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研究生(外文):Wen-chun Fu
論文名稱(外文):The Effects of Infection by Vibrio vulnificus on The Cardiac Activity and Pro-inflammatory Genes Expression in Zebrafish (Danio rerio)
指導教授(外文):Chung-chih Kuo
外文關鍵詞:Autonomic nervous systemHRVspectrum analysisimmune response
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神經系統除了可以調控許多新陳代謝活性,也可對免疫反應進行調控。哺乳類動物的心臟活動可以受到自主神經系統的調控:交感神經系統的活化,可以促進心跳加速;反之,副交感神經系統的活化則會降低心跳速率。交感神經與副交感神經兩大系統相互拮抗作用影響之下,會使得心跳速率產生變化。這樣子的現象稱之為心率變異性。臨床上指出,心率變異性對於偵測病患生理狀態是個非常有用的工具。本論文將針對斑馬魚遭受到創傷弧菌感染之後,其心跳速率,心率變異性和促發炎基因的表現量變化進行觀察。斑馬魚的心電圖在感染前後的不同時間點都被記錄下來,並且利用軟體來進行心跳速率與心率變異性的分析。此外,我們也利用反轉錄-聚合��鏈鎖反應來觀察一些促進發炎反應產生的免疫基因之表現。同時也進行心電圖的記錄,並進行兩者之相關性分析。浸泡在高濃度菌液當中接受感染的斑馬魚,其心跳速率自感染後的三小時起會有增加的趨勢,並隨著感染時間的增加而比對照組有顯著性的上升。另外心率變異性在時域各方面參數(NN50, pNN50, SDNN和RMSSD)以及頻譜方面的分析,受感染的斑馬魚比起對照組也有顯著下降的趨勢。隨著感染時間增加,促發炎基因(TNFa, COX-2, and IL-1b)的mRNA表現量也有顯著上升的趨勢。免疫基因表現也與心跳速率呈現正相關,並與心率變異性的變化呈現負相關,以上的結果顯示了偵測魚類的心率變異性變化可做為一個偵測其生理狀態的便利工具。
Autonomic nervous system is able to regulate a variety of physiological activities, including the cardiovascular function and immune responses. The activation of sympathetic nervous system speeds up the heart rate (HR) and the activation of parasympathetic nervous system slows down the HR. The balances between sympathetic and parasympathetic tone change the intervals between the two neighboring heart beats and induced the heart rate variability (HRV). It has been indicated that the HRV is a useful tool for screening the physiological state of the subject under certain pathological condition. In addition, the stimulation of vagus nerve also reduces the activation of immune cell and the release of pro-inflammatory cytokines. This study investigated the change of HR, HRV and pro-inflammatory genes induced by the infection of Vibrio vulnificus (V. vulnificus) in zebrafish. The electrocardiograms (ECG) of zebrafish were recorded before and after infection for several time points to analyze the changes in HR and HRV. The expressions of pro-inflammatory genes were also monitored with semi-quantitative RT-PCR technique, and the change of HR and HRV were correlated to the gene expressions. The HR increased with the infection time and was significantly higher than the control group after 3 hr of infection. The time-domain measures (NN50, pNN50, SDNN and RMSSD) as well as the spectrum analysis showed that HRV were significantly lower in the infected zebrafish than the control group. The mRNA level of TNFa, COX-2, and IL-1b were also enhanced with the infection time. The expression of pro-inflammatory genes shows positive correlation with the change of HR and negative correlation with the change of HRV. These results suggest that HRV is a convenient method for monitoring the physiological states of zebrafish.
中文摘要 I
Abstract II
目錄 III
圖表目錄 V
Introduction 1
1. Homeostasis and the complex system 1
2. Neuroimmunomodulation 1
2.1. The immune system 1
2.2. Regulation of the immune system by the nervous system 4
3. Heart rate variability (HRV) analysis 8
4. The model for infectious disease 12
4.1. Zebrafish (Danio rerio) 12
4.2. Vibrio vulnificus 13
5. Motivation and Specific aims 14
Materials and Methods 15
1. Animals 15
2. Perfusion and establishment of the recording system 15
3. Bacterial preparation 17
4. Infection of zebrafish 17
5. ECG data analysis 18
6. Semi-quantitative RT-PCR 19
6.1. RNA extraction 19
6.2. Reverse transcription 19
6.3. Polymerase chain reaction (PCR) 20
7. Experimental protocols 20
7.1. The changes of HR and HRV during continuous V. vulnivicus infection 20
7.2. The Correlations between physiological parameters and immune responses. 21
8. Statistical analysis 21
Results 23
1. Electrocardiogram (ECG) of the zebrafish 23
2. The changes of heart rate during V. vulnificus infection. 24
3. The changes of Time-domain measures during V. vulnificus infection. 25
3.1. The effect of V. vulnificus infection on NN50 25
3.2. The effect of V. vulnificus infection on pNN50 26
3.3. The effect of V. vulnificus infection on SDNN 26
3.4. The effect of V. vulnificus infection on RMSSD 27
4. Spectrum analysis of the zebrafish ECG during V. vulnificus infection. 28
5. The relationship between cardiovascular function and immune responses to V. vulnificus infection 28
5.1. The expression of pro-inflammatory cytokine genes during V. vulnificus infection 28
5.2. Correlation between physiological parameters and immune responses 30
Discussion 33
Conclusion remarks 39
Figures 40
Table 55
References 56

Figure 1. The recording system.
Figure 2. Representative ECG of adult zebrafish.
Figure 3. The changes of HR in zebrafish during the infection with different concentrations of V. vulnificus.
Figure 4. The changes of NN50 in zebrafish during the infection with different concentrations of V. vulnificus.
Figure 5. The changes of pNN50 in zebrafish during the infection with different concentrations of V. vulnificus.
Figure 6. The changes of SDNN in zebrafish during the infection with different concentrations of V. vulnificus.
Figure 7. The changes of RMSSD in zebrafish during the infection with different concentrations of V. vulnificus.
Figure 8. The spectrum analysis of the raw ECG in the control and the infected zebrafish (108 CFU/mL).
Figure 9. Induction of pro-inflammatory cytokine gene expressions in zebrafish by infection with V. vulnivicus (108 CFU/mL).
Figure 10. The average expressions of TNFa, COX-2 and IL-1b gene with semi-quantitative RT-PCR in zebrafish after different infection times.
Figure 11. Correlations between gene expressions and changes of heat rate in control and infection groups (108 CFU/mL).
Figure 12. Correlations between gene expressions and changes of NN50 in control and infection groups (108 CFU/mL).
Figure 13. Correlations between gene expressions and changes of pNN50 in control and infection groups (108 CFU/mL).
Figure 14. Correlations between gene expressions and changes of SDNN in control and infection groups (108 CFU/mL).
Figure 15. Correlations between gene expressions and changes of RMSSD in control and infection groups (108 CFU/mL)
Table 1. The PCR primer sequences used for semi-quantitative PT-PCR.
Agelaki, S., Tsatsanis, C., Gravanis, A. and Margioris, A. N. (2002). Corticotropin-releasing hormone augments proinflammatory cytokine production from macrophages in vitro and in lipopolysaccharide-induced endotoxin shock in mice. Infection and Immunity 70, 6068-74.

Altimiras, J. (1999). Understanding autonomic sympathovagal balance from short-term heart rate variations. Are we analyzing noise? Comparative Biochemistry and Physiology - Part A: Molecular & Integrative Physiology 124, 447-460.

Annane, D., Trabold, F., Sharshar, T., Jarrin, I., Blanc, A. S., Raphael, J. C. and Gajdos, P. (1999). Inappropriate sympathetic activation at onset of septic shock . A spectral analysis approach. American Journal of Respiratory and Critical Care Medicine 160, 458-465.

Bross, M. H., Soch, K., Morales, R. and Mitchell, R. B. (2007). Vibrio vulnificus infection: diagnosis and treatment. American Family Physician 76, 539-44.

Campbell, H. A. and Egginton, S. (2007). The vagus nerve mediates cardio-respiratory coupling that changes with metabolic demand in a temperate nototheniod fish. Journal of Experimental Biology 210, 2472-80.

Campbell, H. A., Klepacki, J. Z. and Egginton, S. (2006). A new method in applying power spectral statistics to examine cardio-respiratory interactions in fish. Journal of Theoretical Biology 241, 410-9.

Campbell, H. A., Taylor, E. W. and Egginton, S. (2004). The use of power spectral analysis to determine cardiorespiratory control in the short-horned sculpin Myoxocephalus scorpius. Journal of Experimental Biology 207, 1969-1976.

Campbell, H. A., Taylor, E. W. and Egginton, S. (2005). Does respiratory sinus arrhythmia occur in fishes? Biology Letters 1, 484-7.

Cha, Y. I., Solnica-Krezel, L. and DuBois, R. N. (2006). Fishing for prostanoids: deciphering the developmental functions of cyclooxygenase-derived prostaglandins. Developmental Biology 289, 263-72.

Cotter, P. A. and Rodnick, K. J. (2006). Differential effects of anesthetics on electrical properties of the rainbow trout (Oncorhynchus mykiss) heart. Comparative Biochemistry and Physiology. Part A, Molecular and Integrative Physiology 145, 158-65.

Dempsey, P. W., Vaidya, S. A. and Cheng, G. (2003). The art of war: Innate and adaptive immune responses. Cellular and Molecular Life Sciences 60, 2604-21.

Di Giantomasso, D., May, C. N. and Bellomo, R. (2003). Vital organ blood flow during hyperdynamic sepsis. Chest 124, 1053-9.

Electrophysiology, E. S. o. C. a. N. A. S. o. P. a. (1996). Heart rate variability. Standards of measurement, physiological interpretation, and clinical use. Task Force of the European Society of Cardiology and the North American Society of Pacing and Electrophysiology. European Heart Journal 17, 354-81.

Elenkov, I. J. (2008). Neurohormonal-cytokine interactions: implications for inflammation, common human diseases and well-being. Neurochemistry International 52, 40-51.

Elghozi, J. L. and Julien, C. (2007). Sympathetic control of short-term heart rate variability and its pharmacological modulation. Fundamental and Clinical Pharmacology 21, 337-47.

Ellis, A. E. (2001). Innate host defense mechanisms of fish against viruses and bacteria. Developmental and Comparative Immunology 25, 827-39.

Eskandari, F., Webster, J. I. and Sternberg, E. M. (2003). Neural immune pathways and their connection to inflammatory diseases. Arthritis Research & Therapy 5, 251-65.

Flierl, M. A., Rittirsch, D., Nadeau, B. A., Chen, A. J., Sarma, J. V., Zetoune, F. S., McGuire, S. R., List, R. P., Day, D. E., Hoesel, L. M. et al. (2007). Phagocyte-derived catecholamines enhance acute inflammatory injury. Nature 449, 721-5.

Griffin, M. P., Lake, D. E., Bissonette, E. A., Harrell, F. E., Jr., O'Shea, T. M. and Moorman, J. R. (2005). Heart rate characteristics: novel physiomarkers to predict neonatal infection and death. Pediatrics 116, 1070-4.

Grosser, T., Yusuff, S., Cheskis, E., Pack, M. A. and FitzGerald, G. A. (2002). Developmental expression of functional cyclooxygenases in zebrafish. Proceedings of the National Academy of Sciences of the United States of America 99, 8418-23.
Hansen, M. B. (2003). The enteric nervous system I: organisation and classification. Pharmacology and Toxicology 92, 105-13.

Harris, J. and Bird, D. J. (2000). Modulation of the fish immune system by hormones. Veterinary Immunology and Immunopathology 77, 163-76.

Hsueh, P. R., Lin, C. Y., Tang, H. J., Lee, H. C., Liu, J. W., Liu, Y. C. and Chuang, Y. C. (2004). Vibrio vulnificus in Taiwan. Emerging Infectious Diseases 10, 1363-8.

Ignatowski, T. A., Gallant, S. and Spengler, R. N. (1996). Temporal regulation by adrenergic receptor stimulation of macrophage (M phi)-derived tumor necrosis factor (TNF) production post-LPS challenge. Journal of Neuroimmunology 65, 107-117.

Jault, C., Pichon, L. and Chluba, J. (2004). Toll-like receptor gene family and TIR-domain adapters in Danio rerio. Molecular Immunology 40, 759-71.

Kasahara, M., Suzuki, T. and Pasquier, L. D. (2004). On the origins of the adaptive immune system: novel insights from invertebrates and cold-blooded vertebrates. Trends in Immunology 25, 105-11.

Khalturin, K., Panzer, Z., Cooper, M. D. and Bosch, T. C. (2004). Recognition strategies in the innate immune system of ancestral chordates. Molecular Immunology 41, 1077-87.

Li, J., Barreda, D. R., Zhang, Y. A., Boshra, H., Gelman, A. E., Lapatra, S., Tort, L. and Sunyer, J. O. (2006). B lymphocytes from early vertebrates have potent phagocytic and microbicidal abilities. Nature Immunology 7, 1116-24.

Lin, B., Chen, S., Cao, Z., Lin, Y., Mo, D., Zhang, H., Gu, J., Dong, M., Liu, Z. and Xu, A. (2007). Acute phase response in zebrafish upon Aeromonas salmonicida and Staphylococcus aureus infection: striking similarities and obvious differences with mammals. Molecular Immunology 44, 295-301.

Magnadottir, B. (2006). Innate immunity of fish (overview). Fish and Shellfish Immunology 20, 137-51.

Metz, J. R., Huising, M. O., Leon, K., Verburg-van Kemenade, B. M. and Flik, G. (2006). Central and peripheral interleukin-1beta and interleukin-1 receptor I expression and their role in the acute stress response of common carp, Cyprinus carpio L. Journal of Endocrinology 191, 25-35.

Milan, D. J., Jones, I. L., Ellinor, P. T. and MacRae, C. A. (2006). In vivo recording of adult zebrafish electrocardiogram and assessment of drug-induced QT prolongation. American Journal of Physiology - Heart and Circulatory Physiology 291, H269-73.

Nance, D. M. and Sanders, V. M. (2007). Autonomic innervation and regulation of the immune system (1987-2007). Brain, Behavior, and Immunity 21, 736-45.

Pavlov, V. A., Ochani, M., Gallowitsch-Puerta, M., Ochani, K., Huston, J. M., Czura, C. J., Al-Abed, Y. and Tracey, K. J. (2006). Central muscarinic cholinergic regulation of the systemic inflammatory response during endotoxemia. Proceedings of the National Academy of Sciences of the United States of America 103, 5219-5223.

Pontet, J., Contreras, P., Curbelo, A., Medina, J., Noveri, S., Bentancourt, S. and Migliaro, E. R. (2003). Heart rate variability as early marker of multiple organ dysfunction syndrome in septic patients. Journal of Critical Care 18, 156-63.

Pressley, M. E., Phelan, P. E., 3rd, Witten, P. E., Mellon, M. T. and Kim, C. H. (2005). Pathogenesis and inflammatory response to Edwardsiella tarda infection in the zebrafish. Developmental and Comparative Immunology 29, 501-13.

Rajendra Acharya, U., Paul Joseph, K., Kannathal, N., Lim, C. M. and Suri, J. S. (2006). Heart rate variability: a review. Medical and Biological Engineering and Computing 44, 1031-51.

Randall, D. J. (1962). Effect of an anaesthetic on the heart and respiration of teleost fish. Nature 195, 506.

Reynaud, S., Raveton, M. and Ravanel, P. (2008). Interactions between immune and biotransformation systems in fish: A review. Aquatic Toxicology 87, 139-145.

Roca, F. J., Mulero, I., Lopez-Munoz, A., Sepulcre, M. P., Renshaw, S. A., Meseguer, J. and Mulero, V. (2008). Evolution of the inflammatory response in vertebrates: fish TNF-alpha is a powerful activator of endothelial cells but hardly activates phagocytes. Journal of Immunology 181, 5071-81.

Rosas-Ballina, M., Ochani, M., Parrish, W. R., Ochani, K., Harris, Y. T., Huston, J. M., Chavan, S. and Tracey, K. J. (2008). Splenic nerve is required for cholinergic antiinflammatory pathway control of TNF in endotoxemia. Proceedings of the National Academy of Sciences 105, 11008-11013.
Roy, B., Singh, R., Kumar, S. and Rai, U. (2008). Diurnal variation in phagocytic activity of splenic phagocytes in freshwater teleost Channa punctatus: melatonin and its signaling mechanism. Journal of Endocrinology 199, 471-80.

Schwerte, T., Prem, C., Mairosl, A. and Pelster, B. (2006). Development of the sympatho-vagal balance in the cardiovascular system in zebrafish (Danio rerio) characterized by power spectrum and classical signal analysis. Journal of Experimental Biology 209, 1093-1100.

Sedmera, D., Reckova, M., deAlmeida, A., Sedmerova, M., Biermann, M., Volejnik, J., Sarre, A., Raddatz, E., McCarthy, R. A., Gourdie, R. G. et al. (2003). Functional and morphological evidence for a ventricular conduction system in zebrafish and Xenopus hearts. American Journal of Physiology - Heart and Circulatory Physiology 284, H1152-60.

Seely, A. J. and Macklem, P. T. (2004). Complex systems and the technology of variability analysis. Critical Care 8, R367-84.

Shimizu, H. and Okabe, M. (2007). Evolutionary origin of autonomic regulation of physiological activities in vertebrate phyla. Journal of Comparative Physiology A 193, 1013-9.

Smith, E. M. (2008). Neuropeptides as signal molecules in common with leukocytes and the hypothalamic-pituitary-adrenal axis. Brain, Behavior, and Immunity 22, 3-14.

Sternberg, E. M. (2006). Neural regulation of innate immunity: a coordinated nonspecific host response to pathogens. Nature Reviews Immunology 6, 318-328.

Stewart, R. A., Look, A. T., Kanki, J. P. and Henion, P. D. (2004). Development of the peripheral sympathetic nervous system in zebrafish. Methods in Cell Biology 76, 237-60.

Sullivan, C. and Kim, C. H. (2008). Zebrafish as a model for infectious disease and immune function. Fish and Shellfish Immunology 25, 341-50.

Tanabe, T., Iwamoto, T., Fusegawa, Y., Yoshioka, K. and Shiina, Y. (1995). Alterations of sympathovagal balance in patients with hypertrophic and dilated cardiomyopathies assessed by spectral analysis of RR interval variability. European Heart Journal 16, 799-807.

Taylor, E. W., Jordan, D. and Coote, J. H. (1999). Central control of the cardiovascular and respiratory systems and their interactions in vertebrates. Physiological Reviews 79, 855-916.

Trede, N. S., Langenau, D. M., Traver, D., Look, A. T. and Zon, L. I. (2004). The use of zebrafish to understand immunity. Immunity 20, 367-79.

Uhlar, C. M. and Whitehead, A. S. (1999). Serum amyloid A, the major vertebrate acute-phase reactant. European Journal of Biochemistry 265, 501-23.

von Borell, E., Langbein, J., Despres, G., Hansen, S., Leterrier, C., Marchant-Forde, J., Marchant-Forde, R., Minero, M., Mohr, E., Prunier, A. et al. (2007). Heart rate variability as a measure of autonomic regulation of cardiac activity for assessing stress and welfare in farm animals -- a review. Physiology and Behavior 92, 293-316.

Wang, H., Yu, M., Ochani, M., Amella, C. A., Tanovic, M., Susarla, S., Li, J. H., Wang, H., Yang, H., Ulloa, L. et al. (2003). Nicotinic acetylcholine receptor [alpha]7 subunit is an essential regulator of inflammation. Nature 421, 384-388.

Watzke, J., Schirmer, K. and Scholz, S. (2007). Bacterial lipopolysaccharides induce genes involved in the innate immune response in embryos of the zebrafish (Danio rerio). Fish and Shellfish Immunology 23, 901-5.

Wu, F., Vij, N., Roberts, L., Lopez-Briones, S., Joyce, S. and Chakravarti, S. (2007). A novel role of the lumican core protein in bacterial lipopolysaccharide-induced innate immune response. Journal of Biological Chemistry 282, 26409-17.

Yasuma, F. and Hayano, J. (2004). Respiratory sinus arrhythmia: why does the heartbeat synchronize with respiratory rhythm? Chest 125, 683-90.

Zaccone, G., Mauceri, A., Maisano, M. and Fasulo, S. (2009a). Innervation of lung and heart in the ray-finned fish, bichirs. Acta Histochem 111, 217-29.

Zaccone, G., Mauceri, A., Maisano, M., Giannetto, A., Parrino, V. and Fasulo, S. (2009b). Postganglionic nerve cell bodies and neurotransmitter localization in the teleost heart. Acta Histochemica, doi:10.1016/j.acthis.2009.02.004
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