Blood Vessels In Chick Chorioallantoic Membrane Research Articles

Anti-angiogenic effect of nano-formulated water soluble kaempferol and combretastatin in an in vivo chick chorioallantoic membrane model and HUVEC cells

The present study evaluated the efficacy of nano-formulated water-soluble kaempferol and combretastatin alone and combined against the native kaempferol and combretastatin on angiogenesis. The solvent evaporation method was used to synthesize the nano-formulated water-soluble kaempferol and combretastatin and characterized using various analyses such as dynamic light scattering (DLS) and Fourier-transform infrared (FT-IR) spectroscopy.The anti-angiogenic activity of native, nano-formulated water-soluble kaempferol and combretastatin was investigated by cell viability on HUVEC and A498 cell lines, while chick chorioallantoic membrane (CAM) assay was utilized to assess morphometric and histopathological changes, and mRNA expressions of VEGF-A and FGF2 using qRT-PCR. MTT assay results revealed that the combination of nano-formulated water-soluble kaempferol and combretastatin significantly reduced the cell viability compared to control, individual treatments of native, nano-formulated water-soluble kaempferol, and combretastatin. Morphometric analysis of CAM showed that treatment with nano-formulated water-soluble kaempferol and combretastatin caused a substantial decrease in density, vessel network, branch points, and nets of CAM blood vessels. The histopathological results of CAM showed the irregular shape of blood vessels at the thin stratum of chronic endoderm, and blood capillaries were diminished compared to the control. In addition, the mRNA expression levels of VEGF-A and FGF2 were significantly decreased compared with native forms. Therefore, the findings of this study indicate that nano-formulated water-soluble combretastatin and kaempferol suppress angiogenesis by preventing the activation of endothelial cells and suppressing factors of angiogenesis. Moreover, a combination of nano-formulated water-soluble kaempferol and combretastatin worked much better than individual treatments.

Antiretroviral therapy and HIV-associated cancers: Antiangiogenic effect of efavirenz on chick chorioallantoic membrane

Purpose: To investigate the anti-angiogenic effect of efavirenz in chick chorioallantoic membrane (CAM).Methods: Fertile eggs of the domestic fowl (Gallus gallus, variant Domesticus) were used as the in vivo vascular test environment. Experimental groups were VEGF, thalidomide (negative control), efavirenz (test drug), control 1 (treated with diluent) and control 2 (no treatment). CAM photographs were taken using a microscope camera. CAM blood vessels were scored as either “present” or “absent”.Results: There was association between treatment drugs and growth of CAM blood vessels (p < 0.05) as follows: control 1 – 100 %, control 2 – 67 %, VEGF - 83 %, thalidomide - 17 %, and efavirenz - 0 % (n = 30). Although efavirenz had absolute anti-angiogenicity and presented as a potential anti-cancer drug, there was no significant difference between the anti-angiogenicity of efavirenz (100 %) and thalidomide (83 %) (F = 1.000, p = 0.341).Conclusion: Being a first-line drug in both HAART and combination treatment of HIV-1, efavirenz may be responsible for spontaneous resolution of secondary cancers in HIV-infected patients on HAART regimen.Keywords: Efavirenz, HIV, Cancers, Angiogenesis, Chick chorioallantoic membrane

Methanolic extract of Euchelus asper exhibits in-ovo anti-angiogenic and in vitro anti-proliferative activities

BackgroundThe marine environment is a rich source of bioactive natural products. Many of the marine bioactive compounds have been derived successfully from molluscs. Euchelus asper is a marine mollusc which is commonly found in the intertidal rocky regions of the Mumbai coast. The present study was focused on evaluating the anti-angiogenic and anti- proliferative activities of methanolic extract of Euchelus asper (EAME).MethodsThe anti-angiogenic activity of EAME (50–800 μg/mL) was assessed by chick chorio-allantoic membrane (CAM) model wherein multiple parameters in the CAM blood vessels were analysed through morphometric and histological investigations. In vitro testing of EAME (5–20 μg/mL) included its cytotoxicity against three different cancer cell lines, its effect on cell proliferation by wound healing assay as well as their relevant molecular mechanisms. Statistical analysis was carried out by two-tailed student’s t test for two unpaired groups.ResultsAnalysis of CAM revealed that the extract is effective in reducing the branching points of the 1st order blood vessels or capillaries of CAM. Histological analysis of CAM showed significant decrease in capillary plexus and compartmentalization along with increase in mesodermal blood vessels, thus establishing its anti-angiogenicity. Further, EAME exhibited moderate but significant cytotoxicity against A549 non-small cell lung carcinoma cell line. We also demonstrated that the cytotoxicity of EAME in A549 was associated with its apoptotic activity by subG1 phase arrest. Lastly, EAME significantly reduced A549 proliferation by reducing the expression of Matrix metalloproteinase-2 (MMP-2) and Matrix metalloproteinase-9 (MMP-9).ConclusionOverall, our study suggested that EAME has potential to inhibit tumour angiogenic and proliferative activity and may be a potential source for development of new anti-cancer pharmaceuticals.

Clotrimazole microemulsion and microemulsion-based gel: evaluation of buccal drug delivery and irritancy using chick chorioallantoic membrane as the model.

To investigate the efficacy of clotrimazole microemulsion (CTZ-ME) and its gel form, clotrimazole microemulsion-based gel (CTZ-MBG), for the treatment of oral candidiasis. CTZ-ME and CTZ-MBG were characterized for droplet size and texture, respectively. The ex-vivo permeation study and irritancy assessment of CTZ-ME and CTZ-MBG were performed using chick chorioallantoic membrane (CAM) as the model. Antifungal activity against Candida albicans ATCC 10231 of CTZ-ME and CTZ-MBG was determined by agar diffusion method compared to the blank counterparts. CTZ-ME contained nano-sized droplets and CTZ-MBG had acceptable firmness and spreadability. CTZ-ME exhibited faster CAM permeation of the drug and larger inhibition zone than CTZ-MBG as the increased viscosity of CTZ-MBG resulted in more retardation and higher fluctuations in drug diffusion. As there were no detectable visual changes in CAM blood vessels after applying CTZ-ME or CTZ-MBG, both formulations were non-irritants. CTZ-ME and CTZ-MBG could deliver the drug through CAM, the model for buccal delivery. Additionally, they did not cause irritancy and had effective antifungal activity against C.albicans. The results indicated that CTZ-ME and CTZ-MBG were potential effective antifungal formulations to treat oral candidiasis.

The chick chorioallantoic membrane imaging method as a platform to evaluate vasoactivity and assess irritancy of compounds

To determine if the chick chorioallantoic membrane (CAM) is a potential alternative that is capable of screening test substances for vasoactivity in terms of vessel diameter changes. The CAM was also evaluated as a tool for irritancy screening. Visual assessment of the CAM for irritancy after the application of the test substance or solvent to its surface was made. An imaging based-in-vivo CAM model was developed by imaging CAM blood vessels in a pre-defined area using a semi-automatic image processing and analysis technique to measure blood vessel diameters. Solvents and drugs such as 70% v/v ethanol, normal saline, 5% w/v glucose monohydrate, glycerin, glucagon, N-methylpyrrolidone, nicotine, glyceryl trinitrate, glucagon, propranolol and caffeine were tested on the CAM. Propranolol, nicotine and glycerin were irritants on CAM. Changes in the diameters of fine blood vessels were accurately measured by high resolution image analysis. Vasoconstriction was seen with 70% v/v ethanol while vasodilation was displayed with glucagon and caffeine. The results reflected expected trends with evidence of feedback mechanisms ensuring homeostasis. The CAM model can be applied to assess pharmaceutical and cosmetic formulations in early development work to gain useful insights to potential irritancy and biological effects of components and formulations.

Photothrombic activity of m-THPC-loaded liposomal formulations: Pre-clinical assessment on chick chorioallantoic membrane model

The objective of this study was to evaluate the ability of meso-tetra(hydroxyphenyl)chlorin (m-THPC) encapsulated into liposomal formulations to occlude neovascularization. Two m-THPC formulations including conventional or plain liposomes (Foslip) based on dipalmitoylphosphatidylcholine (DPPC) and the corresponding long-circulating poly(ethylene glycol) (PEG)-modified liposomes (PEGylated liposomes: Fospeg) were evaluated as delivery systems. Using the chick chorioallantoic membrane (CAM) as in vivo model, the fluorescence pharmacokinetic behaviour of encapsulated m-THPC reflecting the rate of the extravasation of the dye from the CAM vasculature and its photothrombic effectiveness were determined. This study was focused on the influence of the drug and/or light doses on the mean retention time of m-THPC within the CAM blood vessels after intravenous injection, and its photothrombic efficacy. Irrespective of the formulations tested and the drug doses injected, similar fluorescence pharmacokinetic profiles were obtained. The fluorescence contrast reached a steady state 30 s after injection. Constant positive values of the fluorescence contrast suggest that m-THPC is confined into the intravascular compartment during the experimental time (500 s). However, the photodynamic therapy assays showed that Foslip appears to be less potent than Fospeg in terms of photothrombic activities on the CAM model. For instance, the light dose necessary to induce the desired vascular damage with Foslip was twice (100 J/cm 2) higher than with Fospeg (50 J/cm 2). It can be inferred that this pre-clinical study showed that the formulation based on PEGylated liposomes technology offers a suitable delivery system for the treatment of choroidal neovascularization associated with age-related macular degeneration.

Photodynamic parameters in the chick chorioallantoic membrane (CAM) bioassay for photosensitizers administered intraperitoneally (IP) into the chick embryo.

The chick chorioallantoic membrane (CAM) assay was used to determine the photodynamic response (PDR) of blood vessels to Photofrin, 5-aminolevulinic acid (ALA), benzoporphyrin derivative monoacid ring A (BPD-MA) and lutetium texaphyrin (Lutex). The photosensitizers were administered systemically via intraperitoneal injection into the chick embryo. Forward stepwise regression analysis of the PDR results enabled the individual contributions of seven experimental variables to be ranked: drug dose, light dose, fluence rate, drug uptake time, vessel type (whether arterioles or venules), vessel diameter, and embryo age. The order of importance of the variables, the PDR profile, was determined for each photosensitizer. Relative contributions of the experimental variables from this study to the CAM PDR were compared with those from our previous study on PDR of CAM blood vessels following topical application of the same photosensitizers. PDR profiles were interpreted in terms of biophysical and biochemical characteristics of the individual photosensitizers and the variation in their interactions with the delivery/distribution environment.

Infrared Imaging of In Vivo Microvasculature Following Pulsed Laser Irradiation

Infrared emission images of the chick chorioallantoic membrane (CAM) microvasculature following pulsed laser irradiation were recorded using a high speed infrared focal plane array camera. A three-dimensional tomographic reconstruction algorithm was applied to compute the initial space-dependent temperature increase in discrete CAM blood vessels caused by light absorption. The proposed method may provide consistent estimates of the physical dimensions of subsurface blood vessels and may be useful in understanding a variety of biomedical engineering problems involving laser-tissue interaction. © 1998 Society of Photo-Optical Instrumentation Engineers.

Laser pulse duration must match the estimated thermal relaxation time for successful photothermolysis of blood vessels

The relationship between photothermal damage to blood vessels of diameter,d, and laser pulse duration,t p, was verified in a series of studies using the chick chorioallantoic membrane (CAM). A total of 879 individual CAM blood vessels (d=50–130μm) was irradiated, using a laser pulse duration of 0.45 or 10 ms. Laser-induced vascular damage was observed in real time, recorded on videotape, and evaluated in a double-blind fashion. Permanent damage was confirmed by inspection 24 h after laser exposure. Under the conditions of this experiment, only when laser pulse durations are approximately equal to the estimated thermal relaxation times (τ) of the CAM microvessels can the critical core intravascular temperature, necessary to destroy vessels irreversibly, be achieved and sustained for sufficient time. Shorter pulse durations are more effective for damaging smaller blood vessels; conversely, longer pulse durations are more effective for damaging larger diameter vessels.