- 學位論文
探討青光眼藥對N-methyl-D-aspartate引起視網膜及神經細胞傷害的保護作用
Protective effects of antiglaucoma drugs to N-methyl-D-aspartate-induced damage in retina and neuronal cells
摘要
報告顯示過度刺激N-methyl-D-aspartate (NMDA)接受體會造成神經細胞的死亡。細胞內過多的鈣離子及一氧化氮也會造成細胞的死亡。 因此本實驗主要是利用實驗動物及N1E-115神經母細胞瘤探討不同種類降眼壓藥物，例如timolol, betaxolol, levobunolol, dipivefrin, brimonidine, latanoprost, carteolol, pilocarpine, dorzolamide, brinzolamide, unoprostone, bimatoprost, travoprost對NMDA誘發神經毒性之保護作用。本實驗中所測試的降眼壓藥濃度是以原藥水濃度，經稀釋成1/100, 1/1000及1/10000後，利用propidium iodide, fura-2-AM測試細胞存活率及細胞內鈣離子的變化。利用組織切片觀察視網膜神經節細胞的形態及數目。並且利用視網膜對3H-arginine的攝取觀察藥物對視網膜神經細胞中iNOS及nNOS的影響。結果顯示所有濃度的betaxolol (162 μM，16.2 μM及1.62 μM )和較高濃度的timolol (58μM及5.8μM)與brimonidine (68μM及6.8μM)能夠增加NMDA造成 N1E-115細胞死亡的存活率。但是其它藥，包括carteolol, levobunolol, pilocarpine, dipivefrin, latanoprost, travoprost, bimatoprost, unoprostone, brinzolamide, dorzolamide 及benzalkonium chloride無法有效防止NMDA造成 N1E-115細胞的死亡。兔子玻璃體注射NMDA經24小時及72小時後，發現NMDA反應24小時後明顯對視網膜神經節細胞(RGCs)造成傷害，反應72小時後傷害性更為顯著。在兔子玻璃體個別注射betaxolol, timolol, brimonidine混合NMDA，反應24小時後發現對NMDA引起視網膜神經節細胞(RGCs)傷害並無顯著保護作用。NMDA會增加N1E-115細胞內游離的鈣離子[Ca2+]i，betaxolol和brimonidine都能抑制NMDA引起N1E-115細胞[Ca2+]i增加，而較高濃度的timolol (58 μM及5.8μM)與dipivefrin (28μM, 2.8μM)及carteolol (680 μM)也會抑制細胞內鈣離子濃度增加。但是其它藥對於NMDA增加N1E-115細胞 [Ca2+]i 的作用則無抑制效果。而加入EGTA螯合細胞外鈣離子，以偵測單純胞內儲存的鈣離子流動，高濃度的betaxolol (162 μM及16.2 μM )、timolol (58 μM及5.8μM)及brimonidine (68μM) 都能抑制NMDA引起胞內儲存的[Ca2+]i增加。而其它藥對於NMDA增加胞內儲存的鈣離子[Ca2+]i 的作用則無抑制效果。兔子視網膜經NMDA反應4小時後，視網膜中neuronal nitric oxide synthase (nNOS) 和inducible nitric oxide synthase (iNOS) 活性明顯增加。而Betaxolol (162 μM, 16.2 μM及1.62 μM)及pilocarpine (408 μM及40.8μM)皆能抑制NMDA誘發nNOS活性的效果。而其它藥對於於NMDA誘發nNOS活性則無抑制效果。此外高濃度的betaxolol (162 μM及16.2 μM) 以及timolol (58 μM及5.8μM)具有抑制NMDA誘發iNOS活性的效果。而其它藥對於於NMDA誘發iNOS活性則無抑制效果。綜合以上結果顯示，betaxolol、timolol及brimonidine皆能降低NMDA引起神經細胞的死亡。但是三種藥的機轉皆不相同，brimonidine主要是降低NMDA誘發細胞內鈣離子增加。timolol則是降低NMDA誘發細胞內鈣離子及iNOS增加。而betaxolol則是同時降低NMDA誘發細胞內鈣離子、iNOS及nNOS增加而達到神經保護的作用，因此betaxolol、timolol及brimonidine可當作治療青光眼疾病之較理想的選擇藥物。
並列摘要
Over activation of the N-methyl-D-aspartate (NMDA) receptor may result in neuronal cell death. Excess calcium influx and nitric oxide (NO) production in cells may also cause cellular deaths. In the present study, we investigated various antiglaucoma drugs including timolol, betaxolol, carteolol, levobunolol, pilocarpine, dorzolamide, brinzolamide, brimonidine, latanoprost, travoprost, bimatoprost, unoprostone and dipivefrin on their protective effects to neurons with animals model and N1E-115 neuroblastoma cells. The commercial antiglaucoma drugs were diluted to 1/100、1/1000、1/10000 from original concentration. The cellular viability and intracellular calcium concentrations were detected using propidium iodide and fura-2-AM. The morphology and numbers of retinal ganglion cells were performed with paraffin section. The effects of antiglaucoma drugs on retinal iNOS and nNOS activities were observed by 3H-arginine uptake. The results indicated that the cell viability was increased by all concentrations of betaxolol (162μM, 16.2μM, 1.62μM) and higher concentrations of timolol (58μM, 5.8μM) and brimonidine (68μM, 6.8μM). NMDA-induced cell deaths were not prevented by all concentrations of carteolol, levobunolol, dipivefrin, pilocarpine, latanoprost, travoprost, bimatoprost, unoprostone, brinzolamide, dorzolamide, and benzalkonium chloride. After Intravitrous injection of NMDA into rabbit eyes, the retinal ganglion cells (RGCs) were obviously damaged after 24 and 72 hr treatment. Intravitrous injection of NMDA with drugs including betaxolol, timolol or brimonidine into rabbit eyes were not preventive the loss of retinal ganglion cells statistically. The NMDA could increase cellular calcium concentration [Ca2+]i in N1E-115 cells. The NMDA-induced [Ca2+]i increase were inhibited by betaxolol and brimonidine, and higher concentrations of timolol (58μM, 5.8μM), dipivefrin (28μM, 2.8μM) and carteolol (680μM). However, other drugs have no effects on NMDA-induced [Ca2+]i increases. In calcium-free buffer, the NMDA-induced [Ca2+]i release from intracellular stores was only inhibited by high concentrations of betaxolol (162μM, 16.2μM), timolol (58μM, 5.8μM) and brimonidine (68μM). In the treatment of 10-7M NMDA for 4 hours, the neuronal nitric oxide synthase (nNOS) and inducible nitric oxide synthase (iNOS) activities were significantly increased in retinas. The NMDA-induced nNOS activities were only inhibited by all concentrations of betaxolol (162μM, 16.2μM, 1.62μM) and higher concentrations of pilocarpine (408μM, 40.8μM). The NMDA-induced iNOS activity was only inhibited by higher concentrations of betaxolol (162μM, 16.2μM) and timolol (58μM, 5.8μM). In the conclusion, we found that betaxolol, timolol and brimonidine prevented NMDA-induced neuroal deaths by distict mechanisms. Brimonidine was mediated by reducing NMDA-induced [Ca2+]i increase. Timolol not only reduced NMDA-induced [Ca2+]i increase but also iNOS activity. In the case of betaxolol, the protective mechanism may be mediated by the inhibition of NMDA-induced [Ca2+]i increase, nNOS and iNOS activities productions. Thus, betaxolol, timolol, and brimonidine may be considered the better drugs than other antiglaucoma drugs in the treatment of glaucoma patients because posses ocular hypotensive and retinal neuroprotective effects simultaneously.