| Background and ObjectiveVascular dementia (VaD) is a cognitive dysfunction syndrome caused by cerebrovascular disease,constituting the second most frequent cause of dementia following Alzheimer’s disease (AD).Bowler et al.proposed the concept of vascular cognitive impairment (VCI) for the need of early diagnosis and preventive treatment to Slow down the development of Vascular dementia.Vascular cognitive impairment (VCI) is the modern term related to vascular burden of the brain,reflecting all encompassing effects of cerebrovascular disease (CVD) on cognition.VCI include all levels of cognitive decline from mild deficits in one or more cognitive domains to a broad dementia-like syndrome. With the rapid progress of China’s aging population, the incidence and prevalence of vascular dementia significantly increased, bringing a heavy burden to the patient’s family and the society.But so far, its pathogenesis is not so clear, and the lack of effective prevention and treatment methods urges even more active exploration and research on the pathogenesis of vascular dementia.The present studies on vascular dementia show that the drugs have no explicit treatment or prevention effect.In a small study of94subjects with recent lacunar stroke,3months of high-dose atorvastatin did not improve cerebral vasoreactivity.Similarly, the Aspirin and antihypertensive drugs does not have a significant role in retarding the progression of dementia compared to placebo.In recent decades the technology of cell replacement therapy based on the research of stem cells, comparing with drug therapy,has aroused widespread concerns.A recent research demonstrated that systemic administration of human cord blood-derived CD34+cells to immunocompromised mice subjected to stroke48hours earlier induces neovascularization in the ischemic zone and provides a favorable environment for neuronal regeneration,hence enhancing migration of neuronal progenitor cells to the damaged area, followed by their maturation and functional recovery. Stem cell-based approaches hold much promise as potential novel treatments to restore neurological function after stroke. Nevertheless, many questions raised by experimental studies particularly related to long-term safety and technical details of cell preparation and administration must be resolved before wider clinical use.In recent years, the domestic and international studies have shown that many factors that can affect the adult endogenous neural regeneration, including some growth factors and neurotransmitters, promoting the proliferation, migration, differentiation, and survival of endogenous neural progenitor cells. Therefore neurological dysfunctions due to cerebral ischemia and other damages can be improved, bringing hopes for future use of endogenous neural progenitor cells as cell replacement therapy, without considering the adverse factors of exogenous cell replacement therapy such as safety considerations and technical problems. The neurogenesis in the subgranular zone of the adult hippocampal dentate gyrus(SGZ) and the rostral subventricular zone(SVZ) after cerebral ischemia is the focus of research at home and abroad. The genes regulating the survival, proliferation and differentiation of neural progenitor cells are the core issue of the research in this field.Domestic and foreign researches reported that traditional Chinese medicine decoction (ginseng Yang Rong Tang),1-3-n-Butylphthalide and Z-Ligustilide can promote the neurogensis in the subgranular zone of the adult hippocampal dentate gyrus, significantly shortening the latency of2VO rat of the chronic cerebral ischemia model in the performance of Morris water maze test, thereby improving cognitive dysfunction induced by chronic cerebral ischemia.Although the neural progenitor cells can migrate from the subgranular zone of the adult hippocampal dentate gyrus towards the granule cell layer, but the new neurons in the hippocampus reach their final position very early. The maximum distance the new cells migrate lies within a range of50to100μm. However, many of them do not seem to migrate at all and remain in the SGZ.Previous studies have shown that in normal rodent brain, many of the new-born neurons of the rostral subventricular zone undergone apoptosis.The surviving new neurons migrate along the rostral migratory stream (RMS) to reach the olfactory bulb, differenciating and maturing into periglomerular neurons and GABAergic granule cells. But in the stimulation of cerebral ischemia and other damage, neural progenitor cells in the subventricular zone increased significantly, and could migrate to the site of injury, and differentiated into the specific neurons of the site of injury.However, the micro-environment changes caused by cerebral ischemia and other damage made the growth of the morphology of the new-born neurons abnormal, and consequently the new-born neurons could not be effectively integrated into the neural circuits of the site of injury involved in the functional repair.But this abnormal could be recovered by FGF-2intervention, suggesting that the functional defects in new-born neurons after cerebral ischemia could be restored to the normal level by the intervention of the exogenous factors,thus playing a functional role in improving neurological dysfunction caused by cerebral ischemia.So it is an urgent need to find effective means to promote the neural progenitor cells to differentiate into neurons, to improve the survival rate, to promote the migration of new neurons to the site of injury. Thus the chance of the use of endogenous neural progenitor cells as cell replacement therapy are increasing, effectively improving neurological dysfunction caused by the conditions.Serum levels of triiodothyronine (T3) and free triiodothyronine (fT3) in patients with vascular dementia (VaD) was significantly lower than patients without dementia. And even between different degrees of vascular dementia,the concentrations of the serum T3and fT3also had significant differences. As compared to patients with Alzheimer’s disease (AD),the serum fT3levels significantly decreased among VaD patients. Patients with vascular cognitive impairment without dementia (VCIND) had lower tetraiodothyronine (T4) and free tetraiodothyronine (fT4) levels than patients with cerebrovascular disease (CVD).And the serum T4and fT4levels of CVD patients was significantly lower than the normal control group with cerebrovascular disease risk factors.The above results showed that the abnormal serum thyroid hormone levels was closely related to the progressive development process of vascular dementia, and may occur in the early stages of cerebrovascular disease without cognitive dysfunction. To strengthen further research on the relationship between thyroid hormone and vascular dementia and the mechanisms invovled is of great significance, and may provide clues for early diagnosis and preventive treatment of vascular dementia in the future.Thyroid hormones (THs) have vital effect on the development of the mammalian central nervous system, exerting great impact on the proliferation, differentiation of the embryonic brain neurons and dendritic growth as well as the process of myelination. In recent years, there is an increasing concerns about the impact of THs on the adult brain.Studied found that the abnormal levels of THs severely affect the proliferation, migration and differentiation process of neural progenitor cells in the subgranular zone of the adult hippocampal dentate gyrus(SGZ) and the rostral subventricular zone(SVZ) of the adult brain, supporting a role for thyroid hormone in the regulation of adult hippocampal neurogenesis and raising the possibility that altered neurogenesis may contribute to the cognitive and behavioral deficits associated with adult-onset hypothyroidism.A number of studies found, whether in the embryonic and adult brain, deficiency in thyroxine can affect all the aspects of proliferation, differentiation, migration and apoptosis of neurons, impair the processes of the axon dendritic growth,synapse formation and myelination development,using the model of gene-knockout and MMI /PTU hypothyroidism, while restoring the thyroid hormone state can reverse the negative role of thyroid hormone deficiency.But the effect of thyroid hormones (THs) on the neurogenesis in the SVZ after cerebral ischemia is rarely reported.Our preliminary study found that T3and T4concentrations decreased significantly in the2VO rat model of chronic cerebral ischemia, and the exogenous supplement of thyroxine can significantly improve T3and T4levels in the brain tissue,significantly shorten the latency of2VO rat in the performance of Morris water maze test as well as improve the cognitive function.And a further study showed that when the2VO rats were injected with T3intraperitoneally for once at the seventh day, BrdU-positive cells in the subventricular zone significantly increased, suggesting that T3can promote the proliferation of the neural progenitor cells in SVZ. However the long-term effects of T3on the neurogensis in the SVZ of the2VO rat is completely unclear. So we are aiming to observe the long effect of T3on the SVZ neurogensis.The procedure of permanent bilateral occlusion of the common carotid arteries was performed to establish a chronic cerebral hypoperfusion condition.Then5-Bromo-2’-deoxyuridine(BrdU) was injected intraperitoneally for7days to label proliferating cells. The rats were sacrificed after T3treatment for7days and14days. Transcardiac perfusion was performed in five rats in each group for immunostaining to determine the number of BrdU-or DCX-positive cells. The tissues of SVZ were dissected from three rats to test the expression of DCX using western blotting.Methods1. Experimental groups:Sprague-Dawley (SD) rats were randomly divided into sham group,2VO and2VO+T3intervention groups(n=8in each group). The procedure of permanent bilateral occlusion of the common carotid arteries was performed to establish a chronic cerebral hypoperfusion condition. Sham group experienced the same surgical procedure except no ligation of the common carotid arteries.The rats in postischemic T3intervention group were injected with T3(10ug/30g Body Weight) intraperitoneally for once daily until killed. The rats in sham group and2VO group were injected with0.9%saline intraperitoneally for once daily until killed.All rats were was injected with5-Bromo-2’-deoxyuridine(BrdU) intraperitoneally for twice daily (50mg/kg BodyWeight) for7days to label proliferating cells.2. Transcardiac perfusion was performed in five rats in each group for immunostaining to determine the number of BrdU-or DCX-positive cells.3. The tissues of SVZ were dissected from three rats to test the expression of DCX using western blotting.4. The positive cells of the immunofluorescence staining were counted by an observer blinded to the experimental group. One slice of every8slices was selected for immunofluorescence staining.A total of six slices of each rat was counted and the total cell count multiplied by eight was considered as the final number of cells used for statistical analyses. Experimental data are expressed as mean±standard deviation (x±s), using SPSS13.0statistical software to test homogeneity of variance and one-way analysis of variance (ANOVA) followed by the LSD post-hoc test is used, with significance determined at P values<0.05.Results1. After T3was delivered for7days, immunofluorescence double staining showed that BrdU, DCX-positive cells is distributed along the SVZ. Some of BrdU-positive cells did not express DCX. BrdU-/DCX-positive cells were enhanced in T3intervention group in comparison with2VO group and sham group. The expression of DCX was also higher in T3intervention group when tested by western blotting.2. After T3was delivered for14days, immunofluorescence double staining showed that BrdU-/DCX-positive cells were also enhanced in T3intervention group in comparison with2VO group and sham group. But BrdU-or BrdU-/DCX-positive cells in2VO14days group diminished remarkably. The western blotting results showed a similar tendency.3.In order to further quantify the differences among the groups, we counted immunofluorescence staining positive cells.The results showed that after T3was delivered for7days, BrdU-positive cells were dramatically higher than those in sham group(P=0.002) and had no significant differences comparing to2VO group(P=0.084). Strikingly,T3intervention group had even more BrdU-positive cells comparing with2VO group and sham group when T3administration14days(P<0.01). In contrast to2VO7-day group, the numbers of BrdU-positive cells in2VO14-day group diminished to58.2%, showing significant difference between groups (P=0.013). And there was no significant difference between T37-day group and T314-day group.4. The number of BrdU-/DCX-positive cells was1502.4±252.6,2636.8±364.6,4316.8±529.9in sham group,2VO7-day group and T37-day group respectively,showing significant differences between groups (F=12.60, P=0.001)And BrdU-/DCX-positive cells were markedly enhanced in T37-day group in comparison with2VO7-day group and sham group(P=0.012,P<0.001). T3intervention group had even more BrdU-/DCX-positive cells comparing with2VO group and sham group when T3administration14days (P<0.001). In contrast to2VO7-day group, the numbers of BrdU-/DCX-positive cells in2VO14-day group diminished to54.0%, showing significant difference between groups(P=0.008). And there was no significant difference between T37-day group and T314-day group.Conclusion1. In this experiment, after T3was delivered for7days, BrdU-/DCX-positive cells in SVZ significantly increased compared with the sham group and2VO7-day group.Although cerebral ischemic injury stimulated BrdU-positive cells and BrdU-/DCX-positive cells,however, this compensatory role showed no significant difference comparing to the sham group, suggesting that the exogenous supplement thyroid hormone can further promote the proliferation of neurogenesis in SVZ after cerebral ischemia.2. When T3was delivered for14days, BrdU-positive cells and BrdU-/DCX-positive cells in2VO group significantly reduced compared with T314-day group and2VO7-day group.And there was no significant difference between T314-day group and T37-day group. These results suggest that T3can significantly improve the survival rate of BrdU-positive cells and BrdU-/DCX-positive cells after chronic cerebral ischemia, which increases the possibility of cell replacement therapy using the endogenous neural progenitor cells, to create opportunities for further functional repair after cerebral ischemic injury. |
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