Motion-Artifact-Free In Vivo Imaging Utilizing Narcotized Avian Embryos In Ovo
Abstract
Purpose: The chick embryo in ovo is a well-accessible and economical in vivo model, but its use in molecular imaging has been limited because of motion artifacts on resulting images. The purpose of this study was to develop a method using narcotics to inhibit motility and to perform motion-artifact-free imaging of living chick embryos in ovo.
Procedures: Chick embryos in ovo were narcotized using three different narcotics: isoflurane, 2,2,2-tribromoethanol, and urethane/α-chloralose. Narcotized embryos were imaged using micro-computed tomography (microCT) at days 10–18 of incubation, and the resulting images were analyzed for reduction of motion artifacts.
Results: All three anesthetics could be used for anesthetizing living chick embryos in ovo, thus allowing the acquisition of motion-artifact-free images.
Conclusions: Our experiments revealed that isoflurane is the best-suited narcotic for single and repeated applications to image living chick embryos in ovo.
Key words: Anesthesia, Motion artifact, Chick embryo, In vivo, In ovo, PET/CT, MicroCT, Model organism
Introduction
Animal models play a crucial role in basic and medical research. Progress in many fields like regenerative medicine, cancer research, infection biology, and drug discovery is strongly dependent on in vivo models to validate in vitro results and to develop new therapeutic approaches. However, conventional rodent and large animal experiments face ethical, practical, and technical issues, thus limiting their usage. The chick embryo in ovo represents an accessible and economical in vivo model, which has long been used in developmental biology, gene expression analysis, and functional experiments. Chicken eggs are available year-round, they are inexpensive, and they can be purchased in any specified quantity. Chicken eggs can be incubated to any stage of interest, thus simplifying experimental design.
Very little has been published on subjecting living chick embryos in ovo to non-microscopy-based molecular imaging technologies, especially positron emission tomography (PET), computed tomography (CT), or the combination of both, i.e., microPET/CT. One major reason might concern the common difficulties in producing motion-artifact-free images, which are overcome with anesthesia in experimental systems other than avian embryos in ovo. There are several recent studies available involving chick embryos and CT or magnetic resonance imaging in which ex vivo/ex ovo approaches were performed or in ovo studies where embryo motility was reduced by exposure of the eggs to low temperatures. As our laboratory aims at both 3D and 4D imaging approaches, i.e., also PET/CT time kinetics, we decided to determine optimum anesthesia procedures for kinetic studies of individual living chick embryos in ovo. This method could solve the problem of motion artifacts on images.
For the most commonly used rodent species like mice and rats, possible routes of anesthesia are straightforward. Blood vessels and respiratory organs are directly accessible for intravascular and inhalational administration, respectively, but these routes are not directly accessible for chick embryos in ovo. Very little information has been published about how to anesthetize chick embryos in ovo for the prevention of motion. For our study, we chose 2,2,2-tribromoethanol (Avertin), urethane/α-chloralose (UC anesthetic), and isoflurane because they already have been described for avian anesthesia. Specifically, the use of isoflurane, the UC anesthetic, and Avertin on chick embryos or chickens has been published, but their use has never been studied systematically for different days of incubation. We assessed the three anesthetics regarding their ability to reduce or inhibit motility, thus presenting a method to acquire motion-artifact-free CT images of chick embryos in ovo.
Materials and Methods
Eggs
Fertilized eggs (weight 60 g ± 2 g) of Gallus gallus domesticus obtained from Geflügel GmbH Boma (Germany) were incubated at 37.7°C ± 0.2°C and a relative humidity of 60 ± 2% (Grumbach BSS300 incubator; Grumbach Brüggeräte GmbH, Germany). Starting at day 4 of incubation, the eggs were automatically rotated (360°) every 6 h. Investigations about the effect of the anesthetics were performed for days 10 to 18 of incubation. Sample sizes of N = 3 eggs plus one control were prepared for each day of incubation.
Anesthetics/Chemicals
Urethane, α-chloralose, tert-amyl-alcohol, 2,2,2-tribromoethanol, and isoflurane were commercially purchased. The UC anesthetic was prepared as a mixture of 450 mg/ml urethane and 45 mg/ml of α-chloralose. The components were dissolved in deionized sterile water and incubated for 15 min in an ultrasonic bath at 30°C and an ultrasound frequency of 35 kHz. Avertin was prepared as stock solution, that is 5.0 g of 2,2,2-tribromoethanol were dissolved in 3.1 ml of tert-amyl-alcohol yielding a final concentration of 1,600 mg/ml of 2,2,2-tribromoethanol. The undiluted stock solution was used for application. Isoflurane is commercially available as ready-to-use solution.
Administration
The UC anesthetic and Avertin were applied as liquids directly onto the chorioallantoic membrane (CAM). Two holes (diameter: 2.5 mm) were drilled into the eggshell, and the CAM was dropped according to the dropped membrane technique. After application, both holes were closed with paraffin wax, and the eggs were incubated at 37.7 ± 0.2°C for approximately 15 min to ensure the full action of the anesthetic agents.
For the isoflurane anesthesia, the commercial veterinary anesthesia system combi-vet® base system was used. It consists of a highly accurate digital flowmeter and a vaporizer. Dräger vaporizers are factory-calibrated individually with interference refractometers at 22°C and at 30°C with a flow of 2.5 L/min of air. Their supported fresh-gas flow range is 0.25 to 15 L/min. The vaporizer was set to deliver an isoflurane concentration of 5% with oxygen as the carrier at a flow set to 0.6 L/min. For narcosis induction, eggs were placed into a commercially available narcosis induction chamber for mice and rats connected to the combi-vet® base system. The narcosis induction chamber has one inlet and one charcoal-filter-connected outlet to guarantee constant narcotic concentration. To reach the desired isoflurane concentration of 5% within the chamber prior to the start of narcosis induction, it was flooded for 5 min with an oxygen flow of 5 L/min and an isoflurane concentration of 5%. An infrared lamp was used to maintain temperature at 37 ± 2°C on the surface of the eggs. The temperature was monitored using a digital thermometer.
Imaging System and Setup
The Siemens Inveon combined microPET/CT scanner was used for image acquisition. CT scans were performed with an X-ray tube voltage of 80 kV and a current of 500 µA. The detector was operated in a four-by-four pixel binning mode. Two bed positions were used, allowing image acquisition of two eggs with only one CT measurement run. The rotation angle of the gantry was 360°, and during rotation 220 projections per bed position and egg were acquired with an exposure time of 300 ms each. These methods resulted in a total acquisition time of approximately 6 min per bed position and egg. Images were displayed and analyzed with the Siemens Inveon Research Workplace Software.
Assessment Criteria
Upon anesthetic treatment and induction time, each egg was candled to visually check for motion of the embryo and imaged by microCT. Impact of anesthesia was evaluated by both image quality and survival of the embryo. The required image quality criterion was stable embryo position within the egg, i.e., no or only very little motion artifacts, and survival time of the embryo of at least 24 h following application of the anesthetics.
Results
The aim of this study was to produce motion-artifact-free images of living chick embryos in ovo, especially by means of microCT. In addition, we aimed at long-term survival to perform time kinetics of the distribution of isotope-labeled molecules in living systems. Artifact-free imaging using untreated living chick embryos in ovo was not possible. Previously used methods like undercooling or freezing chick embryos in ovo resulted in motion-artifact-free images but were not suitable for our applications because undercooling significantly reduces survival rate and slows metabolism, and freezing is an ex vivo approach. Therefore, we tested various narcotics to determine a suitable anesthesia approach for living chick embryos in ovo.
UC Anesthetic
Appropriate amounts of urethane and α-chloralose for a working UC anesthesia were determined. In early days, that is up to day 11 of incubation, mortality of anesthetized chick embryos in ovo was relatively high compared with later days.
Avertin
No data has been available on the use of Avertin for anesthetizing chick embryos in ovo, requiring suitable amounts of the narcotic for a working anesthesia to be determined. As described in “Materials and Methods,” optimum results were obtained by applying undiluted stock solution. By carefully modifying the dose, suitable anesthesia from day 11 until day 18 of incubation was obtained with Avertin. Immobilization was evident within 15 min upon application of the anesthetic. In contrast to the other narcotics, Avertin could not be applied before day 11 of incubation because at earlier days all Avertin concentrations resulting in narcosis were lethal.
Isoflurane
No data has been available on the application of vaporized isoflurane on intact fertilized eggs. Therefore, we had to determine if vaporized isoflurane can be used and, if yes, under what conditions. For this procedure, 10 and 13-day-old embryos were exposed to isoflurane for 15, 30, 45, and 60 min, directly followed by CT measurements. Only exposure times of 30 min and above were sufficient for the acquisition of two motion-artifact-free images of 6 min acquisition time each. For additional images (6 min acquisition time each), longer exposure times to isoflurane were needed. Generally, exposure time to isoflurane had to be relatively long because gas exchange through the porous eggshell is very slow. As determined, it took about 30 min until pharmacologically active amounts of isoflurane were taken up by the chick embryo. Isoflurane exposure times for all stages from days 10 to 18 of incubation are shown in the results. During all experiments with isoflurane, only one embryo died.
Discussion
The aim of this study was to identify anesthetic agents suitable for optimum anesthesia for motion-artifact-free imaging, i.e., a non-lethal dose with no discernable influence on viability. The following three anesthetics were investigated: the UC anesthetic, Avertin, and isoflurane. All agents led to narcosis but with different effects regarding toxicity, usability, and duration. Thereby, a suitable dose of all three anesthetics was dependent on the developmental stage of the embryo, not on the total weight of the fertilized egg.
The UC anesthetic consists of urethane and α-chloralose, both of which are long-term anesthetics. They can be used separately or in combination in animal experiments and are often used in combination because urethane increases the solubility of α-chloralose. Urethane and α-chloralose generate a long-lasting steady level of anesthesia and have only minimal effects on physiology.
Sugiyama and colleagues injected the UC anesthetics into the air sac of chicken eggs and demonstrated that it is possible to anesthetize embryos in ovo in the late stage of incubation (day 16). For anesthetizing embryos in ovo from days 10 to 18 of incubation, we had to carefully adapt and modify the published dose. In our study, the UC anesthetic was very toxic for chick embryos, and lethality was high, especially at early days of incubation (days 10 to 13), even at very low dosages. These observations corroborated previous findings. Nevertheless, we were able to acquire motion-artifact-free images approximately 15 min after the application of appropriate amounts of the UC anesthetic. Due to the long-term narcotic effect of the drugs, it is possible to make repeated CT measurements of anesthetized embryos. In conclusion, we can say that the UC anesthetic is suitable, but each application should be carefully considered. The high mortality of chick embryos after treatment with the UC anesthetic suggests that it should only be used at later days of incubation (days 14 to 18) or if survival of the embryo is not a primary requirement.
2,2,2-Tribromoethanol (Avertin) is most commonly used for the anesthesia of laboratory mice. Avertin anesthesia has never been used for narcosis of chick embryos in ovo. Therefore, we had to cope with several unknown parameters such as survival, duration of anesthesia, and depth of narcosis. In addition, Avertin has several known negative aspects, including inducing necrosis and inflammation. There is also much variation in sensitivity between subjects and a variable time lapse between application of the drugs and attainment of effective narcosis. In this study, deviation also varied considerably, as no correlation between dose, depth of narcosis, weight of eggs, and number of dying embryos could be detected. Furthermore, 24 h after application of the narcotic, we observed that not all embryos fully recovered, as motility was significantly reduced. Avertin always had to be prepared prior to use, and there is a broad range of different protocols, including different recommendations of Avertin concentrations and storage conditions, i.e., a standardized protocol is not available. However, Avertin can be used as an anesthetic for the acquisition of motion-artifact-free images using chick embryos in ovo. Nevertheless, toxic side effects should be considered depending on the topic under investigation. If, for example, survival of the chick embryo is crucial, Avertin should not be used until after day 10 of incubation.
Isoflurane is a volatile anesthetic widely used in veterinary anesthesia. Based on Wojtczak’s observations of hemodynamic effects of isoflurane on chick embryos, we determined optimum exposure times for all days of incubation investigated. Isoflurane was more useful than both the UC anesthetic and Avertin. It was well tolerated, and during all experiments, only one of the chick embryos died. The major advantage of using isoflurane was that no holes had to be drilled into the eggshell, thus minimizing handling effort and mechanical stress. As isoflurane is known to be a very short-acting anesthetic, continuous supply for image acquisition exceeding 15 min was required. Compared to Avertin or the UC anesthetic, isoflurane was the best choice for anesthetizing chick embryos in ovo. The high tolerability of isoflurane made it also possible to repeatedly anesthetize chick embryos in ovo at each day of incubation without killing them, a primary requirement for in vivo studies with multiple CT measurements. No abnormal morphological changes during embryogenesis were observed upon repeated exposure to X-rays, to isoflurane, or to its carrier gas, pure oxygen.
Conclusion
In conclusion, our results show that it is feasible to anesthetize chick embryos in ovo and that anesthesia was an absolute prerequisite for the acquisition of motion-artifact-free images by means of molecular imaging like CT and PET. Regarding tolerability and general handling, isoflurane outperformed both the UC anesthetic and Avertin.