| BackgroundThe prevalence of dementia is increasing year by year. Nowadays, incidence of dementia has been up to5%-20%in individuals over the age of65. The high incidence and high morbidity of dementia have brought heavy financial and mental burden to our society and patient’s family, which has made dementia the21st century’s most serious social problem. But whether it is Alzheimer’s disease (AD) or vascular dementia (VD), the pathogenesis is not clear, which lead to the lack of effective means of prevention and treatment for dementia. Therefore, it is investigating the etiology and pathogenesis of dementia that has become the most important job in dementia studying.Recent clinical researches found abnormal levels of serum thyroid hormone (THs) and normal levels of thyroid stimulating hormone (TSH) in patients with vascular dementia. This changes of thyroid hormones level are consistent with the description for euthyroid sick syndrome (ESS). Euthyroid sick syndrome is a group of clinical syndromes which manifest as abnormal thyroid function in a patient with a severe systemic disease, after a major operation, applied to abrosia or on other conditions, while the thyroid gland itself in euthyroid sick syndrome don’t have pathological changes. People formerly thought that it’s a protection against the injury caused by stress response. However, more and more evidence show that it’s a manifestation of poor clinical prognosis. It was suggested that the euthyroid sick syndrome might be related to the dysfunction of hypothalamus-pituitary-thyroid (HPT) axis. Adverse factors cause functional and structural hypothalamus lesions, disturbing thyroid hormones’ synthesis and metabolic processes. Thyroid hormones can not achieve effective reverse feedback to increase the level of upstream hormones in HPT axis, resulting in hormone levels abnormity and metabolic disorder. In83subjects aged70and over, Ichibangase and his colleagues found that age and serum free T3concentrations had a significantly negative correlation in all subjects; the serum free T3concentrations were significantly lower in rebrovascular dementia than in those without dementia. These findings suggest that cognitive function is related to serum free T3so closely that serum free T3concentrations may be a good indicator reflecting health and cognitive status in patients with vascular dementia.Our previous study have achieved certain results. We prepared animal models of vascular cognitive dysfunction (VCI) induced by chronic cerebral ischemia through2VO methods. By measuring the concentration of thyroid hormones in brain tissue, we found that thyroxine (T4) and T3concentrations decreased at the same time of learning and memory function impairment appeared in2VO rats. Exogenous thyroid hormone significantly improved learning and memory in rats with chronic cerebral ischemia, suggesting that vascular cognitive impairment may be associated with abnormalities of thyroid hormone metabolism in central nervous system. However, the mechanism through which thyroid hormones affect the cognitive function after cerebral ischemia has not been reported. Therefore, it is necessary to clarify the role that thyroid hormones play in improving the vascular cognitive dysfunction, for that will theoretically support he prevention and control of vascular cognitive dysfunction. That will also provide a new possibility for the recovery of cognitive function in patients with dementia.In recent years, more in-depth studies on the pathogenesis of vascular cognitive dysfunction have been conducted. De Jong and his team found that rats still exist spatial learning and memory damage even in the recovery period of chronic cerebral ischemia (6months to1year after the2VO operation), suggesting that memory impairment after2VO is not only caused by the brain hypoperfusion, but also by certain permanent brain damage induced by ischemia. Imai found that neuronal death in CA1region of hippocampus happened on the3rd to7th days after ligation of bilateral carotid arteries; in the second week after the operation, neuronal death in CA1region was present in6%-29%of rats; and then the ratio was increased to55%in the fourth postoperative week. This confirms that2VO may cause neuronal damage in the hippocampus. Numerous studies have observed that chronic cerebral ischemia can result in neuronal apoptosis, which is an important pathogenesis of vascular cognitive dysfunction, by inflammatory response and oxidative stress through depolarization of the hippocampal neurons, imbalance of ion concentration between inside and outside of cells, change of cytoskeleton and membrane structure, dysfunction of mitochondria, toxicity of excitatory amino acid and so on. Neuroprotective agents can reduce stress reaction and calcium overload in ischemic tissues, showing a decrease in neuronal apoptosis in hippocampus and improvement on spatial learning and memory function. These results further prove that it is the neuronal apoptosis in hippocampus caused by chronic cerebral ischemia that lead to a decline in cognitive function in rats.It is well known that thyroid hormones can promote the proliferation and differentiation of progenitors, encourage the growth of dendrites and axons, and facilitates neuronal migration and myelination in embryonic brain tissue, being essential to the maturation of mammalian central nervous system. However, thyroid hormones play vital roles in central nervous system not only in embryos and young individuals, but also in adulthood and even older age. So we can say that thyroid hormones affect individuals throughout their lifetime. Thyroid hormones can regulate the effect of neurotransmitters such as acetylcholine and dopamine, regulate the expression of cytokines and activity of enzyme proteins, affect the energy metabolism of brain through regulating mitochondrial function, and modulate the expansion or contraction of intracranial vessels by controlling signal transduction system such as protein kinases. Hypothyroidism might cause mental and behavior abnormalities in one’s adulthood, while in elderly patients, it was more likely to cause dementia for cortical atrophy. Both of these evidence emphasizes the importance of thyroid hormones for normal brain function. It was reported that T3reduced the emission of lactate dehydrogenase (LDH) and the expression of caspase-3in myocardial tissue which experienced ischemia-reperfusion, suggesting that it can reduce tissue injury and apoptosis under the ischemia-reperfusion condition, In an animal model of ischaemic injury induced for2h by middle cerebral artery occlusion (MCAO) followed by24h reperfusion, intraperitoneal injection of T4at1h after the onset of ischemia and6h after reperfusion reduced the infarct volume significantly through supressing the activation of astrocytes and microglia, promoting the expression of brain-derived neurotrophic factor (BDNF) and glia-derived neurotrophic factor (GDNF), reducing the activity of prooxidative enzymes such as cyclooxygenase-2(COX-2) and inducible nitric oxide synthase (iNOS). Adding T3to culture medium made astrocytes outlive toxic concentrations of glutamate while increasing the survival rate neurons co-cultured with glial cells, suggesting that T3can also provide protection to cultured neurons. However, there haven’t been any evidence on whether thyroid hormones can protect hippocampal cells against chronic cerebral ischemia. Design experiments to find the answer will enable us to know more about the mechanism of thyroid hormones improving cognitive function of2VO rats and help us fix on the significance of hippocampal apoptosis in the pathogenesis of dementia.Studies have shown that the anti-apoptotic protein Bcl-2is a key regulator of programmed cell death in the central nervous system. It was found that a rat hypothyroidism model caused by propylthiouracil (PTU) present down-regulation of anti-apoptotic protein Bcl-2and upregulation of pro-apoptotic protein Bax while increased apoptosis in brain tissue. Thyroid hormones treatment increased the expression of Bcl-2in injured brain tissue and thereby reduced post-traumatic neuronal apoptosis. These results suggest us that thyroid hormone may control the apoptosis in ischemic hippocampus through regulating the expression of Bcl-2. But we still don’t know the effective time and specific pathway of the regulation. In summary, our preliminary studies have proved that rats with chronic cerebral ischemia present significantly lower levels of thyroid hormones in brain and cognitive impairment; exogenous thyroid hormones can improve their cognitive function. In order to further clarify the role of thyroid hormones in repairing vascular cognitive dysfunction and the mechanism, we propose a hypothesis:thyroid hormones improve impaired cognitive function in chronic cerebral ischemia by reducing hippocampal apoptosis; this kind of protection to cell is achieved by upregulating the expression of anti-apoptotic protein Bcl-2. To test this hypothesis, we intend to adopt the animal model of vascular cognitive impairment caused by chronic cerebral ischemia through permanent ligation of bilateral carotid arteries to adult rats. Thyroid hormones intervention group is also designed in the experiment. By Nissl staining, immunofluorescence staining and Western blotting (WB) technique, we suppose to observe the hippocampal apoptosis in different groups and clarify whether thyroid hormones affect cell apoptosis through regulating the expression of Bcl-2. We would like to understand the molecular mechanism of thyroid hormones improving the impaired cognitive function in chronic cerebral ischemia, supporting a new theoretical basis and therapeutic targets for the cognitive recovery in patients with vascular dementia.MethodsI. Experimental groups:50adult SPF male rats were randomly divided into chronic cerebral ischemia group (n=16), T3intervention postischemically group (n=18) and sham-operation group (n=16). Chronic cerebral ischemia group was randomly divided into2VO7days (n=8) and2VO14days (n=8). Similarly, postischemically T3intervention group were randomly divided into7day subgroup (n=10) and14day subgroup (n=8). Sham-operation group were randomly divided into7day subgroup (n=8) and14day subgroup (n=8). Five rats from each subgroup were picked up for tissue staining while the rest were used for Western Blotting (WB) detection. Ⅱ. Nissl staining:Sections underwent Nissl staining were observed14days after the operation to record the tissue structure of hippocampus and cell apoptosis in CA1region.Ⅲ. Immunofluorescence staining:TUNEL staining was carried out7days and14days after the operation. The number of apoptotic cells in dentate gyrus was counted and apoptotic index was calculated for each group.Ⅳ. Western Blotting:The expression of Bcl-2protein in hippocampus of three groups were observed7days and14days after the operation.Ⅴ. Statistical analysis:SPSS13.0was used for statistical analysis. Measurement data were expressed as mean±SEM. Data of three groups were compared with single factor analysis of variance, and the comparison between each two groups was performed with Bonferroni test. P values less than0.05were considered significant.Results:Ⅰ. On the14th day after the operation, sections of sham-operated rats present well-organized structure in hippocampal CA1region and a plenty of pyramid cells arranged in order. Compared with the sham-operation group, the structure of the CA1region after ischemia was significantly loose and messy while the cell layer collapsed and the decreased neurons arranged loosely. T3group present slight disorders in hippocampal structure compared with sham-operation group and remarkable integrity with2VO group, while the number of cells is roughly identical with the sham-operation group.Ⅱ. On the7th day after the operation, the results showed the successive order of apoptosis index of dentate gyrus was2VO group, T3treatment group and sham-operation group, but the difference was not statistically significant (P=0.060). Only a small amount of purplish apoptotic cells appeared in hippocampal dentate gyrus in sham-operation group on the postoperative14days. At the same time, a majority of cells underwent apoptosis in2VO group and nearly half of the cells died in T3treatment groups. By counting and calculating, we found a significant difference of apoptotic index in dentate gyrus among the three groups (P<0.001). Apoptotic index in2V0group was significantly higher than the sham-operation group (P<0.001), while T3treatment group showed lower index than2VO group significantly (P=0.001).III. The expression level of GAPDH was by and large consistent with each other group. On the7th day after the operation, the results showed a descending order of the expression level of Bcl-2protein was T3treatment group,2VO group and sham-operation group; on the14th day, the order was changed to T3treatment group, sham-operation group and2VO group.Conclusion:Thyroid hormones improve impaired cognitive function in chronic cerebral ischemia by reducing apoptosis in the ischemic dentate gyrus and CA1region in hippocampus. This effect is achieved by up-regulating the hippocampal expression of Bcl-2protein. |
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