David W. Boyle

Potential Role of IGF-I in Hypoxia Tolerance Using A Rat Hypoxic-Ischemic Model: Activation of Hypoxia-Inducible Factor 1α

Hypoxia preconditioning and subsequent tolerance to hypoxia-ischemia damage is a well-known phenomenon and has significant implications in clinical medicine. In this investigation, we tested the hypothesis that the transcriptional activation of IGF-I is one of the underlying mechanisms for hypoxia-induced neuroprotection. In a rodent model of hypoxia-ischemia, hypoxia preconditioning improved neuronal survival as demonstrated by decreased hypoxia-ischemia-induced neuronal apoptosis. To study the role of IGF-I in hypoxia tolerance, we used in situ hybridization to examine IGF-I mRNA distribution on adjacent tissue sections. In cerebral cortex and hippocampus, hypoxia preconditioning resulted in an increase in neuronal IGF-I mRNA levels with or without hypoxia-ischemia. To test its direct effects, we added IGF-I to primary neuronal culture under varying oxygen concentrations. As oxygen concentration decreased, neuronal survival also decreased, which could be reversed by IGF-I, especially at the lowest oxygen concentration. Interestingly, IGF-I treatment resulted in an activation of hypoxia-inducible factor 1α (HIF-1α), a master transcription factor for hypoxia-induced metabolic adaptation. To evaluate whether IGF-I transcriptional activation correlates with HIF-1α activity, we studied the time course of HIF-1α DNA binding activity in the same rat model of hypoxia-ischemia. After hypoxia-ischemia, there was an increase in HIF-1α DNA binding activity in cortical tissues, with the highest increase around 24 h. Like IGF-I mRNA levels, hypoxia preconditioning increased HIF-1α DNA binding activity alone or with subsequent hypoxia ischemia. Overall, our results suggest that IGF-I transcriptional activation is one of the metabolic adaptive responses to hypoxia, which is likely mediated by a direct activation of HIF-1α.

On nonlinear random effects models for repeated measurements

Linear random effects models for longitudinal data discussed by Laird and Ware (1982), Jennrich and Schluchter (1986), Lange and Laird (1989), and others are extended in a straight forward manner to nonlinear random effects models. This results in a simple computational approach which accommodates patterned covariance matrices and data insufficient for fitting each subject separately. The technique is demonstrated with an interesting medical data set, and a short, simple SAS PROC IML program based on the EM algorithm is presented.

Fetal Hind Limb Oxygen Consumption and Blood Flow during Acute Graded Hypoxia

ABSTRACT: Hind limb blood flow and O2 uptake, mean blood pressure, and heart rate were measured in six fetal sheep at 127-141 d gestation in the control state and at different levels of hypoxia that were induced by partial occlusion of the maternal terminal aorta. Blood flow was measured by an ultrasonic flow transducer. Control fetal arterial O2 content ([O2]a) in the descending aorta was 3.25 + 0.17 mM. In response to graded acute hypoxia, blood flow first increased (22.2 versus 19.9 mL.min−1.100 g−1, p = 0.003) and then decreased abruptly at approximately 1.5 mM [O2]a.O2 uptake decreased about 12% (14.74 versus 16.71, p = 0.03) as [O2]a decreased to 1.5 mM, and then fell markedly, tending toward zero for [O2]a = 0.9 mM. In the 2.7- to 1.5-mM [O2]a range, heart rate increased above control (194 versus 169 min−1, p = 0.0024), whereas mean blood pressure did not change significantly. For [O2]a < 1.5 mM, heart rate decreased to 148 min−1 (p = 0.0005) and mean blood pressure increased above baseline (55 versus 47 torr, p = 0.0001). In conjunction with previous evidence, these results define a state of acute moderate hypoxia in which the whole fetus can sustain a relatively high rate of oxidative metabolism, and a state of acute severe hypoxia ([O2]a between 1.5 and 1.0 mM) in which O2 uptake by some fetal organs is selectively and markedly decreased.

Measurement of blood flow and oxygen consumption in the pelvic limb of fetal sheep.

Abstract In order to determine blood flow and oxygen consumption in the pelvic limb of fetal sheep, we applied the Fick principle of measurement of oxygen consumption in seven paired experiments in seven fetal sheep under normal conditions and after treatment with pancuronium bromide. Catheterization procedures, which minimized interference with the study limb circulation, avoided changes of catheter tip position during fetal movements, and prevented collateral circulation to and from tissues not located in the pelvic limb, were utilized. Blood flow through the external iliac artery was measured by means of a transit time ultrasonic method. Six sample sets for oxygen content were drawn from the external iliac artery and vein during 45-min control period and repeated after neuromuscular blockade. Normal oxygen consumption under these experimental conditions was determined to be 20.7 ± 1.9 (mean ± SEM) μmole · min-1 · 100 g-1. Neuromuscular blockade caused oxygen consumption to decrease significantly (P < 0.01) by 12% to 18.1 ± 2.1 μmole · min-1 · 100 g-1 and decreased the average coefficient of variation from 15 to 8%. The data demonstrate that spontaneous skeletal muscle activity accounts for a significant amount of oxygen consumption, the level of which can vary widely over brief periods of time. These results suggest that such tissues with significant spontaneous changes in metabolic activity require repeated blood flow measurements with simultaneous determination of substrate arteriovenous differences to best describe metabolism under normal conditions.

Fetal pH Improvement after 24 h of Severe, Nonlethal Hypoxia

The accumulation of hydrogen ions in fetal blood when fetal oxygenation is acutely decreased suggests metabolic instability. However, in 6 chronically prepared fetal sheep whose arterial O2 content and pO2 were abruptly reduced and maintained at about 1.7 mM and 12.6 Torr, respectively, we observed a significant decrease in fetal arterial lactate concentrations and an increase in pH to normal values after 24 h of continuous hypoxia compared with values observed after 5 h. This demonstrates that fetal pH measurements may not detect prolonged intrauterine hypoxia.

Changes in 4E-BP1 and p70S6K phosphorylation in skeletal muscle of the ovine fetus after prolonged maternal fasting: effects of insulin and IGF-I.

This study was conducted to investigate fasting-induced alterations in insulin signaling to the regulatory components of the translation machinery. Insulin (890 mIU/h) and IGF-I (40 nM/h) were infused into a chronically catheterized ovine fetus (0.85 gestation) for 7 h following a 5-d maternal fast. Amino acid and glucose concentrations were clamped to minimize the effects of alterations in circulating substrate concentrations. The IGF-I induced increase in 4E-BP1 phosphorylation (percentage in the γ form) increased from 28% in control to 44% (NS). The insulin-induced increase in 4E-BP1 phosphorylation was more pronounced, and the γ percentage was 56% on average in the insulin group. The insulin-induced increase in 4E-BP1 phosphorylation was lower than in fed animals and did not result in significant changes in eIF4E·4E-BP1 binding or eIF4E·eIF4G binding. Insulin increased PKB/Akt phosphorylation and p70S6K phosphorylation to a similar extent as in fed animals. We conclude that maternal fasting resulted in reduced insulin sensitivity of 4E-BP1 phosphorylation and eIF4F formation. This reduced insulin-induced 4E-BP1 phosphorylation was not due to a global defect in insulin signaling; the defects underlying the reduced basal phosphorylation and insulin-responsiveness of 4E-BP1 in fasted animals may be in signaling components other than, or downstream of, PKB/Akt. Selective inhibition of downstream components of insulin signaling allows fetuses to adapt to nutritional stress by decreasing the anabolic response to insulin and other growth factors, so that more amino acids can be used as oxidative substrate to compensate for shortage of energy due to reduced glucose supply.

Effect of Neuromuscular Blockade on Fetal Oxidative Metabolism

Because fetal neuromuscular blockade has been used during cord blood sampling procedures to prevent fetal movement, we studied the effect of pancuronium bromide on fetal arterial concentrations of glucose, lactate, and oxygen and their umbilical uptake in 6 chronically prepared fetal sheep. We observed a significant increase in the arterial concentrations and decrease in the umbilical uptakes. We suggest that cord blood sampling procedures for fetal assessment of oxidative metabolism not be performed after fetal neuromuscular blockade so that data obtained might best reflect the unperturbed physiological condition of the fetus.

Supplemental Oxygen and Glucose During Prolonged Uterine Blood Flow (UtBF) Reduction Prevents Fetal Growth Restriction in Sheep. |[bull]| 1576

We have shown that 7 days of UtBF reduction in sheep results in a 38% decrease in fetal linear growth rate (LGR) from 6 mm/day to 3.7 mm/day (Am J Physiol, 1996). This decrease in LGR correlates with the fetal concentrations of oxygen and glucose. Subsequent studies, in which fetal PaO2 was increased by providing supplemental oxygen to the ewe during UtBF reduction, failed to prevent the development of fetal growth restriction. The present study was undertaken to test the hypothesis that fetal growth restriction might be prevented by providing supplemental O2 and glucose during UtBF reduction. Six sheep with singleton pregnancies at 115-117 days gestation were prepared with vascular catheters, a fetal crown-rump length measuring device, a vascular occluder on the maternal terminal aorta, and a catheter in the maternal trachea. Fetal LGR was measured daily after surgery. The sheep were recovered for 8-14 days after which serial studies were performed 7 days apart. Following a basal study UtBF was reduced and sheep were supplemented with 100% O2 at 2-2.5 lpm via the maternal tracheal catheter. In addition, a variable glucose infusion was begun through the middle uterine artery with the goal of increasing the fetal arterial glucose concentration by 25-50%. UtBF reduction and O2 and glucose concentrations were checked twice daily between the Basal and Day 7 studies.

Brain Polyubiquitin Up-Regulation in Hypoxic Ischemic Encephalopathy (HIE) and Intrauterine Growth Retardation (IUGR) 1864

Protein ubiquitination plays an important role in rapid degradation of short-lived, denatured or damaged proteins. Previous studies have postulated that ubiquitin expression is up-regulated in the brain after transient global ischemia (Caray et al, 1995). The present study was designed to compare the expression of ubiquitin mRNA level in the fetal sheep brain under conditions of acute cerebral hypoxic ischemia or IUGR induced by 4 to 5 weeks of chronic hypoxia from single umbilical artery ligation. We examined the brains from nine fetal sheep in three different groups: Group 1 (N=3), Fetuses in which single umbilical artery ligation resulted in IUGR. Group 2 (N=3), Fetuses with HIE induced by transient carotid artery occlusion. Group 3 (N=3), control intact fetuses. Haematoxylin-Eosin staining was utilized to confirm histopathologic neuronal injury. mRNA levels were measured by conventional PCR and read by phospho-imaging utilizing Cyber Green II. Statistical comparisons between groups was done by the Wilcoxon rank non-parametric test.

Maternal Oxygen (O2) Supplementation During Prolonged Uterine Blood Flow (Ut BF) Reduction Does Not Prevent Fetal Growth Restriction In Sheep. 234

We have shown that 7 days of fetal hypoxemia due to Ut BF reduction results in a 38% decrease in fetal growth rate (AJP,1996). Further, 25% of the decrease in fetal growth rate was attributed to the level of fetal hypoxemia. We hypothesized that maternal O2 supplementation during Ut BF reduction would improve fetal oxygenation and prevent fetal growth restriction. Sheep were prepared with vascular catheters, a fetal crown-rump length measuring device, a vascular occluder on the maternal terminal aorta, and a catheter in the maternal trachea. Serial studies were performed 7 days apart beginning at 120-130 days gestation. Six sheep (Occluded) underwent Ut BF reduction that was maintained for 7 days after the basal study. Five sheep (Mat O2) underwent Ut BF reduction and in addition were started on 100% O2 at 2-2.5 lpm after the basal study. Results (Mean ± SD): Ut BF decreased by 37% in Occluded sheep and by 38.5% in Mat O2 sheep after the basal study. Fetal linear growth rate decreased in the Occluded sheep from 5.9 ± 2.7 to 2.7 ± 1.7mm/d (p<0.05) after the basal study and from 5.9 ± 1.0 to 3.3 ± 1.5mm/d (p=0.1) in Mat O2 sheep.Table Conclusion: Maternal O2 supplementation reduces the degree of fetal hypoxemia, but does not completely prevent the decrease in fetal growth rate that is associated with prolonged Ut BF reduction.