Regression of ocular neovascularization in response to increased expression of pigment epithelium-derived factor

PURPOSE: Several pharmacologic treatments have been shown to reduce ocular neovascularization when administered before the onset of angiogenic stimuli, but none have been shown to cause regression of already established ocular neovascularization. In this study, the authors tested the effect of adenoviral vectored pigment epithelium-derived factor (PEDF) gene transfer on established neovascularization in transgenic mice with expression of vascular endothelial growth factor (VEGF) in photoreceptors (rho/VEGF mice) and in a model of choroidal neovascularization. METHODS: Two weeks after the onset of VEGF transgene expression in rho/VEGF mice or 2 weeks after laser-induced rupture of Bruch's membrane in wild-type mice, subgroups of mice were killed, and the baseline amount of neovascularization was measured by image analysis. The remainder of the mice received an intravitreous or subretinal injection of adenoviral vector containing a PEDF expression construct (AdPEDF.11) or control vector (AdNull.11). RESULTS: Seven days after injection in rho/VEGF mice or 10 days after injection in the choroidal neovascularization model, the amount of neovascularization in AdPEDF.11-injected eyes was significantly less than the baseline level, indicating that regression of neovascularization had occurred. There was TUNEL staining within choroidal neovascular lesions in eyes injected with AdPEDF.11. Eyes given a subretinal injection of AdNull.11 had TUNEL-positive cells in the retina, but none within areas of choroidal neovascularization. CONCLUSIONS: These data indicate that increased expression of PEDF causes regression of ocular neovascularization by promoting apoptosis of cells within neovascular lesions and possibly represents a new treatment paradigm for patients with established ocular neovascularization.     

Pigment epithelium derived factor as a neuroprotective agent against ischemic retinal injury.

PURPOSE. Pigment epithelium-derived factor (PEDF) is a protein shown to have neurotrophic activity. The purpose of this study was to determine whether PEDF is neuroprotective of retinal neurons that are exposed to transient ischemia-reperfusion. METHODS. Transient retinal ischemia was produced by increasing the intraocular pressure for 45 min in albino rats eyes. Immediately after reperfusion, PEDF was injected intravitreally into the experimental eyes. Injury was evaluated morphologically and by measuring the thickness of the inner retinal layers (IRL) and by counting the number of retinal ganglion cells (RGC) in epon embedded sections. RESULTS. Morphologic and morphometric analysis of the thickness of the IRL and the counting of RGC demonstrated that PEDF injected immediately after reperfusion protected the eyes partially but significantly from the ischemic injury. CONCLUSIONS. Intravitreal injection of PEDF even after the ischemia can ameliorate retinal injury. PEDF may be useful in preventing neuronal degeneration in the inner retina resulting from ischemia.     

缺血再灌注损伤诱导大鼠视网膜细胞凋亡

目的 观察缺血再灌注大鼠视网膜损伤及细胞凋亡情况。 方法 采用升高大鼠眼压到109.725 mm Hg(1 mm Hg=0.133 kPa)持续1 h的方法制作视网膜缺血再灌注模型，采用常规眼球切片观察不同缺血和再灌注时间的视网膜损伤的组织病理改变；采用DNA琼脂糖凝胶电泳法检测视网膜神经元凋亡情况；采用DNA原位末端标记(terminal dUTP nick end labelling, TUNEL)法定位凋亡的视网膜细胞。 结果 缺血30 min 再灌注24、48 h的大鼠视网膜无明显的病理改变；缺血60 min再灌注24、48 h的大鼠视网膜神经节细胞层和内核层细胞明显变薄；缺血60 min再灌注12、24 h的大鼠视网膜有梯状条带。而正常对照组、缺血30 min再灌注24、48 h组及缺血60 min再灌注48 h组大鼠视网膜均无类似表现。TUNEL法显示视网膜内的细胞凋亡主要发生在节细胞和内核层光感受细胞。 结论 大鼠视网膜缺血再灌注主要是导致视网膜神经节细胞层和内核层细胞损伤，细胞凋亡可能是损伤的重要机制。 (中华眼底病杂志, 2002, 18: 296-298)     

Protective effects of selectin ligands/inhibitor (SKK-60060) against retinal ischemia-reperfusion injury

A newly developed selSep;71(3)28 to block P- and L-selectins in vitro. We examined its inhibition of leukocyte-endothelial interactions in vivo against retinal ischemia-reperfusion injury and protective effects on ischemia-induced retinal damage.Retinal ischemia was induced by temporary ligation of the optic sheath for 60 min in anesthetized pigmented rats. SKK-60060 was administered 5 min before reperfusion and 4, 12, 24 and 48 hr thereafter, and leukocyte dynamics in the retinal microcirculation were evaluated using acridine orange digital fluorography. After 7 days of reperfusion, ischemia-induced retinal damage was also assessed histologically.SKK-60060 treatment suppressed leukocyte rolling during the reperfusion period; their numbers in the SKK-60060-treated rats were reduced by 67.0% (P < 0. 01) and 53.2% (P < 0.01) at 12 and 24 hr, respectively. The subsequent leukocyte accumulation was also inhibited in SKK-60060-treated rats; accumulated leukocytes in the SKK-60060-treated rats were reduced by 72.8% (P < 0.01) and 53.4% (P < 0.01) at 12 and 24 hr, respectively. Retinal venous vasodilation in SKK-60060-treated rats were significantly suppressed at each time point (P < 0.05). Histological examination demonstrated protective effects of SKK-60060 on ischemia-induced retinal damage, which were more substantial in the inner retina (P < 0.01).SKK-60060 significantly inhibits the leukocyte rolling along the major retinal veins and their accumulation during the reperfusion period. These results suggest therapeutic potential of SKK-60060 for ischemia-reperfusion injury.     

腺病毒载体介导的色素上皮衍生因子对大鼠视网膜缺血再灌注损伤的保护作用

目的:观察重组腺病毒介导的色素上皮衍生因子(Ad-PEDF)对大鼠视网膜缺血再灌注损伤的保护作用及机制。方法:选用健康大鼠96只,随机分为正常组、缺血再灌注组、缺血再灌注+Ad-CMV组,缺血再灌注+Ad-PEDF组,以前房加压的方法制备大鼠视网膜缺血再灌注模型,缺血再灌注+Ad-CMV组,缺血再灌注+Ad-PEDF组分别玻璃体腔注射Ad-CMV或Ad-PEDF1μL(滴度3.8×109/PFU),每组按照时间点12,24,72,168h,为4亚组,光学显微镜观察视网膜组织切片情况,并测量视网膜内层厚度及神经节细胞层神经节细胞数量。以TUNEL方法观察大鼠视网膜神经节细胞凋亡情况。结果:Ad-PEDF组视网膜内层厚度均超过缺血组及缺血+Ad-CMV组,Ad-PEDF组神经节细胞数目多于缺血组及Ad-CMV组,Ad-PEDF组视网膜神经节细胞凋亡细胞少于缺血组及Ad-CMV组,凋亡程度减轻,上述差异均具有显著性(P<0.05)。结论:腺病毒介导的色素上皮衍生因子玻璃体腔注射能够恢复大鼠视网膜缺血再灌注损伤所致的视网膜内层厚度降低,神经节细胞密度减少,具有保护作用。     

Apoptosis and caspases after ischemia-reperfusion injury in rat retina

PURPOSE: Extensive cell loss in the retinal ganglion cell layer (RGCL) and the inner nuclear layer (INL) was noted in a rat model of retinal ischemia-reperfusion injury by transient elevated intraocular pressure (IOP). The possible involvement of apoptosis and caspases was examined in this model of neuronal loss. METHODS: Transient elevated IOP was induced in albino Lewis rats through the insertion of a needle into the anterior chamber connected to a saline column. Elevated IOP at 110 mm Hg was maintained for 60 minutes. Groups of animals were euthanatized at various times after reperfusion, and their retinas were evaluated by morphology, agarose gel electrophoresis of DNA, in situ terminal deoxynucleotidyl transferase-mediated biotin-deoxyuridine triphosphate nick-end labeling (TUNEL), immunohistochemistry of caspases II (ICH1) and III (CPP32), and morphometry. YVAD.CMK, a tetrapeptide inhibitor of caspases, was used to examine the involvement of caspases. RESULTS: A marked ladder pattern in retinal DNA gel analysis, typical of internucleosomal DNA fragmentation and characteristic of apoptosis, was present 12 and 18 hours after reperfusion. Labeling of nuclei in the RGCL and the inner nuclear layer (INL) by TUNEL was noted between 8 and 18 hours after reperfusion. Histologic and ultrastructural features typical of apoptosis were also observed in the inner retina after ischemia. YVAD.CMK administered during the ischemic period inhibited apoptotic fragmentation of retinal DNA and ameliorated the tissue damage. When administered intravitreally 0, 2, or 4 hours after reperfusion, YVAD.CMK was also effective in preserving the inner retina but had no significant effect when administered 6 or 8 hours after reperfusion. The inner retina showed transient elevated immunoreactivity of caspases II and III 4 and 8 hours after reperfusion. CONCLUSIONS: Retinal ischemia-reperfusion after transient elevated IOP induced apoptosis of cells in the retinal ganglion cell layer and the INL. Caspases may have a pivotal role in the early events of the apoptotic pathway(s). Rescue by using anti-apoptotic agents after ischemia-reperfusion is feasible.     

Inhibition of retinal angiogenesis by PEDF

AIM: To investigate the effect of PEDF on retinal neovascularization in mice. METHODS: 40 7-day-old C57BL/6J mice was exposed to 750mL/L oxygen for 5 days and then to normal situation to produce the murine model of oxygen-induced retinopathy(OIR). One eye of the mouse was regarded as experimental one and the other served as control. Eyes in experimental group received intravitreal injection of PEDF and eyes in control group received intravitreal injection of PBS at postnatal day 12. All mice were executed at postnatal day 17. The changes of retinal vessels of mice were observed by ADPase histochemical technique. The inhibitory effect of PEDF on retinal neovascularization was evaluated by counting the endotheliocyte nuclei of new vessels which extended from retina to vitreous in the tissue slice of HE staining. RESULTS: Neovascularization was reduced, retinal blood vessels distributed regularly and non-perfusion areas were not found in eyes of experimental group compared with control group. The number of endotheliocyte nuclei of new vessels extending from retina to vitreous was significantly less in the eyes of experimental group (10.18±1.74) than that in control group (38.89±2.98) (P <0.01). Retinal toxicity and inflammatory reactions were not found in tissue slice. CONCLUSION: PEDF inhibits retinal angiogenesis in OIR and the feasibility should be determined for use of PEDF in ocular angiogenesis treatment.     

A new method and device to induce transient retinal ischemia in the rat

PURPOSE: To describe and characterize a novel, non-invasive method to induce retinal ischemia in the rat. METHODS: Retinal ischemia was produced by applying a compression force with a suture thread anchored about 2mm behind the limbus of the eye in order to increase intraocular pressure and reduce ocular blood flow. Both ends of the suture thread were connected to weights via a pulley system. The effect of various weights (20, 25, or 35 g) on retinal damage was tested in animals of different body weights (350 and 574 g). Scotopic ERG measurements and histological sections of retinal tissue were used to evaluate possible functional and morphological changes to the retina as a consequence of ischemia followed by reperfusion. RESULTS: Retinal ischemia was achieved by this method with good reproducibility. The degree of retinal damage, as quantified by ERG measurement, correlated with the amount of weight connected to the suture thread. Seven days after 60 minutes of retinal ischemia, the amplitudes of A- and B-waves of eyes compressed with a 20 g weight were 84 +/- 11% and 86 +/- 16% (mean +/- SEM, n = 4, amplitudes before ischemia defined 100%), respectively. The A- and B-wave amplitudes of eyes compressed with 25 g were 69 +/- 7% and 76 +/- 6% (n = 11), respectively. A 35 g treatment produced A- and B-wave amplitudes of 55 +/- 3.9% and 52 +/- 4% (n = 35), respectively. There was no significant difference in the level of ischemic damage in the two groups of animals with different body weights evaluated in this study. At two weeks after 60-minute retinal ischemia, there was a significant decrease (by 31.1%) in the cell density in the retinal ganglion cell layer. CONCLUSIONS: The pulley method is an easy, non-invasive, and highly reproducible technique for inducing retinal ischemia in rats. This procedure may provide a useful animal model of ischemic retinopathy.     

Lipid peroxidation and peroxynitrite in retinal ischemia-reperfusion injury

PURPOSE: To investigate whether lipid peroxides play a role in retinal cell death due to ischemia-reperfusion injury, whether recombinant human thioredoxin (rhTRX) treatment reduces production of lipid peroxides of the retina, and whether such treatment reduces the number of cells expressing c-Jun and cyclin D1. METHODS: Retinal ischemia was induced in rats by increasing the intraocular pressure to 110 mm Hg for 60 minutes. After reperfusion, immunohistochemical staining for lipid peroxide, peroxynitrite, c-Jun, and cyclin D1 and propidium iodide (PI) staining were performed on retinal sections from animals treated intravenously with and without rhTRX, a free radical scavenger. Quantitative analyses of PI-, c-Jun-, and cyclin D1-positive cells were performed after the ischemic insult. Concentration of lipid peroxides in the retina was determined by the thiobarbituric acid assay. RESULTS: Specific immunostaining for lipid peroxides was seen in the ganglion cell layer at 6 hours after reperfusion, in the inner nuclear layer at 12 hours, and in the outer nuclear layer at 48 hours. Time course studies for PI-positive cells in the three nuclear layers coincided with those of specific immunostaining for lipid peroxides. The specific immunostaining was weakened by pre- and posttreatment with 0.5 mg of rhTRX. The number of PI-, c-Jun-, and cyclin D1-positive cells and the concentration of lipid peroxides were significantly decreased by treatment with rhTRX compared with those of vehicle-treated control rats (P: < 0. 01). CONCLUSIONS: Lipid peroxides formed by free radicals may play a role in neuronal cell death in retinal ischemia-reperfusion injury.     

色素上皮衍生因子对大鼠视网膜缺血 再灌注损伤的保护作用

目的已证实色素上皮衍生因子(pigment epithelium-deriv ed factor,PEDF)对中枢神经细胞有抗凋亡作用。本实验评价其对压力诱发的视网膜缺血再灌注的影响。方法经前房灌注维持眼压110 mm Hg (1 mm Hg = 0.133kpa),45 min,建立大鼠视网膜缺血再灌注模型。 随后立即向玻璃体注射10　μ1(0.1μg/μl)PEDF,分别于2d和7d摘除眼球,测量塑 料包埋切片的平均视网膜内层厚度(mean thickness of inner retinal layer,MTIRL) 和视网膜节细胞(retinal ganglion cells,RGC)计数。结果PEDF玻璃体注射7d后治疗组的MTIRL和RGC明显高于对照组[(118.1±5．0) μm对(949±3.0)μm, P<0.05;(6.0±1．0)个/100 μm对(4.5±0.5)个/100 μm, P<0.05]。结论玻璃体内注射PEDF有助于防止视网膜缺血再灌注后神经变性和细胞死亡。(中华眼底病杂志,2001,17:138-140)     

重组人促红细胞生成素对大鼠视网膜缺血-再灌注损伤的保护作用

目的 制作Sprague-Dawley (SD)大鼠视网膜缺血-再灌注(RIR)损伤模型,探讨腹腔内注射重组人促红细胞生成素(rHuEPO)对急性RIR损伤所致的大鼠视网膜神经元损伤的保护作用及其对热休克蛋白72(HSP72)表达的影响.方法 采用前房灌注的方式建立RIR损伤模型,灌注压110 mm Hg(1 mm Hg=0.133kPa),缺血时间1h;腹腔注射rHuEPO.78只SD大鼠随机分组:正常组6只,EPO组、EPO+槲皮黄酮组、RIR组各24只,均以右眼为实验眼.采用免疫组织化学法和末端脱氧核苷酸转移酶介导的dUTP缺口末端标记法(TUNEL)分别测定正常对照组和各实验组大鼠再灌注24h、48 h、72 h和1周视网膜中HSP72及凋亡细胞的表达,观察各组大鼠视网膜病理学改变.结果 ①正常组大鼠视网膜中HSP72表达微弱,各实验组大鼠视网膜中HSP72表达自再灌注12 h开始增强,24h达到高峰,随后逐渐减弱,72 h时表达稍高于正常.再灌注后各时间段,EPO组大鼠视网膜中HSP72表达均高于RIR组、EPO+槲皮黄酮组(P＜0.05).②正常大鼠视网膜中几乎没有凋亡细胞.再灌注后12 h,各实验组大鼠视网膜中可见凋亡细胞,24 h达高峰,48 h后凋亡细胞数逐渐减少;再灌注后各组大鼠视网膜中凋亡细胞数比正常组多(P＜0.05).③再灌注后,RIR组,EPO+槲皮黄酮组大鼠内层视网膜明显水肿,炎性细胞侵入,膜结构逐渐破坏;EPO组大鼠视网膜结构保持相对完整,炎性细胞相对较少.结论 ①HSP72在正常大鼠视网膜中表达微弱,RIR损伤后表达增多.腹腔注射EPO可以明显诱导大鼠视网膜中HSP72的表达增多.②EPO可以减少大鼠RIR损伤后视网膜细胞凋亡,减少视网膜内炎性细胞的浸润,保护视网膜结构,对视网膜具有明显的保护作用.其机制可能与使HSP72表达上调有关.(中国眼耳鼻喉科杂志,2012,12:30-35)     

Pigment epithelium-derived factor suppresses ischemia-induced retinal neovascularization and VEGF-induced migration and growth

PURPOSE: To determine the effect of pigment epithelium-derived factor (PEDF) in a mouse model of ischemia-induced retinal neovascularization and on vascular endothelial growth factor (VEGF)--induced migration and growth of cultured microvascular endothelial cells. METHODS: Human recombinant PEDF was expressed in the human embryonic kidney 293 cell line and purified by ammonium sulfate precipitation and cation exchange chromatography. C57BL/6 mice were exposed to 75% oxygen from postnatal day (P)7 to P12 and then returned to room air. Mice received intravitreal injections of 2 microg PEDF in one eye and vehicle in the contralateral eye on P12 and P14. At P17, mice were killed and eyes enucleated for quantitation of retinal neovascularization. The mitogenic and motogeneic effects of VEGF on cultured bovine retinal and adrenal capillary endothelial cells were examined in the presence or absence of PEDF, using cell counts and migration assays. RESULTS: Two species of human recombinant PEDF, denoted A and B, were purified to apparent homogeneity. PEDF B appeared to comigrate on SDS-PAGE with PEDF from human vitreous samples. Changes in electrophoretic mobility after peptide-N-glycosidase F (PNGase F) digestion suggest that both PEDF forms contain N-linked carbohydrate. Analyses of the intact proteins by liquid chromatography-electrospray mass spectrometry (LC-ESMS) revealed the major molecular weight species for PEDF A (47,705 +/- 4) and B (46,757 +/- 5). LC-ESMS analysis of tryptic peptides indicated that PEDF A and B exhibit differences in glycopeptides containing N-acetylneuraminic acid (NeuAc) and N-acetylhexosamine (HexNAc). Intravitreal administration of either species of PEDF significantly inhibited retinal neovascularization (83% for PEDF A and 55% for PEDF B; P = 0.024 and 0.0026, respectively). PEDF A and B (20 nM) suppressed VEGF-induced retinal microvascular endothelial cell proliferation by 48.8% and 41.4%, respectively, after 5 days (P < 0.001) and VEGF-induced migration by 86.5% +/- 16.7% and 78.1% +/- 22.3%, respectively, after 4 hours (P = 0.004 and P = 0.008, respectively). CONCLUSIONS: These data indicate that elevated concentrations of PEDF inhibit VEGF-induced retinal endothelial cell growth and migration and retinal neovascularization. These findings suggest that localized administration of PEDF may be an effective approach for the treatment of ischemia-induced retinal neovascular disorders.     

PEDF-deficient mice exhibit an enhanced rate of retinal vascular expansion and are more sensitive to hyperoxia-mediated vessel obliteration

Pigment epithelium derived factor (PEDF) is an endogenous inhibitor of angiogenesis. However, its physiological role during vascular development and neovascularization remains elusive. Here we investigated the role of PEDF in normal postnatal vascularization of retina and retinal neovascularization during oxygen-induced ischemic retinopathy (OIR) using PEDF-deficient (PEDF-/-) mice. The beta-galactosidase staining of eye sections from PEDF-/- mice confirmed the expression pattern of endogenous PEDF previously reported in mouse retina. However, strongest staining was observed in the retinal outer plexiform layer. Retinal trypsin digests indicated that the ratio of endothelial cells (EC) to pericytes (PC) was significantly higher in PEDF-/- mice compared to wild type (PEDF+/+) mice at postnatal day 21 (P21). This was mainly attributed to increased numbers of EC in the absence of PEDF. There was no significant difference in the number of PC. We observed an increased rate of proliferation in retinal vasculature of PEDF-/- mice, which was somewhat compensated for by an increase in the rate of apoptosis. Staining of the retinal wholemounts and eye frozen sections indicated postnatal retinal vascularization expansion occurred at a faster rate in the absence of PEDF, and was more prominent at early time points (prior to P21). The retinal vascularization in PEDF+/+ mice reaches that of PEDF-/- mice such that no significant difference in vascular densities was observed by P42. Lack of PEDF had a minimal effect on the regression of hyaloid vasculature and VEGF levels. PEDF-/- mice also exhibited enhanced sensitivity to hyperoxia-mediated vessel obliteration during OIR compared to PEDF+/+ mice despite higher levels of VEGF. However, there was no significant difference in the degree of retinal neovascularization. Our studies indicate that PEDF is an important modulator of early postnatal retinal vascularization and in its absence retinal vascularization proceeds at a faster rate and is more susceptible to hyperoxia-mediated vessel obliteration.     

PEDF抑制鼠视网膜新生血管的实验研究(英文)

目的:观察PEDF对氧诱导的血管增生性视网膜病变动物模型视网膜新生血管的抑制作用。方法:40只7日龄的C57BL/6J新生鼠暴露在750mL/L高氧环境中,然后回到正常空气中建立氧诱导的血管增生性视网膜病变的动物模型。随机取1眼为实验眼,在出氧箱时(12d)玻璃体腔注射PEDF,另1眼为对照组,玻璃体腔注射PBS。所有小鼠均于17d处死,视网膜铺片,ADP酶染色观察视网膜血管情况。HE染色,在光学显微镜下观察并计数突破视网膜内界膜的血管内皮细胞细胞核数目。结果:与对照组相比,实验组视网膜血管分布规则,未见明显的无灌注区形成;实验组突破内界膜的内皮细胞细胞核数目(10.18±1.74)比对照组(38.89±2.98)明显减少(P<0.01) ;组织切片未见视网膜毒性及炎症反应。结论:PEDF能够有效抑制视网膜新生血管的生成。     

电刺激大鼠小脑顶核对视网膜缺血再灌注损伤的保护作用

目的　探讨电刺激大鼠小脑顶核对视网膜缺血再灌注损伤的保护作用。方法　大鼠随机分为缺血再灌注组、电刺激组和假手术组。观察视网膜形态学改变 ;用NADPH黄递酶组织化学染色法 (NADPH NDP)观察视网膜内诱导型一氧化氮合酶 (iNOS)的表达 ;采用TUNEL法检测视网膜细胞凋亡情况。结果　 (1)缺血再灌注组的内视网膜层 (包括内核层、内丛状层、节细胞 )、神经纤维层和内界膜厚度增加 ,尤其是内丛状层厚度明显高于假手术组 (t=3 6 80 ,P <0 0 1) ;电刺激组的内视网膜厚度与假手术组相比 ,差异无显著意义 (t=1 0 6 4 ,P >0 0 5 ) ;(2 )光镜观察可见缺血再灌注组有明显的细胞核染色质致密浓缩、核碎裂等改变 ,电刺激组仅见少量核浓缩及碎裂 ;(3)电刺激组的iNOS阳性的神经节细胞数明显低于缺血再灌注组 ,其差异有显著意义 (t=3 32 6 ,P <0 0 1) ;(4)电刺激组大鼠发生凋亡的视网膜细胞数明显低于缺血再灌注组 ,其差异有显著意义 (t=4 0 38,P <0 0 1)。结论　电刺激大鼠小脑顶核对缺血再灌注所导致的视网膜组织损伤具有保护作用。     

猪源纤维蛋白黏合剂封闭视网膜裂孔的实验研究

目的 研究猪源纤维蛋白黏合剂封闭视网膜裂孔替代眼内充填物的可行性。设计 实验研究。研究对象 45只青紫兰兔（45眼）。方法 将45只青紫兰兔的右眼行玻璃体切割、视网膜造孔和视网膜下注液,建立孔源性视网膜脱离模型。模型建立1周后,将符合条件的42只兔眼随机分为3组,每组14眼,经玻璃体切割视网膜复位,裂孔周围行激光光凝裂孔。蛋白胶组用纤维蛋白胶（广州倍绣生物技术有限公司）封闭视网膜裂孔后,玻璃体腔应用灌注液充填;气体组玻璃体腔应用30%SF6气体充填;单纯激光光凝裂孔组玻璃体腔单纯灌注液充填。术后1、4周分别行间接检眼镜、眼底照相、眼B超、OCT及组织病理学检查。主要指标 视网膜裂孔封闭及视网膜复位情况。结果 术后4周,蛋白胶组、气体组、单纯激光光凝裂孔组视网膜复位分别为12/14眼（85.7%）、13/14眼（92.9%）、5/14眼（35.7%）,前两者复位率均显著高于单纯激光光凝裂孔组（P=0.009,0.036）。结论 在兔眼的实验研究结果显示,国产猪源纤维蛋白黏合剂能够替代眼内充填物起到临时封闭视网膜裂孔的作用。（眼科,2015,24：254-257）     

Heme oxygenase-1 induced in muller cells plays a protective role in retinal ischemia-reperfusion injury in rats

PURPOSE: To investigate the protective roles played by heme oxygenase (HO)-1 and -2 in the rat retina after ischemia-reperfusion injury. METHODS: Retinal ischemia was induced in rats by increasing the intraocular pressure to 110 mmHg for 60 minutes. The expression of HO-1 and -2 in the retina was determined by Western blot, real-time polymerase chain reaction (PCR), and immunohistochemistry. To inhibit the upregulation of HO-1, short interfering (si)RNA of HO-1 was injected intravitreally before ischemia and that of green fluorescent protein (GFP) was used as the control. Muller cell damage was assessed by counting the number of S-100-positive cells. The number of macrophages invading the retina was determined by counting the number of ED-1-positive cells. RESULTS: The expression of HO-1 mRNA and protein was upregulated at 6 hours after reperfusion and peaked at 12 to 24 hours, whereas that of HO-2 was not altered. HO-1 immunoreactivities were detected in Muller cells at 24 hours after reperfusion, and HO-2 immunoreactivities were detected in retinal cells. The HO-1 expression in the retina treated with siRNA of HO-1 was reduced at 12 and 24 hours after reperfusion compared with that injected with siRNA of GFP. The number of S-100-positive cells at 24 hours after reperfusion decreased significantly in retinas treated with HO-1 siRNA (P <0.01). The number of macrophages that had infiltrated the retina was increased in retinas pretreated with the siRNA of HO-1 compared with those treated with siRNA of GFP. On day 14 after reperfusion, HO-1 siRNA-treated retinas showed severe retinal injury and destruction of the retinal architecture. CONCLUSIONS: HO-1 promotes the survival of Muller cells after ischemia-reperfusion injury. Because inhibition of the upregulation of HO-1 resulted in an infiltration of inflammatory cells and destruction of the retina, the authors conclude that HO-1 induced in Muller cells plays a protective role in retinal ischemia-reperfusion.     

豚鼠形觉剥夺性近视眼视网膜蛋白激酶C的变化

摘 要：目的 研究豚鼠形觉剥夺性近视眼形成过程中视网膜蛋白激酶C（PKC）的变化.方法 选取3周龄雄性三色豚鼠48只,用半透明眼罩遮盖豚鼠右眼建立近视眼模型,分为遮盖3、7、14 d组,每组16只豚鼠.每组中8只豚鼠遮盖右眼作为实验眼.以对侧未遮盖眼作为自身对照,剩余8只豚鼠作为正常对照.按预定时间观察豚鼠角膜曲率半径、屈光度和眼轴长度的变化,然后处死豚鼠,取视网膜,采用非放射性方法测定视网膜PKC活性,免疫组化染色定位分析PKC蛋白在视网膜的表达情况.各组间角膜曲率半径、屈光度、眼轴长度、视网膜PKC活性及免疫反应强度比较,采用单因素方差分析;组内左、右眼之间的比较采用t检验：视网膜PKC活性与PKC蛋白免疫反应强度的关系采用相关分析.结果 眼罩遮盖能诱导豚鼠遮盖跟眼轴延长、近视形成,且近视程度随遮盖时间的延长而加深.与对照组比较,各组遮盖眼视网膜PKC活性均升高（P〈0.05）.眼罩遮盖3 d后,遮盖眼视网膜PKC活性升高至（0.28±0.08）units/ml,遮盖7 d时PKC活性进一步升高至（0.43±0.10）units/ml,遮盖14 d时降低为（0.32±0.07）units/ml.免疫组化染色结果显示,PKC蛋白表达于视网膜神经节细胞和内核层细胞,呈棕黄色或棕褐色颗粒.与正常对照眼及自身对照眼比较,遮盖眼视网膜内核层细胞PKC蛋白表达上调（P〈0.05）,神经节细胞PKC蛋白表达无变化.遮盖眼视网膜内核层细胞PKC蛋白表达的变化趋势与PKC活性一致,两者之间有明显的相关关系（r=0.994,P=0.000）.结论 视网膜PKC活性升高参与了豚鼠形觉剥夺性近视的形成.     

葛根素对大鼠视网膜缺血再灌注损伤的保护作用

目的观察葛根素(puerarin)对大鼠视网膜缺血再灌注的保护作用及机制。方法成年Wistar大鼠随机分成对照组、缺血再灌注未治疗组、缺血再灌注葛根素治疗组。采用前房灌注液体形成高眼压而建立RIR模型。治疗组在缺血前30min给予大鼠腹腔内注射葛根素。缺血60min后恢复血流。光镜观察各组视网膜内层厚度以及浸润入视网膜的中性粒细胞数目、神经节细胞数变化;免疫组化法检测Caspase-3蛋白在各组视网膜中的表达。结果葛根素治疗组再灌注6h以后各时间段视网膜内层厚度均较未治疗组视网膜缺血再灌注厚,早期视网膜内层水肿增厚,晚期视网膜神经节细胞数目减少及视神经纤维层萎缩变薄,神经节细胞数目多于未治疗组,而视网膜中的中性粒细胞数目少于未治疗组;Caspase-3蛋白于再灌注后24h达到高峰,但各时间段治疗组表达强度均较未治疗组明显减弱。结论葛根素对视网膜缺血再灌注损伤有治疗作用,抑制缺血再灌注损伤后的炎症反应和Caspase-3蛋白的表达是其可能的保护机制。