This work was supported by the Applied and Engineering Sciences (TTW) (Vidi-project 17543), part of NWO. The authors would like to thank Robert Beurskens, Luxi Wei, and Reza Pakdaman Zangabad from the Department of Biomedical Engineering and Michiel Manten and Geert Springeling from the Department of Experimental Medical Instrumentation for technical assistance, all from the Erasmus MC University Medical Center Rotterdam, the Netherlands.

The authors have nothing to disclose.

This protocol describes three methods on how to obtain and use 5 to 8-day old chicken embryos and their CAM as an in vivo&#160;model to study contrast-enhanced ultrasound imaging and microbubble-mediated drug delivery. The most critical steps for taking 5-day old (section 1.2) and 6 to 7-day old (section 1.3) embryos out of the shell are: 1) make the small hole in the top of the egg to go through the entire eggshell into the air sac before withdrawing egg white; 2) create smooth edges for the large opening in the shell. For the method for taking 8-day old embryos out of the shell (section 1.4) the most critical steps are: 1) Make a sufficient number of indents to create a nice crack along the egg; 2) Keep the egg submerged in PBS. To ensure embryo viability for all methods it is important to keep the egg and its contents at 37 &#176;C. In addition, avoid injecting into a CAM artery. Visually monitoring the heart rate of the embryo during the studies is recommended to ensure embryo vitality. To confirm the exact developmental stage of the embryo, the indication of Hamburger &#38; Hamilton22&#160;can be used.
It is important to prevent damage to the embryo, CAM, and yolk sack. This damage can affect the viability, blood flow, and visibility of the embryo and CAM. In addition, damage to the yolk sack and consequently a low rigidity of the membrane makes an injection into the CAM vessels impossible. A 5-day old embryo has a relatively small air sac so to be able to make a sufficiently large hole in the shell through which the egg content can be removed, 2 mL of egg white needs to be withdrawn. As a result, more space between the eggshell and embryo is created. After withdrawal of the egg white, a piece of tape needs to close off the hole where the needle went in. If egg white still leaks out, apply another piece of tape. Beside this, the application of tape on the hole on the side creates a vacuum inside the egg which prevents the egg content from falling out due to its own weight when the large hole is created in step 1.2.2.8. Damage to the embryo or CAM can also occur when the edge of the eggshell was too sharp or when the egg content is dropped into the weighing boat too rigorously, so the eggshell should be kept very close to the weighing boat. Between day 5 and 6 of development, the CAM starts to attach to the shell membrane32. This attachment increases the risk of damaging the embryo and CAM when taking the egg content out of the eggshell. By opening the egg after injection of PBS into it for a 6 to 7-day incubated egg or in a PBS-filled container as described for an 8-day incubated egg, the risk of damage is reduced. Regarding an injection into a CAM vein: if the first injection fails, a second injection can be done further upstream in the same vein if the damage was minor or in another CAM vein. Separation of the embryo and CAM from the yolk makes the embryo and CAM vessels optically transparent. As a consequence, the embryo loses its primary source of nutrients33. This loss of nutrients could be an explanation for the observed lower heart rate of 80 bpm in comparison to ~190 for a 6-day old embryo that is still in contact with the yolk30&#160;and the reduced survival time of 2 h after this separation procedure. Another factor that can play a role in the reduced heart rate and survival time is the challenge to keep the yolk-separated embryo and CAM vessels at 37 &#176;C. A microscope stage incubator may be of aid. In addition to this, the detachment of the CAM from the yolk likely leads to mechanical changes in the tissue since the membrane tension becomes less. The lower membrane tension may cause an increased inner vessel shear rate which leads to a lower heart rate.
The ex ovo chicken embryo and CAM vessels have some limitations as in vivo model, including short time observations only, for contrast enhanced ultrasound imaging and microbubble-mediated drug delivery studies. Due to the small blood volume of 100&#177;23 &#181;L at day 5 and 171&#177;23 &#181;L at day 634, a maximum volume of ~5 &#181;L can be injected. In the later stages of development (day 7 and older), the vessel stiffness increases and the yolk elasticity decreases. This can complicate a successful injection in older embryos. Once the microbubbles are injected, they circulate for hours because the chicken embryo does not have a fully developed immune system at this stage35. Therefore, microbubbles are not cleared within ~6 min as in humans36,37&#160;making typical ultrasound molecular imaging studies with a 5-10 min waiting period for non-bound targeted microbubbles to be cleared38&#160;not feasible. In order to target microbubbles, suitable ligands able to bind to avian endothelial cells need to be used such as previously described for the angiogenesis marker &#945;v&#946;37. Other aspects to consider for this model are the increased difficulty of separating the embryo and CAM vessels from the yolk in older embryos (&#62; 8 days) and lower hematocrit of ~20%39&#160;in comparison to humans. The latter may affect microbubbles oscillations because it is known that microbubble oscillations are damped in a more viscous environment40. CAM arteries are less oxygenated than CAM veins41,42. This difference should be taken into account when for example studying photoacoustic imaging of blood oxygenation.
The methods described here allow the egg content to be taken out of the eggshell on the day of the ultrasound imaging or drug delivery study, typically at day 5 to 8 of incubation. This is different to existing methods where the egg content is taken out of the shell after a 3-day incubation and further developed as ex ovo&#160;culture18,20,21. The advantages are a higher survival rate of 90% for 5-day, 75% for 6-day, 50% for 7-day, and 60% for 8-day old incubated eggs in comparison to ~50% for 3-day old embryos taken out of the eggshell and further incubated ex ovo1,18&#160;the avoidance of antibiotics during culture18,20&#160;and large sterile incubator for the ex ovo culture. The survival of the 6-to-8-day old embryos is lower because the CAM starts to attach itself to the shell21&#160;which leaves the CAM membrane more prone to rupture upon extraction. The separation of the embryo with the CAM form the yolk is also described making the embryo and CAM optically transparent.
By placing the egg content in different setups, the chicken embryo and CAM can be used for a multitude of ultrasound imaging studies, like IVUS, photoacoustic, without or with ultrasound contrast agents in 2D and 3D. The focus can be on developing new ultrasound pulsing schemes or testing out novel transducers. Besides this, the model can also be used to investigate novel ultrasound contrast agents and their behavior in blood vessels under flow. Since the mechanism of microbubble-mediated drug delivery is still unknown43, the use of the in vivo CAM model may help in elucidating the mechanism by studying the microbubble behavior in relation to the cellular response. Finally, the CAM vessels have proven to be a suitable system to investigate xenograft tumor transplantation44. This creates the possibility to use the CAM vessel as a model to investigate tumor imaging using ultrasound and to investigate the blood flow inside the tumor using CEUS. The tumors are typically grafted on the CAM vessels of 8 or 9-day old embryos1,14,45, for which the embryo&#160;is taken out of the eggshell at day 3 of incubation and further developed ex ovo. The methods described in this protocol could be used to grow embryos in ovo until the day of tumor&#160;grafting.
The authors trust that this paper will be helpful for researchers that want to use chicken embryos and their chorioallantoic membrane (CAM) as an in vivo&#160;model for applications of contrast agents and flow studies.

Ex ovo chicken embryos and the blood-vessel rich chorioallantoic membrane (CAM) have proven to be a suitable model to investigate various biological and biomedical processes like embryogenesis, oncology, and drug delivery1,2,3,4. Ultrasound has been used for imaging of the embryonic heart development4,5&#160;and for activating cavitation nuclei upon injection, such as microbubbles, for vascular drug delivery6,7. Chicken embryos are inexpensive, require less infrastructure and equipment, and have less strict legislation compared to other animal models8. The chicken embryo and CAM vessels are easily accessible after opening the egg whereas this proves to be much more difficult with mammalian embryos and vessels. Besides this, the chicken embryo and CAM vessels provide a heartbeat and a pulsating blood flow. The CAM shows similarities in vessel anatomy with mammals and can be used for drug screening8,9,10. Because of these characteristics, the CAM vessels have also proven to be a suitable model to investigate contrast enhanced ultrasound imaging (CEUS)11,12,13,14,15,16. In addition, the model can be used to optically investigate the behavior of ultrasound contrast agents in an ultrasound field using an ultra-high-speed camera and the effect of acoustic radiation force on propelling, binding and extravasation of drugs7,17,18,19. Although the chicken embryo and CAM are less suitable for long term experiments, they can be beneficial for short term in vivo experiments.
To increase visibility and controllability over the chicken embryo and CAM during experiments, it is important to take the egg content containing the embryo and CAM out of the eggshell18. Previous chicken embryo studies involving ultrasound contrast agents used 5 to 6-day old embryos7,11,12,17,19&#160;and 14 to 18-day old embryos13,14,15,16. Multiple approaches have been described in detail to take the egg content out of the shell18,20,21. However, to the best of our knowledge, the previously published approaches focus on taking the egg content out of the eggshell after 3 days of incubation (i.e., Hamburger &#38; Hamilton (HH) stage 19-2022), and continue the culture ex ovo. This ex ovo culture approach has multiple disadvantages including increased risk of fatalities during culture (~50%)1,18, the use of antibiotics18,20, and decreased total vessel length in comparison to in ovo growth23. Since culturing the embryo within the eggshell is providing the most natural environment, it is easiest to incubate the embryo within the eggshell until the day of the experiment. For this reason, an approach in which the egg content is taken out of the eggshell at 5 to 8 days of incubation would be beneficial especially for experiments on 5 to 8-day old embryos.
In this protocol, we describe three methods to take the egg content out of the eggshell when the embryo is at day 5 to 8 of development (HH 26-3522) allowing the embryo to develop within the eggshell until the day of the experiment. The CAM vessel size ranges from 10-15 &#181;m in diameter, in the smaller capillaries of an 8-day old embryo24,&#160;to 115-136 &#181;m in diameter in the larger vessel of 6 and 8-day old embryos24,25. The three described methods only require basic lab tools and reduce the risk of complications before the experiment has begun, thereby reducing unnecessary costs and labor. We also detail a method to separate the membrane containing the embryo and CAM from the yolk sack making the CAM optically transparent for microscopy studies. Because the membrane containing the embryo and CAM can be pinned down on for example a holder with an acoustic membrane, the setup can also be made acoustically transparent26, allowing the combination of microscopy and ultrasound studies when the light path will be affected by the yolk. Finally, we describe several other ultrasound setups that can be used for ultrasound or CEUS imaging.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Agarose</td>
			<td>Sigma-Aldrich</td>
			<td>A9539</td>
			<td></td>
		</tr>
		<tr>
			<td>Clamp (Kocher clamp)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cling film</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Holder with acoustic membrane (CLINIcell 25 cm2)</td>
			<td>MABIO, Tourcoing, France</td>
			<td>CLINIcell25-50-T FER 00106</td>
			<td></td>
		</tr>
		<tr>
			<td>Demi water</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable plastic Pasteur pipets</td>
			<td>VWR</td>
			<td>612-1747</td>
			<td></td>
		</tr>
		<tr>
			<td>Eggs</td>
			<td colspan="2">Drost Pluimveebedrijf Loenen BV, the Netherlands</td>
			<td>Freshly fertilized</td>
		</tr>
		<tr>
			<td>Fridge 15 &#176;C</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glass capillary needles</td>
			<td>Drummond</td>
			<td>1-000-1000</td>
			<td>Inside diameter: 0.0413 inch</td>
		</tr>
		<tr>
			<td>Heating plate 37 &#176;C</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Humidified incubator 37 &#176;C</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Insect specimen pins</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Metal egg holder</td>
			<td></td>
			<td></td>
			<td>Custom made by Experimental Medical Instrumentation at Erasmus MC. See figure 1 A,B</td>
		</tr>
		<tr>
			<td>Metal weighing boat holder</td>
			<td></td>
			<td></td>
			<td>Custom made by Experimental Medical Instrumentation at Erasmus MC. See figure 1 C,D</td>
		</tr>
		<tr>
			<td>Microinjection system</td>
			<td>FUJIFILM VisualSonics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mineral oil</td>
			<td>Sigma-Aldrich</td>
			<td>M8410-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle, 19 G</td>
			<td>VWR (TERUMO)</td>
			<td>613-5392</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-bufferes saline (PBS), 1x</td>
			<td>ThermoFisher</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish, 1 L</td>
			<td></td>
			<td></td>
			<td>Glass</td>
		</tr>
		<tr>
			<td>Petri dish, 90 mm diameter</td>
			<td>VWR</td>
			<td>391-0559</td>
			<td></td>
		</tr>
		<tr>
			<td>Preclinical animal ultrasound machine (Vevo 2100)</td>
			<td>FUJIFILM VisualSonics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Probe (MS250)</td>
			<td>FUJIFILM VisualSonics</td>
			<td></td>
			<td>30 MHz transmit and 15 MHz receive frequency</td>
		</tr>
		<tr>
			<td>Probe (MS550s)</td>
			<td>FUJIFILM VisualSonics</td>
			<td></td>
			<td>transmission frequency of 40 MHz</td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td>VWR (SWANN-MORTON)</td>
			<td>233-5363</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors, small</td>
			<td>Fine Science Tools (FST)</td>
			<td>14558-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe, 5 mL</td>
			<td>VWR (TERUMO)</td>
			<td>613-0973</td>
			<td></td>
		</tr>
		<tr>
			<td>Table spoon</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tape (Scotch Magic tape)</td>
			<td>Scotch</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue paper</td>
			<td>Tork</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezers large&#160;</td>
			<td>VWR (USBECK Laborger&#228;te)</td>
			<td>232-0107</td>
			<td>See figure 1E</td>
		</tr>
		<tr>
			<td>Tweezers small</td>
			<td>DUMONT Medical, Switzerland</td>
			<td>0103-5/45</td>
			<td>See figure 1F</td>
		</tr>
		<tr>
			<td>Ultrasound contrast agent (custum made F-type)</td>
			<td></td>
			<td></td>
			<td>Produced as described by: Daeichin, V. et al. Microbubble Composition and Preparation for Imaging : In Vitro and In Vivo Evaluation. IEEE TRANSACTIONS ON ULTRASONICS. 64 (3), 555&#8211;567 (2017).</td>
		</tr>
		<tr>
			<td>Ultrasound contrast agent (MicroMarker)</td>
			<td>FUJIFILM VisualSonics, Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasound contrast agent (Definity)</td>
			<td colspan="2">Lantheus medical imaging, United States</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasound gel</td>
			<td>Aquasonic</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Waxi film (Parafilm)</td>
			<td>Parafilm</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Weighing boats (85 &#215; 85 &#215; 24 mm)</td>
			<td>VWR</td>
			<td>611-0094</td>
			<td></td>
		</tr>
	</tbody>
</table>

the preparation of chicken ex ovo embryos and chorioallantoic membrane vessels as in vivo model for contrast-enhanced ultrasound imaging and microbubble-mediated drug delivery studies

The chicken embryo and the blood-vessel rich chorioallantoic membrane (CAM) is a valuable in vivo model to investigate biomedical processes, new ultrasound pulsing schemes, or novel transducers for contrast-enhanced ultrasound imaging and microbubble-mediated drug delivery. The reasons for this are the accessibility of the embryo and vessel network of the CAM as well as the low costs of the model. An important step to get access to the embryo and CAM vessels is to take the egg content out of the eggshell. In this protocol, three methods for taking the content out of the eggshell between day 5 and&#160;8 of incubation are described thus allowing the embryos to develop inside the eggshell up to these days. The described methods only require simple tools and equipment and yield a higher survival success rate of 90% for 5-day, 75% for 6-day, 50% for 7-day, and 60% for 8-day old incubated eggs in comparison to ex ovo cultured embryos (~50%). The protocol also describes how to inject cavitation nuclei, such as microbubbles, into the CAM vascular system, how to separate the membrane containing the embryo and CAM from the rest of the egg content for optically transparent studies, and how to use the chicken embryo and CAM in a variety of short-term ultrasound experiments. The in vivo&#160;chicken embryo and CAM model is extremely relevant to investigate novel imaging protocols, ultrasound contrast agents, and ultrasound pulsing schemes for contrast-enhanced ultrasound imaging, and to unravel the mechanisms of ultrasound-mediated drug delivery.

In this protocol, we describe three methods to take the egg content out of the shell at day 5-8 of incubation (HH 26-3522). Figure 6&#160;shows the egg content in weighing boats after it was taken out of the shell. The 5-day old embryo and CAM (Figure 6A) was taken out using the method described in section 1.2. The 6 and 7-day old embryos and CAM (Figure 6B,C) were taken out using the method described in section 1.3. The 8-day old embryo and CAM (Figure 6D) was taken out using the method described in section 1.4. No bleeding or damage to the embryo or CAM can be observed, indicating that these methods can be used to safely get the egg content out of the shell without harming the embryo or the CAM vessels. When executed correctly, the method for the 5-day old embryos will provide a viable embryo and intact CAM in 90% of all procedures. The viability rate is based on the total number of fertilized eggs successfully extracted from the eggshell.&#160;With the second method, for 6 and 7-day incubated eggs, the chance of a viable embryo and intact CAM is around 75% for 6-day old and around 50% for 7-day old. With the third method described for 8-day old embryos, the chance of a viable embryo and intact CAM is around 60%. Differences in developmental stages between the 5 and 8-day old embryos can be observed which concurs with Hamburger and Hamilton22. Both the size of the embryo and the complexity of the CAM vessels increase during development (Figure 6A-D). Figure 6C shows a thin patch of agarose on top of the egg content that allows the embryo and CAM to be imaged using the ultrasound setup shown in Figure 5C. After the egg content is taken out of the shell the heartbeat of the embryo is visible with the naked eye. The heart rate of these ex ovo&#160;embryos is similar to in ovo embryos at 183 beats per minute (bpm) at day 5 up to ~208 bpm at day 830. When kept humidified and at 37 &#176;C, the embryo will maintain this heart rate for ~5 h in the experimental ultrasound setups.
Multiple complications can occur during the previously described three methods. Figure 7A shows trapped air under the CAM which makes the embryo unsuitable for ultrasound imaging and the pressure of the air bubble(s) can also damage the embryo and/or CAM. This problem arises when the air sac inside the shell does not make contact with the air outside the shell when taking the egg content out of the shell. Figure 7B shows a small leakage of yolk from the yolk sack in the top right of the image. This can occur while taking the egg content out of the shell when the yolk sack gets damaged by sharp edges of the shell or when the yolk sack is penetrated by the tweezers. Leakage of the yolk can affect the visibility of the embryo and the CAM vessels. Figure 7C shows an embryo in which an air bubble is trapped under the CAM. This sometimes occurs in the embryonic development. Another complication which can occur is damage to the vessels. This damage can be created while taking the egg content out of the shell or when performing an injection (Figure 7D). Besides this, the embryo and vessels can also dry out over time (Figure 7E). This occurs when the egg content is not sprinkled with PBS. The drying out of the embryo can result in massive capillary obstructions (Figure 7F) which affects the viability of the embryo. The massive capillary obstructions can also occur during development or when the heartbeat of the embryo is not stable.
After the egg content is taken out of the shell without any complication, the embryo can be injected with, for example, ultrasound contrast agents such as microbubbles (Figure 3C). Figure 8 shows circulating microbubbles in the lumen of the blood vessel upon injection. These microbubbles are carried along with the blood flow and stay present in the blood circulation for several hours (Supplemental Video 1). The presence of these microbubbles in the circulation creates the possibility to perform different types of CEUS and drug delivery experiments7,11,12. The CAM is ideal to investigate novel ultrasound contrast detection methods for which we show three examples. Figure 9A shows high frequency ultrasound subharmonic imaging of a 6-day old chicken embryo in B-Mode and CEUS before and after microbubble injection. Here, the CAM vessels were injected with 5 &#181;L of ultrasound contrast agent and imaging was performed with a preclinical animal ultrasound machine with a MS250 probe (30 MHz transmit and 15 MHz receive frequency, 10% power). Before microbubble injection, contrast can already be seen inside the embryonic heart in the B-Mode images (Figure 9A-I). This phenomenon is due to the presence of a nucleus in the avian red blood cell which increases the contrast of blood in ultrasound imaging5,31. The addition of the microbubbles increased the contrast and the visibility of the embryo, both in the B-Mode and CEUS imaging. Figure 9B shows an optical and a high frequency 3D subharmonic image of a 6-day old embryo and the surrounding vessels. The CAM was injected with 5 &#181;L of ultrasound contrast agent and imaging was performed with a preclinical animal ultrasound machine with MS550s probe (transmission frequency of 40 MHz, peak negative pressure ~300 kPa). These results show that the CEUS imaging combined with a contrast agent can also be used to create high frequency 3D subharmonic images and to image the blood vessels outside the embryo. Figure 9C shows an optical image and an ultraharmonic intravascular ultrasound (IVUS) image made with a custom probe of CAM microvessels of a 6-day old embryo (26 MHz transmit and 39 and 65 MHz receive frequency). CAM vessels were injected with 4 &#177; 1 &#181;L ultrasound contrast agent. The optical image and IVUS image are from the same embryo and same region of interest showing corresponding vessel networks.
The chicken embryo and CAM vessels can also be used to investigate ultrasound-mediated drug delivery for which we show one example. Since the yolk obstructs the light path during imaging, the removal of the yolk sack is necessary to optically investigate drug delivery in the embryo and CAM vessels. For this study, the embryo and CAM were prepared for microscopic imaging as explained in section 2.2 by separating the membrane containing the embryo and CAM from the yolk sack (Figure 4C). In these embryos, the heart rate is stable around 80 bpm and the embryos stay alive for up to 2 h when kept at a 37 &#176;C7. Figure 10&#160;shows an ultrasound and microbubble mediated drug delivery study in endothelial cells of the CAM vessels. Lipid-coated microbubbles, targeted to the vessel wall using &#945;v&#946;3-antibodies and stained with the fluorescent dye DiI7, were injected into the CAM vessels (Figure 10A,C). CAM vessel endothelial cell nuclei were stained with Hoechst 33342 (Figure 10B) and the model drug Propidium Iodide (PI) was used to visualize sonoporation7. Both these dyes were injected simultaneously with the microbubbles. Upon ultrasound treatment (1 MHz, 200 kPa peak negative pressure, single burst of 1000 cycles), PI uptake was observed in the nuclei closest to the targeted microbubbles (Figure 10D). This shows that the ultrasound-induced oscillations of the targeted microbubbles were able to create a pore in the endothelial cell membrane.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62076/62076fig01.jpg" /> 
Figure 1. Embryo preparation equipment. (A-B) top and side view of the metal egg holder and (C-D) top and side view of the metal weighing boat holder. (E-F) tweezers needed to take the egg content out of the shell. Scale in cm. <a href="https://www.jove.com/files/ftp_upload/62076/62076fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62076/62076fig02.jpg" /> 
Figure 2. Embryo removal procedure. (A) Small indent on top of the egg, indicated by the black circle. (B) Small indent 2/3 down the egg, indicated by the black circle. (C) Withdrawing ~2 mL of egg white. (D) Sealed gap on the side with tape. (E) Enlarging the small opening on the top of the egg. (F) The embryo becomes visible after removing part of the shell. (G-H) After rotating the egg 180&#176;, the embryo floats up and will become invisible (arrows indicate moving direction of the embryo). After 1-2 min, the embryo is invisible from the bottom. (I) After scratching the membrane, the egg content drops into the weighing boat.&#160;<a href="https://www.jove.com/files/ftp_upload/62076/62076fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62076/62076fig03.jpg" /> 
Figure 3. Injection of microbubbles into the CAM vessels. (A) Glass capillary needle. Scale in cm. (B) Propidium iodide (PI) solution (left drop) and microbubbles (right drop) prior to aspiration before injection. Needle (outlined in black) can be seen in the top right corner (C) Microbubble injection. The capillary needle tip is positioned inside the lumen of one of the veins (left). Microbubbles, the white cloud indicated with an arrow, are injected and disperse following the blood stream (right). Scale bar represents 1 mm.&#160;<a href="https://www.jove.com/files/ftp_upload/62076/62076fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62076/62076fig04.jpg" /> 
Figure 4. Removing embryo and CAM from yolk sack and placing on holder with acoustic membrane. (A) Holder with acoustic membrane filled with agarose layer. (B) Chicken embryo and CAM vessel in weighing boat before cutting. Dotted line indicates the cutting line around the CAM. (C) Chicken embryo and CAM separated from yolk and pinned down on acoustic membrane. (D) Pinned down chicken embryo with an acoustically and optically transparent membrane in a holder (blue) placed on top of the CAM. The holder can be filled with demi water so a water dipping objective can be used. All scale bars represent 1 cm.&#160;<a href="https://www.jove.com/files/ftp_upload/62076/62076fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/62076/62076fig05.jpg" /> 
Figure 5. Different setups for chicken embryo and CAM ultrasound imaging. (A) Setup for ultrasound imaging from the side. Chicken embryo was placed in a custom-adjusted weighing boat with one acoustically transparent wall and placed in a 37 &#176;C water bath. The ultrasound transducer was positioned on the left (a) side next to the acoustically transparent wall and the laser (b) for photoacoustic imaging on top. (B) Setup for ultrasound imaging from the top. Embryo and CAM were submerged in a beaker of PBS that was placed in a 37 &#176;C water bath. Dashed outline shows the 2 L glass beaker (a) with the 500 mL glass beaker (b) inside. (C) Setup for ultrasound imaging from the top with a movable transducer. A thin agarose pad (dotted line) was placed on top of the embryo with a thin layer of PBS as coupling between the transducer and the agarose surface.&#160;<a href="https://www.jove.com/files/ftp_upload/62076/62076fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/62076/62076fig06.jpg" /> 
Figure 6. Egg content outside the shell. (A) Egg content taken out of the shell after 5 days of incubation. The chorioallantoic membrane (CAM), embryo body (EB), anterior and posterior vitelline veins (*), and appropriate sites for injection (arrowheads) are indicated. (B) Egg content taken out of the shell after 6 days of incubation. The anterior and posterior vitelline veins (*) and appropriate sites for injection (arrowheads) are indicated. (C) Egg content taken out of the shell after 7 days of incubation. A patch of agarose is placed on top to allow for ultrasound imaging. The corners of the agarose patch are indicated with black circles. (D) Egg content taken out of the shell after 8 days of incubation. All scale bars represent 1 cm.&#160;<a href="https://www.jove.com/files/ftp_upload/62076/62076fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/62076/62076fig07.jpg" /> 
Figure 7. Complications which can occur during the procedures with the chicken embryo and CAM model. (A) Air bubbles trapped under the CAM when taking the egg content out of the shell using method 1.2 (5-day old embryo) or 1.3 (6&#160;to 7-day old embryo). (B) Small leakage of yolk indicated with an arrow on the top right (6-day old embryo). (C) Air trapped under the CAM, indicated by the black dotted circle (7-day old embryo). (D) Bleeding, indicated with the black arrows (5-day old embryo. (E) Dried out embryo and CAM (5-day old embryo). (F) Massive capillary obstructions (5-day old embryo). All scale bars represent 1 cm.&#160;<a href="https://www.jove.com/files/ftp_upload/62076/62076fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/62076/62076fig08.jpg" /> 
Figure 8. Microbubbles in CAM blood vessel. The vessel wall is indicated with a dotted line and single microbubbles are indicated with arrows. Scale bar represents 20 &#181;m. The corresponding microscopy recording can be found in Supplemental Video 1.&#160;<a href="https://www.jove.com/files/ftp_upload/62076/62076fig08large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/62076/62076fig09.jpg" /> 
Figure 9. Contrast-enhanced ultrasound imaging in chicken embryos and CAM vessels. (A) Maximum-intensity projection of B-Mode (I, III) and real-time subharmonic (II, IV) images (preclinical animal ultrasound machine with MS250 probe, 30 MHz transmitting and 15 MHz receiving frequency, 10% power) of a 6-day old embryo with a patch of agarose on top. Top images (I, II) show results before and bottom (III, IV) after injection of 5 &#181;L ultrasound contrast agent. Scale bar represents 1 mm. This image has been modified with permission from Daeichin et al.&#160;201511&#160;(B) Optical (left) and 3D subharmonic imaging (right) of a 6-day old chicken embryo with a patch of agarose on top. CAM vessels were injected with 5 &#181;L ultrasound contrast agent and imaging was performed with a high-frequency probe (preclinical animal ultrasound machine with MS550s probe, transmission frequency of 40 MHz, peak negative pressure ~300 kPa, rendered in preclinical animal ultrasound machine 3-D mode). Scale bar represents 5 mm. This image has been modified with permission from Daeichin et al.&#160;201511. (C) Optical image (left) and mean intensity projection of ultraharmonic intravascular ultrasound (IVUS) (right) of the CAM microvasculature of a 6-day old embryo. CAM vessels were injected with 4 &#177; 1 &#181;L contrast agent. Ultraharmonic IVUS imaging was performed with a custom IVUS probe (transmission frequency 35 MHz, peak negative pressure 600 kPa). Both images are made from the same embryo and region of interest. Arrows indicate corresponding vessels in the two images. Scale bar represents 1 mm. This image has been modified with permission from Maresca et al. 201412.&#160;<a href="https://www.jove.com/files/ftp_upload/62076/62076fig09large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 10" class="xfigimg" src="/files/ftp_upload/62076/62076fig10.jpg" /> 
Figure 10. Drug delivery to CAM vessel endothelial cells in 6-day old embryo.&#160;(A) Brightfield image of six &#945;v&#946;3-targeted microbubbles, indicated with white arrows, adhering to the vessel wall before ultrasound treatment. (B) Endothelial cell nuclei fluorescently stained before ultrasound treatment. (C) Fluorescent image of the stained targeted microbubbles, indicated with white arrows, before ultrasound treatment. (D) Uptake of the model drug propidium iodide (PI) into the cell nuclei underneath the targeted microbubbles after ultrasound treatment (1 MHz, 200 kPa peak negative pressure, single burst of 1000 cycles). Scale bar represents 10 &#181;m and applies to all images. This image has been modified with permission from Skachkov et al.&#160;20147.&#160;<a href="https://www.jove.com/files/ftp_upload/62076/62076fig10large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
SUPPLEMENTARY FILES
Supplemental Video 1. Microbubbles in CAM blood vessel. Scale bar represents 20 &#181;m.&#160;<a href="https://www.jove.com/files/ftp_upload/62076/Supplemental Video 1.mp4" target="_blank">Please click here to download this video.</a>

Watch this Scientific Journal Video about The Preparation of Chicken Ex Ovo Embryos and Chorioallantoic Membrane Vessels as In Vivo Model for Contrast-Enhanced Ultrasound Imaging and Microbubble-Media

The Preparation of Chicken Ex Ovo Embryos and Chorioallantoic Membrane Vessels as In Vivo Model for Contrast-Enhanced Ultrasound Imaging and Microbubble-Mediated Drug Delivery Studies

This protocol describes three methods on how to obtain and use 5 to 8-day old chicken embryos and their chorioallantoic membrane (CAM) as an in vivo&#160;model to study contrast-enhanced ultrasound imaging and microbubble-mediated drug delivery.