局部抗青光眼藥、全身性神經保護藥物與抗氧化營養添加物對高眼壓傷害之大鼠視網膜的保護效力研究

Abstract

青光眼是一個多因子的疾病，病變特徵為漸進性視網膜及視神經退化，為造成永久性失明的第二大主因。青光眼的起始及進展都與視網膜缺血病變有關，因此本試驗以高眼壓誘導視網膜缺血之大鼠模式以模擬急性青光眼。 本試驗目的為評估局部點眼抗青光眼藥brinzolamide(AZ)及timolol(TI)；全身性注射型藥物lidocaine(LI)、methylprednisolone(ME)及minocycline(MI)；三種未知抗氧化營養添加物抗氧化物A(18種混合營養添加物)、抗氧化物B(17種混合營養添加物，不含葉黃素)、抗氧化物C(17種混合營養添加物，不含山桑子)，對於高眼壓誘導視網膜缺血傷害的保護效力。試驗分成兩部分。 第一部分：將成年Sprague-Dawley大鼠分成7組：正常控制組(n=6)；視網膜缺血傷害無治療之控制組(n=6)；給予點眼抗青光眼藥或全身性注射型藥物之視網膜缺血傷害治療組AZ(n=6)、TI(n=6)、LI(n=6)、ME(n=6)及MI(n=6)。點眼抗青光眼藥從視網膜缺血傷害結束後開始投予，一天3次，連續投予7天。LI治療組，在缺血傷害前30分鐘靜脈注射lidocaine 1.5 mg/kg的劑量，隨即以2 mg/kg/hr的速率持續靜脈注射直到缺血傷害結束後60分鐘。ME治療組，給予兩次methylprednisolone 30 mg/kg靜脈注射，分別在缺血傷害前2分鐘及缺血傷害結束後馬上給予。MI治療組，視網膜缺血傷害結束後開始投予minocycline腹腔內注射22 mg/kg，一天1次，連續投予7天。利用缺血傷害前1天及傷害後第1天、第3天及第7天的視網膜電波圖，以及缺血傷害後第七天組織病理形態及視網膜的厚度，來評估治療組對視網膜功能及形態的保護效力。選取視網膜功能具保護效力的組別，於缺血傷害後第七天分析氧化壓力及抗氧化酵素活性測試以分析藥物的抗氧化效力，並選取其中最具抗氧化效力的組別進行初步蛋白質體分析，評估藥物對於視網膜保護的分子機制。視網膜電波圖結果顯示局部點眼timolol、全身性給予methylprednisolone及minocycline對於視網膜缺血-再灌流傷害能提供視網膜功能性保護。病理結果顯示局部點眼brinzolamide及lidocaine能提供視網膜內層及外層形態學上的保護。Methylprednisolone及minocycline治療組的氧化壓力較低且抗氧化酵素活性較高，顯示其保護機制可能與其抗氧化活性相關。Methylprednisolone視網膜保護的分子機制包括：減少細胞氧化壓力、增加糖解作用提供視網膜細胞能量、調節炎症反應、減少神經創傷及細胞凋亡、增加視網膜前血液循環及血管舒張。 第二部分：將成年Sprague-Dawley大鼠分成5組：正常控制組(n=6)；視網膜缺血傷害餵食生理食鹽水之控制組(n=6)；視網膜缺血傷害餵食未知抗氧化營養添加物之治療組抗氧化物A(n=6)、抗氧化物B(n=6)及抗氧化物C(n=6)。三個治療組每天餵食抗氧化物A、B及C，缺血傷害前餵食28天，缺血傷害後再餵食28天，總共餵食56天。視網膜電波圖測量於抗氧化物餵食前1天、及餵食後第7、14、21、28、35、42、49、56天。於抗氧化營養添加物餵食後第56天分析氧化壓力及抗氧化酵素活性測試以分析視網膜保護與抗氧化間的關聯性。結果顯示抗氧化物A及抗氧化物C對於視網膜功能具保護效果，氧化壓力較低且抗氧化酵素活性較高，顯示其保護機制與其抗氧化活性相關。 研究結果顯示，局部點眼timolol、全身性給予methylprednisolone及minocycline、抗氧化營養添加物A及C對於視網膜缺血-再灌流傷害能提供視網膜功能性保護，且其保護作用與降低氧化壓力及提升抗氧化酵素活性有關。局部點眼brinzolamide及全身性給予lidocaine提供視網膜形態學上的保護效果。蛋白質體結果顯示，methylprednisolone的藥物作用機制與抗氧化、抗凋亡、抗發炎及調節視網膜血液循環有關。將治療組的視網膜保護效力統整如下：ME>MI>TI>LI>AZ；抗氧化物A(葉黃素+山桑子)≥抗氧化物C(葉黃素)>>抗氧化物B(山桑子)。

Glaucoma, a group of diseases characterized by progressive optic nerve degeneration, is the leading cause of irreversible blindness worldwide. The initiation of the disease and its progression involve an ischaemic-like insult to the ganglion cell axons. Thus, we use a rat model of retinal ischemia–reperfusion (IR) injury in the present study as an acute model of glaucoma. The purpose of this study was to evaluate the retinal protective effect of topical brinzolamide (AZ) and timolol (TI)；intravenous (IV) lidocaine (LI) and methylprednisolone (ME)；intraperitonium (IP) minocycline (MI)；nutritional antioxidant supplements antioxidant A (mixed antioxidants with 18 ingredients), antioxidant B (mixed antioxidants with 17 ingredients, without lutein), antioxidant C (mixed antioxidants with 17 ingredients, without bilberry). The study was separated into two parts. PartⅠ：Adult Sprague-Dawley rats were divided into 7 groups, the normal control (n=6), IR injury (n=6), AZ (n=6), TI (n=6), LI (n=6), ME (n=6) and MI (n=6). In AZ and TI groups, brinzolamide and timolol were administered topically three times a day for 7 days after the IR injury. In LI group, lidocaine bolus (1.5 mg/kg) was IV injected 30 minutes before ischemia and then a constant rate infusion (CRI) with 2 mg/kg/hr was given until 60 minutes after reperfusion. In ME group, methylprednisolone bolus (30 mg/kg) was IV administered twice at 2 minutes before and immediately after ischemia, respectively. In MI group, minocycline at 22 mg/kg was intraperitoneally administered daily for 7 days after the IR injury. Protection of the retina was determined by electroretinogram (ERG) 1 day before and 1, 3, 7 day(s) after the IR injury and by morphometrical histology analysis 7 days after the IR injury. In addition, oxidative stress, the activity of antioxidant enzymes and proteomic analysis were used to investigate the possible mechanisms of the protective effect against IR injury. Our electroretinographic data indicated that topical timolol, methylprednisolone IV and minocycline IP attenuated the effects of retinal ischaemia/reperfusion injury. The retinal histopathology results presented protective effect of topical brinzolamide and lidocaine IV in inner and outer retina. The activity of antioxidant enzymes were higher and the level of oxidative stress was lower in methylprednisolone and minocycline treated groups. The protein expression altered by methylprednisolone include: oxidative stress proteins, glycolysis proteins, immunomodulatary proteins and cytoskeletal proteins. Part Ⅱ：Adult Sprague-Dawley rats were divided into 5 groups, the normal control (n=6), IR injury (n=6), antioxidant A (n=6), antioxidant B (n=6) and antioxidant C (n=6). The antioxidant A, B, C groups were treated by feeding unknown mixed antioxidants daily for 56 days (28 days before and 28 days after the IR injury). The electroretinography of all groups were assessed at 1 day before and 7, 14, 21, 28, 35, 42, 49, 56 days after feeding unknown mixed antioxidants. In addition, oxidative stress and the activity of antioxidant enzymes were used to investigate the antioxidative effect against the IR injury. After IR injury, both the a- and b-waves in the ERG decreased; however, antioxidant A and C treated groups reduced these effects. The activity of antioxidant enzymes was higher and the level of oxidative stress was lower in antioxidant A and C treated groups. These findings suggest that timolol, methylprednisolone, minocycline, antioxidant A and antioxidant C possessed good antioxidative activity and protective effect for the retina. Brinzolamide and lidocaine provided protective effect to preserve the thickness of retina, but not in retinal function. Methylprednisolone had anti-oxidation, anti-apoptotic, neovascularization and inflammatory regulation effects proved by proteomic analysis. Based on these investigations, neuroprotective efficacy of treated groups is listed as follows. Drugs：methylprednisolone > minocycline > timolol > lidocaine > brinzolamide. Antioxidants treated: Lutein + bilberry ≥ lutein >> bilberry.