 bp region with a binding site for DOT6 and TOD6, components of the RPD3 histone deacetylase complex RPD3C(L). We also identified miRNA complementary to a number of the altered genes. Thus, the present study identified molecules in the ovine fetus, which may play a role in the acclimatization response to high-altitude associated LTH.

Correlation of arterial fetal base deficit and lactate changes with severity of variable heart rate decelerations in the near-term ovine fetus

Recent guidelines classify variable decelerations without detail as to degree of depth. We hypothesized that variable deceleration severity is highly correlated with fetal base deficit accumulation. Seven near-term fetal sheep underwent a series of graded umbilical cord occlusions resulting in mild (30 bpm decrease), moderate (60 bpm decrease), or severe (decrease of 90 bpm to baseline <70 bpm) variable decelerations at 2.5 minute intervals. Mild, moderate, and severe variable decelerations increased fetal base deficit (0.21 ± 0.03, 0.27 ± 0.03, and 0.54 ± 0.09 mEq/L per minute) in direct proportion to severity. During recovery, fetal base deficit cleared at 0.12 mEq/L per minute. In this model, ovine fetuses can tolerate repetitive mild and moderate variable decelerations with minimal change in base deficit and lactate. In contrast, repetitive severe variable decelerations may result in significant base deficit increases, dependent on frequency. Modified guideline differentiation of mild/moderate vs severe variable decelerations may aid in the interpretation of fetal heart rate tracings and optimization of clinical management paradigms.

The effect of maternal undernutrition on muscle development in the ovine fetus*

The effect of maternal undernutrition on muscle development of near-term ovine fetuses was studied in 2 experiments. In each experiment, Welsh mountain ewes were randomly assigned to either control (C) or nutrient-restricted (R) groups. In the first experiment, mild maternal undernutrition (85% of daily nutritional requirement (DNR)), and in the second experiment, severe maternal undernutrition (50% of DNR), were imposed between 0 and 70 days of gestation (dg). Controls for both groups were maintained on 100% of their DNR throughout the experiment. In each experiment, ewes were killed at 126 ± 1 dg by an intravenous injection of pentobarbitone. The semitendinosus muscle was dissected, and sections were stained for alkali-stable ATPase. The total number of primary and secondary fibers and the total number of fibers were estimated. In both experiments, there was a significant decrease in primary, secondary, and total fiber numbers (P < 0.05 for each). We speculate that the decrease in primary fiber number in both experiments is due to some of the large, central, and alkali-ATPase-negative (slow) fibers at near-term being secondary fibers. Therefore, the real primary fibers may not be affected.

Central Angiotensin I Increases Swallowing Activity and Oxytocin Release in the Near-Term Ovine Fetus

The brain renin-angiotensin system (RAS) plays an important role in hydromineral and neuroendocrine balance. Although previous studies showed that exogenous angiotensin (Ang) II increased dipsogenic and vasopressin responses in near-term fetuses, little is known about the functional development of fetal endogenous brain RAS in the regulation of body fluid homeostasis. To determine the functional development of the central angiotensin-converting enzyme (ACE) in utero, we investigated the electrocortical (ECoG) activity, swallowing activity, oxytocin (OT) release, and c-fos expression in response to intracerebroventricular Ang I administration in the near-term fetal lamb. Ang I did not change fetal low-voltage (LV) and high-voltage (HV) ECoG temporal distributions, but increased fetal swallowing activity during LV ECoG (1.0 ± 0.1 to 3.5 ± 0.4 swallows/min). Additionally, Ang I evoked an increase in c-fos-immunoreactivity in putative dipsogenic centers, including the supraoptic and paraventricular nuclei of the hypothalamus, accompanied by an increase in fetal plasma OT levels. The expression of c-fos was demonstrated in OT neurons in the hypothalamus. The Ang I-mediated increase in fetal swallowing and plasma OT was inhibited by captopril. These results demonstrate the functional development of the fetal brain ACE system in the last trimester of gestation, which plays an important role in the RAS-mediated dipsogenic response and OT release in the regulation of body fluid homeostasis.

Electrocortical activity in the near-term ovine fetus: Automated analysis using amplitude frequency components

We have designed an automated method for analyzing electrocortical (ECoG) activity in the near-term ovine fetus to process and quantitatively classify large amounts of data rapidly and objectively. Seven chronically catheterized fetal sheep were studied for 8 h each at ~ 0.9 of gestation with continuous recording of ECoG activity using a computerized data acquisition system. Multiple ECoG amplitude and frequency parameters were scored from which we established animal specific parameter cut-off values as well as population based duration cut-off values to distinguish low-voltage/high frequency (LV/HF) and high-voltage/low frequency (HV/LF) state epochs, and indeterminate voltage/frequency (IV/F) and transition period activities. We have shown that the incidence of the predominant LV/HF and HV/LF activity states at 45% and 36% of the time, respectively, is comparable to that previously reported using semi-quantitative techniques with visual analysis. However, the duration of these state epochs is considerably shorter due to the detection of brief periods of IV/F activity which would be difficult to capture using visual analysis. Importantly, our findings in the healthy ovine fetus near-term using this automated ECoG scoring methodology now provide a framework from which to study maturational events in younger animals, and under adverse pregnancy conditions.

Central angiotensin I increases fetal AVP neuron activity and pressor responses

Angiotensin (Ang) II plays a critical role in cardiovascular homeostasis and neuroendocrine regulation. Little is known about whether central angiotensin-converting enzyme (ACE) is functional in the fetal brain. We investigated cardiovascular and neuroendocrinological responses to intracerebroventricular (icv) application of Ang I in the chronically prepared near-term ovine fetus in utero and examined the action sites marked by c-fos expression in the fetal hypothalamus. ACE mRNA was detected in the specific central areas. Intracerebroventricular Ang I significantly increased fetal blood pressure and c-fos expression in the supraoptic nuclei (SON) and the paraventricular nuclei (PVN) in the hypothalamus, accompanied by an increase of fetal plasma arginine vasopressin (AVP). Double labeling demonstrated that AVP neurons in the fetal SON and PVN were expressing c-fos. Captopril, an inhibitor of ACE, significantly suppressed fetal pressor responses and plasma AVP. Double labeling experiments showed colocalization of AT(1) receptor (AT(1)R) and c-fos expression in both SON and PVN following icv Ang I. The results indicate that central endogenous ACE has been functional at least at the last third of gestation and the endogenous brain renin-angiotensin system-mediated pressor responses and AVP release via AT(1)Rs by acting at the sites consistent with the cardiovascular network in the hypothalamus.

Long-term hypoxia enhances ACTH response to arginine vasopressin but not corticotropin-releasing hormone in the near-term ovine fetus

This study tested the hypothesis that long-term hypoxia (LTH) results in enhanced fetal corticotrope sensitivity to the ACTH secretagogues, corticotropin-releasing hormone (CRH), and AVP. Ewes were maintained at high altitude (3,820 m) from 40 to 130-131 days of gestation. Upon return to the laboratory, hypoxia was maintained by maternal nitrogen infusion. Vascular catheters were placed in both LTH (n = 4) and normoxic controls (n = 4). Each fetus received a 15-min infusion of either saline, 100 ng/kg of ovine CRH, or 20 ng/kg of AVP/min over 3 consecutive days in a randomized order. Fetal blood samples were collected at 0, 15, 30, 60, and 90 min after the start of infusion and analyzed for ACTH(1-39), ACTH precursors, and cortisol. Anterior pituitaries were collected from additional noninstrumented fetuses for analysis of vasopressin receptor 1b (V1b) mRNA and protein. Basal plasma concentrations of both ACTH(1-39) and ACTH precursors were higher in LTH fetuses and were not altered by saline infusion. In response to CRH, ACTH(1-39) increased in both groups and was higher in the LTH group compared with control (P < 0.05). When analyzed as sum of ACTH(1-39) released (Delta0-90 min) above basal, CRH released equal amounts of ACTH(1-39) in both groups. In LTH fetuses, AVP evoked a greater ACTH(1-39) release (P < 0.05) when analyzed as an increased sum of ACTH(1-39) (Delta0-90 min) above basal. Both CRH and AVP elicited a release of ACTH precursors with no differences observed between LTH and control. AVP and CRH elicited significant increases in cortisol, which were higher in response to AVP than CRH. V1b mRNA and protein were elevated in the anterior pituitary of LTH fetuses compared with control. LTH significantly increases pituitary sensitivity to AVP. This enhanced sensitivity may be a mechanism of our previously observed enhanced corticotrope function.

Measures of acidosis with repetitive umbilical cord occlusions leading to fetal asphyxia in the near-term ovine fetus

Increasing frequency of severe variable decelerations is associated with acidemia. However, there is little understanding of its development or normalization rate. To quantify the time course of acidemia, 10 near-term fetal sheep underwent a series of mild (1 minute every 5 minutes), moderate (1 minute every 3 minutes), and severe (1 minute every 2 minutes) umbilical cord occlusions (UCO), lasting 1 hour each or until fetal arterial pH decreased to less than 7.00. Each minute of UCO resulted in base deficit (BD) and lactate increases at rates of 0.56 and 0.35 mmol/L per minute, respectively. During a 2 h recovery, BD and lactate normalized at rates of 0.09 and 0.04 mmol/L per minute. BD correlated highly with lactate (r = 0.95; P < .001). Our findings in the near-term ovine fetus suggest that the knowledge of fetal BD deterioration and recovery rates can aid assessing fetal acidemia during labor.

Central cholinergic signal-mediated neuroendocrine regulation of vasopressin and oxytocin in ovine fetuses

BackgroundThe hypothalamic-neurohypophysial system plays a fundamental role in the maintenance of body fluid homeostasis by secreting arginine vasopressin (AVP) and oxytocin (OT) in response to a variety of signals, including osmotic and nonosmotic stimuli. It is well established that central cholinergic mechanisms are critical in the regulation of cardiovascular responses and maintenance of body fluid homeostasis in adults. Our recent study demonstrated that intracerebroventricular (i.c.v.) injection of carbachol elicited an increase of blood pressure in the near-term ovine fetuses. However, in utero development of brain cholinergic mechanisms in the regulation of the hypothalamic neuropeptides is largely unknown. This study investigated AVP and OT neural activation in the fetal hypothalamus induced by central carbachol.ResultsChronically prepared near-term ovine fetuses (0.9 gestation) received an i.c.v. carbachol (3 μg/kg). Fetal blood samples were collected for AVP and OT assay, and brains were used for c-fos mapping studies. I.c.v. carbachol significantly increased fetal plasma AVP and OT concentrations. Intense FOS immunoreactivity (FOS-ir) was observed in the fetal supraoptic nuclei (SON) and paraventricular nuclei (PVN) in the hypothalamus. Double labeling demonstrated that a number of AVP- and OT-containing neurons in the fetal SON and PVN were expressing c-fos in response to central carbachol.ConclusionThe results indicate that the central cholinergic mechanism is established and functional in the regulation of the hypothalamic neuropeptides during the final trimester of pregnancy. This provides evidence for a functional link between the development of central cholinergic mechanisms and hypothalamic neuropeptide systems in the fetus.

Hypoxia Modulation of Caveolin-1 and Vascular Endothelial Growth Factor in Ovine Fetal Membranes

During normal pregnancy, amniotic fluid is absorbed from the amniotic compartment into fetal blood through the intramembranous blood vessels in the fetal membranes. It has been hypothesized that this transport process is mediated by transcytosis of caveolae-like vesicles. Because fetal hypoxia increases intramembranous absorption, the authors explore the effects of hypoxia on the gene expression of caveolin-1, a structural protein of caveolae, in ovine fetal membranes and cultured amnion cells. Near-term ovine fetuses were rendered hypoxic for 4 days. Caveolin-1 mRNA and protein levels were significantly reduced in the amnion and chorion but not in the placenta. In cultured ovine amnion cells incubated in 2% oxygen for 24 hours, hypoxia did not significantly alter caveolin-1 mRNA or protein expression. Vascular endothelial growth factor mRNA levels were increased in response to hypoxia in the fetal membranes as well as in cultured amnion cells. The results indicate that hypoxia does not augment but instead down-regulates or has no effect on caveolin-1 gene expression in the amnion and chorion, suggesting that caveolin-1 may play a role as a negative regulator of amnion transport function under hypoxic conditions.

Tei index for prenatal diagnosis of acute fetal hypoxia due to intermittent umbilical cord occlusion in an animal model

To study the effectiveness of the pulsatility index for veins of ductus venosus (DV-PIV) and the Tei index in a prospective assessment of fetal hypoxic-ischemic brain damage in a near-term ovine fetus model with intermittent umbilical cord occlusion (UCO). Twelve fetal sheep were studied with umbilical cord occlusion performed in the experimental group animals by complete inflation of an occluder cuff for 90 s, every 30 min for approximately 2.5 h. Fetal arterial blood was sampled at 5 min before the first umbilical cord occlusions, approximately 60 s of the first umbilical cord occlusions, and 3 min after each occlusion for blood gas, pH, neuron-specific enolase (NSE) and S100B. Doppler measurements and Doppler echocardiographic examinations were performed 5 min before the first umbilical cord occlusions and 3 min after each successive occlusion. In experimental group animals, UCO caused a large decline in arterial PaO(2) (to approximately 7.70 mmHg, p < 0.01), a modest decline in pH (to approximately 7.24, p < 0.01), and a modest rise in PaCO(2) (to approximately 53.31 mmHg, p < 0.01), with a return more or less to baseline after occluder release. and there was significant change as compared with the control animals (all p < 0.01) with cumulative changes in responses to repetitive cord occlusions. The DV-PIV waveforms, right ventricle (RV) and LV Tei indices, the serum levels of NSE and S100B increased with cord occlusions (all p < 0.05), and were significantly higher than the control animals (all p < 0.05) with a cumulative changes in responses to repetitive cord occlusions. RV and LV Tei indices were significantly correlated with PaO(2) (r = - 0.684, p < 0.01 and r = - 0.725, p < 0.01), PaCO(2) (r = 0.682, p < 0.01 and r = 0.780, p < 0.01), pH (r = - 0.538, p < 0.01 and r = - 0.681, p < 0.01), NSE (r = 0.653, p < 0.01 and r = 0.687, p < 0.01), and S100B (r = 0.606, p < 0.01 and r = 0.640, p < 0.01). Significant but weaker correlations were also present between DV-PIV and the parameters considered. Umbilical cord occlusion during the latter part of the pregnancy, enough to cause significant hypoxemia and acidosis, results in a significant increase of DV-PIV, RV and LV Tei indices, and the serum levels of NSE and S100B. There was a strong correlation between the RV and LV Tei indices and blood gas, pH, and NSE, S100B with hypoxia. Therefore, the Tei index might be an easy and useful quantitative parameter for assessing fetal hypoxic ischemia.

Effects of i.c.v. losartan on the angiotensin II-mediated vasopressin release and hypothalamic fos expression in near-term ovine fetuses

Our previous studies have shown that central administration of angiotensin (ANG II) causes arginine vasopressin (AVP) release in the fetus at 70–90% gestation. This is evidence that the hypothalamic-neurohypophysial system is relatively mature before birth. However, few data exist regarding central ANG receptor mechanisms-mediated AVP response during fetal life. To determine roles of brain ANG receptor subtypes in this response, AT 1 and AT 2 receptor antagonists, losartan and PD123319, were investigated in the brain in chronically prepared ovine fetuses at the last third of gestation. Application of losartan intracerebroventricularly (i.c.v.) at 0.5 mg/kg suppressed central ANG II-stimulated plasma AVP release. Losartan at 5 mg/kg (i.c.v.) demonstrated a significant enhancement of AVP increase to i.c.v. ANG II. Associated with the increase of plasma vasopressin levels, c-fos expression in the hypothalamic neurons was significantly different between the low and high doses of losartan. The low dose losartan markedly reduced the dual immunoreactivity for FOS and AVP in the supraoptic nuclei and paraventricular nuclei after i.c.v. ANG II, whereas the high dose losartan together with ANG II, significantly increased the co-localization of positive FOS in the AVP-containing neurons than that induced by i.c.v. ANG II alone. Central ANG II induced fetal plasma vasopressin increase was not altered by PD123319. The data suggest that losartan in the fetal brain has remarkably different effects based on the doses administrated on central ANG II-related neuroendocrine effects at the late gestation, and that the AT 1 mechanism is critical in the regulation of fetal body fluid homeostasis related to plasma AVP levels.