Morphological differences in cardiovascular anomalies induced by bis-diamine between Sprague-Dawley and Wistar rats

It is known that animals show different responses to the same teratogen between different strains. We examined cardiac malformations in Sprague-Dawley (SD) and Wistar rats induced by bis-diamine, which produced conotruncal anomalies and aortic arch malformations in embryos when administered to the dams, to elucidate the morphological differences and pathogenesis in the two strains. Two hundred milligrams of bis-diamine dissolved in 1% gum-tragacanth was administered to pregnant rats on embryonic day (ED) 9.5, 10.5 and 11.5 in each strain. The embryos were removed on ED 20.5. External appearances, cardiovascular morphology and associated anomalies were examined under a dissecting microscope. An immunohistological study with an anti-N-CAM antibody, an excellent marker for neural crest cells, was performed on ED 12.5 embryos. Isolated aortic arch anomalies were common features of malformations induced by bis-diamine in SD rats and intracardiac defects were found in a small number of the embryos. Wistar rats showed more serious cardiovascular anomalies, such as persistent truncus arteriosus and tetralogy of Fallot, especially when dams were treated on ED 10.5 and isolated arch anomalies were significantly less prevalent than in SD rats. Immunohistology demonstrated that there were fewer N-CAM positive cells in the conotruncal region in Wistar rats than in SD rats. Bis-diamine induced more critical cardiovascular malformations in Wistar rats because neural crest cells, which play an important role in conotruncal septation, were more extensively damaged. Different susceptibility to bis-diamine and/or different time of neural crest cell emigration from the hindbrain might explain those morphological differences.     

Immunohistochemical Distribution of HNK-1 and N-CAM in Rat Embryos Treated with Bis-Diamine

Bis-diamine is known to induce congenital anomalies including cardiac defects, thymic hypoplasia and snout defects in rat embryos. Cardiovascular lesions consist of persistent truncus arteriosus, tetralogy of Fallot and aortic arch anomalies, which are often found in DiGeorge syndrome. In the the present study, we investigated effects of bisdiamine on cardiac neural crest cells, which may be important to the teratogenecity. A single dose of 200 mg bis-diamine was administered to pregnant rats at 10.5 embryonic day (ED). Immunohistochemical studies were performed on embryos at 11.5, 12.5 and 13.5 ED using anti-HNK-1 and anti-N-CAM antibodies. The embryos at 11.5 and 12.5 ED showed the similar distributional patterns of either HNK-1 or N-CAM positive cells in control and bis-diamine treated embryos. N-CAM immunoreactivities in the splanchnic mesoderm were quite weakly detectable in the bis-diamine treated embryos at 12.5 ED. Closure of the neural tube was delayed in the treated embryos at this period. Immunohistochemistry of the control embryos at 13.5 ED demonstrated a continuous chain of N-CAM expression between the neural plexus near the foregut and the fourth aortic arch, while no apparent continuity of N-CAM positive tissue was observed in the bis-diamine treated embryos. These findings suggested that bis-diamine reduced the amount of neural crest cells which appeared to participate in the aortic arch formation or conotruncal septation. The primary effect of bis-diamine in the induction of various congenital anomalies including cardiovascular malformations may be an inhibition of the normal development of the neural tube and neural crest.     

Participation of Neural Crest Cells in Cardiovascular Morphogenesis of Chick Embryos

Neural crest cells appear on top of the neural folds at closure of the neural tube. These cells are capable of migrating and differentiating into multiple tissues. In 1983, Kirby et al., using chimera methodology, reported that cranial neural crest cells participated in aorticopulmonary and truncal septation. Since that time, many experimental studies have been done in this field. In this paper, we report new experimental findings and review four areas of study on neural crest cells, i.e. their general characteristics, participation of neural crest cells in formation of the normal heart, cardiovascular anomalies due to ablation of the neural crest cells, and the relationship between neural crest cells and congenital malformation syndromes, especially the DiGeorge sequence.     

Effects of 13 developmentally toxic chemicals on the migration of rat cephalic neural crest cells in vitro

The inhibition of neural crest cell (NCC) migration has been considered as a possible pathogenic mechanism underlying chemical developmental toxicity. In this study, we examined the effects of 13 developmentally toxic chemicals on the migration of rat cephalic NCCs (cNCCs) by using a simple in vitro assay. cNCCs were cultured for 48 h as emigrants from rhombencephalic neural tubes explanted from rat embryos at day 10.5 of gestation. The chemicals were added to the culture medium at 24 h of culture. Migration of cNCCs was measured as the change in the radius (radius ratio) calculated from the circular spread of cNCCs between 24 and 48 h of culture. Of the chemicals examined, 13‐cis‐retinoic acid, ethanol, ibuprofen, lead acetate, salicylic acid, and selenate inhibited the migration of cNCCs at their embryotoxic concentrations; no effects were observed for acetaminophen, caffeine, indium, phenytoin, selenite, tributyltin, and valproic acid. In a cNCC proliferation assay, ethanol, ibuprofen, salicylic acid, selenate, and tributyltin inhibited cell proliferation, suggesting the contribution of the reduced cell number to the inhibited migration of cNCCs. It was determined that several developmentally toxic chemicals inhibited the migration of cNCCs, the effects of which were manifested as various craniofacial abnormalities.     

Simple in vitro migration assay for neural crest cells and the opposite effects of all‐trans‐retinoic acid on cephalic‐ and trunk‐derived cells

Here, we describe a simple in vitro neural crest cell (NCC) migration assay and the effects of all‐trans‐retinoic acid (RA) on NCCs. Neural tubes excised from the rhombencephalic or trunk region of day 10.5 rat embryos were cultured for 48 h to allow emigration and migration of NCCs. Migration of NCCs was measured as the change in the radius (radius ratio) calculated from the circular spread of NCCs between 24 and 48 h of culture. RA was added to the culture medium after 24 h at embryotoxic concentrations determined by rat whole embryo culture. RA (10 μM) reduced the migration of cephalic NCCs, whereas it enhanced the migration of trunk NCCs, indicating that RA has opposite effects on these two types of NCCs.     

碱性成纤维细胞生长因子对胚胎大鼠神经干细胞增殖分化的影响

目的探讨分离、培养及鉴定胚胎大鼠神经干细胞(NSCs)方法，观察碱性成纤维细胞生长因子(bFGF)对NSCs增殖、分化的影响。方法从大鼠胚胎脑组织中分离出NSCs，用bRGF和胎牛血清诱导其增殖和分化，予BrdU以标记分裂细胞，采用免疫细胞化学鉴定NSCs和分化神经细胞。结果大鼠胚胎脑NSCs在无细胞因子和胎牛血清的培养基中无新生细胞形成，但能在bFGF和血清诱导下形成克隆，并产生nestin和BrdU阳性细胞，贴壁后分化为神经元和星形胶质细胞。结论该方法从大鼠胚胎大脑分离出的细胞具有NSCs特性，即自我更新和多向分化潜能;bFGF是NSCs增殖必须的丝裂原。     

Effects of pure 2,4-dichlorophenoxyacetic acid on cultured rat embryos

2,4-dichlorophenoxyacetic acid (2,4-D), a plant growth regulator, has been used worldwide as a herbicide. Previously we evaluated the prenatal developmental effects of 2,4-D by feeding it to pregnant rats and found that it is maternally toxic and embryolethal, and it induces urogenital malformations in rat fetuses. In the study presented here, we investigated the effects of pure 2,4-D on rat embryos in whole embryo culture. Rat embryos on day 9.5 of gestation were cultured for 48 h at several concentration levels with pure 2,4-D (50-500 microg/mL). 2,4-D caused a concentration-related increase in the incidence of each malformation. Significant decreases in the number of somites were observed at a concentration of 100 microg/mL or more. At the concentration of 100 microg/mL, there was normal yolk sac circulation. This result suggests that 2,4-D has a detrimental effect on somite development and directly damages developing embryos.     

比较胚胎和新生小鼠肠神经嵴干细胞体外增殖分化的实验研究

目的联合应用简化无血清培养基和连续传代法，从胚胎14～17d、新生1d、新生5d小鼠肠管分离培养肠神经嵴干细胞（gut neural crest stem cells，GNCSCs），观察其体外培养过程中增殖、分化的特点，用p75、GFAP、Peripherin、α-Actin作为特异性标志来鉴定GNCSCs及其分化的细胞系，比较胚胎和新生小鼠GNCSCs在生长速度、分化细胞的种类及数量上的差异。方法取3组小鼠的肠管，分离制成单细胞悬液，接种于DMEM／F12完全培养基贴壁培养，在连续传代培养中观察克隆球的形成；将克隆球接种于含血清培养基，观察其分化现象；用免疫细胞化学和免疫荧光法检测克隆球及其分化细胞系特异性标志物的表达。结果部分胚胎和新生小鼠肠管细胞在无血清培养基中连续传代培养形成克隆球。在血清刺激下克隆球可分化为多种形态的细胞。克隆球和分化细胞系的免疫染色均为阳性。结论胚胎和新生鼠肠管内存在具有自我更新能力的GNCSCs，且均可分化为神经元、神经胶质细胞和平滑肌三类细胞。新生鼠较胎鼠GNCSCs生长速度慢，分化的神经元数量减少，神经胶质细胞数量增加，平滑肌细胞无显著变化。     

Effects of supplemental L-methionine on E-64 [trans-epoxysuccinyl-1-leucyl-amido (4-guanido) butane]-induced dysmorphology in rat embryos cultured in vitro

ABSTRACT E‐64 [trans‐epoxysuccinyl‐1‐leucylamido (4‐guanido) butane] is teratogenic, inducing a spectrum of malformations in vivo and producing similar effects in vitro. Numerous studies support the concept that E‐64‐induced malformations result from embryonic nutritional deficiency, without affecting the maternal nutritional status. This has provided a useful model with which to investigate the nutritional requirements of the early embryo, as well as the role of various nutrients in the etiology of congenital defects.
In the current investigation, we examined effects of L‐methionine on E‐64‐induced embryotoxicity in vitro. For these experiments, we cultured rat embryos 9.5 days postconception (p.c.) for 48 hours with E‐64 and/or L‐methionine. We found that the addition of L‐methionine to E‐64‐exposed cultures reduced optic abnormality and increased embryo protein. These results suggest that embryopathy largely results from a deficiency of L‐methionine although E‐64 limits the supply of all amino acids to the embryo. Furthermore, although endocytosis and degradation of proteins by the visceral yolk sac (VYS) supply most amino acids to the embryo, free amino acids may be compensatory when this source is reduced. These results support those of previous investigations that suggest L‐methionine is a limiting nutrient for embryonic development.     

Cardiac surgery in the first year of life: The effect on weight gains of infants with congenital heart disease

Abstract Forty-seven infants with ventricular septal defect ( n = 17), tetralogy of Fallot ( n = 7) and transposition of the great arteries ( n = 23) who had 'corrective' surgery in the first year of life were reviewed with respect to birthweight and pre- and postoperative growth. The mean birthweight for each group was below that of the standard population. There was an overall decrease in growth velocity pre-operatively which was reversed after surgery. At follow up, 12-18 months later (means), most infants had regained at least their birthweight percentile, while the group with ventricular septal defect exceeded it. Consideration is given to the pathophysiological mechanisms contributing to these observations.     

Antisense attenuation of nestin accumulation causes neural tube deformation in rat embryo cultures

ABSTRACT The roles of nestin in neural tube development were studied using immunostaining and antisense experiments in rat embryos. Nestin was detected in the neural tube of embryos of day 10.5 of gestation (E10.5), while no nestin staining was observed in E9.5 embryos in which the neural plate comprised 3 to 4 layers of neuroepithelial cells. As embryos developed, the neural tube became comprised of multiple cell layers and staining was observed in filamentous structures spanning from the ventricular surface to the pial surface of the neural tube. Nestin accumulation was suppressed in the neural tube of embryos treated with nestin antisense oligodeoxynucleotide (ODN). The treated embryos showed two types of neural tube deformation. One type was a thin neural tube which had 3 to 4 layers of neuroepithelial cells and the other was a local distribution of neuroepithelial cells near the basement membrane. While neuroepithelial cells in the neural tube were fewer in embryos treated with nestin antisense ODN than in controls, the percentage of Islet-1-positive neurons relative to the neuroepithelial cells was not different between the treated and control embryos. These results suggested that nestin plays roles in the proliferation of neural tube cells and in the formation and the maintenance of multiple cell layers in the neural tube but not in suppression of development of Islet-1-positive neurons.     

Effect of Chaetochromin A, Chaetochromin D and Ustilaginoidin A, Bis (naphtho-γ-pyrone) Derivatives, on the Mouse Embryo Limb Bud and Midbrain Cells in Culture

Abstract Mouse embryo limb bud (LB) and midbrain (MB) cells were prepared from day 10 mouse embryos and cultured as micromass cell islands for 5 days. Differentiation was determined by the number of stainable foci of differentiated cells, and the concentration at which each compound inhibited the formation of differentiated foci by 50% of the control value (IC₅₀) was estimated according to the method by Flint and Orton (1984). The values of IC₅₀ of L-dopa, which is known as a teratogen, were 24 μig/ml in LB cultures and 12 μ/ml in MB cultures, respectively. Teratogenic hazards of three mycotoxins were compared in this system. Chaetochromin A and chaetochromin D, which were new naphtho-γ-pyrone dimers produced by Chaetomium sp ., indicated strong inhibition (IC₅₀ 0.13-0.24 μ/ml) in LB and MB cell cultures, and ustilaginoidin A, which was a naphtho-γ-pyrone dimer produced by Ustilaginoidea virens , inhibited at higher concentration than chaetochromins by several-folds in LB and MB cultures.     

高氧、维甲酸对胎鼠肺角化细胞生长因子及其受体表达的影响

目的　探讨高氧、维甲酸 (RA)对胎鼠肺成纤维细胞 (LFs)和肺泡II型上皮细胞 (AECII)角化细胞生长因子 (KGF)及其受体 (KGFR)表达的影响。方法　原代培养胎鼠肺LFs及AECII ,待生长至亚汇合状态时 ,随机分为 :空气组 ,空气 +RA组 ,高氧组 ,高氧 +RA组。于培养 2、6、12、2 4、4 8(AECII)和 72h(LFs)时 ,采用半定量RT -PCR方法检测KGF和KGFR的mRNA表达。结果　与空气组比较 ,高氧 2h时 ,胎鼠LFsKGFmRNA表达即明显下降 ,6h时达极点 ,以后下降趋势逐渐减弱 ,至 4 8h后已无显著性差异 ;高氧 2、6、12和 2 4h时 ,其下降幅度分别为 3 0、5 2、2 3和 2 1倍 (P <0 0 5、0 0 1、0 0 1和 0 0 5 )。RA可上调高氧状态下胎鼠LFsKGFmRNA表达 ,与高氧组比较 ,高氧 +RA组在 2、6、12和 2 4h时KGFmRNA表达分别是高氧组的 2 4、3 4、1 7和 1 8倍 (P <0 0 5、0 0 1、0 0 1和 0 0 5 )。高氧对胎鼠AECIIKGFRmRNA表达影响较小 ,与空气组比较 ,仅在高氧 12h和 2 4h时其表达量分别是空气组的 2 3和 1 3倍 (P <0 0 5 ) ;RA对AECIIKGFRmRNA表达没有影响。结论　促进KGF的表达是RA发挥高氧肺损伤保护作用的重要机制之一     

高氧对早产大鼠角化细胞生长因子mRNA及增殖细胞核抗原表达的影响

目的　探讨 85 %高浓度氧暴露对早产新生大鼠肺组织角化细胞生长因子 (KGF)mRNA及增殖细胞核抗原 (PCNA)动态表达的影响。方法　早产SD大鼠生后 1d随机分为空气组、高氧组。高氧组持续暴露 85 %氧气中 ,空气组置于同一室内常压空气中。两组分别于 1、4、7、10、14和 2 1d时 ,提取肺组织RNA ,采用半定量逆转录聚合酶链反应 (RT PCR)测定KGFmRNA ;同时应用免疫组化检测肺组织切片中PCNA。结果　 (1)与空气组相比 ,高氧 1d时 ,KGFmRNA表达无明显差异 (P >0 0 5 ) ;4d时明显增强 ,较空气组增加 3 8倍 (P <0 0 0 1) ;其后开始下降 ,7d时较空气组增加 1 8倍 (P<0 0 5 ) ;10d时两组间无明显差异 (P >0 0 5 ) ;14d时较空气组降低 1 5倍 (P <0 0 1) ,2 1d时复又增高 ,较空气组增加 1 15倍 (P <0 0 1)。 (2 )空气组早产新生大鼠生后 14d内肺组织中均有PCNA表达 ,并且PCNA阳性细胞 90 %以上定位于气道和肺泡上皮细胞 ,2 1d时几乎检测不出其表达 ;与空气组比较 ,高氧组 1～ 4d时肺组织中PCNA表达明显减弱 ,7～ 10d时增强且部分间质细胞也有表达 ,2 1d时PCNA阳性细胞以间质细胞为主。结论　 85 %高氧暴露可促进早产新生大鼠肺组织KGFmRNA及PCNA表达     

少突胶质前体细胞移植治疗早产儿脑白质损伤大鼠模型

目的探讨少突胶质前体细胞(OPCs)移植对治疗早产儿脑白质损伤(WMI)模型大鼠的长期作用。方法将80只3日龄Sprague-Dawley大鼠随机分为假手术组、模型对照组、5 d脑室/白质移植组、9 d脑室/白质移植组、14 d脑室/白质移植组(n=10);除假手术组外其余各组行右侧颈总动脉离断并缺氧80 min制备早产儿WMI模型;采用孕10~12周人胚胎脑组织制备OPCs。各移植组分别在造模后5 d、9 d和14 d将3×105OPCs注入右侧脑室/脑白质中,待大鼠60日龄和90日龄时分别对各组行电镜下脑髓鞘评估和神经功能评估。结果电镜下,大鼠60日龄时各移植组髓鞘损害程度相比模型组略有改善;无论是与同组60日龄大鼠还是与同日龄模型组大鼠比较,90日龄时各移植组的髓鞘均明显增厚,结构破坏更少,其中14 d移植组变化最为明显;但不同移植时间脑室和白质移植组间的髓鞘损害程度未见有明显差异。60及90日龄各移植组大鼠的神经功能缺陷评分(m NSS)均高于假手术组,但均低于模型组(P0.05)。结论 OPCs移植可能对治疗早产儿WMI存在长期效应,延迟移植时间可能增强疗效。