新生期投與dexamethasone對母鼠的長期不良影響
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2018
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地塞米松 (dexamethasone, DEX) 是一種人工合成的醣皮質激素(synthetic glucocorticoid)，目前廣泛做為消炎藥劑(anti-inflammatory)，並用於治療早產兒之呼吸窘迫症 (respiratory distress syndrome)，但多年來研究發現新生期投與DEX (neonatal DEX treatment; NDT) 會導致腦部發育與認知功能的不良影響。海馬迴 (hippocampus) 是空間記憶 (spatial memory) 和連結性學習記憶(associative learning and memory) 的主要腦區，而杏仁核 (amygdala)則是調控情緒的重要腦區，兩者均屬於邊緣系統 (limbic system) 的成員，其功能異常與精神疾病如憂鬱症 (major depression) 及焦慮症 (anxiety disorder) 有重要關聯。
前人研究顯示NDT會導致大鼠的類憂鬱行為 (depression-like behavior) 明顯增加，過去關於NDT的研究多著重於公鼠，較少著墨於母鼠，本研究利用新生期母鼠，模擬臨床上早產兒的投藥方式，以濃度遞減式 (tapering dosage) 於出生後第1~3天 (postnatal day-1 to 3) 連續3天依序投予DEX (0.5 mg/kg, 0.3 mg/kg 和 0.1 mg/kg)，並利用細胞外電刺激紀錄 (in vitro extracellular recording)、西方墨點法 (western blot analysis)、抑制逃避學習實驗 (inhibitory avoidance task; IA) 與強迫游泳行為實驗 (forced swimming tests; FST)，評估NDT對青春期 (6週齡) 與成年期 (10週齡) 的母鼠海馬迴與杏仁核之功能影響。
實驗結果發現在海馬迴和杏仁核，利用高頻電刺激 (high-frequency stimulation; HFS) 誘發的長期增益現象 (long term potentiation; LTP) 中，青春期NDT母鼠的LTP有抑制的情形，而在成年期母鼠的海馬迴和杏仁核以HFS誘發的LTP則有減弱的情形。西方墨點法的結果顯示青春期與成年期母鼠海馬迴與杏仁核的estrogen receptor alpha (ER) 表現量均低於控制組，而estrogen receptor beta (ER) 的表現量與控制組無顯著差異。在檢測海馬迴空間記憶功能的IA行為實驗發現，NDT阻斷了青春期NDT母鼠抑制逃避學習，青春期NDT母鼠滯留潛伏 (retention latencies) 時間低於控制組並有顯著差異，但這種阻斷效果不會持續到成年期。評估杏仁核功能的FST行為實驗發現，青春期與成年期母鼠在FST模式中，不游動時間百分比 (percent time of immobility) 明顯高於控制組，表現出類憂鬱行為的反應增加。為避免NDT所引起的可能的毒性效果 (toxic effect) 對行為實驗數據判讀的干擾，故合併測量其自發性活動行為 (locomotor activity)，結果顯示NDT母鼠在青春期和成年期的總水平移動距離 (total horizontal distance movement) 與控制組相比，並無顯著差異。在青春期和成年期母鼠在細胞外電生理紀錄時投予雌二醇 (estradiol) 可部分恢復杏仁核的HFS誘發之LTP；青春期動物於6週齡時連續四天皮下注射雌二醇 (0.25 mg/kg/day)，成年期動物於10週齡時連續四天皮下注射雌二醇，分別在第四天給藥結束30分鐘後立即進行FST行為實驗，結果發現給予雌二醇的NDT母鼠，其類憂鬱行為降低。青春期母鼠在細胞外電生理紀錄給予雌二醇可部分恢復海馬迴的LTP。在染色質免疫沉澱實驗 (chromatin immunoprecipitation assay; ChIP) 中發現幼年期NDT母鼠 (2週齡) 的海馬迴中H3K9乙醯化 (acetylation) 與控制組相比，NDT母鼠的H3K9乙醯化百分比顯著低於控制組。在NDT組於出生後第一天 同時投與histone deacetylases (HDAC) 抑制劑trichostatin A (TSA)，可使幼年期母鼠海馬迴ER的表現量恢復至與控制組相近，並可使青春期母鼠海馬迴以HFS誘發的LTP恢復，與控制組相比無顯著差異，以及在IA行為實驗發現海馬迴空間性記憶功能恢復，與控制組相比無顯著差異。總結本篇實驗結果，NDT母鼠在青春期與成年期的海馬迴與杏仁核之LTP、ER表現量、空間記憶及情緒功能均產生不良的影響，然而這樣不利的影響會一直持續到成年期，並且這樣的影響可能與H3K9乙醯化下降有關，而於幼年期投予HDAC抑制劑，或於青春期與成年期給予雌二醇，則可有效地降低NDT的長期不良影響。根據本論文的結果發現，NDT對母鼠產生的長期不良影響並直至成年期，這樣的結果可提供給日後相關研究及臨床投藥做為參考。
Synthetic glucocorticoid dexamethasone (DEX) has been widely used as an anti-inflammatory agent and also used to treat respiratory distress syndrome in preterm infants. Previous studies suggested that neonatal DEX treatment (NDT) could alter brain development and cognitive function. Hippocampus is a critical brain area for both spatial and associative learning and memory. Amygdala is an essential region for regulating emotional response and behavior. Both hippocampus and amygdala are also the important components of the limbic system, which exhibit a tight association with mental disorders such as major depression and anxiety disorder. Previous NDT studies often focused on its influence with the male rats only. Because females are more vulnerable to the psychological stress and have higher susceptibility of having mental disorders, it is important and worthy to study the possible long-term adverse effect on female animals. In order to investigate the effects of NDT on the hippocampal and amygdaloid function in female rats, the experimental subjects received subcutaneous injections of a tapering dose of DEX (0.5 mg/kg, 0.3 mg/kg and 0.1 mg/kg) from postnatal day 1 to day 3 (PND1~3). In vitro electrophysiological recording, inhibitory avoidance (IA) and forced swimming test (FST) were used to evaluate the hippocampal and amygdaloid function, particularly the spatial and emotional memory function at juvenile (6 weeks old) and adult (10 weeks old) female rats. We found that NDT could alter high frequency stimulation (HFS) which would induce hippocampal and amygdaloid long-term potentiation (LTP) formation. The expression level of hippocampal and amygdaloid estrogen receptor alpha (ER) was significantly repressed in NDT female rats. The results of this study also showed that NDT blocked IA learning in juvenile NDT female rats which did not sustain to the adulthood. No significant difference in the retention latencies was found between NDT and SAL groups in adult female rats. The long-term adverse effects of NDT on amygdaloid function in both juvenile and adult NDT female rats were investigated by forced swimming test (FST). The FST results demonstrated that the depression-like behaviors were increased in both juvenile and adult NDT female rats as compared with the control group of SAL animals. By using locomotor activity monitoring, we can also study the possible toxic effect of NDT on motor function and spontaneous behavior. The total horizontal distance movement did not reveal any significant difference between the NDT and the control animals. Clearly, the normal HFS induced long-term potentiation (LTP) formation in hippocampus and amygdala would be blocked in juvenile and adult NDT animals. Suprafusion of estradiol could partially restore the hippocampal and amygdaloid LTP formation in NDT female rats, as the systemic administration (6 weeks old or 10 weeks old consecutive four days; 0.25 mg/kg/day; subcutaneous injection) of estradiol could partially attenuate depression-like behavior in both juvenile and adult NDT female rats. More interestingly, our chromation immunoprecipitation (ChIP) results revealed that the level of H3K9 acetylation was significantly decreased in the hippocampus of NDT female rats. Co-administration of HDAC inhibitor trichostatin A (TSA) could restore the expression of hippocampal ER expression in NDT female rats. In addition, TSA was also found to partially restore the hippocampal LTP formation and rescue the hippocampal spatial memory function on IA test in the NDT juvenile female rats. In conclusion, our results strongly suggested that NDT could elicit some long-term adverse effects on the hippocampus and amygdala in NDT female rats, and it is correlated to the decline of H3K9 acetylation, which resulted in the decrease of hippocampal and amygdaloid ER expression. Co-administration of TSA or systemic injection of estradiol could partially restore the hippocampal and amygdaloid function in NDT female rats. These results suggested that the hippocampal and amygdaloid level is closely related to the mechanism that causes ERα to incur the adverse effect of NDT female rats, which might result in adverse consequences in adult age.
Synthetic glucocorticoid dexamethasone (DEX) has been widely used as an anti-inflammatory agent and also used to treat respiratory distress syndrome in preterm infants. Previous studies suggested that neonatal DEX treatment (NDT) could alter brain development and cognitive function. Hippocampus is a critical brain area for both spatial and associative learning and memory. Amygdala is an essential region for regulating emotional response and behavior. Both hippocampus and amygdala are also the important components of the limbic system, which exhibit a tight association with mental disorders such as major depression and anxiety disorder. Previous NDT studies often focused on its influence with the male rats only. Because females are more vulnerable to the psychological stress and have higher susceptibility of having mental disorders, it is important and worthy to study the possible long-term adverse effect on female animals. In order to investigate the effects of NDT on the hippocampal and amygdaloid function in female rats, the experimental subjects received subcutaneous injections of a tapering dose of DEX (0.5 mg/kg, 0.3 mg/kg and 0.1 mg/kg) from postnatal day 1 to day 3 (PND1~3). In vitro electrophysiological recording, inhibitory avoidance (IA) and forced swimming test (FST) were used to evaluate the hippocampal and amygdaloid function, particularly the spatial and emotional memory function at juvenile (6 weeks old) and adult (10 weeks old) female rats. We found that NDT could alter high frequency stimulation (HFS) which would induce hippocampal and amygdaloid long-term potentiation (LTP) formation. The expression level of hippocampal and amygdaloid estrogen receptor alpha (ER) was significantly repressed in NDT female rats. The results of this study also showed that NDT blocked IA learning in juvenile NDT female rats which did not sustain to the adulthood. No significant difference in the retention latencies was found between NDT and SAL groups in adult female rats. The long-term adverse effects of NDT on amygdaloid function in both juvenile and adult NDT female rats were investigated by forced swimming test (FST). The FST results demonstrated that the depression-like behaviors were increased in both juvenile and adult NDT female rats as compared with the control group of SAL animals. By using locomotor activity monitoring, we can also study the possible toxic effect of NDT on motor function and spontaneous behavior. The total horizontal distance movement did not reveal any significant difference between the NDT and the control animals. Clearly, the normal HFS induced long-term potentiation (LTP) formation in hippocampus and amygdala would be blocked in juvenile and adult NDT animals. Suprafusion of estradiol could partially restore the hippocampal and amygdaloid LTP formation in NDT female rats, as the systemic administration (6 weeks old or 10 weeks old consecutive four days; 0.25 mg/kg/day; subcutaneous injection) of estradiol could partially attenuate depression-like behavior in both juvenile and adult NDT female rats. More interestingly, our chromation immunoprecipitation (ChIP) results revealed that the level of H3K9 acetylation was significantly decreased in the hippocampus of NDT female rats. Co-administration of HDAC inhibitor trichostatin A (TSA) could restore the expression of hippocampal ER expression in NDT female rats. In addition, TSA was also found to partially restore the hippocampal LTP formation and rescue the hippocampal spatial memory function on IA test in the NDT juvenile female rats. In conclusion, our results strongly suggested that NDT could elicit some long-term adverse effects on the hippocampus and amygdala in NDT female rats, and it is correlated to the decline of H3K9 acetylation, which resulted in the decrease of hippocampal and amygdaloid ER expression. Co-administration of TSA or systemic injection of estradiol could partially restore the hippocampal and amygdaloid function in NDT female rats. These results suggested that the hippocampal and amygdaloid level is closely related to the mechanism that causes ERα to incur the adverse effect of NDT female rats, which might result in adverse consequences in adult age.
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醣皮質激素, 地塞米松 (dexamethasone), 海馬迴, 杏仁核, 雌激素受體, 類憂鬱行為, 長期增益現象, 表觀遺傳學, 組蛋白乙醯化, Glucocorticoid, Dexamethasone, Hippocampus, Amygdala, Estrogen receptor, Depression-like behavior, Long-term potentiation, Epigenetic, Histone acetylation