以小鼠模式探討新生期地塞米松投藥處理的跨代不良影響
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2023
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地塞米松(dexamethasone, DEX)為一種人工合成的醣皮質素(synthetic glucocorticoid)，一直以來廣泛當作抗發炎藥物使用，同時也用於治療體重過低的早產兒肺部發育不良導致的發炎。然而，許多研究指出新生期地塞米松治療(neonatal dexamethasone treatment, NDT)可能對新生兒的發育與情緒產長期不良影響。腦源性神經滋養因子(brain-derived neurotrophic factor, BDNF)，是神經系統中重要蛋白質，對於腦部的發育(development)、神經細胞的分化(differentiation)、及神經細胞的可塑性(plasticity)都扮演至關重要的角色，同時，憂鬱症患者腦中，BDNF的表現量會顯著低於普通人，因此本研究將BDNF與其專一受體TrkB作為憂鬱的生物標記(biomarker)來檢測NDT誘發的類憂鬱行為(depression-like behavior)。本研究採用C57BL / 6J品系小鼠，在出生後第一~三日(postnatal day1~3, PND1~3)給予遞減式劑量 (tapering dosage)地塞米松皮下注射，第一、第二與第三日注射的劑量分別為0.5、0.3與0.1mg/kg。在六周齡時，利用開放空間試驗(open field test, OFT)與高架十字迷宮試驗(elevated plus maze, EPM)來評測小鼠的類焦慮行為(anxiety-like behavior)，另外OFT也可用於測量小鼠的自發性運動功能(spontaneous activity)。此外，也於平行組(parallel group)以懸尾試驗(tail suspension test, TST)與糖水偏好試驗(sucrose preference test, SPT)測試小鼠的類憂鬱行為。於相同周齡時進行即時聚合酶連鎖反應(real-time polymerase chain reaction, qPCR)測量海馬迴中的Bdnf、TRKB與β-actin等基因表現，並利用西點墨法(western blotting, WB)評估BDNF、TrKB與β-actin的蛋白質表現量。並在十一周齡後配對產生子代(F2 SxS 與 F2 DxD)。並測量生育隻數(litter size)、存活率與性別比來測試NDT是否導致生殖毒性。此外，也在F2六周齡時以TST與SPT測試是否出現跨代類憂鬱行為。於同周齡時，以qPCR測量海馬迴中的Bdnf、TRKB等基因表現，並以WB測量BDNF、TrKB的蛋白質表現量。最後，以TST評估交叉撫育(cross-fostering)是否影響跨代類憂鬱行為。實驗結果表明，F1 NDT小鼠成長至青春期時體重顯著降低，代表有效投藥。OFT結果顯示 NDT小鼠自發運動功能未受影響，NDT雄性小鼠在中間區域時間(center area)顯著增加，顯示NDT會減少雄性小鼠的類焦慮行為，對母鼠則沒有影響。EPM結果則顯示NDT不會對公母鼠的類焦慮行為產生影響。TST結果顯示F1 NDT與F2 DxD公鼠的不掙扎時間皆增加，在母鼠方面則沒有差異。另外，在SPT中，F1 NDT與F2 DxD公鼠對於糖水的偏好程度也顯著下降，在母鼠方面則沒有差異。上述結果顯示NDT會導致跨代類憂鬱行為的增加。qPCR與西點墨法結果顯示在F1 NDT雄性小鼠海馬迴中BDNF與TrkB表現量都減少，雌性則未出現顯著差異；F2 DxD雄性小鼠在海馬迴中TrkB表現量顯著減少，雌鼠則無顯著差異。以上結果表明NDT會導致海馬迴中BDNF與TrkB表現量的減少，以及跨代TrkB表現量的減少。NDT在生殖方面的影響，包括F2 DxD的生育隻數與存活率都顯著減少，代表NDT會對子代的生殖造成影響，F2在性別比則沒有影響。最後，在TST中經過交叉撫育(cross parenting)的F2 SxS-Df小鼠相對於控制組(F2 SxS)，不掙扎時間顯著增加；經過交叉撫育的F2 DxD-Sf小鼠相對於控制組(F2 DxD)， 不掙扎時間則未出現顯著差異。綜上所述，我們推論NDT誘發的跨代憂鬱可能與NDT造成海馬迴中TrkB表現量有密切聯繫，並且母鼠的照料與遺傳因素皆可能參與NDT造成的跨代憂鬱。
Dexamethasone (DEX) is a synthetic glucocorticoid, widely used for its anti-inflammatory properties. It is also applied neonatally as a therapeutic agent to prevent and cure chronic lung diseases in infants with low body weight. However, numerous studies suggest that neonatal dexamethasone treatment (NDT) may have long-term adverse effects on the development and emotional well-being of newborns.Brain-derived neurotrophic factor (BDNF) is a crucial protein within the nervous system, playing an essential role in brain development, neuronal differentiation, and neuronal plasticity. Moreover, patients with depression exhibit significantly lower levels of BDNF in the brain compared to average individuals. Therefore, this study utilized BDNF and its specific receptor tropomyosin receptor kinase B (TrkB, coded by TRKB gene) as biomarkers for depression to examine the depression-like responses induced by NDT.This study subjected C57BL/6J mice to subcutaneous tapering-dose injections of DEX (0.5, 0.3, and 0.1 mg/kg, respectively) from postnatal day one to three (PND1-3). The neonatal dexamethasone-treated (NDT) group mice and control group mice were marked as F1. At six weeks old, animals were subjected to the open field test (OFT) and the elevated plus maze (EPM) to assess their anxiety-like behavior. The open field test was also used to measure the spontaneous activity of animals. Additionally, depression-like behavior in mice was examined in a parallel group using the tail suspension test (TST) and the sucrose preference test (SPT). In addition, the real-time polymerase chain reaction (qPCR) was used to measure the expression of genes such as Bdnf, TRKB, and β-actin in the hippocampus. Western blotting (WB) was employed to evaluate the protein expression levels of BDNF, TrKB, and β-actin. The Offspring of NDT group mice and control mice (F2 DxD and F2 SxS) were born after F1 mice mated with the same group. We measured the litter size, survival rate, and sex ratio to test if NDT led to reproductive toxicity. Furthermore, the TST and SPT were conducted to test for depression-like behavior in the F2 generation at the age of six weeks. In addition, qPCR was used to measure the expression of genes such as Bdnf and TRKB in the hippocampus. WB was used to measure the hippocampal expression of BDNF and TrKB. Lastly, the TST was used to evaluate if cross-fostering influenced transgenerational depression-like behavior.These results indicated that the body weight of the F1 NDT mice was significantly reduced, demonstrating a successful drug administration. The OFT results showed that the spontaneous activity of NDT mice was not affected by NDT. The time spent in the center of the NDT group male mice area significantly increased, indicating that the decrease in anxiety-like behavior in male mice. However, no difference was found in the NDT female mice. The EPM results showed that NDT did not influence anxiety-like behavior in neither male nor female mice. The TST results showed that the immobility time of both F1 NDT and F2 DxD male mice increased, but no differences were observed in female mice. Additionally, in the SPT, both F1 NDT and F2 DxD male mice showed a significant decrease in sucrose preference, but no differences were observed in female mice. These findings suggest that NDT leads to an increase in transgenerational depression-like behavior. The qPCR and Western blotting results showed that the expression of BDNF and TrkB decreased in F1 NDT male mice, but no significant differences were observed in F1 female. In F2 DxD male mice, the expression of TrkB significantly reduced, while no significant differences were found in F2 females. These results indicated that NDT lead to a reduction in the expression of BDNF and TrkB, as well as a transgenerational decrease in the hippocampal expression of TrkB. Regarding NDT effects on the reproduction, both the litter size and survival rate of F2 DxD significantly decreased, suggesting that NDT affects the reproduction of the F1 mice. There was no difference on the gender ratio was observed in F2 mice. In addition, the immobility time of F2 SxS-Df mice increased significantly compared to the control group (F2 SxS) in the TST, while F2 DxD-Sf mice did not show significant differences in immobility time compared to the control group (F2 DxD).In summary, we conclude that the transgenerational depression-like behavior induced by NDT may be associated with the reduce of hippocampal TrkB expression. Moreover, both maternal care and genetic factors could potentially influence cross-generational depression.
Dexamethasone (DEX) is a synthetic glucocorticoid, widely used for its anti-inflammatory properties. It is also applied neonatally as a therapeutic agent to prevent and cure chronic lung diseases in infants with low body weight. However, numerous studies suggest that neonatal dexamethasone treatment (NDT) may have long-term adverse effects on the development and emotional well-being of newborns.Brain-derived neurotrophic factor (BDNF) is a crucial protein within the nervous system, playing an essential role in brain development, neuronal differentiation, and neuronal plasticity. Moreover, patients with depression exhibit significantly lower levels of BDNF in the brain compared to average individuals. Therefore, this study utilized BDNF and its specific receptor tropomyosin receptor kinase B (TrkB, coded by TRKB gene) as biomarkers for depression to examine the depression-like responses induced by NDT.This study subjected C57BL/6J mice to subcutaneous tapering-dose injections of DEX (0.5, 0.3, and 0.1 mg/kg, respectively) from postnatal day one to three (PND1-3). The neonatal dexamethasone-treated (NDT) group mice and control group mice were marked as F1. At six weeks old, animals were subjected to the open field test (OFT) and the elevated plus maze (EPM) to assess their anxiety-like behavior. The open field test was also used to measure the spontaneous activity of animals. Additionally, depression-like behavior in mice was examined in a parallel group using the tail suspension test (TST) and the sucrose preference test (SPT). In addition, the real-time polymerase chain reaction (qPCR) was used to measure the expression of genes such as Bdnf, TRKB, and β-actin in the hippocampus. Western blotting (WB) was employed to evaluate the protein expression levels of BDNF, TrKB, and β-actin. The Offspring of NDT group mice and control mice (F2 DxD and F2 SxS) were born after F1 mice mated with the same group. We measured the litter size, survival rate, and sex ratio to test if NDT led to reproductive toxicity. Furthermore, the TST and SPT were conducted to test for depression-like behavior in the F2 generation at the age of six weeks. In addition, qPCR was used to measure the expression of genes such as Bdnf and TRKB in the hippocampus. WB was used to measure the hippocampal expression of BDNF and TrKB. Lastly, the TST was used to evaluate if cross-fostering influenced transgenerational depression-like behavior.These results indicated that the body weight of the F1 NDT mice was significantly reduced, demonstrating a successful drug administration. The OFT results showed that the spontaneous activity of NDT mice was not affected by NDT. The time spent in the center of the NDT group male mice area significantly increased, indicating that the decrease in anxiety-like behavior in male mice. However, no difference was found in the NDT female mice. The EPM results showed that NDT did not influence anxiety-like behavior in neither male nor female mice. The TST results showed that the immobility time of both F1 NDT and F2 DxD male mice increased, but no differences were observed in female mice. Additionally, in the SPT, both F1 NDT and F2 DxD male mice showed a significant decrease in sucrose preference, but no differences were observed in female mice. These findings suggest that NDT leads to an increase in transgenerational depression-like behavior. The qPCR and Western blotting results showed that the expression of BDNF and TrkB decreased in F1 NDT male mice, but no significant differences were observed in F1 female. In F2 DxD male mice, the expression of TrkB significantly reduced, while no significant differences were found in F2 females. These results indicated that NDT lead to a reduction in the expression of BDNF and TrkB, as well as a transgenerational decrease in the hippocampal expression of TrkB. Regarding NDT effects on the reproduction, both the litter size and survival rate of F2 DxD significantly decreased, suggesting that NDT affects the reproduction of the F1 mice. There was no difference on the gender ratio was observed in F2 mice. In addition, the immobility time of F2 SxS-Df mice increased significantly compared to the control group (F2 SxS) in the TST, while F2 DxD-Sf mice did not show significant differences in immobility time compared to the control group (F2 DxD).In summary, we conclude that the transgenerational depression-like behavior induced by NDT may be associated with the reduce of hippocampal TrkB expression. Moreover, both maternal care and genetic factors could potentially influence cross-generational depression.
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地塞米松, 類憂鬱行為, 海馬迴, 腦源性神經滋養因子, 跨代類憂鬱行為, 母方照護, Dexamethasone, depression-like behavior, hippocampus, brain-derived neurotrophic factor, transgenerational depression-like behavior, maternal care