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Two in ovo T1-weighted MR images of the chick embryo on ID 14, seven days post-implantation on the CAM acquired in an axial slice through the biomaterial scaffold; (a) image without motion artifact and (b) image with motion artifacts that was excluded from further analysis.:
Source publication
Non-invasive assessment of the perfusion capacity of tissue engineered constructs grown on the chorioallantoic membrane by MRI is often hampered by motion artifacts. Therefore, we examined the suitability of three anesthetic regimes for sufficient sedation of the chick embryo. Medetomidine at a dosage of 0.3 mg/kg, was compared to thiopental at 100...
Context in source publication
Context 1
... a sedative effect was clearly observed in our study, residual motion of the chick embryo was not judged sufficient for MRI, as gross motion and strong kicks were still persistent throughout the experiment. /kg and 1 mg/kg, and thiopental at 100 mg/kg, respectively (n = 7). Scores were assigned during each 4 min of videotaping, immediately prior anesthesia (0 min), and every 10 min during anesthesia for one hour and immediately after antagonization where applicable (70 min). ...
Citations
... Anesthesia should be administered during procedures for embryos at developmental stages above ED13 [36,37]. Anesthetic protocols for younger embryos are reported to induce immobilization and avoid motion artifacts during imaging studies [38,39]. ...
... For example, to obtain good quality images, motion-induced image artifacts should be avoided, and this is usually obtained by placing the egg in ice chips between 10 and 90 min. Waschkies et al. [39] further explored the immobilization of the CE, comparing three anesthetic regimes consisting of dropping onto the surface of the CAM medetomidine at a dosage of 0.3 mg/kg, thiopental at 100 mg/kg, and ketamine/midazolam at 50 mg/kg and 1 mg/kg. The study resulted in better performances for medetomidine in terms of reduced motion, time of onset of anesthesia, and anesthesia duration. ...
The chicken embryo has emerged as a valuable model for preclinical studies due to its unique combination of accessibility, affordability, and relevance to human biology. Its rapid development, external growth environment, and clear structural visibility offer distinct advantages over traditional mammalian models. These features facilitate the study of real-time biological processes, including tissue development, tumor growth, angiogenesis, and drug delivery, using various imaging modalities, such as optical imaging, magnetic resonance imaging, positron emission tomography, computed tomography, and ultrasound. The chicken embryo model also minimizes ethical concerns compared to mammalian models, as it allows for early-stage research without the complexity of a fully developed animal. Moreover, its ability to integrate human tumor cells into xenograft models provides a reliable platform for cancer research, enabling high-throughput screening of therapeutic interventions and tracking molecular dynamics in vivo. Advances in molecular imaging techniques further enhance the resolution and depth of data obtained from these studies, offering insights into cellular and molecular mechanisms underlying disease. Given its versatility, cost-effectiveness, and translational potential, the chicken embryo represents a promising tool for advancing preclinical research, particularly in drug development, cancer biology, and regenerative medicine.
... Utilising chick embryos offers simplicity in screening and preparation requirements. This is also coupled with distinct advantages including easy accessibility to their chorioallantoic membranes (CAM), rapid growth, cost-effectiveness, nutritional independence and shared phylogenetic characteristics with mammals [4][5][6][7][8][9]. ...
Goal: This study presents a novel MRI coil design approach explicitly tailored for chick embryo measurements, with the primary objective of improving sensitivity and coverage. Methods: The limitations posed by conventional birdcage coils were addressed by introducing a curvature feature into a standard coil. The performance of the modified coil was assessed using EM simulations and experimental evaluations, which were subsequently validated using a 7 T MRI scanner. A comparative analysis was conducted against a standard quadrature low-pass birdcage coil to evaluate key factors. Results: The proposed coil demonstrated improved SNR and uniformity, particularly in the proximity of the end-rings. These results were consistent with the findings obtained from the simulations. Conclusions: The use of our innovative birdcage coil design holds promise and offers practical potential for in ovo studies.
... 18 F-FDG is rapidly renally excreted in vivo, while in ovo there is no external clearance pathway 8 , which may increase CAM blood radioactivity concentrations and therefore tumor delivery 23 . Additionally, we further optimized the embryo immobilization protocol using the liquid narcotic medetomidine as opposed to cooling, which can lower metabolic rate and other processes governing radiotracer uptake 29,32 . ...
Mouse models are invaluable tools for radiotracer development and validation. They are, however, expensive, low throughput, and are constrained by animal welfare considerations. Here, we assessed the chicken chorioallantoic membrane (CAM) as an alternative to mice for preclinical cancer imaging studies. NCI-H460 FLuc cells grown in Matrigel on the CAM formed vascularized tumors of reproducible size without compromising embryo viability. By designing a simple method for vessel cannulation it was possible to perform dynamic PET imaging in ovo, producing high tumor-to-background signal for both ¹⁸ F-2-fluoro-2-deoxy-D-glucose ( ¹⁸ F-FDG) and (4S)-4-(3- ¹⁸ F-fluoropropyl)-L-glutamate ( ¹⁸ F-FSPG). The pattern of ¹⁸ F-FDG tumor uptake were similar in ovo and in vivo, although tumor-associated radioactivity was higher in the CAM-grown tumors over the 60 min imaging time course. Additionally, ¹⁸ F-FSPG provided an early marker of both treatment response to external beam radiotherapy and target inhibition in ovo. Overall, the CAM provided a low-cost alternative to tumor xenograft mouse models which may broaden access to PET and SPECT imaging and have utility across multiple applications.
... We hypothesize that these differences are due to the immobilization protocol. Here, we used liquid narcotic medetomidine as opposed to cooling, which can lower metabolic rate and other processes governing radiotracer uptake [31,34]. ...
Purpose
Mouse models are invaluable tools for radiotracer development and validation. They are, however, expensive, low throughput, and are constrained by animal welfare considerations. Here, we assessed the chicken chorioallantoic membrane (CAM) as an alternative to mice for preclinical cancer imaging studies.
Methods
Growth of NCI-H460 Fluc tumors on the CAM was optimized using a range of physical and chemical supports. Tumor-bearing eggs were imaged by dynamic ¹⁸ F-2-fluoro-2-deoxy-D-glucose ( ¹⁸ F-FDG) or (4S)-4-(3- ¹⁸ F-fluoropropyl)-L-glutamate ( ¹⁸ F-FSPG) PET/CT following intravenous injection, with mice bearing subcutaneous NCI-H460 Fluc xenografts imaged with ¹⁸ F-FDG for comparison. The dependence of the transporter system xc ⁻ on in ovo ¹⁸ F-FSPG tumor uptake was determined through treatment with imidazole ketone erastin. Additionally, ¹⁸ F-FSPG PET/CT was used to monitor treatment response in ovo 24 h following external beam radiotherapy.
Results
NCI-H460 Fluc cells grown in Matrigel formed vascularized tumors of reproducible size without compromising embryo viability. By designing a simple method for cannulation it was possible to perform dynamic PET imaging in ovo , producing high tumor-to-background signal for both ¹⁸ F-FDG and ¹⁸ F-FSPG. ¹⁸ F-FDG tumor uptake kinetics were similar in ovo and in vivo , with ¹⁸ F-FSPG providing an early marker of both treatment response and target inhibition in CAM-grown tumors.
Conclusions
The CAM provides a low-cost alternative to tumor xenograft mouse models which may broaden access to PET and SPECT imaging. Rapid tumor growth and high-quality PET images that can be obtained with this model suggest its potential use for early radiotracer screening, pharmacological studies, and monitoring response to therapy.
... nonetheless, some studies are required to expand and improve the ce use as an experimental model. critical points on this approach are evaluating the perception of pain [8] and the difficulty of handling the CE since it moves intensively [9]. Some of these limitations can be remedied or reduced using appropriate anaesthetic protocols. ...
... in addition to the administration route, choosing the appropriate drug is extremely important. Since thiopental, for example, seems to not be a good alternative for ces [9], testing new drugs for this model is also necessary. ...
... ces remained with low heart rate until 15 min and low movement until 20 min post-inoculation ( fig. 3). Similar to our results, Waschkies et al. [9] used ketamine/ midazolam anesthesia in ces via caM at 14 di. They observed that although there was a sedative effect, they found gross movement and strong kicks throughout the experiment. ...
Chicken embryos (CE) are an experimental model used as an important life science research tool worldwide, and then, adequate anesthetic protocols must be adopted to avoid the unjustifiable suffering of animals. Thus, our objective was to evaluate different anesthetic protocols in CEs using an easy inoculation route, the shell membrane (SM). We adopted the heart rate by pulse and the CE movements as a parameter of pain by assessing the vase in the chorioallantoic membrane (CAM) through the shell by a sensor of a multiparametric monitor. CEs were distributed into the following groups: (i) association of ketamine (5 mg/CE), midazolam (0.05 mg/CE) and morphine (0.15 mg/CE); (ii) ketamine (5 mg/CE) and xylazine (0.125 mg/CE); (iii) xylazine (0.0125 mg/CE) and morphine (0.15 mg/CE). The stress method used to test the anesthetic potential of the drugs was high temperature stimulation, keeping the CEs 10 cm from the fire of a Bussen nozzle for 30 seconds. In this experimental model, associations between different drugs decreased the pulse and the movement, indicating possible sedation. After treatment, the CE’s submitted to the stress method had the heart rate and movements kept low in the groups ketamine-midazolam-morphine and ketamine-xylazine, while the non-drug-treated group increased heart rate. In a group treated with xylazine-morphine, the heart rate did not decrease, but the movement decreased after the stimulus. As the best results were the combinations of ketamine-midazolam-morphine and ketamine-xylazine, we recommend these associations for use in embryos in the final third of embryonic development in experimental protocols and euthanasia.
... Functional gas challenge is feasible on the CAM [10], when the chicken embryos are sedated with medetomidine [18]. In the present study, tumor phenotypes were characterized using quantitative T1 and T2* as readouts. ...
... Before magnetic resonance imaging, eggs were cooled at 4 • C for 90 min for sedation [17]. Sedation by medetomidine in accordance with a previously published protocol [18] was compared for two kinds of tumor grafts (A549 and MC-38) and one gas challenge (HCHO, see Supplementary Materials Figure S1). MRI was performed on ID 14, 7 days after grafting in A549 (n = 14), H460 (n = 6), and MC-38 graft samples (n = 11) on a 4.7 T cm Bruker PharmaScan system (Bruker BioSpin, Ettlingen, Germany). ...
... No significant change was revealed in either T1 or T2* in MC-38 or H460 grafts, respectively. Moreover, a comparison to medetomidine sedation from a previous study [18] with the cooling sedation regimen revealed, for A549 grafts, that T1 reduction under HCHO challenge was only measured by trend (most individual data points displayed reduced response) because of high data variability under medetomidine, whereas cooling sedation resulted in a significant reduction in T1 for these grafts under HCHO (Supplementary Materials Figure S1). In contrast, for T2* assessment, both sedation regimen resulted in a significant increase under HCHO for A549 grafts (Figure 2 and Supporting Information S2). ...
Tumor grafts grown on the chorioallantoic membrane (CAM) of chicken embryos represent a transition between cell culture and mammalian in vivo models. Magnetic resonance imaging (MRI) started to harness this potential. Functional gas challenge is feasible on the CAM. Using quantitative T1 and T2* mapping, we characterized the response of MC-38 colon, A549, and H460 adeno-carcinoma cell grafts to hypercapnic (HC) and hypercapnic-hyperoxic (HCHO) gas challenges, pertaining to the grafts’ vascular and oxygenation phenotypes. MR imaging revealed that larger T1 and T2* were located in the center of H460 and MC-38 tumors. Quantitative analysis showed a significant reduction in T1 and a significant increase in T2* in response to HCHO for A549 grafts, while H460 and MC-38 tumors did not respond to either gas challenge. Different tumor grafts respond differentially to HC and HCHO conditions. A549 tumor grafts, with higher vessel density and smaller tumor diameter compared with H460 and MC-38 grafts, had a significant response in T1 for HCHO and T2* increased slightly during HC and significantly under HCHO, consistent with a normoxic phenotype and functional vasoreactivity. Therefore, gas challenges enable differential characterization of tumor grafts with respect to their vascular and oxygenation status.
... Starting on DD 31, periodically occurring ECG-like signals were detectable immediately without post-processing. On later DD (32)(33)(34)(35)(36)(37), cardiac signals showed higher amplitudes over time (data not shown). Motion was detectable by nonperiodically, occasionally occurring signals at frequencies of 0.1-3 Hz. ...
In-ovo imaging using ostrich eggs has been described as a potential alternative to common animal testing. The main advantage is its independence from small animal imaging devices as ostrich eggs provide good image quality on regular CT, MRI, or PET used in examinations of humans. However, embryonal motion during dynamic imaging studies produce artifacts. The aims of this study were (1) to explore the feasibility of biomagnetism to detect cardiac signals and embryonal motion and to use these findings (2) to investigate the effect of isoflurane anesthesia on ostrich embryos. A standard magnetoencephalography developed for brain studies was used to detect embryonal signals of ostrich eggs on developmental day 34. Signals were instantly shown on a screen and data were also postprocessed. For assessing the effects of anesthesia, nine ostrich eggs were investigated using isoflurane 6% for 90 min. Biomagnetic signals were recorded simultaneously. A control group consisting of eight different ostrich eggs was also investigated. Cardiac signals similar to electrocardiography were observed in all eggs. Postprocessing revealed frequent motion of embryos without anesthesia. The exposure to isoflurane led to a significant decrease in motion signals in 9/9 ostrich embryos after 8 min. Motion was significantly reduced in the isoflurane group versus control group. There were no isoflurane-related deaths. This study shows that biomagnetism is feasible to detect cardiac signals and motion of ostrich embryos in-ovo. Application of isoflurane is safe and leads to a rapid decrease in embryonal motion, which is an important prerequisite for the implementation of in-ovo imaging using ostrich eggs.
... In addition to the administration route, choosing the appropriate drug is extremely important. Since thiopental, for example, seems to not be a good alternative for CEs (6), testing new drugs for this model is also necessary. ...
Our goal was to evaluate different anaesthetic protocols in CEs using an easy inoculation route via a shell membrane (SM). For this, we adopted the heart rate by pulse and movement of the CE as a parameter of pain by assessing the vase in the chorioallantoic membrane (CAM) directly in the shell. CEs were distributed into the following groups: association of ketamine (5 mg/CE), midazolam (0.05 mg/CE), and morphine (0.15 mg/CE); ketamine (5 mg/CE) and xylazine (0.125 mg/CE); xylazine (0.0125 mg/CE) and morphine (0.15 mg/CE). In this experimental model, associations between different drugs decreased the pulse and the movement, indicating possible sedation. The best result was the association of ketamine (5 mg/CE), midazolam (0.05 mg/CE), and xylazine (0.125 mg/CE) or the association of ketamine (5 mg/CE) and xylazine (0.125 mg/CE). Therefore, we recommend these associations for use in embryos in the final third of embryonic development in experimental protocols and euthanasia.
... The use of anaesthesia was shown to reduce chick embryo movements (Heidrich et al. 2011;Waschkies et al. 2015). Medetomidine has been shown to exert its effect (motion reduction) within 10 min of administration and the effect lasted for at least 30 min (Waschkies et al. 2015) and so was used in the experiment. ...
... The use of anaesthesia was shown to reduce chick embryo movements (Heidrich et al. 2011;Waschkies et al. 2015). Medetomidine has been shown to exert its effect (motion reduction) within 10 min of administration and the effect lasted for at least 30 min (Waschkies et al. 2015) and so was used in the experiment. Medetomidine HCl was purchased (VWR, USA) as a powder and was rehydrated at a concentration of 10 µg/ml of sterile phosphate buffer saline (PBS), filtered through 0.22 µm acrodisc filters, and 100 µl of this solution (containing 1 µg of medetomidine) was injected per embryo into the air cell through the blunt end. Figure 1. ...
1. In developmental embryology in chickens, the cardiovascular system is the first to become functional, the first heart muscular contraction (beat) happens as early as 33 h of incubation of a developmental journey that takes 21 d.
2. An electrocardiogram (ECG) recording system (IX-TA 220) has been used to record the ECG of various species. The following trial describes the use of such a system for recording electrical tracing of the developing heart in chick embryos on d 19 of embryonic development with the electrodes piercing the eggshell in specific locations to a depth of about 2 mm. The recorded ECG offers an opportunity to measure or calculate ECG parameters like those measured/calculated in humans.
3. The use of anaesthesia substantially reduced embryo motion, but may have a transient tachycardia effect on heart rate.
4. This is the first time such a system has been successfully used for measuring heart electrical activities in chick embryos and provides a broader research opportunity in chicken embryo cardio-physiology.
... A more detailed analysis may be provided by additional simulations in a PBPK model. If cooling affects internalization and pharmacokinetics of the ligand, alternative immobilization techniques such as isoflurane vaporization or application of liquid narcotics to the surface may be considered [45,[51][52][53]. ...
Assessment of biodistribution and specific tumor accumulation is essential for the development of new radiopharmaceuticals and requires animal experiments. The HET-CAM (hens-egg test—chorioallantoic membrane) model can be used in combination with the non-invasive imaging modalities PET and MRI for pre-selection during radiopharmaceutical development to reduce the number of animal experiments required. Critical to the acceptance of this model is the demonstration of the quantifiability and reproducibility of these data compared to the standard animal model. Tumor accumulation and biodistribution of the PSMA-specific radiotracer [18F]F-siPSMA-14 was analyzed in the chick embryo and in an immunodeficient mouse model. Evaluation was based on MRI and PET data in both models. γ-counter measurements and histopathological analyses complemented these data. PSMA-specific accumulation of [18F]F-siPSMA-14 was successfully demonstrated in the HET-CAM model, similar to the results obtained by mouse model studies. The combination of MR and PET imaging allowed precise quantification of peptide accumulation, initial assessment of biodistribution, and accurate determination of tumor volume. Thus, the use of the HET-CAM model is suitable for the pre-selection of new radiopharmaceuticals and potentially reduces animal testing in line with the 3Rs principles of animal welfare.
