摘要
背景与目的
从第一台红宝石激光治疗眼病以来,已有十余种激光相继应用于眼科。由于激光治疗方法优越、疗效确切,激光手术在眼科治疗学中已占有重要地位,其中在治疗眼底疾病方面的应用尤为广泛。视网膜激光光凝术可用于治疗视网膜血管性疾病如:糖尿病视网膜病变、视网膜中央静脉阻塞和早产儿视网膜病变;封闭视网膜裂孔;光凝小梁网、虹膜和睫状体治疗青光眼;以及治疗眼内肿瘤等。尤其对于视网膜缺血性疾病,激光光凝术已或为最有效的治疗手段之一。
尽管在临床上已得到广泛的应用并取得了很好的治疗效果,但对其确切机制尚不十分清楚。目前主要有以下几种推测:第一,视网膜光凝后,一部分视网膜被破坏,视网膜面积减少,改善了残余视网膜的营养状态。第二,光凝使外层视网膜萎缩变薄,使脉络膜氧更容易进入内层视网膜,从而改善内层视网膜缺氧状态。第三,光凝破坏了部分的视网膜组织,使缺血的视网膜释放的新生血管因子减少。第四,由于外层血-视网膜屏障破坏,促进了新生血管因子从视网膜向脉络膜扩散。
由于在激光光凝病变视网膜的同时,也会对正常组织细胞造成损害,因此来源于治疗过程中的并发症难以避免,如出血、视网膜膜前膜、视网膜裂孔、脉络膜新生血管及黄斑水肿等。如何在达到治疗效果的同时使损伤达到最小化是现今激光治疗学中普遍关心的问题。
色素上皮层和脉络膜中的黑色素是视网膜光凝术中光吸收的主要部位。在生理的状态下RPE细胞分泌多种细胞因子和生长因子。激光光凝不但可引起色素上皮细胞形态和生长规律的改变,还能够改变其分泌细胞因子和生长因子的质和量。血管内皮生长因子(vascular endothelial growth factor,VEGF)和色素上皮衍生因子(pigment epithelium derived factor,PEDF)分别作为主要的促血管生成因子及抑制血管生成因子在生理性及病理性血管形成中发挥着重要作用。视网膜光凝术能否通过影响这些生长因子和细胞因子的表达来发挥治疗作用,也成为了许多实验研究的焦点。
本实验旨在建立色素家兔视网膜激光光凝模型的基础上,观察视网膜的组织病理学变化及色素上皮的修复过程,并检测激光光凝前后视网膜中色素上皮衍生因子(PEDF)和血管内皮生长因子(VEGF)表达的变化。以进一步探讨视网膜激光光凝术的治疗机制。
材料与方法
15只健康有色家兔随机分为两组:实验组12只(24眼)、正常对照组3只(6眼)。实验组依术后不同时间点分为1d、3d、7d、28d 4个亚组,每组3只(6眼)。对实验组双眼实施氪离子激光光凝术,照射部位选在视盘下方后极部视网膜,距视盘约2PD,激光参数:功率75mW,光斑直径200pm,照射时间0.1s。按不同时间点将兔处死后,随机选取一眼行色素上皮铺片,对侧眼用于病理切片制作。色素上皮铺片观察色素上皮的损伤与修复;HE染色在光镜下观察激光对视网膜和脉络膜造成的组织学改变;免疫组织化学染色方法检测视网膜组织中PEDF及VEGF表达的变化。
所有数据以SPSS12.0统计软件包进行处理,计量资料用(?)±s表示,多组间比较采用单因素方差分析,q检验进行两两比较,以a=0.05作为显著性检验水准。
结果
1.眼底彩照:正常色素兔视网膜透明薄弱,可透见色素层及脉络膜大血管。光凝后出现视网膜水肿,激光斑呈白色:随时间延长,视网膜水肿减轻,光斑逐渐变为灰色,且有相互融合的趋势。
2.色素上皮铺片:正常色素上皮为一单细胞层,细胞大小均一、边界清晰、呈六边形、铺路石状整齐排列。光凝后激光斑周边细胞在大小、形态及色素沉着方面呈现不均一性;光斑内可见细胞坏死、萎缩、缺失,后期通过激光斑周围细胞的增殖、移位填补修复损伤。
3.组织病理学观察:正常视网膜各层结构清晰,细胞完整。激光光凝后光凝斑区域外层视网膜细胞结构破坏,细胞坏死、核碎裂、崩解,炎性细胞浸润,随后色素上皮细胞和成纤维细胞增生修复损伤。
4.免疫组织化学:正常对照组PEDF及VEGF蛋白阳性信号均可见于光感受器细胞层、内颗粒层及神经节细胞层。光凝后视网膜中PEDF蛋白的表达明显升高,3d时达到高峰,之后逐渐下降,至28d时仍高于对照组水平;视网膜组织中VEGF的表达在光凝后1d表达增强,3d时开始下降至正常水平,28d时明显低于对照组。
结论
1.激光光凝可以引起外层视网膜结构破坏、细胞减少,视网膜通过RPE细胞和成纤维细胞增殖修复损伤区。
2.激光光凝可以刺激RPE细胞的增殖,RPE细胞在激光损伤修复过程中发挥着重要作用。
3.激光光凝可以上调视网膜中PEDF的表达,下调VEGF的表达,使血管生成因子与抑制因子达到新的平衡,从而发挥其抑制新生血管的治疗作用。
Background and Objective
Since the first ruby laser was utilized in patient treatment, there have been more than ten kinds of laser used in ophthalmology. Because its superiority process and certain effect, laser treatment has taken important position in ophthalmiatrics, especially in retina diseases. The laser photocoagulation has been used for treatment of retinal vascular diseases, such as diabetic retinopathy, retinal vein occlusions, and retinopathy of prematurity; for sealing of retina holes; for photocoagulation of the trabecular meshwork, iris, and ciliary body in the treatment of glaucoma; and for the treatment of intraocular turners. It is one of the most effective therapy for the retinal ischemia disease.
Despite its widespread clinical use and excellent therapeutic efficacy, the exact mechanism of photocoagulation have not been determined. Presently, there are several presumptions: First, Part of the retina has been destroyed after photocoagulation, the area of the retina decreases, so result in the improvement of nutritional status of the remnant retina. Second, The retina atrophies and thinningzs after photocoagulation, so the diffusion of oxygen from the choriod into the inner retina becomes easier. Accordingly, the hypoxia of the retina is changed. Third, The neovascularization factor decreases because of the destruction of partial retina. Fourth, The damage of the extra-barrier after photocoagulation promotes the diffusion of neovascularization factor from the retina into the choroid.
The normal tissue is also subjected when laser photocoagulation, so complications following the treatment is hard to avoid, such as hemorrahge, epiretina membrane, retina holes, choroidal neovascularization and macular edema. There is now increasing interest in how to minimizing unnecessary retina damage while obtaining therapeutical effect.
The main site of energy absorption in laser photocoagulation is the melanin pigment in the retinal pigment epithelial (RPE) cells and the choroid. Physiologically, RPE cells produces kinds of cytokines and growth factors. Laser photocoagulation can cause RPE cells to change their shape and growth pattern, in addition, the profile of growth factor and cytokine production. Vascular endothelial growth factor (VEGF) and pigment epithelium derived factor (PEDF) working as the primary angiogenic growth factor and angiogenin inhibitor play much important part of the physiological and pathological angiopoiesis, and which is also the focus of recent years research.
This study is to establish the retinal laser photocoagulation model in pigment rabbits. Subsequently, observe the histopathological change of the retina and the reparative process of RPE, and detect expression of PEDF and VEGF in the retina before or after photocoagulation. Accordingly, to approach the therapeutic mechanisms of retina laser photocoagulation.
Materials and Methods
15 normal pigmental rabbits were used and devided into two groups randomly: experiment group and control group, which included 12 rabbits (24 eyes) and 3 rabbits (6 eyes) seperately. The experiment group was devided into four subsets depended on different times: 1d, 3d, 7d, 28d. And each group contained 3 rabbits (6 eyes). Laser photocoagulation was performed (75mW, 200μm, 0.1s) on the both eyes of the experiment rabbits, and the irradiated site located at posterior pole retina, 2PD distanced from the optic disc. The rabbits were killed at intervals, one eye was prepared for RPE whole mounts, and the fellow eye was used for pathological section. The damage and repairation of the RPE was determined in RPE whole mounts; Histopathological effects of the retina and choroid were observed through light microscope; The expression changes of PEDF and VEGF were detected by histochemical stain.
The data were analyzed with statistical software SPSS12.0. Numerical data were expressed with x|-±s. Several groups were compared by simple factor analysis of variance, and every two groups were compared by q test. Results were considered significant at a=0.05.
Results
1. Fundus image: The fundus of nomal pigmental rabbit looked clearing and thin. The pigmentary layer and choroidal vessels could be seen clearly. Retinal edema appeared after photocoagulation, with white color laser spot. Then the retinal edema decreased gradually. The color of laser spot became gray, and there was confluent tendency among the laser spots.
2. RPE whole mounts: RPE was a monolayer tissue. The size and shape of the cells were fairly uniform, which looked like sexangle slabstone lined up in order. After photocoagulation, cells around the laser spot became heterogeneous in size, shape and pigmentation. Cellular necrosis, atrophia and absence could be detected in the laser spot. Then the damage was recovered by the proliferation and aversion of RPE cells around the laser spot.
3. Histopathological examinations: laser photocoagulation caused disorganization of outer retina, cell necrosis, nuclear fragmentation and disintegration, inflammatory cell could be found, Subsequently the proliferous RPE and fibroblast repaired the destroyed areas of retina.
4. Immunohistochemistry: The expression of PEDF and VEGF of the control group could be detected in photoceptor layer, internal granular layer and ganglion cell layer. PEDF protein levels significantly increased after laser photocoagulation. PEDF concentrations were maximum at day 3, and stayed elevated until day 28. Expression of VEGF was increased 1 day after photocoagulation, which declined to the control levels at day 3 and below the control levels statisticly at day 28.
Conclusions
1. Disorganization and cell loss in the outer retina could be detected after laser photocoagulation. Subsequently, the proliferous RPE and fibroblast repaired the destroyed areas of retina.
2. Laser phtocoagulation induced the proliferation of RPE, which play an important parts in the RPE reparative process.
3. Laser photocoagulation induced upregulation of PEDF and downregulation of VEGF, which caused new balance between angiogenesis factor and inhibition factor, so inhibit the neovascularization.
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