Ultra high frequency ultrasound enables real-time visualization of blood supply from chorioallantoic membrane to human autosomal dominant polycystic kidney tissue.

Ultra high frequency (UHF) ultrasound enables the visualization of very small structures that cannot be detected by conventional ultrasound. The utilization of UHF imaging as a new imaging technique for the 3D-in-vivo chorioallantoic membrane (CAM) model can facilitate new insights into tissue perfusion and survival. Therefore, human renal cystic tissue was grafted onto the CAM and examined using UHF ultrasound imaging. Due to the unprecedented resolution of UHF ultrasound, it was possible to visualize microvessels, their development, and the formation of anastomoses. This enabled the observation of anastomoses between human and chicken vessels only 12 h after transplantation. These observations were validated by 3D reconstructions from a light sheet microscopy image stack, indocyanine green angiography, and histological analysis. Contrary to the assumption that the nutrient supply of the human cystic tissue and the gas exchange happens through diffusion from CAM vessels, this study shows that the vasculature of the human cystic tissue is directly connected to the blood vessels of the CAM and perfusion is established within a short period. Therefore, this in-vivo model combined with UHF imaging appears to be the ideal platform for studying the effects of intravenously applied therapeutics to inhibit renal cyst growth.

Phytochemical profiling, cytotoxic, anti-migration, and anti-angiogenic potential of phenolic-rich fraction from Peganum harmala: in vitro and in ovo studies.

Peganum harmala has been extensively employed in Algerian traditional medicine practices. This study aimed to explore the impact of n-butanol (n-BuOH) extract sourced from Peganum harmala seeds on cell proliferation, cell migration, and angiogenesis inhibition. Cytotoxic potential of n-BuOH extract was evaluated using MTT (3-(4,5-dimethylthiazol-2-yl) 2,5 diphenyltetrazolium bromide) assay against human breast adenocarcinoma MCF-7 cells, cell migration was determined using scratch assay, and anti-angiogenic effect was evaluated through macroscopic and histological examinations conducted on chick embryo chorioallantoic membrane. Additionally, this research estimated the phytochemical profile of n-BuOH extract. Fifteen phenolic compounds were identified using Ultra-performance liquid chromatography UPLC-ESI-MS-MS analysis. In addition, the n-BuOH extract of P. harmala exhibited potent antioxidant and free radical scavenging properties. The n-BuOH extract showed potent cytotoxicity against MCF-7 cell with an IC50 value of 8.68 ± 1.58 µg/mL. Furthermore, n-BuOH extract significantly reduced migration. A strong anti-angiogenic activity was observed in the groups treated with n-BuOH extract in comparison to the negative control. Histological analysis confirmed the anti-angiogenic effect of the n-BuOH extract. This activity is probably a result of the synergistic effects produced by different polyphenolic classes.

Chemical composition and anti-angiogenic activity of the essential oil from Blumea eriantha D.C.

Blumea eriantha D.C is a weed from Asteraceae family and is reported to have anticancer activity. The essential oil from the aerial parts was extracted by steam distillation method with the yield of 0.36%. Through GC-MS analysis of the oil, seventeen compounds could be identified by comparing with linear retention indices with the library. Out of the seventeen compounds ß-Caryophylline oxide was isolated by column chromatography with gradient elution and the structure was determined through FT-IR, MS, 1HNMR, 13 C NMR and DEPT. The oil was evaluated for its effect on angiogenesis using Chorioallantoic Membrane Assay (CAM Assay). The concentration dependent antiangiogenic effect was observed with IC 50 value of 19.28 ppm.

Immunometric and functional measurement of endogenous vasoinhibin in human sera.

Introduction: Circulating levels of the antiangiogenic protein vasoinhibin, a fragment of prolactin, are of interest in vasoproliferative retinopathies, preeclampsia, and peripartum cardiomyopathy; however, it is difficult to determine the circulating levels of vasoinhibin due to the lack of quantitative assays. Methods: This study used human serum samples to assess the concentration and bioactivity of vasoinhibin using a novel enzyme-linked immunosorbent assay (ELISA) for human vasoinhibin, which employs an anti-vasoinhibin monoclonal antibody, a human umbilical vein endothelial cell (HUVEC) proliferation assay, and a chick chorioallantoic membrane (CAM) angiogenesis assay. Results: Serum samples from 17 pregnant women without (one group) and with preeclampsia and pregnancy induced hypertension (another group) demonstrated endogenous vasoinhibin concentrations in the range of 5-340 ng/ml. Immunoactive vasoinhibin levels were significantly higher in preeclampsia serum compared to healthy pregnancy serum (mean 63.09 ± 22.15 SD vs. 19.67 ± 13.34 ng/ml, p = 0.0003), as was the bioactive vasoinhibin level as determined by the HUVEC proliferation assay (56.12 ± 19.83 vs. 13.38 ± 4.88 ng/ml, p < 0.0001). There was a correlation between the concentration of vasoinhibin measured by ELISA and the HUVEC proliferation assay (Pearson r = 0.95, p < 0.0001). Healthy serum demonstrated a proangiogenic effect in the CAM assay (p < 0.05, compared to control), while serum from preeclamptic patients demonstrated an antiangiogenic effect (p < 0.05 vs. control), as did recombinant human vasoinhibin and a synthetic circular retro-inverse vasoinhibin analogue (CRIVi45-51). The antiangiogenic effects in the CAM assay and the inhibition of HUVEC proliferation were abolished by addition of the ELISA anti-vasoinhibin monoclonal antibody, but not by mouse IgG. Discussion: These results demonstrate the first quantitation of endogenous vasoinhibin in human sera and the elevation of it levels and antiangiogenic activity in sera from women with preeclampsia. The development and implementation of a quantitative assay for vasoinhibin overcomes a long-standing barrier and suggests the thorough clinical verification of vasoinhibin as a relevant biomarker.

Evaluating the effects of various ethanolic medicinal plant extracts on metastatic breast cancer proliferation, invasion, and expression of a novel potential drug target; CD82 metastatic suppressor protein, and on in vivo angiogenesis using the ex ovo yolk sac membrane (YSM) assay.

PURPOSE: Breast cancer metastasis relies on cellular invasion and angiogenesis facilitated by the downregulation of metastatic suppressor proteins like Cluster of Differentiation 82 (CD82). Currently, no medicines target multiple systems to prevent metastatic progression through CD82 upregulation. This study screened for plant extracts displaying effects on cell proliferation, invasion, and CD82 expression in breast cancer cells, and in vivo angiogenesis, and further correlated between the biological activities and effect on CD82 expression. METHODS: Seventeen ethanolic plant extracts were screened for their effect on cell proliferation (against MDA-MB-231 and MCF-7 breast cancer and Hek293 kidney cells), cell invasion and effect on CD82 expression in metastatic MDA-MB-231 cells. Selected extracts were further evaluated for in vivo anti-angiogenesis. RESULTS: Extracts displayed varying antiproliferative activity against the different cell lines, and those that showed selectivity indexes (SI) > 0.5 against MDA-MB-231 were selected for anti-invasion evaluation. Buddleja saligna Willd. (BS), Combretum apiculatum Sond. (CA), Foeniculum vulgare, Greyia radlkoferi, Gunnera perpensa and Persicaria senegalensis (Meisn.) Soják (PS) displayed 50% inhibitory concentration (IC50) values of 44.46 ± 3.46, 74.00 ± 4.48, 180.43 ± 4.51, 96.97 ± 2.29, 55.29 ± 9.88 and 243.60 ± 2.69 µg/mL, respectively against MDA-MB-231, and compared to Hek293 showed SI of 0.9, 0.7, 1.4, 1.1, 2.2 and 0.5. Significant invasion inhibition was observed at both 20 and 40 µg/mL for BS (94.10 ± 0.74 and 96.73 ± 0.95%) and CA (87.42 ± 6.54 and 98.24 ± 0.63%), whereas GR (14.91 ± 1.62 and 41 ± 1.78%) and PS (36.58 ± 0.54 and 51.51 ± 0.83%), only showed significant inhibition at 40 µg/mL, and FV (< 5% inhibition) and GP (10 ± 1.03 and 22 ± 1.31%) did not show significant inhibition at both concentrations. Due to the significant anti-invasive activity of BS, CA and PS at 40 µg/mL, these extracts were further evaluated for their potential to stimulate CD82. BS showed significant (p < 0.05) reduction in CD82 at 20 and 40 µg/mL (13.2 ± 2.2% and 20.3 ± 1.5% decrease, respectively), whereas both CA and PS at 20 µg/mL increased (p < 0.05) CD82 expression (16.4 ± 0.8% and 5.4 ± 0.6% increase, respectively), and at 40 µg/mL significantly reduced CD82 expression (23.4 ± 3.1% and 11.2 ± 2.9% decrease, respectively). Using the yolk sac membrane assay, BS (59.52 ± 4.12 and 56.72 ± 3.13% newly formed vessels) and CA (83.33 ± 3.17 and 74.00 ± 2.12%) at both 20 and 40 µg/egg showed significant (p < 0.001) angiogenesis inhibition, with BS showing statistical similar activity to the positive control, combretastatin A4 (10 nmol/egg), whereas PS only displayed significant (p < 0.001) angiogenesis stimulation at 40 µg/egg (120.81 ± 3.34% newly formed vessels). CONCLUSION: BS exhibits antiproliferative, anti-invasive, and anti-angiogenic activity despite inhibiting CD82, suggesting an alternative mode of action. CA at 20 µg/mL shows moderate anti-invasive and anti-angiogenic potential by stimulating CD82, while at 40 µg/mL it still displays these properties but inhibits CD82, suggesting an additional mode of action. PS, with the least antiproliferative activity, stimulates CD82 and inhibits angiogenesis at 20 µg/mL but inhibits CD82 and increases angiogenesis at 40 µg/mL, indicating CD82 targeting as a major mode of action. Future studies should explore breast cancer xenograft models to assess the extracts' impact on CD82 expression and angiogenesis in the tumor microenvironment, along with isolating bioactive compounds from the extracts.

The biological characteristics of chicken embryo mesenchymal stem cells isolated from chorioallantoic membrane.

Mesenchymal stem cells (MSCs) derived from fetal membranes (FMs) have the potential to exhibit immunosuppression, improve blood flow, and increase capillary density during transplantation. In the field of medicine, opening up new avenues for disease treatment. Chicken embryo chorioallantoic membrane (CAM), as an important component of avian species FM structure, has become a stable tissue engineering material in vivo angiogenesis, drug delivery, and toxicology studies. Although it has been confirmed that chorionic mesenchymal stem cells (Ch-MSCs) can be isolated from the outer chorionic layer of FM, little is known about the biological characteristics of MSCs derived from chorionic mesodermal matrix of chicken embryos. Therefore, we evaluated the characteristics of MSCs isolated from chorionic tissues of chicken embryos, including cell proliferation ability, stem cell surface antigen, genetic stability, and in vitro differentiation potential. Ch-MSCs exhibited a broad spindle shaped appearance and could stably maintain diploid karyotype proliferation to passage 15 in vitro. Spindle cells were positive for multifunctional markers of MSCs (CD29, CD44, CD73, CD90, CD105, CD166, OCT4, and NANOG), while hematopoietic cell surface marker CD34, panleukocyte marker CD45, and epithelial cell marker CK19 were negative. In addition, chicken Ch-MSC was induced to differentiate into four types of mesodermal cells in vitro, including osteoblasts, chondrocytes, adipocytes, and myoblasts. Therefore, the differentiation potential of chicken Ch-MSC in vitro may have great potential in tissue engineering. In conclusion, chicken Ch-MSCs may be an excellent model cell for stem cell regenerative medicine and chorionic tissue engineering.

Effects of Resveratrol on In Vivo Ovarian Cancer Cells Implanted on the Chorioallantoic Membrane (CAM) of a Chicken Embryo Model.

Ovarian cancer poses a significant threat to patients in its advanced stages, often with limited treatment options available. In such cases, palliative management becomes the primary approach to maintaining a reasonable quality of life. Therefore, the administration of any medication that can benefit patients without a curative option holds potential. Resveratrol, a natural compound known for its in vitro anticancer activities, has generated contrasting results in vivo and human studies. In this study, we aimed to assess the anticancer effects of resveratrol on ovarian cancer cells grown on the chorioallantoic membrane (CAM) of chicken embryos. Two ovarian cancer cell lines, OVCAR-8 and SKOV-3, were cultured in collagen scaffolds for four days before being implanted on the CAM of chicken embryos on day 7. Different doses of resveratrol were applied to the CAM every two days for six days. Subsequently, CAM tissues were excised, fixed, and subjected to histological analysis. Some CAM tumours were extracted to analyse proteins through Western blotting. Our findings indicate that specific doses of resveratrol significantly reduce angiogenic activities, pNF-κB levels, and SLUG protein levels by using immunohistochemistry. These results suggest that resveratrol may have the potential to impact the behaviour of ovarian cancer CAM tumours, thereby warranting further consideration as a complementary treatment option for women with incurable ovarian cancer.

Enhancing bone tissue engineering using iron nanoparticles and magnetic fields: A focus on cytomechanics and angiogenesis in the chicken egg chorioallantoic membrane model.

AIM: To evaluate the potential of iron nanoparticles (FeNPs) in conjunction with magnetic fields (MFs) to enhance osteoblast cytomechanics, promote cell homing, bone development activity, and antibacterial capabilities, and to assess their in vivo angiogenic viability using the chicken egg chorioallantoic membrane (CAM) model. SETTINGS AND DESIGN: Experimental study conducted in a laboratory setting to investigate the effects of FeNPs and MFs on osteoblast cells and angiogenesis using a custom titanium (Ti) substrate coated with FeNPs. MATERIALS AND METHODS: A custom titanium (Ti) was coated with FeNPs. Evaluations were conducted to analyze the antibacterial properties, cell adhesion, durability, physical characteristics, and nanoparticle absorption associated with FeNPs. Cell physical characteristics were assessed using protein markers, and microscopy, CAM model, was used to quantify blood vessel formation and morphology to assess the FeNP-coated Ti's angiogenic potential. This in vivo study provided critical insights into tissue response and regenerative properties for biomedical applications. STATISTICAL ANALYSIS: Statistical analysis was performed using appropriate tests to compare experimental groups and controls. Significance was determined at P < 0.05. RESULTS: FeNPs and MFs notably improved osteoblast cell mechanical properties facilitated the growth and formation of new blood vessels and bone tissue and promoted cell migration to targeted sites. In the group treated with FeNPs and exposed to MFs, there was a significant increase in vessel percentage area (76.03%) compared to control groups (58.11%), along with enhanced mineralization and robust antibacterial effects (P < 0.05). CONCLUSION: The study highlights the promising potential of FeNPs in fostering the growth of new blood vessels, promoting the formation of bone tissue, and facilitating targeted cell migration. These findings underscore the importance of further investigating the mechanical traits of FeNPs, as they could significantly advance the development of effective bone tissue engineering techniques, ultimately enhancing clinical outcomes in the field.

The potential of Paeonia lactiflora pall seeds oil as a pure natural cosmetics raw material: In Vitro findings.

BACKGROUND: As a traditional Chinese herbal medicine, Paeonia lactiflora Pall is rich in various active ingredients such as polysaccharides and total flavonoids while having ornamental value. It has potential application value in the development of food and cosmetics. OBJECTIVE: To study the in vitro efficacy of Paeonia lactiflora Pall seeds oil. METHODS: Firstly, the levels of linolenic acid and linoleic acid in Paeonia lactiflora Pall seeds oil were quantified using gas chromatography. The impact of Paeonia lactiflora Pall seeds oil on the proliferation rate of B16F10 cells was assessed through the CCK-8 method, while the melanin content of B16F10 cells was determined using the sodium hydroxide lysis method. The inhibitory effects of Paeonia lactiflora Pall seeds oil on elastase, collagenase and hyaluronidase were evaluated by biochemical techniques in vitro. Lastly, the hen's egg chorioallantoic membrane test (HET-CAM) was conducted to confirm the absence of eye irritation caused by Paeonia lactiflora Pall seeds oil. RESULTS: Paeonia lactiflora Pall seeds oil within a certain volume concentration range (0.5%-4%) had no effect on the proliferation of B16F10 cells. Paeonia lactiflora Pall seeds oil showed significant inhibition of elastase, collagenase and hyaluronidase. Notably, the highest concentration tested, 4% Paeonia lactiflora Pall seed oil, yielded the most pronounced outcomes without causing any irritation. CONCLUSION: A certain concentration of Paeonia lactiflora Pall seeds oil has a significant effect on decreasing the melanin content in B16F10 cells and inhibiting the activities of elastase, collagenase, and hyaluronidase, which can provide a reference for the development of pure natural cosmetics raw materials.

Intra-allantoic injection of calcium promotes hatching of chick embryos grown in shell-less culture.

The hatch rate of chick embryos cultured outside of the eggshell with 350 mg calcium l-lactate hydrate (CaL) and 3.5 mL water is fourfold greater in cultures in which the chorioallantoic membrane (CAM) surrounds the egg contents by incubation day 17.5 (E17.5) an event which occurs in ovo by E13. It was first investigated whether decreasing the volume of water added with 350 mg CaL would promote CAM expansion due to the smaller volume to enclose. When 350 mg CaL was present, the CAM did not surround the egg contents by E13. By E17.5, the CAM surrounded the egg contents in 53%-74% of cultures; however, CAM expansion was not significantly different when 0, 1, 2, or 3.5 mL water was present. The hatch rate with 2 or 3.5 mL water was greater than 50% but was not improved with less water. Second, it was investigated whether CaL or water inhibits CAM expansion. In the absence of CaL, the CAM surrounded the egg contents in up to two-thirds of cultures by E13, whether 2 mL water was present or not. Thus CaL, but not water, inhibits expansion of the CAM by E13, even though CaL promotes hatching. Finally, it was investigated whether injection of aqueous CaL into the allantoic fluid, in conjunction with not adding CaL to culture hammocks, would promote CAM expansion. Allantoic injection of CaL starting at E13 did not promote CAM expansion at E17.5 but resulted in hatch rates of approximately 30%. Allantoic injection is a novel route for supplementation of calcium in cultured chick embryos.

In vivo high-speed microscopy of microbubbles in the chorioallantoic membrane model.

Rationale: The acoustic stimulation of microbubbles within microvessels can elicit a spectrum of therapeutically relevant bioeffects from permeabilization to perfusion shutdown. These bioeffects ultimately arise from complex interactions between microbubbles and microvascular walls, though such interactions are poorly understood particularly at high pressure, due to a paucity of direct in vivo observations. The continued development of focused ultrasound methods hinges in large part on establishing links between microbubble-microvessel interactions, cavitation signals, and bioeffects. Methods: Here, a system was developed to enable simultaneous high-speed intravital imaging and cavitation monitoring of microbubbles in vivo in a chorioallantoic membrane model. Exposures were conducted using the clinical agent DefinityTM under conditions previously associated with microvascular damage (1 MHz, 0.5-3.5 MPa, 5 ms pulse length). Results: Ultrasound-activated microbubbles could be observed and were found to induce localized wall deformations that were more pronounced in smaller microvessels and increased with pressure. A central finding was that microbubbles could extravasate from microvessels (from 34% of vessels at 1 MPa to 79% at 3 MPa) during insonation (94% within 0.5 ms) and that this occurred more frequently and in progressively larger microvessels (up to 180 µm) as pressure was increased. Following microbubble extravasation, transient or sustained red blood cell leakage ensued at the extravasation site in 96% of cases for pressures ≥1 MPa. Conclusions: The results here represent the first high-speed in vivo investigation of high-pressure focused ultrasound-induced microbubble-microvessel interactions. This data provides direct evidence that the process of activated microbubble extravasation can occur in vivo and that it is linked to producing microvessel wall perforations of sufficient size to permit red blood cell leakage. The association of red blood cell leakage with microbubble extravasation provides mechanistic insight into the process of microvessel rupture, which has been widely observed in histology.

Early Unguided Human Brain Organoid Neurovascular Niche Modeling into the Permissive Chick Embryo Chorioallantoic Membrane.

Engrafting organoids into vascularized tissues in model animals, such as the immunodeficient mouse or chick embryo chorioallantoic membrane (CAM), has proven efficient for neovascularization modeling. The CAM is a richly vascularized extraembryonic membrane, which shows limited immunoreactivity, thus becoming an excellent hosting model for human origin cell transplants. This paper describes the strategy to engraft human brain organoids differentiated at multiple maturation stages into the CAM. The cellular composition of brain organoids changes with time, reflecting the milestones of human brain development. We grafted brain organoids at relevant maturation stages: neuroepithelial expansion (18 DIV), early neurogenesis (60 DIV), and early gliogenesis (180 DIV) into the CAM of embryonic day (E)7 chicken embryos. Engrafted brain organoids were harvested 5 days later and their histological features were analyzed. No histological signs of neovascularization in the grafted organoids or abnormal blood vessels adjacent to the graftings were detected. Moreover, remarkable changes were observed in the cellular composition of the grafted organoids, namely, an increase in the number of glial fibrillary acidic protein-positive-reactive astrocytes. However, the cytoarchitectural changes were dependent on the organoid maturation stage. Altogether, these results suggest that brain organoids can grow in the CAM, and they show differences in the cytoarchitecture depending on their maturation stage at grafting.

Comparison of the angiogenic efficacy of conventional leukocyte- and platelet-rich fibrin versus low-speed advanced platelet-rich fibrin: An in vitro chorioallantoic membrane assay study.

BACKGROUND: Platelet-rich fibrin (PRF) is widely used in periodontics for its wound healing potential. Two major variations of PRF are the original leukocyteand platelet-rich fibrin (L-PRF) and the modified lowspeed advanced PRF (A-PRF). OBJECTIVES: The aim of the present study was to evaluate and compare the conventional L-PRF protocol and the low-speed A-PRF protocol in terms of angiogenic potential of PRF, using an in vivo chorioallantoic membrane (CAM) assay. MATERIAL AND METHODS: Fifteen fertile Giriraja eggs were procured and after a 3-day incubation period, randomly allotted into 3 groups: control; L-PRF; and A-PRF. A total of 20 mL of blood was collected from systemically healthy male volunteers aged 18-24 years, using a standard protocol. The PRF samples were inoculated on the CAM of the eggs. On the 10th day, the eggs were reopened and photographed. The parameters assessed were the number, length, size, and density of blood vessels, as well as the number of junctions formed. The photographs were analyzed using the ImageJ and ProgRes® CapturePro software. RESULTS: Seven days after inoculation, both the A-PRF and L-PRF groups exhibited significantly better results than the control group in terms of the number (59.20 ±6.61 vs. 48.80 ±5.07 vs. 19.20 ±6.98), length (25,000 ±1,813.10 µm vs. 17,000 ±282.90 µm vs. 8,000 ±184.49 µm), size (230,000 ±15,054.00 µm2 vs. 200,000 ±8,295.27 µm2 vs. 150,000 ±4,105.16 µm2), and density (central: 9,100 ±296.78 vs. 5,370 ±272.42 vs. 1,420 ±564.36; peripheral: 9,094 ±400.14 vs. 3,370 ±479.39 vs. 5,420 ±746.73) of blood vessels, as well as the number of junctions formed (52 ±3.81 vs. 41 ±1.58 vs. 33 ±4.64), respectively. CONCLUSIONS: The angiogenic potential was increased by the exposure to both L-PRF and A-PRF. However, A-PRF demonstrated statistically significant benefits in terms of the number, length, size, and density of blood vessels, as well as the number of junctions formed in comparison with the control and L-PRF groups.

Short communication: Characterizing arterial and venous blood gases over the gas exchange surface, the chorioallantoic membrane, of embryonic American alligators (Alligator mississippiensis) at two points of development.

Assessments of arterial and venous blood gases are required to understand the function of respiratory organs in animals at different stages of development. We measured blood gases in the arteries entering and veins leaving the chorioallantoic membrane (CAM) in embryonic alligators (Alligator mississippiensis). The CAM accounts for virtually all gas exchange in these animals, and we hypothesized that the CAM vasculature would be larger in eggs incubated in hypoxia (10% O2 for 50% or 70% of incubation), which would be reflected in a lower partial pressure of CO2 (PCO2). Contrary to this hypothesis, our measurements revealed no effects of hypoxic incubation on PCO2, and seemingly no increase in vascularization of the CAM in response to incubation in 10% O2. PCO2 was lower on the venous side, but only significantly different from arterial blood at 70% of incubation. The calculated blood flow to the CAM increased with development and was lower in both groups of alligators that had been incubated in hypoxia. Future studies should include measurements of blood parameters taken from embryos held in conditions that mirror incubation O2 levels, in combination with direct measurements of CAM artery blood flow.

Experimental Tumor Induction and Evaluation of Its Treatment in the Chicken Embryo Chorioallantoic Membrane Model: A Systematic Review.

The chorioallantoic membrane (CAM) model, generated during avian development, can be used in cancer research as an alternative in vivo model to perform tumorigenesis in ovo due to advantages such as simplicity, low cost, rapid growth, and being naturally immunodeficient. The aim of this systematic review has been to compile and analyze all studies that use the CAM assay as a tumor induction model. For that, a systematic search was carried out in four different databases: PubMed, Scopus, Cochrane, and WOS. After eliminating duplicates and following the established inclusion and exclusion criteria, a total of 74 articles were included. Of these, 62% use the in ovo technique, 13% use the ex ovo technique, 9% study the formation of metastasis, and 16% induce tumors from patient biopsies. Regarding the methodology followed, the main species used is chicken (95%), although some studies use quail eggs (4%), and one article uses ostrich eggs. Therefore, the CAM assay is a revolutionary technique that allows a simple and effective way to induce tumors, test the effectiveness of treatments, carry out metastasis studies, perform biopsy grafts of patients, and carry out personalized medicine. However, unification of the methodology used is necessary.

Calcium Electroporation versus Electrochemotherapy with Bleomycin in an In Vivo CAM-Based Uveal Melanoma Xenograft Model.

Despite recent advancements in the diagnosis and treatment of uveal melanoma (UM), its metastatic rate remains high and is accompanied by a highly dismal prognosis, constituting an unmet need for the development of novel adjuvant therapeutic strategies. We established an in vivo chick chorioallantoic membrane (CAM)-based UM xenograft model from UPMD2 and UPMM3 cell lines to examine its feasibility for the improvement of selection of drug candidates. The efficacy of calcium electroporation (CaEP) with 5 or 10 mM calcium chloride (Ca) and electrochemotherapy (ECT) with 1 or 2.5 µg/mL bleomycin in comparison to monotherapy with the tested drug or electroporation (EP) alone was investigated on the generated UM tumors. CaEP and ECT showed a similar reduction of proliferation and melanocytic expansion with a dose-dependent effect for bleomycin, whereas CaEP induced a significant increase of the apoptosis and a reduction of vascularization with varying sensitivity for the two xenograft types. Our in vivo results suggest that CaEP and ECT may facilitate the adequate local tumor control and contribute to the preservation of the bulbus, potentially opening new horizons in the adjuvant treatment of advanced UM.

Sex-specific transcriptome of the chicken chorioallantoic membrane.

Dimorphism between male and female embryos has been demonstrated in many animal species, including chicken species. Likewise, extraembryonic membranes such as the chorioallantoic membrane (CAM) are likely to exhibit a sex-specific profile. Analysis of the previously published RNA-seq data of the chicken CAM sampled at two incubation times, revealed 783 differentially expressed genes between the CAM of male and female embryos. The expression of some of these genes is sex-dependant only at one or other stage of development, while 415 genes are sex-dependant at both developmental stages. These genes include well-known sex-determining and sex-differentiation genes (DMRT1, HEGM, etc.), and are mainly located on sex chromosomes. This study provides evidence that gene expression of extra-embryonic membranes is differentially regulated between male and female embryos. As such, a better characterisation of associated mechanisms should facilitate the identification of new sex-specific biomarkers.

Effects of neratinib on angiogenesis and the early stage of the embryo using chicken embryo as a model.

Angiogenesis is the process of forming new blood capillaries from pre-existing vessels. Even though it is essential during normal development, it plays a major role in cancer progression. Neratinib is a pan-human epidermal growth factor receptor (HER) inhibitor that has recently been approved for the treatment of HER2-positive breast cancer. However, its effects on angiogenesis and embryogenesis remain unknown. This study examined the antiangiogenic effects of neratinib using the chorioallantoic membrane (CAM) of chicken embryos. We also evaluated neratinib's toxicity during the early stages of normal development using the chicken embryos, primary embryonic fibroblasts (EFBs), and human umbilical vein endothelial cells (HUVEC). Our findings revealed that neratinib significantly inhibited the CAM angiogenesis compared to controls by reducing vessel percentage area and the average vessel length. Furthermore, neratinib downregulated vascular endothelial growth factor (VEGF), a key mediator of angiogenesis. At lower concentrations, neratinib was well-tolerated during early stages of normal development. Additionally, EFBs treated with neratinib showed no morphological or viability changes when compared to controls. However, at the highest concentration tested, neratinib treatment reduced HUVEC cell viability. This effect may be associated with the dysregulation of key apoptotic genes, including caspase-3, caspase-8, caspase-9, and the B-cell lymphoma 2 (Bcl2) gene. Our findings indicate a novel potential application of neratinib as an antiangiogenic agent, exhibiting tolerable toxicity in the early stages of embryogenesis.

Vascularization of the adult mouse lung grafted onto the chick chorioallantoic membrane.

In the later stages of angiogenesis, the vascular sprout transitions into a functional vessel by fusing with a target vessel. Although this process appears to routinely occur in embryonic tissue, the biologic rules for sprout fusion and lumenization in adult regenerating tissue are unknown. To investigate this process, we grafted portions of the regenerating post-pneumonectomy lung onto the chick chorioallantoic membrane (CAM). Grafts from all 4 lobes of the post-pneumonectomy right lung demonstrated peri-graft angiogenesis as reflected by fluorescent plasma markers; however, fluorescent microsphere perfusion primarily occurred in the lobe of the lung that is the dominant site of post-pneumonectomy angiogenesis-namely, the cardiac lobe. Vascularization of the cardiac lobe grafts was confirmed by active tissue growth (p < .05). Functional vascular connections between the cardiac lobe and the CAM vascular network were demonstrated by confocal fluorescence microscopy as well as corrosion casting and scanning electron microscopy (SEM). Bulk transcriptional profiling of the cardiac lobe demonstrated the enhanced expression of many genes relative to alveolar epithelial cell (CD11b-/CD31-) control cells, but only the upregulation of Ereg and Fgf6 compared to the less well-vascularized right upper lobe. The growth of actively regenerating non-neoplastic adult tissue on the CAM demonstrates that functional lumenization can occur between species (mouse and chick) and across the developmental spectrum (adult and embryo).

Pedunculagin and tellimagrandin-I stimulate inflammation and angiogenesis and upregulate vascular endothelial growth factor and tumor necrosis factor-alpha in vivo.

Pedunculagin (PD) and tellimagrandin-I (TL), isolated from Myrciaria cauliflora seeds and Eucaliptus microcorys leaves, respectively, have attracted great attention owing to their relevant biological activities, such as antitumor, antioxidant, and hepatoprotective activities. This study investigated the angiogenic potential of PD and TL using a chick embryo chorioallantoic membrane (CAM) assay. Using the CAM assay, our results showed that both PD and TL promoted a significant increase in the number and caliber of blood vessels, the thickness of the CAM, and the presence of fibroblasts and inflammatory cells. Moreover, an increase of tumor necrosis factor-α and vascular endothelial growth factor was observed in the CAM treated with PD and TL, indicating the induction of angiogenic factors. Thus, the remarkable profile of PD and TL in inducing angiogenesis opens up new perspectives for their potential utilization in different therapeutic approaches involving neovascularization.