Utilization of a chicken embryo membrane model for evaluation of embryonic vascular toxicity of Dorema ammoniacum

Abstract

Objective: Extensive research has been done to assess the efficacy of herbs for treating different disorders. Dorema ammoniacum (D. ammoniacum) is used in folk medicines for various goals. The application of herbs in medicine is accompanied by harmful effects. Chick embryo is considered a suitable model for assessing drugs toxicity. The present study aimed to evaluate the changes in vasculature in chick's extra-embryonic membrane following D. ammoniacum treatment. Alterations in molecular pathways associated with early embryonic angiogenesis such as vascular endothelial growth factor A (VEGF-A) were also evaluated.

Materials and methods: Fertile chicken (Ross 308) eggs were allocated into three similar groups; sham, control and D. ammoniacum groups; in D. ammoniacum group, eggs were inoculated with plant's extract at doses of 50 or 100 mg per kg egg-weight.

Results: Analysis of the extra-embryonic membrane vasculature revealed that D. ammoniacum extract decreases some vascular parameters such as vessels area, total vessels length, vascular branch and increases lacunarity. This herb's vascular toxicity was in a dose-dependent manner. Down-regulation of the expression of VEGF-A was also seen in the extract-treated extra-embryonic membrane.

Conclusion: Vascular toxicity of D. ammoniacum was confirmed by data presented in this paper. We conclude that alteration of vascular parameters and gene expression might finally lead to embryo malformation due to D. ammoniacum consumption. Therefore, the use of this herb must be limited during the fetal growth period especially at doses higher than 50 mg per kg.

Keywords: Angiogenesis; Dorema ammoniacum; Embryo; Fetus; Pathology; VEGF-A.

Figure 1

The vascular networks of the day 4 embryos are presented to illustrate the image manipulations required for the vascular branching pattern analysis. The images are captured from the embryo of the sham group (a-c) and Dorema ammoniacum at doses of 50 (d-f) or 100 (g-i) mg per kg egg-weight. An area of 174 mm² containing 1100·×1970 pixels, was identified at the right-lateral vitelline vascular plexus (a, d, and g). The images were converted to the 8-bit format (b, e, and h). The vascular branching pattern was calculated from the skeletonized pictures (c, f, and i)

Figure 2

The mean capillary area (MCA) quantified from the chick’s extra-embryonic membrane on day 4 of the incubation period. Certain zone is seen inside the right-lateral vitelline vascular plexus (a). The extracted zone has been converted to a binary format (b). Five areas (arrows) without any branch vessels are selected and the percentage of the areas containing black pixels was calculated for quantification of the MCA (c). The black pixels of the image indicate the red color, or blood, in the original image

Figure 3

Embryonated eggs were treated three times at 24, 48 and 72 hr of the incubation period. Sham embryo with normal extra-embryonic membrane vasculature is seen (a). Embryonated egg received Dorema ammoniacum resin-extract at the dosage of 100 mg per kg egg-weight (b). Vascular alteration is demonstrated by the retarded vascular network. A.V.V., anterior vitelline vein; L.V.V., left lateral vitelline vessel; P.V.V., posterior vitelline vein; and R.V.V., right lateral vitelline vessel

Figure 4

Effect of Dorema ammoniacum resin-extract on the early embryonic angiogenesis was assessed by mean capillary area (MCA) quantification. Data are presented for sham (n=10) and Dorema ammoniacum resin-extract 50 (n=10) and 100 (n=10) mg per kg egg-weight. The MCA was reduced in D. ammoniacum-treated group of 100 mg per kg egg-weight (error bars show standard error of mean; *p<0.05, One-Way ANOVA and post hoc Tukey test)

Figure 5

Relative mRNA expression levels of VEGF-A gene in experimental groups. The expression of VEGF-A in the chick extra-embryonic membranes (n=4 per experimental group) was decreased in the Dorema ammoniacum-treated group of 100 mg per kg egg-weight. D. ammoniacum resin-extract was administered at doses of 50 or 100 mg per kg egg-weight (error bars show standard error of mean; *p<0.05, One-Way ANOVA and post hoc Tukey test)