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研究生(外文):Yang-hui Huang
論文名稱(外文):The effect of G-CSF in an animal model of epilepsy
指導教授(外文):Chao-lin LiuWang-tso Lee
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顳葉癲癇(Temporal lobe epilepsy, TLE)為常見的腦部疾病之ㄧ,其不定期的發作會造成病人生活上的不便。紅藻氨酸(kainite; KA)為麩氨酸(glutamate)的類似物,常被用來誘發癲癇的動物模式,其主要導致海馬迴(hippocampus)的損傷尤其是在CA1及CA3的區域。本研究主要利用KA誘發TLE的大鼠動物模式,來探討KA刺激幹細胞增生的時間點以及顆粒球生長激素(Granulocyte colony-stimulating factor; G-CSF)對於KA所誘發TLE動物模式的治療效果及其可能的機制。
我們發現KA可造成海馬迴CA3區域的損傷,並且刺激海馬迴CA3及CA1區域的神經膠細胞增生。利用腹腔注射KA 120分鐘後有68% 的大鼠會達到第六階段的抽慉。我們同時利用5-溴2-脫氧尿嘧啶核苷(5-bromo-2’deoxyurdine; BrdU)來標定新生的幹細胞,結果發現KA所刺激的幹細胞增生能夠持續至少三天,第五天在統計上雖然無顯著增加之結果,但仍可觀察到幹細胞的增生的趨勢。另外,我們利用BrdU及膠纖維酸性蛋白(glial fibrillary acidic protein; GFAP)交叉免疫染色發現,KA所刺激產生的新生幹細胞只有4.82%分化為神經膠細胞,所以KA所造成的神經膠細胞增生並非主要由新生的幹細胞分化而來。此外,我們利用被動閃避學習系統(passive avoidance test)證實,KA會導致動物海馬迴的損傷而造成記憶能力的喪失。為了更進一步尋求治療抽慉所致海馬迴傷害的方法,我們更進一步利用G-CSF刺激TLE動物的幹細胞增生,我們發現經由G-CSF治療過後的TLE動物,其海馬迴CA1以及CA3的周邊會出現大量新生的幹細胞,並且利用被動閃避學習系統證實G-CSF的確能夠改善TLE動物的記憶能力。
Background: Most of the current clinical treatments for temporal lobe epilepsy (TLE) are largely symptomatic and was usually accompanied with serious adverse effects. In the present study, we investigated the possible neurogenesis in an animal model of kainic acid (KA)-induced seizures, and searched for potential treatment for KA-induced hippocampal damage.
Methods and Results: Administration of KA, an analogue of the excitatory amino acid glutamate, can induce seizures in rats, leading to hippocampal lesions closely resembling human TLE. In the current study, we used KA to induce seizures, which resulted in hippocampal lesions, especially in CA3 region. It showed that GFAP positive cells, representative for glia cells, were also increased in CA3 and CA1 areas at one week after KA injection. Additionally, KA-induced seizures were followed by the increase of BrdU-positive cells, which persisted for at least 3 days in both hippocampal CA1 and CA3 regions. However, only 5 % of BrdU-positive cells appeared to be GFAP positive, indicating that only few proliferating stem cells were differentiated into glia cells. Passive avoidance test was used to evaluate the behavioral changes in rats, and it revealed impaired memory in KA-treated rats. When G-CSF was supplemented to these rats, the number of newborn stem cells was relatively increased in peri-CA1 and CA3 areas, and the number of apoptotic neurons in hippocampus at one week post KA administration was attenuated. Furthermore, the memory function of rats treated with G-CSF was also improved.
Conclusion: KA is able to induce neuronal death in hippocampal area and behavioral change in rats. There was also significant neurogenesis persisted for at least 3 days in hippocampal area. G-CSF therapeutic approach, which is able to attenuate the neuronal death and increase the number of stem cells in hippocampus, can be of great potential in the future to treat patients with temporal lobe epilepsy.
中文摘要 v
Abstract vii
I. Introduction 1
The Pathogenesis of Epilepsy 1
Hippocampus and Related Pathways 2
KA as One of the Animal Models of Epilepsy 3
Neural Stem Cells and Their Applications 6
II. Experiment aim 10
III. Experiment flowchart 11
IV. Experiment methods 12
4.1 animals 12
4.2 reagents preparation 12
4.2.1 8% paraformaldehyde 12
4.2.2 0.2M pasphate buffer, pH7.4 12
4.2.3 4% paraformaldehyde in 100mM phosphate buffer 12
4.2.4 BrdU 12
4.2.5 Diethypyrocarbonate (DEPC) water 12
4.2.6 antifreezer 12
4.2.7 blocking reagent 12
4.3 TLE rat model 13
4.4 Onset of KA-induced neurogenesis 13
4.5 Tissue processing 13
4.6 BrdU and Hochest/GFAP double immunohistochemistry 13
4.7 Hematoxylin and Eosin (H&E) Immunohistochemistry 14
4.8 G-CSF treatment 14
4.9 Passive avoidance test 14
V. Expriment results 16
5.1 KA induced the TLE rat model 16
5.2 Effects of KA on hippocampal neuronal degeneration 17
5.3 KA-induced cell proliferation and gliosis induced by KA 17
5.4 Cell differentiation of KA-induced new born cell 18
5.5 G-CSF stimulated stem cell mobilization and homing in the brains of TLE rat model 18
VI.Discussion 20
Seizure induced stem cell proliferation 20
Newborn stem cell differentiation 20
G-CSF therapy in brain injury animal model 21
VII.Figures 23
VIII. Tables 45
IX. Reference 48
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