Impacts Of Thyroxine And Donepezil On Brain Damage In Adult-onset Hypothyroidism

Background:It is likely that all cells in the body are targets for thyroid hormones,one of which are the neurons in the hippocampus. The hippocampus is an important region for memory and emotion in the central nervous system.Studies have revealed adult hypothyroidism could cause the damage of morphologies and functions in the hippocampus. The mechanism is not clear. It might be involved in the alteration of synaptic plasticity,which is a complex physiological process involved by a variety of synaptic proteins.Synaptotagmin-1(syt-1) and SNAP-25(synaptosome-associated protein of 25 k Da)are two important synaptic proteins, highly expressed inside the hippocampal neurons.It had been shown adult hypothyroidism in adult could lead to the expression changes of such synaptic proteins as syt-1 and SNAP-25. The T4 alternative therapy is a conventional means in treating hypothyroidism,which’s treatment standard is to recover the serum thyroid hormone level to normal, but it is still controversial whether it could fully recover the hypothyroidism caused brain damages. Our previous studies reported that after giving regular doses of T4, the serum T3 and T4 levels were returned to normal, but the changed expression of synaptic proteins such as syt-1 and SNAP-25 failed to fully recover, suggesting that it was necessary to look for other more effective ways to treat the hypothyroidism caused brain damage. Donepezil(DON) is a cholinesterase inhibitor, it is widely used for the treatment of mild tomoderate Alzheimer’s disease. It has been confirmed the neuroprotective effects for neurons and these effects are not dependent on acetylcholinesterase inhibition.So it might also have certain therapeutic value towards hypothyroidism caused hippocampal damage.Objective:In the present study, we investigated the ultrastructure of synapses in the hippocampus of adult hypothyroid rats, as well as the expression changes of syt-1 and SNAP-25,aiming to evaluate the impacts of combined T4+DON therapy towards the above outcome.Methods:75 healthy male SD rats, 3 months old, SPF grade, weighed 240-280 g,were randomly divided into five groups: the normal control group(CON), the hypothyroidism group(Hypo), the T4 treatment group(T4), the DON treatment group(DON) and the T4+DON combined treatment group(T4+DON). The CON group was fed with normal water, while the other four groups were given 0.05%(w/v) propylthiouracil(PTU)water every day. The total modeling time was 6 weeks. 4 weeks later the interventions were given. The T4 group was intraperitoneally injected T4(6 μg/100 g body weight),the DON group added 0.005% DON into the drinking water, and the T4+DON group was performed the above administration simultaneously. Meanwhile, the rest three groups(CON,Hypo,DON) were injected the same amount of saline as the alternative.The total treatment time was 2 weeks, and the dosage was adjusted according to the rats’ weights weighed weekly. After weighed the body weight, all the rats were anesthetized, the abdominal aortic blood and the hippocampal tissues were then sampled for the next experiments. The radioimmunoassay was performed to test the serum T3 and T4 levels with the radioimmunoassay kit. Technique of ElectronMicroscope(TEM) was used to observe the hippocampal ultrastructures of each group,Western blot and real-time RT-PCR were performed to analyze the protein and m RNA expressions of syt-1 and SNAP-25 in the hippocampus of each group.Results:1. Body weight changes:There were no significant differences in the body weight among the groups befor modeling(P>0.05). After modeling, the increasing of body weight in the control group was 87.9%, while Hypo group, T4 group,DON group and co-administration group was 36.7%, 59.4%, 33.2% and 58.5%,respectively. The body weights of the rats in other groups were significantly lower compared with the control group, especially in Hypo and DON groups(P < 0.01).2. The levels of serum thyroid hormones:The serum radioimmunoassay test revealed that compared with the CON group, the mean T3(0.23±0.04vs0.42±0.07nmol/L)and T4(22.84±2.50vs75.25±5.89nmol/L) levels of the Hypo group and were significantly lower(P<0.01); the mean T3(0.22±0.05vs0.42±0.07nmol/L)and T4(21.79±2.84vs75.25±5.89nmol/L) levels of the DON group were significantly lower,too. While those of the T4 group and the T4+DON group were close to the CON group, the difference was not statistically significant(P>0.05).3. The hippocampal ultrastructure of synapses:TEM revealed that the structures of presynaptic membrane, synaptic cleft and postsynaptic membrane of the CON group were clear, the specialized belt was obvious, the synaptic vesicles were rich,and exhibited clear and dense-core vesicles. While 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 above injuries in the T4 or DON group were improved, and the performance of the T4+DON group was the most close to the CON group.4. The expression level of syt-1 and SNAP-25:The results of Western blot analysisrevealed that the hippocampal syt-1 protein expression of the Hypo group was significantly reduced than the CON group by 52.3%(P<0.01), the expression of SNAP-25 protein of the Hypo group was significantly increased than the CON group by 57.6%(P<0.01). Real-time RT-PCR results showed that the m RNA level of syt-1 was significantly decreased by 54.2% in the Hypo group(P<0.01). As for SNAP-25, the m RNA levels were found increased in both the Hypo group and the DON group by 36.7% and 32.5%(P=0.025 and 0.029), respectively. The expressions were partially recovered after the T4 treatment, and the T4+DON combined treatment made the expression return to normal.Conclusion:Adult-onset hypothyroidism can cause the damage of hippocampal ultrastructure of synapses, the expression of synaptic protein syt-1 is downregulated, while that of SNAP-25 is upregulated, the T4+DON combined therapy can repair the above injuries,and the roles are better than the single drug treatment.Background:The brain is an important target organ for thyroid hormone(TH) action. It is well known that TH is crucial for the maturity and functional perfection of the brain in fetal and neonatal periods. Either scarce maternal iodine intake or TH deficiency during gestation are related to central nervous system alterations such as cretinism and mental retardation. Meanwhile, there is more and more evidences showed that thyroid hormone is also active in the adult brain, particularly in the anatomic regions that ubserve memory functions, such as the hippocampus. Hypothyroidism in adult rats alters cell number, structure, and functions in the hippocampus and other brain areas.In humans, adult-onset hypothyroidism interferes with a number of cognitive and affective functions, including memory, attention and concentration, language,executive function, and psychomotor speed,depression and anxiety,et al.Levothyroxine(L-T4) alternative therapy is a conventional means in treating hypothyroidism,its treatment objective is to recover the serum thyroid hormone and thyroid-stimulating hormone(TSH) level to normal,but it is still controversial whether patients treated with L-T4 continue to have cognitive or affective deficits. Some studies have shown that L-T4 alternative therapy could fully recover the hypothyroidism caused impairments of cognitive functions and mood status,but many investigators have found some L-T4 treated hypothyroid patients continue to complain of hypothyroid related symptoms such as fatigue, cognitive impairments and mooddisturbances,despite documentation of normal thyroid hormone and TSH levels.Objective:In the current study, we investigated whether L-T4 treated subjects have alterations in cognitive functions and mood status compared to matched healthy control subjects through cognitive and emotional scales tests, and further discussed the impact of L-T4 for hypothyroidism caused cognitive impairments and mood disturbances.Methods:Potential subjects were screened for general health, medicines, drug abuse, thyroid status by history, physical examination, and standard laboratory testing. Two groups of subjects were recruited for these studies:1. Treated hypothyroid group: 30 women(aged 20–45years) with primary hypothyroidism, receiving stable doses of L-T4 as the sole treatment for hypothyroidism for at least 3 months, with documented normal TSH levels during this time. 2. Control group: 30 women(aged 22–46years) with no history of thyroid disease, with no goiter on physical examination, receiving no thyroid hormone therapy,and with documented normal serum TSH and thyroid hormone levels.All subjects underwent the following validated measures:Mini Mental State Examination(MMSE)、Self-Rating Depression Scale(SDS)、Self-Rating Anxiety Scale(SAS). Compared the two groups of scores on MMSE、SDS and SAS.Results:Treated hypothyroid and control subjects were well matched for age, education,and BMI.Individual TSH levels were in the normal range for all hypothyroid an d control subjects. However, mean TSH level was higher in the treated hypothy roid subjects(2.81±0.42vs1.76±0.38 m IU/L, P<0.05).There were no significant di fferences of serum f T3,f T4 levels between two groups. There were much mor e anti-thyroid peroxidase antibodies(TPOAb) positive subjects in the treated hypothyroid group(23/30) than in the control group(1/30). L-T4 treated subjects show ed similar score on MMSE(28.88±1.02vs29.36±1.21, P>0.05),but higher score o n SDS(48.01±4.66vs32.18±2.83,P<0.01) and SAS(50.50±5.15vs30.36±3.72, P<0.01) compared to controls, differences were statistically significant.Conclusion:L-T4 treated hypothyroid subjects would still have disturbances in mood and have normal general recognitive function.Our findings suggest that hypothyroid subjects could require more precise dosing of L-T4 or combining with other drugs to optimize central nervous system functioning.