Exenatide对高糖培养的视网膜神经节细胞的保护作用

目的:评价Exenatide对视神经保护作用,并初步探索其对视网膜神经节细胞(retinal ganglion cells,RGCs)的保护机制。方法:体外培养RGCs,免疫荧光检测RGCs是否表达胰升糖素样肽-1受体(glucagon-like peptide-1receptor,GLP-1R),采用高糖作为细胞凋亡诱导剂建立RGCs凋亡模型,并用不同浓度Exenatide加入细胞培养基中进行干预,用CCK-8检测细胞存活率。结果:免疫荧光结果显示,RGCs存在GLP-1R的表达。CCK-8检测结果显示终浓度为0.5～1μg/L时Exenatide均可促进RGCs存活,其中0.5μg/L浓度较低且已有明显保护作用(P<0.05)。结论:Exenatide注射液对高糖引起的RGCs生存抑制有一定保护作用,这种保护作用很可能通过GLP-1R介导的细胞内保护机制产生。     

补肾活血中药血清对加压纯化培养视网膜神经节细胞PI3K/AKT信号转导通路的影响

目的 研究补肾活血中药血清对加压纯化培养视网膜神经节细胞(retinal ganglion cells,RGCs)凋亡模型PI3K/Akt信号转导通路主要成员PDK及Akt表达的影响,探索补肾活血法保护RGCs的机制.方法 制备补肾活血中药含药血清,体外纯化SD大鼠RGCs,采取开放式压力控制培养系统建立体外加压培养RGCs凋亡模型,以50g·L-1、100g· L-1、200g· L-1血清浓度梯度补肾活血中药血清分别处理.将RGCs分为5组,分别为正常培养组(N组)、对照组(C组)、50 g· L-1补肾活血中药血清组(50 g·L-1BSHX组)、100g· L-1补肾活血中药血清组(100 g·L-1BSHX组)、200g·L-1补肾活血中药血清组(200 g·L-1BSHX组),Annexin V-FITC/PI双染检测细胞凋亡率,实时荧光定量PCR(qRT-PCR)检测补肾活血中药血清对RGCs PDK及AktmRNA表达水平的影响,Western blot检测各组PDK、Akt蛋白表达量.结果 Q-PCR检测各组mRNA结果:C组(0.04±0.01)与N组(1.00±0.04)相比,RGCs中PI3K、Akt的mRNA表达水平下降,差异均有统计学意义(均为P ＜0.05),而50 g·L-1、100 g·L-1、200 g·L-BSHX组(0.18±0.01、0.21±0.02,0.22±0.01、0.36±0.01,0.84±0.10、1.07±0.17)与C组相比,PI3K、Akt mRNA含量逐渐升高,差异均有统计学意义(均为P＜0.05).Western blot检测各组蛋白表达,C组与N组相比,细胞PI3K、Akt的蛋白表达水平下降,差异有统计学意义(P＜0.05),而50 g·L-1、100 g·L-1、200 g·L-BSHX组与C组相比,PI3K、Akt蛋白表达量逐渐升高,差异均有统计学意义(均为P＜0.05).结论 补肾活血中药血清抑制加压诱导的RGCs凋亡,其机制可能与激活PI3K/Akt信号转导通路有关.     

睫状神经营养因子对大鼠急性高眼压眼视网膜神经节细胞的保护作用

背景 青光眼可以引起视网膜神经节细胞(RGCs)凋亡,据报道睫状神经营养因子(CNTF)对外伤性视神经损伤有修复作用,其是否对青光眼视神经病变有保护作用尚少见报道. 目的 观察CNTF对大鼠急性高眼压眼RGCs的保护作用.方法 24只Wistar大鼠双眼采用眼前房平衡盐液加压灌注法建立大鼠急性高眼压模型,造模前2d左眼玻璃体内注入0.5μg CNTF 5μl,右眼以同样的方法注射磷酸钠溶液5μl,另取3只正常大鼠作为正常对照.造模后1、3、7、14 d过量麻醉法处死动物并摘除眼球,制备视网膜组织学切片后采用苏木精-伊红染色法进行形态学观察,光学显微镜下计数RGCs数目;采用免疫组织化学染色法观察RGCs层谷氨酸的表达情况.结果 正常对照组大鼠视网膜各层排列整齐,细胞边界清晰;模型对照组大鼠RGCs细胞膜、细胞核均发现异常改变,有细胞空泡样变;CNTF治疗组大鼠造模后变性的RGCs数量少.与模型对照组比较,造模后3、7、14 d CNTF治疗组RGCs数目明显增加,差异均有统计学意义(均P=0.000).免疫组织化学染色表明,造模后3～7d,CNTF治疗组RGCs层谷氨酸阳性细胞数分别为(5.50±1.04)个／3个高倍视野和(6.00±1.41) 个/3个高倍视野,明显低于模型对照组的(9.00±2.91)个／3个高倍视野和(10.83±1.94)个／3个高倍视野,差异均有统计学意义(均P=0.000),而造模后1d和14 d两组间谷氨酸阳性细胞数的差异均无统计学意义(P=0.578、0.180).结论 CNTF能够下调急性高眼压眼谷氨酸在RGCs中的表达,从而对RGCs提供保护作用.     

脑苷肌肽对rd小鼠视网膜细胞保护作用的研究

目的观察脑苷肌肽(cattle encephalon glycoside and ignotin,CEGI)对体外分离的rd小鼠视网膜细胞的保护作用,旨在探求CEGI对视网膜以及视神经损伤相关疾病的治疗效果,为临床用药提供客观依据和指导。方法采用体外分离rd小鼠视网膜细胞悬液,用MTT和台盼蓝染料排斥法观察CEGI和神经生长因子(NGF)对细胞活性的影响。结果 MTT实验示CEGI8.00mg·L-1、0.04g·L-1、0.20g·L-1和NGF0.04mg·L-1、0.20mg·L-1对视网膜细胞活性有促进作用,尤其以CEGI0.04g·L-1和NGF0.20mg·L-1最为明显,细胞增殖率分别为130.0%±21.8%和124.0%±13.3%。若将二者组合使用,可使细胞增长率达到132.0%±9.1%,与药物空白对照组100.0%±10.1%相比差异均有统计学意义。台盼蓝染料排斥试验结果显示0.04g·L-1CEGI组活细胞比例在除5min外的各时间点,都明显高于对照组,而0.20mg·L-1NGF组也可保护视网膜细胞,主要以短时间点为主(5min～1d),与对照组相比差异均有统计学意义(均为P<0.05)。结论 CEGI有促进视网膜细胞生长发育作用,对rd小鼠视网膜细胞有一定的保护作用。     

玻璃体内注射酵母多糖对视神经夹伤大鼠RGCs的保护作用

目的 观察眼内注射酵母多糖对视神经夹伤大鼠视网膜神经节细胞(RGCs)的保护作用.方法 SD大鼠45只,随机分为3组,以荧光金逆行标记RGCs 1周后,行左眼视神经钳夹伤,右眼为假手术对照.3 d后分别于各对应组大鼠左眼玻璃体腔注射PBS或酵母多糖5μL,所有大鼠存活21 d处死,每组各取5只大鼠行视网膜冰冻切片,余做视网膜铺片,计算RGCs标识率(左眼RGCs数μ右眼RGCs数)× 100%,并行统计学分析.结果 酵母多糖组、PBS组和单纯钳夹组中RGCs标识率分别为:(19.22±2.51)%、(1.86 ±3.09)%和(1.78±2.61)%,酵母多糖组中存活RGCs较单纯钳夹组和PBS组差异有统计学意义(P=0.000),单纯钳夹组和PBS组差异无统计学意义(P=0.925).结论 酵母多糖玻璃体腔注射后,能诱导眼内炎症反应,从而对视神经夹伤大鼠RGCs的存活具有一定的保护作用.     

罗格列酮对高糖诱导视网膜神经节细胞损伤的保护作用

目的 探讨罗格列酮(rosiglitazone,RSG)对高糖诱导视网膜神经节细胞(retinal ganglion cell,RGC)损伤的保护作用.方法 体外培养大鼠RGC细胞株RGC-5细胞,50 mol·L-1葡萄糖孵育细胞诱导损伤.应用CCK-8法测定并计算细胞的生长抑制率,流式细胞仪检测细胞凋亡,全自动氨基酸分析仪测定细胞谷氨酸(glutamic acid,Glu)释放量,测定细胞中丙二醛(malondialdehyde,MDA)含量以及超氧化物歧化酶(superoxide dismutase,SOD)活性.结果 高糖(50 mol·L-1)以时间依赖的方式抑制了RGC-5细胞的生长,高糖处理24 h、48 h和72 h生长抑制率分别为(22.37±3.49)％、(42.18±6.34)％和(57.33±5.39)％(均为P ＜0.05);与高糖组比较,高糖+不同浓度RSG组(0.1×10-6 mol·L-1、10-6 mol·L-1、10×10-6 mol·L-1)处理48 h以剂量依赖的方式降低了高糖诱导的RGC-5细胞生长:生长抑制率高糖组(42.18±6.34)％,高糖+不同浓度RSG组分别为(35.66±4.73)％、(27.35±4.15)％和(25.17±3.42)％(均为P＜0.05).与对照组比较,高糖处理24 h、48 h和72 h以时间依赖的方式促进了细胞凋亡(均为P＜0.05);与高糖组比较,高糖+不同浓度RSG组(0.1×10-6 mol·L-1、10-6 mol·L-1和10×10-6mol·L-1)处理48 h以剂量依赖的方式降低了高糖诱导的RGC-5细胞凋亡:高糖组凋亡率为(31.55±5.34)％,高糖+不同浓度RSG组分别为(23.75±3.72)％、(18.75±2.17)％和(17.53±3.05)％(均为P＜0.05).与对照组比较,高糖组Glu释放量显著增加:2组分别为(85.64±12.75)μg·L-1和(246.84±33.48)μg·L-1(P＜0.05).与高糖组比较,高糖+不同浓度RSG(0.1×10-6mol·L-1、10-6 mol·L-1和10×10-6mol·L-1)处理48 h组Glu的释放以剂量依赖的方式减少:高糖组Glu释放量为(246.84 ±33.48)μg·L-1,高糖+不同浓度RSG组分别为(175.34±23.69)μg·L-1、(117.25±18.76) μg·L-1和(109.34±15.54) μg·L-1(均为P＜0.05).与对照组比较,高糖组SOD活性显著降低:分别为(3.06±0.38) kU·g-1和(0.56±0.07)kU·g-1(均为P＜0.05),而MAD水平显著增加:分别为(5.67±0.76) μmol·g-1和(37.64±4.37) μmol·g-1(均为P＜0.05).与高糖组相比较,高糖+不同浓度RSG处理48 h组细胞中SOD活性以剂量依赖的方式增加(均为P＜0.05),而MAD水平显著降低(均为P＜0.05).结论 RSG抑制了高糖诱导的RGC损伤,其机制与RSG减少了RGC中Glu的释放和抑制氧化应激有关.     

血府逐瘀汤对外伤性视神经损伤大鼠轴浆流动的影响

目的 观察血府逐瘀汤对大鼠外伤性视神经损伤的保护作用.方法 27只健康SD大鼠随机分为A、B、C 3组,均用微型视神经夹制作成单眼视神经夹伤模型,A、B、C 3组分别以蒸馏水、灯盏细辛、血府逐瘀汤灌胃.用30g·L-1荧光金(fluro gold,rG)双上丘注射逆行标记双眼视网膜神经节细胞(retinal ganglion cells,RGCs),计算RGCs存活率.结果 给药4周后进行RGCs计数,A、B、C 3组RGCs的存活率分别为(63.72±12.97)%、(75.75±8.86)%、(88.34±10.34)%,两两比较差异均有统计学意义(PaB=0.048<0.05,PAC=0.0010.01,尸BC=0.026<0.05);对各组左、右眼RGCs数量进行配对比较,差异均具有显著统计学意义(PA=0.000,PR=0.000,Pc=0.005).结论 该造模方法可造成钳夹伤大鼠RGCs一定量的丢失;灯盏细辛和血府逐瘀汤均可提高钳夹伤大鼠视神经RGCs的存活毕,促进轴浆运输,且血府逐瘀汤的作用优于灯盏细辛,这可能是其促进视神经修复、再生的机理之一.     

原花青素对氧化应激人眼小梁网细胞凋亡及细胞色素C释放的影响

目的 研究原花青素对H2O2诱导人眼小梁网细胞(human trabecular meshwork cells,HTMC)凋亡及细胞色素C释放的影响.方法 将HTMC进行传代后随机分为5组.未处理组:正常培养的HTMC;对照组:正常培养的HTMC+H2O2(500μmol·L-1处理lh);3个浓度的原花青素组:正常培养的HTMC+H2O2(500 μmol· L-1处理lh)+原花青素(浓度分别为0.02 g·L-1、0.05 g·L-1、0.10g·L-1).使用Annexin V-FITC检测细胞凋亡情况,Western blot检测HTMC胞浆细胞色素C含量.结果 与未处理组相比,对照组及各原花青素组细胞凋亡率均升高,差异均有统计学意义(均为P＜0.01).与对照组相比,各原花青素组细胞凋亡率均降低,差异均有统计学意义(均为P＜0.01);不同浓度原花青素组间随原花青素浓度增加细胞凋亡率下降,各原花青素组间差异均有统计学意义(均为P＜0.01).与未处理组相比,对照组以及0.02 g·L-1和0.05 g· L-1原花青素组细胞色素C的释放均增加,差异均有统计学意义(均为P＜0.01);而0.10 g·L-1原花青素组与未处理组相比,差异无统计学意义(P＞0.05).与对照组相比,各原花青素组细胞色素C释放均减少,差异均有统计学意义(均为P＜0.01).不同浓度原花青素组间随原花青素浓度增加细胞色素C的释放减少,各组间差异均有统计学意义(均为P＜0.01).结论 外源性原花青素可以降低氧化应激HTMC的凋亡率,减少细胞色素C的释放,有较强的抗氧化作用,并在一定浓度范围内随着浓度的增加其抗氧化作用逐渐增强.     

硫辛酸烟酸二联体对丙烯醛损伤ARPE-19细胞的保护作用

目的 利用丙烯醛建立年龄相关性黄斑变性(age-related macular degeneration,AMD)的细胞损伤模型,并观察硫辛酸烟酸二联体对丙烯醛诱导ARPE-19细胞损伤的保护作用.方法 MTT比色法检测ARPE-19细胞的生长周期.将25μmol·L-1、50 μmol·L-1、100 μmol·L-1丙烯醛加入对数生长期的ARPE-19细胞中处理24 h,复制AMD细胞水平的损伤模型并确定其损伤浓度;预先加入50 μmol·L-1、100 μmol·L-1、150 μmol·L-1硫辛酸烟酸二联体及150 μmol·L-1硫辛酸预处理对数生长期细胞24 h后,再加入已经确定细胞损伤模型的丙烯醛浓度处理24 h,前后两次处理后均用MTT比色法检测细胞活性,HE染色法检测细胞数量和形态改变.结果 ARPE-19细胞24 h内开始贴壁,4～5d细胞生长开始融合,第1-6天后生长分裂迅速进入对数生长期,6d后即开始随着时间延长活性显著下降.当丙烯醛浓度达到50 μmol·L-1时,ARPE-19细胞数量明显减少,细胞肿胀、坏死,胞浆收缩、细胞核聚集;而当硫辛酸烟酸二联体浓度达到100 μmol·L-1时即可以明显减少50 μmol·L-1丙烯醛诱导的ARPE-19细胞活性的降低,防止凋亡小体的形成.MTT结果显示:100 μmol·L-1硫辛酸烟酸二联体+丙烯醛处理组(50 μmol·L-1)抗凋亡效果优于100 μmol·L-1硫辛酸组,但差异无统计学意义(P＞0.05).结论 利用丙烯醛可以诱导ARPE-19细胞损伤来建立AMD细胞损伤的模型,硫辛酸烟酸二联体对丙烯醛诱导的ARPE-19细胞损伤具有保护作用.     

细胞因子信号转导抑制因子3(SOCS3)对视神经损伤大鼠视网膜神经节细胞存活的影响

目的 研究细胞因子信号转导抑制因子3(suppressor of cytokine signaling 3,SOCS3)对视神经损伤大鼠视网膜神经节细胞(retinal ganglion cells,RGCs)存活的影响,并探讨其潜在的分子机制.方法 采用视神经横断手术构建大鼠视神经损伤模型,术后分离RGCs.实验分为视神经损伤组(optic nerve transection,ONT组)和假手术组.采用Western blot和RT-PCR检测SOCS3在两组细胞中的表达.随后将SOCS3 siRNA分别转染假手术组和ONT组RGCs,实验进一步分为空白对照组、阴性对照组和SOCS3沉默组.CCK8和MTT法检测细胞存活,Hoechst 33342荧光染色法和流式细胞技术检测细胞凋亡.进一步将mTOR siRNA和SOCS3 siRNA共转染RGCs,检测细胞存活和细胞凋亡.结果 视神经损伤3d后,ONT组SOCS3表达水平显著高于假手术组(P =0.049),且随损伤时间延长而升高.与空白对照组相比,SOCS3沉默可显著提高视神经损伤后RGCs的存活率[空白对照组与SOCS3沉默组分别为(49.47±7.35)％和(73.24±8.70)％],降低细胞凋亡率[2组分别为(27.25±0.75)％和(10.96±1.07)％]和细胞生长抑制率[2组分别为(23.06±1.43)％和(10.65±1.77)％].Hoechst染色也表明SOCS3沉默可改善视神经损伤诱导的细胞凋亡.同时,SOCS3沉默可显著提高视神经损伤后2周mTOR活性标志蛋白pS6的表达;且与单独沉默SOCS3相比,共同沉默mTOR和SOCS3可降低细胞存活率,提高细胞生长抑制率和细胞凋亡率.结论 SOCS3沉默可以通过上调损伤后期mTOR的活性而促进损伤RGCs的存活并抑制其凋亡.     

Apoptotic retinal ganglion cell death in the DBA/2 mouse model of glaucoma

The DBA/2 mouse has been used as a model for spontaneous secondary glaucoma. We attempted to determine the in vivo time course and spatial distribution of retinal ganglion cells (RGCs) undergoing apoptotic death in DBA/2 mice. Female DBA/2 mice, 3, 9-10, 12, 15, and 18 months of age, received intravitreal injections of Annexin-V conjugated to AlexaFluor 1h prior to euthanasia. Retinas were fixed and flat-mounted. Annexin-V-positive RGCs in the hemiretina opposite the site of injection were counted, and their locations were recorded. Positive controls for detection of apoptotic RGCs by Annexin-V labeling included rats subjected to optic nerve ligation, and C57BL/6 mice subjected to either optic nerve ligation or intravitreal injection of NMDA. To verify that Annexin-V-labeled cells were RGCs, intravitreal Annexin-V injections were also performed on retinas pre-labeled retrogradely with FluoroGold or with DiI. Annexin-V-positive RGC locations were analyzed to determine possible clustering and areas of preferential loss. Annexin-V labeled apoptotic RGCs in eyes after optic nerve ligation, intravitreal NMDA injection, as well as in aged DBA/2 animals. In glaucomatous DBA/2 mice 95-100% of cells labeled with Annexin-V were also FluoroGold- and DiI-positive. This confirms that Annexin-V can be used to specifically detect apoptotic RGCs in rodent retinas. In DBA/2 mice, apoptotic RGC death is maximal from the 12th to the 15th month of age (ANOVA, p<0.001, Fisher's post hoc test) and occurs in clusters. These clusters are initially located in the midperipheral retina and progressively occur closer to the optic nerve head with increasing age. Retrograde axonal transport of FluoroGold in the glaucomatous mouse retina is functional until at least 2-3days prior to initiation of apoptotic RGC death.     

Inflammatory demyelination induces axonal injury and retinal ganglion cell apoptosis in experimental optic neuritis

Optic neuritis is an inflammatory disease of the optic nerve that often occurs in patients with multiple sclerosis and leads to permanent visual loss mediated by retinal ganglion cell (RGC) damage. Optic neuritis occurs with high frequency in relapsing-remitting experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, with significant loss of RGCs. In the current study, mechanisms of RGC loss in this model were examined to determine whether inflammation-induced axonal injury mediates apoptotic death of RGCs. RGCs were retrogradely labeled by injection of fluorogold into superior colliculi of 6-7 week old female SJL/J mice. EAE was induced one week later by immunization with proteolipid protein peptide. Optic neuritis was detected by inflammatory cell infiltration on histological examination as early as 9 days after immunization, with peak incidence by day 12. Demyelination occurred 1-2 days after inflammation began. Loss of RGC axons was detected following demyelination, with significant axonal loss occurring by day 13 post-immunization. Axonal loss occurred prior to loss of RGC bodies at day 14. Apoptotic cells were also observed at day 14 in the ganglion cell layer of eyes with optic neuritis, but not in control eyes. Together these results suggest that inflammatory cell infiltration mediates demyelination and leads to direct axonal injury in this model of experimental optic neuritis. RGCs die by an apoptotic mechanism triggered by axonal injury. Potential neuroprotective therapies to prevent permanent RGC loss from optic neuritis will likely need to be initiated prior to axonal injury to preserve neuronal function.     

Neuroprotective effects of BDNF and GDNF in intravitreally transplanted mesenchymal stem cells after optic nerve crush in mice

AIM: To assess the neuro-protective effect of bone marrow mesenchymal stem cells (BMSCs) on retinal ganglion cells (RGCs) following optic nerve crush in mice. METHODS: C56BL/6J mice were treated with intravitreal injection of PBS, BMSCs, BDNF-interference BMSCs (BIM), and GDNF-interference BMSCs (GIM) following optic nerve crush, respectively. The number of surviving RGCs was determined by whole-mount retinas and frozen sections, while certain mRNA or protein was detected by q-PCR or ELISA, respectively. RESULTS: The density (cell number/mm2) of RGCs was 410.77±56.70 in the retina 21d after optic nerve crush without any treatment, compared to 1351.39±195.97 in the normal control (P<0.05). RGCs in BMSCs treated eyes was 625.07±89.64/mm2, significantly higher than that of no or PBS treatment (P<0.05). While RGCs was even less in the retina with intravitreal injection of BIM (354.07+39.77) and GIM (326.67+33.37) than that without treatment (P<0.05). BMSCs injection improved the internal BDNF expression in retinas. CONCLUSION: Optic nerve crush caused rust loss of RGCs and intravitreally transplanted BMSCs at some extent protected RGCs from death. The effect of BMSCs and level of BDNF in retinas are both related to BDNF and GDNF expression in BMSCs.     

Effects of Erythropoietin-Dextran Microparticle-Based PLGA/PLA Microspheres on RGCs

Purpose. We explored the neuroprotective effects of erythropoietin (EPO)-loaded dextran microparticle-based Poly(DL-lactide-co-glycolide)/Poly(DL-lactide) (PLGA/PLA) microspheres (EPO-dextran PLGA/PLA microspheres) on retinal ganglion cells (RGCs) in optic nerve crush rats for a prolonged period of time. Methods. EPO-dextran PLGA/PLA microspheres were prepared first by a novel solid-in-oil-in-water (S/O/W) technique. Then, the in vitro EPO release profile was assessed. Afterward, the bioactive effect of EPO released from EPO-dextran PLGA/PLA microspheres was explored in vitro on the retinal explants. Lastly, the neuroprotective effects of EPO-dextran PLGA/PLA microspheres on RGCs were evaluated in optic nerve crush rats with TUNEL staining for apoptotic RGCs. The level of glial fibrillary acidic protein (GFAP) expressed in retina was explored by immunohistochemistry staining. Survival RGCs were observed by DiI retrograde labeling using a DiI fluorescent tracer (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate). Results. The results demonstrated that a sustained release of EPO from PLGA/PLA microspheres could last for at least 60 days. EPO released from the microspheres showed as efficaciously neuroregenerative as EPO protein solution on retinal explants (P = 0.2554 for neurite density, P = 0.1004 for neurite length). TUNEL staining revealed that EPO-dextran PLGA/PLA microspheres remarkably reduced RGCs death when compared to the control (untreated) group (P < 0.01 at five days and one week post-crush, P < 0.05 at two weeks post-crush). Increased GFAP expression in retina was reduced greatly in EPO-dextran PLGA/PLA microspheres-administrated rats two weeks post optic nerve crush. DiI retrograde labeling revealed that a single injection of EPO-dextran PLGA/PLA microspheres significantly promoted RGCs survival (P < 0.01 at four and eight weeks post-crush). Conclusions. A single intravitreal injection of EPO-dextran PLGA/PLA microspheres appeared to have a prolonged protective effect on RGCs in optic nerve crush rats. The PLGA/PLA microspheres may be a feasible protein delivery system, such as EPO, to intravitreal injection for retinal degeneration diseases.     

金丝桃素对视神经损伤大鼠视网膜节细胞的保护作用

目的观察金丝桃素对视神经损伤大鼠视网膜节细胞的保护作用。方法24只SD大鼠随机分为正常对照组、单纯夹伤组、生理盐水对照组、金丝桃素治疗组4组,每组6只（12眼）。对所有大鼠行双上丘注射2％荧光金逆行标记节细胞,7d后,对单纯夹伤组、生理盐水对照组、金丝桃素治疗组进行球后视神经钳夹．同时在生理盐水对照组、金丝桃素治疗组玻璃体内分别注入生理盐水和金丝桃素5ul,14d后进行视网膜节细胞的计数。采用SPSS13．0统计软件对所得数据进行t检验。结果视神经夹伤后14d,存活的视网膜节细胞显著减少。单纯夹伤组节细胞存活率为50％,生理盐水对照组节细胞存活率为52％,金丝桃素治疗组节细胞存活率为68％。金丝桃素治疗组相比单纯夹伤组和生理盐水对照组,存活的节细胞明显要多（P〈0．05）。结论玻璃体内注射金丝桃素能减少大鼠视神经损伤后视网膜神经节细胞的死亡率．对视网膜节细胞有保护作用。     

大鼠视神经夹伤模型中莫尼定的视网膜节细胞保护作用

目的 :研究莫尼定对大鼠视神经夹伤模型视网膜神经节细胞的保护作用。方法 :实验用SD大鼠 2 0只随机分为用药组 8只和对照组 12只。所有大鼠右眼用 40 g微型视神经夹紧贴球后夹持视神经 60秒 ,左眼未做夹持。用药组于夹伤前1小时及夹伤后每日腹腔注射莫尼定 1mg/kg ,阴性对照组于夹伤前 1小时及夹伤后每日腹腔注射生理盐水 5ml/kg ,实验观察2 8天。实验结束前 4天双上丘注射 3 %荧光金逆行标记视网膜神经节细胞。做视网膜铺片 ,距离视乳头中心上下左右各2mm拍摄照片 ,使用CPAS图像分析软件做节细胞定量分析 ,节细胞存活率 =右眼节细胞密度 /左眼节细胞密度× 10 0。结果 :用药组、对照组节细胞存活率分别为 61 0 1%和 53 48% ,两者之间存在显著性差异 (P =0 .0 3 5)。结论 :在大鼠视神经夹伤模型中 ,莫尼定具有明显的视网膜节细胞保护作用     

Promoting optic nerve regeneration

Vision is the most important sense for humans and it is irreversibly impaired by axonal damage of retinal ganglion cells (RGCs) in the optic nerve due to the lack of axonal regeneration. The failure of regeneration is partially attributable to factors located in the inhibitory environment of the forming glial scar and myelin as well as an insufficient intrinsic ability for axonal regrowth. Moreover, RGCs undergo apoptotic cell death after optic nerve injury, eliminating any chance for regeneration. In this review, we discuss the different aspects that cause regenerative failure in the optic nerve. Moreover, we describe discoveries of the last two decades demonstrating that under certain circumstances mature RGCs can be transformed into an active regenerative state allowing these neurons to survive axotomy and to regenerate axons in the injured optic nerve. In this context we focus on the role of the cytokines ciliary neutrophic factor (CNTF) and leukemia inhibitory factor (LIF), their receptors and the downstream signaling pathways. Furthermore, we discuss strategies to overcome inhibitory signaling induced by molecules associated with optic nerve myelin and the glial scar as well as the regenerative outcome after combinatorial treatments. These findings are encouraging and may open the possibility that clinically meaningful regeneration may become achievable one day in the future.     

血管内皮生长因子B对小鼠视神经保护作用的研究

目的 探讨血管内皮生长因子B(VEGF-B)在视网膜组织的表达及其对视网膜神经节细胞的保护作用.方法 对照实验研究.35只成年雌性健康C57BL/6小鼠,分为正常对照组,视神经损伤后6 h、1 d、1周、2周组.其中10只鼠用于原位杂交,每组2只鼠;25只鼠用于实时定量逆转录聚合酶链反应(real time RT-PCR),每组5只鼠.采用原位杂交法观察实验鼠视网膜组织VEGF-B的mRNA表达;用real time RT-PCR法观察视网膜组织损伤后不同时间VEGF-B的mRNA定量表达;从双侧上丘行荧光金逆行标记和视网膜神经节细胞计数,评估玻璃体腔内注射重组人VEGF-B(450 mg/L)对视网膜神经节细胞的保护作用.应用SAS统计学软件进行数据分析.对组间real timeRT-PCR检测结果比较采用方差分析,对组间视网膜神经节细胞计数的计量资料比较采用秩和检验.以P<0.05作为差异有统计学意义.结果 小鼠视神经损伤后的视网膜组织VEGF-B表达显著增强,损伤后1周达高峰.玻璃体腔内注射重组人VEGF-B蛋白,可显著增加视网膜神经节细胞的存活数量,分别是单纯视神经损伤组和损伤加玻璃体腔内注射的阴性对照组的1.7倍(t=0.1301,P<0.01)和1.9倍(t=0.001,P<0.01).结论 VEGF-B参与小鼠视神经损伤后的修复,并对视网膜神经节细胞有保护作用.(中华眼科杂志,2009,45:38-42)     

横向定量牵拉法制作大鼠视神经精确损伤模型

目的:采用横向定量牵拉法制作大鼠视神经损伤模型,并利用荧光金逆行标记评价视神经牵拉伤后视网膜节细胞(retinal ganglion cells,RGCs)的存活率.方法:将25只雄性Wistar大鼠随机均分为5组,即假手术组和视神经牵拉伤后1、3、7、14d组.模型组用横向张力计牵拉左眼视神经,假手术组仅暴露左眼视神经但不予牵拉,各组以右眼为正常对照.用荧光金逆行标记,并观察假手术组及视神经牵拉伤后1、3、7、14d组RGCs的密度.结果:正常对照组RGCs形态多呈圆形或椭圆形,边界清楚,细胞外无明显荧光染料渗漏,部分可见明显细胞突起;而视神经牵拉伤后RGCs随时间延长而不断减少,细胞分布不均匀,并可见大量荧光渗漏及小胶质细胞.与正常对照组相比,假手术组RGCs形态和密度无明显差异(P＞0.05);视神经牵拉伤后第1、3、7、14d的RGCs数量进行性减少,且其密度均明显低于正常对照组(P＜0.01);视神经牵拉伤后第1、3、7、14d的RGCs存活率分另别为78.94％±0.92％、60.07％±0.90％、38.92％±1.42％和17.31％±0.97％.结论:横向定量牵拉法可以建立易于量化的视神经损伤模型,为进一步研究视神经损伤后的治疗方法提供有力工具.     

c-Jun expression in surviving and regenerating retinal ganglion cells: effects of intravitreal neurotrophic supply

PURPOSE: To investigate c-jun expression in surviving and axon-regenerating retinal ganglion cells (RGCs) and the effect of intravitreal neurotrophic supply on c-jun expression. METHODS: All animals underwent optic nerve transection (ONT) 0.5 mm behind the eyeball. Some animals underwent a replacement of the optic nerve with an autologous sciatic nerve graft (SNG) to allow axonal regrowth. To provide a neurotrophic supply, a peripheral nerve (PN) segment or brain-derived neurotrophic factor (BDNF)/ciliary neurotrophic factor (CNTF) was applied intravitreally. The time course of c-jun expression was first examined in both surviving and regenerating RGCs. Then, c-jun expression was examined in surviving and regenerating RGCs 3 weeks after intravitreal BDNF/CNTF treatment. Animals with vehicle eye injection were used as the control. Fluorescent dye was used for retrograde labeling of surviving (applied behind the eyeball) and regenerating (applied at the distal end of the SNG) RGCs. All retinas were immunohistochemically stained for c-jun. RESULTS: c-Jun was not detected in normal RGCs, but weak expression was seen in surviving RGCs after ON injury. The proportion of c-jun-positive (+) RGCs among surviving cell population was 52.6% to 86.5% 2 to 6 weeks after ONT. Among regenerating RGCs, more than 80% expressed c-jun in all treatment groups, a proportion that was significantly higher after CNTF treatment (90.7%). In addition, c-jun expression was much stronger in intensity and the c-jun(+) nuclei were much larger in regenerating than in surviving RGCs. CONCLUSIONS: c-Jun expression in RGCs was upregulated after injury. Most regenerating RGCs were c-jun(+), and the intensity of c-jun expression was higher in regenerating than in surviving RGCs. CNTF also upregulated c-jun expression in RGCs.