The Effect Of Maternal Subclinical Hypothyroidism During Pregnancy On Brain Development And Thyroid Related Genes In Rat Offspring

ObjectiveThyroid 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.MethodsTotal 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 T₄ 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 T₄ 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.Results1.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 T₄ 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.Conclusions1.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 T₄ 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.