Quantification of angiogenic characteristics of Naproxen sodium through chorioallantoic membrane assay

Abstract

Introduction: The aim of the current study was to determine the angiogenic/anti-angiogenic effect of Naproxen Sodium and to determine the effective dose of NapS for angiogenesis.

Method: Fertilized eggs (5days old) of chicken were acquired from a local hatchery. They were incubated at 37_oC with humidity of 55-60%. Then, on the 5th day of incubation, a window about 2cm in diameter was created by removing the shell and then the inner shell membrane. It was done under aseptic environment. There were 5 groups designed. Group A was kept as control and was given 0.2ml PBS, group B, C, D, and E were given 0.1ml of 0.086g/100μl, 0.042g/100μl, 0.021g/100μl and 0.011g/100μl of Naproxen Sodium (NapS) respectively. On the sixth day, prepared concentrations of NapS were administered and eggs were again sealed with paraffin film under aseptic conditions. These were placed back in the incubator for next 24 hours. After 24 hours, eggs were taken out from the incubator and images of all groups were made using a DSL camera. The development of blood vessels and other features were observed using Adobe Photoshop version 7.0, then these images were transferred to scan probing image processing (SPIP) software 6.6.2. The diameters of different blood vessels were measured by using expert design software. The parameters include: the diameter and branching system of blood vessels measured as per mm, and categories of blood vessels; i.e., primary, secondary and tertiary blood vessels. Data were analyzed on SPSS statistical software version 22.0 using ANOVA. Dunnett’s test was applied considering P < 0.05 as significant.

Result: Application of Naproxen sodium on chorioallantoic membrane of day six of incubation showed angiogenic effects in high concentration and anti-angiogenic effect in low concentration. Outcomes indicated significant changes in CAM’s design, thinning of primary, secondary and tertiary blood vessels, reduction in surface roughness parameters, and decrease in Abbott curve. Conclusion: The substantial quantities of Naproxen sodium use locally may exhibit anti-angiogenic activity in the same manner those seen in vitro and explain its clinical efficacy.