鼠视网膜感光细胞移植术后超微结构观察

目的 以遗传性视细胞变性动物ＲＣＳ鼠为受体,Ｗｉｓｔａｒ鼠为供体,观察纯视锥、杆细胞移植术后ＲＣＳ鼠视网膜外层超微结构的变化。方法 应用视网膜板层切削技术或准分子激光技术制备供体层状视细胞;视网膜板层外路移植技术,获得视网膜移植动物模型。术后２周及４周时处死动物,眼球常规切片后于光镜及透射电镜下观察受体视网膜。结果 移植的视细胞大多成层排列于受体鼠的视网膜色素上皮层及内颗粒层之间。重新出现的外网状     

准分子激光切削视网膜制备感光细胞片层时存在的一些问题

目的：分析准分子激光切削视网膜制备感光细胞片层时存在的一些问题。方法：以新生小牛视网膜为研究对象,白明胶作载体,行视网膜预定位,液态视网膜膜片法辅片,准分子激光PTK切削方式。结果：发现准分子激光切削时,可以产生感光细胞片层的“锁边”现象;外颗粒层细胞间的离散现象;感光细胞层内细胞核的丢失;同一切削深度下不同植片切削深度不一和同一植片内切削浓度不均等现象。结论：准分子激光切削视网膜时存在的各种问题将会直接影响视网膜移植的手术效果,必须心早解决。     

准分子激光80μm深度切削制备感光细胞片层

目的 探讨PTK切削方式下，准分子激光切削视网膜制备理想感光细胞片层所需的最佳切削深度。方法 以新生小牛视网膜为研究对象，白明胶作载体，液态视网膜铺片法铺片，准分子激光PTK切削方式，共切削3种深度，制备48个感光细胞片层，行光镜和电镜观察。结果 用准分子激光PTK切削方式，80μm切削深度下可以获得较好的视网膜感光细胞片层，光镜和扫描电镜均显示完整的感光细胞节段、外颗粒层、外网状层以及部分残存的内颗粒层细胞，而50μm和100μm深度则分别较浅和较深。结论80μm是准分子激光PTK方式切削视网膜制备感光细胞片层的适宜切削深度。     

准分子激光切削视网膜板层移植术

目的 应用准分子激光切削视网膜获得纯视网膜光感受器层并制作视网膜板层移植的动物模型。 方法 准分子激光切削Wistar大鼠视网膜获得纯视网膜光感受器层,植于成年RCS(Royal College of Surgeons)大鼠视网膜下，术后1个月取材，切片，光镜下观察。 结果 准分子激光切削视网膜可获得均匀整齐的视网膜光感受器层，移植后1个月，光感受器层成活对位良好。 结论 准分子激光切削视网膜光感受器板层移植可获得具有较多移植细胞，并有良好解剖学对位的视网膜移植动物模型。 (中华眼底病杂志,1998,14:209-211)     

视细胞移植术后RCS鼠视网膜谷氨酸的分布

目的 通过纯视细胞移植,观察重建视网膜神经传导通路中兴奋性神经递质谷氨酸的分布和变化。方法取同龄Wistar鼠和皇家外科学院鼠(RCS)为供／受体,准分子激光切削法制备视细胞层,外路途径移入RCS鼠的视网膜下腔,术后两周取RCS鼠手术眼,手术对照眼,RCS对照眼,Wistar鼠眼。分别做冰冻切片,免疫组织化学染色法染色,普通光学显微镜下观察。结果在视细胞移植术后两周,移植视细胞存活,外丛状层重建,与手术对照眼、RCS对照眼相比,受体RCS鼠重建视网膜中在内丛状层和节细胞层Wistar鼠谷氨酸免疫反应阳性的神经纤维相对密度增多。结论视细胞移植不仅可以使RCS鼠视网膜重建正常的解剖结构,而且在移植后的视网膜中观察到兴奋性神经递质谷氨酸的分布接近正常。     

大鼠骨髓间充质干细胞视网膜下移植观察

目的：鉴定骨髓间充质干细胞（mesenchymal stem cells,MSCs)在体外不诱导的条件下视网膜下移植后的定位。方法：体外培养雄性大鼠MSCs，直接作为细胞供体视网膜下移植于成年雄性大鼠视网膜下，4周后将动物处死，取眼球做石蜡切片，用Y染色体鉴定，为做进一步验证，将MSCs用重组腺相关病毒AAV－gfp感染后移植，分别于4、8周取动物眼球作冰冻切片，于荧光显微镜下做绿色荧光蛋白（green fluorescence protein，GFP）表达观察。结果：培养的MSCs集落生长迅速，均一性好；Y染色体原位杂交鉴定表明来源于MSCs的阳性细胞融合入了原来的视网膜结构，在光学显微镜下可分布于视锥，视杆细胞层，双极细胞层及节细胞层；在荧光显微镜下可见GFP标记的阳性细胞存在，分布于视网膜色素上皮层、视锥，视杆细胞层，双极细胞层及节细胞层，细胞形态与结构同周围的视网膜相似；2种标记方法检测到的视网膜结构完整，未见到玫瑰花结样结构。结论：MSCs可在视网膜下移植后4周与原视网膜结构相融合，2种方法检测到的阳性细胞分布于视网膜色素上皮层，视细胞层，双极细胞层及节细胞层。     

明胶载体用于感光细胞片层制备存在的问题

目的：介绍明胶载体用于视网膜切削和制备感光细胞片层时存在的一些问题,方法：以新生小牛视网膜为研究对象,明胶作载体,常规视网膜铺片法铺片,准分子激光PTK切削方式,H－E染色和冰冻切片分析,结果;研究发现,在制备过程中可以出现感光细胞节段脱落,感光细胞核脱落和丢失,视网膜层间和层内分离以及冰冻切片时感光细胞破坏严重等现象,结论：改良明胶载体的供体形式和物理特性是未来视网膜移植 研究中的一个重要课题。     

视网膜定位联合液相铺片法用于感光细胞片层制备研究

制备高质量的感光细胞片层是取得良好视网膜移植手术效果的前提[1] 。为此 ,应首先做到以下几点 :(1)在准分子激光切削时 ,视网膜铺片必须平整 ;(2 )各视网膜植片切削深度应均一 ,即各植片在细胞成分构成上相同或接近 ;(3)激光切削要彻底 ,即整个感光细胞片层均能被全部切削。要达到以上要求 ,必须建立一套完整的视网膜定位、取材及铺片操作程序。笔者自行设计的视网膜定位联合液相铺片法技术 ,经实际应用效果满意。材料与方法一、材料1 视网膜来源 :采用北京市红星屠宰场新出生小牛视网膜。摘取小牛眼球置于冷生理盐水中保存 ,4ｈ内完成眼…     

兔胚胎全层视网膜移植至大鼠视网膜下的观察

视网膜色素变性(RP)是一组能导致进行性感光细胞变性和视觉障碍的遗传病症候群，目前尚无有效治疗方法。近年来研究表明视网膜移植是最接近临床应用治疗RP的方法。当RP进展到晚期，感光细胞和视网膜色素上皮(RPE)细胞都出现变性死亡，单独移植这两种成分都没有作用，此时病变视网膜的修复需要全层视网膜的移植。我们将青紫蓝兔胚胎全层视网膜移植到正常Wistar大鼠和视网膜变性RCS大鼠的视网膜下腔，观察移植物在宿主视网膜下腔的发育状况，从而探讨异种胚胎全层视网膜移植方法的可行性。     

视网膜感光细胞纯化方法的研究

目的　探讨改进获取视网膜纯感光细胞的方法。方法　应用改良的准分子激光切削法和酶分层消化法来分离纯化视网膜感光细胞层。结果　由两种方法获取的视网膜组织片经苏木素 -伊红染色均证实为单一的纯感光细胞层并保持生物活性。结论　改良的准分子激光切削法和酶分层消化法均能获取视网膜纯感光细胞 ,且对细胞损伤小 ,内外节段保持完好 ,可为视网膜的移植创造条件。     

Transplantation of full-thickness retina in the normal porcine eye: surgical and morphologic aspects

PURPOSE: To report a surgical technique for transplantation of full-thickness neuroretinal sheets into the subretinal space of a large animal with a vascularized retina and to establish the light microscopic morphology of such specimens. METHODS: Twelve normal pigs underwent transplantation of a neuroretinal sheet from a neonatal donor into the subretinal space by means of a vitrectomy-based technique. After a survival of 33 to 72 days, eye specimens were studied with a light microscope. RESULTS: In most eyes, the transplants displayed a laminated morphology, with photoreceptor outer segments facing the host retinal pigment epithelium. These grafts had normal outer retinal layers, while the inner layers were less developed. The host retina straddling the graft showed evidence of photoreceptor degeneration, but the inner layers were well preserved. CONCLUSION: Full-thickness neuroretinal sheets can be transplanted to the subretinal space of a large animal eye with a vascularized retina. The grafts survive well and display mostly photoreceptors, which in combination with the well-preserved host inner retina may be of importance in attempts at reconstructing the retina in photoreceptor degenerative disease.     

Transplanted sheets of human retina and retinal pigment epithelium develop normally in nude rats

This study investigated whether transplanted sheets of human fetal retina together with its retinal pigment epithelium (RPE) could develop and maintain their cytoarchitecture after long survival times. Transplant recipients were nine albino athymic nu/nu rats with a normal retina. The donor tissue was dissected from fetuses of 12-17 weeks gestational age. Transplants were analyzed at 5-12 months after surgery by light and electron microscopy, and immunohistochemistry with various antibodies specific for rhodopsin, S-antigen, transducin, neurofilament and synaptophysin. In 4 of 11 transplants, the RPE stayed as a monolayer sheet and supported the development of the retinal sheet with a normal lamination, including photoreceptor inner and outer segments. Cones and rods in the organized transplants were labeled with different photoreceptor markers. Inner and outer plexiform layers, containing cone pedicles and rods spherules, were immunoreactive for synaptophysin. As the recipients had a normal retina, transplant/host integration was not expected. However, at the transplant/host interface, there were sometimes areas without glial barriers, and neurofilament-containing processes could be observed crossing between transplant and host. In other, more disorganized transplants, the RPE cells were partially dispersed or clumped together in clusters. Such transplants developed photoreceptors in rosettes, often with inner and outer segments.In conclusion, sheets of human fetal retina transplanted together with its RPE to the subretinal space of nude rats can develop and maintain perfectly laminated transplants after long survival times, indicating the potential of applying cotransplantation to human patients with retinal diseases.     

Photoreceptor allografts in a feline model of retinal degeneration

· Background: Photoreceptor transplants provide a potential means to restore function in a degenerate retina and/or rescue degenerating host photoreceptors by trophic influences. We have examined photoreceptor allografts in the Abyssinian cat model of hereditary photoreceptor degeneration to determine the viability and influence of such transplants on the host retina. · Methods: Small pieces of 3- to 5-day-old normal kitten retina containing undifferentiated photoreceptors were injected into the subretinal space of adult Abyssinian cats at an early stage of retinal degeneration using standard vitreo-retinal surgical techniques. The retinas were examined by ophthalmoscopy and fundus photography, then by light and electron microscopy at different times after surgery. · Results: Such allografts survive for at least 6 months after surgery. The photoreceptors develop outer segments, invariably in rosettes. The transplants gradually integrate with the host retina but detach the host photoreceptor layer from the retinal pigment epithelium (RPE), which tends to reduce the number of host photoreceptors over the transplant. There is no slowing of the photoreceptor degeneration in neighboring non-detached retina. Inflammation or rejection was not detected. · Conclusion: Undifferentiated, neonatal photoreceptor allografts survive and develop outer segments in the subretinal space of the Abyssinian mutant feline retina. The allografts gradually integrate with the host neural retina without inducing rejection. In the vicinity of the transplant there is increased loss of host photoreceptors, considered to be due to their detachment from the RPE layer. There is no evidence of any rescue of host photoreceptors elsewhere in this mutant retina. Received: 10 November 1997 Revised version received: 19 March 1998 Accepted: 23 March 1998     

Synaptic plasticity in mammalian photoreceptors prepared as sheets for retinal transplantation

PURPOSE: To investigate systematically the early morphologic changes in axon terminals of adult mammalian rod and cone photoreceptors prepared as a sheet for subretinal transplantation. METHODS: An in vitro system was designed to maintain adult porcine retinas for up to 48 hours. Photoreceptor sheets, prepared by vibratome sectioning, and full-thickness retinas were cultured at temperatures similar to those in pretransplantation storage (4 degrees C) and after transplantation (37 degrees C). Changes in the outer nuclear and outer plexiform layers were analyzed, using immunohistochemistry, laser scanning confocal microscopy, and image analysis. RESULTS: Morphologic changes were observed in photoreceptor sheets as early as 10 minutes after incubation. The most significant change was the retraction of photoreceptor axons and terminals toward their cell bodies. Retraction was temperature dependent, being exacerbated at 37 degrees C compared with 4 degrees C, at its maximum by 24 hours of culture, and present in sheets obtained from both superior and inferior retina. The cause of this movement was not preparation techniques associated with vibratome sectioning or gelatin removal. Retraction was also present in full-thickness neural retina incubated at 37 degrees C. Reduction in outer nuclear layer cell counts and thickness were also evident in these preparations, primarily in photoreceptor sheets. CONCLUSIONS: Adult photoreceptor sheets, a potential graft preparation for retinal transplantation, show a rapid retraction of axon terminals toward the cell bodies during culture. Although retraction may impede synaptic integration after transplantation, this intrinsic plasticity could be redirected to stimulate graft-host interaction.     

Structure and function of embryonic rat retinal sheet transplants

PURPOSE: To evaluate retinal sheet transplants in S334ter-line-3 retinal degenerate rats by comparing visual responses recorded electrophysiologically with morphology based on light and electron microscopy. METHODS: S334ter-line-3 retinal degenerate rats (n = 7) received retinal sheet transplants between postnatal days 28 and 31. The donor tissue was derived from transgenic embryonic day 19 (E19) rat retinae expressing human placental alkaline phosphatase (hPAP). Fresh retinal sheets were gently transplanted into the subretinal space of the left eye with the help of a custom-made implantation tool. Selected rats (n = 5) were subjected to electrophysiologic evaluation of visual responses from the superior colliculus about 84-121 days after surgery. Transplanted eyes were processed for light microscopy (LM) and electron microscopy (EM) evaluations. RESULTS: All the transplanted rats that were evaluated for visual responses in the brain showed responses to very low light stimulation (-3.42 to -2.8 log cd/m(2)) of the eye in a small area of the superior colliculus corresponding with the placement of the transplant in the host retina. Histologic evaluation showed that most of the transplants contained well-laminated areas with correct polarity in the subretinal space. Inside the transplant areas, rosettes of photoreceptors with inner and outer segments were found. In the laminated areas, the outer segments of photoreceptors were facing the host retinal pigment epithelium (RPE). Immunohistochemical evaluation of hPAP donor cells revealed areas with specific staining of the transplants in the subretinal space. Electron microscopic evaluation showed a glial demarcation membrane between the host and the transplant, however, processes originating from the transplant were observed inside the host retina. CONCLUSIONS: Sheets of E19 rat retina transplanted into the subretinal space of S334ter-line-3 rats survived without immune rejection and continued to show visual function when tested after 3 months. Well-developed photoreceptors and many synapse types were seen within the transplants. hPAP staining showed a certain degree of integration between the host retina and the transplant suggesting that transplanted photoreceptors contributed to the restored light sensitivity.     

Transplantation of retinal pigment epithelial cells to immature and adult rat hosts: short- and long-term survival characteristics

Retinal pigment epithelial (RPE) cells isolated from 6-8-day-old pigmented Long-Evans rat eyes were successfully grafted onto Bruch's membrane in albino Sprague-Dawley hosts ranging in age from 10 days postnatal to young adulthood. Injections of RPE cells were made into the subretinal space using a lesion paradigm which penetrates through the dorsal surface of the eye cutting through the sclera and choroid. Suspensions of pigmented RPE cells were labeled with Nuclear Yellow prior to transplantation, and at 1 week after grafting, the transplanted RPE cells were found attached to previously denuded areas of Bruch's membrane. The grafted RPE cells were positively identified by double labeling-only those RPE cells with melanosomes in their cytoplasm showed fluorescent Nuclear Yellow labeling; host albino RPE cells showed no nuclear labeling. The grafted RPE cells developed a normal relationship with photoreceptor cell outer segments as seen by electron microscopy. When compared with intact retinas or host control areas there were no significant differences in the thickness of the outer nuclear layer or in the lengths of photoreceptor inner and outer segments underneath the grafted RPE cells for at least 3 months after transplantation, which was the longest survival time examined.