Cell-based therapy for retina degeneration: the promise of a cure

Cell-based therapies in the retina have been associated with the recovery of visual function in animal models of retinal degeneration. This review covers the current status of such therapies with regard to the source of the donor cells, their integration, and their impact on the degenerating host retina. Emphasis is also put on the importance of a careful interpretation of what is meant by "recovery of visual function". Two main approaches are considered here: (1) the use of human embryonic stem cell derived retinal pigment epithelial (RPE) cells to rescue photoreceptors in an animal model of RPE defect; and (2) the use of photoreceptor precursors to repair the degenerating neural retina. The current conclusions are that major hurdles have to be dealt with, such as finding an appropriate and ethically compliant donor cell source that would yield protracted survival and integration of the replacement retinal cells, and that there is no evidence yet that cell-based therapies can allow the long-term preservation or recovery of conscious vision.     

外源性促红细胞生成素对大鼠视网膜脱离光感受器细胞的保护作用

目的 观察外源性促红细胞生成素(EPO)对大鼠视网膜脱离(RD)状态下光感受器细胞的保护作用.方法 采用计算机产生随机数字的方法将162只正常雄性SD大鼠分为正常对照组(NC组)、RD组、RD+磷酸盐缓冲液(PBS)组、RD+EPO 100、200、400 ng组.RD后3 d,采用蛋白免疫印迹(Western blot)检测半胱氨酸蛋白酶3(caspase-3)活性和B细胞淋巴瘤/白血病-XL(bcl-XL)的表达,免疫荧光检测caspase-3活性,脱氧核糖核苷酸末端转移酶介导的缺口末端标记法(TUNEL)检测光感受器细胞凋亡情况.RD后14、28 d,2个月,视网膜组织病理学观察并测量外核层(ONL)厚度.结果 Western bolt检测发现,各组间活化caspase-3条带灰度值差异有统计学意义(F=35.96,P<0.01),bcl-XL条带灰度值差异也有统计学意义(F=30.75,P<0.01).免疫荧光和TUNEL检测发现,caspase-3免疫阳性光感受器细胞数目与TUNEL阳性细胞核趋势表现一致.RD后14 d、2个月,各组间ONL差异有统计学意义(F=21.52,96.25;P<0.01).结论 RD后补充外源性EPO可通过抑制caspase-3活性并增加bcl-XL的表达拮抗凋亡,从而发挥对光感受器细胞的保护作用.     

Preservation of photoreceptors in dystrophic RCS rats following allo- and xenotransplantation of IPE cells

Purpose  To examine whether iris pigment epithelial (IPE) cells transplanted into the subretinal space of Royal College of Surgeons (RCS) rats have the ability to rescue photoreceptors. Methods  Rat IPE (rIPE) or human IPE (hIPE) cells were transplanted subretinally in 23-day-old RCS rats. Sham injection and transplantation of ARPE-19 cells served as controls. After 12 weeks, eyes were evaluated for photoreceptor survival by morphometric analysis and electron microscopy. Results  Morphometric analysis showed photoreceptor rescue in all transplanted and sham-injected animals (number of photoreceptors/300 μm retina±sd: rIPE 41.67 ± 28; hIPE 29.50 ± 16; ARPE-19 36.12 ± 21; sham 16.56 ± 6) compared to age-matched, control rats (number of photoreceptors/300 μm retina±sd: 9.71 ± 4). Photoreceptor rescue was prominent in IPE cell-transplanted rats and was significantly greater than sham-injected eyes (p = 0.02 for rIPE and p = 0.04 for hIPE). Conclusion  Since IPE cells transplanted into the subretinal space have the ability to rescue photoreceptors from degeneration in the RCS rat without any harmful effects, IPE cells may represent an ideal cell to genetically modify and thus carry essential genetic information for the repair of defects in the subretinal space.     

Subretinal transplantation of brain-derived precursor cells to young RCS rats promotes photoreceptor cell survival

The potential use of in vitro-expanded precursor cells or cell lines in brain repair includes transplantation of such cells for cell replacement purposes and the activation of host cells to provide 'self-repair'. Recently, it has been reported that the immortalized brain-derived cell line RN33B (derived from the embryonic rat medullary raphe) survive, integrate and differentiate after subretinal grafting to normal adult rats. Here, it is demonstrated that grafts of these cells survive for at least 6 weeks after implantation into postnatal days 21 and 35 retinas of normal and Royal College of Surgeons rats, a model of retinal degeneration. Implanted cells integrate into the retinal pigment epithelium and the inner retinal layers, and the anterior part of the optic nerve of both normal and Royal College of Surgeons rats. The RN33B cells migrate within the retina, occupying the whole retina from one eccentricity to the other. A significant number of the grafted cells differentiate into glial cells, as shown by the double labelling of the reporter genes LacZ or green fluorescent protein, with several glial markers, including oligodendrocytic markers. Many implanted cells in the host retina were in a proliferative stage judging from proliferative cell nuclear antigen and SV40 large T-antigen immunohistochemistry. Interestingly, there was a promotion of photoreceptor survival, extending over more than 2/3 of the superior hemisphere, in Royal College of Surgeons rats transplanted at postnatal day 21, but not at postnatal day 35. In addition, grafted cells were found in the surviving photoreceptor layer in these rats.     

经巩膜外路至视网膜下腔移植视网膜细胞的实验研究

探讨视网膜光感受器细胞的移植方法及临床意义。方法将16只昆明鼠随机分为A组和B组,每组均8只鼠。于手术显微镜下,用特殊显微注射器穿过巩膜、脉络膜,在A组昆明鼠的视网膜下腔注入视网膜混合细胞,在B组昆明鼠的视网膜下腔注入纯光感受器细胞。于移植术后30、90及180 d摘除实验眼,于光镜下观察移植细胞在视网膜下腔生长的情况。结果大多数标本(13／15)HE染色显示视网膜细胞准确移植在受体眼的视网膜下腔,未见炎性细胞浸润和受体视网膜破坏;且移植到受体视网膜下腔的细胞在术后180 d仍存活。仅少数(2／15)标本可见受体视网膜结构破坏。移植的视网膜混合细胞均形成“玫瑰花”样结构,而移植的纯视网膜光感受器细胞则在视网膜下腔形成整齐的细胞层。结论经巩膜外路至视网膜下腔的显微注射法是较为理想的视网膜下腔注射给药和视网膜细胞移植方式。纯视网膜光感受器细胞移植后的生长状况和功能接近正常生理状态的视网膜组织结构,为临床治疗视网膜变性疾病提供了新途径。     

光感受器与双极细胞间突触在视网膜脱离后的重塑

视网膜脱离（RD）是严重的致盲性眼病。近年来研究发现,RD后光感受器与双极细胞间突触结构会遭到破坏,这一组织学上的变化对RD患者术后视功能的恢复有着重要影响。现就光感受器与双极细胞间突触在RD后的重塑进行综述,探讨RD术后视功能恢复不良的可能机制。     

Gene therapy for choroideremia: in vitro rescue mediated by recombinant adenovirus

Choroideremia (CHM) is an X-linked retinal degenerative disease resulting from a lack of functional Rab Escort Protein-1 (REP-1). As a first step in developing gene-based therapies for this disease, we evaluated the feasibility of delivering functional REP-1 to defective lymphocytes and fibroblasts isolated from individuals with CHM. A recombinant adenovirus delivering the full-length human cDNA encoding REP-1 under the control of a cytomegalovirus promoter was generated. Adenovirus-mediated delivery of REP-1 rescued the defective cells as assessed through protein and enzymatic assays. Ultimately, it may be possible to use virus-mediated delivery of REP-1 to evaluate disease intervention in vivo.     

视网膜下间隙移植经基因修饰的视网膜色素上皮细胞对大鼠光感受器变性及视功能的保护作用

目的 以RCS大鼠作模型,研究经基因修饰的永生化视网膜色素上皮细胞(RPE)视网膜下移植对光感受器变性的保护作用。方法 在绿色荧光蛋白基因逆转录病毒感染的基础上,利用脂质体介导节状神经生长因子(CNTF)表达质粒转移,修饰成人RPE细胞系CRL-2302。将1×105个表达绿色荧光蛋白(GFP)或GFP及CNTF的RPE细胞移植到4～5周龄RCS大鼠右眼视网膜下间隙,左眼不移植或注射。PBS作为对照。术后2、4、6、8、10和12周作荧光显微镜、光镜、电镜及电生理检查。结果 荧光显微镜观察,术后1周移植的人RPE细胞在RCS大鼠视网膜下间隙已扩散到几乎整个眼底,但随时间延长移植的细胞逐渐减少,术后6周仅残留少量移植细胞。光镜及电镜观察显示移植眼保留的光感受器数量明显较对照眼多,凋亡细胞则较对照跟少。此外,移植眼宿主RPE细胞形态较正常,并可见吞噬小体。视网膜电图(ERG)检查结果表明部分移植眼视网膜功能明显较对照眼好。结论 经过基因修饰的RPE细胞移植可延缓RCS大鼠视网膜光感受器变性,为治疗视网膜变性提供了新的途径。(中华眼科杂志,2004,40:552-556)     

The relationship between full field electroretinogram and perimetry-like visual thresholds in RCS rats during photoreceptor degeneration and rescue by cell transplants

Dark-adapted full field electroretinogram (ERG) and visual receptive field thresholds (recorded from the superior colliculus) were correlated in a model of retinal degeneration, the Royal College of Surgeons rat. In both untreated and retinal pigment epithelium cell transplanted rats, optimal correlation was between b-wave amplitude and preserved visual field area with thresholds under a defined level. The work shows that the magnitude of the b-wave can be used to predict the computed area and degree of visual field preservation recorded in the central nervous system. These observations validate using ERG to assess residual visual function and the effect of transplantation.     

Uptake and esterification of vitamin A by RCS rat retinal pigment epithelial cells in primary culture

We investigated the capacity of Royal College of Surgeons (RCS) rat retinal pigment epithelial (RPE) cells to take up all-trans-retinol (ROL) (vitamin A) and to metabolize it into retinyl esters (RE). Cultures of RPE cells were established from RCS and control newborn rats. All-trans-ROL was delivered to the apical surface of the RPE monolayer. Retinoids were analyzed by high-performance liquid chromatography. The cellular retinol-binding protein type I (CRBP-I) was assessed by Western blotting. Before supplementation with ROL, RE were lower in RCS rats. After ROL supplementation, esters increased and reached values that were similar in the two strains, but the increase, expressed relative to the initial value, was higher in RCS rats. The uptake of ROL and the level of CRBP-I were greater in RCS rats. Our results provide evidence of a functional retinol esterifying enzyme in cultured RCS RPE cells and suggest that CRBP-I could play a role in the uptake and esterification of ROL in the RPE cells.     

内源性促红细胞生成素对大鼠脱离视网膜光感受器细胞的保护作用

背景促红细胞生成素（EPO）对多种视网膜疾病模型中视网膜神经元具有一定的保护作用,但EPO对视网膜脱离（RD）后光感受器细胞是否具有保护作用尚不清楚。目的探讨内源性EPO对RD状态下光感受器细胞的保护作用及可能机制。方法利用视网膜下腔注射质量分数1．4％透明质酸钠建立大鼠RD模型,按每组情况各组玻璃体腔内分别单次注射PBS或不同剂量的外源性可溶性EPO受体（EPOsR）,采用计算机产生随机数字法将72只SD大鼠随机平均分为正常对照组、RD组、RD＋PBS组、RD＋EPOsR2、20、200ng组。分别于造模后3d和14d用过量麻醉法处死大鼠并获得大鼠视网膜标本,采用末端脱氧核苷酸转移酶介导的dUTP缺口末端标记（TUNEL）法检测光感受器细胞的凋亡情况,并分别采用Westernblot和免疫荧光法检测视网膜中caspase-3的活性,RD造模后14d进行组织病理学检查并测量外核层（ONL）厚度。结果RD造模后3d,RD组ONL出现凋亡细胞核,玻璃体腔注射EPOsR组光感受器细胞凋亡进一步增加,随着玻璃体腔注射EPOsR的剂量增加,ONL凋亡细胞核有增加趋势。Westernblot和免疫荧光检测结果均显示,各组视网膜caspase-3表达的条带灰度值分别为（0．15±0．04）、（0．49±0．03）、（0．50±0．07）、（0．63±O．03）、（0．69±0．04）、（0．83±0．04）,各组的总体差异有统计学意义（F=76．016,P=0．000）,RD＋EPOsR200ng组的caspase-3活性均强于其他各组,差异均有统计学意义（P〈0．01）。RD造模后14d,正常对照组、RD组、RD＋PBS组、RD＋EPOsR2、20、200ng组的ONL厚度分别为（47．39±3．39）、（33．96±3．54）、（31．83±5．21）、（31．40±2．63）、（24．99±2．06）、（19．30e3．71）μm,总体差异有统计学意义（F=44．733;P=0．000）;EPOsR处理组ONL厚度明显薄于单纯RD组和RD＋PBS组,差异均有统计学意义（P〈0．05）。结论RD状态下,EPOsR通过剂量依赖的方式诱导视网膜细胞的凋亡和caspase-3活性增强,而缺氧状态下视网膜神经上皮的内源性EPO表达增强可通讨抑制casDase-3活性和抗凋亡作用发挥对光感受器细胞的保护作用。     

Neuroimaging of zinc released by depolarization of rat retinal cells

Zinc is associated with glutamatergic pathways in brain and retina, yet its role in neuromodulation remains unknown. High concentrations of reactive zinc in vertebrate photoreceptor terminals suggest a neuromodulatory role in the outer plexiform layer but zinc release has not been demonstrated. Using the membrane-impermeable form of the Zn(2+) sensitive fluorescent dye Newport Green, we have demonstrated increased release of Zn(2+) from the rat retina in response to potassium-induced depolarization of retinal cells. This increase was greatest in the outer retina with densest bands observed in the outer plexiform layer and photoreceptor inner segment regions of rat retinal slices.     

Measuring the cone contribution to the ERG b-wave to assess function and predict anatomical rescue in RCS rats

Subretinal injections of human retinal pigment epithelial (RPE) cells early in the course of retinal degeneration in Royal College of Surgeons (RCS) rats can rescue photoreceptors. Fourteen injected animals were studied using a double flash electroretinogram (ERG): 10 were examined longitudinally and four terminally with immunohistochemistry. The proportion of cone contribution to the ERG b-wave rather than the absolute size of isolated cone response proved to be a reliable indicator of function over time and a predictor of the proportion of cones identified anatomically in the area of optimal photoreceptor rescue.     

在体电穿孔辅助基因转染视网膜色素上皮细胞层和光感受器细胞层的实验研究

目的　探讨在体电穿孔辅助基因转染视网膜色素上皮（RPE）细胞层和光感受器（PR）细胞层的可行性。方法　147只健康雄性Spragne-Dawley(SD)大鼠根据电穿孔刺激模式电压值分为5、10、15、20、25、30、35 V组,右眼视网膜下腔注射增强型绿色荧光蛋白（EGFP）真核表达质粒pEGFP N1为实验眼,左眼注射TE Buffer 为对照眼。各组根据不同转染方向再分为RPE和PR两个亚组。各组除电压不同外,其余参数均为脉宽99 ms,脉冲间期0.5 s,连续5个脉冲。将带有负电荷的质粒在电场作用下,拟转染到RPE细胞层,反向置电极,拟转染到PR细胞层。转染后7 d 取各组视网膜铺片,荧光显微镜下观察EGFP表达强弱、范围及荧光细胞计数分析;采用蛋白质免疫印迹法（Western blot）、实时逆转录聚合酶链反应（RT-PCR）半定量观察EGFP蛋白和mRNA的表达。结果　转染后7 d,荧光显微镜观察发现,各组RPE亚组对照眼RPE细胞层内均未见特异性荧光表达,实验眼可见绿色荧光表达;各组RPE亚组对照眼视网膜铺片均未见荧光表达,实验眼视网膜铺片均可见转染了EGFP的RPE细胞。Western blot检测显示,随电压增加,EGFP蛋白与β-肌动蛋白条带灰度相对比值呈上升趋势。RT-PCR检测显示,各组均产生阳性扩增条带,随电压增高,EGFP mRNA与GADPH mRNA扩增条带灰度相对比值逐渐增高。结论　在体电穿孔方法可以有效辅助基因转染大鼠RPE细胞层。      

Origin of the vertebrate visual cycle: II. Visual cycle proteins are localized in whole brain including photoreceptor cells of a primitive chordate

The visual cycle system in a primitive chordate, ascidian Ciona intestinalis, was studied by whole-mount in situ hybridization and by whole-mount immunohistochemistry. Three visual cycle proteins, Ciona homologue of RGR (Ci-opsin3), CRALBP (Ci-CRALBP), and BCO/RPE65 (Ci-BCO/RPE65) were widely distributed in the brain vesicle and visceral ganglion. To identify the visual cycle system in a primitive chordate, we compared the localization of photoreceptor-specific proteins (visual pigment and arrestin) and visual cycle proteins (Ci-opsin3 and Ci-CRALBP). The ascidian visual cycle is composed of two cellular compartments, the photoreceptors and the brain vesicle, but some photoreceptor cells also contain visual cycle proteins.     

Cortical visual functions can be preserved by subretinal RPE cell grafting in RCS rats

Photoreceptor loss in the Royal College of Surgeons (RCS) rat is limited by transplantation of retinal pigment epithelial cell lines into the subretinal space prior to the onset of major photoreceptor loss. The purpose of this study was to examine to what extent visual cortical function was rescued by such transplantation and how the degree of rescue correlated with threshold responses recorded in the superior colliculus. To achieve this, single unit responses were recorded from the supragranular layers of cortical area, V1, at 7 months of age at a time when the cortex in these animals is normally non-responsive to specific visual stimulation. The best animals gave cortical responses that were very little different from normal. For the whole group studied, of the eight parameters measured for each cell, only three were significantly less well tuned than in normal non-dystrophic rats. In general, better single unit responses in the cortex were obtained with more photoreceptor rescue and this correlated with better threshold responses. These results indicate that discrete central visual responses can be preserved by subretinal transplantation of a cell line which limits chronic loss of input signal associated with progressive photoreceptor loss.     

不同激光强度经瞳孔温热疗法对色素兔视网膜细胞凋亡的影响

目的 观察不同激光能量的经瞳孔温热疗法（TTT）对色素兔视网膜的病理损伤及对细胞凋亡的影响。 方法 将14只有色家兔随机按不同激光能量分为空白组、50、70、90、110、130、150 mW组，每组2只兔4只眼行TTT治疗照射后,分别于24、48 h后取视网膜组织进行光学显微镜检查及用原位末端转移酶标记（TUNEL）技术和流式细胞仪检测细胞凋亡。 结果 直接检眼镜下激光反应斑颜色随能量增加由灰变白逐渐变浅，即由灰白——白——浓白，直径逐渐增大。苏木素 伊红（HE）染色：50~70 mW组光学显微镜下视网膜各层结构无明显改变；90~130 mW组视网膜结构完整,视锥、视杆细胞肿胀，内颗粒层可见少量的固缩核和细胞浆空泡化。以上能量组激光照射后24、48 h，光学显微镜下视网膜结构与空白对照组无明显差别。150 mW组激光照射后24 h视网膜组织各层肿胀、变性，感光细胞内外节部分缺失；而激光照射后48 h视网膜组织可见明显坏死，全层均可见细胞缺失。TUNEL检测结果显示，各能量组外颗粒层均可见阳性细胞，随激光能量的增加而增加，并逐渐累及内颗粒层和神经节细胞层。流式细胞仪检测结果显示，激光照射后24 h，各能量组均可见细胞凋亡峰。 结论 兔视网膜在能量为50~70 mW的TTT照射后，视网膜组织未见明显改变，但感光细胞凋亡显著增加。随着TTT激光能量增加，视网膜组织出现肿胀、变性甚至坏死。细胞凋亡逐渐累及内颗粒层和神经节细胞层。 (中华眼底病杂志, 2006, 22: 249-252)     

超声微泡造影剂介导脑源性神经营养因子联合转染视网膜和视皮质对视神经损伤后视网膜神经节细胞的保护作用

　　目的　观察超声微泡造影剂介导脑源性神经营养因子（BDNF）联合转染大鼠视网膜和视皮质区细胞对视神经损伤后视网膜神经节细胞（RGC）的保护作用。方法　雄性Sprague-Dawley（SD）大鼠88只随机分为正常组（A组）、假手术组（B组）、空白对照组（C组）、单纯眼转染组（D组）、单纯脑转染组（E组）、联合转染组（F组）;A组8只大鼠,B～F组每组16只大鼠。建立钳夹视神经损伤模型,将B～F组大鼠随机分为视神经损伤1、2周亚组,各亚组8只大鼠。B、C组玻璃体腔和视皮质区分别注射磷酸盐缓冲液（PBS）,D、E组玻璃体腔和视皮质区分别注射BDNF质粒（pBDNF）微泡造影剂悬液,F组玻璃体腔和视皮质区同时注射pBDNF微泡造影剂悬液。D～F组注射pBDNF微泡微泡造影剂悬液后,立即用超声辐照相应转染部位。视神经损伤后1、2周,各组行逆行荧光金标记RGC计数;半胱氨酸蛋白酶-3(caspase-3）蛋白免疫组织化学染色,观察其阳性表达情况;图形视网膜电流图（PERG）检测,记录N95振幅。结果　荧光金标记RGC结果显示,各组均可见金黄色荧光散布于视网膜定向铺片上。A~F组间RGC计数差异有统计学意义（F=256.30,P＜0.01）;B~F组视神经损伤1、2周亚组间RGC计数差异也有统计学意义（F=6518,P＜0.01）。光学显微镜观察发现,A、B组大鼠视网膜均未见caspase-3蛋白阳性表达;C~F组均可见主要位于神经节细胞层的caspase-3蛋白阳性表达。PERG检测发现,A~F组间N₉₅振幅差异有统计学意义（F=121.56,P＜0.01）;B~F组视神经损伤1、2周亚组间N95振幅差异也有统计学意义（F=8238,P＜0.01）。结论　超声微泡造影剂介导BDNF联合转染视网膜和视皮质区细胞能抑制视神经损伤后RGC凋亡,提高RGC存活数,保护其视功能。      

Evidence for an enduring ischaemic penumbra following central retinal artery occlusion,with implications for fibrinolytic therapy

The rationale behind hyperacute fibrinolytic therapy for cerebral and retinal arterial occlusion is to rescue ischaemic cells from irreversible damage through timely restitution of tissue perfusion. In cerebral stroke, an anoxic tissue compartment (the “infarct core”) is surrounded by a hypoxic compartment (the “ischaemic penumbra”). The latter comprises electrically-silent neurons that undergo delayed apoptotic cell death within 1–6 h unless salvaged by arterial recanalisation. Establishment of an equivalent hypoxic compartment within the inner retina following central retinal artery occlusion (CRAO) isn't widely acknowledged. During experimental CRAO, electroretinography reveals 3 oxygenation-based tissue compartments (anoxic, hypoxic and normoxic) that contribute 32%, 27% and 41% respectively to the pre-occlusion b-wave amplitude. Thus, once the anoxia survival time (≈2 h) expires, the contribution from the infarcted posterior retina is irreversibly extinguished, but electrical activity continues in the normoxic periphery. Inbetween these compartments, an annular hypoxic zone (the “penumbra obscura”) endures in a structurally-intact but functionally-impaired state until retinal reperfusion allows rapid recovery from electrical silence. Clinically, residual circulation of sufficient volume flow rate generates the heterogeneous fundus picture of “partial” CRAO. Persistent retinal venous hypoxaemia signifies maximal extraction of oxygen by an enduring “polar penumbra” that permeates or largely replaces the infarct core. On retinal reperfusion some days later, the retinal venous oxygen saturation reverts to normal and vision improves. Thus, penumbral inner retina, marginally oxygenated by the choroid or by residual circulation, isn't at risk of delayed apoptotic infarction (unlike hypoxic cerebral cortex). Emergency fibrinolytic intervention is inappropriate, therefore, once the duration of CRAO exceeds 2 h.