內毒素是一種脂多醣（lipopolysaccharide）是來自革蘭氏陰性菌（gram negative bacterium）細胞壁之具有毒性磷酸化醣脂質的衍生物，會透過活化免疫細胞（如巨噬細胞、單核球）開啟NF-κB途徑產生pro-inflammatory cytokine（如interleukin-1β、interleukin-6與tumor necrosis factor-α等）、chemokine、氧自由基、前列腺素等，也可刺激感覺神經C-fiber釋出速激肽（tachykinins）。發炎物質的釋放會造成被感染的宿主引起全身性的發炎反應，以及器官功能喪失等症狀。主要的症狀為血漿外洩增大、黏膜組織皮下水腫、細胞內皮隙的形成與黏液細胞分泌增多等等。Urethan為動物實驗常用麻醉劑，也是α2-腎上腺素受器之拮抗劑，因而可藉此抑制心血管系統的活性，降低血管收縮素（angiotensin）對血壓增加的效果。其也會抑制經由LPS誘發釋放出tumor necrosis factor-α的量。因此本實驗目的是在探討：（1）在LPS誘導的發炎反應在不同時間點所造成之血漿滲漏、水腫、內皮隙的形成的變化等是否涉及α2-adrenoreceptors。（2）以dimethylthiourea（DMTU）前處理，研究LPS引起的血漿滲漏與水腫是否涉及羥自由基機制。實驗中，透過股靜脈注射印地安染劑標定氣管、左右主支氣管、食道之滲漏血管，利用銀染灌流法標定展現內皮細胞間的內皮隙，與另作氣管組織切片，觀察組織結構之變化。實驗數據顯示，內毒素可引發呼吸道與食道嚴重血漿外洩與氣管水腫，以五分鐘時最嚴重，並且造成微血管後小靜脈與收集血小靜脈形成大量內皮隙，透過顯微觀察時可見許多白血球的浸潤與黏附。當以urethan麻醉或前處理DMTU後，則分別可抑制 95 ± 1.7％、88.5 ± 2.5％ 的血漿外洩量與57％、89％ 氣管水腫率，同時 urethan也可減少白血球浸潤與46.8 ± 4.6％ 的內皮隙產生。由實驗結果推論，內毒素可以透過活化α2-腎上腺素受器與促使羥自由基產生，快速的造成內皮隙的形成、增加血管的通透性，進而導致嚴重的血漿外洩與水腫產生。

Endotoxin (lipopolysaccharide, LPS) a chemical component of cell wall of gram-negative bacteria, is an important mediator in pathogenesis of sepsis and acute respiratory distress syndrome. It causes production and release of a wide array of mediators including cytokines, chemokines, oxygen free radicals and nitric oxide from neutrophils, macrophages, endothelial cells and epithelial cells through the NF-κB pathway. LPS increases the permeability of microcirculation, and causes the acute formation of numerous endothelial gaps among venular endothelial cells, resulting in extensive plasma leakage in the inflammatory tissue. Urethan is commonly used as an animal anesthetic for nonrecovery laboratory surgery. It is aslo an α2-adrenoreceptor antagonist, which can suppress the activation of the cardiovascular system and reduce the angiotensin which increases the blood pressure. Urethan or its metabolites protect animals against LPS, in part, by reducing TNF-α release. The aims of the present study to investigate the time-course of vascular permeability in microcirculation of rat trachea, bronchus and esophagus after intravenous application of a high dose of LPS (15 mg/kg), and to reveal the role of urethan (1 g/㎏) and dimethylthiourea (DMTU, 0.375 g/㎏) in inhibition of LPS-induced plasma leakage and edema. India ink was used as a tracer dye to mark leaky microvessels after LPS application. Endothelial gaps were made visible for light microscopy by staining the borders of endothelial cells with silver nitrate. Tracheal sections were stained with toluidine blue to show the subendothelial edema formation. A high dose of LPS was administered intravenously to induce serious plasma leakage and edema and a large number of endothelial gaps formed in postcapillary and collecting venules in the rat trachea and esophagus. The peak values of plasma leakage and edema occurred 5 min after LPS (P<0.01). Urethan anesthesia significantly inhibited LPS-induced plasma leakage by 95 ± 1.7% in various parts of the respiratory tracts and inhibited edema ratio in the trachea by 57%. Urethan was also found to reduce leukocyte infiltration and the number of endothelial gaps by 46.8 ± 4.6%. DMTU pretreatment significantly inhibited plasma leakage by 88.5 ± 2.5% in the respiratory tract and inhibited edema ratio in the trachea by 89% at 5 min after LPS. It is concluded that LPS-induced increase in plasma leakage and edema correlated with the formation of endothelial gaps, and association with activation of alpha 2-adrenergic receptors and hydroxyl free radical production.

【中文摘要】……………………………………………………………1
【Abstract】……………………………………………………………3
【緒論】…………………………………………………………………5
一、內毒素（Endotoxin）…………………………………………… 5
A.內毒素致病機轉………………………………………………………5
B.內毒素耐受性的產生…………………………………………………8
二、Urethan的藥物特性……………………………………………… 9
三、自由基（Free radicals）的作用機轉…………………………11
四、發炎反應引起的血漿外洩……………………………………… 15
五、呼吸道中的杯狀細胞…………………………………………… 18
【研究目的】………………………………………………………… 19
【材料與方法】……………………………………………………… 20
一、實驗動物………………………………………………………… 20
二、藥物與溶液製配………………………………………………… 20
三、動物實驗與標本處理過程……………………………………… 23
A.股靜脈注射內毒素誘發呼吸道、食道發炎反應………………… 23
B.腹膜腔注射 urethan，探討其抑制作用………………………… 24
C.靜脈注射羥自由基清除劑，探討其抑制作用…………………… 24
D.灌流固定…………………………………………………………… 24
E.銀染灌流固定……………………………………………………… 25
F.整體封埋標本製作………………………………………………… 26
G.塑膠組織切片的製作……………………………………………… 26
H.Alcian blue-PAS染色觀察杯狀細胞………………………………27
I.Haematoxylin and Eosin 染色……………………………………28
四、血漿滲漏、水腫程度與內皮隙數目之分析…………………… 28
A.氣管上皮下端水腫程度之量化分析……………………………… 28
B.氣管與食道血漿外洩程度之測定：以印地安染劑標示血管面積密度表示之……………………………………………………………… 29
C.銀染色法標示發炎血管內皮隙計量分析………………………… 30
五、統計分析………………………………………………………… 30
【結果】……………………………………………………………… 31
一、靜脈注射內毒素引發大鼠呼吸道血漿滲漏…………………… 31
二、腹膜腔注射 urethan 對內毒素引起血漿外洩的影響…………33
三、靜脈注射 DMTU 對內毒素引起血漿外洩的影響……………… 35
四、靜脈注射內毒素誘導大鼠氣管黏膜發生之病變……………… 35
五、腹膜腔注射 urethan 對 LPS 引起的大鼠氣管黏膜水腫的抑制效應…………………………………………………………………… 36
六、DMTU 前處理對大鼠氣管黏膜水腫之抑制效應…………………36
七、銀染色法標示內毒素誘發之微循環所形成血管內皮隙……… 37
八、氣管組織 Alcain blue-PAS…………………………………… 39
【討論】……………………………………………………………… 40
一、靜脈注射內毒素引發大鼠呼吸道與食道血漿外洩…………… 40
二、Urethan抑制LPS引發大鼠呼吸道與食道血漿滲漏…………… 41
三、DMTU抑制LPS引發大鼠呼吸道與食道微循環血漿滲漏…………45
四、靜脈注射內毒素引起小靜脈內皮組織之內皮隙血漿滲漏之關係與urethan對其抑制效應………………………………………………47
五、氣管病理組織變化……………………………………………… 48
A.水腫的變化……..………………………………………………… 48
B.白血球與杯狀細胞的反應………………………………………… 50
【結論】……………………………………………………………… 52
【

Armstrong JM, Lefevre-Borg F, Scatton B, and Cavero I 1982. Urethane inhibits cardiovascular responses mediated by the stimulation of alpha-2 adrenoceptors in the rat. J. Pharmacol. Exp. Ther. 223, 524-535
Balligand JL and Cannon PJ 1997. Nitric oxide synthases and cardiac muscle : autocrine and paracrine influences. Arterioscler. Thromb. Vasc. Biol. 17, 1846-1858
Baluk P and McDonald DM 1994. The beta 2-adrenergic receptor agonist formoterol reduces microvascular leakage by inhibiting endothelial gap formation. Am. J. Physiol. 266, L461-L468
Baluk P, Fine NW, Thomas HA, Wei ET, and Mcdonald DM 1998. Anti-inflammatory mystixin peptides inhibit plasma leakage without blocking endothelial gap formation. J. Pharmacol. Exp. Ther. 284, 693-699
Barnes PJ, Chung KF, and Page CP 1998. Inflammatory mediators of asthma: an update. Pharmacol. Rev. 50, 515-596
Bernik TR, Friedman SG, Ochani M, DiRaimo R, Ulloa L, Yang H, Sudan S, Czura CJ, Ivanova SM, and Tracey KJ 2002. Pharmacological stimulation of the cholinergic antiinflammatory pathway. J. Exp. Med. 195, 781-788
Bohuslav J, Kravchenko VV, Parry GCN, Erlich JH, Gerondakis S, Mackman N, and Ulevitch RJ 1998. Regulation of an essential innate immune response by the p50 subunit of NF-kappa B. J. Clin. Invest. 102, 1645-1652
Bolser DC, Del Prado M, O'Reilly S, Mingo G, and Hey JA 1995. Evan's blue dye blocks capsaicin-induced cough and bronchospasm in the guinea pig. Eur. J. Pharmacol. 276, R1-3
Borovikova LV, Ivanova S, Zhang M, Yang H, Botchkina GI, Watkins LR, Wang H, Abumrad N, Eaton JW, and Tracey KJ 2000. Vagus nerve stimulation attenuates the systemic inflammatory response to endotoxin. Nature 405, 458-462

Chuang FC ( 莊豐竹 ) 2004. 6-hydroxydopamine-induced inflammation in respiratory tract and esophagus of rats and it’s inhibition by free radical scavenger. 國立中山大學生物科學所碩士論文.
DeForge LE, Fantone JC, Kenney JS, and Remick DG 1992. Oxygen radical scavengers selectively inhibit interleukin 8 production in human whole blood. J. Clin. Invest. 90, 2123-2129
Devasagayam TP, Kamat JP, Mohan H, and Kesavan PC 1996. Caffeine as an antioxidant: inhibition of lipid peroxidation induced by reactive oxygen species. Biochim. Biophys. Acta 1282, 63-70
Dusser DJ, Djokic TD, Borson DB, and Nadel JA 1989(a). Cigarette smoke induces bronchoconstrictor hyperresponsiveness to substance P and inactivates airway neutral endopeptidase in the guinea pig. Possible role of free radicals. J. Clin. Invest. 84, 900-906
Dusser DJ, Jacoby DB, Djokic TD, Rubinstein I, Borson DB, and Nadel JA 1989(b). Virus induces airway hyperresponsiveness to tachykinins: role of neutral endopeptidase. J. Appl. Physiol. 67, 1504-1511
Farmer P and Pugin J 2000. Beta-adrenergic agonists exert their "anti-inflammatory" effects in monocytic cells through the IkappaB/NF-kappaB pathway. Am. J. Physiol. Lung Cell. Mol. Physiol. 279, L675-L682
Fessler HE, Otterbein L, Chung HS, and Choi AM 1996. Alpha-2 adrenoceptor blockade protects rats against lipopolysaccharide. Am. J. Respir. Crit. Care Med. 154, 1689-1693
Forray C, Bard JA, Wetzel JM, ChiuG., Shapiro E, Tang R, Lepor H, Hartig PR, Weinshank RL, and Branchek TA 1994. The alpha 1-adrenergic receptor that mediates smooth muscle contraction in human prostate has the pharmacological properties of the cloned human alpha 1c subtype. Mol. Pharmacol. 45, 703-708
Fujii Y, Magder S, Cernacek P, Goldberg P, Guo Y, and Hussain SN 2000. Endothelin receptor blockade attenuates lipopolysaccharide-induced pulmonary nitric oxide production. Am. J. Respir. Crit. Care Med. 161, 982-989
Fujihara M, Muroi M, Tanamoto K, Suzuki T, Azuma H, and Ikeda H 2003. Molecular mechanisms of macrophage activation and deactivation by lipopolysaccharide: roles of the receptor complex. Pharmacol. Ther. 100, 171-194
Guo JJ, Wang DS, and Huang HT 2003. Spontaneous remission of edema and regranulation of goblet cells in rat tracheae after capsaicin-induced acute inflammation. Anat. Embryol. (Berl) 206, 301-309
Hara K and Harris RA 2002. The anesthetic mechanism of urethane: the effects on neurotransmitter-gated ion channels. Anesth. Analg. 94, 313-8
Heagy W, Hansen C, Nieman K, and West MA 2003. Evidence for a CD14- and serum-independent pathway in the induction of endotoxin-tolerance in human monocytes and THP-1 monocytic cells. Shock 19, 321-327
Hirano S, Rees RS, Yancy SL, Welsh MJ, Remick DG, Yamada T, Hata J, and Gilmont RR 2004. Endothelial barrier dysfunction caused by LPS correlates with phosphorylation of HSP27 in vivo. Cell Biol. Toxicol. 20, 1-14
Hoffler U, El-Masri HA, and Ghanayem BI 2003. Cytochrome P450 2E1 (CYP2E1) is the principal enzyme responsible for urethane metabolism: comparative studies using CYP2E1-null and wild-type mice. J. Pharmacol. Exp. Ther. 305, 557-564
Hoffler U and Ghanayem BI 2005. Increased bioaccumulation of urethane in CYP2E1-/- versus CYP2E1+/+ mice. Drug Metab. Dispos. 33, 1144-1150
Huang HY and Lai YL 2003. Lipopolysaccharide induces preprotachykinin gene expression. Am. J. Respir. Cell Mol .Biol. 29, 606-612
Ignatowski TA, Kunkel SL, and Spengler RN 2000. Interactions between the [alpha]2-adrenergic and the prostaglandin response in the regulation of macrophage-derived tumor necrosis factor. Clinical Immunology 96, 44-51
Ignarro LJ, Byrns RE, Trinh K, Sisodia M, and Buga GM 2002. Nebivolol: a selective beta(1)-adrenergic receptor antagonist that relaxes vascular smooth muscle by nitric oxide- and cyclic GMP-dependent mechanisms. Nitric Oxide 7, 75-82

Izzo NJ Jr, Seidman CE, Collins S, and Colucci WS 1990. Alpha1-adrenergic receptor mRNA level is regulated by norepinephrine in rabbit aortic smooth muscle cells. PNAS 87, 6268-6271
Kadoi Y, Saito S, Kawahara F, Nishihara F, and Goto F 2002. G-Protein coupled receptor kinase 2 is altered during septic shock in rats. J. Surg. Res. 108, 69-76
Kim JH, Lee SY, Bak SM, Suh IB, Lee SY, Shin C, Shim JJ, In KH, Kang KH, and Yoo SH 2004. Effects o f matrix metalloproteinase inhibitor on LPS-induced goblet cell metaplasia. Am. J. Physiol. Lung Cell. Mol. Physiol. 287, L127-33
Kisley LR, Barrett BS, Bauer AK, Dwyer-Nield LD, Barthel B, Meyer AM, Thompson DC, and Malkinson AM 2002. Genetic ablation of inducible nitric oxide synthase decreases mouse lung tumorigenesis. Cancer Res. 62, 6850-6856
Koh Y, Lee YM, Lim CM, Lee SS, Shim TS, Lee SD, Kim WS, Kim DS, and Kim WD 2001. Effects of heat pretreatment on histopathology, cytokine production, and surfactant in endotoxin-induced acute lung injury. Inflammation 25, 187-196
Kotanidou A, Choi AM, Winchurch RA, Otterbein L, and Fessler HE 1996. Urethan anesthesia protects rats against lethal endotoxemia and reduces TNF-alpha release. J. Appl. Physiol. 81, 2305-2311
Landmann R, Knopf HP, Link S, Sansano S, Schumann R, and Zimmerli W 1996. Human monocyte CD14 is upregulated by lipopolysaccharide. Infect. Immun. 64, 1762-1769
Lim LH, Bochner BS, and Wagner EM 2002. Leukocyte recruitment in the airways: an intravital microscopic study of rat tracheal microcirculation. Am. J. Physiol. Lung Cell. Mol. Physiol. 282, 959-967
Lin YS and Kou YR 2000. Acute neurogenic airway plasma exudation and edema induced by inhaled wood smoke in guinea pigs: role of tachykinins and hydroxyl radical. Eur. J. Pharmacol. 394, 139-148
Margreet RO, Peter C. HH, and Martijn BK 2001. Chlorogenic acid and caffeic acid are absorbed in humans. J. Nutr. 131, 66-71
McDonald DM 1994. Endothelial gaps and permeability of venules in rat tracheas exposed to inflammatory stimuli. Am. J. Physiol. 266, L61-83
McDonald DM, Thurston G, and Baluk P 1999. Endothelial gaps as sites for plasma leakage in inflammation. Microcirculation 6, 7-22
Morimoto A, Hasegawa H, Cheng HJ, Little WC, and Cheng CP 2004. Endogenous {beta}3-adrenoreceptor activation contributes to left ventricular and cardiomyocyte dysfunction in heart failure. Am. J. Physiol. Heart Circ. Physiol. 286, 2425-2433
Nomura T 1975. Letter: Transmission of tumors and malformations to the next generation of mice subsequent to urethan treatment. Cancer Rec. 35, 264-6
Nagase T, Uozumi N, Aoki-Nagase T, Terawaki K, Ishii S, Tomita T, Yamamoto H, Hashizume K, Ouchi Y, and Shimizu T 2003. A potent inhibitor of cytosolic phospholipase A2, arachidonyl trifluoromethyl ketone, attenuates LPS-induced lung injury in mice. Am. J. Physiol. Lung Cell. Mol. Physiol. 284, L720-6
Paller MS, Hoidal JR, and Ferris TF 1984. Oxygen free radicals in ischemic acute renal failure in the rat. J. Clin. Invest. 74, 1156-64
Park SM, Jung HC, Koak IS, Na HY, Woo JS, Jung JS, and Kim YK 2003. Oxidant-induced cell death in renal epithelial cells: differential effects of inorganic and organic hydroperoxides. Pharmaco.l Toxicol. 92(1), 43-50
Pavlov VA, Ochani M, Gallowitsch-Puerta M, Ochani K, Huston JM, Czura CJ, Al-Abed Y, and Tracey KJ 2006. Central muscarinic cholinergic regulation of the systemic inflammatory response during endotoxemia. PNAS 103, 5219-5223
Peters TS and Lewis SJ 1996. Lipopolysaccharide inhibits acetylcholine- and nitric oxide-mediated vasodilation in vivo. J. Pharmacol. Exp. Ther. 279, 918-925
Perkins GD, McAuley DF, Richter A, Thickett DR, and Gao F 2004. Bench-to-bedside review: beta2-Agonists and the acute respiratory distress syndrome. Crit.Care 8, 25-32

Pleiner J, Heere-Ress E, Langenberger H, Sieder AE, Bayerle-Eder M, Mittermayer F, Fuchsjager-Mayrl G, Bohm J, Jansen B, and Wolzt M 2002. Adrenoceptor hyporeactivity is responsible for Escherichia coli endotoxin–induced acute vascular dysfunction in humans. Arterioscler. Thromb. vasc. Biol. 22, 95-100
Pleiner J, Mittermayer F, Schaller G, MacAllister RJ, and Wolzt M 2002. High doses of vitamin C reverse Escherichia coli endotoxin-induced hyporeactivity to acetylcholine in the human forearm. Circulation 106, 1460-1464
Rohrer DK, Desai KH, Jasper JR, Stevens ME, Regula DP Jr, Barsh GS, Bernstein D, and Kobilka BK 1996. Targeted disruption of the mouse beta 1-adrenergic receptor gene: Developmental and cardiovascular effects. PNAS 93, 7375-7380
Schletter J, Heine H, Ulmer AJ, and Rietschel ET 1995. Molecular mechanisms of endotoxin activity. Arch. Microbiol. 164,383-389
Shimizu T, Hirano H, Majima Y, and Sakakura Y 2000. A mechanism of antigen-induced mucus production in nasal epithelium of sensitized rats. A comparison with lipopolysaccharide-induced mucus production. Am. J. Respir. Crit. Care Med. 161, 1648-1654
Simpson P 1985. Stimulation of hypertrophy of cultured neonatal rat heart cells through an alpha 1-adrenergic receptor and induction of beating through an alpha 1- and beta 1-adrenergic receptor interaction. Evidence for independent regulation of growth and beating. Circulation Research, 56, 884-894,
Tasaka S, Ishizaka A, Yamada W, Shimizu M, Koh H, Hasegawa N, Adachi Y, and Yamaguchi K 2003. Effect of CD14 blockade on endotoxin-induced acute lung injury in mice. Am. J. Respir. Cell Mol. Biol. 29, 252-258
Toward TJ and Broadley KJ 2001. Chronic lipopolysaccharide exposure on airway function, cell infiltration, and nitric oxide generation in conscious guinea pigs: effect of rolipram and dexamethasone. J. Pharmacol. Exp. Ther. 298, 298-306
Toward TJ and Broadley KJ 2002. Goblet cell hyperplasia, airway function, and leukocyte infiltration after chronic lipopolysaccharide exposure in conscious Guinea pigs: effects of rolipram and dexamethasone. J. Pharmacol. Exp. Ther. 302, 814-821
Tracey KJ 2002. The inflammatory reflex. Nature 420, 853-859
Walker PD and Shah SV 1990. Reactive oxygen metabolites in endotoxin-induced acute renal failure in rats. Kidney Int. 38(6), 1125-32.
Wang HL and Voelkel NF 1989. Norepinephrine induces lung vascular prostacyclin synthesis via alpha 1-adrenergic receptors. J. Appl. Physiol. 67, 330-338
Wang H, Yu M, Ochani M, Amella CA, Tanovic M, Susarla S, Li JH, Wang H, Yang H, Ulloa L, Al-Abed Y, Czura CJ, and Tracey KJ 2003. Nicotinic acetylcholine receptor alpha7 subunit is an essential regulator of inflammation. Nature 421, 384-8.
Ward PA, Till GO, Kunkel R, and Beauchamp C 1983. Evidence for role of hydroxyl radical in complement and neutrophil-dependent tissue injury. J. Clin. Invest. 72, 789-801
Weiss M, Schneider EM, Tarnow J, Mettler S, Krone M, Teschemacher A, and Lemoine H 1996. Is inhibition of oxygen radical production of neutrophils by sympathomimetics mediated via beta-2 adrenoceptors? J. Pharmacol. Exp. Ther. 278, 1105
Zhang H, Kim YK, Govindarajan A, Baba A, Binnie M, Marco Ranieri V, Liu M, and Slutsky AS 1999. Effect of adrenoreceptors on endotoxin-induced cytokines and lipid peroxidation in lung explants. Am. J. Respir. Crit. Care Med. 160, 1703-1710
Zurovsky Y and Gispaan I 1995. Antioxidants attenuate endotoxin-induced acute renal failure in rats. Am. J. Kidney Dis. 25(1), 51-7