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A new oxidative stress model, 2,2-azobis(2-amidinopropane) dihydrochloride induces cardiovascular damages in chicken embryo.

He RR, Li Y, Li XD, Yi RN, Wang XY, Tsoi B, Lee KK, Abe K, Yang X, Kurihara H - PLoS ONE (2013)

Bottom Line: At this concentration, AAPH was found to significantly reduce the density of blood vessel plexus that was developed in the chorioallantoic membrane (CAM) of HH 35 chick embryos.These results implied that AAPH-induced oxidative stress could impair the whole developmental processes associated with vasculogenesis and angiogenesis.In conclusion, oxidative stress, induced by AAPH, could lead to damage of the cardiovascular system in the developing chick embryo.

View Article: PubMed Central - PubMed

Affiliation: Pharmacy College, Jinan University, Guangzhou, People's Republic of China.

ABSTRACT
It is now well established that the developing embryo is very sensitive to oxidative stress, which is a contributing factor to pregnancy-related disorders. However, little is known about the effects of reactive oxygen species (ROS) on the embryonic cardiovascular system due to a lack of appropriate ROS control method in the placenta. In this study, a small molecule called 2,2-azobis(2-amidinopropane) dihydrochloride (AAPH), a free radicals generator, was used to study the effects of oxidative stress on the cardiovascular system during chick embryo development. When nine-day-old (stage HH 35) chick embryos were treated with different concentrations of AAPH inside the air chamber, it was established that the LD50 value for AAPH was 10 µmol/egg. At this concentration, AAPH was found to significantly reduce the density of blood vessel plexus that was developed in the chorioallantoic membrane (CAM) of HH 35 chick embryos. Impacts of AAPH on younger embryos were also examined and discovered that it inhibited the development of vascular plexus on yolk sac in HH 18 embryos. AAPH also dramatically repressed the development of blood islands in HH 3+ embryos. These results implied that AAPH-induced oxidative stress could impair the whole developmental processes associated with vasculogenesis and angiogenesis. Furthermore, we observed heart enlargement in the HH 40 embryo following AAPH treatment, where the left ventricle and interventricular septum were found to be thickened in a dose-dependent manner due to myocardiac cell hypertrophy. In conclusion, oxidative stress, induced by AAPH, could lead to damage of the cardiovascular system in the developing chick embryo. The current study also provided a new developmental model, as an alternative for animal and cell models, for testing small molecules and drugs that have anti-oxidative activities.

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Effect of AAPH on angiogenesis in the CAM of chick embryos.(A) Representative appearance of CAM treated with saline for 24 h. (B) Representative appearance of CAM treated with 10 µmol/egg of AAPH for 24 h. (C) Statistical chart showing the blood vessel density of CAM from AAPH treated and untreated embryos. (D) The CAMs of embryos were treated with 10 µmol/egg of AAPH for 2–8 hours and then the MDA content of embryos were measured. (E) The thickness of CAM after 12–36 h exposure to AAPH. CAM thickness was calculated as the ratio of weight to area of CAM (mg/cm2). The results are presented as mean ± S.D (n = 10). Statistical significances were evaluated using SPSS13.5 software, presented as *p<0.05, **p<0.01 in comparison with control group. Scale bar = 1 mm.
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pone-0057732-g002: Effect of AAPH on angiogenesis in the CAM of chick embryos.(A) Representative appearance of CAM treated with saline for 24 h. (B) Representative appearance of CAM treated with 10 µmol/egg of AAPH for 24 h. (C) Statistical chart showing the blood vessel density of CAM from AAPH treated and untreated embryos. (D) The CAMs of embryos were treated with 10 µmol/egg of AAPH for 2–8 hours and then the MDA content of embryos were measured. (E) The thickness of CAM after 12–36 h exposure to AAPH. CAM thickness was calculated as the ratio of weight to area of CAM (mg/cm2). The results are presented as mean ± S.D (n = 10). Statistical significances were evaluated using SPSS13.5 software, presented as *p<0.05, **p<0.01 in comparison with control group. Scale bar = 1 mm.

Mentions: Chick embryos (HH 35) were treated with 10 µmol of AAPH solution at volume of 100 µL for 24 h and then the CAMs were photographed (Figures 2A and B). It was found that AAPH treatment lead to a significant reduction in the density of CAM blood vessel plexus produced when compared with control (Figure 2C). Even a shortened exposure time (2 to 8 hours) of AAPH (10 µmol) on the embryos could still significantly elevate malondialdehyde (MDA) contents in CAM (Figure 2D). It was also determined that the thickness of CAM was significantly increased after being exposed to AAPH for more than 12 hours (Figure 2E). The results showed that AAPH caused oxidative damage to the blood vessel plexus in CAM of chick embryos. It implied that AAPH might have affected angiogenesis and vasculogenesis during the growth and development of chick embryos.


A new oxidative stress model, 2,2-azobis(2-amidinopropane) dihydrochloride induces cardiovascular damages in chicken embryo.

He RR, Li Y, Li XD, Yi RN, Wang XY, Tsoi B, Lee KK, Abe K, Yang X, Kurihara H - PLoS ONE (2013)

Effect of AAPH on angiogenesis in the CAM of chick embryos.(A) Representative appearance of CAM treated with saline for 24 h. (B) Representative appearance of CAM treated with 10 µmol/egg of AAPH for 24 h. (C) Statistical chart showing the blood vessel density of CAM from AAPH treated and untreated embryos. (D) The CAMs of embryos were treated with 10 µmol/egg of AAPH for 2–8 hours and then the MDA content of embryos were measured. (E) The thickness of CAM after 12–36 h exposure to AAPH. CAM thickness was calculated as the ratio of weight to area of CAM (mg/cm2). The results are presented as mean ± S.D (n = 10). Statistical significances were evaluated using SPSS13.5 software, presented as *p<0.05, **p<0.01 in comparison with control group. Scale bar = 1 mm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585800&req=5

pone-0057732-g002: Effect of AAPH on angiogenesis in the CAM of chick embryos.(A) Representative appearance of CAM treated with saline for 24 h. (B) Representative appearance of CAM treated with 10 µmol/egg of AAPH for 24 h. (C) Statistical chart showing the blood vessel density of CAM from AAPH treated and untreated embryos. (D) The CAMs of embryos were treated with 10 µmol/egg of AAPH for 2–8 hours and then the MDA content of embryos were measured. (E) The thickness of CAM after 12–36 h exposure to AAPH. CAM thickness was calculated as the ratio of weight to area of CAM (mg/cm2). The results are presented as mean ± S.D (n = 10). Statistical significances were evaluated using SPSS13.5 software, presented as *p<0.05, **p<0.01 in comparison with control group. Scale bar = 1 mm.
Mentions: Chick embryos (HH 35) were treated with 10 µmol of AAPH solution at volume of 100 µL for 24 h and then the CAMs were photographed (Figures 2A and B). It was found that AAPH treatment lead to a significant reduction in the density of CAM blood vessel plexus produced when compared with control (Figure 2C). Even a shortened exposure time (2 to 8 hours) of AAPH (10 µmol) on the embryos could still significantly elevate malondialdehyde (MDA) contents in CAM (Figure 2D). It was also determined that the thickness of CAM was significantly increased after being exposed to AAPH for more than 12 hours (Figure 2E). The results showed that AAPH caused oxidative damage to the blood vessel plexus in CAM of chick embryos. It implied that AAPH might have affected angiogenesis and vasculogenesis during the growth and development of chick embryos.

Bottom Line: At this concentration, AAPH was found to significantly reduce the density of blood vessel plexus that was developed in the chorioallantoic membrane (CAM) of HH 35 chick embryos.These results implied that AAPH-induced oxidative stress could impair the whole developmental processes associated with vasculogenesis and angiogenesis.In conclusion, oxidative stress, induced by AAPH, could lead to damage of the cardiovascular system in the developing chick embryo.

View Article: PubMed Central - PubMed

Affiliation: Pharmacy College, Jinan University, Guangzhou, People's Republic of China.

ABSTRACT
It is now well established that the developing embryo is very sensitive to oxidative stress, which is a contributing factor to pregnancy-related disorders. However, little is known about the effects of reactive oxygen species (ROS) on the embryonic cardiovascular system due to a lack of appropriate ROS control method in the placenta. In this study, a small molecule called 2,2-azobis(2-amidinopropane) dihydrochloride (AAPH), a free radicals generator, was used to study the effects of oxidative stress on the cardiovascular system during chick embryo development. When nine-day-old (stage HH 35) chick embryos were treated with different concentrations of AAPH inside the air chamber, it was established that the LD50 value for AAPH was 10 µmol/egg. At this concentration, AAPH was found to significantly reduce the density of blood vessel plexus that was developed in the chorioallantoic membrane (CAM) of HH 35 chick embryos. Impacts of AAPH on younger embryos were also examined and discovered that it inhibited the development of vascular plexus on yolk sac in HH 18 embryos. AAPH also dramatically repressed the development of blood islands in HH 3+ embryos. These results implied that AAPH-induced oxidative stress could impair the whole developmental processes associated with vasculogenesis and angiogenesis. Furthermore, we observed heart enlargement in the HH 40 embryo following AAPH treatment, where the left ventricle and interventricular septum were found to be thickened in a dose-dependent manner due to myocardiac cell hypertrophy. In conclusion, oxidative stress, induced by AAPH, could lead to damage of the cardiovascular system in the developing chick embryo. The current study also provided a new developmental model, as an alternative for animal and cell models, for testing small molecules and drugs that have anti-oxidative activities.

Show MeSH
Related in: MedlinePlus