臺灣博碩士論文加值系統

長期間歇性低氧，如模擬發生睡眠呼吸中止之情況，與許多心血管疾病之發生息息相關。而間歇性低氧之給予，為一缺氧再獲氧的過程，此氧分壓之改變會經由周邊化學接受器中頸動脈體所偵測，研究顯示，在獲氧的過程中，會有大量反應性氧衍生物釋出，推測此衍生物可能參與於反覆低氧刺激頸動脈體後所引發之化學反射及心血管反應。而目前對於長期間歇性低氧所引發化學反射及心血管反應之連續性變化和機轉，在清醒動物模式下尚未明確。故本實驗欲探討在清醒大鼠給予連續30天長期間歇性低氧暴露下，其化學反射反應為何?而這化學反射與所引發之心血管反應是否有所相關?再者，給予大鼠反應性氧衍生物之驅除物，其在長期間歇性低氧暴露下所引發之反射反應是否有所變化?
實驗採用Sprague-Dawley大鼠於其睡眠期間給予暴露間歇性低氧 (75秒/次，6小時/天，連續30天) 。利用小動物體積描記系統，給予空氣 (氧濃度21%) 連續5分鐘後再給12%急性低氧刺激連續5分鐘，測量清醒大鼠對吸入氣體所引發之呼吸頻率、潮氣容積及肺通氣量變化，視為化學反射反應，且使用血壓無線遙測記錄每日血壓訊號，分析血壓變異性及心率變異性，以評估自主神經功能。此外，給予腹腔注射超氧陰離子基 (MnTMPyP) 及氫氧自由基 (DMTU) 之驅除物，比較這些驅除物對於長期間歇性低氧所造成之化學反射與心血管反應是否有所差異。
本實驗結果顯示，給予間歇性低氧暴露組大鼠相較於空氣組，在第5-6天開始血壓及交感神經活性明顯增加，且同時在化學反射反應有所增加，測量肺組織脂質過氧化反應亦相較於空氣組嚴重，而事先給予超氧陰離子基驅除物，則可抑制間歇性低氧所引發之上述反應，然給予氫氧自由基之驅除物則無法抑制間歇性低氧所引發之化學反射反應，卻可降低血壓及交感神經活性作用。此外，測量各組之基礎代謝率則無顯著改變。由本實驗結果，經在時間點之變化性，可間接推測長期間歇性所引發之血壓上升現象與化學反射反應提升及交感神經活性增強有關，而反應性氧衍生物參與於長期間歇性低氧過程中扮演一重要角色。

The pattern of chronic intermittent hypoxia (CIH) is a characteristic of pathological conditions such as obstructive sleep apnea and is associated with serious cardiovascular morbidity. CIH produces the oscillation of arterial blood oxygen levels which are monitored continuously by peripheral chemoreceptors, especially the carotid bodies. CIH seems to resemble ischemia-reoxygenation, wherein reactive oxygen species (ROS) released during the intervening normoxic periods. Several evidences suggest that ROS may involved in the repeated carotid body stimulation under CIH, which led to chemoreflex and cardiovascular responses. However, the time course changes and ROS-dependent mechanisms in the chemoreflex and cardiovascular responses to CIH are not clear. In this study, we examined whether CIH (6 h/day, for 30 days) affects in chemoreflex and cardiovascular responses in conscious rats and, if so, determine whether superoxide anion and hydroxyl radical play a critical role in mediating these cardiorespiratory responses.
Adult male Sprague-Dawley rats were exposed to CIH (75 s/episode, 6 h/day, for 30 days) during sleep period. Blood pressure signals were measured daily by the telemetry system, which were used to assess the autonomic function by heart rate variability analysis. Ventilatory responses during room air (RA) breathing, an index for tonic chemoreflex activation, and 12% O2 (acute hypoxia) breathing, an index for phasic chemoreflex, were monitored daily. Mean arterial blood pressure (MABP) and normalized low-frequency power of pulses interval spectrogram (LF%), the marker of cardiac sympathetic outflow, were significantly higher in IH-exposed rats, as compared that of RA-exposed rats. Additionally, elevation of the MABP, LF%, and minute ventilation started 5-6 days after IH exposure and lasted until the end of the observation period. Pretreatment with MnTMPyP (a superoxide anion scavenger) prevented CIH-induced these responses and lipid peroxidation in lung tissue. In CIH-exposed rats pretreated with dimethylthiourea (DMTU; a hydroxyl radical scavenger), the enhanced minute ventilation was not significantly affected, whereas the elevated MABP and LF% were reduced. However, no significant difference in the metabolic rate were observed among groups. These results suggest that CIH-induced hypertension is associated with chemoreflex activation and facilitation of sympathetic outflow, and ROS plays an important role in CIH-induced these responses.

目錄

中文摘要…………………………………………………………………………I
英文摘要…………………………………………………………………………II
目錄………………………………………………………………………………III
壹、緒言
重要性……………………………………………………………………．1
背景知識
一、長期間歇性低氧……………………………………………………3
二、長期間歇性低氧對生理之影響……………………………………3
三、長期間歇性低氧引發週邊化學反射反應之變化…………………5
四、長期間歇性低氧引發心血管反應之變化…………………………8
五、長期間歇性低氧引發反應性氧衍生物之產生……………………10
六、反應性氧衍生物參與於長期間歇性低氧所引發心血管反應過
程之可能性……………………………………………………… 12
七、研究目的……………………………………………………………12
貳、 實驗材料與方法
實驗方法
一、實驗動物……………………………………………………………14
二、血壓訊號無線遙測器之埋設手術…………………………………14
三、間歇性低氧模式……………………………………………………15
四、正常氧分壓模式……………………………………………………15
五、血壓之測量…………………………………………………………16
六、自主神經功能之分析………………………………………………16
七、週邊化學反射反應之測量…………………………………………17
八、代謝速率之測量
(1)耗氧率……………………………………………………………18
(2)二氧化碳產出率…………………………………………………19
(3)動物肛溫之測量…………………………………………………19
(4)體重之測量………………………………………………………19
九、肺組織脂質過氧化反應之測定……………………………………20
十、藥品…………………………………………………………………21
十一、實驗執行步驟……………………………………………………22
十二、統計分析…………………………………………………………23
參、 實驗結果
一、大鼠於長期間歇性低氧暴露下對其化學反射反應之影響………24
二、大鼠於長期間歇性低氧暴露下對其心血管反應之影響…………28
三、大鼠於長期間歇性低氧暴露下，肺組織脂質過氧化情形…………33
四、代謝速率……………………………………………………………34
肆、 討論………………………………………………………………………．35
伍、 圖表與說明………………………………………………………………．45
陸、 參考文獻…………………………………………………………………．89

1.Adhikary G, Kline D, Yuan G, Kumar GK, Simonson MS, Cherniack NS, and Prabhakar NR. Gene regulation during intermittent hypoxia: evidence for the involvement of reactive oxygen species. Adv Exp Med Biol 499: 297-302, 2001.

2.Allahdadi KJ, Walker BR, and Kanagy NL. Augmented endothelin vasoconstriction in intermittent hypoxia-induced hypertension. Hypertension 45: 705-709, 2005.

3.Bao G, Metreveli N, Li R, Taylor A, and Fletcher EC. Blood pressure response to chronic episodic hypoxia: role of the sympathetic nervous system. J Appl Physiol 83: 95-101, 1997.

4.Beehler CJ, Ely ME, Rutledge KS, Simchuk ML, Reiss OK, Shanley PF, and Repine JE. Toxic effects of dimethylthiourea in rats. J Lab Clin Med 123: 73-80, 1994.

5.Bisgard GE. Carotid body mechanisms in acclimatization to hypoxia. Respir Physiol 121: 237-246, 2000.

6.Bonigut S, Stebbins CL, and Longhurst JC. Reactive oxygen species modify reflex cardiovascular responses to static contraction. J Appl Physiol 81: 1207-1212, 1996.

7.Cameron NE, Tuck Z, McCabe L, and Cotter MA. Effect of the hydroxyl radical scavenger, dimethylthiourea, on peripheral nerve tissue perfusion, conduction velocity and nociception in experimental diabetes. Diabetologia 44: 1161-1169, 2001.

8.Campen MJ, Shimoda LA, and O'Donnell CP. Acute and chronic cardiovascular effects of intermittent hypoxia in C57BL/6J mice. J Appl Physiol 99: 2028-2035, 2005.

9.Campese VM, Ye S, Zhong H, Yanamadala V, Ye Z, and Chiu J. Reactive oxygen species stimulate central and peripheral sympathetic nervous system activity. Am J Physiol Heart Circ Physiol 287: H695-H703, 2004.

10.Chervin RD and Aldrich MS. Sleep onset REM periods during multiple sleep latency tests in patients evaluated for sleep apnea. Am J Respir Crit Care Med 161: 426-431, 2000.

11.Cistulli PA, Barnes DJ, Grunstein RR, and Sullivan CE. Effect of short-term hormone replacement in the treatment of obstructive sleep apnoea in postmenopausal women. Thorax 49: 699-702, 1994.

12.Cragg PA, Runold M, Kou YR, and Prabhakar NR. Tachykinin antagonists in carotid body responses to hypoxia and substance P in the rat. Respir Physiol 95: 295-310, 1994.

13.Ding Y, Gonick HC, Vaziri ND, Liang K, and Wei L. Lead-induced hypertension. III. Increased hydroxyl radical production. Am J Hypertens 14: 169-173, 2001.

14.Dyugovskaya L, Lavie P, and Lavie L. Increased adhesion molecules expression and production of reactive oxygen species in leukocytes of sleep apnea patients. Am J Respir Crit Care Med 165: 934-939, 2002.

15.Fatemian M, Kim DY, Poulin MJ, and Robbins PA. Very mild exposure to hypoxia for 8 h can induce ventilatory acclimatization in humans. Pflugers Arch 441: 840-843, 2001.

16.Fletcher BL, Dillard CJ, and Tappel AL. Measurement of fluorescent lipid peroxidation products in biological systems and tissues. Anal Biochem 52: 1-9, 1973.

17.Fletcher EC. The relationship between systemic hypertension and obstructive sleep apnea: facts and theory. Am J Med 98: 118-128, 1995.

18.Fletcher EC. Effect of episodic hypoxia on sympathetic activity and blood pressure. Respir Physiol 119: 189-197, 2000.

19.Fletcher EC. Invited review: Physiological consequences of intermittent hypoxia: systemic blood pressure. J Appl Physiol 90: 1600-1605, 2001.

20.Fletcher EC, Bao G, and Li R. Renin activity and blood pressure in response to chronic episodic hypoxia. Hypertension 34: 309-314, 1999.

21.Fletcher EC, Lesske J, Behm R, Miller CC, 3rd, Stauss H, and Unger T. Carotid chemoreceptors, systemic blood pressure, and chronic episodic hypoxia mimicking sleep apnea. J Appl Physiol 72: 1978-1984, 1992.

22.Fletcher EC, Lesske J, Culman J, Miller CC, and Unger T. Sympathetic denervation blocks blood pressure elevation in episodic hypoxia. Hypertension 20: 612-619, 1992.

23.Fletcher EC, Lesske J, Qian W, Miller CC, 3rd, and Unger T. Repetitive, episodic hypoxia causes diurnal elevation of blood pressure in rats. Hypertension 19: 555-561, 1992.

24.Fletcher EC, Miller J, Schaaf JW, and Fletcher JG. Urinary catecholamines before and after tracheostomy in patients with obstructive sleep apnea and hypertension. Sleep 10: 35-44, 1987.

25.Germack R, Leon-Velarde F, Valdes De La Barra R, Farias J, Soto G, and Richalet JP. Effect of intermittent hypoxia on cardiovascular function, adrenoceptors and muscarinic receptors in Wistar rats. Exp Physiol 87: 453-460, 2002.

26.Greenberg HE, Sica A, Batson D, and Scharf SM. Chronic intermittent hypoxia increases sympathetic responsiveness to hypoxia and hypercapnia. J Appl Physiol 86: 298-305, 1999.

27.Guichardant M, Valette-Talbi L, Cavadini C, Crozier G, and Berger M. Malondialdehyde measurement in urine. J Chromatogr B Biomed Appl 655: 112-116, 1994.

28.Heitmann J, Ehlenz K, Penzel T, Becker HF, Grote L, Voigt KH, Peter JH, and Vogelmeier C. Sympathetic activity is reduced by nCPAP in hypertensive obstructive sleep apnoea patients. Eur Respir J 23: 255-262, 2004.

29.Kara T, Narkiewicz K, and Somers VK. Chemoreflexes--physiology and clinical implications. Acta Physiol Scand 177: 377-384, 2003.

30.Kishi T, Hirooka Y, Kimura Y, Ito K, Shimokawa H, and Takeshita A. Increased reactive oxygen species in rostral ventrolateral medulla contribute to neural mechanisms of hypertension in stroke-prone spontaneously hypertensive rats. Circulation 109: 2357-2362, 2004.

31.Kline DD, Yang T, Huang PL, and Prabhakar NR. Altered respiratory responses to hypoxia in mutant mice deficient in neuronal nitric oxide synthase. J Physiol 511: 273-287, 1998.

32.Koehle MS, Foster GE, McKenzie DC, and Sheel AW. Repeated measurement of hypoxic ventilatory response as an intermittent hypoxic stimulus. Respir Physiol Neurobiol 145: 33-39, 2005.

33.Kuo TBJ, Yang CCH, and Chan SHH. Selective activation of vasomotor component of SAP spectrum by nucleus reticularis ventrolateralis in rats. Am J Physiol 272: H485-492, 1997.

34.Lahiri S, Rozanov C, and Cherniack NS. Altered structure and function of the carotid body at high altitude and associated chemoreflexes. High Alt Med Biol 1: 63-74, 2000.

35.Lai CJ, Yang CCH, Hsu YY, Lin YN, and Kuo TBJ. Enhanced sympathetic outflow and decreased baroreflex sensitivity are associated with intermittent hypoxia-induced systemic hypertension in conscious rats. J Appl Physiol, 2006.

36.Lefebvre B, Godin-Ribuot D, Joyeux-Faure M, Caron F, Bessard G, Levy P, and Stanke-Labesque F. Functional assessment of vascular reactivity after chronic intermittent hypoxia in the rat. Respir Physiol Neurobiol 150: 278-286, 2006.

37.Lesske J, Fletcher EC, Bao G, and Unger T. Hypertension caused by chronic intermittent hypoxia--influence of chemoreceptors and sympathetic nervous system. J Hypertens 15: 1593-1603, 1997.

38.Lin AM, Chen CF, and Ho LT. Neuroprotective effect of intermittent hypoxia on iron-induced oxidative injury in rat brain. Exp Neurol 176: 328-335, 2002.

39.Lin AM, Yang CH, and Chai CY. Striatal dopamine dynamics are altered following an intranigral infusion of iron in adult rats. Free Radic Biol Med 24: 988-993, 1998.

40.Ling L, Fuller DD, Bach KB, Kinkead R, Olson EB, Jr., and Mitchell GS. Chronic intermittent hypoxia elicits serotonin-dependent plasticity in the central neural control of breathing. J Neurosci 21: 5381-5388, 2001.

41.McGuire M, Zhang Y, White DP, and Ling L. Chronic intermittent hypoxia enhances ventilatory long-term facilitation in awake rats. J Appl Physiol 95: 1499-1508, 2003.

42.Mohsenin V. Sleep-related breathing disorders and risk of stroke. Stroke 32: 1271-1278, 2001.

43.Nieto FJ, Young TB, Lind BK, Shahar E, Samet JM, Redline S, D'Agostino RB, Newman AB, Lebowitz MD, and Pickering TG. Association of sleep-disordered breathing, sleep apnea, and hypertension in a large community-based study. Sleep Heart Health Study. JAMA 283: 1829-1836, 2000.

44.Parati G, Di Rienzo M, Bonsignore MR, Insalaco G, Marrone O, Castiglioni P, Bonsignore G, and Mancia G. Autonomic cardiac regulation in obstructive sleep apnea syndrome: evidence from spontaneous baroreflex analysis during sleep. J Hypertens 15: 1621-1626, 1997.

45.Partinen M and Guilleminault C. Daytime sleepiness and vascular morbidity at seven-year follow-up in obstructive sleep apnea patients. Chest 97: 27-32, 1990.

46.Peker Y, Hedner J, Kraiczi H, and Loth S. Respiratory disturbance index: an independent predictor of mortality in coronary artery disease. Am J Respir Crit Care Med 162: 81-86, 2000.

47.Peng YJ, Overholt JL, Kline D, Kumar GK, and Prabhakar NR. Induction of sensory long-term facilitation in the carotid body by intermittent hypoxia: implications for recurrent apneas. Proc Natl Acad Sci U S A 100: 10073-10078, 2003.

48.Peng YJ and Prabhakar NR. Reactive oxygen species in the plasticity of respiratory behavior elicited by chronic intermittent hypoxia. J Appl Physiol 94: 2342-2349, 2003.

49.Peng YJ, Rennison J, and Prabhakar NR. Intermittent hypoxia augments carotid body and ventilatory response to hypoxia in neonatal rat pups. J Appl Physiol 97: 2020-2025, 2004.

50.Prabhakar NR. Oxygen sensing by the carotid body chemoreceptors. J Appl Physiol 88: 2287-2295, 2000.

51.Prabhakar NR, Fields RD, Baker T, and Fletcher EC. Intermittent hypoxia: cell to system. Am J Physiol Lung Cell Mol Physiol 281: L524-L528, 2001.

52.Prabhakar NR, Peng YJ, Jacono FJ, Kumar GK, and Dick TE. Cardiovascular alterations by chronic intermittent hypoxia: importance of carotid body chemoreflexes. Clin Exp Pharmacol Physiol 32: 447-449, 2005.

53.Prasad K and Bharadwaj LA. Hydroxyl radical--a mediator of acetylcholine-induced vascular relaxation. J Mol Cell Cardiol 28: 2033-2041, 1996.

54.Pryor WA. Oxy-radicals and related species: their formation, lifetimes, and reactions. Annu Rev Physiol 48: 657-667, 1986.

55.Raha S and Robinson BH. Mitochondria, oxygen free radicals, disease and ageing. Trends Biochem Sci 25: 502-508, 2000.

56.Rangan U and Bulkley GB. Prospects for treatment of free radical-mediated tissue injury. Br Med Bull 49: 700-718, 1993.

57.Rey S, Del Rio R, Alcayaga J, and Iturriaga R. Chronic intermittent hypoxia enhances cat chemosensory and ventilatory responses to hypoxia. J Physiol 560: 577-586, 2004.

58.Schnackenberg CG and Wilcox CS. Two-week administration of tempol attenuates both hypertension and renal excretion of 8-Iso prostaglandin f2alpha. Hypertension 33: 424-428, 1999.

59.Schulz R, Mahmoudi S, Hattar K, Sibelius U, Olschewski H, Mayer K, Seeger W, and Grimminger F. Enhanced release of superoxide from polymorphonuclear neutrophils in obstructive sleep apnea. Impact of continuous positive airway pressure therapy. Am J Respir Crit Care Med 162: 566-570, 2000.

60.Shahar E, Whitney CW, Redline S, Lee ET, Newman AB, Javier Nieto F, O'Connor GT, Boland LL, Schwartz JE, and Samet JM. Sleep-disordered breathing and cardiovascular disease: cross-sectional results of the Sleep Heart Health Study. Am J Respir Crit Care Med 163: 19-25, 2001.

61.Sica AL, Greenberg HE, Ruggiero DA, and Scharf SM. Chronic-intermittent hypoxia: a model of sympathetic activation in the rat. Respir Physiol 121: 173-184, 2000.

62.Smith ML and Muenter NK. Effects of hypoxia on sympathetic neural control in humans. Respir Physiol 121: 163-171, 2000.

63.Somers VK and Abboud FM. Chemoreflexes--responses, interactions and implications for sleep apnea. Sleep 16: S30-S34, 1993.

64.Somers VK, Dyken ME, Clary MP, and Abboud FM. Sympathetic neural mechanisms in obstructive sleep apnea. J Clin Invest 96: 1897-1904, 1995.

65.Stoohs R and Guilleminault C. Cardiovascular changes associated with obstructive sleep apnea syndrome. J Appl Physiol 72: 583-589, 1992.

66.Stornetta RL, Guyenet PG, and McCarty RC. Autonomic nervous system control of heart rate during baroreceptor activation in conscious and anesthetized rats. J Auton Nerv Syst 20: 121-127, 1987.

67.Sun HY, Wang NP, Kerendi F, Halkos M, Kin H, Guyton RA, Vinten-Johansen J, and Zhao ZQ. Hypoxic postconditioning reduces cardiomyocyte loss by inhibiting ROS generation and intracellular Ca2+ overload. Am J Physiol Heart Circ Physiol 288: H1900-H1908, 2005.

68.Tahawi Z, Orolinova N, Joshua IG, Bader M, and Fletcher EC. Altered vascular reactivity in arterioles of chronic intermittent hypoxic rats. J Appl Physiol 90: 2007-2013, 2001.

69.Tirmenstein MA, Pierce CA, Leraas TL, and Fariss MW. A fluorescence plate reader assay for monitoring the susceptibility of biological samples to lipid peroxidation. Anal Biochem 265: 246-252, 1998.

70.Waradekar NV, Sinoway LI, Zwillich CW, and Leuenberger UA. Influence of treatment on muscle sympathetic nerve activity in sleep apnea. Am J Respir Crit Care Med 153: 1333-1338, 1996.

71.Waters KA and Tinworth KD. Depression of ventilatory responses after daily, cyclic hypercapnic hypoxia in piglets. J Appl Physiol 90: 1065-1073, 2001.

72.Wolk R and Somers VK. Cardiovascular consequences of obstructive sleep apnea. Clin Chest Med 24: 195-205, 2003.

73.Xu W, Chi L, Row BW, Xu R, Ke Y, Xu B, Luo C, Kheirandish L, Gozal D, and Liu R. Increased oxidative stress is associated with chronic intermittent hypoxia-mediated brain cortical neuronal cell apoptosis in a mouse model of sleep apnea. Neuroscience 126: 313-323, 2004.

74.Yang CCH, Kuo TBJ, and Chan SHH. Auto- and cross-spectral analysis of cardiovascular fluctuations during pentobarbital anesthesia in the rat. Am J Physiol 270: H575-H582, 1996.

75.Yang CCH, Lai CW, Lai HY, and Kuo TBJ. Relationship between electroencephalogram slow-wave magnitude and heart rate variability during sleep in humans. Neurosci Lett 329: 213-216, 2002.

76.Yang CCH, Shaw FZ, Lai CJ, Lai CW, and Kuo TBJ. Relationship between electroencephalogram slow-wave magnitude and heart rate variability during sleep in rats. Neurosci Lett 336: 21-24, 2003.

77.Young T, Palta M, Dempsey J, Skatrud J, Weber S, and Badr S. The occurrence of sleep-disordered breathing among middle-aged adults. N Engl J Med 328: 1230-1235, 1993.

78.Yuan G, Adhikary G, McCormick AA, Holcroft JJ, Kumar GK, and Prabhakar NR. Role of oxidative stress in intermittent hypoxia-induced immediate early gene activation in rat PC12 cells. J Physiol 557: 773-783, 2004.