Chick Embryo Chorioallantoic Membrane Research Articles

Two new applications in the study of angiogenesis the CAM assay: Acellular scaffolds and organoids.

The chick embryo chorioallantoic membrane (CAM) is a rich vascularized extraembryonic membrane that is commonly used as an in vivo experimental model to study molecules with angiogenic and anti-angiogenic activity, tumor growth and metastasis. Among other applications of the CAM assay, more recently this assay has been used for the study of acellular scaffolds and of organoids, and of their angiogenic capacity. The aim of this review article is to summarize the literature data concerning these two new applications of the CAM assay and to underline the advantages of this assay.

The Chick Embryo Chorioallantoic Membrane Model: A Research Approach for Ex Vivo and In Vivo Experiments.

The chick chorioallantoic membrane (CAM) model has attracted a great deal of interest in pharmaceutical and biological research as an alternative or complimentary in vivo assay to animal models. Traditionally, CAM assay has been widely used to perform some toxicological studies, specifically to evaluate the skin, ocular and embryo toxicity of new drugs and formulations, and to perform angiogenesis studies. Due to the possibility to generate the tumors onto the CAM, this model has also become an excellent strategy to evaluate the metastatic potential of different tumours and to test the efficacy of novel anticancer therapies in vivo. Moreover, in the recent years, its use has considerably grown in other research areas, including the evaluation of new anti-infective agents, the development of biodistribution studies and in tissue engineering research. This manuscript provides a critical overview of the use of CAM model in pharmaceutical and biological research, especially to test the toxicity of new drugs and formulations and the biodistribution and the efficacy of novel anticancer and antiinfective therapies, analyzing its advantages and disadvantages in comparison to animal models. The chick chorioallantoic membrane model shows a great utility in several research areas, such as cancer, toxicology, biodistribution studies and anti-infective therapies. In fact, it has become an intermediate stage between in vitro experiments and animal studies, and, in the case of toxicological studies (skin and ocular toxicity), it has even replaced the animal models.

Reparative Effects of Dandelion Fermentation Broth on UVB-Induced Skin Inflammation.

ObjectiveTo evaluate the efficacy of the dandelion fermentation broth in repairing UVB-induced skin inflammation.MethodsDetection of active ingredients in dandelion fermentation broth and water extract. The antioxidant capacity of dandelion fermentation broth was investigated by in vitro antioxidant experiments. The influence of the broth on the content of inflammatory factors interleukin-6 (IL-6), interleukin-8 (IL-8) and interleukin-1β (IL-1β), and tumor necrosis factor (TNF-α), in human immortalized epidermal cells (HaCaT) is discussed on the basis of a UVB-induced HaCaT damage model. The effects of the broth on the contents of skin barrier-related proteins kallikrein-7 (KLK-7), filaggrin (FLG) and aquaporin (AQP3) in the UVB-induced damage and repair of the HaCaT mechanism are also comprehensively discussed. The effect of DF on the activation of MAPK pathway proteins was detected by PCR. A chicken embryo chorioallantoic membrane test is used to explore the safety of the dandelion fermentation broth.ResultsThe results show that the dandelion fermentation broth is rich inTotal sugar, with good free radical scavenging ability and antioxidant effects; it can regulate the MAPK pathway, reduce the expression of inflammatory factors, adjust the skin barrier factors and good safety.ConclusionDandelion fermentation broth exhibits repairing effect on UVB-induced skin inflammation.

Anti-chloride Intracellular Channel Protein 1 (CLIC1) Antibodies Induce Tumour Necrosis and Angiogenesis Inhibition on In Vivo Experimental Models of Human Renal Cancer.

Chloride intracellular channel protein 1 (CLIC1) is known as a promoter of cancer progression, metastasis, and angiogenesis. Thus, CLIC1 could be a future therapeutic target. This study aimed to evaluate the effect of anti-CLIC1 antibodies on tumour cells and vessels of human renal cell carcinoma (RCC) in rabbit cornea and chick embryo chorioallantoic membrane (CAM) models. Human cc-RCC xenografts on rabbit cornea and CAM surface were performed. Anti-CLIC1 antibodies were applied for 5 consecutive days on both tumor models. We comparatively evaluated treated and untreated tumors by combining ultrasonography with microscopic techniques. RCC implants rapidly recruited blood vessels and had an exponential growth rate on both tumor models. Anti-CLIC1 antibodies suppressed tumor growth by inducing tumor cell necrosis. Tumor vessels regressed rapidly but not completely during anti-CLIC1 antibodies based therapy. Anti-CLIC1 antibodies induced tumor necrosis and tumor vasculature regression in human cc-RCC xenografts in both in vivo experimental models.

Combination of Resminostat with Ruxolitinib Exerts Antitumor Effects in the Chick Embryo Chorioallantoic Membrane Model for Cutaneous T Cell Lymphoma.

Simple SummaryThe combination of Resminostat (HDACi) and Ruxolitinib (JAKi) exerted cytotoxic effects and inhibited proliferation of CTCL cell lines (MyLa, SeAx) in vitro. The aim of the present study was to validate their antitumor effects in vivo using the chick embryo chorioallantoic membrane (CAM) model, which allows quick and efficient monitoring of tumor growth, migration, invasion, and metastatic potential. The drug combination exhibited a significant inhibition of primary tumor size, and inhibited intravasation and extravasation of tumor cells to the liver and lung. It also exerted an inhibitory effect in the migration and invasion of tumor cells and significantly reduced key signaling pathway activation. Our data demonstrate that the CAM assay could be employed as a preclinical in vivo model in CTCL for pharmacological testing, and that the combination of Resminostat and Ruxolitinib exerts significant antitumor effects in CTCL progression that need to be further evaluated in a clinical setting.The combination of Resminostat (HDACi) and Ruxolitinib (JAKi) exerted cytotoxic effects and inhibited proliferation of CTCL cell lines (MyLa, SeAx) in previously published work. A xenograft tumor formation was produced by implanting the MyLa or SeAx cells on top of the chick embryo chorioallantoic membrane (CAM). The CAM assay protocol was developed to monitor the metastatic properties of CTCL cells and the effects of Resminostat and/or Ruxolitinib in vivo. In the spontaneous CAM assays, Resminostat and Ruxolitinib treatment inhibited the cell proliferation (p < 0.001) of MyLa and SeAx, and induced cell apoptosis (p < 0.005, p < 0.001, respectively). Although monotherapies reduced the size of primary tumors in the metastasis CAM assay, the drug combination exhibited a significant inhibition of primary tumor size (p < 0.0001). Furthermore, the combined treatment inhibited the intravasation of MyLa (p < 0.005) and SeAx cells (p < 0.0001) in the organs, as well as their extravasation to the liver (p < 0.0001) and lung (p < 0.0001). The drug combination also exerted a stronger inhibitory effect in migration (p < 0.0001) rather in invasion (p < 0.005) of both MyLa and SeAx cells. It further reduced p-p38, p-ERK, p-AKT, and p-STAT in MyLa cells, while it decreased p-ERK and p-STAT in SeAx cells in CAM tumors. Our data demonstrated that the CAM assay could be employed as a preclinical in vivo model in CTCL for pharmacological testing. In agreement with previous in vitro data, the combination of Resminostat and Ruxolitinib was shown to exert antitumor effects in CTCL in vivo.

Structural Characterization and Tumor Microvascular Inhibition Activity of Total Polysaccharide from Trametes sanguinea Lloyd.

Trametes sanguinea Lloyd total polysaccharide (TsLTP), was obtained by water extraction and ethanol precipitation from T. sanguinea. The structural characterization of TsLTP was elucidated mutually by TsL1 and TsL2, whose mass ratio is 1: 4. TsL1 is mainly composed of mannose, glucose, galactose, and fucose, and consist of T-Linked-Fucp, T-Linked-Manp, T-Linked-Galp, 1,4-Linked-Manp, 1,4-Linked-Glcp, 1,6-Linked-Manp, 1,6-Linked-Galp, 1,3,4-Linked-Glcp, 1,4,6-Linked-Glcp and 1,3,6-Linked-Glcp, with a molar ratio of 2.1: 1.7: 1.4: 1.0: 3.6: 2.0: 8.6: 1.3: 2.2: 1.2, while TsL2 mainly comprise of glucose and consist of T-Linked-Glcp, 1,3-Linked-Glcp, 1,4-Linked-Glcp and 1,4,6-Linked-Glcp, with a molar ratio of 1.0: 2.1: 7.6: 1.4. TsLTP exhibited strong inhibitory effects on the migration, invasion, and tube formation of human umbilical vein endothelial cells (iHUVECs) and chick embryo chorioallantoic membrane (CAM) angiogenesis, whereas no inhibitory activity on human TNBC cell lines. Taken together, our study suggests that TsLTP possesses a significant inhibition of tumor microvascular activity both in vitro and in vivo. The study of TsLTP with novel monosaccharide composition and tumor microvascular inhibitory activity might be a beneficial attempt for application of polysaccharide from the genus Trametes in tumor therapy.

Artemisia vulgaris essential oil nanoemulsions (AVEO-NE), a novel anti-angiogenic agent and safe apoptosis inducer in MCF-7 human cancer cells

Artemisia vulgaris essential oil (AVEO) is known as a therapeutic agent. Several types of investigation have demonstrated its cytotoxic impact on human cancer cells. In the current study, AVEO nanoemulsion (AVEO-NE) was synthesized using an ultrasound-based emulsification method and its cytotoxic, apoptotic, antioxidant, and anti-angiogenic properties were evaluated for the MCF-7 cancer cell line by measuring the cell’s gene expression profile and performing MTT. Finally, the anti-angiogenic activity of AVEO-NE was verified by applying CAM assay analysis. The results showed that AVEO-NE exhibited significant cell-selective cytotoxic, apoptotic, and antioxidant activities by up-regulating Cas-9, CAT, and SOD gene expression. Moreover, the AVEO-NE meaningfully suppressed angiogenesis in MCF-7 cancer cells by down-regulating VEGF gene expression. Finally, the decreased number and length of chick embryo chorioallantoic membrane (CAM) blood vessels verified AVEO-NE’s anti-angiogenic activity. Therefore, AVEO-NE can potentially be employed as a safe cell-dependent anti-breast cancer drug delivery system.

Proanthocyanidins Promote Endothelial Cell Viability and Angiogenesis.

Botanic drugs are reportedly effective in treating ischemic conditions by improving vascular circulation. However, it has been very rare for biomaterial researchers to look into the possibility of using such products in the context of tissue regeneration. This work studied 4 botanic drugs to explore their effects on vascular endothelial cell growth. Human umbilical endothelial cells were cultured in the presence of different doses of astragalus powder extract, astragalus injection, puerarin injection, and proanthocyanidin (PAC). Among the 4 drugs, PAC showed a potent effect on cell viability and stimulated cell growth in a dose-dependent manner. In particular, the PAC under test was able to maintain a high level of cell viability/proliferation comparable with the cells supplemented with the endothelial cell growth medium, at both low and normal serum conditions. Blocking either endothelial cell growth factor receptors or epithelial cell growth factor receptors was ineffective in reducing the stimulatory effect. The PAC released from polyvinyl alcohol cryogels stimulated HUVECs proliferation. The chick embryo chorioallantoic membrane model was further used to test the angiogenicity of PAC, showing that this botanic drug was potent in stimulating vasculature development. This work therefore demonstrates for the first time that PAC is capable of upregulating endothelial cell activity and growth in vitro in the absence of growth factors and that PAC can be loaded and released from drug carriers and can stimulate angiogenesis. These findings suggest the application of PAC in angiogenesis and tissue regeneration.

A New Approach to Inhibiting Triple-Negative Breast Cancer: In Vitro, Ex Vivo and In Vivo Antiangiogenic Effect of BthTx-II, a PLA2-Asp-49 from Bothrops jararacussu Venom.

Phospholipases A2 (PLA2) represent a superfamily of enzymes widely distributed in living organisms, with a broad spectrum of pharmacological activities and therapeutic potential. Anti-angiogenic strategies have become one of the main tools in fighting cancer. In this sense, the present work reports the inhibition of tumor angiogenesis induced by Asp-49 BthTX-II using in vitro, ex vivo and in vivo approaches. We demonstrate that BthTx-II inhibited cell adhesion, proliferation, and migration of human umbilical vein endothelial cells (HUVEC), as well as caused a reduction in the levels of endothelial growth factor (VEGF) during in vitro angiogenesis assays. BthTx-II was also able to inhibit the sprouting angiogenic process, by the ex vivo germination assay of the aortic ring; in addition, this toxin inhibited the migration and proliferation of HUVEC in co-culture with triple-negative breast cancer cells (e.g., MDA-MB-231 cells). Finally, in vivo tumor suppression and anti-angiogenic activities were analyzed using MDA-MB-231 cells with Matrigel injected into the chorioallantoic membrane of chicken embryo (CAM) for 7 days treatment with BthTx-II, showing a considerable reduction in vessel caliber, on the size and weight of tumors. Together, these results suggest an important antiangiogenic and antitumor role for BthTx-II, as a potential prototype for the development of new tools and antitumor drugs in cancer therapy.

The role of α5-nicotinic acetylcholine receptor/NLRP3 signaling pathway in lung adenocarcinoma cell proliferation and migration

α5-nicotinic acetylcholine receptor (α5-nAChR) is involved in tobacco smoking-induced lung carcinogenesis. Increasing evidence has highlighted the importance of inflammation in lung cancer and a strong relationship between smoking and the NLR family pyrin domain containing protein 3 (NLRP3) inflammasome. However, it is unclear whether an inflammation-related effect of α5-nAChR contributes to cigarette smoking-related lung cancer. Here, we identified a functional link between α5-nAChR and NLRP3 inflammasome activation in lung adenocarcinoma (LUAD). The expression of α5-nAChR was correlated with the expression of NLRP3 via STAT3 in LUAD tissues. In vitro, nicotine increased the levels of α5-nAChR, p-STAT3, and NLRP3 inflammasome expression, accompanied by the expression of caspase-1, IL-1β and IL-18. Nicotine-induced activation of p-STAT3 and NLRP3 inflammasome signaling were inhibited by the silencing of α5-nAChR. STAT3 binds to the NLRP3 promoter and α5-nAChR mediates NLRP3 expression via STAT3. Functionally, the combination of nicotine and LPS/ATP could significantly enhance cell proliferation and migration compared to nicotine or LPS/ATP alone. Furthermore, the functional link between α5-nAChR and NLRP3 was confirmed in chicken embryo chorioallantoic membrane (CAM) and mouse xenograft models. Together, these findings reveal a novel nicotine-mediated signaling pathway: nicotine promotes lung cell proliferation and migration via the α5-nAChR/STAT3/NLRP3 axis in lung cancer.

α5-nAChR associated with Ly6E modulates cell migration via TGF-β1/Smad signaling in non-small cell lung cancer.

The α5-nicotinic acetylcholine receptor (α5-nAChR) is closely associated with nicotine-related lung cancer, offering a novel perspective for investigating the molecular pathogenesis of this disease. However, the mechanism by which α5-nAChR functions in lung carcinogenesis remains to be elucidated. Lymphocyte antigen 6 (Ly6) proteins, like snake three-finger alpha toxins such as α-bungarotoxin, can modulate nAChR signaling. Ly6E, a member of the Ly6 family, is a biomarker of poor prognosis in smoking-induced lung carcinogenesis and is involved in the regulation of TGF-β1/Smad signaling. Here, we explored the underlying mechanisms linking α5-nAChR and Ly6E in non-small cell lung cancer (NSCLC). The expression of α5-nAChR was correlated with Ly6 expression, smoking status and lower survival in NSCLC tissues. In vitro, α5-nAChR mediated Ly6E, the phosphorylation of the TGF-β1 downstream molecule Smad3 (pSmad3, a key mediator of TGF-β1 signaling), the epithelial-mesenchymal transition (EMT) markers Zeb1, N-cadherin and vimentin expression in NSCLC cells. The downregulation of Ly6E reduced α5-nAChR, pSmad3, Zeb1, N-cadherin and vimentin expression. Functionally, silencing both α5-nAChR and Ly6E significantly inhibited cell migration compared to silencing α5-nAChR or Ly6E alone. Furthermore, the functional effects of α5-nAchR and Ly6E were confirmed in chicken embryo chorioallantoic membrane (CAM) and mouse xenograft models. Therefore, our findings uncover a new interaction between α5-nAChR and Ly6E that inhibits cancer cell migration by modulating the TGF-β1/Smad signaling pathway in NSCLC, which may serve as a novel target for therapeutic intervention.

Methylglyoxal Scavengers Attenuate Angiogenesis Dysfunction Induced by Methylglyoxal and Oxygen-Glucose Deprivation.

Cerebral endothelial cells play an essential role in brain angiogenesis, and their function has been found to be impaired in diabetes. Methylglyoxal (MG) is a highly reactive dicarbonyl metabolite of glucose formed mainly during glycolysis, and its levels can be elevated in hyperglycemic conditions. MG is a potent precursor of AGEs (advanced glycation end-products). In this study, we investigated if MG can induce angiogenesis dysfunction and whether MG scavengers can ameliorate angiogenesis dysfunction induced by MG. Here, we used cultured human brain microvascular endothelial cells (HBMECs) treated with MG and oxygen-glucose deprivation (OGD) to mimic diabetic stroke in vitro. We also used the MG challenged chicken embryo chorioallantoic membrane (CAM) to study angiogenesis in vivo. Interestingly, administration of MG significantly impaired cell proliferation, cell migration, and tube formation and decreased protein expression of angiogenesis-related factors, which was rescued by three different MG scavengers, glyoxalase 1 (GLO1), aminoguanidine (AG), and N-acetyl cysteine (NAC). In cultured CAM, MG exposure significantly reduced angiogenesis and the angiogenesis-related dysfunction could be attenuated by pretreatment with AG or NAC. Treatment of cultured HBMECs with MG plus OGD increased cellular apoptosis significantly, which could be prevented by exposure to GLO1, AG, or NAC. We also noted that administration of MG increased cellular oxidative stress as measured by reactive oxygen species (ROS) generation, enhanced AGE accumulation, and receptor for advanced glycation end-product (RAGE) expression in the cultured HBMECs, which were partially reversed by GLO1, AG, or NAC. Taken together, our findings demonstrated that GLO1, AG, or NAC administration can ameliorate MG-induced angiogenesis dysfunction, and this can be mainly attributed to attenuated ROS production, reduced cellular apoptosis, and increased levels of angiogenic factors. Overall, this study suggested that GLO1, AG, or NAC may be promising candidate compounds for the treatment of angiogenesis dysfunction caused by hyperglycemia in diabetic ischemic stroke.

A short-term chick embryo in vivo xenograft model to study retinoblastoma cancer stem cells.

Purpose:Cancer stem cells (CSCs) reported in various tumors play a crucial role in tumorigenesis and metastasis of retinoblastoma (Rb). Following the efforts to reduce, replace, and refine the use of mammalian models, we aimed to establish a short-term xenograft for Rb to evaluate the CSC properties of CD133- Rb Y79 cells, using the well-established chick embryo chorioallantoic membrane (CE-CAM) assay.Methods:Y79 cells were cultured, labeled with two different dyes (CM-Dil Y79 and enhanced green fluorescent protein (eGFP)) and sorted for CD133- and CD133 + subsets. Two million cells from each of the labeled groups were transplanted onto the abraded CAM on embryonic day 7 (E7). On E14, the tumor nodule formation on CAM and spontaneous metastasis to the embryos were evaluated by confocal microscopy, in vivo imaging, and histology.Results:Y79 cells formed pink–white raised perivascular nodules with feeder vessels on the CAM with both the types of labeled CD133- cells. CD133- cells, when compared to CD133 + cells, demonstrated significantly larger tumor volume (40.45 ± 7.744 mm3 vs 3.478 ± 0.69 mm3, P = 0.0014) and higher fluorescence intensity (CM-Dil: AUF = 6.37 × 107 ± 7.7 × 106 vs 1.08 × 107 ± 1.6 × 106; P < 0.0001; eGFP: AUF = 13.94 × 104 ± 2.54 × 104 vs AUF = 1.39 × 104 ± 0.4 × 104; P = 0.0003). The metastatic potential of CD133- cells was also observed to be higher as noted by in vivo imaging and histopathology.Conclusion:This study highlights that CE-CAM is a feasible alternative nonmammalian model for evaluating tumorigenicity and metastatic potential of Y79 CSCs. Increased tumorigenicity and metastatic potential of CD133- subset of tumor cells substantiate their CSC properties.

Novel sulfonamide-chalcone hybrid stimulates inflammation, angiogenesis and upregulates vascular endothelial growth factor (VEGF) in vivo.

Chalcones and sulfonamides are well-known chemical groups associated with several biological activities such as antibiotic, anti-inflammatory, and antitumor activities. Over the past few decades, a series of sulfonamide-chalcone hybrids have been synthesized and assessed to develop compounds with interesting biological properties for application in disease therapy. In the present study, a new sulfonamide-chalcone hybrid μ - (2,5-dichloro-N-{4-[(3E)-4-(3-nitrophenyl) buta-1,3-dien-2-yl] phenyl} benzene sulfonamide), or simply CL185, was synthesized, and its angiogenic activity was assessed using the chick embryo chorioallantoic membrane (CAM) assay at different concentrations (12.5, 25, and 50μg/μL). To further investigate the role of CL185 in the angiogenic process, we evaluated the levels of vascular endothelial growth factor (VEGF) in all treated CAMs. The results showed that all concentrations of CL185 significantly increased tissue vascularization (p<0.05) as well as the parameters associated with angiogenesis, in which inflammation was the most marked phenomenon observed. In all CAMs treated with CL185, VEGF levels were significantly higher than those in the negative control (p<0.05), and at the highest concentration, VEGF levels were even higher than in the positive control (p<0.05). The pronounced angiogenic activity displayed by CL185 may be related to the increase in VEGF levels that were stimulated by inflammatory processes observed in our study. Therefore, CL185 presents a favorable profile for the development of drugs that can be used in pro-angiogenic and tissue repair therapies.

Matteucinol combined with temozolomide inhibits glioblastoma proliferation, invasion, and progression: an in vitro, in silico, and in vivo study.

Glioblastoma is the most prevalent and malignant brain tumor identified in adults. Surgical resection followed by radiotherapy and chemotherapy, mainly with temozolomide (TMZ), is the chosen treatment for this type of tumor. However, the average survival of patients is around 15 months. Novel approaches to glioblastoma treatment are greatly needed. Here, we aimed to investigate the anti-glioblastoma effect of the combination of matteucinol (Mat) (dihydroxyflavanone derived from Miconia chamissois Naudin) with the chemotherapeutic TMZ in vitro using tumor (U-251MG) and normal astrocyte (NHA) cell lines and in vivo using the chick embryo chorioallantoic membrane (CAM) assay. The combination was cytotoxic and selective for tumor cells (28 μg/mL Mat and 9.71 μg/mL TMZ). Additionally, the combination did not alter cell adhesion but caused morphological changes characteristic of apoptosis in vitro. Notably, the combination was also able to reduce tumor growth in the chick embryo model (CAM assay). The docking results showed that Mat was the best ligand to the cell death membrane receptor TNFR1 and to TNFR1/TMZ complex, suggesting that these two molecules may be working together increasing their potential. In conclusion, Mat-TMZ can be a good candidate for pharmacokinetic studies in view of clinical use for the treatment of glioblastoma.

Cardiac glycosides from Digitalis lanata and their cytotoxic activities.

Cardiac glycosides (CGs) are good candidates as drug leads in the treatment of cancer because of their structural diversities and potent biological activities. In this study, fifteen CGs including three new ones (1–3) were isolated from Digitalis lanata Ehrh. Their structures were elucidated by HRESIMS, NMR spectroscopic methods, including homonuclear and heteronuclear coupling constant analysis, and acid-catalyzed hydrolysis and derivatization analysis of the sugar chain. The cytotoxic activities of these CGs were evaluated against three human cancer cell lines (A549, HeLa and MCF-7 cell lines), and all of them showed strong activities at nanomolar scale. The flow cytometric analysis indicated that compound 1 induced cell cycle arrest in the G2/M phase. Transcriptome analysis revealed a panel of possible targets for compound 1. RT-PCR and western blot experiments showed that 1 significantly inhibited the expression of vasohibin-2 (VASH2). Moreover, compound 1 restrained angiogenesis in a concentration-dependent manner in the chick embryo chorioallantoic membrane (CAM) model.

Low-dose melittin is safe for intravitreal administration and ameliorates inflammation in an experimental model of uveitis

Uveitis is a group of sight-threatening ocular inflammatory disorders, whose mainstay of therapy is associated with severe adverse events, prompting the investigation of alternative treatments. The peptide melittin (MEL) is the major component of Apis mellifera bee venom and presents anti-inflammatory and antiangiogenic activities, with possible application in ophthalmology. This work aims to investigate the potential of intravitreal MEL in the treatment of ocular diseases involving inflammatory processes, especially uveitis. Safety of MEL was assessed in retinal cells, chick embryo chorioallantoic membranes, and rats. MEL at concentrations safe for intravitreal administration showed an antiangiogenic activity in the chorioallantoic membrane model comparable to bevacizumab, used as positive control. A protective anti-inflammatory effect in retinal cells stimulated with lipopolysaccharide (LPS) was also observed, without toxic effects. Finally, rats with bacille Calmette-Guerin- (BCG) induced uveitis treated with intravitreal MEL showed attenuated disease progression and improvement of clinical, morphological, and functional parameters, in addition to decreased levels of proinflammatory mediators in the posterior segment of the eye. These effects were comparable to the response observed with corticosteroid treatment. Therefore, MEL presents adequate safety profile for intraocular administration and has therapeutic potential as an anti-inflammatory and antiangiogenic agent for ocular diseases.

The Chicken Embryo Chorioallantoic Membrane (CAM): A Versatile Tool for the Study of Patient-Derived Xenografts.

Patient-derived xenografts represent the gold standard in pre-clinical research models. The chicken embryo chorioallantoic membrane (CAM) is used in functional studies for studying biological processes such as blood vessel development and embryogenesis, biocompatible material testing, and more recently three-dimensional patient-derived xenograft (PDX) tumor modeling. We describe here a detailed method used to readily engraft established mouse PDX and primary patient tumor specimens on the CAM with as little as 25mg of tissue per embryonated egg.

The Chicken Embryo Chorioallantoic Membrane as an In Vivo Model for Photodynamic Therapy.

For many decades the chicken embryo chorioallantoic membrane (CAM) has been used for research as an in vivo model in a large number of different fields, including toxicology, bioengineering, and cancer research. More specifically, the CAM is also a suitable and convenient model system in the field of photodynamic therapy (PDT), mainly due to the easy access of its membrane and the possibility of grafting or growing tumors on the membrane and, interestingly, to study the PDT effects on its dense vascular network. In addition, the CAM is simple to handle and cheap. Since the CAM is not innervated until later stages of the embryo development, its use in research is simplified compared to other in vivo models as far as ethical and regulatory issues are concerned. In this review different incubation and drug administration protocols of relevance for PDT are presented. Moreover, data regarding the propagation of light at different wavelengths and CAM development stages are provided. Finally, the effects induced by photobiomodulation on the CAM angiogenesis and its impact on PDT treatment outcome are discussed.

Use of Chicken Embryos as an Angiogenesis Model for Central Nervous System Malignant Tumor Research.

To demonstrate the usability of chicken chorioallantoic membrane (CAM) as an angiogenesis model for the development and treatment of malignant tumors of the central nervous system. A fresh tumor tissue piece taken from Glioblastoma patients, a malignant tumor of the central nervous system, was transferred to the CAM of chicken embryos and left to incubate in the incubator and their development was monitored. After examining the results of the study macroscopically, CAM tissue samples were evaluated both histochemically and immunohistochemically in terms of angiogenic factors VEGF (Vascular Endothelial Growth Factor), bFGF (basic Fibroblast Growth Factor) and PDGF (Platelet Derived Growth Factor). According to histochemical findings obtained from our study when compared with control embryos, blood vessels, fibroblast count and inflammatory infiltration were observed more in the tumor transplanted groups, especially in the tumordeveloping CAM region. There was also intense pleomorphism and marked hypercellularity in the cells. In our immunohistochemical findings, it was determined that bFGF, PDGF, VEGF staining intensities were higher in tumor transplanted groups compared to control groups, and this elevation was more pronounced in the tumor-developing region. As a result, it has been shown that the chicken embryo CAM model may be a suitable in vivo model for cancer angiogenesis studies. The protocol we created in this study will be a source for projects related to the use of therapeutic agents in cancer angiogenesis.