妊娠期母体亚临床甲减对后代智力和脑发育相关基因表达影响的动物实验研究
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摘要
目的
甲状腺激素对于中枢神经系统的分化和发育具有十分广泛而显著的影响,胎儿期和出生后早期甲状腺激素缺乏将造成不可逆的脑损伤,引起严重的感觉、运动障碍和智力低下,其发病时间、严重程度及开始甲状腺激素治疗的时间等因素均与脑损害的发生和恢复密切相关。造成胎儿甲状腺激素缺乏的原因包括妊娠期母体甲状腺激素的减少如甲状腺功能减退症(甲减)、胎儿甲状腺激素减少如先天性甲状腺缺如以及因妊娠期碘缺乏导致的母体和胎儿甲状腺激素均减少。母体临床甲减及碘缺乏对胎儿和后代智力的影响已经明确。妊娠期母体亚临床甲减导致后代智力障碍目前已有数篇报导。妊娠期母体亚临床甲减的患病率为2%～3%,如此庞大的女性患病人群存在,提示甲状腺激素缺乏带来胎儿第一阶段脑发育影响的普遍性和重要性。但是母体亚临床甲减对后代脑发育影响的机制研究尚未见报道。本实验在成功建立亚临床甲减孕鼠动物模型的基础上,观察母鼠亚临床甲减对仔鼠行为学改变的影响,并从脑发育相关基因表达方面探讨母体亚临床甲减导致的胎儿脑发育障碍的机制。
方法
60只SPF级雌性Wistar大鼠随机分成3组(每组n=20)。①正常对照组:行假手术、皮下等量注射生理盐水,作为阴性对照;②亚临床甲减组:手术切除甲状腺,于背部皮下植入微渗泵(osmotic minipump),泵中的L-T4以1.0μg/(100g·d)浓度持续泵入,当血清TSH高于正常、但TT_4水平在正常范围时,提示亚临床甲减鼠建模成功。十天后与正常的雄鼠合笼交配,雌鼠镜下阴道涂片发现精子确定为妊娠0天,记为E0。自E15天后L-T4剂量调整为1.05μg/(100g·d)持续泵入至仔鼠生后10d,将分娩当日定为P0天;③完全甲减组:阳性对照组、手术完全切除甲状腺,皮下等量注射生理盐水,作为阳性对照。母鼠于E10、E13、E17、P1时眶后静脉丛取血,测定血清TSH、TT_4。仔鼠出生后P3、P7、P21冰上断头取海马组织,以Real-time PCR方法检测BDNF、NCAM mRNA表达量,以Westernblot方法检测BDNF、Rap1蛋白表达量,并与同日龄对照组比较。仔鼠出生后P35断头取脑,以HE染色、尼氏染色方法观察亚临床甲减仔鼠脑病理改变及神经细胞形态学改变,仔鼠出生后P40行Morris水迷宫行为学检测并与同日龄对照组比较。应用SPSS12.0软件处理和分析数据。
结果
1、亚临床甲减孕鼠模型的制备:孕鼠血清TSH、TT_4水平结果显示:与假手术组比较,各孕龄(E10、E13、E17、P1)亚临床甲减组表现为血清TT_4水平未见明显改变,而TSH水平明显升高(P＜0.05);与完全甲减组比较,各孕龄亚临床甲减组表现为血清TT_4水平明显升高,TSH水平明显降低(P＜0.05),符合亚临床甲减激素血清学改变。
2、亚临床甲减孕鼠后代发育及激素水平:正常对照组仔鼠活动好,体重增加快,毛发发育正常;亚临床甲减组仔鼠各方面未见明显异常;甲减组仔鼠睁眼较对照组迟3～5天,体形偏小,尾短,行动迟缓,步态不稳,毛发出现迟缓且稀疏,体重增加缓慢,明显低于正常对照(1日龄P＞0.05,21日龄P＜0.01)。40日龄进行Morris水迷宫测试时,亚临床甲减仔鼠血清激素水平和对照组无统计学差异;完全甲减组仔鼠体重低,血清TT_4水平显著低于同日龄对照组(P＜0.01),TSH较同日龄对照明显升高(P＜0.01)。
3、Morris水迷宫行为测定结果:定位航行试验中,随着训练次数的增加,各组的逃避潜伏期总的趋势是不断缩短,第9次训练结束,即学习训练结束时,对照组逃避潜伏期为18.23±4.27s,亚临床甲减组为35.63±5.95s,甲减组为55.16±5.15s,组间逃避潜伏期比较有显著性差异(P＜0.05)。空间搜索试验中,在P44(撤离平台当日)时,正常对照组仔鼠在靶象限内游泳的时间明显长于其它象限,亚临床甲减组仔鼠在靶象限内游泳的时间也长于其它象限,与对照组比较无显著性差异(P＞0.05),而完全甲减组仔鼠,在各个象限内游泳的时间差别不明显,与对照组比较有显著性差异(P＜0.01)。在P49天(撤离平台24h后),空间探索测量大鼠的长时空间记忆能力实验中,正常对照组仔鼠在靶象限内游泳的时间仍明显长于其它象限,而亚临床甲减组仔鼠在靶象限内游泳的时间与其它象限游泳时间无明显差异,与对照组比较有显著性差异(P＜0.05)。完全甲减组后代,在各个象限内游泳的时间差别仍不明显,与对照组比较有显著性差异(P＜0.05)。
4、亚临床甲减组仔鼠海马CA1区HE染色可见神经细胞排列较整齐,层次较清楚,无核固缩,其细胞形态同正常对照组仔鼠相比无明显差异;亚临床甲减组仔鼠海马尼氏小体染色的平均光密度、积分光密度低于正常对照组(P＜0.05),但是,明显多于完全甲减组(P＜0.01)。
5、用实时定量PCR方法测定海马组织的BDNF mRNA水平结果显示,与正常对照组仔鼠相比,亚临床甲减组仔鼠在出生后3d海马BDNF mRNA表达量下降约0.73倍,差异有统计学意义(P＜0.05),出生后7d、21d海马BDNF mRNA表达量与正常对照组间无显著差异(P＞0.05);甲减组仔鼠海马BDNF mRNA表达量3d、7d和21d均明显低于亚临床甲减组和对照组,差异有统计学意义(P＜0.01)。与正常对照组仔鼠相比,亚甲减组仔鼠在出生后3d、7d及21d海马NCAM mRNA表达量有升高的趋势,但差异无显著性(P＞0.05);与亚临床甲减组和对照组比较,甲减组仔鼠海马NCAM mRNA表达量在3d、7d和21d均明显升高(P＜0.01)。
6、用Western印迹法测定仔鼠海马组织的BDNF、Rap1蛋白水平。检测结果显示,与正常对照组比较,亚临床甲减组仔鼠海马BDNF蛋白表达量在仔鼠出生后3d、7d明显降低,降低幅度分别为33%(P＜0.01)、29%(P＜0.05)。21d时与正常对照组差异无统计学意义(P＞0.05);与亚临床甲减组和对照组比较,完全甲减组仔鼠海马BDNF蛋白的表达量在各时间点均明显下降(均P＜0.01)。与正常对照组比较,亚临床甲减组后代仔鼠海马内Rap1蛋白表达量在出生后7d时出现显著升高,升高幅度为42%(P＜0.05),21d时继续升高,升高幅度为59%(P＜0.01);与亚临床甲减组和对照组比较,完全甲减组仔鼠海马Rap1蛋白的表达量在3d时即出现升高,升高幅度为20%(P＞0.05),7d和21d继续升高,分别升高42%、58%,差异有统计学意义(均P＜0.05)。
结论
1、通过手术切除甲状腺,于背部皮下植入L-T4微渗泵,孕前至E15天L-T4剂量1.0μg/(100g·d),E15天后L-T4剂量1.05μg/(100g·d)持续泵入至仔鼠生后10d,能够成功建立亚临床甲减孕鼠模型。模型稳定,为后部分的研究工作奠定了基础。
2、妊娠期亚临床甲减仔鼠出生后P35海马尼氏小体染色的平均光密度、积分光密度均明显减少。通过Morris水迷宫的行为学实验指标,从不同角度验证了不仅母体甲减可以导致后代学习记忆能力的减低,母体亚临床甲减也可以导致仔鼠的空间学习记忆能力受损。
3、妊娠期亚临床甲减孕鼠能引起后代仔鼠出生后早期(P3)大脑海马DBNFmRNA表达水平下降,P3、P7 BDNF蛋白水平表达下降;P7和P21大脑海马Rap1蛋白表达水平增加,表明二者共同参与了妊娠期母体亚临床甲减对后代学习记忆功能的损伤。
Objective
Thyroid hormone plays a key role in the differentiation and development of central nervous system.Thyroid hormone deficiency can cause the delayed neuropsychological development,even cretinism.Recently more attention was paid by specialists to the impact of maternal thyroid insufficiency in early pregnancy on fetal neuropsychological development of phase 1.The main etiology of thyroid insufficiency is hypothyroidism,especially subclinical hypothyroidism.Some investigators reported the prevalence of subclinical hypothyroidism during pregnancy was 2%to 3%.These indicate that the effect of maternal thyroid insufficiency on fetal brain development is common.Clinical trials have showed that mild thyroid insufficiency such as subclinical hypothyroidism and hypothyroxinemia during pregnancy can affect fetal neuropsychological development.The present study aimed to explore whether the neurodevelopment damage was associated with the expression of genes regulated by thyroid hormone in the rat offspring.
Methods
Total of 60 SPF female nulliparous Wistar rats,weighing 200-220g at the start of each experiment were used.Three groups of rats,control(sham operated),subclinical hypothyroidism(thyroidectomized-thyroxine,Sub),and hypothyroidism (thyroidectomized,Hypo,used as a positive control) were established.Subclinical hypothyroidism rats(Sub,n=20) were induced by the surgical ablation of the thyroid gland(thyroidectomy) under 10%of Chloral hydrate-aldehyde after a 7 day adaptation period.Rats were provided with 0.1%(w/v) calcium lactate in the drinking water after the surgery and maintained on normal rat chow.Four weeks after surgery,as serum T4 concentrations fell below the level of detection of the assay(＜1μg/dL) and body weight kept stasis rats were implanted with osmotic minipumps(ALZET(?),models 2001 or 2002,delivering 1.0 or 0.5μl/h,respectively,Alza Corp.,Palo Alto,CA) under the dorsal skin of the animals.The osmotic minipumps delivered at a constant rate of levo-thyroxine(L-T4) at a dose of 1.0μg per 100g body weight(BW) per day from the 10th day before mating to the 14th day after conception,and then were followed by 1.05μg per 100g BW per day from the 15th day after conception to the 10th day postpartum.These dosages resulted in elevated serum thyrotropin(TSH) level and normal total T_4 level(subclinical hypothyroidism rats).Hypothyroidism rats(Hypo, n=20) were surgically thyroidectomized with no L-T4 treatment.Euthyroid sham-operated(control,n=20) rats were submitted to surgery as described above,but without ablation of the thyroid gland and infused with placebo solution.Serum total T_4 and TSH are measured according to the manufacturer's instructions using chemiluminescence immunoassay.Their pups were decapitated at P3,P7 and P21. Hippocampus were collected and detected for BDNF and Rap1 protein expression by western blotting and for BDNF and NCAM mRNA expression by real-time PCR.The content changes of Nissl body within hippocampus neuron endochylema,stained by Toluidine blue at P35 of their pups were observed.On P40,Morris water maze was used for evaluating short-term memory and long-term memory.
Results
1.Maternal and pup assessment:During the period of prenatal,there were significant differences in serum TSH levels between the sham control and sub group dams and between the sub and Hypo group dams(P＜0.05 for all comparisons).There were no significant differences in total T_4 levels between the sham control and sub group dams.There were no significant differences in litter sex ratio or pup weight at P1 between the sham control and sub groups,but there were significant differences in litter size between the sub and hypo groups.
2.Morphological study:The average optical density and integral optical density of Nissl body in neuron cells in hippocampus CA1 area were significantly less in maternal subclinical hypothyroidism pups than normal group(P＜0.05) but more than that of the hypothyroidism group(P＜0.01).
3.Morris water maze test:The mean escape latency did not differ between any of the groups on first three days of testing in Morris water maze.On day 44 there were significant differences in the mean latency between the sham control and sub group pups and between the sub and hypo group pups.In probe trials,there were no significant differences among groups in swimming velocity.Long-term memory testing done more than 24h after training(day 49) showed that Sub group pups were not able to remember a fixed platform position,and spend less time in the proper quadrant as compared to nomal controls(P＜0.05 for all comparisons)
4.BDNF and Rapl expression in the hippocampus:BDNF mRNA expression by real-time PCR was downregulated at P3 in the Sub groups(P＜0.05) under our experimental conditions.Western blot analysis showed that the level of expression of BDNF was lower in sub group than that in sham control group(P＜0.05).The expression of Rap1 was slightly increased at the early time(before P7) but there was no significant difference between control and sub group.A statistically significant up-regulation was observed in sub group at P7 and P21 when compared to sham control group(P＜0.05).
5.NCAM expression was found homogeneously upregulated in hypothyroid pups at P3,P7 and P21,but no significant differences in the levels of NCAM mRNA were observed in the hippocampus between the sub group and sham control rat offspring.
Conclusions
1.We successfully established maternal subclinical hypothyroidism rat model by thyroidectomy and osmotic minipumps for the first time.Thyroidectomized rats were infused with placebo or L-T4.The osmotic minipumps delivered at a constant rate of L-T4 at a dose of 1.0μg per 100g body weight per day from the 10th day before mating to the 14th day after conception,and then were followed by 1.05μg per 100g BW per day from the 15th day after conception to the 10th day postpartum,These dosages resulted in elevated serum TSH level and normal total T_4 level(subclinical hypothyroidism).
2.Maternal subclinical hypothyroidism could disturb learing and memory and other cognitive performances of their pups.The average optical density and integral optical density of Nissl body in neuron cells in hippocampus CA1 area were decreased significantly in maternal subclinical maternal hypothyroidism group.
3.Both gene expression and protein level in BDNF in hippocampus of pups of subclinical hypothyroid dams decreased at the early time(P3,P7).The expression of Rap1 protein was higher than that of control offspring at P21.No change was observed in the levels of NCAM mRNA.The present results indicated that the memory retardance of pups of subclinical hypothyroidism dams likely stemed from the alterations in expression of target genes directly regulated by thyroid hormone.
甲状腺激素对于中枢神经系统的分化和发育具有十分广泛而显著的影响,胎儿期和出生后早期甲状腺激素缺乏将造成不可逆的脑损伤,引起严重的感觉、运动障碍和智力低下,其发病时间、严重程度及开始甲状腺激素治疗的时间等因素均与脑损害的发生和恢复密切相关。造成胎儿甲状腺激素缺乏的原因包括妊娠期母体甲状腺激素的减少如甲状腺功能减退症(甲减)、胎儿甲状腺激素减少如先天性甲状腺缺如以及因妊娠期碘缺乏导致的母体和胎儿甲状腺激素均减少。母体临床甲减及碘缺乏对胎儿和后代智力的影响已经明确。妊娠期母体亚临床甲减导致后代智力障碍目前已有数篇报导。妊娠期母体亚临床甲减的患病率为2%～3%,如此庞大的女性患病人群存在,提示甲状腺激素缺乏带来胎儿第一阶段脑发育影响的普遍性和重要性。但是母体亚临床甲减对后代脑发育影响的机制研究尚未见报道。本实验在成功建立亚临床甲减孕鼠动物模型的基础上,观察母鼠亚临床甲减对仔鼠行为学改变的影响,并从脑发育相关基因表达方面探讨母体亚临床甲减导致的胎儿脑发育障碍的机制。
方法
60只SPF级雌性Wistar大鼠随机分成3组(每组n=20)。①正常对照组:行假手术、皮下等量注射生理盐水,作为阴性对照;②亚临床甲减组:手术切除甲状腺,于背部皮下植入微渗泵(osmotic minipump),泵中的L-T4以1.0μg/(100g·d)浓度持续泵入,当血清TSH高于正常、但TT_4水平在正常范围时,提示亚临床甲减鼠建模成功。十天后与正常的雄鼠合笼交配,雌鼠镜下阴道涂片发现精子确定为妊娠0天,记为E0。自E15天后L-T4剂量调整为1.05μg/(100g·d)持续泵入至仔鼠生后10d,将分娩当日定为P0天;③完全甲减组:阳性对照组、手术完全切除甲状腺,皮下等量注射生理盐水,作为阳性对照。母鼠于E10、E13、E17、P1时眶后静脉丛取血,测定血清TSH、TT_4。仔鼠出生后P3、P7、P21冰上断头取海马组织,以Real-time PCR方法检测BDNF、NCAM mRNA表达量,以Westernblot方法检测BDNF、Rap1蛋白表达量,并与同日龄对照组比较。仔鼠出生后P35断头取脑,以HE染色、尼氏染色方法观察亚临床甲减仔鼠脑病理改变及神经细胞形态学改变,仔鼠出生后P40行Morris水迷宫行为学检测并与同日龄对照组比较。应用SPSS12.0软件处理和分析数据。
结果
1、亚临床甲减孕鼠模型的制备:孕鼠血清TSH、TT_4水平结果显示:与假手术组比较,各孕龄(E10、E13、E17、P1)亚临床甲减组表现为血清TT_4水平未见明显改变,而TSH水平明显升高(P＜0.05);与完全甲减组比较,各孕龄亚临床甲减组表现为血清TT_4水平明显升高,TSH水平明显降低(P＜0.05),符合亚临床甲减激素血清学改变。
2、亚临床甲减孕鼠后代发育及激素水平:正常对照组仔鼠活动好,体重增加快,毛发发育正常;亚临床甲减组仔鼠各方面未见明显异常;甲减组仔鼠睁眼较对照组迟3～5天,体形偏小,尾短,行动迟缓,步态不稳,毛发出现迟缓且稀疏,体重增加缓慢,明显低于正常对照(1日龄P＞0.05,21日龄P＜0.01)。40日龄进行Morris水迷宫测试时,亚临床甲减仔鼠血清激素水平和对照组无统计学差异;完全甲减组仔鼠体重低,血清TT_4水平显著低于同日龄对照组(P＜0.01),TSH较同日龄对照明显升高(P＜0.01)。
3、Morris水迷宫行为测定结果:定位航行试验中,随着训练次数的增加,各组的逃避潜伏期总的趋势是不断缩短,第9次训练结束,即学习训练结束时,对照组逃避潜伏期为18.23±4.27s,亚临床甲减组为35.63±5.95s,甲减组为55.16±5.15s,组间逃避潜伏期比较有显著性差异(P＜0.05)。空间搜索试验中,在P44(撤离平台当日)时,正常对照组仔鼠在靶象限内游泳的时间明显长于其它象限,亚临床甲减组仔鼠在靶象限内游泳的时间也长于其它象限,与对照组比较无显著性差异(P＞0.05),而完全甲减组仔鼠,在各个象限内游泳的时间差别不明显,与对照组比较有显著性差异(P＜0.01)。在P49天(撤离平台24h后),空间探索测量大鼠的长时空间记忆能力实验中,正常对照组仔鼠在靶象限内游泳的时间仍明显长于其它象限,而亚临床甲减组仔鼠在靶象限内游泳的时间与其它象限游泳时间无明显差异,与对照组比较有显著性差异(P＜0.05)。完全甲减组后代,在各个象限内游泳的时间差别仍不明显,与对照组比较有显著性差异(P＜0.05)。
4、亚临床甲减组仔鼠海马CA1区HE染色可见神经细胞排列较整齐,层次较清楚,无核固缩,其细胞形态同正常对照组仔鼠相比无明显差异;亚临床甲减组仔鼠海马尼氏小体染色的平均光密度、积分光密度低于正常对照组(P＜0.05),但是,明显多于完全甲减组(P＜0.01)。
5、用实时定量PCR方法测定海马组织的BDNF mRNA水平结果显示,与正常对照组仔鼠相比,亚临床甲减组仔鼠在出生后3d海马BDNF mRNA表达量下降约0.73倍,差异有统计学意义(P＜0.05),出生后7d、21d海马BDNF mRNA表达量与正常对照组间无显著差异(P＞0.05);甲减组仔鼠海马BDNF mRNA表达量3d、7d和21d均明显低于亚临床甲减组和对照组,差异有统计学意义(P＜0.01)。与正常对照组仔鼠相比,亚甲减组仔鼠在出生后3d、7d及21d海马NCAM mRNA表达量有升高的趋势,但差异无显著性(P＞0.05);与亚临床甲减组和对照组比较,甲减组仔鼠海马NCAM mRNA表达量在3d、7d和21d均明显升高(P＜0.01)。
6、用Western印迹法测定仔鼠海马组织的BDNF、Rap1蛋白水平。检测结果显示,与正常对照组比较,亚临床甲减组仔鼠海马BDNF蛋白表达量在仔鼠出生后3d、7d明显降低,降低幅度分别为33%(P＜0.01)、29%(P＜0.05)。21d时与正常对照组差异无统计学意义(P＞0.05);与亚临床甲减组和对照组比较,完全甲减组仔鼠海马BDNF蛋白的表达量在各时间点均明显下降(均P＜0.01)。与正常对照组比较,亚临床甲减组后代仔鼠海马内Rap1蛋白表达量在出生后7d时出现显著升高,升高幅度为42%(P＜0.05),21d时继续升高,升高幅度为59%(P＜0.01);与亚临床甲减组和对照组比较,完全甲减组仔鼠海马Rap1蛋白的表达量在3d时即出现升高,升高幅度为20%(P＞0.05),7d和21d继续升高,分别升高42%、58%,差异有统计学意义(均P＜0.05)。
结论
1、通过手术切除甲状腺,于背部皮下植入L-T4微渗泵,孕前至E15天L-T4剂量1.0μg/(100g·d),E15天后L-T4剂量1.05μg/(100g·d)持续泵入至仔鼠生后10d,能够成功建立亚临床甲减孕鼠模型。模型稳定,为后部分的研究工作奠定了基础。
2、妊娠期亚临床甲减仔鼠出生后P35海马尼氏小体染色的平均光密度、积分光密度均明显减少。通过Morris水迷宫的行为学实验指标,从不同角度验证了不仅母体甲减可以导致后代学习记忆能力的减低,母体亚临床甲减也可以导致仔鼠的空间学习记忆能力受损。
3、妊娠期亚临床甲减孕鼠能引起后代仔鼠出生后早期(P3)大脑海马DBNFmRNA表达水平下降,P3、P7 BDNF蛋白水平表达下降;P7和P21大脑海马Rap1蛋白表达水平增加,表明二者共同参与了妊娠期母体亚临床甲减对后代学习记忆功能的损伤。
Objective
Thyroid hormone plays a key role in the differentiation and development of central nervous system.Thyroid hormone deficiency can cause the delayed neuropsychological development,even cretinism.Recently more attention was paid by specialists to the impact of maternal thyroid insufficiency in early pregnancy on fetal neuropsychological development of phase 1.The main etiology of thyroid insufficiency is hypothyroidism,especially subclinical hypothyroidism.Some investigators reported the prevalence of subclinical hypothyroidism during pregnancy was 2%to 3%.These indicate that the effect of maternal thyroid insufficiency on fetal brain development is common.Clinical trials have showed that mild thyroid insufficiency such as subclinical hypothyroidism and hypothyroxinemia during pregnancy can affect fetal neuropsychological development.The present study aimed to explore whether the neurodevelopment damage was associated with the expression of genes regulated by thyroid hormone in the rat offspring.
Methods
Total of 60 SPF female nulliparous Wistar rats,weighing 200-220g at the start of each experiment were used.Three groups of rats,control(sham operated),subclinical hypothyroidism(thyroidectomized-thyroxine,Sub),and hypothyroidism (thyroidectomized,Hypo,used as a positive control) were established.Subclinical hypothyroidism rats(Sub,n=20) were induced by the surgical ablation of the thyroid gland(thyroidectomy) under 10%of Chloral hydrate-aldehyde after a 7 day adaptation period.Rats were provided with 0.1%(w/v) calcium lactate in the drinking water after the surgery and maintained on normal rat chow.Four weeks after surgery,as serum T4 concentrations fell below the level of detection of the assay(＜1μg/dL) and body weight kept stasis rats were implanted with osmotic minipumps(ALZET(?),models 2001 or 2002,delivering 1.0 or 0.5μl/h,respectively,Alza Corp.,Palo Alto,CA) under the dorsal skin of the animals.The osmotic minipumps delivered at a constant rate of levo-thyroxine(L-T4) at a dose of 1.0μg per 100g body weight(BW) per day from the 10th day before mating to the 14th day after conception,and then were followed by 1.05μg per 100g BW per day from the 15th day after conception to the 10th day postpartum.These dosages resulted in elevated serum thyrotropin(TSH) level and normal total T_4 level(subclinical hypothyroidism rats).Hypothyroidism rats(Hypo, n=20) were surgically thyroidectomized with no L-T4 treatment.Euthyroid sham-operated(control,n=20) rats were submitted to surgery as described above,but without ablation of the thyroid gland and infused with placebo solution.Serum total T_4 and TSH are measured according to the manufacturer's instructions using chemiluminescence immunoassay.Their pups were decapitated at P3,P7 and P21. Hippocampus were collected and detected for BDNF and Rap1 protein expression by western blotting and for BDNF and NCAM mRNA expression by real-time PCR.The content changes of Nissl body within hippocampus neuron endochylema,stained by Toluidine blue at P35 of their pups were observed.On P40,Morris water maze was used for evaluating short-term memory and long-term memory.
Results
1.Maternal and pup assessment:During the period of prenatal,there were significant differences in serum TSH levels between the sham control and sub group dams and between the sub and Hypo group dams(P＜0.05 for all comparisons).There were no significant differences in total T_4 levels between the sham control and sub group dams.There were no significant differences in litter sex ratio or pup weight at P1 between the sham control and sub groups,but there were significant differences in litter size between the sub and hypo groups.
2.Morphological study:The average optical density and integral optical density of Nissl body in neuron cells in hippocampus CA1 area were significantly less in maternal subclinical hypothyroidism pups than normal group(P＜0.05) but more than that of the hypothyroidism group(P＜0.01).
3.Morris water maze test:The mean escape latency did not differ between any of the groups on first three days of testing in Morris water maze.On day 44 there were significant differences in the mean latency between the sham control and sub group pups and between the sub and hypo group pups.In probe trials,there were no significant differences among groups in swimming velocity.Long-term memory testing done more than 24h after training(day 49) showed that Sub group pups were not able to remember a fixed platform position,and spend less time in the proper quadrant as compared to nomal controls(P＜0.05 for all comparisons)
4.BDNF and Rapl expression in the hippocampus:BDNF mRNA expression by real-time PCR was downregulated at P3 in the Sub groups(P＜0.05) under our experimental conditions.Western blot analysis showed that the level of expression of BDNF was lower in sub group than that in sham control group(P＜0.05).The expression of Rap1 was slightly increased at the early time(before P7) but there was no significant difference between control and sub group.A statistically significant up-regulation was observed in sub group at P7 and P21 when compared to sham control group(P＜0.05).
5.NCAM expression was found homogeneously upregulated in hypothyroid pups at P3,P7 and P21,but no significant differences in the levels of NCAM mRNA were observed in the hippocampus between the sub group and sham control rat offspring.
Conclusions
1.We successfully established maternal subclinical hypothyroidism rat model by thyroidectomy and osmotic minipumps for the first time.Thyroidectomized rats were infused with placebo or L-T4.The osmotic minipumps delivered at a constant rate of L-T4 at a dose of 1.0μg per 100g body weight per day from the 10th day before mating to the 14th day after conception,and then were followed by 1.05μg per 100g BW per day from the 15th day after conception to the 10th day postpartum,These dosages resulted in elevated serum TSH level and normal total T_4 level(subclinical hypothyroidism).
2.Maternal subclinical hypothyroidism could disturb learing and memory and other cognitive performances of their pups.The average optical density and integral optical density of Nissl body in neuron cells in hippocampus CA1 area were decreased significantly in maternal subclinical maternal hypothyroidism group.
3.Both gene expression and protein level in BDNF in hippocampus of pups of subclinical hypothyroid dams decreased at the early time(P3,P7).The expression of Rap1 protein was higher than that of control offspring at P21.No change was observed in the levels of NCAM mRNA.The present results indicated that the memory retardance of pups of subclinical hypothyroidism dams likely stemed from the alterations in expression of target genes directly regulated by thyroid hormone.
引文
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16 Harman,A.M.,J.Rodger,A.Ahmat,C.Thomas,C.Bartlett,P.Chen,S.A.Dunlop,and L.D.Beazley.PSA-NCAM is up-regulated during optic nerve regeneration in lizard but not in goldfish.Exp Neurol.2003;182:180-5.
17 Parkash,J.and G.Kaur.Potential of PSA-NCAM in neuron-glial plasticity in the adult hypothalamus:role of noradrenergic and GABAergic neurotransmitters.Brain Res Bull.2007;74:317-28.
18 Wakabayashi,Y.,S.Uchida,H.Funato,T.Matsubara,T.Watanuki,K.Otsuki,M.Fujimoto,A.Nishida,and Y.Watanabe.State-dependent changes in the expression levels of NCAM-140 and LI in the peripheral blood cells of bipolar disorders,but not in the major depressive disorders.Prog Neuropsychopharmacol Biol Psychiatry.2008;32:1199-205.
19 Lowe,B.,H.A.Avila,F.R.Bloom,M.Gleeson,and W.Kusser.Quantitation of gene expression in neural precursors by reverse-transcription polymerase chain reaction using self-quenched,fluorogenic primers.Anal Biochem.2003;315:95-105.
20 Messaoudi,E.,K.Bardsen,B.Srebro,and C.R.Bramham.Acute intrahippocampal infusion of BDNF induces lasting potentiation of synaptic transmission in the rat dentate gyrus.J Neurophysiol.1998;79:496-9.
21 Vissio,RG,M.M.Canepa,and M.C.Maggese.Brain-derived neurotrophic factor (BDNF)-like immunoreactivity localization in the retina and brain of Cichlasoma dimerus (Teleostei,Perciformes).Tissue Cell.2008;40:261-70.
22 Ohira,K.,N.Funatsu,S.Nakamura,and M.Hayashi.Expression of BDNF and TrkB receptor subtypes in the postnatal developing Purkinje cells of monkey cerebellum.Gene Expr Patterns.2004;4:257-61.
23 Tapia-Arancibia,L.,E.Aliaga,M.Silhol,and S.Arancibia.New insights into brain BDNF function in normal aging and Alzheimer disease.Brain Res Rev.2008;59:201-20.
24 Linnarsson,S.,A.Bjorklund,and P.Ernfors.Learning deficit in BDNF mutant mice.Eur J Neurosci.1997;9:2581-7.
25 Gomez-Pinilla,R,V.So,and J.P.Kesslak.Spatial learning induces neurotrophin receptor and synapsin I in the hippocampus.Brain Res.2001;904:13-9.
26 Gorski,J.A.,S.A.Balogh,J.M.Wehner,and K.R.Jones.Learning deficits in forebrain-restricted brain-derived neurotrophic factor mutant mice.Neuroscience.2003;121:341-54.
27 Dragunow,M.,P.Hughes,S.E.Mason-Parker,P.Lawlor,and W.C.Abraham.TrkB expression in dentate granule cells is associated with a late phase of long-term potentiation.Brain Res Mol Brain Res.1997;46:274-80.
28 Wang,Y.,B.Su,and Z.Xia.Brain-derived neurotrophic factor activates ERK5 in cortical neurons via a Rap1-MEKK2 signaling cascade.J Biol Chem.2006;281:35965-74.
29 Altieri,M.,F.Marini,R.Arban,G Vitulli,and B.O.Jansson.Expression analysis of brain-derived neurotrophic factor (BDNF) mRNA isoforms after chronic and acute antidepressant treatment.Brain Res.2004;1000:148-55.
30 Gomez-Palacio-Schjetnan,A.and M.L.Escobar.In vivo BDNF modulation of adult functional and morphological synaptic plasticity at hippocampal mossy fibers.Neurosci Lett.2008;445: 62-7.
31 Frech,M.,J.John,V.Pizon,P.Chardin,A.Tavitian,R.Clark,F.McCormick,and A.Wittinghofer.Inhibition of GTPase activating protein stimulation of Ras-p21 GTPase by the Krev-1 gene product.Science.1990;249:169-71.
32 Hallberg,B.,S.I.Rayter,and J.Downward.Interaction of Ras and Raf in intact mammalian cells upon extracellular stimulation.J Biol Chem.1994;269:3913-6.
33 Hogan,C,N.Serpente,P.Cogram,C.R.Hosking,C.U.Bialucha,S.M.Feller,V.M.Braga,W.Birchmeier,and Y.Fujita.Rapl regulates the formation of E-cadherin-based cell-cell contacts.Mol Cell Biol.2004;24:6690-700.
34 Selcher,J.C.,E.J.Weeber,J.Christian,T.Nekrasova,GE.Landreth,and J.D.Sweatt.A role for ERK MAP kinase in physiologic temporal integration in hippocampal area CA1.Leam Mem.2003;10:26-39.
35 Soule,J.,E.Messaoudi,and C.R.Bramham.Brain-derived neurotrophic factor and control of synaptic consolidation in the adult brain.Biochem Soc Trans.2006;34:600-4.
36 Morozov,A.,LA.Muzzio,R.Bourtchouladze,N.Van-Strien,K.Lapidus,D.Yin,D.G Winder,J.P.Adams,J.D.Sweatt,and E.R.Kandel.Rapl couples cAMP signaling to a distinct pool of p42/44MAPK regulating excitability,synaptic plasticity,learning,and memory.Neuron.2003;39:309-25.
37 Asha,H.,N.D.de Ruiter,M.G Wang,and I.K.Hariharan.The Rapl GTPase functions as a regulator of morphogenesis in vivo.EMBO J.1999;18:605-15.
38 Tsygankova,O.M.,A.Saavedra,J.F.Rebhun,L.A.Quilliam,and J.L.Meinkoth.Coordinated regulation of Rapl and thyroid differentiation by cyclic AMP and protein kinase A.Mol Cell Biol.2001;21:1921-9.
39 Hisata,S.,T.Sakisaka,T.Baba,T.Yamada,K.Aoki,M.Matsuda,and Y.Takai.Rap1-PDZ-GEF1 interacts with a neurotrophin receptor at late endosomes,leading to sustained activation of Rapl and ERK and neurite outgrowth.J Cell Biol.2007;178:843-60.
40 Huang,C.C.,J.L.You,M.Y.Wu,and K.S.Hsu.Rap1-induced p38 mitogen-activated protein kinase activation facilitates AMPA receptor trafficking via the GDI.Rab5 complex.Potential role in (S)-3,5-dihydroxyphenylglycene-induced long term depression.J Biol Chem.2004;279:12286-92.
41 Reedquist,K.A.,E.Ross,E.A.Koop,R.M.Wolthuis,F.J.Zwartkruis,Y.van Kooyk,M.Salmon,CD.Buckley,and J.L.Bos.The small GTPase,Rapl,mediates CD31-induced integrin adhesion.J Cell Biol.2000;148:1151-8.
42 Altschuler,D.L.and F.Ribeiro-Neto.Mitogenic and oncogenic properties of the small G protein Raplb.Proc Natl Acad Sci USA.1998;95:7475-9.
43 Jorgensen,O.S.Neural cell adhesion molecule (NCAM) as a quantitative marker in synaptic remodeling.Neurochem Res.1995;20:533-47.
44 Thiery,J.P.,R.Brackenbury,U.Rutishauser,and GM.Edelman.Adhesion among neural cells of the chick embryo.Ⅱ.Purification and characterization of a cell adhesion molecule from neural retina.J Biol Chem.1977;252:6841-5.
45 Schmid,R.S.,R.D.Graff,M.D.Schaller,S.Chen,M.Schachner,J.J.Hemperly,and P.F.Maness.NCAM stimulates the Ras-MAPK pathway and CREB phosphorylation in neuronal cells.J Neurobiol.1999;38:542-58.
2 de Escobar,GM.,M.J.Obregon,and F.E.del Rey.Maternal thyroid hormones early in pregnancy and fetal brain development.Best Pract Res Clin Endocrinol Metab.2004;18:225-48.
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5 Pop,V.J.,J.L.Kuijpens,A.L.van Baar,G Verkerk,M.M.van Son,J.J.de Vijlder,T.Vulsma,W.M.Wiersinga,H.A.Drexhage,and H.L.Vader.Low maternal free thyroxine concentrations during early pregnancy are associated with impaired psychomotor development in infancy.Clin Endocrinol.1999;50:149-55.
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30 Auso,E.,R.Lavado-Autric,E.Cuevas,F.E.Del Rey,G.Morreale De Escobar,and P.Berbel.A moderate and transient deficiency of maternal thyroid function at the beginning of fetal neocorticogenesis alters neuronal migration.Endocrinology.2004;145:4037-47.
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38 Iglesias,T,J.Caubin,H.G Stunnenberg,A.Zaballos,J.Bernal,and A.Munoz.Thyroid hormone-dependent transcriptional repression of neural cell adhesion molecule during brain maturation.EMBO J.1996;15:4307-16.
1 Alzoubi,K.H.,N.Z.Gerges,A.M.Aleisa,and K.A.Alkadhi.Levothyroxin restores hypothyroidism-induced impairment of hippocampus-dependent learning and memory:Behavioral,electrophysiological,and molecular studies.Hippocampus.2009;19:66-78.
2 Anderson,G.W.,C.M.Schoonover,and S.A.Jones.Control of thyroid hormone action in the developing rat brain.Thyroid.2003;13:1039-56.
3 滕卫平.论母体甲状腺激素不足对胎儿神经智力发育的影响.中华内分泌代谢杂志.2005:21:97-98.
4 Zoeller,R.T.Transplacental thyroxine and fetal brain development.J Clin Invest.2003;111:954-7.
5 Wikner,B.N.,L.S.Sparre,C.O.Stiller,B.Kallen,and C.Asker.Maternal use of thyroid hormones in pregnancy and neonatal outcome.Acta Obstet Gynecol Scand.2008;87:617-27.
6 Thompson,J.,S.E.Moore,and F.S.Walsh.Thyroid hormones regulate expression of the neural cell adhesion molecule in adult skeletal muscle.FEBS Lett.1987;219:135-8.
7 Auso,E.,R.Lavado-Autric,E.Cuevas,F.E.Del Rey,G.Morreale De Escobar,and P.Berbel.A moderate and transient deficiency of maternal thyroid function at the beginning of fetal neocorticogenesis alters neuronal migration.Endocrinology.2004;145:4037-47.
8 Chan,S.,S.Kachilele,C.J.McCabe,L.A.Tannahill,K.Boelaert,N.J.Gittoes,T.J.Visser,J.A.Franklyn,and M.D.Kllby.Early expression of thyroid hormone deiodinases and receptors in human fetal cerebral cortex.Brain Res Dev Brain Res.2002;138:109-16.
9 Haddow,J.E.,G.E.Palomaki,W.C.Allan,J.R.Williams,G.J.Knight,J.Gagnon,C.E.O'Heir,M.L.Mitchell,R.J.Hermos,S.E.Waisbren,J.D.Faix,and R.Z.Klein.Matemal thyroid deficiency during pregnancy and subsequent neuropsychological development of the child.N Engl J Med.1999;341:549-55.
10 Pop,V.J.,J.L.Kuijpens,A.L.van Baar,G.Verkerk,M.M.van Son,J.J.de Vijlder,T.Vulsma,W.M.Wiersinga,H.A.Drexhage,and H.L.Vader.Low maternal free thyroxine concentrations during early pregnancy are associated with impaired psychomotor development in infancy.Clin Endocrinol(Oxf).1999;50:149-55.
11 Pop,V.J.,E.P.Brouwers,H.L.Vader,T.Vulsma,A.L.van Baar,and J.J.de Vijlder.Maternal hypothyroxinaemia during early pregnancy and subsequent child development:a 3-year follow-up study.Clin Endocrinol (Oxf).2003;59:282-8.
12 Evans,I.M.,A.K.Sinha,M.R.Pickard,P.R.Edwards,A.J.Leonard,and R.P.Ekins.Maternal hypothyroxinemia disrupts neurotransmitter metabolic enzymes in developing brain.J Endocrinol.1999;161:273-9.
13 Donahue,D.A.,E.J.Dougherty,and L.A.Meserve.Influence of a combination of two tetrachlorobiphenyl congeners (PCB 47;PCB 77) on thyroid status,choline acetyltransferase (ChAT) activity,and short-and long-term memory in 30-day-old Sprague-Dawley rats.Toxicology.2004;203:99-107.
14 Ernfors,P.,C.Wetmore,L.Olson,and H.Persson.Identification of cells in rat brain and peripheral tissues expressing mRNA for members of the nerve growth factor family.Neuron.1990;5:511-26.
15 Zoeller,R.T.and J.Rovet.Timing of thyroid hormone action in the developing brain:clinical observations and experimental findings.J Neuroendocrinol.2004;16:809-18.
16 Harman,A.M.,J.Rodger,A.Ahmat,C.Thomas,C.Bartlett,P.Chen,S.A.Dunlop,and L.D.Beazley.PSA-NCAM is up-regulated during optic nerve regeneration in lizard but not in goldfish.Exp Neurol.2003;182:180-5.
17 Parkash,J.and G.Kaur.Potential of PSA-NCAM in neuron-glial plasticity in the adult hypothalamus:role of noradrenergic and GABAergic neurotransmitters.Brain Res Bull.2007;74:317-28.
18 Wakabayashi,Y.,S.Uchida,H.Funato,T.Matsubara,T.Watanuki,K.Otsuki,M.Fujimoto,A.Nishida,and Y.Watanabe.State-dependent changes in the expression levels of NCAM-140 and LI in the peripheral blood cells of bipolar disorders,but not in the major depressive disorders.Prog Neuropsychopharmacol Biol Psychiatry.2008;32:1199-205.
19 Lowe,B.,H.A.Avila,F.R.Bloom,M.Gleeson,and W.Kusser.Quantitation of gene expression in neural precursors by reverse-transcription polymerase chain reaction using self-quenched,fluorogenic primers.Anal Biochem.2003;315:95-105.
20 Messaoudi,E.,K.Bardsen,B.Srebro,and C.R.Bramham.Acute intrahippocampal infusion of BDNF induces lasting potentiation of synaptic transmission in the rat dentate gyrus.J Neurophysiol.1998;79:496-9.
21 Vissio,RG,M.M.Canepa,and M.C.Maggese.Brain-derived neurotrophic factor (BDNF)-like immunoreactivity localization in the retina and brain of Cichlasoma dimerus (Teleostei,Perciformes).Tissue Cell.2008;40:261-70.
22 Ohira,K.,N.Funatsu,S.Nakamura,and M.Hayashi.Expression of BDNF and TrkB receptor subtypes in the postnatal developing Purkinje cells of monkey cerebellum.Gene Expr Patterns.2004;4:257-61.
23 Tapia-Arancibia,L.,E.Aliaga,M.Silhol,and S.Arancibia.New insights into brain BDNF function in normal aging and Alzheimer disease.Brain Res Rev.2008;59:201-20.
24 Linnarsson,S.,A.Bjorklund,and P.Ernfors.Learning deficit in BDNF mutant mice.Eur J Neurosci.1997;9:2581-7.
25 Gomez-Pinilla,R,V.So,and J.P.Kesslak.Spatial learning induces neurotrophin receptor and synapsin I in the hippocampus.Brain Res.2001;904:13-9.
26 Gorski,J.A.,S.A.Balogh,J.M.Wehner,and K.R.Jones.Learning deficits in forebrain-restricted brain-derived neurotrophic factor mutant mice.Neuroscience.2003;121:341-54.
27 Dragunow,M.,P.Hughes,S.E.Mason-Parker,P.Lawlor,and W.C.Abraham.TrkB expression in dentate granule cells is associated with a late phase of long-term potentiation.Brain Res Mol Brain Res.1997;46:274-80.
28 Wang,Y.,B.Su,and Z.Xia.Brain-derived neurotrophic factor activates ERK5 in cortical neurons via a Rap1-MEKK2 signaling cascade.J Biol Chem.2006;281:35965-74.
29 Altieri,M.,F.Marini,R.Arban,G Vitulli,and B.O.Jansson.Expression analysis of brain-derived neurotrophic factor (BDNF) mRNA isoforms after chronic and acute antidepressant treatment.Brain Res.2004;1000:148-55.
30 Gomez-Palacio-Schjetnan,A.and M.L.Escobar.In vivo BDNF modulation of adult functional and morphological synaptic plasticity at hippocampal mossy fibers.Neurosci Lett.2008;445: 62-7.
31 Frech,M.,J.John,V.Pizon,P.Chardin,A.Tavitian,R.Clark,F.McCormick,and A.Wittinghofer.Inhibition of GTPase activating protein stimulation of Ras-p21 GTPase by the Krev-1 gene product.Science.1990;249:169-71.
32 Hallberg,B.,S.I.Rayter,and J.Downward.Interaction of Ras and Raf in intact mammalian cells upon extracellular stimulation.J Biol Chem.1994;269:3913-6.
33 Hogan,C,N.Serpente,P.Cogram,C.R.Hosking,C.U.Bialucha,S.M.Feller,V.M.Braga,W.Birchmeier,and Y.Fujita.Rapl regulates the formation of E-cadherin-based cell-cell contacts.Mol Cell Biol.2004;24:6690-700.
34 Selcher,J.C.,E.J.Weeber,J.Christian,T.Nekrasova,GE.Landreth,and J.D.Sweatt.A role for ERK MAP kinase in physiologic temporal integration in hippocampal area CA1.Leam Mem.2003;10:26-39.
35 Soule,J.,E.Messaoudi,and C.R.Bramham.Brain-derived neurotrophic factor and control of synaptic consolidation in the adult brain.Biochem Soc Trans.2006;34:600-4.
36 Morozov,A.,LA.Muzzio,R.Bourtchouladze,N.Van-Strien,K.Lapidus,D.Yin,D.G Winder,J.P.Adams,J.D.Sweatt,and E.R.Kandel.Rapl couples cAMP signaling to a distinct pool of p42/44MAPK regulating excitability,synaptic plasticity,learning,and memory.Neuron.2003;39:309-25.
37 Asha,H.,N.D.de Ruiter,M.G Wang,and I.K.Hariharan.The Rapl GTPase functions as a regulator of morphogenesis in vivo.EMBO J.1999;18:605-15.
38 Tsygankova,O.M.,A.Saavedra,J.F.Rebhun,L.A.Quilliam,and J.L.Meinkoth.Coordinated regulation of Rapl and thyroid differentiation by cyclic AMP and protein kinase A.Mol Cell Biol.2001;21:1921-9.
39 Hisata,S.,T.Sakisaka,T.Baba,T.Yamada,K.Aoki,M.Matsuda,and Y.Takai.Rap1-PDZ-GEF1 interacts with a neurotrophin receptor at late endosomes,leading to sustained activation of Rapl and ERK and neurite outgrowth.J Cell Biol.2007;178:843-60.
40 Huang,C.C.,J.L.You,M.Y.Wu,and K.S.Hsu.Rap1-induced p38 mitogen-activated protein kinase activation facilitates AMPA receptor trafficking via the GDI.Rab5 complex.Potential role in (S)-3,5-dihydroxyphenylglycene-induced long term depression.J Biol Chem.2004;279:12286-92.
41 Reedquist,K.A.,E.Ross,E.A.Koop,R.M.Wolthuis,F.J.Zwartkruis,Y.van Kooyk,M.Salmon,CD.Buckley,and J.L.Bos.The small GTPase,Rapl,mediates CD31-induced integrin adhesion.J Cell Biol.2000;148:1151-8.
42 Altschuler,D.L.and F.Ribeiro-Neto.Mitogenic and oncogenic properties of the small G protein Raplb.Proc Natl Acad Sci USA.1998;95:7475-9.
43 Jorgensen,O.S.Neural cell adhesion molecule (NCAM) as a quantitative marker in synaptic remodeling.Neurochem Res.1995;20:533-47.
44 Thiery,J.P.,R.Brackenbury,U.Rutishauser,and GM.Edelman.Adhesion among neural cells of the chick embryo.Ⅱ.Purification and characterization of a cell adhesion molecule from neural retina.J Biol Chem.1977;252:6841-5.
45 Schmid,R.S.,R.D.Graff,M.D.Schaller,S.Chen,M.Schachner,J.J.Hemperly,and P.F.Maness.NCAM stimulates the Ras-MAPK pathway and CREB phosphorylation in neuronal cells.J Neurobiol.1999;38:542-58.
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