| Objective Adult-onset hypothyroidism is a common endocrine disease that can cause cognitive impairments,synaptic plasticity may be involved in the neural mechanisms of it.A growing body of studies have suggested that T4 monotherapy may not fully restore adult-onset hypothyroidism induced impairments.This study was to investigate the effects of T4 treatment alone and T4 plus donepezil(DON)on synaptic plasticity in the hippocampus and prefrontal cortex(PFC)of adult hypothyroid rats.MethodsSixty healthy male SD rats,were randomly divided into five groups,namely the control group,the hypothyroid group,the DON treatment group,the T4 treatment group and the T4 combined DON treatment group.A hypothyroid rat model was induced by adding the anti thyroid drug propylthiouracil(with a concentration of 0.05%)to their drinking water,for a tolal time of six weeks.From the fifth week,the T4 group was injected with T4(6 ?g/100 g body weight)intraperitoneally,0.005%DON was added into the drinking water in the DON group,T4 and DON was performed as above simultaneously in the combined treatment group.The levels of serum T3,T4 and TSH were measured by radioimmunoassay.Concentration of hippocampal ACh and the activity of AChE were determined colorimetrinally.Levels of hippocampal Syt-1 and SNAP-25 were determined by immunohistochemistry.Transmission electron microscope(TEM)was used to observe the prefrontal ultrastructures of each group.Acetylcholine content in the PFC was measured by the modified method of Hestrin.Results(1)Body weight:There were no significant differences in body weight of rats among the five groups before experiment(P>0.05);after the experiment,the body weight in the control group,hypothyroid group,donepezil group,thyroxine treatment group and the combined treatment group increased by 49.29%,21.28%,19.53%,31.90%and 32.44%respectively.The weights of SD rats in the other groups were significantly lower than that of the control group after experiment(P<0.05).(2)Serum concentrations of the hormones:The serum T3 and T4 levels were significantly lower(P<0.001)and TSH levels were higher(P<0.001)in SD rats of Hypo and DON groups than those in the controls.Both T4 and T4 plus DON treatment restored T3,T4 and TSH levels,which were not significantly different from the control values(P>0.05).(3)ACh levels in the hippocampus:The ACh was significantly decreased by 26.98%in the hypothyroid rats(P=0.027),and the content was found to restore to control values(P=0.212,0.860 and 0.255,respectively)by DON,T4 or T4 plus DON treatment.Comparng with the Hypo group,the ACh content of the DON treatment group increased by 16.58%,but the difference was not statistically significant(P=0.321;the content of ACh in the T4 and T4 plus DON groups increased by 34.07%and 54.92%,respectively,and the differences were statistically significant(P=0.046,P=0.002).(4)AChE activity in the hippocampus:The AChE activity were significantly decreased by 34.35,40.13 and 39.12%(P= 0.028,0.014 and 0.016,respectively)in the Hypo,DON and T4 plus DON groups comparing to rats in the control group,and the AChE activity was found to restore to control value by T4 administration(P>0.05).(5)Protein Levels of Syt-1 in hippocampus:The immunoreactivity distributions of Syt-1 were similar among these groups.Punctate spots of reaction product distributed in every layer of CA1,CA3 and DG subregions.The OD values of five layers of CA1,CA3 and DG subfields in the Hypo,DON and T4 groups were significantly lower compared to the corresponding layers in the control group(P<0.05).i.e.CA1-SO,CA1-SR,CA3-SL,CA3-SR,and DG-ML.In the T4 plus DON group,the OD values in all layers were similar to those in the C group.Comparing with the Hypo group,the Syt-1 immunoreactive products OD values in the above mentioned layers of the DON group are higher than that of the Hypo group,but there were no significant differences(P>0.05);the OD values in the hippocampal CA1-SO,CA1-SR,CA3-SL,CA3-SR of the T4 treatment group were significantly higher than those in Hypo group,the differences were statistically significant(P<0.05);and the OD values in the hippocampal CA1-SO,CA1-SR,CA3-SL,CA3-SR and DG-ML of the T4 plus DON treatment group were significantly higher than that of the corresponding layers of the Hypo group,the differences were statistically significant(P<0.01).(6)Protein Levels of SNAP-25 in hippocampus:The OD values of SNAP-25 in CA3-SO,CA3-SR and in all layers of CA1 and DG were significantly higher in Hypo and DON groups compared to the corresponding layers in the control group(P<0.01).In the T4 group,the OD values were significantly higher compared to the control group in all layers of CA1 and CA3-SO(P<0.01).In the T4 plus DON group,the OD values in all layers were similar to those in the control group(P>0.05).Comparing with the Hypo group,the SNAP-25 immunoreactive products of OD values in the above mentioned layers of the DON group are lower than that of the Hypo group,but there were no significant differences(P>0.05);the OD values in the hippocampal CA3-SR and DG-ML,DG-PL of the T4 treatment group were significantly lower than those in Hypo group,the differences were statistically significant(P<0.05);and the OD values in the hippocampal CA1-SO,CA1-SR,CA1-SLM,CA3-SO,CA3-SR and DG-ML,DG-PL of the T4 plus DON treatment group were significantly lower than that of the corresponding layers of the Hypo group,the differences were statistically significant(P<0.05).(7)Prefrontal ultrastructures:TEM revealed that in the hypothyroid rats,the neurons displayed a scanty cytoplasm with few organelles,the mitochondria were characterized by highly fractured and degenerated cristae and a clear vacuolation,the endoplasmic reticulum(ER)was greatly dilated.The presynaptic membrane and the postsynaptic membrane of the Hypo group were fused,the synaptic vesicles were significantly reduced,and almost no clear-type synaptic vesicles could be seen.The morphologic changes of myelin sheath in the hypothroid rats showed delamination and broken,with thinner thickness and irregular outline.The above injuries in the T4 or DON group were improved,exhibited mild disruptions,the prefrontal ultrastructures of the T4 plus DON group was the most close to the control group.(8)Content of ACh in the rat PFC:Hypothyroidism decreased neurotransmitter ACh content by 29.8%in the PFC of adult rats,as compared with the rats in the control group(P=0.030).The content of ACh could be restored to control levels(P=0.237,0.291 and 0.524,respectively)by administration of DON,T4 or T4 plus DON.Comparng with the Hypo group,the ACh content of the DON,T4 and T4 plus DON groups increased by 20.98%,22.26%and 30.95%,respectively,the differences were not statistically significant(P=0.274,0.946 and 0.581,respectively).Conclusions1.Adult-onset hypothyroidism induces alterations of ACh level and AChE activity in the hippocampus,as well as decreased ACh content in the PFC.In the hippocampus and the PFC,both ACh levels and AChE activity can be restored by T4 administration.2.Adult-onset hypothyroidism induces marked ultrastructural changes,including the neurons,the synapses and the myelin sheath in the PFC.These alterations are reversible by T4 administration,while the T4 combined with DON therapy is more effective than the single drug alone.3.In the hippocampus,the adult-onset hypothyroidism induces expression alterations of both Syt-1 and SNAP-25,which can not be fully restored by T4 administration.Significantly,co-administration of T4 and DON lead to normalization of the levels. |
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