Trophic factors GDNF and BDNF improve function of retinal sheet transplants

The aim of this study was to compare glial-derived neurotrophic factor (GDNF) treatment with brain-derived neurotrophic factor (BDNF) treatment of retinal transplants on restoration of visual responses in the superior colliculus (SC) of the S334ter line 3 rat model of rapid retinal degeneration (RD). RD rats (age 4-6 weeks) received subretinal transplants of intact sheets of fetal retina expressing the marker human placental alkaline phosphatase (hPAP). Experimental groups included: (1) untreated retinal sheet transplants, (2) GDNF-treated transplants, (3) BDNF-treated transplants, (4) none surgical, age-matched RD rats, (5) sham surgery RD controls, (6) progenitor cortex transplant RD controls, and (7) normal pigmented rat controls. At 2-8 months after transplantation, multi-unit visual responses were recorded from the SC using a 40 ms full-field stimulus (−5.9 to +1 log cd/m²) after overnight dark-adaptation. Responses were analyzed for light thresholds, spike counts, response latencies, and location within the SC. Transplants were grouped into laminated or rosetted (more disorganized) transplants based on histological analysis. Visual stimulation of control RD rats evoked no responses. In RD rats with retinal transplants, a small area of the SC corresponding to the position of the transplant in the host retina, responded to light stimulation between −4.5 and −0.08 log cd/m², whereas the light threshold of normal rats was at or below −5 log cd/m² all over the SC. Overall, responses in the SC in rats with laminated transplants had lower response thresholds and were distributed over a wider area than rats with rosetted transplants. BDNF treatment improved responses (spike counts, light thresholds and responsive areas) of rats with laminated transplants whereas GDNF treatment improved responses from rats with both laminated and rosetted (more disorganized) transplants. In conclusion, treatment of retinal transplants with GDNF and BDNF improved the restoration of visual responses in RD rats; and GDNF appears to exert greater overall restoration than BDNF.     

Cell replacement and visual restoration by retinal sheet transplants

Retinal diseases such as age-related macular degeneration (ARMD) and retinitis pigmentosa (RP) affect millions of people. Replacing lost cells with new cells that connect with the still functional part of the host retina might repair a degenerating retina and restore eyesight to an unknown extent. A unique model, subretinal transplantation of freshly dissected sheets of fetal-derived retinal progenitor cells, combined with its retinal pigment epithelium (RPE), has demonstrated successful results in both animals and humans. Most other approaches are restricted to rescue endogenous retinal cells of the recipient in earlier disease stages by a ‘nursing’ role of the implanted cells and are not aimed at neural retinal cell replacement. Sheet transplants restore lost visual responses in several retinal degeneration models in the superior colliculus (SC) corresponding to the location of the transplant in the retina. They do not simply preserve visual performance – they increase visual responsiveness to light. Restoration of visual responses in the SC can be directly traced to neural cells in the transplant, demonstrating that synaptic connections between transplant and host contribute to the visual improvement. Transplant processes invade the inner plexiform layer of the host retina and form synapses with presumable host cells. In a Phase II trial of RP and ARMD patients, transplants of retina together with its RPE improved visual acuity.In summary, retinal progenitor sheet transplantation provides an excellent model to answer questions about how to repair and restore function of a degenerating retina. Supply of fetal donor tissue will always be limited but the model can set a standard and provide an informative base for optimal cell replacement therapies such as embryonic stem cell (ESC)-derived therapy.     

MHC expression in fragment and full-thickness allogeneic embryonic retinal transplants

Background: The study was carried out to evaluate the expression of major histocompatibility complex (MHC) molecules in retinal transplants with different tissue integrity. Methods: Twelve adult rabbits received an allogeneic subretinal neuroretinal transplant, in the form of either fragmented embryonic cells or a complete full-thickness embryonic retina. A controlled transvitreal approach was used for both transplantation types. The grafts were examined histologically after 31 or 49 days with hematoxylin and eosin staining and immunohistochemical analysis of MHC class I and class II expression. Results: All five fragment transplants developed into rosettes. Two of them displayed MHC class I-labeled cells, and four MHC class II-labeled cells. The cells were concentrated on the scleral side of the graft, and there was also a marked increase of labeled cells in the choroid. MHC labeling was often associated with defects in the retinal pigment epithelium. Six of the seven full-thickness grafts displayed a laminated morphology with well-developed retinal layers. The seventh consisted of rosettes. None of these grafts displayed MHC class I- or class II-labeled cells. Conclusions: The findings suggest that host immune response against fragmented and intact neuroretinal grafts is different, indicating tissue integrity as one factor affecting graft-host immune interactions. The absence of immune response in full-thickness grafts is encouraging and important in the struggle to find therapies for retinal degenerative disease. Received: 17 November 1999 Revised: 6 January 2000 Accepted: 18 January 2000     

Short-term study of retinal pigment epithelium sheet transplants onto Bruch's membrane

The purpose of this study is to investigate the survival and behaviour of retinal pigment epithelium sheets transplanted onto hydraulically debrided Bruch's membrane. Uncultured retinal pigment epithelium sheets obtained from male cats and sandwiched between two gelatin sheets were transplanted onto the tapetal area of female cats after native retinal pigment epithelium was debrided. For controls, the gelatin carrier was transplanted after debridement. Each transplant or control specimen was analyzed histologically and immunohistochemically. Transplanted male retinal pigment epithelial cells were identified by in situ labelling of the cat Y chromosome. Over half of the transplants appeared as retinal pigment epithelium multilayers in the subretinal space. Retinal pigment epithelium pigment dispersion into the subretinal space was seen in most of the transplants, and retinal pigment epithelium pigment infiltration into the neural retina was seen in all 7-day survival transplants. A few condensed darkly stained retinal pigment epithelium nuclei and Terminal Transferase dUTP Nick End Labelling-positive retinal pigment epithelium cells were observed in all transplants. Cellular retinaldehyde-binding protein was present up to day-7 in most transplanted RPE cells. In both transplant and control specimens, the antibody against the Ki-67 nuclear antigen labelled a few retinal pigment epithelium cells at day-3. Terminal Transferase dUTP Nick End Labelling-positive outer nuclear layer nuclei were most frequently observed at day-1 but were much less frequent at day-3 in both transplants and controls. The survival and effectiveness of retinal pigment epithelium sheet transplants appeared similar to the retinal pigment epithelium microaggregates transplants conducted previously in this model.     

Intraocular retinal transplants

Embryonic rat retinae transplanted into the anterior chamber of adult rat eyes of the same or different strain survive and grow. Light and electron microscopic studies show that the transplants undergo histogenetic differentiation, resulting in the development of mature inner and outer layer neurons and Müller glial cells. Vascular connections develop between the host iris and the retinal transplant. These initial observations indicate that retinal transplantation to a recipient eye is a procedure which offers ample opportunities for the study of problems related to neural development, retinal plasticity and repair.     

大鼠胚胎视网膜干细胞的分离培养

目的分离培养大鼠胚胎视网膜干细胞。方法从胎龄第16d的SD大鼠视网膜神经上皮分离视网膜细胞并进行悬浮培养,观察细胞增殖以及自发分化情况,采用免疫细胞化学方法检测Nestin、β-Tubulin、GFAP和Recoverin的表达。结果原代细胞可形成悬浮生长的神经球,传代后能形成新的细胞球。原代及传代细胞大部分表达神经干细胞标记物Nestin,分化后的细胞部分表达神经元标记物β-Tubulin或神经胶质细胞标记物GFAP,少数细胞表达光感受器细胞标记物Recoverin。结论分离培养的SD胚鼠视网膜神经干细胞可以在体外扩增并具有多分化潜能。     

Adult rabbit retinal transplants

PURPOSE: To study the survival of adult retinal grafts prepared in a physiologically optimized way. METHODS: Twenty-three rabbits received an adult full-thickness rabbit retinal transplant positioned under the host retina, using a vitrectomy technique. The transplants were prepared using a procedure based on a previously described in vitro model used for physiological experiments on the adult retina. Five rabbits received a fragmented graft. All grafts were prelabeled with 4',6-diaminidin-2-phenylindoldihydrochloride (DAPI) to allow identification. The eyes were examined by light and fluorescence microscopy 6 to 174 days after surgery. To assess the amount of cell death in the graft before actual transplantation, in vitro experiments were performed. The extent of cell death in retinas prepared by the optimized protocol was examined and compared with a simpler preparation previously used successfully for embryonic grafts. The amount of cell death in the in vitro experiments was evaluated using a fluorescent green nucleic acid stain that penetrates dying cells. RESULTS: In 21 of the 23 animals that received full-thickness grafts prepared in an optimized way, the transplant survived. Sixteen grafts, including all four with a 174-day survival time, displayed normal morphology, with all retinal layers preserved. The fragmented grafts survived poorly. The in vitro experiments showed minimal cell death in retinas prepared according to the optimized protocol, whereas control retinas displayed extensive cell death after 5 hours. CONCLUSIONS: The results showed that it is possible to transplant adult retina in the rabbit and that the grafts survive well if they are prepared under physiologically optimized conditions and the integrity of the grafted tissue is kept intact.     

MHC expression in syngeneic and allogeneic retinal cell transplants in the rat

· Background: The major histocompatibility complexes, MHC class I and II, are found only sparsely or not at all in the retina. Since the eye is immunoprivileged, we decided to investigate how the MHC class I and II antigens were influenced by a retinal transplant and whether this could be correlated to rejection of the transplant. · Methods: Fetal neural retinas of Sprague-Dawley (SD) rats were implanted in the subretinal space of adult Lewis and SD rats. After 5 weeks the retinas and the transplants were evaluated with antibodies against MHC class I and II antigens as well as microglia. · Results: In the syngeneic transplants no upregulation of MHC class I antigen was seen and no MHC class II-positive cells could be detected. In the allogeneic transplants, on the other hand, there was marked upregulation of MHC class I antigen. Numerous MHC class II antigen-positive cells were seen in the subretinal transplant but also in the host retina. · Conclusion: Allogeneic retinal transplants seem to grow and thrive just as well as syngeneic transplants, but in the former there is considerable upregulation of MHC expression. Our interpretation of these results is that the allogeneic transplants are recognized as non-self, but that there is also something that modifies this reaction of the immune system at this level, preventing the rejection that would normally ensue. Received: 8 September 1997 Revised version received: 16 February 1998 Accepted: 27 February 1998     

RPE transplants stabilize retinal vasculature and prevent neovascularization in the RCS rat.

Previous reports indicate that in the Royal College of Surgeons (RCS) rat a decline in the retinal vessel density accompanies the loss of the normal architecture of the deep bed. This begins at about three months with neovascularization that originates in the deep vessel bed and develops in the direction of the retinal pigment epithelial (RPE) cells by four months. A surgical technique has been developed recently for the transplantation of healthy RPE cells into the subretinal space of the RCS rat, resulting in the rescue of photoreceptor cells. This permits evaluation of the possibility that such transplants also protect the retinal vessels. We report for the first time: (1) the stabilization of the normal retinal vasculature by maintenance of the density and architecture of the deep vessel bed; and (2) prevention of neovascularization of the RPE by the surgical transplantation of healthy RPE cells into the subretinal space of the RCS rat. More specifically, we show a maintenance of the deep vessel bed density under the transplant in contrast to a significant reduction in the vessel density that had taken place in corresponding areas in nongrafted and sham injected controls at four months of age. The vessel density in the transplanted group is statistically different from the nongrafted and the sham injected groups. We also report a significant decline in the number of neovascularization profiles around the transplant site of the RPE-grafted RCS retina. We also note that the pathological changes in the vasculature of the RCS rat occur in a predictable central to peripheral gradient.     

Transplantation of neuroblastic progenitor cells as a sheet preserves and restores retinal function

Diseases affecting the outer retina are incurable once photoreceptors are lost, and these diseases usually cause retinal pigment epithelium (RPE) dysfunction. However, the inner retina can remain functional for some time, even though retinal remodeling occurs as compensation for photoreceptor loss. If the damaged part can be replaced with neuroblastic progenitor and RPE cells as sheets with a beneficial effect on function, vision loss may be prevented and vision may be restored. This review presents an overview of the research of transplanting sheets of neural retina, with or without its RPE, to the subretinal space. In different animal models of retinal degeneration, retinal transplants can morphologically reconstruct a damaged retina, and restore visual sensitivity. Good morphological integration of transplants with the host retina can occur, whereas other transplants exhibit a glial barrier. Synaptic connections between transplant and host have been indicated by transsynaptic tracing. Retinal transplants can restore and preserve visual responses in a small area of the superior colliculus corresponding to the placement of the transplant in the retina. The beneficial effect of retinal transplantation likely involves two mechanisms: trophic effects, e.g., rescue of host cones; and synaptic connectivity between transplant and host retina.     

Transplantation of rat embryonic stem cell-derived retinal cells restores visual function in the Royal College of Surgeons rats

Aim of study

To evaluate the feasibility of transplantation of embryonic stem cell (ESC)-derived retinal cells in the treatment of retinal degeneration.

Materials and methods

Rat ESCs were isolated and induced into retinal progenitor cells (RPCs) in vitro, which were subsequently induced into retinal pigment epithelium cells (RPEs) and photoreceptors (PRCs). All cells were identified by Western blot detection of their specific markers. RPEs and PRCs were, respectively, injected into the retina of Royal College of Surgeons (RCSs) rats. Control group was injected with PBS. Post-transplantation visual function was determined by electroretinography (ERG). The histology of the whole eye was compared by H&E staining.

Results

RPEs and PRCs were successfully derived from rat ESCs through the two-step differentiation as indicated by the presence of ESC- (Oct-3/4, Nanog, TRA-1-60 and TRA-1-81), RPC- (Rx, Mitf, Pax6 and Chx10), RPE- (RPE65 and keratin) and PRC-specific markers (blue opsin, red/green opsin, recoverin and rhodopsin) in Western blot. The amplitude of ERG a- and b-wave in RPE- and PRC-transplanted groups at week 2 and 10 after transplantation was markedly higher compared with PBS controls. Retinal injury and vascular stress response was not detected in any of the RCS rats after transplantation.

Conclusion

The developed stepwise protocol can derive retinal cells from ESCs. Transplantation of these retinal cells can restore visual function of RCS rats. Our study provides evidence for potential clinical application of ESC-based cell therapy for retinal degeneration.

     

Photoreceptor function of retinal transplants implicated by light-dark shift of S-antigen and rod transducin.

The aim was to demonstrate functional properties of transplanted histologically normal photoreceptors. Subretinal intact-sheet transplants of fetal E17-E20 rat retinas to light-damaged albino rat eyes were fixed in light or dark, 2 to 42 weeks after transplantation, and stained immunohistochemically for certain phototransduction proteins. In light adapted transplants, transducin was predominantly found in inner segments of parallel-organized photoreceptors. Transducin shifted to the outer segments with dark-adaptation. S-antigen distribution was opposite to transducin. Rhodopsin distribution did not change. The shift of signal transduction proteins correlated to the light conditions indicates that normal phototransduction processes were established in photoreceptors of transplanted retinal sheets.     

Survival of long-term retinal cell transplants

PURPOSE: If retinal cell transplants are to be used in the management of retinal degeneration, they will need to survive in the eye for long time. This study assesses the fate of neural retinal transplants in the eye after long survival times. METHODS: Fragmented pieces of neural retinas from embryonic day 15 rabbits were transplanted to the subretinal space of adult animals of the same strain. The transplants were allowed to survive for up to 583 days prior to sacrifice and light microscopic examination. RESULTS: In most grafts, both neural and glial cells survived, however, some of the grafts seemed to contain predominantly glial cells. Only when the photoreceptors in the graft were apposed to the host pigmented epithelium did they survive. Otherwise, the neuronal components of the grafts were largely cells typically found in the inner retina. The long-term transplants were much thinner than short-term ones. Even after long survival times, the transplants lacked vascularization. No inflammatory cells were seen in or around the grafts. CONCLUSION: The results suggest that the cells of the inner retina survive for long periods after transplantation, but the photoreceptor cells seem to need the support of the host pigmented epithelium for long-term survival. It may therefore be important to have a transplantation technique where the graft photoreceptors can be placed in apposition with the host pigment epithelium.     

CuZn superoxide dismutase transgenic retinal transplants

· Background: The morphology of retinal transplants is believed to depend on the extent of mechanical disruption of the donor tissue during the surgical procedure and on local factors of the host environment. We hypothesized that oxidative stress during donor tissue preparation and implantation further affects transplant development and investigated the effects of CuZn superoxide dismutase (SOD) overexpression on the survival and morphological development of mouse embryonic retinal transplants. · Methods: Retinae and livers from embryonic day 14–15 SOD overexpressing transgenic mice and CBA control mice were harvested under sterile conditions. In order to identify transgenic mouse embryos, the embryonic livers were analyzed via nondenaturing gel-electrophoresis for the presence of the human SOD protein. Neural retinae were transplanted as fragmented tissue into the subretinal space of albino BALB/c mice. At 4–8 weeks following transplantation, the grafted eyes were fixed in Bouin’s solution and processed for histological analysis. · Results: Both SOD transgenic and control retinal transplants had developed all retinal layers except for a ganglion cell layer and exhibited a similar extent of rosette formation. Computer-assisted, quantitative assessment of retinal graft volumes revealed a significant, around 58% increase in size of SOD transgenic transplants compared with controls. · Conclusions: Enhanced intracellular SOD levels do not seem to influence retinal transplant morphology as detected by light microscopy. However, volumes of the SOD trangenic transplants were found to be increased compared to control grafts. Received: 9 May 1997 Revised version received: 13 May 1998 Accepted: 17 June 1998     

Full-thickness retinal transplants: a review

Embryonic full-thickness rabbit neuroretinal sheets were transplanted to the subretinal space of adult hosts. This was accomplished by using a new transplantation technique involving vitrectomy and retinotomy. The grafts were followed from 10 to 306 days after surgery and were then examined by different histological techniques. In the light microscope, the transplants were seen to develop the normal retinal lamination and fusion with the host retina, especially after long survival times. Ultrastructurally, normal photoreceptor outer segments, well integrated with the host retinal pigment epithelium, were found. Growth cones were present in the zone of fusion between graft and host retina. Immunohistochemical labeling revealed many of the normal retinal components not previously found in retinal transplants, and graft-host connections between neurons in the rod pathway were seen. The morphology of vibratome-sectioned neuroretinal sheets as well as adult full-thickness grafts was also examined. These transplantation types showed less of the normal morphology compared with embryonic full-thickness grafts. The immunogenicity of embryonic full-thickness and fragmented grafts was compared using major histocompatibility complex immunolabeling. Fragmented grafts elicited a response from the host immune system similar to a chronic transplant rejection. This reaction was absent in the full-thickness grafts which is in accordance with their good long-term survival.     

Correlation of retinal function with retinal histopathology following ischemia-reperfusion in rat eyes

PURPOSE: To investigate the relationship between retinal function and histopathology following retinal ischemia-reperfusion in rats. METHODS: Retinal ischemia was induced in 27 Sprague-Dawley rats by raising the intraocular pressure for 60- or 90-minutes. Two weeks after the retinal ischemia-reperfusion, ganzfeld stimuli of different luminances (maximum 0.51 log cd-s/m2) were used to elicit full-field ERGs from the right (experimental) and left (control) eyes. The amplitudes of the scotopic b-waves were measured, and intensity-response curves were plotted. The b-wave amplitudes at the initial peak (VbIP), the dip (Vb(dip)), and the maximum stimulus intensity (Vb(max)) were measured. The animals were sacrificed after the ERG recordings to determine the surviving retinal cells in the inner nuclear layer (INL). RESULTS: The log values of the VbIP, Vb(dip) and Vb(max) were linearly correlated with the number of surviving cells in the INL (r = 0.928, 0.905, and 0.913 for VbIP, Vb(dip) and Vb(max), respectively; p < 0.0001 for each). The slope of the regression line for the Vb(dip) was significantly steeper than that for the VbIP and Vb(max) (p < 0.005). The regression line for the Vb(dip) reached undetectable level of the b-wave (<1 microV) even though approximately 25% of the cells still remained in the INL. CONCLUSIONS: The amplitude of the scotopic b-wave is a sensitive index of the surviving INL cells. However, if one selects the stimulus intensity at the dip, the functional loss would be exaggerated in spite of the presence of cells in the INL.     

Development of glycine-accumulating neurons in retinal transplants

Previous studies have shown that the fetal retina not only survives transplantation but also continues to develop and differentiate in the host eye. Several structural and functional proteins have been demonstrated in the transplanted retinas, and the presence of such proteins has been taken as evidence for the capability of retinal transplants to function. Glycine is an important inhibitory neurotransmitter and is found in a large number of the retinal neurons. Uptake of glycine rather than de novo synthesis is the main source of glycine in glycinergic neurons. The present study examined whether glycine-accumulating neurons develop normally in rabbit retina transplants. Embryonic day (E) 15 rabbit retinas were transplanted into the eyes of adult rabbits of the same strain. Transplants were allowed to survive for various times so that the grafts attained the equivalent ages of (donor age + survival time) E 19, 22 and 29 and postnatal days (PN) 2, 5, 9, 12, 19 and 58. On formaldehyde-fixed cryostat sections of these transplants, glycine-accumulating neurons were demonstrated by immunohistochemistry by using an antibody against one of the glycine transporters: GLYT1. Immunoreactivity was first detected 2 days before birth and increased with age until it reached its mature level at PN 19. The immunoreactivity was found in cells belonging to the inner retinal layers, and in plexiform layers of the transplant equivalent to the normal inner and the outer plexiform layers. In places these cells integrated well with similar cells in the host. In the host retina, the immunoreactivity was found in proximal cell layers of the inner nuclear layer, in certain bipolar cells, and in the inner and the outer plexiform layers. The immunoreactivity was preserved even in the degenerated retina overlying the retinal graft. In conclusion, the present study demonstrates that glycine-accumulating neurons develop, integrate and survive in retinal transplants.     

Progress in retinal sheet transplantation

The aim of retinal transplantation is to prevent blindness and to restore eyesight, i.e. to rescue photoreceptors or to replace damaged photoreceptors with the hope of re-establishing neural circuitry. A promising experimental paradigm is the sub-retinal transplantation of sheets of fetal retina, with or without its attached retinal pigment epithelium (RPE), into recipient rats with retinal degeneration. Sheets of fetal retina have already developed their primordial circuitry. Such transplants can develop lamination resembling a normal retina dependent on the presence of healthy RPE either from the host or from the graft. In several retinal degeneration models, transplants have been shown to restore visually evoked responses in an area of the superior colliculus corresponding to the placement of the transplant in the retina. The functional effect of transplants may be due to transplant/host connectivity and/or rescue of host photoreceptors. In summary, sheets of fetal retina can morphologically repair an area of a degenerated retina, and there is evidence to suggest that transplants form synaptic connections with the host and restore visual responses in rats with retinal degeneration.     

胚胎和生后大鼠视网膜干细胞在体内和体外的分化差异

目的 研究胚胎和生后大鼠视网膜干细胞（RPCs）在体内和体外的分化.方法 取胚胎18 d和生后2周SD大鼠视网膜,分别用悬浮法及贴壁法培养.传至第1代后转入分别含10 ng/ml睫状神经营养因子（CNTF）、20 ng/ml表皮生长因子（EGF）、10 ng/ml碱性成纤维细胞生长因子（bFGF）、10%视黄酸、10%胎牛血清的DMEM：F12中进行诱导分化,贴壁后2、3、5、6 d的细胞及胚胎18 d,生后1、3、5、7、14 d的大鼠视网膜切片分别行nestin、vimentin、opsin及GFAP的二步法免疫组织化学染色.结果 大鼠的RPCs在悬浮时呈球状生长,nestin染色阳性,悬浮培养传至第1代,贴壁培养传至第5代.CNTF、bFGF、EGF及视黄酸组在培养至6 d时可呈神经元样细胞改变,有轴突,但持续出现nestin阳性.生后2周的RPCs无法诱导成opsin阳性细胞.结论 RPCs可在体外培养并传代,其在分化过程中形态学的改变先于功能学变化.光感受器细胞的体外诱导较困难.     

GDNF家族成员artemin在大鼠视网膜神经节细胞中的表达及其功能

目的观察胶质细胞系源性神经营养因子（GDNF）家族成员artemin在正常大鼠视网膜神经节细胞（RGCs）中的分布、表达及其功能。方法体外培养出生1～3d SD乳鼠的视网膜神经上皮细胞,免疫荧光染色检测RGCs中artemin的定位及表达,实时定量聚合酶链反应（real—time PCR）对RGCs中artemin进行定量检测。并用40mmol／L葡萄糖处理细胞12h后,再进行上述检测,观察高糖对于RGCs中artemin表达的影响。结果免疫荧光的结果证实了培养的RGCs出现Thy1．1抗体阳性的红色荧光,且多重荧光标记显示RGCs同时存在显示绿色荧光的artelnin抗体和红色荧光的Thy1．1抗体,表明artemin在RGCs中有分布。正常对照组中Thy1．1抗体阳性的细胞为（442±9）个／高倍视野,而40mmol／L高糖培养组中为（263±7）个／高倍视野,差异有统计学意义（P〈0．05）。Real—time PCR对大鼠RGCs中artemin的mRNA水平进行定量分析,40mmol／L葡萄糖处理视网膜神经细胞12h后,artemin在视网膜神经细胞及RGCs的表达明显下降（P〈0．05）。结论研究发现在原代培养的大鼠视网膜神经细胞及RGCs巾均有artemin的表达,并且在高糖作用下表达下降,为进一步研究artemin对受损RGCs的保护作用提供了思路。