Basic research for clinical application of retina and optic nerve regeneration by transplantation

1)视网膜神经节细胞的变性过程：激光共聚焦扫描显微镜在一张图像上可以同时观察到位于不同焦平面上的树突和轴突，没有任何模糊。共聚焦激光扫描显微镜不需要对平板视网膜进行垂直切片，我们可以根据其重建的垂直图像清晰而容易地识别出合型细胞或非合型细胞。2)视网膜神经节细胞轴突再生：我们认为即使是成年的老龄视网膜神经节细胞在损伤后也具有轴突再生的能力，神经营养因子可能增强了这种能力。因此，神经营养因子在神经视网膜和视神经顽固性疾病的药物治疗中可能具有实际应用价值。3)可能移植病例的观察：使用扫描激光检眼镜（SLO）检查了一例Best’s黄斑营养不良患者的眼底。使用更多的氩蓝色激光，在中央凹处可以看到一个黑暗的区域。弱亮反射区与检眼镜观察到的炒蛋病变相对应。在氩绿激光下，在中央凹处观察到高度亮斑。后极炒蛋灶上方可见强烈的明亮反射。氦氖激光显示出高度明亮的斑块，与后极黄色沉积物的有序环状分布相对应。二极管激光显示，在炒蛋损伤处有暗斑和亮斑的斑驳外观。SLO在贝斯特黄斑黄斑营养不良的黄斑特殊受影响层提供了形态学增强。4)利用禽类胚胎再生视网膜的分析：鸟类松果体是一种光接受器官，被认为具有生物钟的功能。禽类松果体细胞是分泌初级光感受器，前人的研究表明，发育中的鹌鹑松果体有两种类型的光感受器，一种是视紫红质样免疫反应性光感受器，另一种是碘样免疫反应性光感受器。在体内和离体实验中，在鹌鹑松果体中发现的视紫红样免疫反应细胞比碘样免疫反应细胞数量多得多。本研究将胚胎鹌鹑的松果体细胞与视网膜细胞共培养，并用生物膜滤膜将二者分离，发现视网膜细胞所产生的扩散物质在体外强烈促进松果体碘样免疫反应细胞的出现。这种视网膜源性因子的作用是细胞类型特异性的，因为它对松果体视紫红质样免疫反应细胞的分化没有影响。视网膜细胞培养物比其他胚胎脑培养物（如脑细胞培养物）具有更强的碘素促进作用。视网膜因子的产生似乎是受发育调控的，因为来自较老胚胎（E13及以上）的视网膜细胞没有这种影响。这些因子可能通过刺激碘样免疫反应表型的表达而作用于松果体前体细胞。从培养的视网膜细胞中获得的条件培养基的初步表征表明，该因子是一种稳定的多肽，可能具有低分子量。松果体-视网膜培养系统为分析外界环境对细胞分化的影响提供了一个良好的实验系统。5)视网膜再生生理学：将成年蝾螈眼分离的视网膜细胞植入塑料培养皿中，培养皿表面涂有各种细胞粘附物质。在不含血清的Leibovitz’s L-15培养基中培养。与未涂布的培养皿相比，无论视网膜细胞类型如何，刀豆蛋白A （ConA）和聚l -赖氨酸（PLL）都能产生相对较好的细胞附着。另一方面，细胞外基质（ECM）成分如胶原蛋白、纤维连接蛋白和层粘连蛋白倾向于抑制细胞附着。在cona和pll包被的培养皿中，神经节细胞比其他视网膜细胞存活的时间更长，并且随着培养时间的推移，神经突也延长了。发现ConA对神经节细胞的神经突生长的促进程度远高于PLL。神经突分支在ConA上也出现得更频繁。从这些观察结果来看，ConA可能是研究体外视网膜再生的有用工具。少

1) Degeneration process of retinal ganglion cells : By means of confocal laser scanning microscope, both dendritic and axonal processes located in different focal planes can be observed at a single image without any blur. Without sectioning flat-mounted retinas vertically, confocal laser scanning microscope enabled us to identify either on-type cells or off-type cells clearly and easily on the basis of their reconstructed vertical image.2) Axonal regeneration of retinal ganglion cells : We concluded that even adult aged retinal ganglion cells had the ability of axonal regeneration after injury and that neurotrophic factors might enhanced these abilities. Therefore neurotrophic factors might have practicable appiocations in drug treatments for intractable disease of the neural retina and optic nerve.3) Observation of possible cases for transplantation : Using a scanning laser ophthalmoscope (SLO) , the ocular fundus of a patient with Best's vitelliform maculardystrophywas examined. With … More the argon blue laser, a dark area was seen in the fovea. Areas of weakly bright reflex were seen corresponding to the scrambled-egg lesion observed by ophthalmoscope. With the argon green laser, highly bright spots were observed at the foveola. Intense bright reflex was seen over the scrambled-egg lesion at theposterior pole. The helium-neon laser revealed highly bright patches corresponding to the orderly ring distribution of the yellowish deposits at the posterior pole. The diode laser revealed a mottled appearance with dark and bright patches in the scrambled-egg lesion. SLO provided a morphologic enhancement in the specifically affected layrs of the maculs in Best's vitelliform macular dystrophy.4) Analysis of retinal regeneration using avian embryo : The avian pineal is a photoreceptive organ and is believed to function as a circadian clock. Avian pineal cells are secretory rudimentary photoreceptors, and previous studies have demonstrated that there are two types of photoreceptors in developing quail pineals, one of which is rhodopsin-like immunoreactive and the other iodopsin-like immunoreactive. Much larger number of rhodopsin-like immunoreactive cells than of iodopsin-like immunoreactive cells were found in quail pineals, both in vivo and in vitro. In the present study we co-cultured pineal cells of embryonic quails with retinal cells but separated the two with a biomembrane filter We found that diffusible material produced by the cultured retinal cells intensely promotes the appearance of pineal iodopsin-like immunoreactive cells in vitro. This effect of retina-derived factors) is cell-type specific, since there is no effect on the differentiation of pineal rhodopsin-like immunoreactive cells. Retinal cell cultures had much more intensive iodopsin-promoting effect than other embryonic brain cultures such as cerebral cell cultures. The production of the retinal factors) seems to be developmentally regulated, since retiual cells from older embryos (E13 and older) did not have such effects. The factors) possibly act on pineal precursor cells by stimulating the expression of the iodopsin-like immunoreactive phenotype. Preliminary characterization of conditioned medium obtained from cultured retinal cells shows that the factor is a stable polypeptide, probably of low molecular weight. The pineal-retina culture system win provide a good experimental system to analyze the effect of extrinsic environments on cell differentiation.5) Physiology of the retinal regeneration : Retinal cells dissociated from adult newt eyes were seeded onto plastic dishes which were coated with various cell adhesion substances. They were cultured in Leibovitz's L-15 medium with no serum. Concanavalin A (ConA) and poly-L-lysine (PLL) caused relatively good cell attachment, regardless of retinal cell types, in comparison with uncoated dishes. On the other hand, extracellular matrix (ECM) components such as collagen, fibronectin and laminin tended to inhibit the cell attachment. In both ConA-and PLL-coated dishes, ganglion cells survived for longer periods than other retinal cells and extended neurites with time in culture. The outgrowth of neurites by ganglion cells was found to be promoted to a far greater degree by ConA than PLL.Branching of neurites also appeared to occur more frequently on ConA.From these observations, ConA may be an useful tool for investigating retinal regeneration in vitro. Less