Chicken embryos, as a classical developmental model, are used in our lab to assess developmental cardiotoxicities following exposure to various environmental contaminants. Exposure methods and morphological/functional assessment methods established are described in this manuscript.
Chicken embryos are a classical model in developmental studies. During the development of chicken embryos, the time window of heart development is well-defined, and it is relatively easy to achieve precise and timely exposure via multiple methods. Moreover, the process of heart development in chicken embryos is similar to mammals, also resulting in a four-chambered heart, making it a valuable alternative model in the assessment of developmental cardiotoxicities. In our lab, the chicken embryo model is routinely used in the assessment of developmental cardiotoxicities following exposure to various environmental pollutants, including per- and polyfluoroalkyl substances (PFAS), particulate matter (PMs), diesel exhaust (DE) and nano materials. The exposure time can be freely selected based on the need, from the beginning of development (embryonic day 0, ED0) all the way to the day prior to hatch. The major exposure methods include air-cell injection, direct microinjection, and air-cell inhalation (originally developed in our lab), and the currently available endpoints include cardiac function (electrocardiography), morphology (histological assessments) and molecular biological assessments (immunohistochemistry, qRT-PCR, western blotting, etc.). Of course, the chicken embryo model has its own limitations, such as limited availability of antibodies. Nevertheless, with more laboratories starting to utilize this model, it can be used to make significant contributions to the study of developmental cardiotoxicities.
The chicken embryo is a classic developmental model, which has been used for over two hundred years1. The chicken embryo model has various advantages compared to traditional models. First of all, as early as over 70 years ago, the normal development of the chicken embryo had been illustrated very clearly in the Hamburger-Hamilton staging guide2, in which a total of 46 stages during chicken embryo development were defined with precise time and morphological characteristics, facilitating detections of abnormal development. Additionally, the chicken embryo model has other features such as being relatively low-cost and redundant in quantity, relatively accurate exposure-dose controls, an independent, closed system within the shell and easy manipulation of the developing embryo, all of which guarantees its potential to be used as a powerful toxicological assessment model.
In cardiotoxicity, the chicken embryo features a four chambered heart, similar to mammalian hearts but with thicker walls, allowing easier morphological assessments. Additionally, the chicken embryo allows for developmental inhalation exposure, which is not possible in mammalian models: during the later stage of development, the chicken embryo will transition from internal respiration to external respiration (getting oxygen via the lung); the latter requires that the embryo penetrates the air cell membrane with the beak, and starts to breathe air3, making the air cell a mini-inhalation chamber. Utilizing this phenomenon, the toxicological effects of gas contaminants on the heart (and other organs) may be assessed without the need of dedicated inhalation chamber instruments.
In this manuscript, several exposure/endpoint assessment methods are described, all of which serve to make the chicken embryo a powerful tool in the assessment of development cardiotoxicity following exposure to environmental contaminants.
All procedures described were approved by the Institutional Animal Care and Use Committee (IACUC) of Qingdao University. In our lab, the eggs were incubated in two incubators. Eggs were held upright in the incubator and randomly placed on the shelves. The incubation conditions for the eggs were as follows: incubation temperature started at 37.9 °C, and gradually decreased to 37.1 °C as incubation proceeded; the humidity started at 50% and gradually increased to 70%.
1. Exposure methods
NOTE: Exposure of environmental contaminants to chicken embryos may be achieved in several ways. In this section, three routinely used methods are described in detail.
2. Endpoint assessment methods
NOTE: Following exposure of contaminant-of-interest to the developing embryo, several toxicity parameters can be evaluated, including cardiotoxicity. In this section, two frequently used specific methods are described in detail.
Exposure results
Air cell injection
Air cell injection can effectively expose developing chicken embryos to various agents, which may be subsequently detected in the collected samples (serum, tissue, etc.) of embryos/hatchling chickens. Here is an example, in which perfluorooctanoic acid (PFOA) was air-cell injected, and serum PFOA concentrations were then determined with Ultra-performance liquid chromatography-mass spectrometry. The serum concentrations corresponded with the injected doses, indicating the effectiveness of this procedure (Figure 6).
Microinjection
Microinjection may expose the developing embryos to agents that may not effectively penetrate the inner membrane, or with a short duration of action, such as lentivirus. Here is an example, in which lentivirus was injected at embryonic day two with this method and then significant green fluorescence was observed in the heart of embryonic day 15 embryos, indicating the effectiveness of lentivirus transfection (Figure 7).
Air cell infusion
Air cell infusion is a novel method, which may work very well for small amount of gas/aerosol inhalation exposure during the initiation stage of external respiration. Here is an example, in which diesel exhaust was infused into air cell at embryonic day 18 and 19, resulting in significant fibrotic changes in the cardiac as well as pulmonary tissues (Figure 8).
Endpoint assessment results
Electrocardiography results
Due to the limitation of two electrodes, only 3 channels of electrocardiography may be shown. But they are sufficient to distinguish r waves, thus they may be used for functional assessments. In a real-life example, electrocardiography of chickens exposed to diesel exhaust indicated significantly shortened R-R interval, indicating functional changes (Figure 9).
Histopathology results
Our method of right ventricular wall thickness assessment was successfully used in several studies5, 7,8,9,10,11,12. In one of our previous studies, diesel exhaust exposure resulted in thickened right ventricular wall (Figure 10).
Figure 1: Demonstration of air cell injection. An undeveloped fertile egg is shown in the picture, but embryos at all different stages may be exposed with this method. Please click here to view a larger version of this figure.
Figure 2: Demonstration of microinjection. An early embryo is shown in the picture, which is the preferred exposure time point for this method, but other time points may also be tried. Please click here to view a larger version of this figure.
Figure 3: Demonstration of air cell infusion. A late-stage embryo undergoing internal pipping is shown in the picture, which is the preferred exposure time point for this method. Four stages of the operation were shown. 1: Intact embryo. 2: Two holes have been made. 3: Infusion is being performed. The PVF sampling bag is also shown at the bottom left. 4: Infusion is finished, holes sealed with tape. Please click here to view a larger version of this figure.
Figure 4: Demonstration of electrocardiography. Top left panel showed how a hatchling chicken was anesthetized and undergoing electrocardiography measurement. Top right panel shows the electrocardiography instrument with the electrodes attached. Bottom panel shows a representative electrocardiography acquired from the chickens. Please click here to view a larger version of this figure.
Figure 5: Demonstration of histopathological assessment of right ventricular wall thickness (Hematoxylin and eosin staining). (A) Demonstration of the cutting position of chicken hearts prior to embedding. (B) Demonstration of the right ventricular wall thickness measurement. Scale bars represent 1000 µm. Blue circles demonstrate the seven measurement points on the inner right ventricular wall. Red circle demonstrates a measurement point on the outer right ventricular wall. Arrow demonstrates the anatomic landmark for the appropriate cross-section position. This figure has been modified from Jiang et al. Toxicology. 293 (1-3), 97-106 (2012)7. Please click here to view a larger version of this figure.
Figure 6: Serum concentration of perfluorooctanoic acid from hatchling chickens following air cell injection with 0, 0.5, 1 or 2 mg/egg kg perfluorooctanoic acid prior to incubation. The resulting serum concentrations corresponded with the injected doses, indicating the effectiveness of air cell injection. This figure has been modified from Jiang et al. Toxicology. 293 (1-3), 97-106 (2012)7. Please click here to view a larger version of this figure.
Figure 7: Demonstration of lentivirus transfection efficacy following microinjection exposure (Direct observation following cryo-sectioning). Left panels showed light field images, while right panels showed green fluorescence for the same tissue sections. Embryonic day two chicken embryos were injected with lentivirus or control, and then incubated until embryonic day 15. The hearts were frozen-sectioned and directly visualized under fluorescent microscope. (A) Control group, little green fluorescence was present. (B) Lentivirus exposed group, significant green fluorescence was observed, indicating the effectiveness of lentivirus transfection following microinjection. Scale bars represent 125 µm. This figure has been modified from Zhao et al. Environmental Toxicology and Pharmacology. 56, 136-144 (2017)11. Please click here to view a larger version of this figure.
Figure 8: Demonstration of the effectiveness of air cell infusion. Chicken embryos were infused with diesel exhaust at embryonic day 18 and 19, and then the hatched chickens were kept for 0, 1 or 2 weeks and then sacrificed. The heart tissues were assessed with Masson Trichrome staining for fibrotic lesions. Arrows showed the fibrotic lesions (blue staining). *: statistically different from control (P<0.05 from analysis of variance and least significant difference tests). Scale bars represent 150 µm. This figure has been modified from Jiang et al. Environmental Pollution. 264, 114718 (2020)8. Please click here to view a larger version of this figure.
Figure 9: Demonstration of the effectiveness of electrocardiography. Chicken embryos were infused with diesel exhaust at embryonic day 18 and 19, and then the hatched chickens were kept for 0, 1 or 2 weeks and then electrocardiography was performed. Significantly shortened R-R intervals were observed in the chickens exposed to diesel exhaust via air cell infusion, indicating the effectiveness of the method. *: statistically different from control (P<0.05 from analysis of variance and least significant difference tests). This figure has been modified from Jiang et al. Environmental Pollution. 264, 114718 (20208. Please click here to view a larger version of this figure.
Figure 10: Demonstration of the effectiveness of right ventricular wall thickness measurement (Hematoxylin and eosin staining). Chicken embryos were infused with diesel exhaust at embryonic day 18 and 19, and then the hatched chickens were kept for 1 week, and then histological assessment of the right ventricular wall thickness was performed. A: Representative pictures of the heart cross-sections. Note the presence of anatomical marker in all the right ventricles (In older chickens, the marker tends to be a bit longer at desired position, which does not affect the accuracy of measurements). B: Quantification of the right ventricular wall thickness, which were firstly converted to actual length with standard slides, and then normalized with whole heart weight thus were represented in the form of um/ug. Blue arrows: two ends of the free right ventricular wall. Red arrows: the middle points of the right ventricular wall. Black arrows: anatomical marker. *: statistically different from control (P<0.05 from analysis of variance and least significant difference tests). Scale bars represent 1000 µm. This figure has been modified from Jiang et al. Environmental Pollution. 264, 114718 (2020)8. Please click here to view a larger version of this figure.
The chicken embryo has been a classical model in developmental studies for 200 years1. Our methods presented in this manuscript have been used in the assessment of several environmental contaminants, including perfluorooctanoic acid, particulate matter, and diesel exhaust with success5, 7,8,9,10,11,12. With these methods, developmental cardiotoxicity was indicated cost-effectively and clearly. Furthermore, it is not difficult to expose chicken embryos with other compounds-of-interest and assess potential developmental cardiotoxicity.
The air-cell injection method is a classical method used previously in many studies13,14,15, which is convenient and effective. Compared to other developmental exposure methods, such as rodent models16,17,18, it features direct exposure into a closed system, which greatly reduces the variabilities due to maternal effects and varied excretion. Microinjection is an enhancement of the air-cell injection method, ensuring definitive exposure on or in the vicinity of developing early embryo, which may achieve similar effects as in utero injections in rodent models19,20. Comparing to in utero injections, our method allows visual confirmation of the injection with relatively easy manipulation steps, and accurate injection is easily achieved by controlling for the egg weight, which is not possible in the in utero injection, where the actual quantity and weight of embryos are not easily acquired. The infusion method is mainly for assessment of inhaled agents on the pulmonary system, but cardiotoxicity and pulmonary toxicity often co-occur. This method takes advantage of the air cell, into which a small amount of gas or aerosol are infused, allowing continuous inhalation of gas/aerosol without the need of specific inhalation chambers. Counterpart rodent models need to use relatively large amounts of gas/aerosol and large, expensive inhalation instruments21,22.
The two routinely tested endpoints in our lab, electrocardiography and histomorphometrical assessment of right ventricular wall thickness, represent functional and morphological changes following toxicant exposure, respectively. The assessment of right ventricular wall thickness has specific advantages in getting a comprehensive understanding of the right ventricular wall, as the traditional echocardiography-based assessment on right ventricle is usually challenging and not very accurate, due to the asymmetrical and complex crescent shape of right ventricle23. Our method may help to overcome this inaccuracy by supplementing with additional information about the right ventricular wall thickness at a representative position. Currently it is all manual, in the future, the measurements may be made automatically and the number of measurement points may be increased considerably, further improving the accuracy of this method.
Chicken embryo-based developmental models have several advantages in toxicological studies, such as the ability to deliver a relatively accurate exposure dose, an independent exposure system within the shell, and easy manipulation of the developing embryo. With respect to cardiotoxicity, chickens have relatively large hearts and thick ventricular walls, allowing easy histomorphometrical assessments. There are some shortcomings, such as availability of antibodies/primers and extra cage space requirements comparing to rodents if rearing chickens after hatch. Nevertheless, the chicken embryo is still a good alternative toxicological model to be used for potential developmental cardiotoxicity assessments.
The authors have nothing to disclose.
This work was supported by National Natural Science Foundation of China (Grant No. 91643203, 91543208, 81502835).
4% phosphate buffered formaldehydefixative | Biosharp, Hefei, China | REF: BL539A | |
75% ethanol | Guoyao,Shanghai,China | CAS:64-17-5 | |
Biosignaling monitor BL-420E+ | Taimeng, Chengdu, China | BL-420E+ | |
Candling lamp | Zhenwei, Dezhou, China | WZ-001 | |
Disposable syringe | Zhiyu, Jiangsu, China | ||
Egg incubator | Keyu,Dezhou, China | KFX | |
Electrical balance | OHAUS, Shanghai, China | AR 224CN | |
Electro-thermal incubator | Shenxian, Shanghai, China | DHP-9022 | |
Ethanol absolute | Guoyao,Shanghai,China | CAS:64-17-5 | |
Fertile chicken egg | Jianuo, Jining, China | ||
Hematoxylin and Eosin Staining Kit | Beyotime, Bejing, China | C0105 | |
Histology paraffin | Aladdin, Shanghai, China | P100928-500g | Melt point 52~54°C |
Histology paraffin | Aladdin, Shanghai, China | P100936-500g | Melt point 62~64°C |
IV catheter | KDL, Zhejiang, China | The catheters have to be soft, plastic ones. | |
Lentivirus | Genechem, Shanghai, China | The lentivirus were individually designed/synthesized by Genechem. | |
Masson's trichrome staining kit | Solarbio, Beijing, China | G1340 | |
Metal probe | Jinuotai, Beijing, China | ||
Microinjector (5 uL) | Anting,Shanghai, China | ||
Microscope | CAIKON, Shanghai, China | XSP-500 | |
Microtome | Leica, Germany | HistoCore BIOCUT | |
Microtome blade | Leica,Germany | Leica 819 | |
Pentobarbitual sodium | Yitai Technology Co. Ltd., Wuhan, China | CAS: 57-33-0 | |
Pipetter(10ul) | Sartorius, Germany | ||
Povidone iodide | Longyuquan, Taian, China | ||
Scissor | Anqisheng,Suzhou, China | ||
Sterile saline | Kelun,Chengdu, China | ||
Sunflower oil | Mighty Jiage, Jiangsu, China | Any commerical sunflower oil for human consumption should work | |
Tape | M&G, Shanghai, China | ||
Tedlar PVF Bag (5L) | Delin, Dalian, China | ||
Vortex mixer | SCILOGEX, Rocky Hill, CT, US | MX-F | |
Xylene | Guoyao,Shanghai,China | CAS:1330-20-7 |