Cardiovascular Research

Cardiovascular Research 1997 34(3):525-528; doi:10.1016/S0008-6363(97)00065-5
© 1997 by European Society of Cardiology

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Copyright © 1997, European Society of Cardiology

Cardiac output distribution in the chick embryo from stage 36 to 45

Twan L.M. Mulder^a,*, Jolanda C. van Golde^a, Frits W. Prinzen^b and Carlos E. Blanco^a

^aDepartment of Pediatrics, University Hospital Maastricht, Maastricht, Netherlands
^bDepartment of Physiology, Maastricht University, Maastricht, Netherlands

^* Corresponding author. Department of Pediatrics–Neonatology, University Hospital Maastricht, P.O. Box 5800, 6202 AZ Maastricht, Netherlands. Tel.: +31 (43) 3877249; fax: +31 (43) 3875246; e-mail: amul@skin.azm.nl

Received 12 December 1996; accepted 5 February 1997

	Abstract

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Objective: The distribution of cardiac output to different organs is well described in the mammalian fetus. Chick embryos are not often used in perinatal cardiovascular research and therefore it is not known whether they can serve as an animal model for this purpose. In this study we documented cardiac output distribution in chick embryos at increasing incubation time. Methods: Fertilized eggs from day 10 to 19 with an incubation time of 21 days were studied in 3 increasing incubation time groups (10–13, 14–16 and 17–19 days). For the experiment, the egg was placed in a holder in an incubator. The egg was opened at the air cell and a small vein of the chorioallantoic membrane was catheterized. Twenty thousand fluorescent 15 µm microspheres in 0.2 ml were injected. After 5 min, the embryo was sacrificed and the different organs were dissected and digested for microsphere isolation and subsequent fluorescence analysis. Results: The chorioallantoic membrane, which is the placenta equivalent of the chick embryo, received a relatively large fraction of the combined cardiac output: 52.08% (interquartile range [IQR] 12.67%) on days 10–13 and 40.95% (IQR 27.24%) on days 17–19. Relatively small fractions were distributed: to the heart 2.03% (IQR 1.58) on days 10–13 and 3.18% (IQR 1.95) on days 17–19, and to the brain 3.20% (IQR 1.80) on days 10–13 and 5.02% (IQR 3.39) on days 17–19. As incubation time advanced, the fraction of the combined cardiac output to the chorioallantoic membrane and yolk-sac decreased significantly in favor of the heart and brain. Conclusion: This distribution shows great similarity to the one found in the mammalian fetus. The chick embryo is an attractive model for perinatal cardiovascular research.

KEYWORDS Development; Microspheres, fluorescent; Circulation; Chicken, embryo

	1 Introduction

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
A fetus needs large amounts of oxygen and nutrients for development and growth. The distribution of oxygen and nutrients to the fetal organs is dependent on gas exchange area, transport vehicle and cardiac output. In the mammalian fetus, a large fraction of the cardiac output is directed to the placenta, where gas exchange and nutrients are provided. The distribution of fetal cardiac output (CO) has been studied in sheep [1, 2], primate [3] and llama fetuses [4] using radioactive microspheres, both under physiological conditions and abnormal events associated with birth (e.g., asphyxia due to umbilical cord occlusion or hypoxia) [5]. The early chick embryo in particular is a well-known animal model for cardiovascular research with regard to cardiac development [6], angiogenesis [7], cardiovascular pharmacology and toxicology [8, 9] and cardiovascular mechanics [10]. With regard to cardiovascular responses in the late chick embryo (e.g., cardiovascular responses to perinatal events) not much is known.

The chick embryo is not mammalian and the main difference is the absence of placenta. In the chick embryo, gas exchange takes place in the chorioallantoic membrane (CAM). This is a well-vascularized membrane attached to the shell of the egg. This membrane, which is in direct contact with the environmental air through micropores in the shell, is the vehicle for gas exchange. Therefore, the CAM can be seen as a placenta equivalent [11]. The embryonic circulation in the CAM greatly resembles the umbilical cord circulation in the mammalian fetus [12]. Furthermore, the fetal shunts over ductus venosus, foramen ovale and ductus arteriosus are also present in the chick embryo. The distribution of the CO in the chick embryo was summarily documented by Rahn, Matalon and Sotherland using radio-active microspheres [13]. They reported on the distribution for CAM, yolk-sac and total embryo in 18 chick embryos from day 17–19 incubation time.

For several years now, fluorescent microspheres have been used to determine organ blood flow and CO distribution as an alternative to radioactive microspheres [14]. As far as we know, this method has not been used in chick embryos. In this study we used fluorescent microspheres, injected into a chorioallantoic vein, to determine the CO distribution in the chick embryo.

The aim of this study was: (a) to test the possibility of determining CO distribution in the chick embryo using fluorescent microspheres; (b) to examine the changes in CO distribution in the chick embryo during development; and (c) to evaluate whether the chick embryo is a suitable model for perinatal cardiovascular research.

	2 Methods

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Fertilized eggs of White Leghorns were incubated in a commercial incubator. The eggs were rotated constantly to avoid adhesions between the embryo and its membranes [15]. Incubation time until hatching for these eggs is 21 days. Since we wanted to study developmental changes we used chick embryos ranging from day 10 to day 19 of incubation time, corresponding to stages 36–45 according to Hamburger and Hamilton [16]. Three different incubation time groups were studied: 10–13, 14–16 and 17–19 days.

2.1 Preparation
To determine the distribution of the CO, a chorioallantoic vein was catheterized to inject fluorescent microspheres. Both catheterization of the chorioallantoic vein and the injection of fluorescent microspheres were performed in a clinical infant incubator, provided with a light microscope (WILD 3M, magnification 10x). Temperature and humidity were maintained constant at 38°C and 60%, respectively. The eggs were candled to identify the air cell and the shell was opened with an electrical saw above the level of the CAM. After opening, the egg was placed in a holder under the microscope. Now the outer shell membrane was visible. By moistening it with a NaCl 0.9% solution, the vessels of the CAM became visible. Without damaging the chorioallantoic membrane vessels, the outer shell membrane was removed. A chorioallantoic vein of approximately the same diameter was identified through its bright red color and erythrocyte stream direction. The vein was lifted using sutures, opened with the hooked tip of a 30-Gauche needle and a polyethylene catheter, which was stretched by heat to a diameter of 100 µm, was inserted. The catheter, flushed with a heparin solution (10 IU/ml), was connected to a 1.0 ml syringe and fixed to the eggshell with clay.

2.2 Protocol
Five minutes after catheterization, 20 000 fluorescent microspheres (Fluospheres^® Molecular Probes Inc., Eugene, OR, USA) with a diameter of 15 µm (0.2 ml of a 100 000 microspheres/ml 0.05% Tween 80 solution) were injected in 1 minute. The dye used was orange. After 5 min, the chick embryos were decapitated and the chorioallantoic membrane, brain, heart, lungs, intestine, liver and yolk-sac were dissected.

2.3 Method of measurement
We determined fluorescence in whole organs and in the remaining carcass. The tissues were placed in test tubes and digested in a 2M ethanolic-KOH solution for 48 h at 60°C. The microspheres were isolated from the homogenate by repeated centrifugation, removal of the supernatant and rinsing as described by van Oosterhout [17]. In the final pellet the dye was extracted by adding 3 ml of 2-(2-ethoxyethoxy)ethylacetate and fluorescence was counted by fluorimetry using a LS-50B fluori-spectrometer (Perkin Elmer). Since during fluorimetry all samples had the same volume (3 ml), the absolute fluorescence measured, corrected for background, expressed the fraction of CO that the tissue received.

2.4 Analysis of data
All data were processed using SPSS statistical software. Since not all data obtained were normally distributed, we found it appropriate to express the data as median with interquartile range (p25–p75). A non-parametric sample test (Mann-Whitney U) was applied to compare the CO distribution within the 3 different groups at the different incubation times. Statistical significance was defined as P<0.05.

	3 Results

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
The placement of the catheter into the chorioallantoic vein required skill and practice.

The major problems encountered were: (a) difficulty in inserting the catheter into the vessel lumen and (b) bleeding due to damage to the vessel. Our present success rate is about 80%.

We determined the CO distribution in a total of 72 chick embryos at different incubation times: at days 10–13 (n=22), at days 14–16 (n=24) and at days 17–19 (n=26). None of the instrumented chick embryos showed pipping or hatching.

In the chick embryo, a large fraction of the CO (median range for the three groups 41–52%) was directed to the CAM. Relatively small fractions were directed to the heart (median range 2.0–3.8%), brain (median range 3.2–5.0%), lungs (median range 0.5–1.0%), intestine (median range 2.0–3.8%), and liver (median range 1.5–3.3%). The fraction to the yolk-sac in the 10–13 day group is substantial (median 11.9%), but decreases rapidly with increasing incubation time (P<0.01) (Fig. 1).

View larger version (32K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Cardiac output distribution for increasing incubation time. Bars represent the median, the range is the interquartile range (p25–p75). *Significant change (P<0.05) compared to 10–13 day group. Significant change (P<0.01) compared to 10–13 day group. Significant change compared to 14–16 day group (P<0.05).

 
The CO distribution to the various organs changed with increasing incubation time. The fractions to the heart, brain, intestine and carcass increased significantly (P<0.05) with increasing incubation time, whereas the fractions to CAM and yolk-sac decreased (Fig. 1).

	4 Discussion

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
In this study, we determined the CO distribution of the chick embryo in the second half of incubation time. Early in this period, there were two vascular beds that received the most important percentage of the combined CO: the CAM and the yolk-sac, both important for gas exchange and nutrition. However, later in incubation time, these percentages, although remaining important, decreased in favour of other organs and tissue.

Oxygen and carbon dioxide concentrations in the air cell of the egg change during incubation time. Oxygen concentration falls from 21% at day 4 to 12% at day 20 and carbon dioxide concentration increases from 0.6% at day 4 to 6% at day 20 [18]. Therefore, the opening of the egg's air cell could have had consequences for the embryonic blood gases, with a subsequent effect on CO distribution. In a previous study we have shown that pO₂, pCO₂ and pH did not significantly change after opening of the air cell [19].

Rudolph and Heymann introduced the radioactive microsphere technique for the study of circulation in the fetal lamb [20]. This technique is now regarded as the gold standard for determination of the CO, CO distribution and organ blood flow. Recently, the fluorescent microsphere technique was introduced and validated, using the same principles as Rudolph and Heymann. The accuracy is similar to that of the radio-active microsphere method [17, 21]. This method presents the following advantages: it does not require radioactivity and when being used in conjunction with histological techniques, offers the possibility of examining regional distributions within organs. At present, microsphere isolation and analysis are relatively time-consuming [17], but new techniques will be available in the near future. In our study, we chose to use fluorescent microspheres to determine CO distribution and we can now report that it is suitable for use in chick embryos; injections are possible thanks to downsizing the catheter, and isolation of the microspheres and extraction of the dye are possible from chick embryo tissues.

Due to difficult access to the chick embryo and its limited blood volume, it was impossible to take a reference blood sample simultaneously with injection of the microspheres. It was also impossible to place a flowprobe and catheterize the same embryo simultaneously. Therefore, we cannot present the absolute organ flow values of individual animals. In a previous study we measured absolute blood flow to the CAM in chick embryos from day 10 to day 16 of incubation using a Transonic flowprobe [19], and since from the present data we know the fraction of the CO to the CAM, we can estimate the mean combined cardiac output of the chick embryo from day 10–16. We calculated a mean combined CO of 472 ml/kg·min (range 330–582), which did not change with advancing incubation time. This is similar to the late-gestation mammalian fetus [2].

The distribution of the CO in the chick embryo (16–19 day group) resembles the CO distribution in the near-term sheep fetus [2]. In the chick embryo the fractions of the CO directed to the heart, brain, intestine and carcass are similar to the fractions found in the sheep fetus (heart 3 vs. 3%; brain 5 vs. 3%; intestine 6 vs. 6%; carcass 39 vs. 30%). Also the fraction to the CAM (41%) is similar to the fraction to the placenta (44%) in the sheep fetus. The fractions to the lungs differ. In the chick embryo the lungs receive 1.0% of the CO while in the sheep fetus the lungs receive 11.81%.

We had expected to observe a higher fraction of the CO to the lungs in the older embryos since internal pipping and air breathing in the chick embryo starts at around day 20. However, when opening the air cell in our experiments no internal pipping was seen.

With increasing incubation time, we observed a change in CO distribution. The fractions to the heart, brain, liver, intestine and carcass increased significantly (P<0.05). This could be explained by: (a) a relatively lower resistance of the vascular beds in these organs—a lower vascular resistance can be the result of organ growth with subsequent growth of the vascular bed [1]; (b) the result of an increase in metabolic activity of these organs, demanding a greater fraction of CO controlled by local vaso-active substances producing vasodilatation [1]; and (c) an increase in vascular resistance in the vascular bed of the CAM and yolk-sac.

Recently Hu observed in early chick embryos that the CO distribution between the embryo and the extraembryonic vascular bed changed from 18.7 versus 81.3% at stage 18 to 34.2 versus 65.8% at stage 24 [22]. Our data showed a further shift from 36 versus 64% at days 10–13 (stages 36–39) to 57 versus 43% at days 17–19 of incubation (stages 43–45). This indicates that during development of the chick embryo the fraction of the CO directed to the embryo is continuously increasing. These developmental changes were also documented in fetal sheep by Rudolph and Heymann [1]. They demonstrated in fetal lambs from 0.6 gestational age to term a significant increase in the fraction of the CO to the lungs, intestine, and brain at the expense of the placenta.

Most perinatal cardiovascular studies are performed on sheep because of the following advantages: (a) its size facilitates catheterization of fetus and (b) manipulation of its uterus does not readily trigger labor [23]. However, there also are some disadvantages: (a) the ewe has a long gestation (150 days); (b) it is expensive; (c) instrumentation of the fetal lamb in utero can be difficult with some failure rate; and (d) it is always necessary to operate 2 animals: the fetus and the mother animal. Furthermore, mammals present a close interaction between the fetus and the mother. Maternal factors can affect the fetus. The chick embryo is cheap, easily available and independent of its mother, providing an attractive model to study responses without any influence from maternal factors.

In summary, the CO distribution in the chick embryo as determined in this study shows a close resemblance to that of the mammalian fetus. Therefore, chick embryos could be considered as a feasible model for further perinatal cardiovascular research. Furthermore, the fluorescent microspheres technique is appropriate to determine CO distribution. This study provides basic cardiovascular data, allowing for the design of future studies that will help in understanding both the consequences of hypoxia or asphyxia and their underlying mechanisms during fetal cardiovascular development.

Time for primary review 26 days.

	Acknowledgements

 
The authors thank Johan Hekking, Department of Anatomy-Embryology, for his assistance in instrumenting the chick embryo and Anita Rousseau, Department of Physiology, for her assistance in analyzing the fluorescence.

	References

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 

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