吲哚菁绿对人视网膜色素上皮细胞的影响

目的　研究吲哚菁绿 (ICG)对培养的人视网膜色素上皮 (RPE)细胞的影响 ,为黄斑裂孔手术中ICG着染并剥除视网膜内界膜寻求合适的质量浓度和作用时间。方法　以质量浓度分别为 2 0、1 0、0 5、0 2 5mg/mLICG分别作用于培养的人RPE细胞 ,用四唑盐比色法 (MTT)、电镜及免疫组织化学方法从形态、功能方面观察RPE细胞的损伤。结果　 ( 1)MTT检测ICG对RPE细胞活性的抑制与时间无关 (F =1 96,P =0 173 3 ) ,与质量浓度有明显相关性 (F =80 96,P =0 0 0 0 1)。ICG质量浓度小于或等于 0 5mg/mL时 ,RPE细胞活性与阴性对照组无明显差异 (P >0 0 5 )。( 2 )电镜观察以上质量浓度ICG对RPE细胞形态均有损伤 ,以变性为主 ,表现为细胞器肿胀且与质量浓度呈正相关。( 3 )以上 4种质量浓度ICG作用RPE细胞后 ,各组细胞增殖核抗原 (PCNA)表达与阴性对照组无明显差异 (P >0 0 5 )。结论　质量浓度≤ 0 5mg/mL时ICG可安全地着染视网膜内界膜。     

吲哚青绿辅助剥离内界膜后在黄斑裂孔患者眼底残留的观察

视网膜内界膜薄而透明,手术过程中难以辨认;内界膜染色有助于将内界膜剥离干净。目前比较常用的内界膜染色剂是吲哚青绿(ICG)。Gale等[1],Iriyama等[2]通过体外实验发现,ICG对视网膜神经节细胞(RGCs)和视网膜色素上皮(RPE)细胞有毒性,并且这种毒性表现为浓度依赖性和时间依赖性     

人视网膜色素上皮细胞对吲哚青绿的吸收与代谢

目的 研究人视网膜色素上皮（RPE）细胞对吲哚青绿 (ICG) 的摄 取和代谢规律以及ICG 在细胞内的亚细胞定位。 方法 将RPE细胞与0.25 mg/ml ICG在37℃暗箱中共同孵育1、4、24 h后,应用透射电子显微镜观察细胞浆内ICG颗粒和细胞超微结构改变。荧光显微镜下观察RPE细胞与ICG共同孵育1、2、4、8、12、24、48 h 后细胞浆内ICG的自发荧光。应用紫外/可见光分光光度计测定梯度稀释的ICG 溶液805 nm吸 光度［A，旧称光密度（OD）］值，绘制ICG 溶液浓度的标准曲线，计算ICG浓度与 A 值的相关公式。RPE细胞与ICG共同孵育1、2、4、8、12、24、48、72 h后检测上清液805 nm A值，代入公式，计算细胞对ICG 的吸收情况。RPE细胞与ICG共同孵育24 h后，1周 内每次取1个样本进行透射电子显微镜和荧光显微镜检查，记录RPE细胞对ICG的代谢周期。 结果 ICG 进入细胞后，在细胞浆内均匀分布。0.25 mg/ml ICG作用RPE 细胞 24 h后，对细胞超微结构无显著影响。RPE对ICG的吸收随孵育时间延长逐渐增加。共同孵育24 h后，RPE细胞对ICG的摄取达高峰，以后由于细胞对其缓慢代谢而逐渐下降。1周 后RPE细胞内仍有微量ICG残留。 结论 RPE细胞通过主动转运摄取ICG。I CG在RPE细胞内代谢周期较长。这一特点是ICG在玻璃体手术中对视网膜具有潜在毒性的机制之一。（中华眼底病杂志，2004，20：179-181）     

黄斑裂孔内界膜剥离术中应用吲哚青绿对视网膜的影响

吲哚青绿（ICG）可选择性地染色内界膜，使无色透明的内界膜清晰可见，剥离内界膜手术变得简单、快捷。有关黄斑裂孔手术中使用ICG对视网膜有无毒性尚存在较多争议。体外实验和活体应用结果显示吲哚青绿对视网膜的色素上皮细胞和神经节细胞均有一定毒性，且ICG对视网膜的毒性损害存在剂量依赖性和时间依赖性。在黄斑裂孔手术中应用ICG辅助剥离内界膜时，应注意降低ICG浓度，减少ICG与视网膜接触时间；应快速注射在黄斑区，然后立即将玻璃体腔内ICG完全冲洗干净；术中在气液交换后再使用ICG染色内界膜，使ICG与视网膜的接触局限在后极部视网膜；用少量重水或粘弹剂先封堵黄斑裂孔，以减少ICG与黄斑裂孔底部RPE接触的机会。     

低浓度氯喹保护成年小鼠视网膜神经节细胞抵抗N-甲基-D-天冬氨酸的兴奋性毒性作用

目的:观察不同浓度氯喹对N-甲基-D-天冬氨酸(NMDA)损伤模型小鼠RGC的保护作用及可能调控机制。方法:健康雄性C57/BL6小鼠54只,随机分为氯喹低浓度组、氯喹高浓度组、PBS对照组,每组均为18只。氯喹低浓度组、氯喹高浓度组小鼠分别按体重10、100 mg/kg剂量腹腔注射氯喹;PBS组小鼠腹腔注射等体积PBS。腹腔注射1次/d,连续2d后,氯喹低浓度组、氯喹高浓度组小鼠左眼玻璃体腔注射5 nmol/L NMDA,对侧眼注射等体积PBS。建模后7d,视网膜铺片RGC染色,计数RGC存活数目;建模后9、10d,分别行视动反应和ERG检查;建模后11 d,行视网膜免疫荧光染色检测各组小鼠视网膜胶质纤维酸性蛋白(GFAP)荧光表达和实时荧光定量PCR(RT-PCR)检测各组小鼠视网膜GFAP、TNF-α、IL-6 mRNA表达。各组间RGC密度、视敏度、光负性反应(PhNR)波振幅比较采用单因素方差分析。结果:建模后7d,与PBS对照组小鼠RGC密度比较,氯喹低浓度组显著升高,差异有统计学意义(F=54.41,P<0.01);氯喹高浓度组降低,但差异无统计学意义(F=1.18,P>0.05)。氯喹低浓度组小鼠视敏度、PhNR波振幅均高于PBS对照组,差异有统计学意义(F=9.10、17.60,P<0.05、<0.01)。氯喹低浓度组小鼠视网膜GFAP绿色荧光表达明显少于PBS对照组、氯喹高浓度组,差异有统计学意义(F=23.66,P<0.05)。低浓度氯喹组小鼠视网膜GFAP(F=110.20)、IL-6(F=167.60)、TNF-α(F=17.78)mRNA表达较高浓度氯喹组、PBS对照组显著下降,差异有统计学意义(P<0.010、<0.001、<0.010)。结论:低浓度氯喹对NMDA损伤所致RGC丢失有保护作用,其作用机制可能与抑制胶质细胞活化与炎症反应有关;高浓度氯喹会加重RGC凋亡。     

脑源性神经营养因子对小鼠视网膜Müller细胞谷氨酸转运体表达的调控

谷氨酸是视网膜主要的兴奋性神经递质,然而在病理性浓度升高时,对视网膜神经节细胞(RGC)具有兴奋毒性作用[1].L-谷氨酸/L-天门冬氨酸转运体(GLAST)是表达在视网膜Müller细胞上最主要的谷氨酸转运体[5],在维持谷氨酸平衡和保护RGC免受谷氨酸兴奋毒性作用中发挥着重要作用[3].     

脑源性神经营养因子对小鼠视网膜Müller细胞谷氨酸转运体表达的调控

谷氨酸是视网膜主要的兴奋性神经递质,然而在病理性浓度升高时,对视网膜神经节细胞(RGC)具有兴奋毒性作用[1].L-谷氨酸/L-天门冬氨酸转运体(GLAST)是表达在视网膜Müller细胞上最主要的谷氨酸转运体[5],在维持谷氨酸平衡和保护RGC免受谷氨酸兴奋毒性作用中发挥着重要作用[3].     

脑源性神经营养因子对小鼠视网膜Müller细胞谷氨酸转运体表达的调控

谷氨酸是视网膜主要的兴奋性神经递质,然而在病理性浓度升高时,对视网膜神经节细胞(RGC)具有兴奋毒性作用[1].L-谷氨酸/L-天门冬氨酸转运体(GLAST)是表达在视网膜Müller细胞上最主要的谷氨酸转运体[5],在维持谷氨酸平衡和保护RGC免受谷氨酸兴奋毒性作用中发挥着重要作用[3].     

Cyclosporine玻璃体腔内注射对异种视网膜色素上皮移植的排异抑制作用

目的 观察兔眼视网膜下腔植入人胚眼视网膜色素上皮（retinal pigment epithe-lium,RPE）后不同时期的眼底和组织学改变。研究环胞菌素A（Cyclosporines,CsA）玻璃体腔内注射能否抑制人胚眼RPE在兔眼视网膜下腔中诱导的异种移植排异反应。方法 人胚眼色素上皮片和浓缩色素上皮细胞悬液植入36只兔眼的视网膜下腔,其中16眼为对照组,用于观察排异的自然转规。分别7d(8眼）和30d(8眼）后获取组织标本。另20眼为实验组。RPE移植后,每周一次玻璃体腔内注射CsA 1mg(12眼）或CsA0.1mg(8眼）。视网膜和视神经的毒性反应使用ERG进行检查。结果 人胚眼RPE片和浓缩的RPE细胞均能在视网膜下腔短期存活。移植的RPE与视细胞结合良好并显示吞噬功能。排异反应发生时间约在术后10～30d。对照组中7d的排异发生率为0/8;30d排异发生率为7/8。排异发生后荧光造影中移植区为高荧光区,组织切片中显示有大量的组织细胞积聚。CsA1mg组30d排异发生率为0/12,0.1mg组为5/8。ERG波幅的下降与CsA剂量和注射次数呈正相关。结论 异种RPE视网膜下腔移植在无免疫抑制剂的条件下,只能短期存活。CsA玻璃体腔中注射能抑制异种RPE移植的排异反应但易引起明显的视网膜毒性反应。     

乳酸林格氏液和平衡盐液对角膜内皮影响的临床观察

目的　探讨乳酸林格氏液和平衡盐液在小切口白内障摘除人工晶状体植入术中对角膜内皮的影响。方法　 45例 (46只眼 )老年性白内障患者随机分为两组 ,分别以乳酸林格氏液 (2 3例 )和平衡盐液 (2 2例 )为灌注液。术式为非超声乳化小切口白内障摘除人工晶状体植入术。术前和术后 3个月行角膜内皮显微镜检查 ,观察角膜内皮细胞密度、平均内皮细胞面积、六边形细胞百分率和变异系数的改变。结果　 41例患者 (91% )完成了术后随访。乳酸林格氏液组 (n =2 0 )角膜内皮细胞密度较术前减少 5 2 5± 192个 /m m2 ;平均盐液组 (n =2 1)减少 5 0 5±187个 /mm2 ,二者无显著性差异 (P >0 .0 5 )。乳酸林格氏液组平均内皮细胞面积较术前增加 (95 .1± 42 .6 ) μm2 ;平衡盐液组增加 (85 .9± 2 6 .4)μm2 ,二者无显著性差异 (P >0 .0 5 )。乳酸林格氏液组六边形细胞百分率较术前下降(11.1± 4.9) % ;平衡盐液组下降 (0 .9± 0 .2 ) % ,二者之间存在显著性差异 (P <0 .0 1)。乳酸林格氏液组变异系数较术前增加 (0 .12± 0 .0 3) ;平衡盐液组增加 (0 .0 5± 0 .0 4) ,二者之间存在显著性差异 (P <0 .0 5 )。结论　角膜内皮细胞形态学分析较细胞密度和平均面积的改变能更灵敏的反映角膜内皮的变化。平衡盐液对角膜内皮的     

Effects of retinal glial cells on isolated rat retinal ganglion cells

PURPOSE: The effect of retinal glial cells on retinal ganglion cell (RGC) survival was investigated in cocultures of pure, isolated retinal glial cells with pure, isolated RGCs. METHODS: RGCs from 2-day-old rats were cocultured for 48 hours, avoiding direct contact between cell types, with either nonconfluent retinal glial cells from 3-day-old rats or confluent retinal glial cells from 3-day-old, 12-day-old, or 1-year-old rats. Survival of RGCs was evaluated by flow cytometry. Amino acids were determined in culture medium. The effects of glutamate antagonists, 6-cyano-7-nitroquinoxaline-2,3-dione and MK801, a nitric oxide (NO) scavenger, 2-(4-carboxyphenyl)-4,4,5,5tetramethylimidazoline-1-oxyl-3-oxide potassium salt (c-PTIO), and an NO synthase inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME), were examined. RESULTS: Nonconfluent retinal glial cells significantly reduced the survival of small and large RGCs, but confluent retinal glial cells reduced the survival of only small RGCs, regardless of the rat's age at the time of retinal glial cell harvesting. Profiles of some amino acids significantly varied, depending on the culture condition. Cocultures of RGCs with nonconfluent retinal glial cells released significantly more glutamate into the medium than cocultures of RGCs with confluent retinal glial cells or RGCs in pure culture. The glutamate antagonists improved the survival of RGCs cocultured with nonconfluent retinal glial cells, especially when the two were administered in combination, and in the case of large RGCs. c-PTIO and L-NAME, also improved the survival of RGCs cocultured with nonconfluent retinal glial cells. CONCLUSIONS: Adverse effects of retinal glial cells on the survival of RGCs varied by size of the RGCs and retinal glial cell confluence. Glutamate and NO may be involved in retinal glial cell-related antisurvival effects.     

Müller细胞在视网膜损伤中对视神经节细胞影响的研究进展

Müller细胞是视网膜中主要神经胶质细胞，贯穿视网膜全层。尽管近年来针对Müller细胞功能的研究较多，但是Müller细胞和视网膜神经节细胞(retinal ganglion cells，RGCs)的相互作用关系仍不完全清楚。目前已知生理状态下Müller细胞的许多功能和RGCs密切相关，例如Müller细胞已经证实为神经元祖细胞的来源，调节视网膜细胞间质K⁺水平和谷氨酸代谢，维持视网膜内能量代谢和营养支持等。在视网膜损伤时，Müller细胞相关功能对RGCs的影响也十分重要。因此，本文就此研究进展进行综述，以期为视网膜中视神经的保护治疗提供新的思路。     

Notch-1调控视网膜前体细胞向视网膜神经节细胞分化

目的研究Notch-1对视网膜前体细胞(RPC)向视网膜神经节细胞(RGC)分化的调控作用。方法分离培养胚胎14 d龄Sprague-Dawley大鼠的RPC，实验组和对照组分别用含有Notch-1反义寡核苷酸链和无关序列寡核苷酸链的培养液进行诱导分化14 d，倒置相差显微镜每天观察细胞的生长和分化情况，Thy1.1标记RGC并进行计数。结果实验组和对照组的RPC都能分化为多种视网膜细胞类型，包括Thy1.1阳性的RGC，但两组RPC向RGC分化的百分比不同。实验组和对照组RGC的百分比分别为(16.57±4.31)%和(31.19±6.90)%，两组比较差异有统计学意义（t=9.84,P＜0.001）。结论Notch-1对RPC的分化具有负向调控作用，阻断Notch-1能促进RPC向RGC分化。(中华眼底病杂志, 2007, 23: 101-103)     

实验性视网膜脱离复位后的视网膜细胞凋亡

目的研究视网膜脱离(retinaldetachment,RD)复位后的视网膜细胞凋亡的发生情况,探讨RD后视功损害的机制。方法14只成年灰兔,分为正常对照组1只,RD阳性对照组1只,实验组12只分为4组,视网膜下注射透明质酸钠建立RD模型,后分别观察1周、2周、3周、4周,观察期满取眼球进行组织切片。采用TUNEL法和细胞计数的方法进行细胞凋亡观察。结果正常对照组视网膜细胞凋亡标记基本为阴性。脱离的视网膜在内、外核层发现凋亡标记阳性细胞。在复位的视网膜上观察到凋亡标记阳性细胞,主要分布在内、外核层和神经节细胞层,并随着复位时间而变化(P<0.01)(凋亡细胞计数:1周10.50±3.41,2周6.90±2.42,3周5.50±2.07,4周1.78±1.56)。细胞计数显示实验组除观察4周组外,凋亡细胞数与正常视网膜和脱离时的视网膜分别相比,差异均有高度显著性(P<0.01)。结论RD复位后的视网膜细胞有细胞凋亡的发生,是视网膜功能恢复不良的重要原因。     

Stem cells and retinal repair

Retinal stem cells (RSCs) are multipotent central nervous system (CNS) precursors that give rise to the retina during the course of development. RSCs are present in the embryonic eyecup of all vertebrate species and remain active in lower vertebrates throughout life. Mammals, however, exhibit little RSC activity in adulthood and thus little capacity for retinal growth or regeneration. Because CNS precursors can now be isolated from immature and mature mammals and expanded ex vivo, it is possible to study these cells in culture as well as following transplantation to the diseased retina. Such experiments have revealed a wealth of unanticipated findings, both in terms of the instructive cues present in the mature mammalian retina as well as the ability of grafted CNS precursors to respond to them. This review examines current knowledge regarding RSCs, together with other CNS precursors, from the perspective of investigators who wish to isolate, propagate, genetically modify, and transplant these cells as a regenerative strategy with application to retinal disease.     

Dispersion of retinal pigment epithelial cells from experimental retinal holes

The effects of cryotherapy, transcleral massage, or directed irrigation on exposed retinal pigment epithelial (RPE) cells at the base of an experimental retinal hole were studied in living rabbit eyes. Cryotherpy or massage with or without vitrectomy failed to release RPE cells or result in preretinal membrane formation. Directed irrigation dispersed viable RPE cells into the vitreous and produced cellular precipitates with membranes on the inferior retina, although the membranes tended to regress within 6 weeks after surgery. These results suggest that healthy RPE cells at the base of retinal tears are not easily released into the vitreous and may not contribute to the formation of preretinal membranes found in proliferative vitreoretinopathy.This work was supported in part by a grant from MedChem Products, Woburn, Mass     

视网膜脱离状态下色素上皮细胞的凋亡变异

目的探讨视网膜脱离(RD)后色素上皮(RPE)细胞的凋亡.方法制造有色眼家兔的RD模型,以流式细胞仪(FCM)技术分析RD后28天内RPE细胞的凋亡变化.结果RD后8hRPE细胞凋亡显著增加,第7天达顶峰,凋亡细胞平均数由正常的131.67±35.67上升至1196.2±238,凋亡率由正常的0.70%±0.21%上升至5.98%±1.21%,然后缓慢下降,第28天仍显著高于正常.结论RD可导致RPE细胞的凋亡.RD后28天内,随时间延长,RPE凋亡增加.     

视网膜神经节细胞保护的药物治疗

视网膜神经节细胞(RGCs)是青光眼、眼外伤等神经退行性病变的主要损伤细胞,它的死亡常导致视功能的不可逆性损害。     

Molecular and cellular reactions of retinal ganglion cells and retinal glial cells under centrifugal force loading

PURPOSE: To investigate changes in retinal ganglion cell (RGC) survival and morphology, retinal glial cell effects on RGC survival, and changes in mRNA expression during centrifugal force loading using a newly developed device. METHODS: Changes in RGC survival and morphology were examined when isolated RGCs from 2-day-old rats were loaded with centrifugal force equivalent to 16, 28, or 33 mm Hg. The effects of cocultured retinal glial cells on RGC survival were studied in the presence of centrifugal force equivalent to 16 and 28 mm Hg for 48 hours. The microarray method and real-time polymerase chain reaction confirmed changes in mRNA expression when RGCs and retinal glial cells were loaded with centrifugal force equivalent to 28 mm Hg for 24 hours. RESULTS: The survival of isolated RGCs and the number of neurites were significantly decreased by centrifugal force loading. Conversely, there was no significant change in the survival of isolated retinal glial cells. The survival of cocultured RGCs was significantly better than that of isolated RGCs. In contrast to the numerous changes in the mRNA expression of retinal glial cells subjected to centrifugal force loading, there was no significant change in the mRNA expression of RGCs. CONCLUSIONS: The developed device may have potential for use as an in vitro model of RGC damage. The response to centrifugal force loading varies according to cell type, and the marked changes in the mRNA expression of retinal glial cells may be involved in the improvement of RGC survival.