Maternal n-3 fatty acid deficient diet on hypothalamic-pituitary-adrenal axis responses and feedback regulation to stress in postpartum rats and in the offspring later in life

本論文主要探討母鼠在懷孕泌乳期攝取n-3缺乏飼料對產後及子代下視丘－腦下垂體－腎上腺軸對壓力反應及回饋調控和憂鬱、焦慮行為之影響。二十二碳六烯酸(22:6n-3, DHA)為影響腦部神經發育生長與維持功能的重要因子。母體在懷孕泌乳期即子代的大腦發育時期，DHA會透過胎盤及泌乳的方式給予子代。在母鼠懷孕泌乳時期餵食含葵花油的n-3缺乏飼料，或補充魚油做為n-3正常組別到母鼠犧牲，子代在離乳後皆餵食相同的chow diet直到十週大犧牲。實驗結果發現在母鼠懷孕泌乳時期攝取n-3缺乏飼料會造成其照顧子代及主動泌乳行為的表現顯著下降且對壓力回饋調控的程度較差，在產後焦慮及憂鬱的程度也較高。在母鼠懷孕泌乳時期餵食n-3缺乏飼料，其子代在出生及離乳期的體重都較輕。子代大腦發育期缺乏DHA在給予急性束縛壓力後，其肛溫及血清中糖皮質固醇的濃度顯著較高且海馬中的糖皮質固醇受體的蛋白表現有下降的趨勢；而在糖皮質固醇受體促進劑(agonist)的作用下，利用急性束縛壓力及促腎上腺皮質激素釋放激素刺激也發現其血清中糖皮質固醇的濃度也顯著較高；在行為實驗表現焦慮及憂鬱的程度也較高。此外，在糖皮質固醇受體促進劑(agonist)壓力誘導的實驗發現親代攝取n-3缺乏飼料，其子代雌鼠對壓力的反應較雄鼠敏感。然而雄鼠行為表現上較為焦慮及憂鬱。綜合以上所述，在母鼠懷孕泌乳期攝取n-3缺乏飼料，會增加下視丘-腦下垂體-腎上腺軸對壓力反應的過度活化及降低其負回饋調控，導致母鼠產後及子代成鼠憂鬱、焦慮的表現。This thesis was to examine whether a maternal n-3 fatty acid-deficient diet during pregnancy and lactation altered the hypothalamic-pituitary-adrenal (HPA) axis responses and negative feedback regulation leading to anxiety and depression behaviors in postpartum rats and in the offspring later in life. Docosahexaenoic acid (DHA, 22:6n-3), is specifically enriched in the brain and is essential for normal neurological function. Most DHA accumulation in the brain occurs during brain development and is supplied via the placenta to the fetus and in the breast-fed milk to the pup. Pregnant rats were fed a sunflower oil-based n-3 fatty acid-deficient diet or the same diet supplemented with fish oil as an n-3 fatty acid-adequate diet during pregnancy and lactation. The maternal rats were maintained on the same diet till sacrificed at postpartum day 30. The pups, after weaning at postnatal day 22, were fed chow diet till sacrificed at 10-week-old. We found that the maternal rats fed an n-3 fatty acid-deficient diet showed significant lower licking/grooming and arch-back nursing behavior, increased in sensitivity to dexamethasone suppression following restraint stress-induced serum corticosterone levels, and enhanced depressive-like behavior in the forced-swimming test and anxiety-like behavior in the plus-maze test compared to the maternal rats fed an n-3 fatty acid-adequate diet. Exposure to the maternal n-3 fatty acid-deficient diet during the brain development was resulted, at newborn and weaning, in a significant reduced offspring body weight. DHA deficiency during the brain development significantly increased and prolonged restraint stress-induced changes in colonic body temperature and serum corticosterone levels, caused a significant increase in sensitivity to dexamethasone suppression with corticotrophin releasing hormone- or restraint stress-induced serum corticosterone III changes, and enhanced depressive-like behavior in the forced-swimming test and anxiety-like behavior in the plus-maze test in later life. In addition, DHA deficiency during the brain development in female offspring significantly increased in sensitivity to dexamethasone suppression with restraint stress-induced serum corticosterone changes than the male offspring. However, the male offspring compared to females, showed higher depressive-like behavior in the forced-swimming test and anxiety-like behavior in the plus-maze test. These results suggest that a maternal n-3 fatty acid-deficient diet during pregnancy and lactation leads to excessive HPA responses and dysregulation to stress and elevated behavioral indices of depression and anxiety in postpartum rats and the offspring later in life.摘要 ......................................................................................................................... I Abstract ..................................................................................................................... II 目錄 ............................................................................................................................IV 表次 …………………………………………………………………………………VI 圖次 ………………………………………………………………………………...VII 第一章 緒論(Introduction) ....................................................................................... 1 一、二十二碳六烯酸 ....................................................................................... 1 二、二十二碳六烯酸與憂鬱症 ........................................................................ 1 三、下視丘-腦下垂體-腎上腺路徑與憂鬱 ...................................................... 2 四、海馬GR、c-Fos及 BDNF與壓力的關係 ............................................... 3 五、親代泌乳照顧行為.................................................................................... 5 六、性別差異在壓力反應及HPA axis的影響 ............................................... 6 第二章、實驗目的與假設 ....................................................................................... 7 第三章 材料與方法(Materials and Methods) ........................................................... 9 一、實驗設計 ................................................................................................... 9 二、實驗用飼料配方與分析 .......................................................................... 10 三、母鼠照顧子代及泌乳行為(maternal care behavior) ........................ 13 四、抬高型十字迷宮實驗 (elevated plus-maze) ..................................... 13 五、強迫游泳實驗 (forced swim test) ........................................................... 14 六、束縛壓力實驗(Restraint stress) ............................................................... 15 七、Dexamethasone suppression test .............................................................. 15 八、Dexamethasone/corticotropin-releasing hormone (Dex/CRH) test ........................................................................................................................ 16 九、腎上腺皮質素酵素免疫分析 .................................................................. 16 十、海馬中GR、c-Fos及BDNF蛋白表現分析 .......................................... 17 十一、脂肪酸檢測分析.................................................................................. 22 十二、統計分析 ............................................................................................. 24 第四章 實驗結果(Result) ...................................................................................... 25 一、 母鼠懷孕泌乳期攝取n-3脂肪酸缺乏飼料對子代泌乳行為、HPA axis負回饋調控及產後焦慮憂鬱之影響 .............................................................. 25 (一) 母鼠懷孕泌乳期攝取n-3脂肪酸缺乏飼料對子代照顧及泌乳行為之影響 ..................................................................................................... 25 (二) 母鼠懷孕泌乳期腦部缺乏DHA對HPA axis負回饋調控之影響 26 (三) 母鼠懷孕泌乳期腦部缺乏DHA對其產後焦慮之影響 ................. 26 (四) 母鼠懷孕泌乳期腦部缺乏DHA對其產後憂鬱之影響 ................. 27 V 二、子代大腦發育時期缺乏DHA對成鼠HPA axis壓力反應及負回饋調控功能、憂鬱焦慮行為之影響 .............................................................................. 27 (一) 母鼠懷孕泌乳期給予n-3缺乏飼料對子代體重之影響 ............. 27 (二) 大腦發育時期缺乏DHA之成鼠面對急性壓力之肛溫變化 ...... 27 (三) 大腦發育時期缺乏DHA之成鼠面對急性壓力之corticosterone濃度變化 ..................................................................................................... 28 (四) 大腦發育時期缺乏DHA之成鼠HPA axis負回饋調控功能之影響 (DEX-CRH test) ...................................................................................... 29 (五) 大腦發育時期缺乏DHA之成鼠HPA axis負回饋調控功能之影響 (DEX-restraint test) .................................................................................. 29 (六) 子代大腦發育時期缺乏DHA對雄性成鼠壓力相關蛋白質表現之影響 30 (七) 大腦發育時期缺乏DHA對成鼠焦慮程度之影響 ..................... 31 (八) 大腦發育時期缺乏DHA對成鼠憂鬱程度之影響 ..................... 31 第五章 討論(Disscusion) ....................................................................................... 32 一、實驗動物之建立 ..................................................................................... 32 二、母鼠懷孕泌乳期攝取n-3脂肪酸缺乏飼料對其HPA axis負回饋調控功能、產後焦慮及憂鬱之影響 .......................................................................... 33 三、母鼠懷孕泌乳期攝取n-3脂肪酸缺乏飼料對照顧子代及泌乳行為之影響 ........................................................................................................................ 34 四、母鼠懷孕泌乳期攝取n-3脂肪酸缺乏飼料對其子代體重之影響 ......... 34 五、子代大腦發育期缺乏DHA對大鼠遭遇壓力其HPA axis活性之反應 . 35 六、子代大腦發育期缺乏DHA對大鼠遭遇壓力之回饋調控之影響 .......... 36 七、子代大腦發育期缺乏DHA對雄性成鼠遭遇壓力之相關蛋白表現之影響 ........................................................................................................................ 37 八、子代大腦發育期缺乏DHA對大鼠焦慮及憂鬱之影響 ......................... 38 九、結論 ......................................................................................................... 39 圖 (Figure) ............................................................................................................. 40 參考文獻 (reference).............................................................................................. 50 VI 表次 Table 1. Composition of the experimentaldiet .......................................................... 11 Table 2. ................................................................................................................... 12 Fatty acids composition of the n-3 fatty acid deficient diet, chow diet and fish oil ... 12 圖次 Fig.1 Number of dams used and assignment of offspring exposed to an n-3 fatty acid-deficient alone or the same diet supplemented with fish oil as an n-3 fatty acid-adequate diet to each test. ....................................................................... 10 Fig 2. Effect of n-3 fatty acid-deficient diet or the same diet supplemented with fish oil as an n-3 fatty acid-adequate diet during pregnancy and lactation on maternal licking/grooming and nursing behaviors in pups. ............................................ 40 Fig 3. Effect of n-3 fatty acid-deficient diet or the same diet supplemented with fish oil as an n-3 fatty acid-adequate diet during pregnancy and lactation on HPA negative feedback regulation in the Dexamethasone suppression following restraint-induced stress test in postpartum rats on day 30 of lactation.............. 41 Fig 4. Effect of n-3 fatty acid-deficient diet or the same diet supplemented with fish oil as an n-3 fatty acid-adequate diet during pregnancy and lactation on anxiety-like behavior in the elevated plus-maze test in postpartum rats on day 15 of lactation. ............................................................................................... 41 Fig 5. Effect of n-3 fatty acid-deficient diet or the same diet supplemented with fish oil as an n-3 fatty acid-adequate diet during pregnancy and lactation on depression-like behavior in the forced swimming test in postpartum rats on day 17 of lactation. ............................................................................................... 42 Fig 6. Effect of n-3 fatty acid-deficient diet or the same diet supplemented with fish oil as an n-3 fatty acid-adequate diet during pregnancy and lactation on offspring body weight .................................................................................... 42 Fig 7. Effect of exposure of rats to an n-3 fatty acid-deficient diet or the same diet supplemented with fish oil as an n-3 fatty acid-adequate diet during brain development on changes in colonic temperature during 60 min restraint-induced stress in the 10-week-old offspring ................................................................. 43 Fig 8. Effect of exposure of rats to an n-3 fatty acid-deficient diet or the same diet supplemented with fish oil as an n-3 fatty acid-adequate diet during brain development on HPA axis responses to restraint-induced stress in the 10-week-old offspring. ................................................................................... 44 Fig 9. Effect of exposure of rats to an n-3 fatty acid-deficient diet or the same diet supplemented with fish oil as an n-3 fatty acid-adequate diet during brain development on HPA negative feedback regulation in the Dexamethasone suppression following CRH stimulation test in the 10-week-old offspring. ..... 45 Fig 10. Effect of exposure of rats to an n-3 fatty acid-deficient diet or the same diet supplemented with fish oil as an n-3 fatty acid-adequate diet during brain VIII development on HPA negative feedback regulation in the Dexamethasone suppression following restraint-induced stress test in the 10-week-old offspring. ....................................................................................................................... 47 Fig 11. Effect of exposure of rats to an n-3 fatty acid-deficient diet or the same diet supplemented with fish oil as an n-3 fatty acid-adequate diet during brain development on hippocampal stress related protein expression in the 10-week-old male offspring. ........................................................................... 48 Fig 12. Effect of exposure of rats to an n-3 fatty acid-deficient diet or the same diet supplemented with fish oil as an n-3 fatty acid-adequate diet during brain development on anxiety-like behavior in the elevated plus-maze test in the 10-week-old offspring. ................................................................................... 49 Fig 13. Effect of exposure of rats to an n-3 fatty acid-deficient diet or the same diet supplemented with fish oil as an n-3 fatty acid-adequate diet during brain development on depression-like behavior in the forced swimming test in the 10-week-old offspring. ................................................................................... 4