Effects Of Levothyroxine Treatment On Offspring Brain Development In Pregnant Rats With Subclinical Hypothyroidism

ObjectiveSubclinical 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.MethodsSeventy 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.Results1.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.