| Background and objective As an important part of health care in obstetrics,ultrasound plays an irreplaceable role in the diagnosis of fetal malformation.Obstetric ultrasound can be applied in early phase and highly repeatable.Its safety has drawn widespread attention.With the increasing demand for better image quality,the output power of the ultrasonic machine at the diagnostic level also increased considerably.At present,real-time three-dimensional ultrasound has been gradually carried out in clinical practice.Experimental studies have shown that low-frequency ultrasound can produce biological effects on humans and animals.However,there are few studies on the safety of real-time three-dimensional obstetric ultrasound today.Improved output power of the real-time three-dimensional ultrasound is also related to the problem of continuous irradiation,and some non-medical institutions even make fetus facial photo album for commercial profit,illustrating the abuse of real-time three-dimensional ultrasound.The present study investigated the effect of real-time three-dimensional ultrasound irradiation on the offspring's body growth and neural development by different exposure time in mid-gestation pregnant rats.The offspring's learning and memorizing abilities were also evaluated by water maze and open field test.The ultrastructure and apoptosis related gene expression of hippocampus and fetus were observed by transmission electron microscopy,immunohistochemistry and Western blot.The objectives are to investigate the effects of different doses of real-time three-dimensional ultrasound on the brain function of offspring,and to provide experimental reference for the safe application of real-time three-dimensional ultrasound.Methods 1.Model of real-time three-dimensional ultrasound irradiation: Female SD rats of pregnant 16 days,which were randomly divided into 4 groups by exposure time: 0-minute group(control group),10-minute group,20-minute group and 30-minute group.The rats received continuous real-time three-dimensional ultrasound irradiation.2.The expression of hippocampus apoptosis related genes Bcl-2,Bax,Cyt-c,Caspase-9,Caspase-3 in fetal rats were examined after 24 hours exposure to the real-time three-dimensional ultrasound by immunofluorescence and Western blot.3.The ultrastructural changes of neurons in the hippocampus of fetal rats were observed by transmission electron microscopy(TEM).The ultrastructural changes of nerve cells and synapses in the hippocampus of the experimental groups were observed 24 hours after birth.4.The permeability of blood brain barrier in hippocampus was detected by lanthanum nitrate tracing technique,and the results were observed by transmission electron microscope.5.The behavior activity of pregnant rats was observed after anesthesia,and the duration of pregnancy of pregnant rats and the number of birth were analyzed.6.Then the growth and neurobehavioral development of neonatal rat were observed.7.Then 7 weeks old neonatal rats were selected to take Morris water maze test forevaluating learning and memory abilities.Results 1.The expressions of Bax,Cyt-c(cytosol),Caspase-9 and Caspase-3 in the 20 min group and 30 min group were increased in comparsion with the control group.Compared with the control group,the expression of promote apoptotic gene in 10 min group was not significantly different.Compared with the control group,the expression of anti-apoptotic gene Bcl-2 in the hippocampus of the 10 min group did not change significantly,but the trend of decrease was observed in the 20 min and 30 min groups.The Bcl-2/Bax ratios in 20 min and 30 min groups were decreased than those in the control group.The Bcl-2/Bax ratio in 10 min group was not significantly different from that in the control group.2.There was no obvious change in the ultrastructure of nerve cells and synapses in control group and 10 min group,but the 20 min and 30 min groups showed cell apoptosis.3.Lanthanum nitrate tracing technique and transmission electron microscope observation showed that the structure of blood-brain barrier was damaged at irradiation above 20 min,and the damage of blood-brain barrier was more serious with time increase.4.In 20 min group,the pericyte was intact.The cytoplasm,mitochondria and endoplasmic reticulum of pericytes showed no cavitation changes,and the cell membranes were intact without damage.The distribution of lanthanum nitrate particles was found between the extracellular vascular processes and the damage of the vascular endothelial cells under irradiation with 30 min.The pericytes of capillaries were intact.5.In the control and 10 min group,there were fewer glial cells,less active cells,less neurites,and less mitochondria and endoplasmic reticulum in the cytoplasm.In 20 min group,the tracer lanthanum nitrate particles were distributed around the glial cells,but not in the glial cells.The mitochondria in the cytoplasm were intact,the endoplasmic reticulum extended and the structures were clear.the number of glial cells in 30mingroup increased significantly,the state was active,the cell process was prolonged,and the process of contact with capillaries was increased.6.In 30-minute group,the days of opened ears and eyes,tooth eruption were later than those of control group(P < 0.05),but there were no significant difference between 10-minute group,20-minute group and control group(P > 0.05).The standard days of rat turned over on plane,heard astonished,turned over in air appeared and cliff parry after birth in 20-minute group and 30-minute group were later than those in control group(P < 0.05).7.Open field test indicated that movement duration in central area of 30-minute group was less than that of control group(P < 0.05).8.In Morris water maze test: The escape latencies of rats in 20 min and 30 min groups were significantly longer than those of the control group(P<0.05);the time spent percent in the target quadrant and the numbers of crossing the plate in 20 min and 30 min groups were significantly lower than those of the control group(P<0.05),whereas there were no differences between the 10 min group and the control group(P>0.05).Conclusions 1.Real-time three-dimensional ultrasound exposure during mid-gestation can activate Mitochondrial mediated apoptosis pathway.2.Long time real-time three-dimensional ultrasound exposure during mid-gestation has effect on offspring' brain ultrastructure,including increased blood-brain barrier permeability,activation of pericytes and proliferation of glial cells.3.Long time real-time three-dimensional ultrasound exposure during gestation could delay the growth and neurobehavioral development of the rat offspring,and reduce their learning and memorizing abilities. |
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