实时三维超声对晚孕胎鼠大脑神经细胞的生物学影响
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摘要
超声波用于诊断疾病及产前诊断已近45年的历史,其对人体产生的生物效应及安全性始终是人们关注的焦点,经过多年的研究人们认识到诊断级超声波辐照剂量存在着时间-强度关系,即大剂量、高强度的超声对机体组织可瞬间产生损害作用,小剂量、低强度超声波长时间辐照也会对人体组织产生不良后果。超声安全问题实际上就是产科胚胎和胎儿的超声检查安全的问题,因为在早孕期只要几个胚胎细胞受损,就有可能造成严重的不良后果,如畸形和缺陷,因此有许多超声工作者对超声的生物学损害做了大量研究。随着电子与计算机技术的不断进步超声在产科领域的检查类型也不断更新,早期使用的是二维B型超声(黑白超声)用以观察胎儿的切面图像,随后又发明了频谱多普勒和彩色多普勒用以观察脐带血流及胎儿心血管的血流情况。上述的几种超声检查技术已有大量的研究成果,也基本得出了相应的结论,近年来由于三维超声和四维超声(实时三维超声)不断用于产前超声诊断,总的超声辐照时间延长,为胎儿的安全性带来了潜在的危害,因此,对后两种超声检查技术作用于胚胎后所产生的生物学效应及其危害做相应的研究是一项紧迫的任务。以往的学者对超声的生物学损害主要是做临床上的病例对照研究和队列研究,自韩国学者Oh等人应用凋亡细胞做研究后认为超声辐照后检测细胞凋亡率可作为确定超声检查是否安全的一个生物学指标。从此各国学者应用电镜技术或多种组织化学方法来确定超声辐照后组织器官中的细胞凋亡率,以获得各自研究的相关数据。如应用透射电镜技术方法、应用原位末端脱氧核苷酸转移酶标记法(TUNEL)、应用Caspase酶测定法间接确定凋亡细胞情况等。美国学者Ang等人运用组织化学手段来确定孕小鼠经超声辐照后对胎鼠大脑神经元迁移的影响。这些研究为机体超声生物学效应的研究开辟了新的领域。
本研究分为三个部分,第一部分是利用光学显微镜、透射电子显微镜技术观察孕小鼠接受不同时间段实时三维超声辐照后对胎鼠大脑神经细胞的生物学影响,主要观察病理损害和凋亡细胞情况等形态学改变。第二部分是利用原位末端脱氧核苷酸转移酶标记法、Caspase3,9酶活性测定来确定实时三维超声辐照后小鼠大脑神经细胞的凋亡情况及相关酶的表达。第三部分是在实时三维超声辐照前给予孕16d母鼠DNA复制标记物BrdU,标记胎鼠大脑脑室附近的未分化神经细胞,不同时间段实时三维超声辐照后在生后第10d观察幼鼠那些被BrdU标记的大脑神经细胞的迁移情况。
第一部分实时三维超声对晚孕胎鼠大脑神经细胞形态学的影响
目的:探讨不同实时三维超声辐照剂量对晚孕胎鼠大脑神经细胞形态学改变的观察。
方法:30只孕鼠随机等量分配到对照组、假辐照30min组、辐照5min组、辐照10min组、辐照20min组及辐照30min组。辐照组在孕鼠孕16 d时行实时三维超声辐照。假辐照组实施辐照操作,但仪器不发射超声波。每组选生后24h乳鼠10只,取大脑右侧顶叶做标本,透射电镜下观察。其余乳鼠于生后10 d行灌流固定,取大脑标本,HE染色光镜观察。
结果:光镜结果:对照组、假辐照组、辐照5min组及辐照10min组光镜观察未见明显异常,辐照20min组及30min组,神经细胞排列紊乱,部分神经细胞坏死。电镜结果:对照组、假辐照组、辐照5min组未见明显异常;辐照10min组可见部分线粒体膨大,嵴断裂,内质网呈空泡化,偶见凋亡细胞;辐照20min、30min组线粒体与粗面内质网异常改变增多,细胞核异染色质凝集、边集,观察到典型的凋亡小体,凋亡细胞明显增多。
结论:实时三维超声辐照超过10min可引起大脑神经细胞形态改变和凋亡细胞增加。
第二部分实时三维超声辐照后TUNEL法及Caspase3、Caspase9免疫组织化学技术观察小鼠大脑组织中细胞凋亡变化
目的:探讨实时三维超声辐照后小鼠大脑细胞凋亡及Caspase3、Caspase9的表达及其意义。
方法:30只孕鼠随机等量分配到对照组、假辐照30min组、辐照5min、辐照10min、辐照20min及辐照30min组。辐照组在孕鼠孕16 d时行实时三维超声辐照,假辐照组实施辐照操作,但仪器不发射超声波。每组选生后10d乳鼠10只,行灌流固定,取大脑标本,HE染色光镜观察、原位末端标记(TUNEL)法检测凋亡大脑细胞、比色法测定Caspase3和Caspase9活性。
结果:TUNEL法标记结果:辐照5min组凋亡细胞表达率与对照组和假辐照组间比较差异无统计学意义。辐照10min组凋亡细胞开始增多,辐照20min、30min组凋亡细胞表达率明显增强,与其他各组相比差异具有统计学意义;Caspase3和Caspase9结果:Caspase3平均光密度及Caspase9阳性细胞百分数均随着辐照时间的延长而增强、增多,除对照组与假辐照组比较无统计学意义外,其他各组两两比较均有统计学意义。
结论:实时三维超声辐照可引起小鼠大脑神经细胞凋亡增加,相关凋亡基因Caspase3、Caspase9蛋白合成增加,活性升高,诱发神经细胞凋亡。
第三部分三维超声辐照后对晚孕胎鼠大脑神经细胞迁移的影响
目的:对晚孕小鼠进行不同时间段实时三维超声辐照后观察幼鼠大脑神经细胞迁移的情况。
方法:30只孕鼠随机等量分配到对照组、假辐照30min组、辐照5min、辐照10min、辐照20min及辐照30min组。辐照组在孕鼠孕16 d时行实时三维超声辐照,在辐照前24h、16h、8h分别在孕鼠腹腔内注射BrdU50mg/kg,假辐照组和对照组也在孕16d时每8h腹腔注射5-溴脱氧尿苷嘧啶(BrdU)50mg/kg,共注射3次。假辐照组实施辐照操作,但仪器不发射超声波。每组选生后10d乳鼠10只,行灌流固定,取大脑标本,HE染色光镜观察,行抗BrdU免疫组化染色,观察大脑皮层各部BrdU阳性细胞数及分布情况,并比较它们之间的差异。
结果:辐照组BrdU阳性细胞到达大脑皮质浅层减少,停留在深层较多,随着辐照时间的延长,此种情况越来越明显,对照组和假辐照组之间比较无统计学意义,对照组与各辐照组,假辐照组与各辐照组比较均有统计学意义。大脑皮质深层(6～10层)BrdU+细胞阳性率的构成比较对照组、假辐照组与各辐照组间差别有统计学意义(χ2=139.1, P<0.01),随辐照时间的延长,大脑皮质深层BrdU+阳性细胞构成有增加的趋势。
结论:实时三维超声辐照晚孕胎鼠时间过长可影响大脑神经细胞的迁移。
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 Mice
Objective: 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 mice
Objective : 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.
本研究分为三个部分,第一部分是利用光学显微镜、透射电子显微镜技术观察孕小鼠接受不同时间段实时三维超声辐照后对胎鼠大脑神经细胞的生物学影响,主要观察病理损害和凋亡细胞情况等形态学改变。第二部分是利用原位末端脱氧核苷酸转移酶标记法、Caspase3,9酶活性测定来确定实时三维超声辐照后小鼠大脑神经细胞的凋亡情况及相关酶的表达。第三部分是在实时三维超声辐照前给予孕16d母鼠DNA复制标记物BrdU,标记胎鼠大脑脑室附近的未分化神经细胞,不同时间段实时三维超声辐照后在生后第10d观察幼鼠那些被BrdU标记的大脑神经细胞的迁移情况。
第一部分实时三维超声对晚孕胎鼠大脑神经细胞形态学的影响
目的:探讨不同实时三维超声辐照剂量对晚孕胎鼠大脑神经细胞形态学改变的观察。
方法:30只孕鼠随机等量分配到对照组、假辐照30min组、辐照5min组、辐照10min组、辐照20min组及辐照30min组。辐照组在孕鼠孕16 d时行实时三维超声辐照。假辐照组实施辐照操作,但仪器不发射超声波。每组选生后24h乳鼠10只,取大脑右侧顶叶做标本,透射电镜下观察。其余乳鼠于生后10 d行灌流固定,取大脑标本,HE染色光镜观察。
结果:光镜结果:对照组、假辐照组、辐照5min组及辐照10min组光镜观察未见明显异常,辐照20min组及30min组,神经细胞排列紊乱,部分神经细胞坏死。电镜结果:对照组、假辐照组、辐照5min组未见明显异常;辐照10min组可见部分线粒体膨大,嵴断裂,内质网呈空泡化,偶见凋亡细胞;辐照20min、30min组线粒体与粗面内质网异常改变增多,细胞核异染色质凝集、边集,观察到典型的凋亡小体,凋亡细胞明显增多。
结论:实时三维超声辐照超过10min可引起大脑神经细胞形态改变和凋亡细胞增加。
第二部分实时三维超声辐照后TUNEL法及Caspase3、Caspase9免疫组织化学技术观察小鼠大脑组织中细胞凋亡变化
目的:探讨实时三维超声辐照后小鼠大脑细胞凋亡及Caspase3、Caspase9的表达及其意义。
方法:30只孕鼠随机等量分配到对照组、假辐照30min组、辐照5min、辐照10min、辐照20min及辐照30min组。辐照组在孕鼠孕16 d时行实时三维超声辐照,假辐照组实施辐照操作,但仪器不发射超声波。每组选生后10d乳鼠10只,行灌流固定,取大脑标本,HE染色光镜观察、原位末端标记(TUNEL)法检测凋亡大脑细胞、比色法测定Caspase3和Caspase9活性。
结果:TUNEL法标记结果:辐照5min组凋亡细胞表达率与对照组和假辐照组间比较差异无统计学意义。辐照10min组凋亡细胞开始增多,辐照20min、30min组凋亡细胞表达率明显增强,与其他各组相比差异具有统计学意义;Caspase3和Caspase9结果:Caspase3平均光密度及Caspase9阳性细胞百分数均随着辐照时间的延长而增强、增多,除对照组与假辐照组比较无统计学意义外,其他各组两两比较均有统计学意义。
结论:实时三维超声辐照可引起小鼠大脑神经细胞凋亡增加,相关凋亡基因Caspase3、Caspase9蛋白合成增加,活性升高,诱发神经细胞凋亡。
第三部分三维超声辐照后对晚孕胎鼠大脑神经细胞迁移的影响
目的:对晚孕小鼠进行不同时间段实时三维超声辐照后观察幼鼠大脑神经细胞迁移的情况。
方法:30只孕鼠随机等量分配到对照组、假辐照30min组、辐照5min、辐照10min、辐照20min及辐照30min组。辐照组在孕鼠孕16 d时行实时三维超声辐照,在辐照前24h、16h、8h分别在孕鼠腹腔内注射BrdU50mg/kg,假辐照组和对照组也在孕16d时每8h腹腔注射5-溴脱氧尿苷嘧啶(BrdU)50mg/kg,共注射3次。假辐照组实施辐照操作,但仪器不发射超声波。每组选生后10d乳鼠10只,行灌流固定,取大脑标本,HE染色光镜观察,行抗BrdU免疫组化染色,观察大脑皮层各部BrdU阳性细胞数及分布情况,并比较它们之间的差异。
结果:辐照组BrdU阳性细胞到达大脑皮质浅层减少,停留在深层较多,随着辐照时间的延长,此种情况越来越明显,对照组和假辐照组之间比较无统计学意义,对照组与各辐照组,假辐照组与各辐照组比较均有统计学意义。大脑皮质深层(6～10层)BrdU+细胞阳性率的构成比较对照组、假辐照组与各辐照组间差别有统计学意义(χ2=139.1, P<0.01),随辐照时间的延长,大脑皮质深层BrdU+阳性细胞构成有增加的趋势。
结论:实时三维超声辐照晚孕胎鼠时间过长可影响大脑神经细胞的迁移。
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 Mice
Objective: 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 mice
Objective : 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.
引文
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