实验研究
ENGLISH ABSTRACT
新型视网膜缺血-再灌注损伤模型的建立及评价
温鑫
袁敏而
李成
曾婕婷
林晓峰
作者及单位信息
·
DOI: 10.3760/cma.j.cn115989-20190404-00162
Establishment and evaluation of a novel retinal ischemia-reperfusion injury model
Wen Xin
Yuan Miner
Li Cheng
Zeng Jieting
Lin Xiaofeng
Authors Info & Affiliations
Wen Xin
State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-Sen University, Guangzhou 510060, China (Wen Xin is now in Department of Ophthalmology, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou 510120, China)
Yuan Miner
State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-Sen University, Guangzhou 510060, China
Li Cheng
State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-Sen University, Guangzhou 510060, China
Zeng Jieting
State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-Sen University, Guangzhou 510060, China
Lin Xiaofeng
State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-Sen University, Guangzhou 510060, China
·
DOI: 10.3760/cma.j.cn115989-20190404-00162
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摘要

目的探索在新西兰兔视网膜中央动脉阻塞(CRAO)模型中行经睫状体平坦部玻璃体切割术(PPV)联合反搏术构建新型视网膜缺血-再灌注(RIR)损伤模型。

方法选用20只健康成年新西兰兔,按照随机数字表法随机分为2个组,每组10只:激光组单纯行视网膜动脉激光光凝术,反搏术组于光凝术后行PPV联合反搏术,均取右眼为实验眼,左眼作为正常对照组。通过荧光素眼底血管造影(FFA)、玻璃体腔氧分压(PO 2)评估灌注恢复情况,观察视网膜电图(ERG)的振荡电位(OPs)变化评估视网膜功能改变,苏木精-伊红染色法观察视网膜结构改变。

结果反搏术组术中即可观察到视网膜灌注恢复;术后2 h,FFA检查示所有眼视网膜动静脉完全恢复灌注,早期即见视网膜动脉充盈,随后静脉充盈,充盈时间无延迟,无血流中断。不同时间点反搏术组、激光组和正常对照组PO 2百分数总体比较,差异均有统计学意义( F 分组=330.87, P<0.001; F 时间=985.70, P<0.001),其中激光后、术后不同时间点反搏术组玻璃体腔PO 2百分数分别为(18.67±6.29)%、(38.82±1.48)%、(57.33±4.25)%、(84.51±3.91)%和(89.20±2.97)%,高于激光组的(23.24±1.95)%、(31.44±3.29)%、(40.21±3.05)%、(43.65±3.82)%和(58.07±2.93)%,差异均有统计学意义(均 P<0.05)。不同时间点反搏术组、激光组和正常对照组OPs百分数总体比较,差异均有统计学意义( F 分组=164.09, P<0.001; F 时间=447.91, P<0.001),其中术后3 d、7 d、2周和1个月反搏术组OPs百分数分别为(47.23±2.73)%、(70.79±3.09)%、(78.39±3.63)%、(76.69±4.08)%和(82.18±1.78)%,较激光组的(46.83±2.89)%、(55.32±1.58)%、(51.08±4.02)%、(52.32±6.59)%和(53.46±6.46)%升高,差异均有统计学意义(均 P<0.05)。反搏术组视网膜内层结构破坏较小,有髓神经纤维层(MFL)结构疏松,大量空泡状改变。激光组MFL、内丛状层、内核层和外丛状层结构紊乱,Müller细胞神经纤维破坏。

结论在新西兰兔CRAO模型中行PPV联合反搏术可构建新型RIR损伤模型。

视网膜疾病;缺血-再灌注损伤;玻璃体切割术;动物模型;反搏术
ABSTRACT

ObjectiveTo explore the feasibility to establish a novel retinal ischemia reperfusion (RIR) injury model by applying pars plana vitrectomy (PPV) combined with retinal vascular counterpulsation in the central retinal artery occlusion (CRAO) model of New Zealand rabbit.

MethodsTwenty New Zealand adult rabbits were randomly divided into two groups by random number table, 10 for each group.Rabbits in the laser group were treated with retinal laser photocoagulation only, while rabbits in the counterpulsation group were treated with PPV combined counterpulsation.The right eye of each New Zealand rabbit was used as the experimental eye and the left eye was used as the normal control (the normal control group). Fundus fluorescence angiography (FFA) , oxygen partial pressure (PO 2) in vitreous cavity was performed to assess the recovery status of perfusion.Scotopic 3.0 oscillatory potentials (OPs) in electroretinogram (ERG) were used to evaluate the retinal function, and retinal pathological sections were used to evaluate the structural changes in the retina.The use and care of the animals complied with the Statement of the Association for Research in Vision and Ophthalmology (ARVO), and the study was approved by the Animal Research Committee of Zhongshan Ophthalmic Center, Sun Yat-sen University.

ResultsIn the counterpulsation group, retinal reperfusion was observed during counterpulsation processure.FFA examination at 2 hours after counterpulsation showed reperfusion of retinal blood stream in all the eyes.Retinal artery filling, followed by venous filling was observed in the early stage, with no delay in filling and no interruption of blood flow.The percentage of vitreous PO 2 was significantly different among the counterpulsation group, the laser group and the normal control group at different time points ( F group=330.87, P<0.001; F time=985.70, P<0.001). The percentages of vitreous PO 2 in the counterpulsation group at different time points was (18.67±6.29)%, (38.82±1.48)%, (57.33±4.25)%, (84.51±3.91)% and (89.20±2.97)%, which were significantly higher than that in the laser group ([23.24±1.95]%, [31.44±3.29]%, [40.21±3.05]%, [43.65±3.82]% and [58.07±2.93]%) (all at P<0.05). The percentage of OPs was significantly different among the counterpulsation group, the laser group and the normal control group at different time points ( F group=164.09, P<0.001; F time=447.91, P<0.001). The percentages of OPs of counterpulsation group at different time points after laser and after PPV were (47.23±2.73)%, (70.79±3.09)%, (78.39±3.63)%, (76.69±4.08)% and (82.18±1.78)%, which were significanthy higher than that in the laser group ([46.83±2.89]%, [55.32±1.58]%, [51.08±4.02]%, [52.32±6.59]% and [53.46±6.46]%) ( all at P<0.05). There was a lesser damage in inner retinal structure in pathological section in the counterpulsation group.The myelinated nerve fiber layer (MFL) was loose and a mass of vacuolar changes were observed in MFL.The structure of MFL, inner plexiform layer, inner and outer plexiform layer in the laser group were disordered, and the Müller cell nerve fibers were destroyed in the laser group.

ConclusionsA novel type of RIR injury model can be established by applying PPV combined with counterpulsation in the CRAO model of New Zealand rabbit.

Retinal disease;Retinal ischemia-reperfusion;Vitrectomy;Animal model, disease;Counterpulsation
Lin Xiaofeng, Email: nc.defudabe.usys.liamfoaixnil
引用本文

温鑫,袁敏而,李成,等. 新型视网膜缺血-再灌注损伤模型的建立及评价[J]. 中华实验眼科杂志,2020,38(07):566-572.

DOI:10.3760/cma.j.cn115989-20190404-00162

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视网膜缺血-再灌注(retinal ischemia reperfusion,RIR)损伤可发生于视网膜动静脉阻塞、急性闭角型青光眼、糖尿病视网膜病变等缺血性视网膜疾病 [ 1 ]。目前,RIR的损伤机制尚未完全明确,是国内外研究的热点之一。RIR模型是研究其发病机制的基础,建立接近临床病理过程的动物模型对于减少RIR损伤和保护视功能有重要意义。理想的RIR模型应当是视网膜动脉阻断血流后再恢复灌注,而对视网膜其他组织没有损伤,且再灌注时间具有可控性。目前常用的RIR模型中血管结扎模型手术难度高,易损伤血管及其周围组织,且从血管外部结扎无法模拟血管内部因素所致血管阻塞的病理状态;管内段视神经损伤模型、视神经结扎模型、线栓法模型和高眼压模型对视神经均有不同程度损伤;视网膜动脉激光光凝后自发再灌注模型的再灌注时间不可控 [ 2 ]。这些模型均无法满足理想RIR模型的要求。经睫状体平坦部玻璃体切割术(pars plana vitrectomy,PPV)联合术中血管切开、眼压调控等操作可治疗视网膜中央动脉阻塞(central retinal artery occlusion,CRAO) [ 3 ]。在应用PPV治疗眼底病变的临床研究中如果在术中降低眼内灌注压,将玻璃体切割头置于视盘凹陷前,增大负压抽吸,可观察到视网膜动脉扩张,随后动脉和静脉充盈,血流灌注增加,当停止负压抽吸时,动脉变窄,血管充盈减少。本研究中将此种方法命名为视网膜血管反搏术(简称反搏术)。PPV广泛应用于临床,具有较高的安全性和可控性。本研究中探讨采用PPV联合反搏术制作RIR损伤动物模型的可行性,以期构建一种模拟血管内部阻塞后再灌注病理过程且再灌注时间可控的RIR动物模型。
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备注信息
A
林晓峰,Email: nc.defudabe.usys.liamfoaixnil
B
所有作者均声明不存在利益冲突
C
广东省自然科学基金项目 (2018A030313585)
眼科学国家重点实验室专项经费项目 (30306020240020130、3030902113030)
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