Intermittent Umbilical Cord Occlusion Research Articles

Bidirectional Ductal Shunting and Preductal to Postductal Oxygenation Gradient in Persistent Pulmonary Hypertension of the Newborn.

Background: The aim was to evaluate the relationship between the direction of the patent ductus arteriosus (PDA) shunt and the pre- and postductal gradient for arterial blood gas (ABG) parameters in a lamb model of meconium aspiration syndrome (MAS) with persistent pulmonary hypertension of the newborn (PPHN). Methods: PPHN was induced by intermittent umbilical cord occlusion and the aspiration of meconium through the tracheal tube. After delivery, 13 lambs were ventilated and simultaneous 129 pairs of pre- and postductal ABG were drawn (right carotid and umbilical artery, respectively) while recording the PDA and the carotid and pulmonary blood flow. Results: Meconium aspiration resulted in hypoxemia. The bidirectional ductal shunt had a lower postductal partial arterial oxygen tension ([PaO2] with lower PaO2/FiO2 ratio—97 ± 36 vs. 130 ± 65 mmHg) and left pulmonary flow (81 ± 52 vs. 133 ± 82 mL/kg/min). However, 56% of the samples with a bidirectional shunt had a pre- and postductal saturation gradient of < 3%. Conclusions: The presence of a bidirectional ductal shunt is associated with hypoxemia and low pulmonary blood flow. The absence of a pre- and postductal saturation difference is frequently observed with bidirectional right-to-left shunting through the PDA, and does not exclude a diagnosis of PPHN in this model.

Apoptosis in the Ovine Fetal Brain Following Placental Embolization and Intermittent Umbilical Cord Occlusion.

The purpose of this study was to compare the regional distribution of apoptotic cells in the near term ovine fetal brain caused by prolonged moderate hypoxia, as seen in placental insufficiency, and intermittent severe hypoxia, as seen in umbilical cord compression, which may then contribute to adverse neurodevelopment in the postnatal life. We hypothesized that apoptosis in the fetal brain will be increased in response to both prolonged moderate hypoxia and intermittent severe hypoxia. Twenty-one near term (126-127 days) sheep were divided into 3 groups: control (CON; n = 7), placental embolization (EMB; n = 7), and umbilical cord occlusion (UCO; n = 8). The EMB group had microsphere injections into the umbilical arterial circulation until the oxygen content was at 50% of baseline value. The UCO group had complete cord occlusion for 2 minutes every hour, 6 times a day for 2 consecutive days. At 4 pm on day 2, the animals were euthanized; fetal brains were fixed and prepared for apoptosis staining using the terminal uridine deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay method. In the cerebellar white matter, there was a 3-fold increase in the number of TUNEL positive cells per 1000 cells in both EMB and UCO animals as compared to CON (P = .017). There was also a significant increase in the frontal cortical grey matter (layers 1-3) in EMB animals as compared to CON (P = .014). As such, apoptosis in the near term fetal sheep brain is altered with both sustained moderate hypoxia and intermittent severe hypoxia in the latter part of pregnancy, with potential for long-term neurological sequelae.

Accelerated acidosis in response to variable fetal heart rate decelerations in chronically hypoxic ovine fetuses

Due to limitations of technology, clinicians are typically unable to determine if human fetuses are normoxic or moderately, chronically hypoxic. Risk factors for chronic hypoxia include fetal growth restriction, which is associated with an increased incidence of oligohydramnios and thus a risk for umbilical cord occlusion (UCO) and variable fetal heart rate (FHR) decelerations. At delivery, fetal growth restriction infants (<3rd percentile) have nearly twice the incidence of low Apgar scores and umbilical pH <7.0. Despite the risks of oligohydramnios and intermittent UCO, there is little understanding of the acid/base responses rates of chronically hypoxic fetuses to variable FHR decelerations as might occur during human labor. We sought to compare the increase in base deficit (BD) among chronically hypoxic as compared to normoxic ovine fetuses in response to simulated mild, moderate, and severe variable FHR decelerations. Near-term ovine fetuses were chronically prepared with brachial artery catheters and an inflatable umbilical cuff occluder. Following a recovery period, normoxic (n = 9) and spontaneously hypoxic (n = 5) fetuses were identified (arterial O2 saturation ≤55%). Both animal groups underwent graded, 1-minute occlusions every 2.5 minutes with 1 hour of mild (∼30 beats/min [bpm] decrease from baseline), 1 hour of moderate (∼60 bpm decrease from baseline), and up to 2 hours of severe (∼90 bpm decrease from baseline) variable FHR decelerations until fetal arterial pH reached 7.00, when occlusions were stopped. Repetitive UCO resulted in development of acidosis (pH <7.0) in both groups. Hypoxic and normoxic fetuses demonstrated similar BD increases in response to both mild (0.39, interquartile range [IQR] 0.28-0.45 vs 0.26, IQR 0.01-0.30 mEq/L/10 min, P = .25) and severe (1.97, IQR 1.50-2.43 vs 1.51, IQR 0.97-2.45 mEq/L/10 min, P = .63) variable decelerations. However, moderate variable decelerations increased BD in hypoxic fetuses at 2.5 times the rate of normoxic fetuses (0.97, IQR 0.52-1.72 vs 0.39, IQR 0.23-0.47 mEq/L/10 min, P = .03). During the recovery period, hypoxic fetuses cleared BD slower than normoxic fetuses (0.08 ± 0.02 vs 0.12 ± 0.03 mEq/L/min, P = .02). In comparison to normoxic fetuses, hypoxic fetuses can more rapidly progress to significant metabolic acidosis in response to moderate FHR variable decelerations, and more slowly recover with in utero resuscitation, likely a consequence of impaired placental function and fetal physiologic responses.

Microtubule-Associated Protein 2 and Synaptophysin in the Preterm and Near-Term Ovine Fetal Brain and the Effect of Intermittent Umbilical Cord Occlusion

We have determined the change in immunoreactivity (IR) for microtubule-associated protein 2 (MAP-2) and synaptophysin (SYN) as markers for dendritic and presynaptic nerve development, respectively, in the ovine fetal brain with advancing gestation and in response to intermittent umbilical cord occlusion (UCO), which might then contribute to adverse neurodevelopment. Fetal sheep (control and experimental groups preterm at 111-115 and near term at 132-138 days of gestation; term = 145 days) were studied over 4 days with UCOs performed by inflation of an occluder cuff for 90 seconds every 30 minutes for 3 to 5 hours each day. Animals were then euthanized and fetal brains assessed for IR of MAP-2 and SYN. In control animals, the IR of SYN increased in the gray matter with advancing gestation consistent with a developmental increase in presynaptic vesicles and/or nerve terminals as expected; however, the IR of MAP-2 decreased in all brain regions studied, suggesting concurrent refinement in dendritic branching and spine development. Intermittent UCO as studied with marked but limited hypoxemia resulted in a decrease in IR of SYN for the brain regions of the preterm animals when protein turnover is higher and indicates decreased presynaptic vesicle formation; whereas, MAP-2 IR was selectively increased in the hippocampus CA1 and thalamus of the near-term animals, consistent with reactive dendritic change and heightened vulnerability for neuronal injury. As such, intermittent cord compressions in the ovine fetus can impact protein markers for dendritic and presynaptic nerve development depending on their timing, which might then lead to alterations in synapse formation and neuronal circuitry.

The Impact of Chronic Intrauterine Inflammation on the Physiologic and Neurodevelopmental Consequences of Intermittent Umbilical Cord Occlusion in Fetal Sheep

To determine the effect of intrauterine inflammation on fetal responses to umbilical cord occlusion (UCO). In pregnant sheep, lipopolysaccharide (LPS) or saline (SAL) was infused intra-amniotically for 4 weeks from 80 days of gestation (d). At 110 d, fetuses were instrumented for UCOs (5 × 2-minutes, 30-minute intervals: LPS + UCO, n = 6; SAL + UCO, n = 8) or no UCO (sham, n = 6) on 117 and 118 d. Tissues were collected at 126 d. Fetal physiological responses to UCO were similar between LPS + UCO and SAL + UCO. Histologic chorioamnionitis and increased amniotic fluid interleukin 8 (IL-8) were observed in LPS + UCO pregnancies (versus SAL + UCO, P < .05). CNPase-positive oligodendrocyte number in the cerebral white matter was lower in LPS + UCO and SAL + UCO than sham (P < .05); there was no effect on astrocytes or activated microglia/macrophages. Two of the SAL + UCO fetuses had white matter lesions; none were observed in LPS + UCO or sham. Chronic pre-existing intrauterine inflammation did not exacerbate fetal brain injury induced by intermittent UCO.

Cord around the neck syndrome

A nuchal cord (or Cord-Around-the Neck (CAN)) occurs when the umbilical cord becomes wrapped around the fetal neck 360 degrees. Nuchal cords are very common, the incidence of nuchal cord increases with advancing gestation from 12% at 24 to 26 weeks to 37% at term [1]. Most are not associated with perinatal morbidity and mortality. In some fetuses and newborns CAN may cause problems, especially when the cord is tightly wrapped around the neck. The cluster of cardiorespiratory and neurological signs and symptoms associated with unique physical features that occur secondary to tight cord-round-the-neck has been referred to as 'tCAN syndrome' (tight Cord Around the Neck Syndrome) [2]. A small number of studies have shown that nuchal cord and or tCAN can affect the outcome of delivery and may have long-term effects on the infant [3] and but as a causative factor for stillbirth it is debatable [4,5]. However, some case reports of postmortem findings on stillbirths show negative pathology reports and tight cord around the neck being the only cause of death [6]. It is the unique physical features of tCAN syndrome that distinguishes it from birth asphyxia even though there are many similarities between these two conditions. Umbilical cord abnormalities are considered as one of the causative factor for birth asphyxia. The manifestation of tCAN symptomatology seems to happen both in the presence of normal and depressed AGPAR scores [7]. Umbilical cord compression due to tCAN may cause obstruction of blood flow first in thin walled umbilical vein, while infant’s blood continues to be pumped out of baby through the thicker walled umbilical arteries thus causing hypovolemia and hypotension resulting in acidosis [8]. Anemia [9] and mild respiratory distress may occur. Some of these infants may also have facial and conjuctival petechiae [10] and rarely petechiae of the neck and upper part of the chest and skin abrasion of neck [11] where the cord was tightly wrapped and facial suffusion [12], all of which can also be seen in some postmortem findings of stillbirth infants who had tCAN [Archana Bargaje, personal communication]. If born alive, some of these infants may also be somewhat obtunded with a low tone and have transient feeding difficulties. These findings raise the possibility of transient encephalopathy, which may lead to long-term complications. A stillbirth attributed to a cord problem should have evidence of cord obstruction or circulatory compromise. Other potential causes of stillbirth need to be excluded prior to labelling cord abnormalities as the causative factor, since cord abnormalities seen in more than a third of all normal live births. The tCAN Syndrome may conceptually be similar to strangulation which may result in non lethal problems or death. The pathophysiological mechanisms of strangulation injuries (lethal and non lethal) involves venous, arterial obstruction (arterial spasm due to carotid pressure) in the neck and vagal collapse (increased parasympathetic tone) [13]. This can lead to cerebral stagnation, hypoxia, and unconsciousness, which, in turn, produces loss of muscle tone. The same pathophysiology of strangulation may possibly be applicable to tCAN syndrome in neonates. A study on potentially asphyxiating conditions and spastic cerebral palsy in infants of normal birth weight showed evidence of association of tCAN in children with quadriplegia [14]. Intermittent umbilical cord occlusion in preterm and near term sheep caused a decline in pO2 and pH, and higher PCO2 and altered brain protein synthesis/degradation [6]. Whether human fetal intermittent strangulation by tCAN have similar brain protein alterations and thus long-term effects remains to be seen. Using specific placental histologic criteria for umbilical blood flow restriction in unexplained stillbirth Parast et al [4] showed significant correlation of placental changes of “minimal histologic criteria” with cord accidents (as tCAN is part of cord accidents). Nuchal cords showed highest rates of thrombosis-related placental histopathology and fetal thrombotic vasculopathy and thrombosis seems to be highly specific for cord related stillbirths [4,5].

The Effect of Intermittent Umbilical Cord Occlusion on Elastin Composition in the Ovine Fetus

This study aimed to determine the effect of varying degrees of intermittent umbilical cord occlusion (UCO) on arterial elastin composition. Over 4 days, chronically catheterized late gestation fetal sheep received 5 total UCO per day lasting 1 min/h (mild group: n = 6), 2 min/h (moderate group: n = 4), 3 min/h (severe group; n = 6); or no occlusion (control group: n = 7). Each group was evaluated for elastin content of the carotid and superior mesenteric artery (SMA), the arterial pressure response to UCO, and plasma cortisol concentration. Elastin content of the carotid artery was significantly increased by severe UCO (9.5 µg/mg versus 6.4 µg/mg; P < .05) and insignificantly increased in mild and moderate groups, whereas UCO had no effect on elastin content of the SMA. This dose- and site-dependent response of the vasculature appears attributable to the hemodynamic changes that accompany UCO.

Noninvasive fetal electrocardiography following intermittent umbilical cord occlusion in the preterm ovine fetus

To investigate whether a noninvasive fetal electrocardiography (fECG) system can identify cardiovascular responses to fetal hypoxaemia and validate the results using standard invasive fECG monitoring techniques.Prospective cohort study.Biological research facilities at The University of Southampton.Late gestation ovine fetuses; n = 5.Five fetal lambs underwent implantation of vascular catheters, umbilical cord occluder and invasive ECG chest electrodes under general anaesthesia (3% halothane/O(2)) at 119 days of gestation (term approximately 147 days of gestation). After 5 days of recovery blood pressure, blood gases, glucose and pH were monitored. At 124 and 125 days of gestation following a 10-minute baseline period a 90-second cord occlusion was applied. Noninvasive fetal ECG was recorded from maternal transabdominal electrodes using advanced signal-processing techniques, concurrently with invasive fECG recordings.Comparison of T:QRS ratios of the ECG waveform from noninvasive and invasive fECG monitoring systems.Our fECG monitoring system is able to demonstrate changes in waveforms during periods of hypoxaemia similar to those obtained invasively, which could indicate fetal distress.These findings may indicate a future use for noninvasive electrocardiography during human fetal monitoring both before and during labour in term and preterm pregnancies.

BDNF and TrkB in the Preterm and Near-term Ovine Fetal Brain and the Effect of Intermittent Umbilical Cord Occlusion

We have determined the developmental change in immunoreactivity for brain-derived neurotrophic factor and its high-affinity tyrosine kinase receptor, TrkB, in the ovine fetal brain with advancing gestation and in response to intermittent umbilical cord occlusion, which might then contribute to adverse neurodevelopment. Fetal sheep (control and experimental groups at 0.75 and 0.90 of gestation) were studied over 4 days with umbilical cord occlusions performed in the experimental group animals by complete inflation of an occluder cuff for 90 seconds every 30 minutes for 3 to 5 hours each day. Animals were then euthanized and the fetal brains perfusion fixed and prepared for subsequent histology with the distribution of brain-derived neurotrophic factor and tyrosine kinase receptor immunoreactivity determined by immunohistochemistry. In the control group animals brain-derived neurotrophic factor immunoreactivity decreased in the gray matter, thalamus, and hippocampus but increased in the white matter, while tyrosine kinase receptor immunoreactivity decreased in all regions (most P < .01), with advancing gestation consistent with the developmental change from neurogenesis/gliagenesis to myelination over this time period. Intermittent umbilical cord occlusion as studied with severe but limited hypoxemia resulted in a variable decrease in brain-derived neurotrophic factor and tyrosine kinase receptor immunoreactivity for all brain regions in the preterm animals (most P < .01) when protein turnover is higher, but a selective increase in brain-derived neurotrophic factor immunoreactivity in the hippocampus of the near-term animals consistent with a heightened vulnerability for necrotic/apoptotic injury at this time. As such, brain-derived neurotrophic factor-tyrosine kinase receptor in the ovine fetal brain may be altered with intermittent hypoxic insults over the latter part of pregnancy with potential for longer term neurologic consequences.

Relationship between ductus venosus pulsatility index,Tei index,the serum levels of neuron specific enolase,S100B and intermittent umbilical cord occlusion of the near-term hircine fetus

目的 探讨胎羊间断性脐带闭塞后静脉导管搏动指数(ductus venosus pulsatility index,DVPI)、Tei指数评估胎儿缺氧缺血性脑损伤的应用价值.方法 7只晚孕山羊作为实验组,于妊娠116～130 d对胎羊行官内外科手术,用套囊充气闭合器完全夹闭脐带,每次夹闭90 s,间隔30min,重复5次.于实验前5 min、每次夹闭后3 min应用彩色多普勒血流显像技术检测DVPI、Tei指数,抽取胎羊脐动脉血做血气分析,应用电化学发光法测定脐血神经元特异性烯醇化酶(neuron speeific enolase,NSE)、S100B蛋白含量,同时分析这些指标的相关性.另选5只晚孕山羊作为对照组,只手术不夹闭脐带. 结果 实验组胎羊脐动脉血PaO2和pH随夹闭时间下降,PaCO2、DVPI、右心室和左心室Tel指数、脐血NSE和S100B均随夹闭时间而升高(P均＜0.05).实验组夹闭后PaO2低于对照组,夹闭后36 min开始pH值低于对照组,PaCO2高于对照组;从夹闭后36 min开始实验组NSE和S100B蛋白较对照组升高(P均G0.05).实验组DVPI、右心室Tel指数、左心室Tei指数、脐血NSE、S100B含量与血pH值呈负相关(Υ分别为-0.358、-0.538,-0.681、-0.388、-0.539,P均＜0.05),与血PaO2呈负相关(Υ分别为-0.435、-0.684、-0.725、-0.602,-0.447,P均＜0.01),与血PaCO2呈正相关(Υ分别为0.699、0.682、0.780、0.477、0.527,P均GO.01).实验组DVPI、右心室和左心室Tei指数与血清NSE含量呈正相关(Υ分别为0.394、0.653、0.687,P均＜0.05),与血清S100B含量呈正相关(Υ分别为0.359、0.606、0.640,P均＜0.05).结论 Tei指数与血气指标及血清NSE、S100B的相关性较好,可能成为预测胎儿缺氧缺血性脑损伤简便而有效的指标。

“Retracted: Changes in the Tei index during acute fetal hypoxemia in the near‐term ovine fetus after intermittent umbilical cord occlusion”

AbstractRetraction: The following article from Ultrasound in Obstetrics and Gynecology, Changes in the Tei index during acute fetal hypoxemia in the near‐term ovine fetus after intermittent umbilical cord occlusion by G. Lu, Z. Wang, S. He, B. Li, Z. Huang, Q. Chen, P. Jin, R. Su, published online on July 18 2007 in Wiley InterScience (www.interscience.wiley.com), has been retracted by agreement between the journal Editor‐ in‐Chief, Yves Ville and John Wiley and Sons, Ltd. The retraction has been agreed because the article shows significant overlap with another paper published by the group in Prenatal Diagnosis, entitled “Tei index for prenatal diagnosis of acute fetal hypoxia due to intermittent umbilical cord occlusion in an animal model” (Prenat Diagn 2007; 27: 817–823). Copyright © 2009 ISUOG. Published by John Wiley &amp; Sons, Ltd.

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.

The Production of Hydroxyl Radicals in the Fetal Lamb Brain Resulting from Occlusion of the Umbilical Circulation and the Transplacental Effect of MCI-186 to Inhibit Hydroxyl Radical Production

The present study evaluated hydroxyl radical production in fetal lamb brain during and after umbilical cord occlusion and examined the effects of injecting MCI-186 (3-metyl-1-phenyl-2-pyrazolin-5-one; Edaravone), a hydroxyl radical scavenger, into the maternal circulation. In 11 chronically instrumented lambs, intermittent total umbilical cord occlusions 1 min out of every 3 min for 1 h and 10-min persistent total umbilical cord occlusion were performed with brain microdialysis using 5 mM of sodium salicylate. In the remaining four lambs, 60 mg of MCI-186 was administered into the maternal circulation from shortly before the end of 10-min persistent total umbilical cord occlusion. Concentrations of 2,3-dihydroxy-benzoic acid (2,3-DHBA), produced by hydroxyl radical reactions with sodium salicylate, were measured in perfusate by HPLC. Concentration of 2,3-DHBA in perfusate was 23.05 +/- 10.95 nM before umbilical cord occlusion. Levels of 2,3-DHBA tended to increase slightly during and after intermittent umbilical cord occlusion, and were significantly increased by the end of 10-min occlusion (40.06 +/- 21.36 nM) and after occlusion (93.74 +/- 29.17 nM). Infusion of MCI-186 suppressed 2,3-DHBA concentration to 29.35 +/- 14.95 nM after occlusion. Administration of MCI-186 into the maternal circulation reduces hydroxyl radical production induced by umbilical cord occlusion in the fetal lamb brain.

Transplacental effects of allopurinol on suppression of oxygen free radical production in chronically instrumented fetal lamb brains during intermittent umbilical cord occlusion

Objective. To evaluate the transplacental effect of allopurinol, which acts as a xanthine oxidase inhibitor and free radical scavenger, on inhibiting the production of superoxides during intermittent partial umbilical cord occlusion.Methods. Using four chronically instrumented fetal lambs, ewes received 400 mg allopurinol over a period of two hours. Concentrations of allopurinol and oxypurinol in blood samples from mothers and fetuses and fetal brain microdialysis perfusate were measured by HPLC. In another three cases the production of superoxide during intermittent umbilical cord occlusion was studied by measurement of chemiluminescence in perfusate before and after administration of Allopurinol.Results. (i) Allopurinol concentration in mothers had reached equilibrium by 30 min after starting administration and maintained a concentration about 6 μg/ml. Allopurinol concentration in fetuses increased gradually and reached 2.25 ± 0.54 μg/ml at 120 min; (ii) Oxypurinol concentration in both mothers and fetuses increased during administration of allopurinol; (iii) Concentrations of allopurinol and oxypurinol in the perfusates reached 0.32 ± 0.12 μg/ml, 0.53 ± 0.22 μg/ml at 120min respectively; and (iv) Administration of allopurinol significantly suppressed superoxide production during intermittent partial umbilical cord occlusion.Conclusion. These results demonstrated a good transfer of allopurinol from mother to fetus and suggested the possibility of intrauterine treatment to inhibit fetal brain damage resulting from increased oxygen free radicals.

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.

Necrotic cell injury in the preterm and near-term ovine fetal brain after intermittent umbilical cord occlusion

ObjectiveThis study was undertaken to determine the extent of necrotic cell death as a measure of neurologic injury in the preterm and near-term ovine fetal brain in response to intermittent umbilical cord occlusion (UCO) with severe, but limited hypoxia and no cumulative acidosis to ensure longer-term survival. Study designFetal sheep (control and experimental groups at 0.75 and 0.90 of gestation) were studied over 4 days with UCOs performed in the experimental group animals by complete inflation of an occluder cuff for 90 seconds every 30 minutes for 3 to 5 hours each day. Animals were then euthanized and the fetal brain perfusion-fixed and prepared for subsequent histology and assessment of necrotic cell injury by using standard staining with hematoxylin and eosin (H&E), and with a novel fluorescent marker, Fluoro-Jade B, that targets degenerating neurons. ResultsIn both preterm and near-term animal groups, UCO caused a large decline in arterial Po2 (to ∼7 mm Hg), a modest decline in pH (to ∼7.30), and a modest rise in Pco2 (to ∼61 mm Hg) (all P<.01), but with a return to control values after the occluder release and no cumulative acidosis over each day of study. Overall, very low levels of H&E-stained necrotic-appearing cells and Fluoro-Jade B–stained positive cells were observed across all brain regions studied with values not significantly different from zero, excepting that for the gray matter of the preterm control (by H&E staining), preterm and near-term cord occlusion (by H&E and Fluoro-Jade B staining), and the thalamus of the near-term cord occlusion (by H&E staining) animals. Although there were no differences in the levels of H&E-stained necrotic-appearing cells and Fluoro-Jade B–stained positive cells between respective control and cord occlusion group animals for most of the brain regions studied, a significant increase in Fluoro-Jade B–stained positive cells was observed in the gray matter of both the preterm and near-term cord occlusion animals (P<.05). ConclusionIntermittent cord occlusion insult with severe but limited fetal hypoxemia and no cumulative acidosis, was generally well tolerated in both the preterm and near-term animal groups as assessed by measures of necrotic cell injury throughout the brain with minimal evidence for such. However, compensatory mechanisms which are protective for the brain may become limited with repetitive hypoxia insult over time as suggested by the low level of Fluoro-Jade B–stained positive cells noted in the gray matter tissues for both occlusion groups.

Extracellular glutamate levels and neuropathology in cerebral white matter following repeated umbilical cord occlusion in the near term fetal sheep

Umbilical cord occlusion causes fetal hypoxemia which can result in brain injury including damage to cerebral white matter. Excessive glutamate release may be involved in the damage process. This study examined the relation between extracellular glutamate levels in the cerebral white matter of the ovine fetus during and after intermittent umbilical cord occlusion and the degree of resultant fetal brain injury. Fetal sheep underwent surgery for chronic catheterisation and implantation of an intra-cerebral microdialysis probe at 130 days of gestation (term ∼147 days). Four days after surgery (day 1), seven fetuses were subjected to 5×2 min umbilical cord occlusions, and on the following day (day 2) they were subjected to either 4 or 5×4 min umbilical cord occlusions; seven fetuses served as controls. Microdialysis samples were collected before, during and after the umbilical cord occlusions to determine extracellular glutamate levels in the cerebral white matter. Fetal blood gas status was measured and the fetal electrocorticogram was recorded continuously. During the periods of umbilical cord occlusions on both days 1 and 2, fetal arterial oxygen saturation, arterial partial pressure of oxygen and arterial pH decreased ( P<0.05) while arterial partial pressure of carbon dioxide increased ( P<0.05). All fetuses showed episodes of isoelectric electrocortical activity during umbilical cord occlusions on both days 1 and 2. In fetuses with patent microdialysis probes there were marked increases of glutamate efflux in the cerebral white matter following umbilical cord occlusion. Fetal brains were removed at autopsy on day 5 and subjected to histological assessment. Brain damage was observed in all fetuses exposed to cord occlusion, particularly in the periventricular white matter, with the most extensive damage occurring in the fetuses with the greatest increases in glutamate levels. We conclude that, in the unanesthetised fetus in utero, glutamatergic processes are associated with umbilical cord occlusion-induced brain damage in the cerebral white matter.

Apoptosis in the preterm and near term ovine fetal brain and the effect of intermittent umbilical cord occlusion

Programmed cell death or apoptosis plays a central role during the development of the brain, but can also be activated by hypoxic/ischemic insult. The purpose of the present study was to determine the regional distribution of apoptotic cells in the preterm and near term ovine fetal brain and thus in relation to the maturation of neurobehavioural activity, and the effect of intermittent umbilical cord occlusion (UCO), which might then contribute to adverse neurodevelopment. Fetal sheep (control and experimental groups at 0.75 and 0.90 of gestation) were studied over 4 days with UCOs performed in the experimental group animals by complete inflation of an occluder cuff for 90 s every 30 min for 3 to 5 h each day. Animals were then euthanized and the fetal brain perfusion-fixed and prepared for subsequent histology and apoptosis staining using the TUNEL assay method. The number of TUNEL positive cells for both the preterm and near term control group animals was low but with a significant regional hierarchy whereby values were higher in the cerebellar peduncle and cortex and lower in the cortical grey and white matter, hippocampus, and pons. While the apoptotic indices (expressed as TUNEL positive cells/1000 cells or high powered field) for most brain regions were not significantly changed between the preterm and near term control group animals, that for the hippocampus and pons were increased ∼5- and 4-fold, respectively, (both P<0.05), in the near term animals. Intermittent UCO with severe but limited hypoxemia and no cumulative acidosis to ensure longer term survival, had no significant effect on apoptotic indices in the brains of either the preterm or near term animals, although hippocampal values for both occlusion groups were increased ∼2–3-fold. Levels of apoptosis noted for the ovine fetal brain at 0.75 to 0.90 of gestation are thus low and likely approaching the basal levels of later life, but there are regional differences and changes over this period although little change in response to intermittent cord occlusion as studied, with implications for behavioural state activity and antenatal hypoxic insults in the brain’s development.

Effect of intermittent umbilical cord occlusion on fetal respiratory activity and brain adenosine in late-gestation sheep.

It was hypothesized that intermittent umbilical cord occlusion (UCO) would inhibit ovine fetal breathing movements (FBM) in association with increased cerebral adenosine levels. To test this hypothesis, on two successive days during late gestation (133-134 days; term = 146 days), microdialysis samples were collected from the brains of 10 chronically instrumented fetal sheep during 2-h periods of complete UCO induced every 30 min (Day 1: 2-min UCOs; Day 2: 4-min UCOs). Control fetuses (n = 10) underwent no UCO. Tracheal pressure was measured throughout. This regimen resulted in a decrease in fetal arterial PO2 (PaO2) during each UCO to 7.3 +/- 0.8 mmHg (P<0.01; Day 1) and 8.4 +/- 1.1 mmHg (P<0.01; Day 2). Throughout each UCO period, fetal arterial pH (pHa) decreased to 7.28 +/- 0.02 (P<0.01; Day 1) and 7.11 +/- 0.07 (P<0.01; Day 2). The hourly incidence of FBM decreased significantly only on Day 2, from 38.6 +/- 4.1% to 4.1 +/- 1.6% (P<0.01). The frequency of deep isolated inspiratory efforts increased from 4.7 +/- 2.0 h(-1) to 17.6 +/- 6.1 h(-1) (P<0.05; Day 1) and from 2.2 +/- 0.9 h(-1) to 33.6 +/- 4 h(-1) (P<0.01; Day 2). The amplitude of both FBM and deep isolated inspiratory efforts increased during the UCO periods on both days. The concentration of cerebral extracellular fluid (ECF) adenosine during UCO increased by 219 +/- 215% (P<0.05; Day 1) and 172 +/- 107% (P<0.05; Day 2) over the baseline periods. In conclusion, the severity of the inhibitory effect of repeated UCO on FBM depends, in part, on the length of the occlusions. The inhibition of FBM during intermittent UCO may be mediated by the increase in ECF adenosine in the fetal brain. Furthermore, FBM and deep isolated inspiratory efforts appear to be regulated by different mechanisms.

Oxidative stress in the fetal lamb brain following intermittent umbilical cord occlusion: a path analysis

Objective To evaluate the relative contribution of cord occlusion length intervals between occlusions and experimental duration on oxidative stress in the fetal lamb brain. Design Acute, partially exteriorised fetal lambs with intermittent total cord occlusion. Setting The Vivarium of Westmead Hospital, University of Sydney, Australia and The Chinese University of Hong Kong. Main outcome measures Arterio-venous differences in the concentration of organic hydroperoxides, measured in paired samples of carotid arterial and jugular venous blood, as an index of oxidative stress in the brain. Methods Thirty-two fetal lambs were exposed to graded hypoxia, induced by intermittent total umbilical cord compression of 30 seconds, 60 seconds and 90 seconds duration, occurring every minute for a total of 27 occlusions over 81 minutes. Three sham experiments were also performed. In addition to organic hydroperoxides, carotid arterial blood samples were also assayed in 15 animals (two sham) for oxygen saturation, pH, hypoxanthine, xanthine and urate concentrations. A causal model for oxidative stress was defined: occlusions leading to hypoxia with a rise in hypoxanthine; reperfusion during intervals between occlusions leading to the accelerated production of xanthine and uric acid and the generation of oxygen free radicals, which in turn, are responsible for the rise in lipid peroxidation. Path analysis was performed to assess the strength of the relationships between these variables and the cord occlusion length, the interval between occlusions and the duration of the experiment. Results Sham experiments showed no change in organic hydroperoxide production. Following 30-second umbilical cord occlusions a sixfold drop in mean organic hydroperoxides was observed between carotid arterial and jugular venous levels. In contrast, following occlusions of 60 seconds duration (or longer) a median 20-fold increase in organic hydroperoxide production was observed. Path analysis revealed a strong indirect pathway from occlusion length → hypoxanthine → urate and weak positive pathways from oxygen saturation→ urate and from interval between occlusions → urate. After accounting for these pathways reflecting oxidative stress, a strong direct path remained from time from first occlusion → organic hydroperoxide production. Conclusions Peroxidation of lipids in the brain occurs under conditions of severe hypoxia and reperfusion associated with intermittent umbilical cord occlusions of 60 seconds or longer. The path analysis supported the causal model as originally defined, with the exception that the indirect pathway via pH was found to be trivial.