缺氧預處理與抗氧化物質對大鼠下位腦幹及結狀神經節內神經元及非神經元組成元素之影響

Effects of mild hypoxic preconditioning and antioxidant on the neuronal and non-neuronal elements in the lower brainstem and nodose ganglion of rats

魏一華(I-Hua Wei)

指導教授 ： 謝正勇

共同指導教授 ： 溫振源

國立臺灣大學/醫學院/解剖學研究所/博士(2005年)

https://doi.org/10.6342/NTU.2005.00006

摘要

一氧化氮(nitric oxide, NO)為一種自由基氣體，經由細胞內一氧化氮合成酶(NOS)將L-精胺酸(L-arginine)轉換成果胺酸(citrulline)過程中，同時將其釋放出來。NO在特定狀況下具有調控正常生理運作及造成細胞劇烈毒性的能力。據過去研究發現周邊神經損傷及嚴重缺氧均會改變神經元胞膜上NMDA受器的活性，進而誘使一氧化氮等自由基大量增生及影響相關神經化學物質的代謝路徑，導致神經細胞退化或死亡。然而，近年來生理、生化方面的研究則指出若先將鼠腦切片置於非致死性缺氧/缺血環境進行預處理(precondition)，則對於隨後的嚴重缺氧/缺血環境介入，反而具有神經保護的能力。本實驗旨在利用組織化學、免疫組織化學法及西方免疫墨點法，闡明神經元及非神經元組成元素在缺氧預處理(mild hypoxic preconditioning; MHPC)合併及未合併嚴重缺氧(severe hypoxic exposure; SHE)或周邊軸突夾傷(peripheral nerve crush injury; PNCI)後，胞體內相關神經化學物質的改變及其與神經退化之關連。實驗擬同時探討抗氧化物質¾綠茶萃取物(EGCG)的施予，對於結狀神經節內之感覺神經元是否具良好神經保護(neuroprotection)的效果。
本實驗首先將大鼠置放於海拔5500公尺缺氧艙中28天進行缺氧預處理，探討單純缺氧預處理對感覺神經元及運動神經元相關神經化學物質表現之影響。實驗結果發現，缺氧預處理後，結狀神經元內一氧化氮合成酶(nitric oxide synthase, NOS)、錳-超氧化物歧化酶(Mn-superoxide dismutase, Mn-SOD)、銅-鋅超氧化物歧化酶(Cu/Zn-superoxide dismutase, Cu/Zn-SOD)及鈣離子結合蛋白－Calbindin D-28K、parvalbumin免疫組織化學反應皆有呈現上揚的表現，同時其熱休克蛋白70(Heat Shock Protein 70 kDa, HSP70)免疫組織化學反應則有大量上揚的表現。於缺氧預處理後八週觀察，發現結狀神經節內並無細胞死亡的現象發生。在舌下神經核及迷走神經背運動核之運動神經元並未觀察到類似的結果。實驗進一步將缺氧預處理後之實驗動物置放於高海拔10000公尺缺氧艙中4小時進行嚴重缺氧處理，以探討大鼠經缺氧預處理合併嚴重缺氧後所造成之影響。實驗結果發現，大鼠經嚴重缺氧後於結狀神經節內可看到大量NOS表現，且此一免疫反應表現的多寡與神經細胞死亡間，具有正向關連。實驗同時發現，嚴重缺氧後亦會逐漸提升錳-超氧化物歧化酶、銅-鋅超氧化物歧化酶、鈣離子結合蛋白－Calbindin D-28K、parvalbumin及熱休克蛋白70免疫反應表現。缺氧預處理合併嚴重缺氧後，結狀神經節內上述相關神經化學分子表現的消長及細胞存活的數量，皆較單獨嚴重氧處理者明顯。
為測試缺氧預處理合併周邊軸突夾傷後所造成之影響，因此將缺氧預處理後之實驗動物單側舌下及迷走神經軸突分別進行夾傷處理30秒，實驗結果發現於受損側核區皆可看到強烈之NOS免疫反應表現及細胞死亡。實驗同時發現，一氧化氮大量生成亦會抑低膽鹼乙醯移轉酶(choline acetyltransferase, ChAT)及提升抑鈣素基因相關胜肽(calcitonin gene-related peptide, CGRP)、熱休克蛋白 70免疫組織化學反應。另外，以OX-42、ED-1及glia fibrillary acidic protein (GFAP)免疫組織化學染色分別標誌小神經膠細胞及星狀神經膠細胞，發現受損核區神經膠細胞皆有大量活化的表現。單獨周邊軸突夾傷組受損核區相關神經化學分子表現的消長、神經膠細胞活化的狀況及細胞死亡的數量，皆較缺氧預處理合併周邊軸突夾傷處理組明顯。據上述實驗結果推測，嚴重缺氧處理或周邊軸突夾傷會過度誘發一氧化氮合成酶，藉由干擾細胞能量生合成狀態、神經傳導功能及自由基與抗氧化酵素間之平衡，促使更多受損神經元死亡。然而，若經缺氧預處理後將可大量減少因嚴重氧處理或周邊軸突夾傷後大腦內一氧化氮的過度生成，進而減少細胞損傷及死亡，顯示其在平衡氧化壓力及執行細胞保護功能的作用中，扮演相當重要的角色。
綠茶萃取物(EGCG)處理的實驗結果則發現，綠茶萃取物確能有效減少經嚴重缺氧後受損結狀神經元NOS之免疫表現，同時亦能減緩神經元死亡進程，且此效果與注射劑量成正比。本實驗首次提供綠茶萃取物於缺氧處理上具良好神經保護功能之形態學證據，同時也提供使用綠茶萃取物預防因氧化傷害所引發之相關神經病變上，一詳實且詳盡之參考資料。

關鍵字

缺氧預處理 ； 綠茶萃取物

並列摘要

Nitric oxide (NO) is a free radical gas, which is synthesized from L-arginine and citrulline by nitric oxide synthase (NOS). It can both mediate normal physiological events and be highly toxic under certain conditions. Previous studies have shown that either peripheral nerve injury (PNI) or hypoxia will alter the NMDA receptor activity and result in neuronal degeneration or cell death via the over production of NO. However, recent physiological and biochemical studies have shown that a sublethal hypoxic/ischemic preconditioning of the brain slice will protect against later challenge to the acute hypoxic/ischemic treatment. We employed the histochemical, immunohistochemical and western blotting methods to study the relationships between the neurochemical expression and neuronal degeneration of neuronal and non-neuronal elements following mild hypoxic preconditioning (MHPC) with or without severe hypoxic exposure (SHE) or peripheral nerve crush injury (PNCI) in the rats. Furthermore, an antioxidant substance such as EGCG was used to test if it has the potential effect of protection on the sensory neurons of the nodose ganglion (NG) in the acute hypoxic rats.
The experimental rats were exposed at the high altitude (5,500 m) chamber for 28 days to test the effect of MHPC on the expression patterns of related neurochemicals of sensory and motor neurons. The results showed that NOS, Mn-superoxide dismutase (Mn-SOD), Cu/Zn-superoxide dismutase (Cu/Zn-SOD) and calcium binding protein-calbindin D-28K, parvalbumin immunoreactivities in the NG were increased, while the heat shock protein 70 kDa (HSP70) immunohistochemical staining were significantly up-regulated. The neuronal loss was not detected in the NG 8 weeks following MHPC. Similar findings were not detected in the motor neurons either in the hypoglossal nucleus or dorsal motor nucleus of the vagus. To test the effect of MHPC coupled with SHE, the MHPC animals were placed in the high altitude chamber at 10,000 m for 4h. The results showed that NOS expression was drastically increased in the NG neurons; its reactivities were in proportion to the severity of neuronal damage. The immunoreactivities of Mn-SOD, Cu/Zn-SOD, calbindin D-28K, parvalbumin and HSP70 were gradually increased. The expression patterns of related neurochemicals and the numbers of neuronal survival were all more significant in the MHPC coupled with SHE treated rats as compared with those of SHE only.
To test the effect of MHPC coupled with PNCI, the MHPC animals whose left vagus and hypoglossal nerves were crushed for 30 sec, respectively. The results revealed that both the NOS expression and cell loss were significantly increased in the lesioned nuclei. In addition, PNCI will also suppress the immunoreactivities of choline acetyltransferase (ChAT) and up-regulate the reactivities of calcitonin gene-related peptide (CGRP) and HSP70 via the over production of NO. The current results also elucidated that activated microglia and astrocytes were marked expressed in the lesioned nuclei using OX-42, ED-1 and glia fibrillary acidic protein (GFAP) methods, respectively. The expression patterns of related neurochemicals, activated status of glial cells and the numbers of neuronal loss were all more significant in PNCI rats as compared with those of MHPC coupled with PNCI. The results of the current study implied that over expression of NOS might severely damage the neuronal elements; this could subsequently block the metabolic pathway and neurotransmitter function as well as the balanced status of the free radical/anti-oxidative defense system. Furthermore, MHPC coupled with SHE or PNCI might have the potential effect to protect against SHE or PNCI-induced NO overexpression in the brain and prevent from damaging neurons or cell death. MHPC played an important role on the balance of the oxidative stress and enhanced the protection of neurons.
The current study also showed that EGCG would indeed suppress NOS expression and reduce the neuronal damage in the NG neurons of acute hypoxic rats; this effect was dose-dependent. The present study not only provides the first morphological evidence concerning the neuroprotective effect of EGCG but also increases the current knowledge for the potential therapeutic use of EGCG to prevent the related neuropathies induced by oxidative damage.

並列關鍵字

EGCG ； mild hypoxic preconditioning

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