Embryonic Hypoxia Research Articles

Abnormal Myocardial and Coronary Vasculature Development in Experimental Hypoxia

Oxygen availability is one of the necessary prerequisites for normal embryonic development. In our previous study we found that quail embryos incubated under hypoxic conditions (16% O(2)) die at embryonic day (ED) 9 with signs of heart failure. By ED4 and ED6 we found thinner ventricular wall and increased capillary density. We thus hypothesized that the cause of death would lie in severe myocardial and coronary maldevelopment. ED6 and 7 hypoxic hearts had thinner ventricular wall, especially left. There was a simultaneous increase in capillary density, most pronounced in the interventricular septum. This site corresponds to an area of tissue hypoxia and ensuing increased angiogenesis, and also formation of ventricular conduction system. Hypoxia had a positive effect on normal sequence of maturation of the conduction system evaluated by optical mapping at ED7. In sections from ED9 hypoxic hearts we found, in addition to thinner ventricular walls, irregularities in development of coronary tree (missing coronary ostia, absence of one coronary artery, and irregular arterial wall). This deficiency was due to decreased myocyte proliferation rather than to increased apoptosis. By Indian ink injection through the left ventricle we found in normoxic hearts regular coronary branching pattern, while in the hypoxic ones there was often only an irregular plexus. Embryonic hypoxia thus leads to increased capillarity and trabeculation to minimize diffusion distance. In the subsequent period there is a failure in organization of vascular plexus into normal vasculature, resulting in thin compact myocardium that likely leads to heart failure and embryonic death.

Ventilatory chemosensitivity and thermogenesis of the chicken hatchling after embryonic hypercapnia

Hypoxia during incubation results in hatchlings with a reduced thermogenic capacity and a blunted ventilatory (V (E)) chemosensitivity (Szdzuy, K., Mortola, J.P., 2007b. Ventilatory chemosensitivity of the 1-day-old chicken hatchling after embryonic hypoxia. Am. J. Physiol. (Regul. Integr. Comp. Physiol.) 293, R1640-R1649). We asked if similar effects occurred with embryonic hypercapnia, that is, with a non-hypoxic sustained stimulation of the chemoreceptors. White Leghorn chicken eggs were incubated at 38 degrees C either in air (controls, C) or in 4% CO(2) from embryonic day 5 (4% CO(2)), hatching included. The 4% CO(2) embryos hatched about 12h later than C, with similar body weight. On the day of hatching the thermogenic capacity, assessed from the changes in oxygen consumption (.V(O2)) during 1h at 30 degrees C, increased from the early (about 3h old) to the late hours (about 20 h old), and was similar between 4% CO(2) and C. Ventilatory chemosensitivity was evaluated from the changes in (.V(E)) and in ventilatory equivalent (.V(E)/.V(O2)) during acute hypoxia (15 and 10% O(2), 20 min each) or hypercapnia (2 and 4% CO(2), 20 min each). Both at the early and late hours (.V(E)) chemosensitivity was lower in 4% CO(2) than in C. The .V(E)/.V(O2) responses of 4% CO(2) in hypoxia and hypercapnia averaged, respectively, about 45 and 60% of C. A separate set of eggs incubated in 2% CO(2) gave results qualitatively intermediate between C and 4% CO(2). We conclude that prenatal hypercapnia does not compromise the newborn's thermogenesis, but, like hypoxia, affects the development of respiratory control, resulting in a blunted chemosensitivity.

Abnormal myocardial and coronary vasculature development under hypoxia leads to embryonic heart failure and death

Oxygen availability is one of the prerequisites for embryonic development. In our previous study we found that quail embryos incubated under hypoxic conditions (16%O2) die at embryonic day (ED) 9 with signs of heart failure. By ED4 and 6 we found thinner ventricular wall and increased capillary density. We thus hypothesized that the cause would lie in severe myocardial and coronary maldevelopment.ED6 and 7 hypoxic hearts had thinner ventricular wall. There was a simultaneous increase in capillary density, most pronounced in the interventricular septum. This site corresponds to an area of tissue hypoxia and ensuing increased angiogenesis, and also formation of conduction system. Hypoxia had a positive effect on normal sequence of maturation of the conduction evaluated by optical mapping at ED7.In sections from ED9 hypoxic hearts we found irregularities in development of coronary tree. This deficiency was due to decreased myocyte proliferation rather than to increased apoptosis. By Indian ink injection we found in normoxic specimens regular coronary branching, while in the hypoxic ones there was often only an irregular plexus.Embryonic hypoxia thus leads to increased capillarity and trabeculation to minimize diffusion distance. In the subsequent period there is a failure in organization of vascular plexus into normal vasculature, leading to heart failure. Supported by MSMT VZ 206100‐3

The effect of hypoxia in development

There is increasing evidence that the oxygen supply to the human embryo in the first trimester is tightly controlled, suggesting that too much oxygen may interfere with development. The use of hypoxia probes in mammalian embryos during the organogenic period indicates that the embryo is normally in a state of partial hypoxia, and this may be essential to control cardiovascular development, perhaps under the control of hypoxia-inducible factor (HIF). A consequence of this state of partial hypoxia is that disturbances in the oxygen supply can more easily lead to a damaging degree of hypoxia. Experimental mammalian embryos show a surprising degree of resilience to hypoxia, with many organogenic stage embryos able to survive 30-60 min of anoxia. However, in some embryos this degree of hypoxia causes abnormal development, particularly transverse limb reduction defects. These abnormalities are preceded by hemorrhage/edema and tissue necrosis. Other parts of the embryo are also susceptible to this hypoxia-induced damage and include the genital tubercle, the developing nose, the tail, and the central nervous system. Other frequently observed defects in animal models of prenatal hypoxia include cleft lip, maxillary hypoplasia, and heart defects. Animal studies indicate that hypoxic episodes in the first trimester of human pregnancy could occur by temporary constriction of the uterine arteries. This could be a consequence of exposure to cocaine, misoprostol, or severe shock, and there is evidence that these exposures have resulted in hypoxia-related malformations in the human. Exposure to drugs that block the potassium current (IKr) can cause severe slowing and arrhythmia of the mammalian embryonic heart and consequently hypoxia in the embryo. These drugs are highly teratogenic in experimental animals. There is evidence that drugs with IKr blockade as a side effect, for example phenytoin, may cause birth defects in the human by causing periods of embryonic hypoxia. The strongest evidence of hypoxia causing birth defects in the human comes from studies of fetuses lacking hemoglobin (Hb) F. These fetuses are thought to be hypoxic from about the middle of the first trimester and show a range of birth defects, particularly transverse limb reduction defects.

Ventilatory chemosensitivity of the 1-day-old chicken hatchling after embryonic hypoxia

We investigated the effects of sustained embryonic hypoxia on the neonatal ventilatory chemosensitivity. White Leghorn chicken eggs were incubated at 38 degrees C either in 21% O(2) throughout incubation (normoxia, Nx) or in 15% O(2) from embryonic day 5 (hypoxia, Hx), hatching time included. Hx embryos hatched approximately 11 h later than Nx, with similar body weights. Measurements of gaseous metabolism (oxygen consumption, Vo(2)) and pulmonary ventilation (Ve) were conducted either within the first 8 h (early) or later hours (late) of the first posthatching day. In resting conditions, Hx had similar Vo(2) and body temperature (Tb) and slightly higher Ve and ventilatory equivalent (Ve/Vo(2)) than Nx. Ventilatory chemosensitivity was evaluated from the degree of hyperpnea (increase in Ve) and of hyperventilation (increase in Ve/Vo(2)) during acute hypoxia (15 and 10% O(2), 20 min each) and acute hypercapnia (2 and 4% CO(2), 20 min each). The chemosensitivity differed between the early and late hours, and at either time the responses to hypoxia and hypercapnia were less in Hx than in Nx because of a lower increase in Ve and a lower hypoxic hypometabolism. In a second group of Nx and Hx hatchlings, the Ve response to 10% O(2) was tested in the same hatchlings at the early and late hours. The results confirmed the lower hypoxic chemosensitivity of Hx. We conclude that hypoxic incubation affected the development of respiratory control, resulting in a blunted ventilatory chemosensitivity.

Organ growth in chicken embryos during hypoxia: Implications on organ “sparing” and “catch-up growth”

The primary aim of this study was to establish whether or not embryonic hypoxia selectively affects the growth of specific organs. Chicken embryos were incubated either in normoxia (Nx) or in hypoxia (15% O2 from embryonic day E5, Hx). The length of the beak and third toe (as indexes of skeletal growth) and the weights of internal organs (eyes, brain, heart, lungs, liver, kidneys, stomach, and intestines) were collected at E14, E17, E19, and E20. Hypoxia reduced embryonic body weight (BW). At any given age, the specific weight (organ weight/BW) of some organs in Hx was higher, and that of others was lower, than in Nx. However, almost all differences disappeared when organ weights were compared as function of BW, rather than at fixed chronological ages. The important exception was the chorioallantoic membrane (CAM), the mass of which in Hx developed out of proportion. In a third group of embryos, hypoxic until E14 and normoxic thereafter, there was no post-hypoxic catch-up growth, differently from what known to occur postnatally. A possible interpretation is that catch-up growth does not depend on the age of the embryo but on its BW. In conclusion, at least in the chicken embryo and for the level of hypoxia tested, hypoxia has no selective effects on the growth of specific organs, except for the CAM. Qualitative differences in the weight response to hypoxia among organs observed at any given age can be explained largely by the effects of the blunted growth on the growth trajectory of the individual organs.

Role of A1 adenosine receptors in mitigating the effects of hypoxia on embryonic and cardiac development

Hypoxia exposure during embryonic development is associated with intra-uterine growth retardation and is induced by placental insufficiency, maternal smoking, and other factors. One possible mediator of intra-uterine hypoxia effects is the nucleoside adenosine whose levels rise markedly in hypoxia. Adenosine binds A1 adenosine receptors (A1ARs) which are expressed in cardiac and nervous tissue. An in utero model of hypoxia was used to examine acute and long term effects of embryonic hypoxia exposure. When pregnant dams were placed in a chamber to maintain 10% O2 for 6 to 96 hours, developing embryos displayed widespread tissue hypoxia (less than 10 mm of Hg). Hypoxic exposure of wild type embryos as well as A1AR knockout embryos (A1AR+/− and A1AR−/−) resulted in embryonic growth inhibition and a reduction in heart size. A1AR −/− embryos were the most adversely affected including greater growth inhibition, more embryos with severe growth retardation (50% reduction in size), greater percent of reabsorbed embryos, and thinner ventricular walls. One target of A1AR signaling may be hypoxia-inducible factor 1a (HIF1a) since A1AR−/− embryos have much less HIF1a protein after hypoxia compared to A1AR +/− littermates. These results indicate that A1AR activity contributes to the protection of the embryo during hypoxic insults and may mediate this effect through stabilization of HIF1a protein. Supported by NIH (HL58442).

Abnormal coronary tree development in embryonic hypoxia leads to heart failure and embryonic lethality

In our previous studies we found that quail embryos incubated under hypoxic conditions (16 % O2) die at embryonic day (ED) 9 with signs of heart failure. By ED 4 and 6 we found thinner ventricular wall and increased capillary density. We thus hypothesized that the cause of death would lie in severe coronary maldevelopment. We analyzed serial histological sections of normoxic and hypoxic hearts from ED 6, 7, 8, and 9. In ED 6 and 7 hypoxic hearts we found thinner wall in all compartments, especially the left ventricle. Simultaneously we found an increase in capillary density in interventricular septum. This site corresponds to an area of tissue hypoxia and ensuing increased angiogenesis. In sections from ED 9 hypoxic hearts we found irregularities in development of coronary tree (missing coronary ostia, absence of one coronary artery, and thinner arterial wall). By injection of ink through the left ventricle to the coronary arteries we found in normoxic specimens regular coronary branching pattern, while in the hypoxic ones there was only irregular plexus. In conclusion, hypoxia leads to increased capilarization and trabecularization to minimize diffusion distance. In the subsequent period there is a failure in organization of vascular plexus into normal vasculature, leading to heart failure and embryonic death. Supported by grant No. 0021620806 from the Ministry of Education of the Czech Republic.

P99. Effect of Hypoxic Stress on Vertebral Growth Plates

BACKGROUND CONTEXT: An adequate animal model to study the effects of hypoxic stress on vertebral end plates has not been developed. Vascular compromise of the thoracic spine can lead to scoliosis and has been reported in animal models by De Sallis et al. (1981) and congenital malformations due to embryonic hypoxia have been demonstrated in mice by Rivard (1986). We observed from previous studies that photodynamic therapy (PDT), which delivers a hypoxic insult to targeted tissue, can up-regulate VEGF when applied to normal bone.

The Relationship Between Cleft Lip, Maxillary Hypoplasia, Hypoxia and Phenytoin

Cleft lip (CL) is a common malformation that has both genetic and exogenous causes. The main pharmaceutical cause is exposure to phenytoin during early facial development in the 5th to 6th weeks of gestation. Phenytoin also causes CL if administered to pregnant rats during the period of early facial development. Evidence is presented that in the pregnant rat, a teratogenic dose of phenytoin slows the early embryonic heart and causes a prolonged period of embryonic hypoxia. It is proposed that this hypoxia, through an undefined downstream mechanism, leads to the development of CL. The involvement of hypoxia in the pathogenesis of CL is in agreement with studies in mouse strains with a spontaneous rate of CL in which exposure to hypoxia has been shown to increase the rate and hyperoxia to decrease the rate. Other exogenous risk factors during pregnancy for human CL include maternal cigarette smoking, residence at high altitude and exposure to corticosteroids. It is suggested that these exposures all involve an increased risk of embryonic hypoxia. It has been proposed that phenytoin affects the embryonic heart by inhibition of the human-ether-a-go-go (HERG) potassium channel. Phenytoin also inhibits sodium and calcium channels and these properties may also be involved in the observed effect on the embryonic heart. Phenytoin-induced bradycardia leading to embryonic hypoxia may be an important mechanism by which phenytoin causes birth defects.

Phenytoin teratogenicity: Hypoxia marker and effects on embryonic heart rhythm suggest an hERG-related mechanism

The antiepileptic drug phenytoin (PHT) is a human and animal teratogen. The teratogenicity has been linked to PHT-induced embryonic cardiac arrhythmia and hypoxic damage during a period when regulation of embryonic heart rhythm is highly dependent on a specific K(+) ion current (I(Kr)). PHT has been shown to inhibit I(Kr). The aims of this study were to investigate whether teratogenic doses cause embryonic hypoxia during and after the I(Kr) susceptible period and to further characterize PHT effects on embryonic heart rhythm. Pregnant C57BL mice were administered the hypoxia marker pimonidazole followed by PHT or saline (controls) on GD 10 or GD 15. The embryos were fixed and sectioned, and the immunostained sections were analyzed with a computer assisted image analysis. Effects of PHT (0-250 microM) on heart rhythm in GD 10 embryos cultured in vitro were videotaped and then analyzed by using a digitalization technique. PHT dose-dependently increased the hypoxia staining (6- and 11-fold after maternal dosing of 100 and 150 mg/kg, respectively) during the period I(Kr) is expressed and functional (GD 10). In contrast, there were no differences between the PHT doses in hypoxia staining, and much less pronounced hypoxia after this period (GD 15). With increasing PHT concentrations, increased length of the interval (bradycardia) and large variations in length between individual heartbeats (arrhythmia) were recorded. PHT induced bradycardia/arrhythmia and severe embryonic hypoxia during the I(Kr) susceptible period, supporting the idea of an I(Kr)-arrhythmia-hypoxia-related teratogenic mechanism.

Teratogenicity by the hERG potassium channel blocking drug almokalant: use of hypoxia marker gives evidence for a hypoxia-related mechanism mediated via embryonic arrhythmia

The rapid component of the delayed rectifying potassium ion current ( I Kr), plays an important role in cardiac repolarization. In rats, potent I Kr channel blocking drugs cause similar stage-specific malformations (such as orofacial clefts and digital reductions) on gestational days (GDs) 10–14 as after periods of embryonic oxygen deprivation (hypoxia). The idea of a hypoxia-related teratogenic mechanism is supported by studies using rat embryos cultured in vitro. These studies show that the embryonic heart reacts with concentration-dependent bradycardia, arrhythmia, and cardiac arrest when exposed to I Kr blockers on GDs 10–14. The main purpose of this study was to investigate whether previously shown teratogenic doses on GD 11 and 13 of the selective I Kr blocker almokalant (ALM) induce hypoxia in rat embryos in vivo by using the hypoxia marker pimonidazole hydrochloride (PIM). Rats were orally dosed with almokalant or tap water on GD 11 (150 μmol/kg), 13 (50 μmol/kg), or 16 (800 μmol/kg), followed by PIM intravenously 30 min later. Two hours after the PIM dose, the embryonic heart activity was videotaped and analysed, and the embryos were fixed, sectioned, and immunostained. Computer-assisted image analysis showed a two- and threefold increase in hypoxia staining in embryos exposed to teratogenic doses of ALM on GDs 11 and 13. Embryonic arrhythmia was observed in almokalant groups on these GDs, but not in controls. In contrast, dosing on GD 16, with a much higher dose (800 μmol/kg), caused neither hypoxia nor any effects on heart rhythm. The results support the I Kr-related arrhythmia–hypoxia hypothesis, by showing that the potent I Kr-blocking drug, almokalant, (1) causes severe embryonic hypoxia and arrhythmia at stages (GDs 11 and 13) when developmental toxicity could be induced and I Kr is functional and (2) does not cause hypoxia or affect heart rhythm at a developmental stage when I Kr is suppressed (GD 16) and potent I Kr blockers do not induce developmental toxicity.

Possible mechanism of congenital malformations induced by exposure of mouse preimplantation embryos to mitomycin C

ICR mice were treated intraperitoneally with mitomycin C at 5 mg/kg on day 3 of gestation. On day 18 of gestation, fetuses of treated dams were inspected for external, skeletal and visceral malformations. At 6 or 12 hr after mitomycin C treatment, the blastocysts were obtained from the uteri of treated dams and the degenerated cells within inner cell mass (ICM) and trophectoderm (TE) tissues were examined microscopically. On day 5, 8, 11, or 18 of gestation, the uteri of treated dams were obtained and those including embryos/fetuses and placentae were examined histologically. Finally, on each of gestational days 5-14, the blood of the treated dams was collected and the hematological parameters determined. Pre- and postimplantation losses in the dams treated with mitomycin C were significantly increased; increased frequency of abdominal wall defects and lumbar ribs in term fetuses, decreased fetal weight, and increased placental weight were noted as well. No significant increase in visceral malformations was found in term fetuses treated with mitomycin C. Frequency of degenerated cells within ICM and TE of blastocysts from dams treated with mitomycin C was significantly increased as compared with the controls. In dams treated with mitomycin C, decidua developed insufficiently and the trophoblast giant cell layer was not separated from the uterine lumen by maternal components; hemorrhage from the denuded trophoblast giant cell layer into the uterine lumen was noted. The number of erythrocytes, as well as hemoglobin concentration, hematocrit, and the percentage of reticulocytes in blood of dams treated with mitomycin C were significantly lower from days 6-12 of gestation, as compared with controls. The results of the present study showed that an increase in number of degenerated cells within blastocysts results in preimplantation loss and both maternal and embryonic hypoxia during major organogenesis results in postimplantation loss and congenital fetal malformations.

Application of Phonocardiography for Detecting Hypoxia-Induced Cardiovascular Adaptation in the Chicken

Phonocardiography was evaluated as a noninvasive technique for diagnosis of cardiovascular adaptation and disease in broiler chickens. Heart sounds (HSs) were compared in a fast-growing (FG) commercial broiler line that is highly susceptible to chronic right heart failure resulting from pulmonary hypertension syndrome (PHS) and in a non-selected slow-growing (SG) broiler line that is resistant to PHS. HSs were analyzed in broilers reared in hypobaric hypoxia (HYP) and normobaric (CON) conditions. PHS was induced by a combination of embryonic hypoxia and HYP exposure. HSs were recorded with a microphone placed at the thoracic inlet of each chicken. Electrocardiograms were used to mark the sampling interval for the first, second, and total HS. Digitized HS signals were analyzed for peak frequency, mean peak frequency, and band width. Birds were examined for PHS lesions when 6 wk of age, at the end of each experiment. HSs were compared by line and treatment (Experiment 1) or by treatment and week (Experiment 2). In addition, HS frequencies were analyzed within the HYP treatment group for differences between birds with severe or no gross PHS lesions. HS frequencies generally decreased with age and were also lower in the FG than the SG line. Hypobaric exposure decreased all HS frequencies in the SG line and components of the first HSs in the FG line. The SG line did not develop gross lesions of PHS. In the FG line, significant differences in HS frequencies were observed between HYP and CON groups but not between PHS- and PHS+ broilers. Frequency changes described in humans with PHS were not observed. Further development to maximize the resolution of the HS waveforms and improved matching of the sampling interval to the electrical or hemodynamic output of the chicken heart may allow its use as a diagnostic tool for differentiating broilers with normal cardiac function or physiologic adaptation from those with cardiovascular disease.

Effects of hypoxia on embryonic development in two Ambystoma and two Rana species.

Oxygen available to amphibian embryos fluctuates widely and is often very low. We investigated the effects of oxygen partial pressure (1. 3-16.9 kPa) on embryonic development and hatching of two salamander (Ambystoma) and two frog (Rana) species. In Ambystoma, chronic hypoxia resulted in slowed development, delayed hatching, and embryos that were less developed at the time of hatching. Although hypoxia was not lethal to embryos, temporary developmental abnormalities were observed in Ambystoma at oxygen partial pressures of 3.8 kPa and below. Posthatching survival decreased below 3.3 kPa. In Rana, hypoxia did not affect developmental rate, presumably because hatching occurs at a very early stage of development relative to Ambystoma. However, Rana embryos hatched sooner in hypoxia than in normoxia, resulting in less developed embryos at the time of hatching. The results suggest that embryonic hypoxia may negatively affect survival and fitness in these species.

Prenatal toxicity studies in rats and rabbits with the calcium channel blocker diproteverine

In prenatal toxicity studies, diproteverine, a calcium channel blocker with demonstrated antianginal properties, produced an unusual pattern of digital, heart, tail, and vertebral defects in rat fetuses from mothers treated during the major period of organogenesis, but only a very low incidence of heart abnormalities was seen in the rabbit. Heart changes were rarely seen in association with digital defects. The findings were consistent with those seen with other calcium channel blockers and add weight to the suggestion of Danielsson and colleagues (5) that digital malformations are a class effect for this type of compound, the effects being related to reduced uteroplacental blood flow. In addition, it is proposed that cardiovascular malformations are also a class response with calcium channel blockers. The distribution of fetal death and hemorrhages and the varying association between cardiovascular, digital, and tail abnormalities seen in the rat with increasing doses of diproteverine fits the pattern of changes reported following hypoxia in the chick embryo. Reduced uteroplacental blood flow with resultant embryonic hypoxia secondary to pharmacologic action is considered a probable mechanism of action for the induction of abnormalities produced by calcium channel blockers.

Water Vapor and Oxygen Exchange of Praying Mantis (Tenodera aridifolia sinensis) Egg Cases

Mantid egg cases must be capable of preventing desiccation of the developing eggs for 5-6 mo while allowing adequate respiratory gas exchange for timely hatching. This investigation examined water vapor, oxygen, and thermal exchange of Chinese mantis (Tenodera aridifolia sinensis) egg cases. Water vapor conductance was 0.277 mg · d⁻¹ · mmHg⁻¹. Results of experiments with altered vapor pressure gradients, moving air, and hypobaria were consistent with water vapor loss by simple gaseous diffusion. Experiments at 100% relative humidity showed a linear increase in the internal egg case temperature with development, peaking more than 0.3° C above ambient temperature before hatching. Egg case O₂ consumption increased with increasing hatchling mass. Oxygen consumption at 25° C increased linearly with incubation time and for a case producing 1 g of hatchlings peaked at 537.5 μL · h⁻¹. The Q10's for O₂ consumption between 15° and 25° C were typical of insects. Calculations using diffusive gas exchange equations indicate the high resistance to water vapor flux does not result in embryonic hypoxia.