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研究生：	陳昊 Chen, Hao
論文名稱：	壓力和缺乏心理層面的控制感可影響血腦障壁的完整性、海馬迴中粒線體細胞的自噬以及誘發憂鬱傾向的行為 Stress and lack of psychological aspect of control may induce depression-like behavior through its impact to the blood-brain barrier integrity and autophagy activity in the hippocampus
指導教授：	游一龍 Yu, Lung
學位類別：	碩士 Master
系所名稱：	醫學院 - 生理學研究所 Department of Physiology
論文出版年：	2020
畢業學年度：	108
語文別：	英文
論文頁數：	33
中文關鍵詞：	心理控制、血腦屏障、自噬、情緒、髓鞘形成
外文關鍵詞：	psychological control, blood-brain-barrier, autophagy, emotional
相關次數：	點閱：93 下載：4
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長期受到壓力源的影響可能會對認知功能和情緒造成損害，而更有文獻提出在面對壓力時，缺乏心理層面的控制感更會對其生理造成嚴重的影響。然而壓力是透過怎樣的機制去損害認知和情緒仍然是未知的。在這項研究中，我們利用給予腳部電擊的方式來模擬8週大的小鼠受到壓力的情形，並透過觀察工具行為的方式，來建立心理層面控制感的有無。在連續10天腳部電擊的刺激中，成對的老鼠每天會受到每小時60次隨機的電刺激，並且每次持續至7秒。在電擊過程中，執行鼠若做出工具行為則能提早停止電擊時間，而共軛鼠則受到與執行鼠相同的電擊時間，但其行為無法提早停止電擊。另外未受到電擊刺激的組別則作為對照組。在10天的電擊刺激結束後，利用強迫游泳實驗發現共軛鼠有類似憂鬱的情形，而透過頸靜脈注射伊文思藍評估血腦障壁的完整性發現共軛鼠與執行鼠相比其血腦障壁完整性有下降的趨勢。同樣的利用西方墨點法發現形成血腦障壁相關的緊密連接蛋白ZO-1和claudin-5在共軛鼠中表現量與執行鼠相比也有較低的情形。雖然在共軛鼠大腦中檢測TNF-α的濃度沒有差異，但仍然有看到發炎因子IL-6在大腦中表現量與執行鼠相比有上升的趨勢。有趣的是，透過西方墨點法評估在海馬迴中LC3II / I的比例和p62的表現量發現共軛鼠的自噬與執行鼠相比之下有較高的趨勢，而透過藥物調控海馬迴中的自噬可改善共軛鼠的情緒損傷。最後我們利用螢光染色發現在執行鼠的齒狀迴中髓鞘鹼性蛋白的表現量與共軛鼠相比下所涵蓋的面積比例較大，而這可能是免於情緒損傷的原因之一。這些結果表明在面對壓力時，缺乏心理控制感所造成情緒損傷可能是透過破壞血腦障壁的完整性，進而增加大腦中細胞因子的含量，而使得海馬迴中自噬的上升；而在面對壓力時，心理層面的控制感可能透過增加齒狀迴中髓鞘鹼性蛋白的含量來避免情緒上的損傷。

Various forms of stressors may exert negative impact on cognitive function, while the biological substrates and underpinnings are mostly unclear. In this study, a 10-day foot-shock (60 random-arranged shocks/hr/day, 0.5 mA, lasting at best 7 seconds, each) stressor regimen was used in the 8-week-old male C57BL/6 mice receiving an operant protocol contingent on the termination of the on-going foot-shock (executive group) or non-contingent on such termination (yoke group). Another batch of mice received no foot-shock regimen serving as a control group. After the 10-day foot-shock regimen, the yoke mice showed depression-like behavior and increased immobility duration in the forced swimming test. To access the integrity of blood brain barrier, intra-jugular vein Evans Blue injection and western immunoblotting were used to detect the expression of zonula occludens-1 (ZO-1) and claudin-5. The yokes, but not executive mice, showed deteriorated blood brain barrier integrity after the conclusion of the 10-day foot-shock regimen. Likewise, western immunoblotting results further revealed trend higher expression in interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) in the yoke mice as compared with the control mice. Moreover, the yoke mice demonstrated increased autophagy activity in hippocampus, as indicated by higher light chain 3 (LC3)II/I ratios and p62 expression, as compared to the “executive” controls. Furthermore, manipulation of autophagy in the hippocampus with rapamycin and 3-Methyladenine (3-MA), demonstrated the causal relationship of depression-like behavior and autophagy. Interestingly, the executive mice were found to increase the myelination in the dentate gyrus compared to the control mice. Our finding demonstrated the mechanism by which stress leads to emotional deficits, and the psychological aspect of control can avoid these damages by increasing the myelination in the dentate gyrus.

Table of contents 
Abstract (Chinese) ----------------------------II 
Abstract (English) ---------------------III 
Introduction -----------------------------1 
Materials and Methods 
1. Animals ---------------------5 
2. The model of psychological aspect of control ----------5 
3. Object location test ------------------5 
4. Y maze ------------------------------6 
5. Forced swimming ---------------6 
6. Evaluation of BBB Permeability ----------------------6 
7. Western Blotting ------------------------------------7 
8. Immunofluorescence and quantification --------------9 
9. Cannula implantation and microinjection into the hippocampus -----------9 
10. Statistical analysis ---------------11 
Results 
1. The executive mice terminated the foot shock earlier through the establishment of psychological control under stress --------------13 
2. Depression-like behavior was observed in yoke mice after 10-day foot shock regimen --------------------13 
3. Damages in the blood-brain barrier was observed in yoke mice after 10-day foot shock regimen ------------------14 
4. The level of cytokines was upregulated in brain after 10-day foot shock regimen in yoke mice ---------------------15 
5. Local autophagy flux in hippocampus was upregulated after 10-day foot shock regimen in yoke mice -----------15 
6. Focal manipulation of autophagy with 3-MA in hippocampus prevented the emotional deficit ------------------16 
7. Increase in dentate gyrus myelination was observed in executive mice after 10-day foot shock regimen ----------17 
Discussion --------------------------17 
References ---------------------------20 
 
Figure Legend
Figure 1. Timeline of the study design and a photo of the model of psychological aspect of control -------------------------------------------------------------------------------------26
Figure 2. The executive mice terminated the foot shock period earlier through the establishment of psychological control under stress --------------------------------27
Figure 3. Depression-like behavior was observed in yoke mice after the 10-day foot shock regimen ------------------------------------------------------------------------------------28
Figure 4. Lack of psychological control induced blood-brain barrier leakage by decreasing tight junction protein expression in yoke mice --------------------------------------29
Figure 5. The level of cytokines was upregulated in brain after 10-day foot shock regimen in yoke mice ------------------------------------------------------------------------------30
Figure 6. Activation of autophagy in hippocampus in yoke mice lacking psychological control after 10-day foot shock regimen ----------------------------------------------31
Figure 7. Reduction of depression-like behavior was observed by inhibiting autophagy in hippocampus after 10-day foot shock regimen in yoke mice-----------------------32
Figure 8. The psychological aspect of control established during the 10-day regimen led to an increase in the myelination of the dentate gyrus (DG) in executive mice-----33 

                                    

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