| 摘要: | 過去有許多研究指出當身體面臨缺血、缺氧或是壓力時，常出現包括有口渴、血壓上升以及尿量減少等體液失衡的症狀。而位於下視丘中室旁核(paraventricular nucleus; PVN)與視上核(suproptic nucleus; SON)內的大細胞神經元，曾被暗喻與此些症狀的產生有關。SON和PVN兩神經核內，目前已知含多種神經胜肽分泌物，這些分泌物在身體處於壓力時的表現，以及彼此相互間的關係為何？除血管加壓素(vasopressin；VP)外，目前仍鮮少有報告說明。故本篇論文的研究目的，主是要想了解當暴露在低壓缺氧的情況下，PVN 和 SON內大細胞神經元中神經胜肽分泌物變化的情形。本研究將動物分成處於正常環境氣壓的控制組，及放置於缺氧艙中的實驗組動物(低壓缺氧的狀況設定為，相當於9000公尺，氧分壓為48托耳的環境下)。動物經缺氧七小時後，再細分成把老鼠立即犧牲(0h)，以及存活四小時 (4h)、存活一天(1d)、存活三天(3d)、存活七天(7d)再把老鼠犧牲的實驗組。以免疫組織化學染色法、西方墨點法以及影像分析等方法來做探討：1) 低壓缺氧是否會激發PVN 和 SON內大細胞神經元的活性。2)被激發的大細胞神經元內含何種神經胜肽分泌物(如，VP、腎上腺髓質素及抑鈣素基因相關胜肽素等)。3)這些神經胜肽分泌物是否受一氧化氮的調控。4)這些神經胜肽分泌物間彼此的相互關係。我們得到以下的結果。
首先經由即刻早期基因(immediately early gene)- c-Fos的免疫組織化學染色結果發現，暴露於缺氧環境後，不論是PVN或是SON內具 c-Fos免疫反應的神經元，於存活 0～4小時有大量被誘發增加的情形出現，但存活七天則幾乎回復到正常的狀態。此表示低壓缺氧確實會激發PVN 和 SON內大細胞神經元的活性，且給予回氧後會漸漸恢復正常。另外，經免疫組織化學染色，或是西方墨點法以及影像分析的統計結果，發現不管是位於PVN或是SON內具VP免疫反應的神經元，在暴露於缺氧環境後，存活 0～4小時，均有大量被誘發增加的情形。在PVN內VP的免疫反應平均約增加24% ~46 %，而SON內VP的免疫反應平均約增加33% ~76 %，但存活7天則幾乎回復到正常的狀態。而PVN或是SON內具 nNOS的免疫反應神經元似乎也有類似的發展趨勢存在。在暴露於缺氧環境下存活0～4小時後，均有大量被誘發增加的情形。在PVN內 nNOS的免疫反應平均增加約有49% ~ 73 %，而SON內 nNOS的免疫反應平均增加約有 56% ~ 99 %。
經雙重免疫組織化學染色標誌法來看，這些被激發出來具c-Fos免疫反應的神經元與VP或是nNOS的神經元間的關係。結果發現暴露於缺氧環境後存活四小時，在PVN，只有21.5±2.95％ 具c-Fos/nNOS 雙重免疫反應，或14.3±1.14％ 具 c-Fos/VP 雙重免疫反應。在SON神經元有類似的表現：c-Fos/VP (13.0±1.03 %) 及c-Fos/nNOS (23.1±3.98%)。雙重免疫組織化學染色同步的結果比例不高，顯示兩神經核中另有神經胜肽分泌物被激發，所以我們進行腎上腺髓質素(adrenomedullin；AM)及抑鈣素基因相關胜肽素(calcitonin gene related peptide；CGRP)等的西方墨點法以及影像分析，來了解缺氧對此些物質的影響。結果顯示此兩者的變化與上述幾種的免疫反應有相類似的分布趨勢。
經過去研究得知一氧化氮(Nitric oxide；NO)是一種氣體分子，它會透過神經訊息傳遞的方式來影響下視丘垂體路徑以調節體液平衡，但對於VP的調控究竟是抑制或是促進，至今仍有爭論。所以我們於進行缺氧之前，事先給予一氧化氮合成酶拮抗劑- N硝基左旋精胺酸(NG-nitro-L-arginine； LNNA)，來阻斷nNOS的生成，以了解 NO對 VP的調控情形。實驗結果顯示，有給予LNNA的組別，於低壓缺氧後存活1天，在PVN和SON中，nNOS的免疫反應相較於只有缺氧的組別有下降的趨勢，而VP的表現有增加的趨勢。所以我們認為在正常狀態下NO的生成對VP應有抑制的作用，以維持體液平衡。然而這作用是透過旁泌作用(paracrine)或是自泌作用(autocrine)來調節呢？ 經雙重免疫組織化學染色標誌法結果發現，具nNOS免疫反應的神經元，在PVN和SON，約只有10.9 ±2.19%和 14.3±1.12% 同時也具VP免疫反應，表示NO調節VP的分泌自泌作用不高，較可能經由旁泌作用來進行。另外，此些 nNOS/VP共存的神經元數目經低壓缺氧處理後數目 (11.2±2.25% 和 15.3±0.85% )也並無明顯增加。顯示在PVN 與 SON內這些 nNOS/VP共存的神經元可能不受缺氧的影響。
綜合以上研究結果知道，缺氧確實會激發下視丘內SON 與PVN 內大細胞神經元神經胜肽分泌物的生成(如，VP、AM和CGRP)，但缺氧並非對此些神經核內所有的神經元造成影響。而於正常環境下NO可能扮演調控下視丘內VP的生成，但於缺氧後VP的大量生成將造成類似高山症的症狀產生，所以下視丘內的NO將被迫也增加，以便回饋抑制VP的生成。此些結果將可提供對於低壓缺氧狀態下，下視丘內PVN與 SON中神經胜肽分泌物變化進一步的了解。
In the past, many researches have found that the body faced to lack of the blood, anoxia or pressure, often appeared to the condition of body fluid disequilibrium, such as thirsty, blood pressure rising and decrease of the urine. The magnocellular neurons of the paraventricular (PVN) and supraoptic (SON) nucleus in the hypothalamus have been implicated in aforementioned symptoms. Although we already know to contain various peptides in the PVN and SON of the hypothalamus, the role of those peptides plays in which interaction (exception vasopressin) still obscure so far. Thus, the purpose of this paper is using immunohistochemistry、western blotting and image analyzer to understand the effect of the hypobaric hypoxia on the change of the secretory products of the magnocellular neuron in the PVN and SON.
The first, we were used the c-fos as a marker, c-Fos as an immediately early gene, to identify the activated neurons following altitude exposure. At 0-4 hrs following altitude exposure, many neurons in the PVN and SON were induced to express Fos-like immunoreactivity. At 7 days, the expression of Fos-like immunoreactivity had subsided and was comparable to the corresponding controls.
In addition, the neuron numbers of the vasopressin (VP)-like immunoreactivity in the PVN and SON were also marked increase after 0-4 hrs following altitude exposure by using immunohistochemistry and western blotting. After image analyser statistical results to find out was increased around 24 ~46% in PVN and 33~76% in SON. At 7 days, the expression of VP-like immunoreactivity has parallel distribution of c-Fos-like immunoreactiviy which gradually fades away to return a normal value. In nitric oxide synthase (nNOS) immunoreactivity, we were found that a marked increase of the nNOS-like neurons in PVN or SON after 0-4 hrs following altitude exposure. Statistical results was shown that the immunoreactiviy optical density was increased around 49 ~ 73 % in PVN and 56~ 99% in SON.
Dual immunostaining for Fos/VP and Fos/ nNOS showed that Fos-like positive neurons in the PVN and SON was colocalized to VP or nNOS after 0-4 hrs following altitude exposure, but each other colocalization ration was not high (in PVN about 21.5 ± 2.95% for Fos/ nNOS and 14.3 ± 1.14% for Fos/VP；in SON about 23.1 ± 3.98% for Fos/ nNOS and 13.0 ± 1.03% for Fos/VP). The less frequency of the abovementioned neuronal subpopulations implies that still have other peptides in the hypothalamus may be activated following altitude exposure exception the VP and nNOS. Consequently, we were shown that the calcitonin gene related peptide (CGRP) and adrenomedullim (AM) immunoreactivity were also increase in PVN and SON after 0-4 hrs following altitude exposure.
Up to now, we well know that nitric oxide (NO) is a gas molecule that regulates the fluid balance through the signal neurotransmission in the hypothalamo- neurohypophysial system. In this present study, we pretreated with LNNA (60 mg/kg, i.p.) before exposed to hypobaric hypoxia to determinate the role of NO in the regulation of vasopressin expression in the magnocellular neurons of the paraventricular nucleus (PVN) and supraoptic nucleus (SON) following hypobaric hypoxia. We were found that nakedly decrease nNOS immunoreactivity comparable to the hypobaric hypoxic rats, but VP expression is markedly increased.
The present study showed that hypobaric hypoxia may induced the expression of the c-Fos gene, AM and CGRP in the PVN and SON. Meanwhile this induced was also significantly increasing the nNOS which may play a role of the negative feedback to inhibit the VP release. These results suggest that the hypobaric hypoxia may activate various peptides in the PVN and SON to regulate the fluid balance. |
