林中天張本恆臺灣大學：鄭詠月Jeng, Yung-YueYung-YueJeng2007-11-282018-07-092007-11-282018-07-092006http://ntur.lib.ntu.edu.tw//handle/246246/59981本實驗的目的為評估兩種具有神經保護功能的藥物: Brimonidine及 Phenylbutyrate (PBA)對於大鼠視網膜缺血傷害(retinal ischemic injury)是否有保護能力及其保護效力。在成年Sprague-Dawley大鼠，先利用螢光追蹤器Fluorogold(FG)放置在四疊體上丘上方以達成回溯性標定視網膜神經節細胞(RGCs)。七天之後，以高眼壓方式製造大鼠視網膜缺血。藥物治療分為三組，視網膜缺血一小時前點眼投與0.2% Brimonidine於大鼠左眼；視網膜缺血前30分鐘，以腹腔注射100 mg/kg或400 mg/kg PBA；視網膜缺血後一個小時，以腹腔注射400 mg/kg PBA。在視網膜缺血後七天，將大鼠犧牲，取下視網膜後，在標準的16個區域計算FG-labeled RGCs數量，評估存活的RGCs數目。另一方面，也是在視網膜缺血後七天，將眼球取下固定製作組織病理切片。120 mmHg，45分鐘的視網膜缺血會造成FG-labeled RGCs 18.7%的喪失。但是缺血前給予0.2% Brimonidine點眼的視網膜只有喪失6.74%的FG-labeled RGCs。在缺血前給予高劑量或低劑量的PBA可以幾乎100%的保護RGCs但是缺血後給予PBA對於RGCs卻沒有保護效果。缺血前給予Brimonidine點眼治療及缺血前給予PBA都可以有意義的減少視網膜、內層視網膜、內叢狀層厚度的消失及減少神經節細胞層及內核層細胞的喪失。這些結果都可以揭示Brimonidine與PBA對視網膜缺血是有保護能力。The purpose of this study was to investigate the effects of two neuroprotective agents: Brimonidine and Phenylbutyrate (PBA), in retinal ischemic injury of rats. In adult Sprague-Dawley rats, RGCs were retrograde labeled with fluorescent tracers Fluorogold (FG) applied to both superior collicoli. Seven days later, the high intraocular pressure was induced. Medications were divided into three groups: one hour prior to retinal ischemia, 0.2% Brimonidine was instilled on the left eye; 30 minutes prior to retinal ischemia, 100 mg/kg or 400 mg/kg PBA were injected intraperitoneally; one hour after retinal ischemia, 400 mg/kg PBA was injected intraperitoneally. Rats were processed 7 days later and densities of surviving RGCs were estimated by counting FG-labeled RGCs in 16 standard regions of each retina. On the other hand, histological outcomes were also determined at 7 days after retinal ischemia. 120 mm Hg for 45 minutes ischemic injury caused 18.7% loss of FG-labeled RGCs and after the treatment of Brimonidine only 6.74% of FG-labeled RGCs were lost. Almost 100% RGCs could survive by pre-ischemic treatments of PBA but post-ischemic treatment of PBA cannot rescue RGCs from IR injury. Pre-ischemic treatment with Brimonidine and pre-ischemic treatment of PBA significantly reduced loss of thickness of retina, inner retina, and inner plexiform layer and reduced loss of cells in the ganglion cell layer and inner nuclear layer. Although post-ischemic treatment of PBA cannot rescue cells in ganglion cell layer but can reduce loss of thickness of retina, inner retina, and inner plexiform layer and reduced loss of cells in the inner nuclear layer. These results indicate that both Brimonidine and PBA have neuroprotective effects on retinal ischemia.ABSTRACT 5 第一章 緒 論 6 第二章 文獻回顧 8 2.1 青光眼與治療 8 2.2 青光眼所造成的視網膜及視神經神經病變 (GLAUCOMATOUS NEUROPATHY) 10 青光眼視神經病變之病理生理機制 11 2.2.1 神經營養性因子缺乏 12 2.2.2 麩氨酸對神經原之神經興奮性毒 12 2.2.3 自由基的聚集所造成的傷害 13 2.2.4 一氧化氮的神經毒性 13 2.2.5 細胞凋亡(apoptosis) 15 2.3 神經保護藥物 (NEUROPROTECTIVE AGENTS) 16 2.3.1 鈣離子通道阻斷劑 (Ca2+ channel blockers, CCB) 16 2.3.2 降眼壓藥物 (Antiglaucoma medications) 18 2.3.3 NMDA antagonist 18 2.3.4 抗氧化劑 19 2.3.5 一氧化氮合成酶抑制劑 19 2.3.6 神經營養物質 (Neurotrophins) 19 2.4 ALPHAL-2腎上腺受器致效劑的神經保護功能 20 2.5 研究青光眼之動物模式 (ANIMAL MODELS OF GLAUCOMA) 22 2.5.1 雷射誘發青光眼 22 2.5.2 高張溶液注入鞏膜上靜脈 23 2.5.3 燒灼鞏膜上靜脈 23 2.5.4 視網膜缺血之動物模式 (Animal model of retinal ischemia) 25 2.6 視網膜缺血再灌流之病理生理學 32 2.7 視網膜神經節細胞與視覺傳遞 34 2.8 神經原追蹤器回溯性標定RGCS 39 2.8.1 回溯性標定視網膜神經節細胞的原理 39 2.8.2 視網膜神經原標定物質 41 2.8.3 Fluorogold回溯性標定RGCs之操作方式 43 2.9 苯基丁酸鈉 (SODIUM 4-PHENYLBUTYRATE, NAPB, PBA)之神經保護功能 45 2.9.1 NaPB與尿素循環障礙 46 2.9.2 Phenylbutyrate與神經退化性疾病相關研究 47 第三章 研究材料與方法 54 3.1 動物、麻醉方式及藥物投與 54 3.2 回溯性標定視網膜神經節細胞 54 3.3 實驗分組 56 3.4 高眼壓模式誘發視網膜缺血再灌流 56 3.5 計算視網膜神經節細胞數 57 3.6 型態計量學分析 59 3.7 統計分析 60 第四章 實驗結果 61 第一節 回溯性RGCS螢光標定 61 4.1.1 Fluorogold回溯性標定RGCs 61 4.1.2 視網膜缺血造成RGC喪失 (retinal ischemia group) 61 4.1.3 alpha-2 致效劑: Brimonidine 62 4.1.4 Pre-ischemia 100 mg/kg PBA 63 4.1.5 Pre-ischemia 400 mg/kg PBA 65 4.1.6 缺血後1小時全身投與400 mg/kg PBA 66 第二節 型態計量學結果 72 4.2.1 正常視網膜組織學分析 72 4.2.2 視網膜缺血後視網膜各層厚度減少 72 4.2.3 視網膜缺血前1小時點眼投與Brimonidine 73 4.2.4 Pre-ischemia 100 mg/kg PBA (PI 100) 74 4.2.5 Pre-ischemia 400 mg/kg PBA (PI 400) 74 4.2.6 Post-ischemia 400 mg/kg PBA (Post ischemia 400) 75 第五章 討論 79 第六章 結論 87 參考文獻 884308059 bytesapplication/pdfen-US青光眼視網膜缺血大鼠4苯丁酸鈉GlaucomaRetinal ischemiaRatSodium 4-Phenylbutyratethesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/59981/1/ntu-95-R90629018-1.pdf