Chorioallantoic Capillaries Research Articles

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.

Regional differences in diffusive conductance/perfusion ratio in the shell of the hen's egg

Are both gas exchange and gas tensions uniform in different regions of the developing hen's egg? To answer this question we measured the O 2 uptake and CO 2 production of the whole egg, and at the same time the O 2 and CO 2 tensions of the air cell. The gas exchange ratio (R) of the whole egg differed from R calculated from air cell P O 2 and P CO 2 values, in agreement with the findings of Visschedijk [Br. Poultry Sci. 9: 173–184 (1968)], who measured gas exchange separately over both the air cell region and the remainder of the egg. We constructed a diffusive shell conductance/perfusion (G/Q̇) line on the O 2-CO 2 diagram from a blood nomogram for the chick embryo in late development [Olszowka et al., Fed. Proc. 46: 512 (1987)], and used this to analyze our results. The G/Q̇ ratio for the area of shell over the air cell differs from that for the remainder of the egg. Our analysis permits us to calculate, for each area, the regional shell conductance, blood flow, and O 2 and CO 2 tensions in the gas spaces between the shell and the chorioallantoic capillaries.

The analysis of PO2 difference between air space and arterialized blood in chicken eggs with respect to widely altered shell conductance.

The gas exchange of chicken eggs takes place by molecular diffusion. The diffusion barrier between ambient atmosphere and erythrocyte hemoglobin of the gas exchanger (the vascularized chorioallantoic membrane) is conveniently divided into two parts by the air space in the fibrous shell membranes; i.e., the outer barrier (mainly the porous eggshell) and the inner barrier (the chorioallantoic membrane and the chemical reaction with hemoglobin). In contrast to the alveolar-arterial Po2 difference in vertebrate lungs, the difference of Po2 between the air space and the arterialized blood in the allantoic vein (delta PAo2.Pao2) is large in chick embryos. The present study analyzed the delta PAo2.Pao2 in relation to the diffusing capacity of the chorioallantoic membrane (inner barrier) and physiological shunt in the allantoic circulation with respect to widely altered diffusive conductance of the shell (outer barrier). The shell diffusive conductance (Go2) was altered of the beginning of incubation, and the O2 consumption (Mo2) was measured on day 16. The Mo2 increased hyperbolically with increasing Go2, reached a maximum at control values of Go2 and decreased with further increases in Go2. From Go2 and Mo2, the air space Po2 was determined. The delta PAo2.Pao2 was increased in eggs with augmented Go2 (from about 50 torr in control eggs to 70 torr in conductance-increased eggs). The diffusing capacity and allantoic shunt which produce a given delta PAo2.Pao2 were estimated employing a microcomputer performing the Bohr integration procedure so that a calculated Pao2 agreed with the measured Pao2. The allantoic shunt is not more than 20%; 10% is likely. The diffusing capacity becomes maximum in intact control eggs and is decreased at both lowered and augmented Go2. At lowered Go2, diffusion limitation is responsible for about 90% of delta Pao2.Pao2 even in the presence of a 10% shunt. The diffusion limitation to blood oxygenation decreases as Go2 increases, but it is still predominant at augmented Go2. In control eggs, the resistance of the inner barrier to O2 diffusion is about 1.7-fold that of the shell (outer barrier) which agrees with the previous reports.

Estimation of the transfer coefficients of oxygen and carbon monoxide in the boundary of human and chicken red blood cells by a microphotometric method.

The reaction rates of O2 and CO with the human and chicken red blood cell (RBC) were measured by using a microphotometric apparatus. In the experiments on the human RBC, a small amount of RBCs were put in an air-tight reaction cuvette. Gas mixtures containing various concentrations of O2 and CO were sequentially injected into the cuvette and the change in O2 and CO saturation of hemoglobin was measured from the change in transmission of the RBCs at 402 and 416.5 nm. The reaction rate of CO with RBCs was significantly influenced by photodissociation of carboxyhemoglobin (COHb). To eliminate this, a short-pass filter (400 to 435 nm) and a sector (100 Hz) were used. By comparing the measured reaction rates of O2 and CO with the theoretical rates obtained from the numerical solutions of the partial differential equations of the diffusions of O2 and CO, the transfer coefficients of O2 and CO (eta O2 and eta CO) in the RBC boundary, including the RBC membrane and water layer around the RBC, were estimated. Both the values showed good agreement, ranging from 0.3 to 2.5 x 10(-6) cm.sec-1.Torr-1. Furthermore, the chorioallantoic capillary of chicken embryo was used for the measurements of the reaction rates of O2 and CO with RBC through the capillary membrane. The reaction rates of O2 and CO in the chorioallantoic capillary were slower than those obtained in the human RBC. By comparing the measured reaction rates and the numerical solutions, the eta O2 and eta CO in the boundary, including the capillary membrane, plasma, and RBC membrane, were estimated. These two values ranged from 0.1 to 0.4 x 10(-6) cm.sec-1.Torr-1 and showed good agreement. These results suggest that the diffusion rates for O2 and CO across the capillary and RBC membrane are similar.

Oxygen availability and growth of the chick embryo

Two experiments were performed using White Leghorn eggs incubated in 50% humidity, at 38° centigrade, and an average barometric pressure 747 Torr. In one experiment, eggs in the experimental group were each half covered with a neoprene membrane, reducing the diffusing capacity by approximately 20%, and incubated in 21% O 2. In the second experiment, the experimental eggs were incubated (uncovered) in 60% O 2. Control eggs were incubated (uncovered) in 21% O 2 simultaneously with the experimental eggs in each study. A relationship between egg surface area (calculated from weight of the freshly laid egg) and embryo weight at 18 days was confirmed for all three groups. When comparisons were made among the groups, there was significant retardation of embryonic growth in the half-covered eggs; in contrast, embryos from eggs incubated in 60% O 2 were significantly heavier at 18 days than control eggs incubated in air. The fact that embryo growth can be accelerated by incubation of the egg in 60% O 2 suggests that the oxygen tension in blood leaving the chorioallantoic capillaries normally limits the rate of embryonic growth for the first 18 days of incubation in this species.

Effect of O2 and CO2 in N2, He, and SF6 on chick embryo blood pressure and heart rate.

Arterial pressure of chick embryos was measured electromanometrically to investigate the effect of altered gaseous environments on blood pressure (BP) and heart rate (HR). The experiments were made in eggs incubated for 14-16 days at 38 degrees C without impeding the diffusive respiratory gas exchange through the shell and chorioallantois. In air, the HR was counted 260-270 beats/min and the BP increased from 14/7 Torr at day 14 to 21/12 Torr at day 16. Both the BP and HR decreased with hypoxia, whereas hyperoxia affected a slight increase in BP and little change in HR. Hypercapnia decreased the HR and tended to enhance a systolic maximum pressure. The effect of hypoxia was augmented markedly in the presence of hypercapnia and vice versa. When N2 was replaced with helium (He), the effect of hypoxia was mitigated significantly. On the contrary, replacement of N2 with sulfur hexafluoride (SF6) augmented the effect of hypoxia. Because the respiratory gas exchange of the egg takes place by diffusion through the shell and chorioallantoic capillaries, the effect of He and SF6 atmospheres on BP and HR is attributed to an altered diffusivity of O2 and CO2 in these inert gases.

The effect of deoxygenation rate of the erythrocyte on oxygen transport to the cardiac muscle.

Up to the present time O2 delivery to the cardiac muscle has been studied theoretically by many authors (1, 2, 11, 12) using a Krogh’s cylinder model. However, relatively little attention has been paid to the influence of deoxygenation of red blood cells (RBC) on the O2 delivery. Mochizuki (8) measured the O2 dissociation rate of oxygenated hemoglobin and RBC by using a rapid flow method, and found that the deoxygenation rate of RBC was proportional to the PO2 difference between RBC and surrounding medium, suggesting that the diffusion inside RBC and across the cell membrane was a rate limiting factor. The rate factor, Fc’, which is given by dividing the O2 quantity taken up by 1 ml RBC by the PO2 difference between RBC and surrounding medium, was 0.02 – 0.03 sec-1•mmHg-1. Recently, Tazawa et al (10) reported that the deoxygenation rate of RBC in the chorioallantoic capillary of chick embryos ranged from 0.008 to 0.009 sec-1•mmHg-1. In contrast to our observations, Lawson and Forster (5) previously described that the PO2 difference between RBC and plasma in tissue was negligibly small, when the rate factor measured by them in a RBC suspension mixed with hydrosulfite (4) was used.

Oxygen analyses of chicken embryo blood

According to data obtained previously on the blood gas tensions and the oxygen dissociation curve of chicken embryos, the arteriovenous oxygen saturation difference in the allantoic circulation has been conjectured fairly large. In order to confirm this conjecture as well as to check the validity of the in vitro dissociation curve, both the blood oxygen capacity and content in allantoic artery and vein were measured. The in vivo O 2 saturation measured here resulted in a similar value to that estimated from the dissociation curve. The O 2 content in allantoic vein ranges from about 7 to 11.5 vol % during the 10th to the 18th days of incubation and that in artery is pronouncedly low in a range of 1 to 2.5 vol%, suggesting that the blood flow rate through the body tissues is fairly larger than that through the gas exchange capillary plexus. Then, the distribution of blood flow was estimated from the analyzed data based on a model of blood circulation and some assumptions. In connection with this estimation, the diffusing capacity for deoxygenation in the tissues was speculated to be much larger than that for oxygenation in the chorioallantoic capillaries.

Estimation of contact time and diffusing capacity for oxygen in the chorioallantoic vascular plexus

The contact time of erythrocyte in the chorioallantoic capillaries of chicken embryos was estimated by referring to the oxygénation rate measured with a microphotometer. The chorioallantoic membrane was excised from an incubated egg and the S O2 of blood in it's capillary was changed by varying the gas composition around the membrane from the venous blood P o2 level to the air space gas. The oxygénation time (te) required to attain the level of arterialized blood So o2 was measured as the contact time (tc) in the capillaries, which resulted in 0.87, 0.74, 0.57, 0.49 and 0.36 sec for 10, 12, 14, 16 and 18 days of incubation, respectively. The obtained te value coincided with the contact time calculated from the CO diffusing capacity referring to the reaction rate of CO with oxygenated erythrocyte. Using the te value, the diffusing capacity for 0 2 in the chorioallantoic capillary was calculated; 1.1, 2.2, 4.4, 6.0 and 6.6 x 10 −3 ml min −1 mm Hg −1 for the same incubation days as above. The capillary blood volume (Vc) was also estimated, which increased from about 16 to 35 μd during development from 10 to 18 days. The values of D o2 and Vc converted per kg weight of embryo at the days near hatching were similar to those per kg body weight estimated in human lung

Oxygenation and deoxygenation velocity factors of chorioallantoic capillary blood.

The oxygenation and deoxygenation rates of capillary blood of chicken embryo were measured precisely with a microphotometric reaction apparatus. The velocity factors expressed as Fcox and Fcdeox in ml O2-ml RBC-1-S-1-mmHg-1 were calculated as functions of oxygen saturation. They were similar to results previously obtained in human blood using the rapid flow apparatus. The continuous registration of the reaction with oxygen enables us to estimate the equilibrium time of blood when passing through the chorioallantoic capillary. The value obtained was almost identical to the contact time indirectly assessed from the CO reaction. The diffusing capacity of the chorioallantoic capillary plexus of the 16-day-old embryos was estimated as 7.3 x 10(-3) ml-min-1mmHg-1 by using the values of the Fcox and the equilibrium time determined here together with the values of the blood flow and hematocrit which had been obtained in the previous experiments.

Oxygen dissociation curve for chorioallantoic capillary blood of chicken embryo.

Oxygen dissociation curves for blood in the chorioallantoic capillary of chicken embryos were determined using a microphotometric apparatus made for measuring the reaction velocity of a red blood cell with oxygen and carbon monoxide. The modified Hill's equations expressing the dissociation curve during development were calculated by two methods. P50's at pH of 7.4 were found to be 60.0, 54.4, 46.2, 33.1, and 28.6 mmHg for 10, 12, 14, 16 and 18 days of incubation, respectively. Although the Bohr factor did not show a clear relation to age, the oxygen affinity and the oxygen capacity tended to increase with the lapse of days, and the power of heme-to-heme interaction, to decrease with age. The findings imply that there is a respiratory adaptation of embryos during development.

Measurement of oxygenation and deoxygenation of a single red cell of chicken embryo by means of a microphotometer.

In order to overcome difficulties inherent in the flow reaction apparatus so far used for kinetic studies of red cells, we have developed a technique of microphotometry. Since in a microphotometer a small light beam of about 10 u is transmitted through a single red cell, the reactions with O2 and CO can be measured by flushing a reacting gas onto the red cells. In the previous studies (1,2), the method of reaction measurement was elaborated and the CO reaction was determined in the red cells existing in the chorioallantoic capillary of chicken embryo. Since then, by using the same method and material, we have measured the oxygenation and deoxygenation rates and examined the velocity of combination and dissociation of O2 due to the Bohr effect.

Reaction velocity of carbon monoxide with blood cells in the chorioallantoic vascular plexus of chicken embryos

A velocity factor (Fc: ml CO −1 red cells-sec −1mm Hg −1) representing a reaction rate of CO with red blood cells in the chorioallantoic capillary of chicken embryos was determined using a microphotometer reaction apparatus. The chorioallantoic membrane was excised from an incubated egg together with the shell membranes, and after placement on a slide in the reaction cuvette the shell membranes were peeled off. The embryos were of 10, 12, 14, 16 and 18 of incubation. pco in the gas mixture ranged from O.7 to 4.5 mm Hg and P O 2 , varied from 46 to 355 mm Hg. The Fc values were almost constant for each incubation day and independent of the P CO level, but gradually decreased during development. Increases in P O 2 , in the CO gas mixture slowed down the reaction rate and the relation of Fc to P O 2 , was empirically expressed as Fc=2,31/(P O 2 +232). A contact time was estimated as approximately O.5 sec by referring to the Fc and the other data.

Microscopic observation of the chorioallantoic capillary bed of chicken embryos

Using a method of rapid freezing and freeze-drying, the chorioallantoic vascular plexus which plays an important role in embryonic gas exchange was investigated microscopically in connection with a microphotometric method for measuring reaction rates of O 2 or CO with the red cells in the chorioallantoic capillaries. The capillary bed forms a thin layer on the outer surface of the chorioallantoic membrane and has a fine reticulate formation, unlike that of the mammalian alveolar capillary. Length of the capillary between bifurcations is approximately the size of the blood cell. Therefore, the cells flowing zigzag through the capillaries are deformed within its walls. The number of blood cells in the capillary bed increases with the progress of incubation. These observations may relate to the daily increase in diffusing capacity in embryonic gas exchange and somewhat effect the reaction rate.

Hypothermal effect on the gas exchange in chicken embryo

Hypothermal effects on metabolic rate and blood gas properties were studied in chicken embryos. Hypothermia was applied in two manners; 1) the eggs were acutely exposed to 30 °C for 2 hr before measurement and 2) the incubation temperature was lowered to 35.5 °C from the beginning. V̇ O2 and V CO2 were notably decreased by both hypothennal procedures, but retardation of embryonic development V̇ CO 2 were notably decreased by both hypothermal procedures, but retardation of embryonic develepment was predominantly significant only in embryos incubated at 35.5°C. The retardation in development in the latter case seemed to be caused by the long-lasting decrease in metabolic rate. In other words, metabolic rate and blood gas properties were comparable with those of the control embryos (incubated at 38°C) whose development stage was similar to that of the embryos incubated at 35.5°C. On the other hand, acute hypothermia made by exposure to 30°C caused the increase in P O2, notable decrease in P CO2 and increase in pH of arterialized blood and mixed venous blood. These changes might be attributed to the marked decrease in metabolic rate and the unaltered facility of the gas exchange in the chorioallantoic capillaries during the acute hypothennal period.