scholarly journals The Chorioallantoic Membrane Assay in Nanotoxicological Research—An Alternative for In Vivo Experimentation

Nanomaterials ◽  
2020 ◽  
Vol 10 (12) ◽  
pp. 2328
Author(s):  
Christoph R. Buhr ◽  
Nadine Wiesmann ◽  
Rachel C. Tanner ◽  
Jürgen Brieger ◽  
Jonas Eckrich
Keyword(s):  
Imaging Techniques ◽  
Chorioallantoic Membrane ◽  
Drug Carriers ◽  
Human Organism ◽  
Cam Assay ◽  
In Ovo ◽  
In Vivo Models ◽  
Toxicological Profile ◽  
Application Fields

Nanomaterials unveil many applicational possibilities for technical and medical purposes, which range from imaging techniques to the use as drug carriers. Prior to any human application, analysis of undesired effects and characterization of their toxicological profile is mandatory. To address this topic, animal models, and rodent models in particular, are most frequently used. However, as the reproducibility and transferability to the human organism of animal experimental data is increasingly questioned and the awareness of animal welfare in society increases at the same time, methodological alternatives are urgently required. The chorioallantoic membrane (CAM) assay is an increasingly popular in ovo experimental organism suitable for replacement of rodent experimentation. In this review, we outline several application fields for the CAM assay in the field of nanotoxicology. Furthermore, analytical methods applicable with this model were evaluated in detail. We further discuss ethical, financial, and bureaucratic aspects and benchmark the assay with other established in vivo models such as rodents.

scholarly journals Monitoring of tumor growth and vascularization with repetitive ultrasonography in the chicken chorioallantoic-membrane-assay

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Jonas Eckrich ◽  
Philipp Kugler ◽  
Christoph Raphael Buhr ◽  
Benjamin Philipp Ernst ◽  
Simone Mendler ◽  
...  
Keyword(s):  
Tumor Growth ◽  
Tumor Imaging ◽  
Imaging Techniques ◽  
Chorioallantoic Membrane ◽  
Cam Assay ◽  
In Ovo ◽  
Tumor Research ◽  
Xenograft Models ◽  
Cost Efficient

Abstract The chorioallantoic-membrane (CAM)-assay is an established model for in vivo tumor research. Contrary to rodent-xenograft-models, the CAM-assay does not require breeding of immunodeficient strains due to native immunodeficiency. This allows xenografts to grow on the non-innervated CAM without pain or impairment for the embryo. Considering multidirectional tumor growth, limited monitoring capability of tumor size is the main methodological limitation of the CAM-assay for tumor research. Enclosure of the tumor by the radiopaque eggshell and the small structural size only allows monitoring from above and challenges established imaging techniques. We report the eligibility of ultrasonography for repetitive visualization of tumor growth and vascularization in the CAM-assay. After tumor ingrowth, ultrasonography was repetitively performed in ovo using a commercial ultrasonographic scanner. Finally, the tumor was excised and histologically analyzed. Tumor growth and angiogenesis were successfully monitored and findings in ultrasonographic imaging significantly correlated with results obtained in histological analysis. Ultrasonography is cost efficient and widely available. Tumor imaging in ovo enables the longitudinal monitoring of tumoral development, yet allowing high quantitative output due to the CAM-assays simple and cheap methodology. Thus, this methodological novelty improves reproducibility in the field of in vivo tumor experimentation emphasizing the CAM-assay as an alternative to rodent-xenograft-models.

scholarly journals Extending the Applicability of In Ovo and Ex Ovo Chicken Chorioallantoic Membrane Assays to Study Cytostatic Activity in Neuroblastoma Cells

2021 ◽  
Vol 11 ◽  
Author(s):  
Miguel Angel Merlos Rodrigo ◽  
Berta Casar ◽  
Hana Michalkova ◽  
Ana Maria Jimenez Jimenez ◽  
Zbynek Heger ◽  
...  
Keyword(s):  
High Throughput Screening ◽  
Neuroblastoma Cells ◽  
Chorioallantoic Membrane ◽  
Stage Iv ◽  
Metastatic Potential ◽  
Significant Inhibition ◽  
Cam Assay ◽  
Anticancer Activities ◽  
In Ovo

PurposeThe chick chorioallantoic membrane (CAM) assay can provide an alternative versatile, cost-effective, and ethically less controversial in vivo model for reliable screening of drugs. In the presented work, we demonstrate that CAM assay (in ovo and ex ovo) can be simply employed to delineate the effects of cisplatin (CDDP) and ellipticine (Elli) on neuroblastoma (Nbl) cells in terms of their growth and metastatic potential.MethodsThe Nbl UKF-NB-4 cell line was established from recurrent bone marrow metastases of high-risk Nbl (stage IV, MYCN amplification, 7q21 gain). Ex ovo and in ovo CAM assays were optimized to evaluate the antimetastatic activity of CDDP and Elli. Immunohistochemistry, qRT-PCR, and DNA isolation were performed.ResultsEx ovo CAM assay was employed to study whether CDDP and Elli exhibit any inhibitory effects on growth of Nbl xenograft in ex ovo CAM assay. Under the optimal conditions, Elli and CDDP exhibited significant inhibition of the size of the primary tumor. To study the efficiency of CDDP and Elli to inhibit primary Nbl tumor growth, intravasation, and extravasation in the organs, we adapted the in ovo CAM assay protocol. In in ovo CAM assay, both studied compounds (CDDP and Elli) exhibited significant (p < 0.001) inhibitory activity against extravasation to all investigated organs including distal CAM.ConclusionsTaken together, CAM assay could be a helpful and highly efficient in vivo approach for high-throughput screening of libraries of compounds with expected anticancer activities.

scholarly journals Monitoring of Tumor Growth and Vascularization with Repetitive Ultrasonography in the Chicken Chorioallantoic-Membrane-Assay.

2020 ◽  
Author(s):  
Jonas Eckrich ◽  
Philipp Kugler ◽  
Christoph Raphael Buhr ◽  
Benjamin Philipp Ernst ◽  
Simone Mendler ◽  
...  
Keyword(s):  
Tumor Growth ◽  
Tumor Angiogenesis ◽  
Imaging Techniques ◽  
Tumor Development ◽  
Three Dimensional ◽  
Chorioallantoic Membrane ◽  
Shielding Effect ◽  
Cam Assay ◽  
In Ovo ◽  
Cost Efficient

Abstract Background: The chorioallantoic-membrane (CAM)-assay is used for versatile experimentation and eligible for the analysis of tumor angiogenesis, development and metastasis. In contrast to rodent xenograft models, the CAM-assay does not require breeding of immunodeficient strains for tumor experimentation due to native immunodeficiency. This allows xenografts to grow on the non-innervated CAM without pain or impairment for the embyo.Taking into account the variability of multidirectional tumor growth, limited size monitoring capability is a major disadvantage of the CAM-assay as the enclosure of the tumor in ovo by the eggshell only allows for two-dimensional monitoring from above. The small size and the eggshell’s shielding effect further challenge established imaging techniques. We report the eligibility of ultrasonographic imaging for repetitive monitoring of tumor growth and vascularisation in the CAM-assay.Methods: Chicken eggs were placed in an incubator and cut open laterally on day three. On day seven a three-dimensional tumor was placed onto the CAM. Ultrasonographic imaging was then repetitively performed starting from day twelve. On day 14 the tumor was excised, fixed and histologically analyzed using light microscopy.Results: Tumor volume and vascularization were repetitively visualized using a commercial ultrasonographic scanner, allowing a longitudinal monitoring of tumor growth and tumor angiogenesis. Findings in ultrasonographic imaging significantly correlated with results obtained in histological analysis. Conclusion: Ultrasonography is cost efficient and widely available. It allows repetitive in ovo imaging and thereby enables visualization of tumor development. This increases the applicability of the CAM-assay as an alternative to xenograft rodent models in tumor research.

scholarly journals Depletion of Embryonic Macrophages Leads to a Reduction in Angiogenesis in the Ex Ovo Chick Chorioallantoic Membrane Assay

Cells ◽  
2020 ◽  
Vol 10 (1) ◽  
pp. 5
Author(s):  
Hanna Tay ◽  
Charis Du Cheyne ◽  
Kristel Demeyere ◽  
Jurgen De Craene ◽  
Lobke De Bels ◽  
...  
Keyword(s):  
Chorioallantoic Membrane ◽  
Collagen Type I ◽  
Type I ◽  
Cam Assay ◽  
In Vivo Models ◽  
Chick Chorioallantoic Membrane ◽  
Angiogenesis Assay ◽  
Fold Reduction ◽  
Phosphate Buffered Saline

Macrophages play an important but poorly understood role in angiogenesis. To investigate their role in vessel formation, relevant in vivo models are crucial. Although the chick chorioallantoic membrane (CAM) model has been frequently used as an angiogenesis assay, limited data are available on the involvement of chicken macrophages in this process. Here, we describe a method to deplete macrophages in the ex ovo chick CAM assay by injection of clodronate liposomes and show that this depletion directly affects vascularisation of collagen onplants. Chicken embryos were injected intravenously with either clodronate or phosphate-buffered saline (PBS) liposomes, followed by placement of collagen type I plugs on the CAM to quantify angiogenic ingrowth. Clodronate liposome injection led to a significant 3.4-fold reduction of macrophages compared with control embryos as measured by immunohistochemistry and flow cytometry. Furthermore, analysis of vessel ingrowth into the collagen plugs revealed a significantly lower angiogenic response in macrophage-depleted embryos compared with control embryos, indicating that chicken embryonic macrophages play an essential function in the development of blood vessels. These results demonstrate that the chick CAM assay provides a promising model to investigate the role of macrophages in angiogenesis.

scholarly journals The Role of Pre-Clinical 3-Dimensional Models of Osteosarcoma

2020 ◽  
Vol 21 (15) ◽  
pp. 5499
Author(s):  
Hannah L. Smith ◽  
Stephen A. Beers ◽  
Juliet C. Gray ◽  
Janos M. Kanczler
Keyword(s):  
Three Dimensional ◽  
Chorioallantoic Membrane ◽  
3D Models ◽  
In Ovo ◽  
Future Directions ◽  
3 Dimensional ◽  
In Vivo Animal Models

Treatment for osteosarcoma (OS) has been largely unchanged for several decades, with typical therapies being a mixture of chemotherapy and surgery. Although therapeutic targets and products against cancer are being continually developed, only a limited number have proved therapeutically active in OS. Thus, the understanding of the OS microenvironment and its interactions are becoming more important in developing new therapies. Three-dimensional (3D) models are important tools in increasing our understanding of complex mechanisms and interactions, such as in OS. In this review, in vivo animal models, in vitro 3D models and in ovo chorioallantoic membrane (CAM) models, are evaluated and discussed as to their contribution in understanding the progressive nature of OS, and cancer research. We aim to provide insight and prospective future directions into the potential translation of 3D models in OS.

Nerve-dependent accumulation of myosin light chain 3 in developing limb musculature

Development ◽  
1987 ◽  
Vol 101 (4) ◽  
pp. 673-684
Author(s):  
P.A. Merrifield ◽  
I.R. Konigsberg
Keyword(s):  
Neural Tube ◽  
Light Chain ◽  
Limb Development ◽  
Myosin Light Chain ◽  
Chorioallantoic Membrane ◽  
In Ovo ◽  
Fast Myosin ◽  
Limb Musculature

Myosin alkali light chain accumulation in developing quail limb musculature has been analysed on immunoblots using a monoclonal antibody which recognizes an epitope common to fast myosin light chain 1 (MLC1f) and fast myosin light chain 3 (MLC3f). The limb muscle of early embryos (i.e. up to day 10 in ovo) has a MLC profile similar to that observed in myotubes cultured in vitro; although MLC1f is abundant, MLC3f cannot be detected. MLC3f is first detected in 11-day embryos. To determine whether this alteration in MLC3f accumulation is nerve or hormone dependent, limb buds with and without neural tube were cultured as grafts on the chorioallantoic membrane of chick hosts. Although differentiated muscle develops in both aneural and innervated grafts, innervated grafts contain approximately three times as much myosin as aneural grafts. More significantly, although aneural grafts reproducibly accumulate normal levels of MLC1f, they fail to accumulate detectable levels of MLC3f. In contrast, innervated grafts accumulate both MLC1f and MLC3f, suggesting that the presence of neural tube in the graft promotes the maturation, as well as the growth, of muscle tissue. This is the first positive demonstration that innervation is necessary for the accumulation of MLC3f that occurs during normal limb development in vivo.

scholarly journals In Vivo Modulation of Angiogenesis and Immune Response on a Collagen Matrix via Extracorporeal Shockwaves

2020 ◽  
Vol 21 (20) ◽  
pp. 7574 ◽  
Author(s):  
Diana Heimes ◽  
Nadine Wiesmann ◽  
Jonas Eckrich ◽  
Juergen Brieger ◽  
Stefan Mattyasovszky ◽  
...  
Keyword(s):  
Gene Expression ◽  
Immune Response ◽  
Chorioallantoic Membrane ◽  
Extracorporeal Shock Wave Therapy ◽  
Collagen Matrix ◽  
Cam Assay ◽  
Immunological Responses ◽  
Tissue Integration ◽  
Extracorporeal Shock Wave

The effective management of tissue integration and immunological responses to transplants decisively co-determines the success of soft and hard tissue reconstruction. The aim of this in vivo study was to evaluate the eligibility of extracorporeal shock wave therapy (ESWT) with respect to its ability to modulate angiogenesis and immune response to a collagen matrix (CM) for tissue engineering in the chorioallantoic membrane (CAM) assay, which is performed with fertilized chicken eggs. CM were placed on the CAM on embryonic development day (EDD) 7; at EDD-10, ESWT was conducted at 0.12 mJ/mm2 with 500 impulses each. One and four days later, angiogenesis represented by vascularized area, vessel density, and vessel junctions as well as HIF-1α and VEGF gene expression were evaluated. Furthermore, immune response (iNOS2, MMP-9, and MMP-13 via qPCR) was assessed and compared between ESWT- and non-ESWT-groups. At EDD-14, the vascularized area (+115% vs. +26%) and the increase in vessel junctions (+751% vs. +363%) were significantly higher in the ESWT-group. ESWT significantly increased MMP-9 gene expression at EDD-11 and significantly decreased MMP-13 gene expression at EDD-14 as compared to the controls. Using the CAM assay, an enhanced angiogenesis and neovascularization in CM after ESWT were observed. Furthermore, ESWT could reduce the inflammatory activity after a latency of four days.

scholarly journals Automated Segmentation and Object Classification of CT Images: Application toIn VivoMolecular Imaging of Avian Embryos

2013 ◽  
Vol 2013 ◽  
pp. 1-10 ◽  
Author(s):  
Alexander Heidrich ◽  
Jana Schmidt ◽  
Johannes Zimmermann ◽  
Hans Peter Saluz
Keyword(s):  
Image Analysis ◽  
Image Segmentation ◽  
Bone Growth ◽  
Imaging Techniques ◽  
Long Bone ◽  
Automated Segmentation ◽  
Chick Embryos ◽  
In Ovo

Background. Although chick embryogenesis has been studied extensively, there has been growing interest in the investigation of skeletogenesis. In addition to improved poultry health and minimized economic loss, a greater understanding of skeletal abnormalities can also have implications for human medicine. Truein vivostudies require noninvasive imaging techniques such as high-resolution microCT. However, the manual analysis of acquired images is both time consuming and subjective.Methods. We have developed a system for automated image segmentation that entails object-based image analysis followed by the classification of the extracted image objects. For image segmentation, a rule set was developed using Definiens image analysis software. The classification engine was implemented using the WEKA machine learning tool.Results. Our system reduces analysis time and observer bias while maintaining high accuracy. Applying the system to the quantification of long bone growth has allowed us to present the first truein ovodata for bone length growth recorded in the same chick embryos.Conclusions. The procedures developed represent an innovative approach for the automated segmentation, classification, quantification, and visualization of microCT images. MicroCT offers the possibility of performing longitudinal studies and thereby provides unique insights into the morpho- and embryogenesis of live chick embryos.

scholarly journals Evaluation of the Anti-angiogenic Activity of Saponins from Maesa lanceolata by Different Assays

2012 ◽  
Vol 7 (9) ◽  
pp. 1934578X1200700
Author(s):  
Kenn Foubert ◽  
Annelies Breynaert ◽  
Mart Theunis ◽  
Rita Van Den Bossche ◽  
Guido R.Y. De Meyer ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Chorioallantoic Membrane ◽  
Rat Aorta ◽  
Cam Assay ◽  
Plant Metabolites ◽  
Angiogenic Activity ◽  
Test Systems ◽  
Aorta Ring

Angiogenesis, in which a vascular network is established from pre-existing vessels, is a complex multistep process. Mechanisms underlying angiogenesis can be investigated using a variety of in vitro, ex vivo and in vivo approaches. Evaluation of several promising plants and plant metabolites, including terpenoids, revealed promising anti-angiogenic activity. Since the maesasaponins displayed anti-angiogenic activity in the chick chorioallantoic membrane (CAM) assay, their activity was further investigated in several test systems. The rat aorta ring assay was compared with the placental vein assay and then selected for the ex vivo investigation of the saponins. Besides their effect on the viability of HUVEC, the anti-angiogenic capacity of the compounds was also investigated in an in vivo zebrafish assay. The activity of the saponins in the viability assay was more pronounced than in the rat aorta ring assay and similar to the effect observed in the CAM assay. The use of different test systems, however, implies different results in the case of saponins.

scholarly journals Nymphaeol-A Isolated from Okinawan Propolis Suppresses Angiogenesis and Induces Caspase-Dependent Apoptosis via Inactivation of Survival Signals

2013 ◽  
Vol 2013 ◽  
pp. 1-9 ◽  
Author(s):  
Ikumi Tsuchiya ◽  
Takahiro Hosoya ◽  
Motoko Ushida ◽  
Kazuhiro Kunimasa ◽  
Toshiro Ohta ◽  
...  
Keyword(s):  
Screening Program ◽  
Biological Activities ◽  
Umbilical Vein ◽  
Chorioallantoic Membrane ◽  
Tube Formation ◽  
Mitogen Activated Protein Kinase ◽  
Cam Assay ◽  
Prenylated Flavonoids

Propolis, a resinous substance that honeybees collect to protect their beehive from enemies, is reported to have various biological activities. In our screening program to search for antiangiogenic compounds from propolis, the ethanol extracts of Okinawan propolis (EEOP) showed significant antiangiogenic activities in a tube formation assay with human umbilical vein endothelial cells (HUVECs)in vitroat 3.13 μg/mL and chorioallantoic membrane (CAM) assayin vivoat 25 μg/egg. To elucidate the active compounds of EEOP and their mode of action, we isolated some prenylated flavonoids from EEOP and found that nymphaeol-A had the strongest antiangiogenic activity among them. Nymphaeol-A significantly reducedin vivoneovessel formation in the CAM assay at 25 μg/egg. At the molecular level, nymphaeol-A markedly inactivated mitogen-activated protein kinase/ERK kinase 1/2 (MEK1/2) and extracellular signal-regulated kinase 1/2 (ERK1/2), whose molecular activations signal new vessel formation in HUVECs. In addition, nymphaeol-A dose- and time-dependently induced caspase-dependent apoptosis in tube-forming HUVECs. Taken together, nymphaeol-A was shown to inhibit angiogenesis at least in part via inactivation of MEK1/2–ERK1/2 signaling and induction of caspase-dependent apoptosis. Okinawan propolis and its major component, nymphaeol-A, may be useful agents for preventing tumor-induced angiogenesis.

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