| It has been nearly 45 years since ultrasound had been used to diagnose diseases and prenatal examination. The safety of ultrasound is always the focus of many investigators which concentrate on. Ultrasound has a time-dose relationship,namely large-dose,high-intensity ultrasound could injury the body instantly,while small-dose,low intensity ultrasound harm the body in a quite longer period of time. The safety of ultrasound is in fact the question of prenatal and fetal ultrasound bioeffect. The damage of several embryonic cells in early gestation will lead to serious outcome, such as abnormalities and defects. As computer technology progresses some newly-made ultrasound machines are employed in the field of obstetrics. Old-fashionable types of two-dimensional scanners (B Mode) could visualize fetal cross-sections, spectral Doppler and color Doppler machines are able to evaluate the umbilical flow and fatal cardiovascular dynamics. Many studies about the ultrasound safety are based on the old type ultrasound machines, which have less output than"state of art"machines. Recent years the trend of using three dimensional (3D) and four dimensional (4D) ultrasound for prenatal examination poses a potential impact to fetuses. It is an imperative task to investigate the safety of modern machines for ultrasound workers. A variety of clinical parameters have been evaluated in infants exposed to ultrasound. These studies were designed as nonrandomized clinical studies and randomized controlled trials. In 2000 Oh et al established the biological indicator for the radiation and safety of diagnostic ultrasound using apoptosis. Since then various scholars began to observe the apoptosis rate in the tissues and organs after exposure to ultrasound by using electron microscopy and a few kinds of histochemistry, including terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), Caspase family etc. American scholars Ang et al extended the bioeffect study through their test of ultrasound wave impacting neuronal migration in mice.Our study contains three parts. The first part utilizes microscopy, transmission electron microscopy and TUNEL methods to observe the morphological changes and neuronal apoptosis after exposure to live three-dimensional ultrasound. The second part mainly investigates the apoptosis and Caspase 3,9 expression in mouse cortex induced by live three-dimensional ultrasound exposure. The third part inspects the effect of live three-dimensional ultrasound wave on neuronal migration within the embryonic cerebral cortex in mice by using BrdU injection to label neurons generated at embryonic day 16 and destined for the superficial cortical layers on postnatal day 10.Part I Effect of Diagnostic Live Three-Dimensional Ultrasound on Morphological Changes of Embryo-Cerebral Cells in Late Pregnant MiceObjective: To study the effect of live three-dimensional ultrasound aniso-irradiation on morphological changes of embryo-cerebral cells in late pregnant mice.Methods: Thirty pregnant mice were randomly divided into 6 groups, unexposed group, pseudo-exposed group, 5min-exposed group, 10min-exposed group, 20min-exposed and 30min-exposed group, and 5 mice in each group. Exposed mice were irradiated under the system's probe for 5 to 30 min on pregnant day 16. Ten fetuses of each group 24h after birth were selected; the right parietal lobes were observed with transmission electron microscope. On the 10th day after birth, the other fetuses of each group were perfused and fixed with 4 % paraformaldehyde; brain slices were made and stained with HE.Results: 1) Light microscope: there were no evident abnormal changes in unexposed groups, pseudo-exposed group, 5min-exposed group and 10min-exposed group .The disorder of nerve cell arrangement and cell necrosis were observed in 20min-exposed group and 30min-exposed group. 2) Electron microscope: there were no obvious abnormal changes in unexposed group, pseudo-exposed group, and 5min-exposed group. There were some mitochondrion enlargement; crista breakage, vacuolization of rough endoplasmic reticulum and a few apoptosis cell were seen in 10min-exposed group. More abnormal mitochondria, rough endoplasmic reticulums and apoptosis cells were observed in 20min-exposed and 30min-exposed groups,nucleus condensed,typical apoptotic bodies were seen。Conclusion:Live three-dimensional ultrasound irradiation for more than 10 minutes may result in abnormal neuron morphological changes and apoptosis in fetal mouse cerebrum.Part II Study on Apoptosis of Neurons in Mouse Cerebrum by using TUNEL and Expression of Caspase3, 9 after Live Three-dimensional Ultrasound Radiation.Objective: To study the effect of live three-dimensional ultrasound radiation on apoptosis of embryo-cerebral cells and expression of caspase3,caspase 9 in late pregnant mice.Methods: Thirty pregnant mice were randomly divided into 6 groups, unexposed group, pseudo-exposed group, 5min-exposed group, 10min-exposed group, 20min-exposed and 30min-exposed group, and 5 mice in each group. Exposed mice were irradiated under the system's probe for 5 to 30 min on pregnant day 16. On the 10th day after birth, the pups of each group were perfused and fixed with 4 % paraformaldehyde; brain slices were made and stained with HE or terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). Colorimetric method was used to measure caspase 3,9 activity.Results: 1) Tunel method: comparing with un-exposed group and pseudo-exposed group, positive rate of apoptosis in 5min-exposed group had no significant statistically difference (P>0.05), but in 10min-exposed group, apoptosis positive cells were increased and the findings became remarkable in 20min-exposed and 30min-exposed groups which showed a significant statistically difference (P<0.01) compared with other four groups. 2) Caspase3, 9 positive cells were detected in all groups and were remarkably increased in ultrasound exposed group.Conclusion:Live three-dimensional ultrasound irradiation for longer duration may result in higher caspase3,9 activity that lead to excessive neuron apoptosis in fetal mouse cerebrum.Part III Influence of Live Three-dimensional Ultrasound on neuronal migration in miceObjective : To observe the influence of live three-dimensional ultrasound irradiation on neuronal migration in mice.Methods: Thirty pregnant mice were randomly divided into 6 groups, unexposed group(control group), pseudo-exposed group, 5min-exposed group, 10min-exposed group, 20min-exposed and 30min-exposed group, 5 mice in each group. Mice in exposed group were irradiated under the system's probe for 5 to 30 min on pregnant day 16. Each pregnant mouse received i.p. injection of 50 mg/kg BrdU 24h,16h and 8h before beginning ultrasound exposure. Pregnant mouse in pseudo-exposed group and control group were also given i.p. injection of 50 mg/kg BrdU on pregnant day 16,once every 8h, 3 times in total. On the 10th day after birth, the pups of each group were perfused and fixed with 4 % paraformaldehyde; brain slices were made and stained with HE or incubated in anti-BrdU. BrdU+ cells were counted in each slice and compared.Results: Exposed groups showed a consistent overall pattern of a small number of BrdU+ cells situated in the upper cortical layers and a great number in the lower layers. There was an increasing tendency of this finding as ultrasound exposure prolonged. When compared with control group and pseudo-exposed group, there was a statistically significant difference (p<0.05). The proportion of ectopic BrdU+ cells in the deeper cortex (within bins 6~10) in each exposed group demonstrated a statistically significant difference(χ2=139.1,P<0.01) when was compared with control group and pseudo-exposed group. Increasing the exposure time would increase the proportion of BrdU+ cells to a higher percentage. Conclusion: Prenatal exposure to live three-dimensional ultrasound for longer duration may impact neuronal migration in mice. |
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