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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
... 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.
... 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.
... To obtain high-resolution images, motion artifacts due to sudden movements of the chick embryo should be prevented. Anesthetics have been delivered to the chick embryo through fumigation or topical administration [98][99][100]. Alternatively, eggs could be pre-cooled at 4°C for at least 1 hour prior to imaging [83]. ...
Introduction: Advancements in cancer management and treatment are associated with strong preclinical research data, in which reliable cancer models are demanded. Indeed, inconsistent preclinical findings and stringent regulations following the 3Rs principle of reduction, refinement, and replacement of conventional animal models currently pose challenges in the development and translation of efficient technologies. The chick embryo chorioallantoic membrane (CAM) is a system for the evaluation of treatment effects on the vasculature, therefore suitable for studies on angiogenesis. Apart from vascular effects, the model is now increasingly employed as a preclinical cancer model following tumor-grafting procedures.
Areas covered: The broad application of CAM tumor model is highlighted along with the methods for analyzing the neoplasm and vascular system. The presented and cited investigations focus on cancer biology and treatment, encompassing both conventional and emerging nanomaterial-based modalities.
Expert opinion: The CAM tumor model finds increased significance given the influences of angiogenesis and the tumor microenvironment in cancer behavior, then providing a qualified miniature system for oncological research. Ultimately, the establishment and increased employment of such a model may resolve some of the limitations present in the standard preclinical tumor models, thereby redefining the preclinical research workflow.
... Anesthesia was given topically to the CAM using medetomidine solution 1 mg/mL diluted to 1:100 in saline (0.9% NaCl) at a dosage of 0.3 mg/kg. as described by Waschkies et al. 24 Three types of the blood vessels differing in diameter were identified: a primary vessel (i.e. the largest vessel), second-order vessels branching from the primary vessel, and third-order vessels branching from second-order vessels. 15 A precise micrometer was used to measure vessel diameter with the assistance of video-editing software (Fig. 2). ...
Objective
Previous research has shown that effective application of angiolytic lasers in microlaryngeal surgery is determined by wavelength, pulse width (PW), and fluence. Recently, a 445‐nm (blue) laser (BL) has been developed with a potentially greater hemoglobin absorption than previous lasers. The chick chorioallantoic membrane (CAM) represents a suitable model for testing various settings to find out the most optimal settings of this laser. This study used the CAM model to examine whether successful photoangiolytic effects could be obtained using BL.
Methods
Seven hundred and ninety three third‐order vascular segments of viable CAM were irradiated using BL via 400‐μm diameter fiber, 1 pulse/second, with PW and power varied systematically at standardized fiber‐to‐vessel distances of 1 and 3 mm. Outcome measures including vessel ablation rate (AR), rupture rate (RR), and visible tissue effects were analyzed using Chi‐square test.
Results
Energy levels of 400, 540, and 600 mJ (per pulse) were most effective for vessel ablation. A working distance of 3 mm resulted in higher ablation and less vessel rupture compared with 1 mm at these optimal energy levels. At 3 mm, a longer PW resulted in higher AR. At 1 mm, AR increased with shorter PW and higher power. The 1‐mm working distance resulted in lower tissue effects than 3 mm.
Conclusion
Findings in this study showed that BL was effective in vessel ablation using relevant combination of working distance, PW, and energy levels. To obtain high AR, longer working distance plus longer PW was required and if working distance was reduced, shorter PW should be set.
Level of Evidence
NA. Laryngoscope, 2021
... At the time of writing, no studies could be found reporting the change, if any, in vascular resistance to topical ketamine on the CAM; however, the change in vascular resistance should be equal in all test subjects if a consistent method is used, allowing comparisons between materials on the CAM to be made. 151 The injection of contrast agent in this study also necessitated the use of a surgical microscope, which may not be accessible for all researchers. Woloszyk et al. 149 MRI in ovo using gadolinium-based contrast agent and µCT post-perfusion with 'Microfil' to provide an overall 3D assessment of functional blood flow and quantification of vascularisation. ...
... Histology was used in this study to give further detail at the cellular level, allowing a complete assessment of the interaction and angiogenic response of the CAM to the scaffolds. Medetomidine was used by Woloszyk et al. 149 to sedate the chicks in ovo for MRI in this study, as it has been found by Waschkies et al. 151 that medetomidine (0.3 mg/kg) applied topically to the CAM was more effective than ketamine/midazolam or thiopental at reducing movement of the chick, and therefore motion artefacts, for 30 min which was sufficient time to generate MRI images safely. ...
The chick chorioallantoic membrane (CAM) model has been around for over a century, applied in angiogenic, oncology, dental and xenograft research. Despite its often perceived archaic, redolent history, the CAM assay offers new and exciting opportunities for material and growth factor evaluation in bone tissue engineering. Currently, superior/improved experimental methodology for the CAM assay are difficult to identify, given an absence of scientific consensus in defining experimental approaches including, timing of inoculation with materials and the analysis of results. In addition, critically, regulatory and welfare issues impact upon experimental designs. Given such disparate points, this review details recent research using the ex vivo CAM assay and ex vivo organotypic culture to advance the field of bone tissue engineering and, highlights potential areas of improvement for their application based on recent developments within our group and the tissue engineering field.
