Near-term Ovine Fetus Research Articles

3394: Doppler ultrasonography combined with maternal serum VIP and NPY permit evaluation of acute fetal hypoxemia in the near-term ovine fetus

Objectives: To study the effectiveness of Doppler ultrasonography combined with maternal serum VIP and NPY determination in a prospective assessment of fetal hypoxemia. Methods: Fetal sheep were studied with umbilical cord occlusion (UCO) performed by complete in inflation of an occluder cuff for 90 seconds every 30 minutes for approximately 2.5 hours. Fetal and maternal arterial blood was sampled for blood gas and pH, VIP and NPY, respectively. Doppler measurements were performed for Pulsatility indices (PI). Results: UCO caused a large decline in arterial PaO2 (p < 0.01), modest decline in pH (p < 0.01) and modest rise in PaCO2 (p < 0.01), with cumulative changes in responses to repetitive cord occlusions, which were significantly changed compared with the control (all p < 0.01). Umbilical artery, renal artery and ductus venosus PI values, maternal serum VIP and NPY increased with cord occlusions (all p < 0.05), and rose compared with the control (all p < 0.05) with cumulative changes in responses to repetitive cord occlusions. There was a strong correlation between PI, maternal serum VIP, NPY values and PaO2, PaCO2 and pH. Conclusions: Doppler ultrasonography combined with maternal serum VIP and NPY determination might be an easy and useful quantitative parameter for assessing acute fetal hypoxia in fetuses with intermittent umbilical cord compression.

Effects of intracerebroventricular losartan on angiotensin II‐mediated pressor responses and c‐fos expression in near‐term ovine fetus

The renin-angiotensin system plays an important role in cardiovascular control. Intracerebroventricular (i.c.v.) angiotensin (ANG) II causes a reliable pressor response in the fetus at 90% gestation. To determine the roles of brain AT1 and AT2 receptors in this response, the effects of the central AT1 and AT2 receptor antagonists losartan and PD123319 were investigated in chronically prepared near-term ovine fetuses. Losartan at 0.5 mg/kg (i.c.v.) abolished central ANG II-induced pressor responses. High-dose losartan (5 mg/kg, i.c.v.) showed a potentiation of the pressor response to i.c.v. ANG II, accompanied by bradycardia. Associated with the pressor responses, c-fos expression in the cardiovascular controlling areas was significantly different between the low and high doses of losartan. These areas included the subfornical organ, median preoptic nucleus, organum vasculosum of the lamina terminalis, and paraventricular nuclei in the forebrain, and the tractus solitarius nuclei, lateral parabrachial nuclei in the hindbrain. Low-dose losartan markedly reduced c-fos in these areas after i.c.v. ANG II, while the high-dose losartan together with ANG II elicited a much stronger FOS-immunoreactivity in these areas than that induced by i.c.v. ANG II alone. This is a novel finding, that c-fos expression in the brain can be both activated and inhibited under the same condition. Central ANG II-induced fetal pressor responses were not altered by PD123319 (0.8 mg/kg). These results indicate that i.c.v. losartan at a high and a low dose has strikingly different effects on central ANG II-induced pressor responses in fetuses at late gestation, and that the AT1 mechanism plays an important role in fetal cardiovascular regulation.

Long-term hypoxia represses the expression of key genes regulating cortisol biosynthesis in the near-term ovine fetus

Basal plasma ACTH(1-39) concentrations are elevated in long-term hypoxic (LTH) fetal sheep. This study was designed to determine whether the expression of genes regulating cortisol biosynthesis was altered after LTH. Pregnant ewes were maintained at high altitude (3,820 m) from day 30 of gestation to near term, when the animals were transported to the laboratory. Reduced PO2 was maintained by nitrogen infusion through a maternal tracheal catheter. On days 137-141, fetal adrenal glands were collected from LTH and normoxic control fetuses. Real-time PCR was used to quantify mRNA for steroidogenic acute regulatory protein, 17alpha-hydroxylase (CYP17), 21-hydroxylase (CYP21), cholesterol side-chain cleavage (CYP11A1), 3beta-hydroxysteroid dehydrogenase type II (HSD3B2), and the ACTH receptor. We analyzed mRNA by slot-blot hybridization and also quantified mRNA for transcription factors necessary for adrenocortical development by quantitative real-time PCR: steroidogenic factor 1 and dosage-sensitive sex reversal, adrenal hypoplasia congenital, critical region on the X chromosome (DAX-1). Protein was quantified by Western blot analysis. Adrenal mRNAs for CYP17, CYP11A1, and the ACTH receptor were significantly reduced in LTH fetal sheep compared with levels shown in controls. Similarly, CYP11A1 protein and CYP17 protein were reduced in the LTH group. CYP21, steroidogenic acute regulatory protein, HSD3B2, steroidogenic factor 1, and DAX-1 expressions were not altered in response to LTH. We conclude that expression of two key steroidogenic enzymes (CYP17, CYP11A1) regulating cortisol biosynthesis and the ACTH receptor is lower in response to LTH. This likely represents an adaptive response to LTH, to prevent excessive cortisol production that would restrict fetal growth and potentially induce preterm delivery.

The Association of Cardiovascular Responses with Brain c-fos Expression after Central Carbachol in the Near-Term Ovine Fetus

Central cholinergic mechanisms play important roles in the control of cardiovascular responses. However, in utero development of brain cholinergic mechanism in regulation of arterial pressure before birth is largely unknown. This study investigated cardiovascular responses to central application of carbachol in fetuses and determined functional development of the central cholinergic systems controlling fetal pressor responses in utero. Chronically prepared near-term ovine fetuses (90% gestation) received an injection of carbachol intracerebroventricularly (i.c.v.). Fetal cardiovascular responses were measured, and the brains were used for c-fos mapping studies. In response to carbachol injection i.c.v., fetal systolic, diastolic, and mean arterial pressure (MAP) immediately increased, accompanied by a bradycardia. The maximum increase of MAP was at 30 min after the i.c.v. injection of carbachol and lasted 90 min. Associated with the pressor response, the neuronal activity marked with c-fos was enhanced significantly in the fetal anterior third ventricle (AV3V) region (including the median preoptic nucleus and organum vasculosum of the lamina terminalis) in the forebrain, and in the area postrema, lateral parabrachial nucleus, nucleus tractus solitary, and rostral ventrolateral medulla in the hindbrain. These results indicate that the central cholinergic mechanism is functional in the control of fetal blood pressure at the last third of gestation, and the central AV3V region and hindbrain have been intact relatively during in utero development in sheep at 90% gestational stage.

Mechanisms of Meconium Passage: Cholinergic Stimulation of Electromechanical Coordination in the Fetal Colon

Fetal gastrointestinal function develops in utero, with evidence of enhanced motility near-term. Although colonic passage of meconium in utero may be associated with fetal maturation or stress, little is known of the mechanisms potentiating motility. We assessed the effect of bethanechol, a cholinergic prokinetic agent, on colonic muscle muscular contractile and electromyogram (EMG) activity in the near-term ovine fetus. Near-term (130 days, n = 8) singleton ovine fetuses were chronically prepared with vascular catheters and three sets of miniature strain gauges and bipolar EMGs on the serosal surface of the transverse colon, left colic flexure, and distal colon. Following a 60-minute control period, fetuses received intravenous bethanechol (60 microg/kg, Low-Beth; 120 microg/kg, High-Beth) at 60 and 180 minutes. Colonic activity was recorded digitally and analyzed for short-duration (2<SHORT<15 seconds) and long-duration (15<LONG<120 seconds) strain gauge and EMG contractions. Data were expressed as means +/- SEM and analyzed using one-way analysis of variance (ANOVA) and paired t test. During the control period, there was significantly greater SHORT versus LONG strain gauge contractions in all segments (P <.05). As compared to control values, Low-Beth and High-Beth significantly increased SHORT strain gauge contractions in the transverse colon (160 +/- 13 to 201 +/- 36 and 307 +/- 74 spikes/30 min, respectively, P <.05), although not in left colic flexure or distal colon. Bethanecol did not affect LONG strain gauge contractions. SHORT-EMG and LONG-EMG spike bursts did not change in response to bethanecol (280 +/- 20, 59 +/- 2 spikes/30 min, respectively). Cholinergic stimulation of fetal sheep colonic activity at 0.9 gestation occurs in the transverse colon, but not the more distal left colic flexure or distal colon. The increased strain gauge, but not EMG activity, suggests that cholinergic stimulation improves electromechanical coordination in the fetal colon. We speculate that cholinergic-induced delivery of gastrointestinal contents to the distal colon evokes local contractile/expulsive mechanisms resulting in meconium passage.

Long-term hypoxia enhances proopiomelanocortin processing in the near-term ovine fetus

Secondary stressors in long-term hypoxic (LTH) fetal sheep lead to altered function of the hypothalamic-pituitary-adrenal axis. Although ACTH is considered the primary mediator of glucocorticoid production in fetal sheep, proopiomelanocortin (POMC) and 22-kDa pro-ACTH (22-kDa ACTH) have been implicated in the regulation of cortisol production in the ovine fetus. This study was designed to determine whether POMC expression and processing are altered after LTH. Pregnant ewes were maintained at high altitude (3,820 m) from day 30 of gestation to near term, when the animals were transported to the laboratory. Reduced Po2 was maintained by nitrogen infusion through a maternal tracheal catheter. On days 139-141, fetal anterior pituitaries were collected from normoxic control and LTH fetuses. We measured POMC and corticotrophin-releasing factor type 1 receptor (CRF1-R) mRNA using quantitative real-time PCR, and we used Western blot analysis for quantitation of ACTH, ACTH precursor, and CRF1-R proteins. We measured plasma ACTH1-39 using a two-site immunoradiometric assay specific for ACTH1-39. Plasma ACTH precursors were measured by ELISA. Anterior pituitary POMC mRNA levels were not different between groups, whereas CRF1-R levels were significantly higher in the LTH anterior pituitaries compared with control (P<0.05). In contrast, protein levels of POMC, CRF1-R, 22-kDa ACTH, and ACTH1-39 were significantly lower in the LTH group. Plasma concentrations of both ACTH precursors and ACTH1-39 were significantly elevated in LTH fetuses, whereas the ratio of plasma precursors to ACTH was significantly lower. We conclude that LTH results in enhanced POMC processing and/or release to ACTH and increased hypothalamic drive.

Central angiotensin II AT1 receptors mediate fetal swallowing and pressor responses in the near-term ovine fetus

Swallowed volumes in the fetus are greater than adult values (per body weight) and serve to regulate amniotic fluid volume. Central ANG II stimulates swallowing, and nonspecific ANG II receptor antagonists inhibit both spontaneous and ANG II-stimulated swallowing. In the adult rat, AT1 receptors mediate both stimulated drinking and pressor activities, while the role of AT2 receptors is controversial. As fetal brain contains increased ANG II receptors compared with the adult brain, we sought to investigate the role of both AT1 and AT2 receptors in mediating fetal swallowing and pressor activities. Five pregnant ewes with singleton fetuses (130 +/- 1 days) were prepared with fetal vascular and lateral ventricle (LV) catheters and electrocorticogram and esophageal electromyogram electrodes and received three studies over 5 days. On day 1 (ANG II), following a 2-h basal period, 1 ml artificial cerebrospinal fluid (aCSF) was injected in the LV. At time 4 h, ANG II (6.4 microg) was injected in the LV, and the fetus was monitored for a final 2 h. On day 3, AT1 receptor blocker (losartan 0.5 mg) was administered at 2 h, and ANG II plus losartan was administered at 4 h. On day 5, AT2 receptor blocker (PD-123319; 0.8 mg was administered at 2 h and ANG II plus PD-123319 at 4 h. In the ANG II study, LV injection of ANG II significantly increased fetal swallowing (0.9 +/- 0.1 to 1.4 +/- 0.1 swallows/min; P < 0.05). In the losartan study, basal fetal swallowing significantly decreased in response to blockade of AT1 receptors (0.9 +/- 0.1 to 0.4 +/- 0.1 swallows/min; P < 0.05), while central injection of ANG II in the presence of AT1 receptor antagonism did not increase fetal swallowing (0.6 +/- 0.1 swallows/min). In the PD-123319 study, basal fetal swallowing did not change in response to blockade of AT2 receptor (0.9 +/- 0.1 swallows/min), while central injection of ANG II in the presence of AT2 blockade significantly increased fetal swallowing (1.5 +/- 0.1 swallows/min; P < 0.05). ANG II caused significant pressor responses in the control and PD-123319 studies but no pressor response in the presence of AT1 blockade. These data demonstrate that in the near-term ovine fetus, AT1 receptor but not AT2 receptors accessible via CSF contribute to dipsogenic and pressor responses.

Induced fetal depressor or pressor responses associated with c- fos by intravenous or intracerebroventricular losartan

Previous fetal studies have indicated depressor responses of intravenous (i.v.) administration of angiotensin antagonists. However, little is known of central effects of angiotensin blockers on fetal cardiovascular controlling. The cardiovascular effects of central administration of the angiotensin-1 (AT 1) and angiotensin-2 (AT 2) receptor antagonists, losartan and PD123319, were investigated in the chronically catheterized near-term ovine fetuses. Intravenous losartan produced within 1.5 min a significant and persistent depressor response [maximum Δ mean arterial pressure (MAP)=9 mm Hg] without altering fetal heart rate. Intracerebroventricular (i.c.v.) administration of losartan (1–5 mg/kg) increased fetal arterial pressures (ΔMAP=9–14 mm Hg). Central application of losartan (1 mg/kg) also increased fetal heart rate (maximum Δ heart rate=33 beat per minute). Losartan increased c- fos expression in the median preoptic nucleus and paraventricular nuclei in the forebrain, and the tractus solitarius nuclei, the lateral parabrachial nuclei, and the ventrolateral medullabrain. These brain sectors are with abundant AT 1 receptors and have been demonstrated in the involvement in cardiovascular regulation. In contrast, intracerebroventricular injection of the AT 2 receptor antagonist PD123319 had no effect on fetal arterial pressure and heart rate. The results demonstrate strikingly functional differences of losartan on the fetal cardiovascular regulation in central and peripheral sides.

Structural proteins during brain development in the preterm and near-term ovine fetus and the effect of intermittent umbilical cord occlusion

ObjectiveThe purpose of this study was to determine the immunoreactivity of selected structural proteins in the preterm and near-term ovine fetal brain and the response to intermittent umbilical cord occlusion as a measure of altered cellular growth. The intermediate filament proteins nestin, vimentin, and glial fibrillary acidic protein was used as markers for astroglial maturation and astrogliosis, and myelin basic protein as a marker for oligodendrocytes and myelin formation. Study designFetal sheep (control and experimental groups at 0.75 and 0.90 of gestation) were studied over 4 days; umbilical cord occlusion was performed in the experimental group by complete inflation of an occluder cuff for 90 seconds every 30 minutes for 3 to 5 hours each day. Animals were then killed, and the fetal brain was perfusion fixed and processed for immunohistologic examination of the gray and white matter. Immunoreactivity was quantified with an image analysis system and expressed as the fractional area positive stain for each protein. ResultsIn both preterm and near-term animal groups, umbilical cord occlusion caused a large decline in arterial Po2 (to approximately 7 mm Hg), a modest decline in pH (to approximately 7.30), and a modest rise in Pco2 (to approximately 61 mm Hg; all P < .01), with a return to control values after the occluder release and no cumulative acidosis over each day of study. Vimentin and glial fibrillary acidic protein immunoreactivity showed reciprocal changes, with vimentin decreased and glial fibrillary acidic protein increased in both the gray and white matter of the control group from 0.75 to 0.90 of gestation, which can be attributed to the transition of radial glia into mature astrocytes. Myelin basic protein immunoreactivity increased approximately 3-fold in the white matter of the control group with advancing gestation, which likely reflected active oligodendrocyte differentiation and increased myelination at this time of development. Intermittent umbilical cord occlusion over 4 days resulted in an approximately 60% decrease in nestin, vimentin, and glial fibrillary acidic protein immunoreactivity, which was qualitatively similar for both the gray and white matter and likely indicative of altered protein synthesis and/or degradation, but only in the preterm group and with no change in myelin basic protein immunoreactivity. ConclusionThere is considerable change in the immunoreactivity of structural proteins within the ovine fetal brain over the latter part of gestation and consistent with a high rate of protein turnover, as previously reported. Intermittent umbilical cord occlusion as studied with minimal evidence for necrotic cell injury appears capable of altering selected protein synthesis/degradation, more so in younger animals when protein turnover is higher, which might then impact on the brain's development.

Unopposed appetite (orexigenic) mechanisms in the near-term ovine fetus: central leptin does not inhibit sucrose ingestion

Objective: Leptin is produced in adipocytes and is present in the term fetus. In the adult, leptin acts centrally to inhibit neuropeptide Y-induced carbohydrate intake. We sought to examine if central leptin alters fetal ingestion of oral sucrose in the near-term ovine fetus.Methods: Five pregnant ewes and fetuses were prepared with fetal vascular, sublingual and intracerebroventricular (ICV) catheters and esophageal electromyogram electrodes, and studied at 132 ± 1 days' gestation (term 145–150 days). Following a 2-h baseline period, 10% sucrose was infused sublingually (0.25 ml/min) for the duration of the study. At time 4 h, leptin (0.075 mg/kg) was administered ICV and fetal swallowing was monitored for an additional 6 h.Results: During the basal period, fetal swallowing averaged 0.7 ± 0.1 swallows/min. Fetal swallowing increased significantly in response to 10% sucrose (1.2 ± 0.1 swallows/min; p < 0.05). In response to ICV leptin, fetal swallowing remained significantly elevated at 2, 4 and 6 h (1.3 ± 0.4, 1.4 ± 0.3 and 1.5 ± 0.2 swallows/min, respectively; p < 0.05 vs. control).Conclusions: These results indicate that central leptin inhibition of sucrose ingestion is not functional in the near-term fetus. We speculate that a leptin-mediated anorexigenic response is not present at birth, such that unopposed appetite stimulatory mechanisms in the newborn may facilitate rapid newborn weight gain despite high body fat levels.

Brain C-FOS expression and pressor responses after I.V. or I.C.V. angiotensin in the near-term ovine fetus

Fetal brain c-fos and cardiovascular responses after i.v. or i.c.v. angiotensin II administrations was determined in the near-term ovine fetuses. Both routes of angiotensin II markedly increased fetal mean arterial pressure. The latency of pressor responses by i.v. angiotensin II administration was shorter than by the i.c.v. route. The increased fetal mean arterial pressure was greater and transient by the i.v. route in comparison to that caused by i.c.v. angiotensin II administration. Following the i.v. administration of angiotensin II, the fetal heart rate was significantly decreased. Associated with fetal pressor responses and bradycardia, c-fos expression induced by i.v. angiotensin II was in the paraventricular nuclei (PVN) of the hypothalamus, and the area postrema, the tractus solitarius nuclei, and the lateral parabrachial nuclei in the brain stem. After i.c.v. angiotensin II administration, fetal blood pressure was also increased in association with the intensive c-fos expression in the PVN and the hindbrain. However, fetal heart rate was not affected by the central injection of angiotensin II. These results indicate that the central pathways between the forebrain circumventricular organs and the PVN have developed, and suggest that the neural activity in the hindbrain associated with bradycardia may be linked to the baroreflex. In the face of i.c.v. angiotensin II, sympathetic activation may play a predominant role in pressor responses. Taken together, these results suggest that central and peripheral angiotensin II-induced fetal pressor responses may be mediated by separate mechanisms, and these regulatory mechanisms start to function by near-term or early.

Sweet taste adaptation in the near-term ovine fetus

Sweet taste adaptation in the near-term ovine fetus

Neuropeptide Y administered into cerebral ventricles stimulates sucrose ingestion in the near-term ovine fetus

Objective In adults, nutrient intake is controlled by opposing actions of appetite stimulants (eg, neuropeptide Y [NPY]) and suppressors (eg, leptin). Because NPY may exert a preferential role in mediating adult carbohydrate intake, we sought to determine the effect of central NPY on near-term fetal carbohydrate ingestion. Study design Five pregnant ewes and fetuses were prepared with fetal vascular, sublingual, and intracerebroventricular catheters, electrocorticogram, and esophageal electromyogram electrodes and studied at 131±2 days' gestation. After a 2-hour baseline period, 10% sucrose was infused sublingually for the duration of the study. At 4 hours' time, NPY was injected into the fetal cerebral ventricles and fetal swallowing monitored for an additional 6 hours. Results During the basal period, mean (±SEM) swallowing averaged 0.8±0.1 swallows per minute. Fetal swallowing increased significantly in response to sublingual sucrose (1.3±0.1 swallows/min, P = .001), and further significantly increased at 4 to 6 hours after NPY injection into the cerebral ventricles (1.8±0.3, P = .001). Conclusion These results indicate central NPY stimulation of fetal ingestion beyond that resulting from sublingual 10% sucrose. The in utero development of NPY-induced ingestive behavior may be in preparation for high neonatal caloric intake.

In utero development of central ANG-stimulated pressor response and hypothalamic fos expression

Central renin–angiotensin system (RAS) is as important as the peripheral RAS in the control of the cardiovascular homeostasis in the adult. However, previous fetal studies on angiotensin II (ANG II)-induced cardiovascular responses focused exclusively on the peripheral side. Thus, few data exist characterizing the in utero development of central angiotensin-mediated pressor responses. The present study determined cardiovascular responses to central application of ANG II in the chronically prepared near-term ovine fetus, and determined the action sites marked by c-fos expression in the fetal hypothalamus following intracerebroventricular (icv) injection of ANG II in utero. ANG II significantly increased fetal systolic, diastolic, and mean arterial pressure (MAP) within 5 min after injection of this peptide into the brain. Adjusted fetal MAP against amniotic pressure was also increased by icv ANG II, associated with increased c-fos in the central putative cardiovascular area—the paraventricular nuclei (PVN). Application of ANG II also induced intense c-fos expression in the supraoptic nuclei (SON), accompanied by a significant increase of fetal plasma vasopressin (AVP) levels, while maternal blood pressure (BP) and plasma AVP concentration were not changed. These results indicate that the central ANG II-mediated pressor response is functional at the last third of gestation, acting at the sites consistent with the cardiovascular neural network in the hypothalamus.

Intravenous angiotensin induces brain c-fos expression and vasopressin release in the near-term ovine fetus.

The effect of intravenous angiotensin II (ANG II) on fetal brain c-fos expression and arginine vasopressin (AVP) release was studied in the near-term ovine fetus. Fetuses with chronically implanted catheters received an intravenous infusion of ANG II or saline. Fetal plasma AVP concentrations were significantly increased after the peripheral administration of ANG II, with peak levels (3-fold) at 30 min after the intravenous infusion. There was no change in fetal plasma osmolality, sodium, and hematocrit levels between the control and experimental groups or between the periods before and after the infusion of ANG II. Intravenous ANG II administration induced Fos immunoreactivity (Fos-IR) in the circumventricular organs and the median preoptic nucleus of the fetal brain. Fos-IR was also demonstrated in the fetal supraoptic nuclei (SON). Double labeling demonstrated that the AVP-containing neurons in the SON were expressing c-fos in response to intravenous ANG II. These results indicate that the peripheral ANG II in the fetus may play a significant role in stimulating the central hypothalamic-neurohypophysial system during late gestation. It supports the hypothesis that circulating ANG II may act at the fetal AVP neurons in the hypothalamus in body fluid balance via the circumventricular organs, which are situated outside the blood-brain barrier, and the central neural pathway between these two brain structures has been relatively established in utero, at least at near-term.

Effects of antenatal steroids on ischemic brain injury in near-term ovine fetuses

Background: Hypoxia/ischemia in utero can result in brain damage to the fetus and newborn. Antenatal steroids are a routine part of the management of women who develop premature labor. Pretreatment of young postnatal rats with dexamethasone before hypoxic/ischemic insults has been reported to attenuate brain injury. However, the effects of antenatal steroids on ischemic brain injury in fetuses have not been investigated. Objective: We examined the effects of maternally administered antenatal corticosteroids on ischemic brain injury in near-term ovine fetuses. Methods: Chronically instrumented fetuses at 122 days of gestation were studied 12 h after the last of four 4 mg dexamethasone, or placebo injections were given over 48 h to the ewes. Groups were dexamethasone/ischemic, placebo/ischemic and sham-treated control. Fetuses were exposed to 30 min of carotid occlusion (ischemia) or no occlusion (control) and 72 h of reperfusion. Whole brain coronal sections stained with Luxol fast blue-hematoxylin-eosin were scored for white matter and cerebral cortical lesions. Both areas received pathological scores of 0 to 5 reflecting the degree of injury (0=0%, 1=1–10%, 2=11–50%, 3=51–90%, 4=91–99% and 5=100%). Bilateral carotid blood flow also was measured before, during and after brain ischemia in the dexamethasone/ischemic and placebo/ischemic groups. Results: White matter (WM) and cerebral cortical scores did not differ between the dexamethasone/ischemic and placebo/ischemic (WM: 3.0±1.9 and 2.9±1.7; cortex: 3.1±1.7 and 2.6±1.8, mean±S.D.) groups. White matter and cerebral cortical scores were higher in the dexamethasone/ischemic (WM: 3.0±1.9, P<0.02; cortex: 3.1±1.7, P<0.005) and placebo/ischemic (WM: 2.9±1.7, P<0.006; cortex: 2.6±1.8, P<0.007) than control (WM: 0.2±0.4; cortex: 0.2±0.4) group. Carotid blood flow was relatively higher ( P<0.05) after 24, 48 and 72 h of reperfusion in the dexamethasone/ischemic than placebo/ischemic group. Conclusions: We conclude that maternal pretreatment with antenatal dexamethasone did not attenuate ischemic brain injury in the fetus, and that carotid blood flow was higher during reperfusion in fetuses of dexamethasone than placebo-treated ewes, most likely secondary to decreases in arterial oxygen tension.

Cerebral blood flow and metabolism in relation to electrocortical activity with severe umbilical cord occlusion in the near-term ovine fetus

Objective: The purpose of this study was to determine the change in cerebral blood flow and substrate metabolism in relation to electrocortical activity in the near-term ovine fetus with repeated umbilical cord occlusion of a severe degree. Study Design: Eight near-term fetal sheep were studied through a 2-hour control period, a 6-hour experimental period with repeated cord occlusion of 4 minutes' duration every 90 minutes, and a 16-hour recovery period. Regional cerebral blood flow was measured with the microsphere technique before, during, and after the first cord occlusion; blood flow in the superior sagittal sinus, the cerebral perfusion pressure, and the electrocortical activity were monitored continuously. Brachiocephalic arterial and sagittal venous blood were sampled at selected time points for blood gas and pH, oxygen content, and glucose and lactate levels. Results: Severe umbilical cord occlusion as studied resulted in profound hypoxemia with modest hypercapnia and acidemia, to a similar degree with each insult, but with a return to preocclusion values after occluder release. Glucose values also fell acutely with each cord occlusion by approximately 30% but showed an overall increase through the experimental period, from 0.80 to 1.44 mmol/L; lactate values showed an increase, from 1.21 to 6.10 mmol/L (both P 1 <.01). Fetal electrocortical activity was disrupted markedly, with an abrupt flattening of the electrocortical amplitude by 1.5 minutes of each cord occlusion on average and with an overall increase in indeterminate state activity during the experimental and through the recovery periods. Cerebral blood flow increased approximately 2.5- and 2.8-fold, as measured at 2 and 3.5 minutes during the first cord occlusion (both P <.01) and with the regional flow increase greater in the subcortex and brainstem. Cerebral extraction of oxygen fell toward zero, as measured at 2 minutes during the second and fourth occlusions (P <.05) with oxygen uptake no longer measurable; glucose extraction was now increased approximately 2-fold (P <.05), which indicates that anaerobic metabolism of glucose must be the predominant source of energy at this time. Superior sagittal sinus blood flow also increased in all animals, approximately 1.4- and 1.6-fold at 2 and 3.5 minutes of the first cord occlusion, but much less than the corresponding increase in arterial inflow; the increase was in response to subsequent occlusions was further reduced. Conclusion: Severe umbilical cord occlusion in the near-term ovine fetus results in a rapid decrease in the availability of oxygen to the brain. The low PO2 gradient from blood to tissue rate limits for oxygen consumption by 2 minutes of insult (despite the marked increase in blood flow) and signals the shift to anaerobic metabolism, the suppression in electrocortical activity, and the probable shutdown of other energy-using processes. (Am J Obstet Gynecol 2003;188:961-72.)

Cerebral leucine uptake and protein synthesis in the near-term ovine fetus: relation to fetal behavioral state.

Behavioral/sleep state activity may impact on synthetic processes within the brain, thus accounting for the developmental change in such activity and suggesting a role in the brain's growth and development. We have therefore determined the cerebral uptake of leucine and [(14)C]leucine during continuous tracer infusion as measures of leucine metabolism in relation to behavioral state activity, as well as the regional flux of leucine into brain tissue in the ovine fetus near term. The cerebral fractional protein synthetic rate and the absolute protein synthetic rate averaged approximately 20%/day and approximately 1 g/day, respectively, as measured for the whole brain, which is considerably higher than anticipated protein accretion and indicates a high rate of protein turnover with protein synthesis closely linked to protein degradation. Measures of protein synthesis were significantly higher in the pituitary gland, which may be attributed to the active synthesis and export of peptide hormones from this region. Cerebral leucine and [(14)C]leucine uptakes averaged approximately 630 and approximately 1,000 nmol. 100 g(-1). min(-1), with the latter higher than leucine unidirectional flux and thus supporting a degree of leucine oxidation by the brain. Cerebral leucine metabolism as studied was affected by behavioral state activity, with uptake measurements for both leucine and [(14)C]leucine significantly increased during the high-voltage electrocortical/non-rapid eye movement state by 1.7-fold and 2.8-fold, respectively, indicating that protein synthesis and degradation must also be increased at this time, and supporting a role for behavioral state activity in the brain's growth and development.

Effect of oral sucrose on ingestive behavior in the near-term ovine fetus

Background: Dipsogen-mediated ingestion matures acutely in late gestation because the preterm fetus may demonstrate absent responses to putative dipsogens. Although central appetite-mediated ingestive behavior is functional near term, it is unknown whether peripheral mechanisms for stimulation of appetite also are functional. In the adult, sweet taste stimulates and potentiates ingestive behavior. We sought to determine whether oropharyngeal sucrose exposure stimulates ingestive behavior in the near-term ovine fetus. Study Design: Time-dated pregnant ewes with near-term singleton fetuses (n = 6) were chronically prepared with fetal vascular and sublingual catheters and esophageal electromyogram electrodes and studied at 129 ± 1 days of gestation. After an initial 2-hour baseline period, successive solutions of distilled water and 2.5%, 10%, and 40% sucrose were infused sublingually (0.25 mL/min), each for 2 hours. Maternal and fetal arterial blood samples were drawn at timed intervals. Results: During the basal period, fetal swallowing averaged 0.9 ± 0.1 swallows per minute. Swallowing did not change in response to distilled water (0.9 ± 0.2 swallows per minute) but significantly increased after sublingual infusion of 2.5% sucrose (1.3 ± 0.1 swallows per minute), 10% sucrose (1.8 ± 0.1 swallows per minute), and 40% sucrose (1.3 ± 0.1 swallows per minute, P =.001). There were no significant changes in other fetal or maternal parameters. Conclusions: The stimulation of fetal swallowing in response to sublingual sucrose infusion suggests that taste-mediated ingestive behavior is functional in the near-term fetus and that both central and systemic appetite mechanisms are intact near term. Fetal swallowing increased in response to an increase in sucrose concentration to peak at 10% and then decreased with further rises in concentration, possibly mediated by aversive fetal reaction to a high-intensity sucrose concentration. ( Am J Obstet Gynecol 2002;187:898–901.)

The role of adenosine in regulation of cerebral blood flow during hypoxia in the near-term fetal sheep.

The aim of this study was to determine in the near-term ovine fetus the role of adenosine in the basal regulation of cerebral blood flow and in the increases in cerebral blood flow in response to acute hypoxic insult. We measured cerebral blood flow in chronically instrumented fetal sheep (127-135 days gestation, term approximately 145 days) using laser Doppler flowmetry probes implanted in the parietal cortices. Hypoxia was administered for 30 min by lowering the ewe's inspired oxygen to 10-12 % during an infusion of either saline or theophylline, a non-specific adenosine receptor antagonist. The theophylline infusion was begun 30 min prior to and ended 30 min after the completion of the hypoxic insult. The administration of theophylline had no significant effect on cerebral blood flow during the baseline period. During control hypoxic periods, cerebral blood flow increased by approximately 45 %. During theophylline experiments, however, there was no significant increase in cerebral blood flow during hypoxia. In the control experiments, cerebral blood flow returned to baseline levels during the recovery period, while in the theophylline experiments cerebral blood flow fell below baseline levels. We conclude that, in the near-term ovine fetus, adenosine plays a minimal role in the regulation of basal cerebral blood flow. However, these data are strong evidence for the involvement of adenosine in increased fetal cerebral blood flow during an acute hypoxic insult. Finally, adenosine may also play an important role in the maintenance of fetal cerebral blood flow immediately following hypoxic insult.