| Background:Vascular dementia (VaD) is one of the major types of senile dementia, also the most common cause of dementia, which brought eavy burden of financial and spiritual to the family and society. VaD has become an important research focus in elderly medical field. Recently, vascular cognitive impairment(VCI) has been paid more and more attention by researchers and VaD is one of the types of VCI. Researchs show that the possible mechanisms of VCI are excitatory amino acid toxicity, energy failure, inflammation, nerve cell necrosis, loss and neuronal apoptosis, but the exact pathogenesis is not very clear, so lack the effective prevention and treatment measures. Therefore, actively explore and study the etiology and pathogenesis of dementia has become a top priority.The protophase work done by our colleagues showed that rats with merely chronic cerebral ischemia showed central thyroid dysfunction and cognitive impairment. But when the rats were supplied exogenous thyroxin in the begin of experiment, their water maze results were normal, which suggested that study and memory impairment of rats with chronic cerebral ischemia were associated with thyroid dysfunction in brain. However, the mechanism of thyroid hormone affect brain cognitive function after ischemia has not been reported, so further research is necessary to clarify the role of thyroid hormone in VCI. The dogma that neurons is not production in adult animals brain has been over turned. The study on neural stem cells (NSCs) is becoming a hot field of neuroscience abroad and have achieved encouraging results. Adult neurogenesis has been consistently observed in two regions of the adult brain:the subgranular zone (SGZ) of the dentate gyrus (DG) in the hippocampus and the subventricular zone (SVZ) of the lateral ventricle. Neurons born in the SGZ differentiate and integrate into the local neural network as granule cells of the dentate gyrus. Neurons born in the SVZ migrate through the rostral migratory stream and become granule neurons and periglomerular neurons of the olfactory bulb. The present study on NSCs after ischemia mainly in two ways:NSC transplantation and endogenous NSC research. In recent years, The experimental studies of NSCs transplantation for treatment brain injury has achieved good results, but may occur the hybrid cells phenomenon during transplantation and cause tumors or other issues after transplantation, so there is some distance from clinical application.Although there is only a low level neurogenesis in normal adult animals brain, but precursor cells may be actived to proliferate and differentiate into neurons and glial cells by ischemia and neural factors, repair damaged nerve function. Recently, scientists interested in the role of neurogenesis to improve cognitive function after cerebral ischemia. The generation of newborn neurons within the adult hippocampus has been suggested to play an important role in hippocampal-dependent learning and memory. Several studies found that the proliferation of neural progenitor cells increased 10 times in SGZ after cerebral ischemia. More studies show that the proliferation of progenitor cells beginning in 3-4 days after ischemia, reaching a peak in 7-10 and then returning to normal levels within 3-5 weeks after ischemia, which is essential for the recovery of cognitive function. However, the nerve regeneration of ischemic-induced is a compensatory mechanism of brain, which is not enough, so the further explore on the regulatory mechanism of neurogenesis is need to provide a new target for treatment in cognitive function.Thyroid hormone plays a critical role in neurodevelopment. In the developing nervous system, thyroid hormone exerts significant influences on both neuronal and glial progenitors, regulating their proliferation, survival and differentiation. Deficiency of thyroid hormone during critical periods of brain development is associated with profound, and often irreversible, morphological defects that contribute to severe cognitive and neurological impairment. Although the developmental effects of thyroid hormone have been well established, its influence on the adult brain is relatively poorly understood. The latest data show that THs regulates neurogenesis of the adult mammalian brain. Hypothyroidism decrease the survival and differentiation of adult dentate granule cell progenitors, these decreased could be rescued by restored euthyroid status, as well as reduce the mitotic activity of adult SVZ progenitor cells in hypothyroidism animals, but the rapid treatment with T3 fully restored the number of progenitor cells, suggesting a direct effect of THs on. proliferation. Therefore, thyroid hormone can regulate the endogenous neurogenesis of adult animal.In summary:the cognitive dysfunction after chronic cerebral ischemia cuold be restored through neurogenesis of hippocampus. However the nerve regeneration of ischemic-induced is a compensatory mechanism of brain, which is not enough. The thyroid hormones can enhance endogenous neurogensis in adult animals, but it is unknow whether thyroid hormone could affect the neurogenesis after chronic cerebral ischemia.Objective:To clarify the mechanism of thyroid hormone improve vascular cognitive impairment, combined with our previous work and literature datas, we propose the hypothesis:Thyroid hormones improve vascular cognitive impairment may through increase neurogenesis of hippocampal SGZ progenitor cells. Therefor, the aim of our study is to investigated the effects of thyroid hormone on proliferation of stem cells in the SGZ. We get vascular cognitive impairment animal model by permanent occlusion of bilateral common carotid arteries, set long-term and immediate intervention of thyroid hormone treatment, and observe the effect of thyroid hormone on proliferation of adult dentate granule cell progenitors after chronic cerebral ischemia by immunofluorescence staining method. Methods:1. Adult male Sprague-Dawley (SD) rats (weighing 250-300 g, n=30) were randomly divided into 2 groups:Triiodothyronine (T3) continuous treatment group (n=15) and short treament group (n=15). Each group was randomly divided into 3 subgroups:sham group (n=5), chronic cerebral ischemia group (n=5) and T3 treatment after chronic cerebral ischemia group (n=5). Adult rats were housed at 22-23℃under a 12:12-h light dark cycle and had access to food and water ad libitum.2. Prior to surgery, rats were fasted 24h, allowde to water. The animals were anaesthetized with 10% chloral hydrate (4ml/kg, IP). The common carotid arteries were exposed via amidline ventral cervical incision, carefully separated from their sheaths and vagal nerves, and permanently doubly ligated with silk below the origin of the external carotid artery. The incision was then sutured. The same procedure was performed on the sham group but without actual ligation.3. T3 continuous intervention groups:Animals received intraperitoneally T3 (10μg/30g) or vehicle daily for 7 days. All animals in the proliferation experiments received intraperitoneal 5-bromo-2'-deoxyuridine (BrdU,50 mg/kg) twice daily for 5 days and were sacrificed 24h following the last BrdU injection. In T3 short treament group:Animals received a single T3 (10μg/30g) or vehicle injection on 7d after surgey. All animals received a single BrdU (150mg/kg) injection 8h following the T3 injection and were sacrificed 2 h later.4. Animals were deeply anesthetized with 10% chloral hydrate and transcardially perfused with 0.9% saline solution, followed by 4% paraformaldehyde. The brains were then harvested and kept in the same fixative overnight at 4℃. Then the brains were transferred in 15% sucrose at the next day and later cryoprotected in 30% sucrose until they sunk. Frozen the brains with liquid nitrogen and saved.5. Sections were denatured in 2 N HCl for 30 min at 37℃, then the sections were rinsed in 0.1 M boric acid for 10 min, blocked in 0.3% Triton X-100 for 30 min and in sheep serum for 1h at 37℃. Afterwards the sections were incubated in primary antibody anti-BrdU (1:100) overnight at 4℃and in the secondary antibody for 2h at 37℃. Glycerol were mounted. Fluorescence microscope image acquisition.6. Quantitation was done by an experimenter blind. BrdU-positive cells within dentate gyrus were counted as being in the SGZ and hilar (10 sections/animal) using a 200×objective. BrdU-positive cells in SVZ was counted by an experimenter blind (5 sections/animal) using a 40×objective.7. All the numerical data have been statistically analyzed by SPSS 13.0 for windows statistical package.All data were presented as mean±standard deviation. Differences between groups were analyzed by using One-way analysis of variance (ANOVA), and multiple comparisons were analyzed by LSD method when P values less than 0.05. Welch method was used when equal variances not assumed, and multiple comparisons was analyzed by Dunnett's T3 method. P<0.05 was regarded as statistical significance.Result:1. Immunofluorescence staining showed that the BrdU-positive cells distributed in the dentate gyrus of hippocampus every group, the most were seen in the granule cells underlying. Celll nuclear shapes and sizes were round or oval, some clustered distribution and some single distribution. There are also visible distribution of BrdU-positive cells in SVZ of the dorsolateral nucleus.2. There was an increase in BrdU-positive cells of SGZ in T3 continuous treatment group compared to the ischemic group (p=0.005) and sham-operated group (p<0.001) at 7d after surgey. There was also a slight increase in the number of neuroblasts in the ischemic SGZ compared to sham-operated group (p=0.047).3. Quantification of BrdU-incorporation at day 7 postsurgery revealed a strong inseased precursor cell proliferation of SGZ in rats with T3 injection on 10h before death compared to the ischemic group (p=0.015) and sham-operated group (p<0.001). After ischemia, a slight insease in ischemia group than sham-operated group (p=0.007).4. In addition to effect the proliferation of hippocampal neural progenitor cells, the short T3 treatment also increased BrdU incorporation after a 2h BrdU pulse in SVZ. at day 7 postsurgery compared with ischemia group (p<0.001) or sham group (p<0.001).Conclusion1. Rats treatd with T3 for 7d or 10h all increased the proliferation of adult hippocampal dentate granule cell progenitors after chronic cerebral ischemia, and a slight increase compared to proliferation stimulated by ischemia alone.2. The 10h treatment with T3 increased the proliferation of progenitors in SGZ after chronic cerebral ischemia suggest a direct action of T3 on stem cell activity.3. The 10h treatment with T3 increased the proliferation of progenitors in SVZ after chronic cerebral ischemia. |
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