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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 exposure on heart size and weight of chick embryos.(A) Representative appearance of hearts harvested from 14-day-old (HH 40) embryos treated with saline, 1.8 and 2.6 µmol of AAPH. (B) Statistical chart showing the embryonic heart weight after AAPH exposure. Results presented as mean ± S.D. (n = 10). Analyzed by SPSS13.5 software, **p<0.01 compared with control. Abbreviation: LV, left ventricle; RV, right ventricle. Scale bar = 1 mm.
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pone-0057732-g006: Effect of AAPH exposure on heart size and weight of chick embryos.(A) Representative appearance of hearts harvested from 14-day-old (HH 40) embryos treated with saline, 1.8 and 2.6 µmol of AAPH. (B) Statistical chart showing the embryonic heart weight after AAPH exposure. Results presented as mean ± S.D. (n = 10). Analyzed by SPSS13.5 software, **p<0.01 compared with control. Abbreviation: LV, left ventricle; RV, right ventricle. Scale bar = 1 mm.

Mentions: It was determined that AAPH (10 µmol/egg) exposure for 2 hours significantly elevated MDA content in the HH35 embryonic heart (Figure 5A) and ORAC level was significantly decreased (Figure 5B). Interestingly, ORAC level (antioxidant indicator) was recovered after 8 hour AAPH treatment. In addition, the size of the developing heart was found larger following lower doses of AAPH exposure. This was especially obvious when the HH 35 embryos were treated with 1.8 or 2.6 µmol/egg AAPH for 5 consecutive days (Figure 6A). However, exposing AAPH on alternative days at the same concentrations did not significantly affect the heart as compared with controls (Figure 6A). Consistent with the increase in heart size, there was a corresponding increase in heart weight (Figure 6B). However, it happened only if AAPH was given every day rather than on alternative days (i.e. AAPH exposure continually maintained at high levels). Haematoxylin-eosin (H&E) stained histological vertical sections of embryonic hearts were obtained from hearts shown in Figure 6A. We measured the thickness of the left and right ventricular walls and interventricular septum of AAPH (1.8 or 2.6 µmol/egg) treated heart at HH 40 (Figure 7). It was found that the left ventricular wall and interventricular septum were significantly thicker (Figures. 7B’–B’’ and C’–C’’) than the control (Figures. 7A’–A’’) in a dose-dependent manner (Figure 7D). This explained the increased heart size and weight following AAPH treatment (Figure 6). Interestingly, the wall thickness of right ventricle was not altered by AAPH treatment (Figures. 7A’’’, B’’’, C’’’ and D). Further experiments had performed on analysis of myocytes size, and results found that average myocytes size is increased in left ventricle and interventricular septum of both AAPH treatment groups (Figure 8 A–C, A’–C’). However, cell size did not vary in right ventricle between control and AAPH groups (Figure 8 A’’–C’’). Therefore, heart enlargement caused by AAPH was probably due to myocardic cell hypertrophy.


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 exposure on heart size and weight of chick embryos.(A) Representative appearance of hearts harvested from 14-day-old (HH 40) embryos treated with saline, 1.8 and 2.6 µmol of AAPH. (B) Statistical chart showing the embryonic heart weight after AAPH exposure. Results presented as mean ± S.D. (n = 10). Analyzed by SPSS13.5 software, **p<0.01 compared with control. Abbreviation: LV, left ventricle; RV, right ventricle. Scale bar = 1 mm.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0057732-g006: Effect of AAPH exposure on heart size and weight of chick embryos.(A) Representative appearance of hearts harvested from 14-day-old (HH 40) embryos treated with saline, 1.8 and 2.6 µmol of AAPH. (B) Statistical chart showing the embryonic heart weight after AAPH exposure. Results presented as mean ± S.D. (n = 10). Analyzed by SPSS13.5 software, **p<0.01 compared with control. Abbreviation: LV, left ventricle; RV, right ventricle. Scale bar = 1 mm.
Mentions: It was determined that AAPH (10 µmol/egg) exposure for 2 hours significantly elevated MDA content in the HH35 embryonic heart (Figure 5A) and ORAC level was significantly decreased (Figure 5B). Interestingly, ORAC level (antioxidant indicator) was recovered after 8 hour AAPH treatment. In addition, the size of the developing heart was found larger following lower doses of AAPH exposure. This was especially obvious when the HH 35 embryos were treated with 1.8 or 2.6 µmol/egg AAPH for 5 consecutive days (Figure 6A). However, exposing AAPH on alternative days at the same concentrations did not significantly affect the heart as compared with controls (Figure 6A). Consistent with the increase in heart size, there was a corresponding increase in heart weight (Figure 6B). However, it happened only if AAPH was given every day rather than on alternative days (i.e. AAPH exposure continually maintained at high levels). Haematoxylin-eosin (H&E) stained histological vertical sections of embryonic hearts were obtained from hearts shown in Figure 6A. We measured the thickness of the left and right ventricular walls and interventricular septum of AAPH (1.8 or 2.6 µmol/egg) treated heart at HH 40 (Figure 7). It was found that the left ventricular wall and interventricular septum were significantly thicker (Figures. 7B’–B’’ and C’–C’’) than the control (Figures. 7A’–A’’) in a dose-dependent manner (Figure 7D). This explained the increased heart size and weight following AAPH treatment (Figure 6). Interestingly, the wall thickness of right ventricle was not altered by AAPH treatment (Figures. 7A’’’, B’’’, C’’’ and D). Further experiments had performed on analysis of myocytes size, and results found that average myocytes size is increased in left ventricle and interventricular septum of both AAPH treatment groups (Figure 8 A–C, A’–C’). However, cell size did not vary in right ventricle between control and AAPH groups (Figure 8 A’’–C’’). Therefore, heart enlargement caused by AAPH was probably due to myocardic cell hypertrophy.

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