Maternal Subclinical Hypothyroidism Rats Impairs Spatial Learning And Memory In Offspring By Disrupting Balance Of The TrkA/p75^NTR Signaling Pathway

Objective:Thyroid hormones play an indispensable and important role in the development of the fetal brain.If the deficiency of thyroid hormone occurs in the mother during pregnancy,it will cause significant and irreversible damage to the development of the fetal nervous system,which could lead to the declined in offspring's ability to learn and memory.Several authoritative epidemiological investigations have found that mothers with maternal subclinical hypothyroidism?SCH?may cause neurological and intellectual developmental disorders in their offspring.However,the right amount of levothyroxine?L-T₄?replacement therapy at the right time can effectively improve this adverse effect.In the early stage of this research group,we found that the offspring of maternal SCH rats showed a decline in spatial learning and memory,through Morris water-maze?MWM?and long-term potentiation?LTP?experiments.However,the mechanism is still unclear.Nerve growth factor?NGF?participates in a variety of physiological activities during the development of the nervous system,such as neuron survival,proliferation,differentiation,anti-apoptosis,promotion of axon and dendrite growth,and enhancement of synaptic plasticity and so on,playing an important role in fetal brain development.NGF-mediated tropomyosin receptor kinase?Trk?A signaling pathway kicks in promoting neuronal proliferation and inhibits the p75 neurotrophy receptor(p75^NTR)signaling pathway to exert anti-neuronal apoptosis.It has been reported that the expression of NGF in the cerebral cortex and cerebellum of offspring of perinatal hypothyroidism?OH?rats is reduced,thereby inhibiting the expression of the TrkA signaling pathway,promoting the expression of the p75^NTR signaling pathway,and then destroying the balance of TrkA/p75^NTR signaling pathway.However,it is unclear whether there is an imbalance in the TrkA/p75^NTR signaling pathway during hippocampal development in maternal SCH rats'offspring.The purpose of this study was to investigate the relationship between neurocognitive impairment in offspring and the balance of NGF-related TrkA/p75^NTR signaling pathway in the hippocampus of offspring of SCH rats during pregnancy,and to investigate whether L-T₄ replacement therapy during pregnancy can improve hippocampal development in pups by improving the balance of TrkA/p75^NTR signaling pathway and determine the optimal intervention time.Methods:1.Female adult Wistar rats were randomly divided into six groups?n=15 per group?:?1?Control?CON?group:rats were underwent thyroidectomy sham operation and were given a daily subcutaneous injection of 50?l/?100g?d?of normal saline in the neck;?2?OH group:rats were underwent thyroidectomy and were given a daily subcutaneous injection of 50?l/?100g?d?of normal saline in the neck to establish the OH model;?3?SCH group:using the successfully established OH rat model,OH rats were injected with 1.00?g/?100g?d?of L-T₄ solution subcutaneously in the neck daily to establish the SCH model;?4?E10 treatment group:using the successfully established SCH rat model,the dose of L-T₄ solution was adjusted to 1.25?g/?100g?d?starting from day 10 of pregnancy?E10?to establish a subclinical hypothyroidism L-T₄ E10 treatment model;?5?E13 treatment group:using the successfully established SCH rat model,the dose of L-T₄ solution was adjusted to 1.25?g/?100g?d?starting from day 13 of pregnancy?E13?,to establish a subclinical hypothyroidism L-T₄ E13 treatment model;?6?E17 treatment group:using the successfully established SCH rat model,the dose of L-T₄ solution was adjusted to 1.25?g/?100g?d?starting from day 17 of pregnancy?E17?to establish a subclinical hypothyroidism L-T₄ E17 treatment model;2.Serum thyroid stimulating hormone?TSH?and total thyroxine?TT₄?levels were measured before and after pregnancy.If the models were successfully built,the next experiment could be carried out.3.Western Blot was used to detect the expression of NGF,proNGF,NGF/TrkA and NGF/p75^NTR signaling pathway-related proteins in hippocampus of pups on postnatal day 7?PND7?and postnatal day 40?PND40?from each group.4.Immunofluorescence method was used to locate and detect the expression of Neuronal nuclear antigen?NeuN?,phospho-cAMP response element-binding protein?p-CREB?and cleaved cysteinyl aspartate specific proteinase-3?cleaved caspase-3?in CA1 region of hippocampus of pups on PND7 and PND40 from each group.5.Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling?TUNEL?and 4',6-diamidino-2-phenylindole?DAPI?were used to detect the level of neuronal apoptosis in hippocampal CA1 region of PND7 and PND40 pups.Results:1?Modeling of female rats in SCH group,OH group and three treatment groupsTwo thyroid function tests of female rats before and after pregnancy showed that compared with the CON group,the TSH levels of female rats in the SCH group and the OH group were significantly increased?p<0.05?,and the TT₄ levels of female rats in the OH group were significantly decreased?p<0.05?.There was no significant difference between the SCH group and the CON group in the levels of TT₄?p>0.05?.Before L-T₄ treatment,the levels of thyroid function in three treatment groups?E10group,E13 group and E17 group?were similar to those in the SCH group?p>0.05?.There were no significant differences between the three treatment groups?E10 group,E13 group and E17 group?and the CON group after L-T₄ treatment in the levels of thyroid function?p>0.05?.It is verified that the modeling of each group was successful,and the next experiment could be carried out.2.Expression of NGF,proNGF,NGF/TrkA and NGF/p75^NTR signaling pathway-related proteins in PND7 pups?1?Western experiments showed that in the hippocampus of PND7 pups,compared with the CON group,the expression of proNGF in the OH group and the SCH group was significantly increased,the expression of NGF in the OH group,the SCH group and the E17 group was significantly decreased,as well as the ratio of NGF/proNGF in the OH group,the SCH group,the E17 group and the E13 group was significantly decreased?p<0.05?.?2?Western experiments showed that in the hippocampus of PND7 pups,compared with the CON group,the expression of NGF/TrkA signaling pathway-related proteins?p-TrkA,p-ERK1/2,and p-CREB?in the OH group and the SCH group was significantly decreased?p<0.05?;the expression of p-TrkA and p-CREB in the E10 and E13 groups was not significantly different from those in the CON group?p>0.05?;the expression of p-ERK1/2 in the E10 group was not significant compared to the CON group;and there was no statistical difference in the total protein levels among the six groups?p>0.05?.Immunofluorescence staining was used to locate and detect the expression of p-CREB in the hippocampal CA1 region,and the results were consistent with the results of Western experiments.?3?Western experiments showed that in the hippocampus of PND7 pups,compared with the CON group,the expression of NGF/p75^NTR signaling pathway-related proteins(p75^NTR,p-JNK,p53,Bax,and cleaved caspase-3)in the OH group and the SCH group was significantly increased?p<0.05?;the expression of Bax and cleaved caspase-3 in the E10 group and the E13 group was not significantly different from those in the CON group?p>0.05?;the expression of p75^NTR in the E10 group was not significantly different from those in the CON group?p>0.05?;the expression levels of p53 and p-JNK in the E10 group,the E13 group and the E17 group was significantly higher than those in the CON group?p<0.05?;and there was no statistical difference in the total JNK among the six groups?p>0.05?.Immunofluorescence staining was used to locate and detect the expression of cleaved caspase-3 in the hippocampal CA1 region,and the results were consistent with the results of Western experiments.TUNEL experiments showed that compared with the CON group,the apoptosis of hippocampal neurons in the OH group,the SCH group,and the E17 group was significantly increased,and no significant changes were observed in the E13 group and E10 group.3.Expression of NGF,proNGF,NGF/TrkA,NGF/p75^NTR signaling pathway related proteins in PND40 pupsAll of the Western experiments,immunofluorescence staining,and TUNEL experiments showed that in the hippocampus of PND40 pups,compared with the CON group,the expression levels of NGF,proNGF,NGF/TrkA and NGF/p75^NTR signaling pathways-related proteins in the OH group,the SCH group,and the three treatment groups were not statistically different?p>0.05?.No significant changes were observed in hippocampal neurons apoptosis.Conclusion:1.Compared with the CON rats,maternal SCH inhibits the expression of NGF/TrkA signaling pathway,activates the expression of NGF/p75^NTR signaling pathway and disequilibrated the TrkA/p75^NTR signaling pathway on hippocampus of the offspring.This may lead to decrease the neural proliferation and increase the neural apoptosis on hippocampus of PND7 offspring,which affects the neurocognitive abilities of their offspring.2.L-T₄ treatment from early pregnancy could ameliorate the adverse effects of maternal SCH on neurodevelopment,and the optimal time of treatment should start before E10.3.The abnormal expression of NGF-related proteins in the hippocampus of offspring from maternal SCH may be transient and the influence could be recovered at PND40.