胎儿生长受限SD大鼠模型的建立和相关信号转导机制*☆

背景：研究发现,胎儿生长受限可造成胎儿代谢的适应性改变及多种内分泌激素系统重整,导致胎儿生长发育迟缓以及成年后胰岛素抵抗的发生。但胎儿生长受限演变为胰岛素抵抗的分子机制尚不明确。目的：探讨胎儿宫内生长受限发生胰岛素抵抗的潜在分子机制,为早期诊断和临床干预成年后代谢性疾病提供理论依据。方法：SD大鼠分成胎儿生长受限仔鼠组和正常对照仔鼠组。胎儿生长受限仔鼠组大鼠构建胎儿生长受限SD大鼠动物模型。提取各组仔鼠胎盘组织蛋白质,应用特异性抗磷酸化抗体进行免疫共沉淀和Western blot方法对有丝分裂素激活蛋白激酶信号途径上各蛋白激酶及其抑制剂的活性进行分析。结果与结论：在胎儿生长受限仔鼠组中,有丝分裂素激活蛋白激酶信号转导途径中细胞外信号调节激酶1/2、应激活化蛋白激酶/c-Jun氨基末端激酶及Raf-1蛋白激酶活性比正常对照组显著降低(P < 0.05),p38有丝分裂素激活蛋白激酶活性两组比较差异无显著性意义(P > 0.05);相反,有丝分裂素激活蛋白激酶信号转导途径中各抑制剂在胎儿生长受限仔鼠组中表达水平较正常对照组中表达显著升高(P < 0.05)。结果表明,胎儿生长受限的发生与有丝分裂素激活蛋白激酶及细胞凋亡信号传导机制异常密切相关。     

妊娠末期高浓度氧对生长受限胎鼠神经元的保护作用

目的 探讨高压氧(HBO)和常压高浓度氧(NBH)对胎儿生长受限(FGR)大鼠大脑神经元的保护作用. 方法 40只孕12d大鼠按照随机数字表法分为5组,每组8只,分别为:(1)对照组:不结扎子宫血管;(2)FGR组,采用子宫血管缩窄方法建立FGR模型;(3)高压氧疗组(F+HBO组):建立FGR模型后给予高压氧治疗,压力达0.2 MPa; (4)常压高浓度氧疗组(F+NBH组):建立FGR模型后给予常压高浓度氧治疗,氧浓度达900 mL/L; (5)低浓度氧疗组(F+LCO组):建立FGR模型后给予300 mL/L浓度氧治疗.3个氧疗组从孕14d开始,持续治疗7d,每天80 min.孕21d时剖宫产娩出新生鼠,各组再随机取10只进行组织病理学检查. 结果 FGR组0d、7d、14d新生鼠大脑皮层及海马回神经元密度、尼氏染色阳性细胞密度明显减少,凋亡细胞明显增多,与假手术对照组比较,差异均有统计学意义(P＜0.05).F+HBO组和F+NBH组0d、7d、14d新生鼠大脑皮层及海马回神经元密度、尼氏染色阳性细胞数明显增加,凋亡细胞明显减少,与FGR组相比,差异均有统计学意义(P＜0.05).上述指标在F+ HBO组与F+ NBH组、F+LCO组与FGR组间差异无统计学意义(P＞0.05). 结论 妊娠末期高浓度氧对胎鼠大脑皮层和海马回神经元具有保护作用.     

Decreased CRH mRNA expression in the fetal guinea pig hypothalamus following maternal nutrient restriction

The regulation of corticotropin-releasing hormone (CRH) mRNA expression following maternal nutrient restriction was examined in the fetal hypothalamus. Pregnant guinea pigs were food restricted for 48 h or fed normally during late gestation. After nutrient restriction, CRH mRNA levels in the hypothalamic paraventricular nucleus of the fetus were determined using in situ hybridization and were found to be significantly decreased (P<0.0001) compared to controls. In conclusion, we have successfully sequenced the coding sequence of the guinea pig CRH gene, and have shown that a short period (48 h) of maternal nutrient restriction inhibits CRH mRNA expression in the fetal hypothalamus.     

Effects of maternal dietary protein restriction on growth of the brain and body in the rat

We studied the growth of the brain and body in rats born of dams fed a low-protein (8% casein) diet ad lib beginning 5 weeks prior to mating and continuing throughout gestation and lactation. Control dams were fed an isocaloric 25% casein diet. Litters were culled at birth to 8 pups. At birth, brain weights and body weights of pups of protein restricted dams were similar to those of control pups. During the period of lactation, pups of restricted dams exhibited severely retarded body growth but only mildly retarded brain growth resulting in an elevated brain/body weight ratio. This relative macroeephaly was maximal at 10–20 days of age, declining completely by 35 days of age. The relative macroeephaly could not be accounted for by increased retention of water in the brains of the malnourished rats. Following weaning, pups were maintained ad lib on the diets fed their mothers. At adulthood brain/body weight ratios were normal in the protein restricted group.     

Gestational protein restriction induces CA3 dendritic atrophy in dorsal hippocampal neurons but does not alter learning and memory performance in adult offspring

Studies have demonstrated that nutrient deficiency during pregnancy or in early postnatal life results in structural abnormalities in the offspring hippocampus and in cognitive impairment. In an attempt to analyze whether gestational protein restriction might induce learning and memory impairments associated with structural changes in the hippocampus, we carried out a detailed morphometric analysis of the hippocampus of male adult rats together with the behavioral characterization of these animals in the Morris water maze (MWM). Our results demonstrate that gestational protein restriction leads to a decrease in total basal dendritic length and in the number of intersections of CA3 pyramidal neurons whereas the cytoarchitecture of CA1 and dentate gyrus remained unchanged. Despite presenting significant structural rearrangements, we did not observe impairments in the MWM test. Considering the clear dissociation between the behavioral profile and the hippocampus neuronal changes, the functional significance of dendritic remodeling in fetal processing remains undisclosed.     

Late gestational maternal serum cortisol is inversely associated with fetal brain growth

To analyze the association between fetal brain growth and late gestational blood serum cortisol in normal pregnancy.Blood total cortisol was quantified at delivery in 432 Chinese mother/child pairs. Key inclusion criteria of the cohort were: no structural anomalies of the newborn, singleton pregnancy, no alcohol abuse, no drug abuse or history of smoking no hypertensive disorders and no impairment of glucose tolerance and no use of steroid medication during pregnancy. Differential ultrasound examination of the fetal body was done in early (gestational day 89.95 ± 7.31), middle (gestational day 160.17 ± 16.12) and late pregnancy (gestational day 268.89 ± 12.42). Newborn's cortisol was not correlated with any of the ultrasound measurements during pregnancy nor with birth weight. Multivariable regression analysis, considering timing of the ultrasound examination, the child's sex, maternal BMI, maternal age, maternal body weight at delivery, the timing of cortisol measurement and maternal uterine contraction states, revealed that maternal serum total cortisol was significantly negative correlated with ultrasound parameters describing the fetal brain: late biparietal diameter (R2=0.512, p=0.009), late head circumference (R2=0.498, p=0.001), middle biparietal diameter (R2=0.819, p=0.013), middle cerebellum transverse diameter R2=0.76, p=0.014) and early biparietal diameter(R2=0.819, p=0.013). The same analysis revealed that birth weight as well as ultrasound parameters such as abdominal circumference and femur length were not correlated to maternal cortisol levels. In conclusion, our study demonstrates that maternal cortisol secretion within physiological ranges may be inversely correlated to fetal brain growth but not to birth weight. It remains to be demonstrated whether maternal cortisol secretion negatively influencing fetal brain growth translates to adverse neurological outcomes in later life.     

Maternal caloric restriction spares fetal brain polyunsaturated fatty acids in Wistar rats

There is increasing interest in the role of developmental programming; however, the impact on fetal oxidative stress and brain fatty acid levels has been relatively unexplored. Recent reports have shown that caloric restriction regimens in adult animals reduce the occurrence of chronic diseases by reducing the oxidative stress and altering the long chain polyunsaturated fatty acids (LCPUFA). The present study examined whether caloric restriction during pregnancy alters oxidative stress and essential fatty acid metabolism in mother and offspring at birth. Pregnant female rats were fed either a standard chow (C, n = 7) or were calorie restricted (CR, n = 7) by feeding 60% of the intake of the control. Oxidative stress marker (malondialdehyde) and polyunsaturated fatty acid profiles in brain and liver were analyzed in both dams and offspring. Total weight gain during pregnancy was lower (p < 0.01) in the CR group as compared to the control group but did not affect the litter size and weight. Brain malondialdehyde levels were lower (p < 0.05) in dams from the CR group. There was no change in brain and liver LCPUFA levels in both male and female offspring in the CR group. Most of the polyunsaturated fatty acids were reduced (p < 0.05) in plasma and brain in the CR dams. Caloric restriction during pregnancy did not alter LCPUFA metabolism in the offspring suggesting that during maternal caloric restriction mothers own stores are mobilized to provide docosahexaenoic acid and arachidonic acid for fetal brain development.     

Glucocorticoid‐induced fetal brain growth restriction is associated with p73 gene activation

Fetal exposure to excessive amounts of glucocorticoids (GCs) hampers proper brain development. The molecular mechanism(s) underlying these GCs effects are not well understood. We explored the impact of fetal exposure to maternal GCs on fetal brain expression of p63 and p73 transactivation (TA) and dominant negative (ΔN) gene variants that promote neural cell death (TA) and cell survival programs (ΔN). The fetoplacental enzyme 11β‐hydroxysteroid dehydrogenase 2, which shields fetuses from maternal glucocorticoids, was inhibited throughout pregnancy by daily injection of carbenoxolone to pregnant dams. The expression of p63 and p73 gene variants and proteins was monitored by real‐time rtPCR and Western blot in the brains of male and female fetuses. Carbenoxolone administration led to an overall enhanced level of corticosterone in the amniotic fluid of both male and female fetuses at late pregnancy. These enhanced corticosterone levels were associated with a significant reduction in fetal brain weights and a significant increase in TAp73 mRNA and p73 protein levels. However, the expression levels of TAp63 mRNA and p63 proteins were either suppressed or unaffected. The pro‐neural survival gene variant ΔNp73 was significantly reduced in female and enhanced in male fetal brains, whereas ΔNp63 was significantly reduced in the brains of both genders. These data suggest that the GCs‐induced negative impact on fetal brain development likely is due, at least in part, to their action of the pro‐neural cell death gene variant TAp73 and to the modulation of the pro‐survival ΔNp63 and ΔNp73 gene variants in a gender‐dependent fashion. © 2012 Wiley Periodicals, Inc.     

Depression in pregnancy is associated with decreased glutathione peroxidase activity in fetal cord blood.

The investigation of fetal cord blood (FCB) during child delivery has created a novel topic in the field of psychiatric research. The umbilical vein receives nutrients and oxygen from the mother’s circulation and transports them to the fetal circulation. Investigating fetal cord blood during delivery is beneficial for understanding the fetal environment. Depression in pregnancy is associated with medical and emotional burdens. In this study, we aimed to investigate glutathione peroxidase (Gpx) and myeloperoxidase (MPO) activity in the FCB of depressed mothers and healthy controls. Our study included 45 depressed mothers and 59 healthy controls. The FCB samples were collected from the umbilical vein during delivery. We found that Gpx levels were significantly decreased in the FCB of depressed mothers than healthy controls, medians were 0.14 U/ml and 0.16 U/ml respectively, Z: −3.567 and p < 0.001. MPO levels were similar in both groups, medians were 1.0 U/L and 1.2 U/L respectively, Z: −1.837 and p:0.066. Depression in pregnancy may be associated with decreased antioxidant levels, and this condition may cause an oxidative load, which may lead to improper brain development. Future studies should be performed in larger samples to clarify our preliminary results.     

Association of protein S p.Pro667Pro dimorphism with plasma protein S levels in normal individuals and patients with inherited protein S deficiency

A dimorphism in PROS1 gene (c.A2,001G, p.Pro667Pro) has been associated with significantly reduced levels of both free and total protein S in carriers of the GG genotype. It is not known how the GG genotype could influence PS levels in normals, whether it could influence the levels of protein S in carriers of mutations in PROS1 gene and whether this genotype acts as an isolated or additive risk factor for venous thrombosis. With this as background, we evaluated the association of p.Pro667Pro dimorphism with free and total protein S centrally measured in a panel of 119 normal controls, 222 individuals with low protein S and 137 individuals with normal PS levels belonging to 76 families with protein S deficiency enrolled in the ProSIT study. Transient expression of recombinant wild type protein S and p.Pro667Pro protein S was performed to evaluate the role of the A to G transition at position 2001 in vitro. The p.Pro667Pro polymorphism was also expressed together with a p.Glu67Ala variant to assess a possible influence on protein S levels in protein S deficient subjects. Free and total protein S levels were significantly lower in normal women. In normal women only was the GG genotype associated with significantly lower free protein S levels in comparison to AA and AG genotypes (P=0.032). No significant influence of GG genotype was observed in patients, either with known mutations or with low protein S levels. These data were confirmed by in vitro transient expression, showing no difference in secretion levels of the p.Pro667Pro variant (even in association with the p.Glu67Ala mutation), compared to the wild type protein S. The genotype in itself was neither a significant risk factor for venous thrombosis nor a risk modifier in patients with known mutations.     

Antenatal taurine reduces cerebral cell apoptosis in fetal rats with intrauterine growth restriction*

From pregnancy to parturition, Sprague-Dawley rats were daily administered a low protein diet to establish a model of intrauterine growth restriction. From the 12 th day of pregnancy, 300 mg/kg taurine was daily added to food until spontaneous delivery occurred. Brain tissues from normal neonatal rats at 6 hours after delivery, neonatal rats with intrauterine growth restriction, and neo-natal rats with intrauterine growth restriction undergoing taurine supplement were obtained for fur-ther experiments. The terminal deoxyribonucleotidyl transferase (TdT)-mediated biotin-16-dUTP nick-end labeling assay revealed that the number of apoptotic cells in the brain tissue of neonatal rats with intrauterine growth restriction significantly increased. Taurine supplement in pregnant rats reduced cell apoptosis in brain tissue from neonatal rats with intrauterine growth restriction. Immu-nohistochemical staining revealed that taurine supplement increased glial cell line-derived neuro-trophic factor expression and decreased caspase-3 expression in the cerebral cortex of intrauterine growth-restricted fetal rats. These results indicate that taurine supplement reduces cell apoptosis through the glial cell line-derived neurotrophic factor-caspase-3 signaling pathway, resulting in a protective effect on the intrauterine growth-restricted fetal rat brain.     

Regulation of bovine activated protein C by protein S: the role of the cofactor protein in species specificity

The anticoagulant activity of bovine activated Protein C was observed to exhibit species specificity when tested in the plasmas from cow, dog, rabbit, and human. The relative effectiveness in descending order was cow, dog, human, and rabbit. When bovine Protein S was added to each of these plasmas, the species specificity of bovine activated Protein C was lost. No activated Protein C cofactor activity could be detected in either rabbit or human plasma. Bovine activated Protein C enhanced the rate of inactivation of both rabbit and human Factor Va in serum. The rate of activated Protein C catalyzed inactivation of rabbit and human Factor Va could be enhanced by the addition of bovine Protein S. These results indicated that the species specificity of the anticoagulant activity of bovine activated Protein C is mediated by a cofactorenzyme interaction rather than an enzyme-substrate interaction. These results further demonstrated that the anticoagulant activity of activated Protein C is due, in part, to the inactivation of Factor Va.     

血浆蛋白C、蛋白S与脑梗死的相关性研究

目的研究血浆蛋白C、蛋白S与脑梗死发病的关系。方法对64例脑梗死患者及15例正常对照组进行血浆蛋白C(PC)、蛋白S(PS)检测,并分析其与脑梗死严重程度、年龄及其它脑梗死危险因素之间的关系。结果实验组血浆PC浓度(4.97±1.82μg/ml)与对照组血浆PC浓度(4.74±1.95μg/ml)之间差别无统计学意义;首发组血浆PC浓度(5.28±1.85μg/ml)与复发组血浆PC浓度(4.03±1.40μg/ml)之间差别有统计学意义(P=0.016);实验组血浆PS浓度(7.47±3.87μg/ml)与对照组血浆PS浓度(12.06±3.99μg/ml)之间差别有统计学意义,中老年组血浆PS浓度(6.58±3.33μg/ml)与青年组血浆PS浓度(9.28±4.33μg/ml)之间差别有统计学意义。结论PC降低是复发性脑梗死的危险因素之一;游离PS降低是脑梗死的危险因素;PS水平的降低导致PC功能的降低,进而凝血功能障碍,导致脑梗死的发生。     

Reductions in the plating efficiency of the fetal neural precursor cells following maternal alcohol consumption

Maternal alcohol abuse has been associated with reduced neural cell number and abnormal cell differentiation and organization in many regions of the developing mammalian brain. Experiments were conducted to determine whether changes in the neural precursor cells could be detected in primary culture following maternal alcohol consumption during the early proliferative period of the fetal brain growth spurt. Alcohol was administered to pregnant mice in the drinking water from days 11 to 19 of gestation. There was no alcohol-related increase in the incidence of fetal mortality or malformation, but fetal body and brain weights were reduced. When disaggregated cells from the day 19 fetal neopallium were grown in culture, there was a reduction in the number of astroglial colonies yielded in 42% of alcohol-exposed brains. This effect was expressed as a reduction in the absolute plating efficiency (APE) of the neural precursor cells. The APE was reduced as much as 80% in severely affected brains. There was no alcoholrelated difference in the in vitro morphogenesis of the astroglial colonies. Observations of the proliferative neural cells in situ suggest that there is an impaired recruitment of all neural cell types, but that the reduced APE reflects primarily a proportionate increase in the number of immature neurons among the cells obtained from the fetal neopallium. It appears that a prolongation of mitosis may be resulting in a general developmental delay in the fetal neocortex.     

Fetal growth restriction caused by MIMT1 deletion alters brain transcriptome in cattle

We examined levels of gene expression in the brains of bovine fetuses carrying a truncated MIMT1 allele, MIMT1^Del, shown to cause late abortion and stillbirth as a result of fetal growth restriction. MIMT1 is a non-protein coding gene that forms part of the imprinted PEG3 (paternally expressed gene 3) domain. Microarray analysis of brain cortex samples from mid-gestation MIMT1^Del/WT bovine fetuses and wild-type siblings was performed to study the effect of fetal growth restriction on brain gene expression. Statistical analysis revealed 134 genes with increased mRNA levels and 22 with reduced levels in MIMT1^Del/WT fetuses. Gene set enrichment analysis identified a relatively small number of significant functional clusters representing three major biological processes: response to oxidative stress, angiogenesis, and epithelial cell proliferation. Gene expression microarray analyses identified increased expression of VIPR2, HTRA1, S100A4 and MYH8 in fetuses carrying the deletion and decreased expression of DRD2, ADAM18, miR345, ZNF585A. ADAM18, DRD2 and S100A4 are known to be involved in prenatal brain development. ZNF585A, miR-345, VIPR2, HTRA1, and MYH8 are known to be involved in cell growth and differentiation, but any role in neural developmental has yet to be elucidated.     

Diurnal rhythm of cortisol during late pregnancy: associations with maternal psychological well-being and fetal growth

Maternal psychological functioning during pregnancy affects both maternal and fetal well-being. The hypothalamic-pituitary-adrenal (HPA) axis provides one mechanism through which maternal psychosocial factors may be transduced to the fetus. However, few studies have examined maternal psychological factors or birth outcomes in relation to the diurnal pattern of cortisol across the day. The current study examined maternal psychological well-being, parity status, and birth weight in relation to the maternal cortisol diurnal rhythm in a group of 98 low-risk pregnant women (51 primiparae). At 36 weeks gestation, participants completed both pregnancy-specific and general self-report measures of psychological functioning and provided saliva samples at 8:00, 12:00, and 16:00h on 2 consecutive working days for the assay of cortisol. The expected diurnal decline in salivary cortisol was observed. Higher trait anxiety was associated with a flatter afternoon decline for all mothers. For primiparae, steeper morning cortisol declines were associated with lower infant birth weight. The findings suggest that regulation of the HPA axis may differ by parity status with downstream implications for fetal growth and development.