Standardizing ordinal subadult age indicators: Testing for observer agreement and consistency across modalities.

Skeletal and dental data for subadult analyses obtained from dry bones or various types of medical images, such as computed tomography (CT) scans or conventional radiographs/x-rays, should be consistent and repeatable to ensure method applicability across modalities and support combining study samples. The present study evaluates observer agreement of epiphyseal fusion and dental development stages obtained on CT scans of a U.S. sample and the consistency of epiphyseal fusion stages between CT scans and projected scan radiographs/scout images (U.S. CT sample), and between dry bones and conventional x-rays (Colombian osteological sample). Results show that both intra- and interobserver agreements of scores on CT scans were high (intra: mean Cohen's kappa=0.757-0.939, inter: mean Cohen's kappa=0.773-0.836). Agreements were lower for dental data (intra: mean Cohen's kappa=0.757, inter: mean Cohen's kappa=0.773-0.0.820) compared to epiphyseal fusion data (intra: mean Cohen's kappa=0.939, inter: mean Cohen's kappa=0.807-0.836). Consistency of epiphyseal fusion stages was higher between dry bones and conventional x-rays than between CT scans and scout images (mean Cohen's kappa=0.708-0.824 and 0.726-0.738, respectively). Differences rarely surpassed a one-stage value between observers or modalities. The complexity of some ossification patterns and superimposition had a greater negative impact on agreement and consistency rates than observer experience. Results suggest ordinal subadult skeletal data can be collected and combined across modalities.

Combined computed tomography and position-resolved X-ray diffraction of an intact Roman-era Egyptian portrait mummy.

Hawara Portrait Mummy 4, a Roman-era Egyptian portrait mummy, was studied with computed tomography (CT) and with CT-guided synchrotron X-ray diffraction mapping. These are the first X-ray diffraction results obtained non-invasively from objects within a mummy. The CT data showed human remains of a 5-year-old child, consistent with the female (but not the age) depicted on the portrait. Physical trauma was not evident in the skeleton. Diffraction at two different mummy-to-detector separations allowed volumetric mapping of features including wires and inclusions within the wrappings and the skull and femora. The largest uncertainty in origin determination was approximately 1.5 mm along the X-ray beam direction, and diffraction- and CT-determined positions matched. Diffraction showed that the wires were a modern dual-phase steel and showed that the 7 × 5 × 3 mm inclusion ventral of the abdomen was calcite. Tracing the 00.2 and 00.4 carbonated apatite (bone's crystalline phase) reflections back to their origins produced cross-sectional maps of the skull and of femora; these maps agreed with transverse CT slices within approximately 1 mm. Coupling CT and position-resolved X-ray diffraction, therefore, offers considerable promise for non-invasive studies of mummies.

Relocation during incubation of endothelial nuclei in the chick chorioallantois.

Early in incubation the nuclei of the chick chorioallantoic capillaries are randomly distributed around the capillary lumen; later most of them are located on the portion of the capillary surface opposite the inner shell membrane. This is one of the complex of processes that results in progressive thinning of the diffusion pathway for gases between the external environment and the blood of the embryo. The present study quantified this nuclear "relocation". Our data show a progressive relocation of the endothelial nuclei from the tenth through the sixteenth day at an average rate of 6% per day.

Effects of brief hypoxia and hyperoxia on tissue trace element levels in the developing chick embryo.

Brief hypoxia or hyperoxia has been shown to affect growth and metabolism of chick embryos during the late stages of development. The objective of this study was to alter the availability of oxygen to chick embryos developing in ovo and to determine the effects on tissue zinc, copper, iron and manganese levels. On day 15 of incubation fertile chicken eggs were divided into three groups: 15% O2 (hypoxic), 60% O2 (hyperoxic) and 21% O2 (normoxic) and incubated under these conditions for 72 h to day 18. Hypoxia reduced embryo, heart, brain and liver wet weights, whereas hyperoxia increased embryo, heart, lung and liver wet weights compared to normoxic controls. Chorioallantoic membrane (CAM) wet weight was increased by hypoxia and reduced by hyperoxia. Livers from hyperoxic embryos contained more zinc, iron and manganese and less copper than livers from hypoxic or normoxic embryos. Tissue concentrations of zinc, copper, iron and manganese were reduced in brains from hyperoxic compared to hypoxic or normoxic embryos. Hyperoxia increased the zinc and copper concentrations in CAM, whereas hypoxia reduced zinc and iron levels. The contents of zinc and copper were increased in hyperoxic compared to normoxic or hypoxic lungs. Hearts from hyperoxic embryos had more zinc, copper and manganese than hypoxic or normoxic hearts. Hypoxic yolk sac contained more zinc and manganese than hyperoxic or normoxic yolk sac. Except for yolk sac, the trace element content of tissues from normoxic embryos increased from day 15 to day 18 of incubation in concert with tissue growth. We conclude that the availability of oxygen to the developing chick embryo affects tissue trace element levels through its effects on tissue growth, as a result of adaptation by specific tissues to different oxygen tensions, or via effects on the regulation of trace element uptake and assimilation by the tissues.

Prenatal oxidative stress: I. Malondialdehyde in hypoxic and hyperoxic chick embryos.

Evidence suggests a positive correlation between metabolic rate (VO2), or ambient oxygen (O2) tension, and the rate of formation of free radicals from O2. We have previously demonstrated that the rates of growth, VO2, protein and DNA accumulation, and the activity of cytochrome oxidase (a key mitochondrial respiratory enzyme), are increased significantly by exposing the chick embryo to 72 h of hyperoxia (60% O2) late in incubation. To test the hypothesis that the chick embryo responds to a prenatal alteration in O2 availability in such a way as to protect its tissues from oxidative damage, we have used the thiobarbituric acid assay to estimate lipid peroxidation (a major form of free radical damage) in selected organs from chick embryos exposed to altered O2 availability. We found significantly higher concentrations of malondialdehyde (MDA, a secondary product of lipid peroxidation) in liver than in chorioallantoic membrane, brain, or heart. However, embryos exposed to brief (72 h) hypoxia (15% O2) or hyperoxia (60% O2) late in incubation, or 48 h of such exposure followed by 24 h of incubation in pure O2, exhibited no significant difference in MDA levels compared to normoxic (21% O2) controls in any of the tissues examined. We conclude that the increase in aerobic metabolism induced in the chick embryo by 3 days of hyperoxia is not accompanied by an increase in lipid peroxidation. We postulate that the chick embryo adapts to hyperoxia in such a way as to escape additional free radical damage, perhaps by increasing the capacity of its antioxidant defenses to compensate for a potential increase in the rate of free radical generation.

O2 effect on composition of chick embryonic heart and brain.

Heart ventricles from chick embryos incubated in 60% O2 (hyperoxia) on the 16th through the 18th days of incubation were 21% heavier than those from control embryos maintained in 21% O2 (normoxia). Heart ventricles from embyros incubated in 15% O2 (hypoxia) were 8% lighter than controls. Changes in ventricular weight were accompanied by proportional changes in protein content (21% more in hyperoxic ventricles; 8% less in hypoxic ventricles). Ventricular tissue DNA content showed a significant increase in hyperoxia. Tissue protein/DNA ratios were significantly higher in hyperoxia and lower in hypoxia. These data suggest that increased O2 availability led to hypertrophy of chick embryo ventricular cells and an increase in the level of DNA synthesis. Cytochrome oxidase activity per mg DNA was 15-25% higher in hyperoxic ventricles than in hypoxic ventricles. This result is consistent with our previous findings that alterations in O2 availability affect the O2 consumption rate of the chick emryo in ovo, and it provides direct evidence that a phenomenon repeatedly observed in vitro is of importance in vivo. In contrast to the heart, O2 availability did not affect the wet weight, protein or DNA contents, or cytochrome oxidase activity of the chick embryo brain.

Matching of maternal and fetal flow ratios in the sheep placenta.

Local interaction of maternal and fetal placental blood flows was studied in two groups of unanaesthetized near-term sheep. Five sheep were exposed to a simulated dive to 100 feet of seawater (4.03 atmospheres) for 25 min. Six fetuses received an infusion of noradrenaline (6.8 micrograms/[kg x min]). Radioactive microspheres were administered simultaneously to mother and fetus before (control) and after (test) the experimental manipulation. Maternal and fetal relative activities, defined as % of total placental radioactivity divided by % of total placental weight, were calculated for 1-g pieces of cotyledonary tissue under control and test conditions. Pieces of cotyledons were defined as matched if the direction of change in relative activity from control to test was the same for mother and fetus. In the absence of an interaction between the maternal and fetal placental circulations, the probability of a piece of cotyledon being matched is 0.5. In each series of experiments the proportion of all cotyledon pieces having maternal and fetal relative activities that changed in the same direction was significantly greater than 0.5. Thus, the majority of the placental mass responds to a physical or chemical perturbation of the fetus in such a way that changes in relative perfusion are qualitatively matched in the adjacent maternal and fetal placental circulations.

Modulation of growth and metabolism of the chick embryo by a brief (72-hr) change in oxygen availability.

Growth of the chick embryo is accelerated by brief (72-hr) exposure to 60% O2 beginning on day 16 of incubation, and acute (2-3 hr) hyperoxia stimulates oxygen consumption (VO2). However, the increment in VO2 that accompanies acute exposure to 60% O2 is only about half that required to account for the degree of growth acceleration observed during 72 hr of hyperoxia. We tested the hypothesis that the magnitude of the oxygen-induced change in embryonic metabolism depends on the length of exposure by making daily measurements of embryonic VO2 during brief (72-hr) exposure to 60% or 15% O2. White leghorn eggs were incubated in 21% O2 for 15 days. On day 16 the experimental eggs were switched to 60% or 15% O2; control eggs were maintained in 21% O2. Oxygen consumption and CO2 production (VCO2) were measured daily. Embryo and organ weights were compared on day 18. Wet and dry weights of briefly hyperoxic embryos were significantly greater than those of normoxic controls. The relative increase in wet weight was significant for the heart and liver but not the brain. Oxygen consumption increased 7% relative to control after 24 hr of hyperoxia and 17% after 72 hr; VO2/gm embryo was 9% greater than control on day 18. Normal growth deceleration was exaggerated by hypoxia; mean wet and dry weights of the briefly hypoxic embryos were significantly less than those of controls. Organ wet weights also showed growth retardation, although the relative decrease in brain weight was not significant. Body water concentration increased in briefly hypoxic embryos. Oxygen consumption was 13% less than control after 24 hr and 17% less after 72 hr.(ABSTRACT TRUNCATED AT 250 WORDS)

Stimulatory and persistent effect of acute hyperoxia on respiratory gas exchange of the chick embryo.

The hypothesis that oxygen availability limits growth of the normal chick embryo late in development predicts that an increase in oxygen availability would accelerate the rate of growth and, therefore, metabolism. We tested the prediction concerning metabolism by comparing the oxygen consumption (VO2) and carbon dioxide production (VCO2) of 14-18 day embryos acutely exposed to either 50% or 100% O2 with those of normoxic (21% O2) controls. Two hours of hyperoxia produced increases in both VO2 and VCO2; however, repeated measurements over time in normoxia also demonstrated a significant increase in gas exchange, presumably due to normal growth of the embryos. After correcting for the increase in VO2 due to growth, there was no effect of 60% O2 on day 14. Thereafter the stimulatory effect of 60% O2 increased gradually, reaching 6.1% on day 18. VCO2 was 4 to 6% higher in embryos acutely exposed to 60% O2 than in normoxic controls throughout the observation period, although the difference was significant only on day 18. The VO2 of embryos acutely exposed to 100% O2 was not significantly different from that observed in 60% O2, and was still significantly elevated 3 h after the eggs were returned to 21% O2. We conclude that acute hyperoxia late in incubation elicits an increase in embryonic VO2 and VCO2, with little or no effect on the respiratory exchange ratio, and that the stimulation of gas exchange by 100% O2 persists after the embryo is returned to normoxic conditions. These findings support the hypothesis that oxygen availability limits growth and metabolism of the normoxic chick embryo late in development.

Monosaccharide uptake by erythrocytes of the embryonic and adult chicken.

Rates of monosaccharide uptake by adult and 10-18 day old embryonic chicken erythrocytes were quantitated. The rate of carrier-mediated, stereospecific transport decreased 28% from day 10 to day 14 of incubation and was unchanged thereafter. At no time, however, did the rate of carrier-mediated transport by embryonic erythrocytes differ significantly from that of the adult cells. The rate of transfer by simple diffusion was 3-5 fold faster in embryonic than in adult erythrocytes. Uptake by simple diffusion decreased slightly as the embryo developed. Chronic hyperoxic incubation (70% O2) had little influence on total monosaccharide uptake by embryonic erythrocytes.

Stimulation of growth of the chick embryo by acute hyperoxia.

We have previously demonstrated that continuous incubation in elevated concentrations of ambient oxygen leads to accelerated growth of the chick embryo. We now report that a similar growth response is elicited by acute (72 h) exposure beginning on the 16th day of incubation. White Leghorn eggs were paired by initial weight and incubated in air for 15 days in forced-draft incubators. Embryos were sampled on days 11, 13 and 15 (experiments 1, 2, 3) or 13, 14 and 15 (experiment 4), freed of all extra-embryonic membranes and weighed. On day 16 the experimental group was switched to 60% O2. The control group was maintained in air. A portion of the remaining eggs from each group was opened on days 16, 17 and 18 (experiment 4), or alternatively all the remaining eggs were opened at the end of the 18th day of incubation (experiments 1, 2, 3). Linear regression analysis of growth curves obtained by plotting log wet embryo weight (g) vs log incubation age (d) showed a significantly greater rate of growth (slope) for days 15-18 in embryos exposed to 60% O2. These results support the hypothesis that growth of the chick embryo is normally limited by the availability of oxygen.

Cardiovascular effects of induced decompression sickness in sheep fetus.

Ten chronically catheterized near-term sheep fetuses were used to study the effect of induced intravascular bubbling on fetal placental vascular dynamics and fetal regional blood flows. Fetal blood pressure and heart rate were measured, and radioactive microspheres were administered to fetuses before and 5, 10, 15, and 20 min after a simulated no-decompression dive to 100 fsw (4 ATA) for 25 min. A decrease in brain blood flow was the only effect observed 5 min after decompression. Fifteen minutes later arterial hypertension was evident. Eight fetuses displayed arrhythmia, but there was no significant change in heart rate, myocardial blood flow, or resistance. There was a reduction in blood flow, and a concomitant increase in vascular resistance, in the gut, kidneys, placental membranes, and skeletal muscle, as well as in the brain. Total placental blood flow was unchanged throughout the postdive period, although placental vascular resistance was elevated 20 min after surfacing. Analysis of the placental blood flow at the cotyledonary level in 6 animals revealed no uniform response to decompression. The results are consistent with the hypothesis that the instrumented fetus suffers widespread vascular embolization and disruption of organ blood flows. We conclude that the observed changes in the fetal cardiovascular system, however, are not precipitated by an initial effect of decompression on the fetal placental circulation.

Organ growth in chick embryos incubated in 40% or 70% oxygen.

Incubation of eggs in 60% oxygen has been shown to enhance growth of the chick embryo. To determine whether oxygen accelerates growth over a range of concentrations, eggs were incubated in 40% or 70% O2. Control eggs, pair-matched by initial weight, were simultaneously incubated in room air (21% O2). Embryo and organ weights from matched pairs of eggs were compared on incubation days 14, 16 and 18 (40% O2) or 10, 12, 14, 16 and 18 (70% O2). Embryos incubated in 40% O2 displayed a pattern of growth enhancement similar to that previously reported for 60% O2. Accelerated embryonic growth was maintained through day 18. The heart showed the greatest percentage increase in weight over control, exceeding that of the whole embryo on days 16 and 18. The brain displayed significant enhancement only on day 16. Weight of the liver was unaffected by hyperoxia. Embryos in 70% O2 exhibited accelerated growth in all of the tissues examined early in incubation. Growth rate of the hyperoxic embryos then declined, so that embryo weight on day 18 did not differ from control. The brain, heart, eye, and proventriculus plus gizzard from 70% O2 embryos weighed significantly less than controls on day 18. Growth inhibition was most striking in the heart; heart/body weight ratio of 70% O2 embryos was significantly less than control throughout the observation period. The results confirm the growth accelerative effect of oxygen and suggest that the degree of growth enhancement is proportional to the ambient oxygen concentration. Accelerated growth cannot be maintained, however, in 70% O2.

Placental vascular responses to 6-keto-prostaglandin E1 in the near-term sheep.

6-Keto-prostaglandin E1 (6-keto-PGE1) is a biologically active, stable metabolite of prostaglandin I2 (PGI2). It has vasoactive properties similar to those of PGI2 and it has been shown to decrease the resistance of the renal, mesenteric, and pulmonary vascular beds. PGI2 is synthesized by the pregnant uterus and is vasoactive in the ovine placenta. The effects of 6-keto-PGE1 on uterine and placental blood flow in pregnant ewes were determined for comparison with those of PGI2. Near-term ewes and their fetuses were chronically catheterized to permit the measurement of regional blood flow by the radioactive microsphere method. In six sheep a 5-minute maternal jugular infusion of 3.25 micrograms/kg/min of 6-keto-PGE1 decreased mean arterial blood pressure from 89 +/- 4.8 to 63 +/- 7.1 mm Hg. Uterine vascular resistance decreased from 0.55 +/- 0.11 to 0.35 +/- 0.05 peripheral resistance units (PRU), but maternal cotyledonary resistance increased from 0.19 +/- 0.04 to 0.27 +/- 0.03 PRU. In five sheep a fetal intravenous infusion of 18 micrograms/min of 6-keto-PGE1 decreased mean fetal blood pressure from 43 +/- 2 to 29 +/- 2 mm Hg. Cotyledonary vascular resistance increased from 0.30 +/- 0.02 to 0.55 +/- 0.09 PRU . kg-1. In these sheep there were no significant changes in maternal uterine, renal, or cotyledonary blood flows. These results indicate that 6-keto-PGE1 is similar to PGI2 in that it produces maternal cotyledonary vasoconstriction, hypotension, and vasodilation in other organs.

Effect of PGI2 on the response of the ovine placenta to norepinephrine.

We have examined the effects of PGI2, 50 microgram/kg, on norepinephrine induced placental vasoconstriction in 6 chronically catheterized near-term sheep. Regional blood flows were measured with radioactive microspheres. Control flows were measured. Norepinephrine was than infused at 50 microgram/min throughout the experiment. After 15 min the blood flows were again measured and PGI2 was then added to the infusate at 50 microgram/min. In 15 min regional blood flows were again measured and the PGI2 infusion was stopped. Regional blood flows were measured for the last time 15 min later. The renal and nonplacental uterine vasculatures behaved in a predictable manner. There was constriction with norepinephrine but PGI2 opposed the effects of norepinephrine and decreased the resistance towards the normal levels. The placenta did not behave as did the other organs. Norepinephrine increased placental resistance but PGI2 did not decrease the resistance and severely depress the placental blood flows. PGI2 does not appear to oppose norepinephrine induced placental vasoconstriction.

Vascular response of the fetal placenta to local occlusion of the maternal placental vasculature.

Local regulation of fetal placental blood flow was studied in 6 near-term sheep. Maternal blood flow was reduced or eliminated to 6-17% of the placenta by ligation or embolization with non-radioactive microspheres. Maternal and fetal placental blood flows were measured, using radioactively-labelled microspheres, before and after vascular occlusion. The change in fetal placental blood flow was significantly different in the occluded as compared to the non-occluded cotyledons. Occlusion of the maternal placental vasculature was associated with a 40% decline in the adjacent fetal placental flow after 24 h. These data support the concept of a local regulatory interaction between fetal and maternal placental circulations wherein fetal placental blood flow is dependent upon the adjacent maternal flow.

Responses of fetal sheep to simulated no-decompression dives.

The effect of simulated standard no-decompression dives to 60 and 100 ft of seawater was tested in 12 near term sheep carrying 16 fetuses. In the immediate postdive period there were no significant changes in fetal blood pressure or fetal placental or renal blood flow, but the maternal blood pressure was elevated and the maternal placental blood flow was depressed. Six surgically prepared fetuses were dived to 100 ft. Five died within 20 min of ascent and the sixth suffered severe cardiac arrhythmia and hypotension. At autopsy all fetuses were observed to have massive bubbling in the arterial system and heart. Five fetuses were dived to 100 ft without surgery. Two were alive 3 h later and no bubbles were present at autopsy, and three were born alive at term. With the 60-ft dives, three fetuses were subjected to surgery and all suffered massive bubbling. Two fetuses were dived to 60 ft without surgery; one was alive after 3 h and the other was born alive at term. We conclude that surgery and monitoring result in the formation of postdive gas bubbles that would not otherwise appear.