探討懷孕母鼠分別暴露於酯多醣或3,5-二甲基苯胺對胎鼠腦部發育之影響

大腦發育包含了神經分化、遷移、排列與網路形成，這些過程受到許多內在與外在因子於空間與時間上有順序性的調控。然而胎兒的發育容易因母體所接觸到外在環境的危險因子而受到影響，生物性的危險因子例如細菌，而化學性危險因子則包括例如殺蟲劑、食品添加物、香菸、空氣汙染等。胎兒大腦發育與調控的過程若受到外在因素的干擾，往往會導致發育的大腦畸形或功能失常。例如母體在懷孕期間，若受到細菌性的子宮內感染會增加神經發育異常的機率，並可能導致精神疾病的發生。許多文獻指出，子宮內感染會引發發育中神經系統的發炎反應，並導致胎兒腦神經退化與腦部功能缺陷，但其致病機制目前尚不明確。本實驗所選用的單環芳香胺為一普遍存在於環境中的有機化合物，香菸的煙霧、染劑、殺蟲劑以及汽機車燃料等皆含有單環芳香胺。先前研究顯示，一些特定的單環芳香胺代謝產物會與DNA鍵結並產生活性氧物種(reactive oxygen species, ROS)，導致細胞死亡。本研究將分別探討神經發炎或單環芳香胺是否會對胎兒大腦發育產生影響。於神經發炎部分，本實驗於母鼠懷孕第14天時進行腹腔注射脂多醣 (lipopolysaccharide , LPS) 以誘發發炎反應模仿子宮內感染；而於單環芳香胺部分，實驗採用細胞毒性顯著之3,5-二甲基苯胺 (3,5-dimethylaniline, 3,5-DMA) ，並於母鼠懷孕第15、16、17天時經由皮下注射。母鼠犧牲後，分析LPS或3,5-DMA對母鼠基本生理參數的影響。胎兒方面，除了組織切片染色觀察外，兩者皆在懷孕期間注射BrdU用以觀察神經細胞新生狀況。為了觀察胎兒大腦皮質發育期間細胞遷移分層情況，本實驗使用抗體如Tbr1、Ctip2、 Satb2等來標定不同分層之細胞。實驗結果顯示，注射LPS造成母鼠白血球上升、脾臟腫大以及胎鼠存活數下降，而LPS對於胎鼠腦部細胞的增生並無顯著影響，但本研究發現LPS誘發之子宮內發炎反應會使胎鼠皮質腫脹、腦組織細胞密度下降，且會減少大腦皮質內第五層與第六層之神經細胞數目，並嚴重影響其細胞遷移，實驗也證實此影響會延續至胚胎大腦發育後期。另外於神經發炎部分，文獻指出神經發炎反應會產生ROS，造成神經損傷，因此本研究同時假設若抑制ROS生成，可能會降低其對胎鼠腦部發育造成之影響。本實驗在注射LPS之前1小時，對母鼠進行腹腔注射ROS抑制劑：N-acetylcysteine (NAC) ，結果顯示NAC可以有效降低由LPS所引起之母鼠的白血球上升、脾臟腫大與胎鼠大腦皮質發育異常，不論是大腦皮質型態以及第五層、第六層神經細胞的數目與遷移情況皆較接近控制組，顯示NAC達到保護的作用。另外，母鼠注射高濃度的3,5-DMA後導致血液中紅血球數目明顯下降、白血球數目大量的增加，且胚胎體重明顯下降。3,5-DMA也造成胚胎大腦皮質厚度減少、皮質內細胞數目下降以及新生細胞數目減少，並嚴重使皮質內第二層、第五層、第六層神經細胞數目減少與其遷移分布情形。

關鍵字

腦部發育；脂多醣；3,5-二甲基苯胺、

並列摘要

The brain is not only a control center of the body but a part of way that the body can communicate with outside environments. Normal development of the brain is well defined spatial and temporal events, which includes neuron generation, migration, organization and patterning. Disruption of these steps results in malformation of the brain. Many factors could affect embryo development during pregnancy, including biological or chemical factors. Maternal bacterial infections during pregnancy represent a significant risk factor in neuropsychiatric disorders with a presumed neurodevelopmental failure. Neuroinflammation, has been reported to be highly associated with numbers of neurological and pathological diseases. Epidemiological studies suggest that intrauterine infection acts as a crucial role in inducing neuroinflammatory response, which would lead to neurodegeneration and functional deficits in fetal brain. However, little is known in the mechanism regarding to possible changes induced by neuroinflammation in the developing fetal brain. For chemical aspects, monocyclic aromatic amines (MAAs) are a group of chemicals ubiquitous presence in the environment, exposure assessment indicate that most individuals experience lifelong exposure to these compounds from several sources, such as tobacco smoke, colorants, pesticides and motor fuels. A class of specific MAAs, for example, 3,5-dimethylaniline (3,5-DMA), their bioactivated products generate covalent DNA adducts and induce the production of reactive oxygen species (ROS), which consequently leads to cell death. Thus, this study plans to clarify the possible biological machinery underling the inflammation or 3,5-DMA caused deficits in brain development and hypothesize that MAAs may perturb fetal brain development. We mimicked the maternal infection by intraperitoneal injections of lipopolysaccharide (LPS) in a dose dependent manner at 14 days of gestation. On the other side, 3,5-DMA was injected subcutaneously at 14 days of gestation. To label the neuron generation, BrdU was injected subcutaneously during gestation. Moreover, cortical laminate markers, such as Tbr1, Ctip2, Satb2, were labelled to investigate the migration events during brain development. The other maternal or fetal physiological and histochemical parameters were examined as well. Results show that LPS induced the inflammatory response in increasing the number of white blood cell and the size of the spleen significantly. Furthermore, no significant differences were observed in cell proliferation of the fetal brain, but the migration of fetal brain in high dose of LPS is disturbed. Previous findings implies that neuroinflammatory response may induce the reactive oxygen species (ROS) generation, which further leads to neural injury. Accordingly, we hypothesize that these LPS-induced abnormalities in migration pattern in the fetal brain may due to the production of ROS. To prove the hypothesis, ROS scavengers, N-acetylcysteine (NAC) was applied into pregnant rats one hour previous to LPS injection. The data show that NAC can rescue the disrupted brain development. This study suggest that neuroinflammation interfere the migration of brain development during pregnancy, which could be reversed in the presence of NAC. On the other side, 3,5-DMA increased the number of white blood cell and reduced the number of red blood cell, also the lighter embryo weight was observed. Furthermore, 3,5-DMA reduced cell proliferation of the fetal brain, and the migration of neuron in fetal brain was disturbed seriously.

並列關鍵字

lipopolysaccharide；3,5-dimethylaniline；brain development；fetal brain

參考文獻

1. Koibuchi, N. and T. Iwasaki, Regulation of brain development by thyroid hormone and its modulation by environmental chemicals. Endocr J, 2006. 53(3): p. 295-303.

2. Ajuwon, O.R., O.O. Oguntibeju, and J.L. Marnewick, Amelioration of lipopolysaccharide-induced liver injury by aqueous rooibos (Aspalathus linearis) extract via inhibition of pro-inflammatory cytokines and oxidative stress. BMC Complement Altern Med, 2014. 14: p. 392.

3. Boksa, P., Effects of prenatal infection on brain development and behavior: a review of findings from animal models. Brain Behav Immun, 2010. 24(6): p. 881-97.

4. Archer, T., Effects of exogenous agents on brain development: stress, abuse and therapeutic compounds. CNS Neurosci Ther, 2011. 17(5): p. 470-89.

5. Esmat, A., et al., Dichloroacetonitrile induces oxidative stress and developmental apoptotic imbalance in mouse fetal brain. Environ Toxicol Pharmacol, 2012. 33(1): p. 78-84.

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探討懷孕母鼠分別暴露於酯多醣或3,5-二甲基苯胺對胎鼠腦部發育之影響

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