摘要
前言
亚临床甲状腺功能减退症(Subclinical hypothyroidism,SCH)又称亚临床甲减,在一般人群的总体患病率是4-10%,妊娠期妇女患病率为5%。母体甲减,特别是碘缺乏病引起的母体和胎儿甲减可以导致儿童的智力缺陷(克汀病)早已被认识。Man及其同事首次发现轻微的母亲甲减就可引起后代智力评分(intelligence quotient,IQ)降低,1999年Haddow和Pop等学者发现：母亲妊娠时TSH升高或FT4降低会导致后代智力评分降低,本课题组的研究结果也提示妊娠16～20周妇女单纯亚临床甲减,单纯低T4血症和单纯TPOAb阳性皆可以使后代25-30月时的智力和运动发育评分降低8-10分。但是母亲亚临床甲减引起后代智力评分降低的确切机制还不清楚,本研究拟通过动物实验探讨左旋甲状腺素(L-T4)治疗孕期母鼠亚临床甲减改善仔鼠脑发育的有效性及治疗的最佳时期,并探讨其作用机制。
方法
75只Wistar雌鼠称重并麻醉后行甲状腺完全切除术,一个月后眶后静脉采血检测甲功,当血清TSH高于正常,但TT4小于最小值(<1μg/dL)时,提示甲状腺切除干净,成功建立临床甲减(CH)模型。其中15只临床甲减鼠皮下注射含有BSA的PBS液；60只以0.95μg/100g·d剂量皮下注射L-T4,9天后眶后静脉采血检测甲功,当血清TSH高于正常、而TT4水平在正常范围时,提示亚临床甲减大鼠(SCH)模型建立成功。另15只Wistar雌鼠行甲状腺切除假手术做为正常对照组(C),皮下注射含有BSA的PBS液。各组模型建立成功后同时与正常雄鼠合笼交配,雌：雄=2：1,当次日晨发现雌鼠阴道栓或阴道涂片镜下发现精子确定为妊娠0天,记为E0。60只亚临床甲减孕鼠模型,其中15只孕鼠继续以0.95μg/100g·d剂量皮下注射L-T4至产后21天(P21)；其余45只大鼠分别在妊娠第10天(E10)(n=15)、第13天(E13)(n=15)及第17天(E17)(n=15)开始以1.25μg/100g·d剂量皮下注射L-T4到P21；所有母鼠分别于孕前、E10、E13、E17、E19眶后静脉丛取血,测定血清TSH、TT4,在E14-E16天腹腔注射溴脱氧尿苷(BrdU)20mg/kg·d共3天。分娩当天记为P0。P3时每窝仔鼠择优存留8只,P3、P7及P40时监测仔鼠体重,对仔鼠脑发育相关的指标进行检测。P45应用水迷宫实验检测仔鼠学习记忆能力。Nissl染色法在光学显微镜下观察大脑皮质和海马形态学改变；应用免疫组化染色检测BrdU的分布,从而判断妊娠早期大脑皮质细胞的迁移位置；应用免疫组化或Western blot进行Reelin、肌腱蛋白-C、L1细胞粘附分子(LICAM)及层粘连蛋白的半定量分析。
结果
1、血清TSH、TT4水平结果显示
与假手术组比较,亚临床甲减组表现为血清TSH水平明显高于同期假手术组(P<0.05),而TT4水平未见明显改变(P>0.05),符合亚临床甲减激素血清学改变。E10治疗组、E13治疗组及E17治疗组在治疗之前TSH升高(P<0.05),TT4无明显改变(P>0.05),仍处于亚临床甲减状态,治疗后孕鼠血清激素水平示TSH及TT4与正常对照组孕鼠均无明显区别(P>0.05)。
2、发育期各组仔鼠的体重及激素水平比较
P3及P7时亚临床甲减组仔鼠体重明显低于正常对照组仔鼠(P<0.05),P40时与正常对照组无明显区别(P>0.05)；与完全甲减组仔鼠相比,P3时高于完全甲减组(P<0.05),但P7及P40时无明显区别(P>0.05)；甲减组仔鼠体形偏小,体重增加缓慢,在P3、P7及P40时体重均低于正常对照组仔鼠(P<0.05)。E10治疗组、E13治疗组及E17治疗组仔鼠体重在P3、P7及P40与正常对照组仔鼠均无明显区别(P>0.05),明显高于完全甲减组。40日龄进行甲功测试时发现各组仔鼠血清TSH激素水平及血清TT4激素水平均无统计学差异(P>0.05)。
3、Morris水迷宫行为测定指标分析
甲减组及亚临床甲减组逃避潜伏期与对照组比较均有显著性差异(P<0.05),到第5天时逃避潜伏期与对照组比较仍有显著性差异(P<0.05)。E10治疗组仔鼠及E13治疗组仔鼠在各时段训练的逃避潜伏期与正常对照组仔鼠均无明显差别(P>0.05),但E17开始治疗组各时段训练的逃避潜伏期均比正常对照组仔鼠延长,差异显著(P<0.05)。
4、各组仔鼠皮质及海马病理形态学变化
Nissl染色光镜结果显示7日龄正常对照组仔鼠躯体感觉皮质各层神经细胞排列致密整齐,Ⅳ层见典型的桶状结构,Ⅴ层见大的锥体细胞,各细胞层间界限清晰,亚临床甲减组及完全甲减组仔鼠躯体感觉皮质则无这些典型的表现。E17治疗组也是如此,但E10开始治疗及E13开始治疗组仔鼠表现与正常对照组仔鼠无明显差别；40日龄仔鼠也有类似结果。皮质厚度测量结果显示P7时各组仔鼠之间均无明显差别(P>0.05),P40时甲减组仔鼠皮质比正常对照组仔鼠薄(P=0.000)。正常对照组仔鼠海马CA1区锥体细胞层界限清晰,E10治疗组及E13治疗组仔鼠表现与正常对照组仔鼠无明显差别,E17治疗组、亚临床甲减组及完全甲减组仔鼠海马CA1区神经元层间界限不清。亚临床甲减组及完全甲减组仔鼠海马锥体层(py)增厚,辐射层(ra)较薄(P<0.05),E17治疗组与之类似,但E10治疗组及E13治疗则与正常对照组相似,厚度无统计学差异(P>0.05)。
5、大脑细胞迁移情况
亚临床甲减组及临床甲减组仔鼠BrdU标记的细胞分布更广泛,白质内出现BrdU标记的细胞,统计结果显示Ⅵ层及皮质下白质内BrdU标记细胞的数量明显多于正常对照组仔鼠,Ⅱ-Ⅲ层减少(P<0.05),神经细胞移行于与出生日期不相应的异常位置；E17治疗组仔鼠表现与亚临床甲减组及完全甲减组仔鼠相似,但E10治疗组及E13治疗组则与正常对照组仔鼠相似(P>0.05)。
6、各组仔鼠大脑Reelin及肌腱蛋白-C表达量的变化
免疫组化染色发现：7日龄及40日龄甲减组、亚临床甲减组及E17治疗组仔鼠躯体感觉皮质及海马Reelin的表达量明显低于正常对照组(P<0.05),E10及E13治疗组与正常对照组无明显区别。7日龄甲减组、亚临床甲减组及E17治疗组仔鼠躯体感觉皮质肌腱蛋白-C表达量明显高于正常对照组(P<0.05)；40日龄各组之间均无明显区别。
7、仔鼠皮质和海马组织的L1CAM、层粘连蛋白(Laminin)水平
Western blotting检测皮质结果显示,与正常对照组比较,E17治疗组、亚临床甲减组及甲减组仔鼠皮质L1CAM三个主要多肽的蛋白表达量在出生后3d(P3)明显升高(P<0.05),但E10及E13治疗组则与正常对照组无区别；P7时甲减组L1CAM 140kDa多肽的表达明显多于正常对照组(P<0.05),其他各组间无差异；P40时L1CAM的三个主要多肽在各组间均无明显差异。海马检测结果显示,与正常对照组比较,甲减组仔鼠皮质L1CAM三个多肽蛋白表达量在出生后3d(P3)明显升高,E10、E13、E17及亚临床甲减组L1CAM三个多肽与正常对照组之间均无明显区别(P>0.05)；P7及P40时海马L1CAM的三个主要多肽在各组间的表达均无明显差异。皮质及海马检测结果显示层粘连蛋白在各组之间均无明显区别(P>0.05)。
结论
1、孕期母鼠亚临床甲减会影响后代的正常发育,孕第10天(E10)、E13和E17开始应用L-T4治疗母鼠至甲功正常,都可以促进仔鼠体重的正常发育。
2、母体甲减及亚临床甲减可以导致后代学习记忆能力的严重损伤,若在E13之前进行L-T4治疗则对后代仔鼠行为学测定指标无明显影响；若在E17之后开始L-T4治疗则仍会对后代仔鼠行为学测定指标产生影响。
3、亚临床甲减母鼠的后代学习和记忆能力的下降可能与细胞构筑、神经细胞的迁移及定位异常有关,E13之前开始L-T4治疗能够阻止神经细胞迁移及定位紊乱。
4、甲状腺功能不足时细胞构筑、神经细胞迁移及定位的异常与迁移相关分子Reelin的下降、肌腱蛋白-C的上升及L1CAM的上升有关,而与层粘连蛋白无关。
Objective
Subclinical hypothyroidism(SCH) is a common clinical problem for which the overall prevalence is 4-10% in the general population and up to 5% in women during pregnancy. Evidence had been accumulating,especially maternal iodine deficiency (ID),that maternal clinical hypothyroidism (CH) alters optimal fetal neurodevelopment, such as cretinism. Man and co-workers first suggested in 1969 that mild maternal hypothyroidism alone was associated with lower intelligence quotients (IQs) in the offspring.In 1999, Haddow and Pop et al.found that higher maternal thyrophin (TSH) or lower maternal free thyroxine (FT4) concertration during early pregnancy are associated with impaired psychomotor development in early infancy.At 16-20 weeks gestation, maternal SCH, hypothyroxinaemia or euthyroidism with elevated TPOAb titres were all statistically significant predictors of lower motcr and intellectual development at 25-30 months.The intimate mechanisms by which maternal SCH during pregnancy influence the neurodevelopment of offspring is unknown. The present study aimed to investigate the effects and the optimal period of treatment with levothyroxine (L-T4) in early maternal SCH on the intelligence development of the progeny.
Methods
Seventy five rats were thyroidectomized after weighing and anesthetizing. The models of CH were established and thyroid ablation completely as serum T4 concentrations fell below the level of detection of the assay(<1μg/dL). Fifteen CH rats were injected subcutaneouly phosphate buffered solution (PBS) containing bovine serum albumin (BSA).Sixty CH rats were injected subcutaneouly L-T4 at a dose of 0.95μg per 100g body weight (BW) per day for nine days.Blood samples were collected from the vena orbitalis posterior, and sera were analyzed for thyroid function. The models of SCH were established with elevated serum TSH level and normal total T4 (TT4) level.Then fifteen SCH rats were injected subcutaneouly L-T4 at a dose of 0.95μg per 100g BW per day until postnatal d 21 (P21).The other 45 SCH rats treated with L-T4 at embryonic d 10 (E10) (n=15),embryonic d 13 (E13)(n=15)or embryonic d 17 (E17) (n=15)at a dose of 1.25μg per 100g BW per day until P21.Another fifteen euthyroid sham-operated rats (C) were injected subcutaneouly PBS containing BSA. These rats were mated with normal male Wistar rats (female:male=2:1),and the next day a vaginal smear was obtained and analyzed under a microscope to search for spermatozoa. Rats with smear positive for spermatozoa were considered mated, and the first day after mating was referred to as embryonic d 0 (E0).They were injected intraperitoneally for 3 days with BrdU (20 mg/kg per day in physiological saline; Sigma, USA) from E14 to E16.The day of birth was referred to as postnatal d 0 (P0). Blood was obtained from dams of the six main experimental groups on the mornings of progestation, E10, E13,E17 and E19 for the determination of TT4 and TSH concentrations.At P3 each litter was culled to eight pups and pups were weaned at P21. Detection correlated index of the developing brain of progeny at P3,P7 and P40.The method of the water maze test is used to assess spatial learning and memory in pups. Nissl violet-stained coronal sections coronal sections showing the cytoarchitecture of the barrel cortex of the primary somatosensory cortex and hippocampus. The radial distribution of BrdU-labeled cells was observed by immunohistochemistry stain.The semiquantitative analysis of Reelin, tenascin C,L1CAM and laminin were detected by immunohistochemistry stain or western blotting.
Results
1.During pregnancy, maternal plasma TT4 concentration of the SCH dams were similar with control dams and TSH concentration of the SCH dams increased significantly than control dams.Dams with E10,E13 and E17 had significantly higher TSH levels and similar TT4 compared with controls before treatment. Sesum TSH and TT4 of E10 dams were not different from control dams at E13 after treatment,and so were E13 dams at E17 and E17 dams at E19.
2.Pups with SCH had significantly lower BW than control pups at P3 and P7,and were similar to controls at P40. At the same time, SCH pups had significantly higher BW at P3 and similar BW at P7 and P40 compared with CH pups.CH pups were small and their BW increased slowly.Not only at P3 and P7 but also at P40,the BW of CH pups were all lower than control pups.However, BW did not differ among the groups of E10,E13,E17 and control pups during development process.Moreover, BW of E10, E13 and E17 pups were significantly higher than that of CH pups at P3,P7 and P40 (except for E10 pups at P40).There were similar TSH and TT4 levels among the six groups of pups at P40.
3.Significantly longer escape latencies were observed for CH and SCH pups, compared with controls in the water maze test.The escape latencies of CH and SCH pups were still longer than control pups on the fifth day.The latencies from E10 and E13 pups were comparable to those from control pups, however the E17 pups took markedly more time than controls to find the hidden plateform during all 4 days of testing.
4.The major features in the control pups were typical barrels that can be seen in layer IV, large pyramids in layer V, and easily distinguishable borders within all layers in barrel cortex.In contrast, these features are less prominent in the progeny of CH and SCH pups.These changes were also seen in the E17 pups,whereas these features were normal in the E10 and E13 pups.No statistically significant differences were found in the thickness of the neocortex among the six groups at P7. However, the thickness of the barrel cortex of CH pups was thinner than controls at P40.There were changes in the hippocampus in CH, SCH and E17 pups.As in the barrel cortex, the gross cytoarchitecture of E10 and E13 pups was similar to that of control pups both at P7 and at P40.In CA1,the pyramidal cell layer showed clear-cut borders with the adjacent strata. The thickness of stratum pyramidale and stratum radiatum did not differ among the groups of E10,E13 and control pups.In contrast, in CA1 of CH, SCH and E17 pups, the border of the pyramidal layer with the strata oriens was more blurred.In addition, the stratum radiatum was thinner and the stratum pyramidale was thicker than controls in CA1 both at P7 and at P40.
5.The radial distribution of BrdU-labeled cells in the primary somatosensory cortex was abnormal in pups of CH, SCH and E17 dams, with heterotopic BrdU-labeled cells at locations different from those corresponding to their birth date at P40.In these pups,the percentage of BrdU-labeled cells decreased in layerⅡ-Ⅲ, and increased in layerⅥand subcortical white matter, whereas no differences were found between E10 and E13 pups and control pups.
6.The level of expression of the Reelin in cerebral cortex and hippocampus was lower in CH,SCH and E17 groups than that in sham control group (P<0.05)at P7 and P40 with immunohistochemistry stain and there were not different among E10, E13 and controls.Tenascin C expression was found homogeneously upregulated in cerebral cortex of CH, SCH and E17 pups at P7, but no significant differences in the levels of tenascin C among the six groups of pups at P40.
7. Western blot analysis showed that the level of expression of the three major L1CAM polypeptides in cerebral cortex was higher in CH, SCH and E17 groups than that in sham control group (P<0.05) at P3 and there were not different among E10, E13 and controls.The expression of 140kDa polypeptides was increased at P7 in cerebral cortex of CH pups but there was no significant difference between control and othter groups.A statistically significant up-regulation was observed in hippocampus of CH at P3 when compared to sham control group (P<0.05) with the three major L1CAM polypeptides. No differences were found the six groups of pups in cerebral cortex or hippocampus at P7 or P40.The the level of expression of laminin were comparable among the six groups of pups at P3,P7 and P40.
1.Maternal SCH influence the normal development of BW, and treatment of SCH at E10,E13 and E17 improve the BW of SCH pups.
2.Maternal SCH could disturb learning and memory performances.Treatment with L-T4 in early maternal SCH before E13 improves the learning and memory performances in the developing brain of the progeny.
3.Early maternal SCH alters cerebral cortex cytoarchitecture and the cell migration of cerebral cortex of the progeny, and treatment with L-T4 in early maternal SCH(before E13)improves the cerebral cortex cytoarchitecture in the developing brain of the progeny. Maybe the results induced to the impairment of learning and memory of pups.
4. The lower Reelin, higher tenascin C and L1CAM in cerebral cortex or hippocampus may contribute to the alteration of cytoarchitecture and cell migration when thyroid function was insufficiency.
引文
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