治療黃斑部破孔的內界膜剝離手術中，會透過輔助染劑indocyanine green (ICG)、brilliant blue G (BBG)，trypan blue (TB) 來進行手術，然而這些染劑對於視網膜有毒性疑慮。本實驗利用人類視網膜色素上皮細胞株(ARPE-19)，搭配現行手術之清洗液balanced salt solution (BSS)及降低毒性之清洗液fetal bovine serum (FBS)，搭配前處理及後處理兩種清洗方式，透過細胞存活率、生長速率曲線、細胞內氧化壓力、粒線體膜電位變化、粒線體質量變化，來進行毒性及解毒實驗探討。
實驗結果顯示，ICG染劑濃度小於2.5 mg/ml，BBG及TB皆無細胞毒性反應。在細胞生長速率曲線測試，當ICG及BBG濃度大於1 mg/ml會有顯著之細胞生長停滯的現象，TB在所有的測試濃度下，皆無顯著之細胞生長停滯現象。在細胞內氧化壓力測試，ICG、BBG 、TB在各種實驗濃度下皆有顯著之氧化壓力上升，但是在FBS介入及清洗液前處理皆會有顯著之氧化壓力緩解 。
本研究中發現，(1)內界膜剝離術中所使用的染劑ICG、BBG，TB皆會對視網膜細胞造成負面影響；(2)FBS具有比BSS更好的緩解效果；(3)沖洗液前處理對於染劑所造成之負面效果有較好之舒緩效果。透過本研究結果，可應用於未來臨床上進行內界膜剝離術之參考。

Aims:
To investigate and compare the cytotoxicity of indocyanine green (ICG)、brilliant blue G (BBG) and trypan blue (TB) using ARPE-19 cells that have been pre-treated/post-treated with balanced salt solution (BSS) or fetal bovine serum (FBS).
Methods:
The cultured human retina pigment epithelium (RPE) ARPE-19 cells were pre-treated/post-treated with BSS or FBS (represent the autologous serum in clinic) in parallel with cells being soaked with various concentrations of ICG, BBG, and TB. Cells were then assessed for viability, growth rate, reactive oxygen species (ROS) level, mitochondrial (MT) membrane potential (Δψ) and MT mass as cytotoxic indices. For the FBS pre-treated cells, only ROS was examined.
Results:
Using the MTT assay, cytotoxicity seemed to appear when the dye concentration was above 2.5mg/ml for ICG but no cytotoxicity for BBG and TB at tested concentrations used. Cell growth was arrested at a concentration 1mg/ml when ICG or BBG were present but no arrest at any of the tested concentrations was found for TB with the cell-growth curve was slowest for ICG. Cellular ROS levels increased at all concentrations of all dyes, but the increasing slopes were decreased after FBS post-treatment washout.
Conclusions:
As a rinse buffer FBS performs much better than BSS in terms of cell rescue, which agrees with a clinical report when autologous whole blood was applied to macular hole surgery. However, FBS pre-treatment seems to be much better than FBS use as washout buffer in post-treatment.

致謝 i
目錄 ii
中文摘要 vi
英文摘要 vii
第一章 簡介 1
1.1 研究背景與動機 1
1.2 研究方法 3
1.3 研究假設 4
1.4 研究重要性 4
第二章 文獻回顧 5
2.1染劑應用於黃斑部破孔 5
2.2染劑毒性 5
2.3替代染劑 6
2.4解毒洗清液 7
2.5毒性原理 8
第三章 研究材料方法 9
3.1實驗溶液材料配製 9
3.1.1 Phosphate Buffered Saline (PBS)配製 9
3.1.2 Bovine serum albumin (BSA)配製 9
3.1.3DCF配製 10
3.1.4MTT配製 10
3.1.5培養基配製 10
3.2細胞培養 11
3.2.1細胞培養（cellculture） 11
3.2.2細胞繼代培養（subculture） 11
3.2.3實驗細胞種植 12
3.3實驗染劑 13
3.3.1染劑ICG 13
3.3.2染劑BBG 13
3.3.3染劑TB 13
3.3.4染劑使用步驟 13
3.3.5細胞染色前處理及後處理 14
3.4 研究方法 15
3.4.1 MTT毒性測試 15
3.4.2生長曲線 15
3.4.3細胞氧化壓力測試 16
3.4.4粒線體質量及電位測試 16
3.4.5殘留染劑吸收光譜量測 17
3.4.6染色能力測試 18
3.4.7細胞型態觀測 18
3.4.8白蛋白影響效益 19
3.4.9細胞吞噬能力測試 19
3.4.10系統性文獻回顧方法 19
3.4.11統計方法 20
第四章 結果 21
4.1 MTT毒性測試 21
4.2生長曲線 21
4.3 ROS分析 22
4.4 粒線體質量及電位測試 23
4.5殘餘染料測量 23
4.6細胞形態學檢查 24
4.7白蛋白效應 25
4.8 細胞吞噬能力測定 25
4.9染色能見度測試 26
第五章 討論 27
5.1染劑使用現況 27
5.2研究結果 27
5.3研究比較 28
5.4研究限制 29
5.5角色定位 29
5.6其他降低毒性方法 29
5.7未來發展 30
第六章 結論 31
第七章 圖表 32
圖一、細胞毒性 32
圖二、生長曲線 33
圖三、氧化壓力變化 34
圖四、粒線體膜電位及粒線體質量 35
圖五、染劑殘留光譜 36
圖六、ICG處理之細胞型態 37
圖七、BBG處理之細胞型態 38
圖八、TB處理之細胞型態 39
圖九、BSA對於氧化壓力之變化效果 40
圖十，吞噬能力測試 41
圖十一、染色能力測試 42
表一、染色能力比較表 43
表二、文獻分析 44
文獻參考 47
附件:發表論文 54

Abdelkader, E., and N. Lois. 2008. Internal limiting membrane peeling in vitreo-retinal surgery. Surv Ophthalmol 53:368-396.
Ando, F., K. Sasano, N. Ohba, H. Hirose, and O. Yasui. 2004a. Anatomic and visual outcomes after indocyanine green-assisted peeling of the retinal internal limiting membrane in idiopathic macular hole surgery. Am J Ophthalmol 137:609-614.
Ando, F., K. Sasano, F. Suzuki, and N. Ohba. 2004b. Indocyanine green-assisted ILM peeling in macular hole surgery revisited. Am J Ophthalmol 138:886-887.
Baker, K.J. 1966. Binding of sulfobromophthalein (BSP) sodium and indocyanine green (ICG) by plasma alpha-1 lipoproteins. Exp Biol Med (New York, N.Y.) 122:957-963.
Balayre, S., M. Boissonnot, B. Fernandez, N. Quellard, P. Babin, and P. Dighiero. 2005. Ultrastructural study of epiretinal membrane stained by trypan blue: 15 case reports. J FR OPHTALMOL 28:159-167.
Barot, M., M.R. Gokulgandhi, and A.K. Mitra. 2011. Mitochondrial dysfunction in retinal diseases. Curr. Eye Res. 36:1069-1077.
Beatty, S., H. Koh, M. Phil, D. Henson, and M. Boulton. 2000. The role of oxidative stress in the pathogenesis of age-related macular degeneration. Survey of ophthalmology 45:115-134.
Boyer, N.P., D. Higbee, M.B. Currin, L.R. Blakeley, C. Chen, Z. Ablonczy, R.K. Crouch, and Y. Koutalos. 2012. Lipofuscin and N-retinylidene-N-retinylethanolamine (A2E) accumulate in retinal pigment epithelium in absence of light exposure: their origin is 11-cis-retinal. The Journal of biological chemistry 287:22276-22286.
Brooks, H.L., Jr. 2000. Macular hole surgery with and without internal limiting membrane peeling. Ophthalmology 107:1939-1948; discussion 1948-1939.
Burk, S.E., A.P. Da Mata, M.E. Snyder, R.H. Rosa Jr, and R.E. Foster. 2000. Indocyanine green–assisted peeling of the retinal internal limiting membrane. Ophthalmology 107:2010-2014.
Cai, J., K.C. Nelson, M. Wu, P. Sternberg, Jr., and D.P. Jones. 2000. Oxidative damage and protection of the RPE. Prog Retin Eye Res 19:205-221.
Chang, A.A., M. Zhu, and F. Billson. 2005. The interaction of indocyanine green with human retinal pigment epithelium. Invest Ophthalmol Vis Sci 46:1463-1467.
Cherrick, G.R., S.W. Stein, C.M. Leevy, and C.S. Davidson. 1960. Indocyanine green: observations on its physical properties, plasma decay, and hepatic extraction. Journal of Clinical Investigation 39:592-600.
Da Mata, A.P., S.E. Burk, R.E. Foster, C.D. Riemann, M.R. Petersen, M. Nehemy, and J.J. Augsburger. 2004. Long-term follow-up of indocyanine green assisted peeling of the retinal internal limiting membrane during vitrectomy surgery for idiopathic macular hole repair. Ophthalmology 111:2246-2253.
Eckardt, C., U. Eckardt, S. Groos, L. Luciano, and E. Reale. 1997. [Removal of the internal limiting membrane in macular holes. Clinical and morphological findings]. Der Ophthalmologe : Zeitschrift der Deutschen Ophthalmologischen Gesellschaft 94:545-551.
Enaida, H., T. Hisatomi, Y. Goto, Y. Hata, A. Ueno, M. Miura, T. Kubota, and T. Ishibashi. 2006a. Preclinical investigation of internal limiting membrane staining and peeling using intravitreal brilliant blue G. Retina (Philadelphia, Pa.) 26:623-630.
Enaida, H., T. Hisatomi, Y. Hata, A. Ueno, Y. Goto, T. Yamada, T. Kubota, and T. Ishibashi. 2006b. Brilliant blue G selectively stains the internal limiting membrane/brilliant blue G-assisted membrane peeling. Retina (Philadelphia, Pa.) 26:631-636.
Enaida, H., and T. Ishibashi. 2008. Brilliant blue in vitreoretinal surgery. Dev Ophthalmol 42:115-125.
Engelbrecht, N.E., J. Freeman, P. Sternberg, Jr., T.M. Aaberg, Sr., T.M. Aaberg, Jr., D.F. Martin, and B.D. Sippy. 2002. Retinal pigment epithelial changes after macular hole surgery with indocyanine green-assisted internal limiting membrane peeling. Am J Ophthalmol 133:89-94.
Feron, E.J., M. Veckeneer, R. Parys-Van Ginderdeuren, A. Van Lommel, G.R. Melles, and P. Stalmans. 2002. Trypan blue staining of epiretinal membranes in proliferative vitreoretinopathy. Arch Ophthalmol 120:141-144.
Fox, I.J., and E.H. Wood. 1960. Indocyanine green: physical and physiologic properties. Proceedings of the staff meetings. Mayo Clinic 35:732-744.
Gale, J.S., A.A. Proulx, J.R. Gonder, A.J. Mao, and C.M. Hutnik. 2004. Comparison of the in vitro toxicity of indocyanine green to that of trypan blue in human retinal pigment epithelium cell cultures. Am J Ophthalmol 138:64-69.
Gandorfer, A., C. Haritoglou, C.A. Gass, M.W. Ulbig, and A. Kampik. 2001. Indocyanine green-assisted peeling of the internal limiting membrane may cause retinal damage. Am J Ophthalmol 132:431-433.
Gass, C.A., C. Haritoglou, M. Schaumberger, and A. Kampik. 2003. Functional outcome of macular hole surgery with and without indocyanine green-assisted peeling of the internal limiting membrane. Graefes Arch Clin Exp Ophthalmol 241:716-720.
Grisanti, S., A. Altvater, and S. Peters. 2008. Safety parameters for indocyanine green in vitreoretinal surgery. Dev Ophthalmol 42:43-68.
Haritoglou, C., K. Eibl, M. Schaumberger, A.J. Mueller, S. Priglinger, C. Alge, and A. Kampik. 2004a. Functional outcome after trypan blue-assisted vitrectomy for macular pucker: a prospective, randomized, comparative trial. Am J Ophthalmol 138:1-5.
Haritoglou, C., A. Gandorfer, M. Schaumberger, S.G. Priglinger, A.J. Mueller, C.A. Gass, and A. Kampik. 2004b. Trypan blue in macular pucker surgery: an evaluation of histology and functional outcome. Retina (Philadelphia, Pa.) 24:582-590.
Hernández, F., N. Alpizar-Alvarez, and L. Wu. 2014. Chromovitrectomy: an Update. Journal of ophthalmic & vision research 9:251-259.
Horio, N., and M. Horiguchi. 2004. Effect on visual outcome after macular hole surgery when staining the internal limiting membrane with indocyanine green dye. Arch Ophthalmol 122:992-996.
Jain, S., K. Kishore, and Y.R. Sharma. 2013. Progressive atrophy of retinal pigment epithelium after trypan-blue-assisted ILM peeling for macular hole surgery. Indian J Ophthalmol 61:235-237.
Kadonosono, K., N. Itoh, E. Uchio, S. Nakamura, and S. Ohno. 2000. Staining of internal limiting membrane in macular hole surgery. Arch Ophthalmol 118:1116-1118.
Kelly, N.E., and R.T. Wendel. 1991. Vitreous surgery for idiopathic macular holes. Results of a pilot study. Arch Ophthalmol 109:654-659.
Kodjikian, L., T. Richter, M. Halberstadt, F. Beby, F. Flueckiger, M. Boehnke, and J.G. Garweg. 2005. Toxic effects of indocyanine green, infracyanine green, and trypan blue on the human retinal pigmented epithelium. Graefes Arch Clin Exp Ophthalmol 243:917-925.
Kung, Y.-H., and T.-T. Wu. 2013. The effect of autologous serum on vitrectomy with internal limiting membrane peeling for idiopathic macular hole.
J Ocul Pharmacol Ther 29:508-511.
Kunikata, H., T. Abe, H. Murata, Y. Sagara, R. Wakusawa, H. Sato, M. Yoshida, N. Fuse, and M. Tamai. 2006. Hypothermia of 8 degrees C protects cultured retinal pigment epithelial cells and retinal ganglion cells against trypan blue toxicity. Am J Ophthalmol 141:754-756.
Kunikata, H., H. Tomita, T. Abe, H. Murata, Y. Sagara, H. Sato, Y. Wada, N. Fuse, Y. Nakagawa, M. Tamai, and K. Nishida. 2007. Hypothermia protects cultured human retinal pigment epithelial cells against indocyanine green toxicity.
J Ocul Pharmacol Ther 23:35-39.
Kwok, A.K., T.Y. Lai, W.W. Li, D.T. Yew, and V.W. Wong. 2004. Trypan blue- and indocyanine green-assisted epiretinal membrane surgery: clinical and histopathological studies. Eye 18:882-888.
Li, K., D. Wong, P. Hiscott, P. Stanga, C. Groenewald, and J. McGalliard. 2003. Trypan blue staining of internal limiting membrane and epiretinal membrane during vitrectomy: visual results and histopathological findings. Br J Ophthalmol 87:216-219.
Liang, F.Q., and B.F. Godley. 2003. Oxidative stress-induced mitochondrial DNA damage in human retinal pigment epithelial cells: a possible mechanism for RPE aging and age-related macular degeneration. Exp Eye Res 76:397-403.
Lim, S.L., and M.T. Dunbar. 2000. Update on current surgical management of idiopathic macular holes. Clinical eye and vision care 12:51-60.
Mao, Y., and S.C. Finnemann. 2013. Analysis of Photoreceptor Outer Segment Phagocytosis by RPE Cells in Culture. Methods Mol Biol 935:285-295.
McCannel, C.A., J.L. Ensminger, N.N. Diehl, and D.N. Hodge. 2009. Population Based Incidence of Macular Holes. Ophthalmology 116:1366-1369.
Meier, P.J. 1995. Molecular mechanisms of hepatic bile salt transport from sinusoidal blood into bile. Am J Physiol Renal Physiol 269:G801-812.
Miceli, M.V., M.R. Liles, and D.A. Newsome. 1994. Evaluation of oxidative processes in human pigment epithelial cells associated with retinal outer segment phagocytosis. Exp Cell Res 214:242-249.
Molday, R.S., and K. Zhang. 2010. Defective lipid transport and biosynthesis in recessive and dominant Stargardt macular degeneration. Prog Lipid Res 49:476-492.
Morales, M.C., V. Freire, A. Asumendi, J. Araiz, I. Herrera, G. Castiella, I. Corcostegui, and G. Corcostegui. 2010. Comparative effects of six intraocular vital dyes on retinal pigment epithelial cells. Invest Ophthalmol Vis Sci 51:6018-6029.
Naithani, P., N. Vashisht, S. Khanduja, S. Sinha, and S. Garg. 2011. Brilliant blue G-assisted peeling of the internal limiting membrane in macular hole surgery. Indian J Ophthalmol 59:158-160.
Nakamura, H., K. Hayakawa, S. Sawaguchi, and T. Gaja. 2005. Removal of retinal indocyanine green dye by autologous serum irrigation in macular hole surgery. Retina (Philadelphia, Pa.) 25:736-741.
Narayanan, R., M.C. Kenney, S. Kamjoo, T.H. Trinh, G.M. Seigel, G.P. Resende, and B.D. Kuppermann. 2005. Toxicity of indocyanine green (ICG) in combination with light on retinal pigment epithelial cells and neurosensory retinal cells. Curr. Eye Res. 30:471-478.
Penha, F.M., M. Pons, P. Costa Ede, E.B. Rodrigues, M. Maia, M.E. Marin-Castano, and M.E. Farah. 2013a. Effect of vital dyes on retinal pigmented epithelial cell viability and apoptosis: implications for chromovitrectomy. Ophthalmologica. Journal international d'ophtalmologie. Int J Ophthalmol Clin Res. Zeitschrift fur Augenheilkunde 230 Suppl 2:41-50.
Penha, F.M., M. Pons, E.F. Costa, N.M. Barros, E.B. Rodrigues, E.B. Cardoso, E. Dib, M. Maia, M.E. Marin-Castano, and M.E. Farah. 2013b. Retinal pigmented epithelial cells cytotoxicity and apoptosis through activation of the mitochondrial intrinsic pathway: role of indocyanine green, brilliant blue and implications for chromovitrectomy. PLoS One 8:e64094.
Perrier, M., and M. Sebag. 2003. Trypan blue-assisted peeling of the internal limiting membrane during macular hole surgery. Am J Ophthalmol 135:903-905.
Peters, S., A. Altvater, S. Bopp, R. Vonthein, P. Szurman, M.S. Spitzer, M. Warga, M. Lueke, K.U. Bartz-Schmidt, and S. Grisanti. 2007. Systematic evaluation of ICG and trypan blue related effects on ARPE-19 cells in vitro. Exp Eye Res 85:880-889.
Proulx, A.A., and J.R. Gonder. 2007. Comparison of in vitro toxicity of single vs double exposures of indocyanine green in human retinal pigment epithelial cell cultures. Am J Ophthalmol 143:353-354.
Remy, M., S. Thaler, R.G. Schumann, C.A. May, M. Fiedorowicz, F. Schuettauf, M. Gruterich, S.G. Priglinger, M.M. Nentwich, A. Kampik, and C. Haritoglou. 2008. An in vivo evaluation of Brilliant Blue G in animals and humans. Br J Ophthalmol 92:1142-1147.
Rezai, K.A., L. Farrokh-Siar, E.M. Gasyna, and J.T. Ernest. 2004. Trypan blue induces apoptosis in human retinal pigment epithelial cells. Am J Ophthalmol 138:492-495.
Rodrigues, E.B., M. Maia, F.M. Penha, E. Dib, G.B. Melo, A. Maia, and M.E. Farah. 2013. Staining properties of brilliant blue depending on different incubation times and solvents in humans. Ophthalmologica. Journal international d'ophtalmologie. Int J Ophthalmol Clin Res. Zeitschrift fur Augenheilkunde 230 Suppl 2:68-72.
Rodrigues, E.B., and C.H. Meyer. 2008. Meta-analysis of chromovitrectomy with indocyanine green in macular hole surgery. Ophthalmologica. Journal international d'ophtalmologie. Int J Ophthalmol Clin Res. Zeitschrift fur Augenheilkunde 222:123-129.
Rodrigues, E.B., C.H. Meyer, S. Mennel, and M.E. Farah. 2007. Mechanisms of intravitreal toxicity of indocyanine green dye: implications for chromovitrectomy. Retina (Philadelphia, Pa.) 27:958-970.
Rodrigues, E.B., C.H. Meyer, J.C. Schmidt, and P. Kroll. 2003. Trypan blue stains the epiretinal membrane but not the internal limiting membrane. Br J Ophthalmol 87:1431-1432.
Sheidow, T.G., K.J. Blinder, N. Holekamp, D. Joseph, G. Shah, M.G. Grand, M.A. Thomas, J. Bakal, and S. Sharma. 2003. Outcome results in macular hole surgery: an evaluation of internal limiting membrane peeling with and without indocyanine green. Ophthalmology 110:1697-1701.
Sippy, B.D., N.E. Engelbrecht, G.B. Hubbard, S.E. Moriarty, S. Jiang, T.M. Aaberg, Jr., T.M. Aaberg, Sr., H.E. Grossniklaus, and P. Sternberg, Jr. 2001. Indocyanine green effect on cultured human retinal pigment epithelial cells: implication for macular hole surgery. Am J Ophthalmol 132:433-435.
Smiddy, W.E., W. Feuer, and G. Cordahi. 2001. Internal limiting membrane peeling in macular hole surgery. Ophthalmology 108:1471-1476; discussion 1477-1478.
Sparrow, J.R., E. Gregory-Roberts, K. Yamamoto, A. Blonska, S.K. Ghosh, K. Ueda, and J. Zhou. 2012. The bisretinoids of retinal pigment epithelium. Prog Retin Eye Res 31:121-135.
Sparrow, J.R., Y. Wu, C.Y. Kim, and J. Zhou. 2010. Phospholipid meets all-trans-retinal: the making of RPE bisretinoids. J Lipid Res 51:247-261.
Tang, P.H., M. Kono, Y. Koutalos, Z. Ablonczy, and R.K. Crouch. 2013. New insights into retinoid metabolism and cycling within the retina. Prog Retin Eye Res 32:48-63.
Vote, B.J., M.K. Russell, and B.C. Joondeph. 2004. Trypan blue assisted vitrectomy. Retina (Philadelphia, Pa.) 24:736-738.
Wells, J.A., and Z.J. Gregor. 1996. Surgical treatment of full-thickness macular holes using autologous serum. Eye 10:593-599.
Winkler, B.S., M.E. Boulton, J.D. Gottsch, and P. Sternberg. 1999. Oxidative damage and age-related macular degeneration. Mol Vis 5:32-32.
Yuen, D., J. Gonder, A. Proulx, H. Liu, and C. Hutnik. 2009. Comparison of the in vitro safety of intraocular dyes using two retinal cell lines: a focus on brilliant blue G and indocyanine green. Am J Ophthalmol 147:251-259.e252.