延腦鼻端腹外側核（rostral ventrolateral medulla; RVLM）是一個在腦幹死亡的過程中，可以反映出腦幹調控心血管能力的“生與死”訊號的神經核區。因此，RVLM是一個適合探討腦幹死亡過程中細胞分子機制的神經核區。由於在台灣每年有許多有機磷中毒的案例發生，所以我們利用大鼠的美文松（一種有機磷農藥）中毒模式來研究腦幹死亡的機制。本篇論文想要探討以下兩個假設，第一個假設是在美文松中毒所引起的腦幹死亡模式中，是否透過刺激RVLM內phosphoinositide 3-kinases (PI3K)/Akt/phosphatase and tensin homologue deleted on chromosome ten (PTEN)訊息傳遞路徑，並藉由核轉錄因子（NF-κB）的活化來促進一氧化氮合成酶第一亞型（nitrict oxide synthase I; NOS I）或是第二亞型（NOS II）的表現？第二個假設是RVLM內cyclic adenosine monophosphate-dependent protein kinase A (PKA)是否透過不經由蕈毒鹼受體（muscarinic receptor）活化的訊息傳遞路徑參與在美文松中毒所引起的心血管反應？
我們以Sprague-Dawley品系大鼠為實驗對象，實驗結果顯示，當微量注射美文松（10 nmol）至大鼠的RVLM中會誘發RVLM內NF-κB，PI3K，Akt與PTEN的活性增加，以及NOS II與過亞硝酸根（peroxynitrite）的表現增加。當我們阻斷RVLM內NF-κB， PI3K或是Akt的活性後，發現能有意義的加強並延長“生與死”訊號在一開始的增加，以及恢復美文松所引起的低血壓現象，並且能夠抑制美文松所誘導的NOS II與nitrotyrosine的表現。另外，當我們阻斷了RVLM內PI3K或Akt之後，發現亦能夠阻止美文松所誘發的NF-κB活化。然而，當我們阻斷PTEN的功能後，反而有意義地減少美文松在一開始所引起的“生與死”訊號的增加，以及減少美文松誘發的Akt活化。因此，我們認為在RVLM內PI3K/Akt訊息傳遞路徑在我們的美文松中毒引起的腦死模式裡扮演“pro-death”的角色，並且是透過增進NF-κB的活化，來促進RVLM內NOS II與peroxynitrite的表現。然而，PTEN在這個過程中扮演著拮抗者的角色。
在第二個假設探討中，當我們微量注射美文松至大鼠的RVLM內，會誘發RVLM內PKA活性有意義地增加；然而，合併注射美文松與蕈毒鹼受體的抑制劑之後，並不影響美文松誘導的PKA的活化。當我們阻斷RVLM內PKA的活性後，發現能有意義地減少美文松在一開始所引起的“生與死”訊號的增加，並且能夠抑制美文松所誘導的NOS I的表現。因此，我們認為在RVLM內，不需要蕈毒鹼受體活化的PKA在美文松中毒引起的腦死模式裡扮演“pro-life”的角色，並且是透過增進RVLM內NOS I與protein kinase G（PKG）的表現來調控美文松引起的心血管反應。
本篇論文證實了美文松同時會引起不同的機制，藉由個別刺激muscarinic receptor-independent/PKA以及PI3K/Akt/NF-κB路徑來調控NOS I 與NOS II的表現，並引起在“pro-life”與“pro-death”時期的心血管反應。本篇論文在腦幹死亡過程中，腦幹對心血管調控的機制提供了進一步的細胞分子層次探討，並且對於未來有機磷中毒與腦幹死亡治療方法與策略，提供了新的思維。

As the origin of a “life-and-death” signal that reflects central cardiovascular regulatory failure during brain stem death, the rostral ventrolateral medulla (RVLM) is a suitable neural substrate to evaluate the cellular mechanism of this fateful phenomenon. Based on a clinically relevant animal model that employed the organophosphate pesticide mevinphos (Mev) as the experimental insult, this study evaluated two hypotheses. First, transcriptional upregulation of nitric oxide synthase I or II (NOS I or II) gene expression by nuclear factor-κB (NF-κB) on activation of phosphoinositide 3-kinases (PI3K)/Akt/phosphatase and tensin homologue deleted on chromosome ten (PTEN) cascade in the RVLM underlies brain stem death. Second, muscarinic receptor-independent activation of cyclic adenosine monophosphate-dependent protein kinase A (PKA) in the RVLM is involved in the cardiovascular responses exhibited during Mev intoxication.
In Sprague-Dawley rats, our results showed that microinjection bilaterally of Mev (10 nmol) into RVLM induced a progressive augmentation in NF-κB, PI3K, Akt or PTEN activity that paralleled the increase in NOS II or peroxynitrite level in RVLM. Loss-of-function manipulations that included pharmacological blockade, gene knockdown, or immunoneutralization of NF-κB, PI3K or Akt in RVLM significantly potentiated and prolonged the initial increase in “life-and-death” signal, reversed the cardiovascular depression, and blunted the augmented expression of NOS II or nitrotyrosine on induced by Mev. Blockade of PI3K or Akt in RVLM also significantly blunted the Mev-induced activation of NF-κB in the RVLM. However, immunoneutralization of PTEN in RVLM significantly diminished the increase in “life-and-death” signal and potentiated the increase in Akt activity. We conclude that the PI3K/Akt cascade plays a “pro-death” role in our Mev intoxication model of brain stem death by upregulating NF-κB/NOS II/peroxynitrite in the RVLM, subject to antagonism by PTEN in this process.
Microinjection bilaterally of Mev (10 nmol) into the RVLM induced a significantly augmentation in PKA activity in ventrolateral medulla that was not antagonized by coadministration of a nonselevtive or selective muscarinic receptor inhibitor. However, pharmacological blockade PKA in RVLM significantly blunted the initial increase in “life-and-death” signal and the accompanying augmentation of NOS I expression in the ventrolateral medulla exhibited during Mev intoxication. We conclude that a muscarinic receptor-independent activation of PKA plays a “pro-life” role in our Mev intoxication model of brain stem death by up regulating NOS I/PKG in the RVLM.
According to this study, we proved that Mev stimulates different mechanism, muscarinic receptor-independent/PKA and PI3K/Akt/NF-κB, to regulate NOS I and NOS II expression respectively, and induces cardiovascular responses during “pro-life” and “pro-death” phases. This information should provide further insights on the cellular mechanism of central cardiovascular regulation during the progression towards brain stem death, and offer news vistas in our search for therapeutic remedies or management strategies against fatal organophosphate poisoning and brain stem death.

摘要 I
Abstract IV
Content VII
List of Figures and Tables X
Abbreviations XIV
Chapter 1. Introduction 1
1.1 Brain stem death 2
1.2 Power density of vasomotor components of systemic arterial pressure signals 2
1.3 Rostral ventrolateral medulla (RVLM) 3
1.4 Organophosphate 4
1.4.1 Mechanism of action 4
1.4.2 Classification 5
1.4.3 Clinical symptoms 5
1.4.4 Clinical therapy 5
1.4.5 Mevinphos 6
1.5 Mevinphos intoxication model of brain stem death 6
1.6 Nuclear factor-kB (NF-kB) 7
1.7 Phosphoinositide 3-kinases (PI3K) 7
1.7.1 PI3K classification 8
1.7.2 PI3K signal transduction 8
1.8 Akt 9
1.9 Phosphatase and tensin homologue deleted on chromosome ten
(PTEN) 9
1.10 Cyclic adenosine monophosphate-dependent protein kinase A (PKA) 10
Chapter 2. Rationale and Research Aim 16
2.1 Rationale 17
2.2 Research aim 17
Chapter 3. Materials and Methods 19
3.1 General preparation of animals 20
3.2 Recording of cardiovascular parameters 20
3.3 Microinjection of test agents into the RVLM 21
3.4 Collection of tissue samples from ventrolateral medulla 22
3.5 Protein excitation 22
3.6 Measuremant of NF-kB transcription activation 23
3.7 Measurement of enzyme activity 23
3.7.1 Measurement of PI3K activity 23
3.7.2 Measurement of Akt activity 23
3.7.3 Measurement of PTEN activity 24
3.7.4 Measurement of PKA activity 24
3.8 Western blot analysis 24
3.9 Electrophoretic mobility shift assay (EMSA) 25
3.10 Real-time polymerase chain reaction (real-time PCR) 25
3.11 Statistical analysis 26
Chapter 4. “Pro-death” Role for PI3K/Akt/NF-kB Cascade at the RVLM and Its Antagonism by PTEN in a Mevinphos Intoxication Model of Brain Stem Death 30
4.1 Introduction 31
4.2 Results 31
4.3 Discussion 37
Chapter 5. Muscarinic Receptor-independent Activation of PKA in RVLM Underlies the “Pro-life” Phase of Cardiovascular Responses during Mevinphos Intoxication Model of Brain Stem Death 71
5.1 Introduction 72
5.2 Results 72
5.3 Discussion 75
Chapter 6. Conclusion and Future Work 81
6.1 Conclusion 82
6.2 Future work 83
References 86
Appendix 108

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