Ultrastructural changes in embryoic neuroepithelial cells caused by passive smoking in golden hamsters at different periods of pregnancy: A randomized controlled trial*⋆

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Abstract

BACKGROUND:

Tobacco smoke exposure is recognized as a health risk for pregnant women and it is increasingly evident that tobacco smoke affects the development of brain. Recently, associations between maternal smoking during pregnancy and subsequent mental health problems in offspring have been reported.

OBJECTIVE:

To observe the effect of passive smoking on the morphology of nerve tissues and the ultrastructure of neuroepithelial cells during embryogenesis in golden hamster at different pregnant period.

DESIGN:

A randomized control study.

SETTING:

Department of Histology and embryology, Qingdao University.

MATERIALS:

Adult golden hamsters, including 40 males and 40 females that had not delivered, weighing (105±5) g, were provided by Shenyang Changsheng Biotechnology, Co., Ltd. At 20: 00 – 21: 00, one male and one female were matched in each cage, and their mating was observed. The vaginal swabs were examined the next day and the day of positive sperm was taken as embryonic day 1 (E1).

METHODS:

The experiment was completed in the Department of Histology and Embryology of Qingdao University from September 2001 to September 2003. ① Abnormality caused by smoking, grouping and model establishment: A total of 40 healthy pregnant golden hamsters were randomly divided into control group (n =20) and experimental group (n =20). The hamsters in the experimental group were exposed to tobacco smoke from embryonic day 4 to 7, 3 times per day, continuously 1 hour per time, 1 cigarette per golden hamster, for 4 consecutive days in the self-made chamber. The animals in the control group were given the same conditions as those in the experimental group except exposure to smoke. ② Observation with transmission electron microscope: According to different gestational ages, the experimental group and the control group were all divided 4 subgroups (Groups A, B, C and D) respectively, and 5 hamsters in each subgroup. The pregnant golden hamsters were anaesthetized with 1 g/L pentobarbital sodium at 12: 00 and 18: 00 at E8, 8: 00 at E9 and 8: 00 at E10, and all the pregnant uteruses were divulsed under the stereomicroscope. The development of the neural plate, neural groove and neural tube were observed. Meanwhile, the amount of normal embryos and abnormal embryos including the neural tube defect ratios were recorded. ③ Electron microscopic specimen preparation and observation: Three embryos of each group ad libtium were fixed. The alternations of neuroepithelial ultrastructures were observed with transmission and scanning electron microscopes.

MAIN OUTCOME MEASURES:

① The incidences of abnormality of nervous system development were observed under stereomicroscope and scanning electron microscope in smoking group and the control group; ② Alternations of neuroepithelial ultrastructures were observed with transmission electron microscope.

RESULTS:

All the 40 pregnant golden hamsters were involved in the final analysis. ① Manifestations and incidence of nervous system dysplasia: Passive smoking could induce dymorphogenesis during neurulation, which mainly presented as growth retardation, spina bifida and failure of formation of neural tubes; The incidences of the nervous system dysplasia in the experimental groups [20%(10/49), 27%(14/51), 32% (19/59), 27% (17/63)] were higher than those in the corresponding control groups [0, 2% (1/57), 4% (2/53), 4% (2/52), P < 0.01]. ② Histomorphological changes at different time points after spermatiation observed with transmission and scanning electron microscopes: In the control group, the embryos formed C-shape columned embryos, anterior and posterior neuropores were all closure at 10: 00 on E10; In the experimental group, unfused anterior and posterior neuropores still could be found, and some embryos presented spina bifida at 10: 00 on E10. In the control group, neuroepithelial cell arranged tidily and closely, the boundary of the cells was clear, the free surface of neuroepithelial had a mass of long and regular microvillus, and the surface of mesenchymal cell around the neuroepithelium had many processes which mutually related at 12: 00 on E8. In the experimental group, the neuroepithelial cells arranged irregularly and the intercellular spaces became wide at 12: 00 on E8. The apical portion of many neuroepithelial cells bulged into the lumen and many microvilli were shorted and swollen. The quantity of the microvillus reduced gradually, evenly disappeared with the increasing of gestational age at 18:00 on E8. Under transmission electron microscope, the neuroepithelial cells in experimental embryos arranged irregularly. There were many visible materials in the intercellular space which increased the breadth and anomaly. It was apparent that passive smoking evoked major alterations in neuroepithelial cytoarchitecture. Junctional complex reduced. Many microvilli were shorted and swollen, even the apical portion of many neuroepithelial cells bulged, and abscised into the lumen. A lot of vacuolation appeared in the cytoplasm of neuroepithelia and mesenchymal cell around the neuroepithelium. The cristae of mitochondria reduced even disappeared, and some mitochondria became elongate. Irregular nuclear, increased heterochromatin and karyopycnosis/karyorrhexis were observed easily. Perinuclear cisternae partially swelled and embraced tangible material (maybe the material from nuclear). Some death cells separated into a lot of apoptotic bodies. Some apoptotic bodies were found in the cytoplasm of other healthy-looking or healthy cells.

CONCLUSION:

Passive smoking may induce degeneration, apoptosis, and cells loss in the neural epithelium, thereby result in failure of formation and differentiation of neural tube. It is an important way by which passive smoking caused neural tube defects.

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