Multiple Skeletal Anomalies of Sprague Dawley Rats following Prenatal Exposure to Anastatica hierochuntica, as Delineated by a Modified Double-Staining Method

Anastatica hierochuntica (A. hierochuntica) is a plant that originates from Middle Eastern countries. This herb is commonly consumed by pregnant women to ease the process of childbirth. However, consumption of A. hierochuntica during the prenatal period may disrupt foetal development. In this study, we aimed to evaluate the potential effects of four different doses (0, 250, 500 and 1000 mg/kg) of A. hierochuntica aqueous extract (AHAE) on the skeletal development of Sprague Dawley rat foetuses. The AHAE was administered from gestational day (GD) 6 till GD20. We also aimed to produce a simplified and reproducible skeletal staining procedure for proper skeletal assessment of full-term Sprague Dawley rat foetuses. Skeletal structures were stained using a modified method that utilised Alcian Blue 8GX and Alizarin Red S dyes. The staining procedure involved fixation, skinning, evisceration, cartilage staining, bone staining and clearing. Our modified staining technique has successfully showed a clear demarcation between the bone and cartilage components, which enabled objective assessment of the skeletal ossification following administration of AHAE. Some skeletal anomalies such as sacrocaudal agenesis and maxillary defect (cleft lip) were observed in 250 and 1000 mg/kg groups, respectively. These findings indicate potential toxicity effects of AHAE on the developing foetuses.