鼠光感受器细胞层移植的方法及观察

目的 研究视锥和视杆细胞与周围组织间在解剖结构及是生理等方面的关系。方法 取２０只同龄Ｗｉｓｔａｒ和ＲＣＳ鼠作为供体和受体,利用视见解发削技术取得视锥及视杆细胞层,并植入ＲＣＳ鼠眼视网膜下。术后２周时摘除术眼,于光镜下观察交摄影。结果 术后２周,在视网膜完全复位的受体眼,移植的纯视锥和视杆细胞成活,可见移植细胞位于网膜下腔的视网膜色素上皮（ｒｅｔｉｎａｌ ｐｉｇｍｅｎｔ ｅｐｉｔｈｅｌｉｕｍ,ＲＰ     

视细胞移植后RCS鼠视网膜超微结构观察

自八十年代以来视网膜移植在世界上蓬勃兴起 ,为临床上视网膜色素变性和黄斑病变等难治性眼病的治疗开辟了一条全新的道路。我们通过观察皇家外科学院 (royal college of sur-geon,RCS)大鼠 [1 ] 在接受正常 Wistar鼠的视细胞层后重建的视网膜超微结构 ,以了解视细胞移植术后 ,移植 Wistar鼠视细胞 ,RCS鼠视网膜超微结构的变化。1　材料和方法供体动物选用 Wistar雄性鼠 (体重约 2 0 0 g) ,受体鼠为雄性 RCS鼠 ,出生后 6 0 d,体重 180～ 2 0 0 g( 级实验动物 ,北京医科大学实验动物部提供 )。移植视细胞片的制备及视细胞移植见文献 [2 …     

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。     

Wistar鼠和RCS鼠视网膜内一氧化氮合酶的分布

目的 观察正常Ｗｉｓｔａｒ大鼠和患遗传性视网膜病变的ＲＣＳ鼠视网膜内一氧化氮合酶（ＮＯＳ）的分布。方法 ７４天Ｗｉｓｔａｒ鼠和７４天ＲＣＳ鼠各６眼分为２组,冰冻切片,用ＮＡＤＰＨ黄递酶组织染色法（ＮＤＰ）,光镜下观察。结果 一氧化氮合酶主要存在于Ｗｉｓｔａｒ鼠,ＲＣＳ鼠的无长突细胞内,在双侧锯齿缘、赤道部和后极部内丛层均有分布,二者间的数量有差异。结论 一氧化氮合酶分布在Ｗｉｓｔａｒ鼠,ＲＣＳ鼠的无长突细胞,但视细胞萎缩对含一氧化氮合酶的阳性无长突细胞数量有影响。     

视细胞移植后重建视网膜中一氧化氮合酶的分布

近年发现一氧化氮 (ｎｉｔｒｉｃｏｘｉｄｅ ,ＮＯ)是一类新的神经递质 ,在神经系统中起到一种信使作用[1,2 ] 。Ｌ精氨酸在一氧化氮合酶 (ｎｉｔｙｉｃｏｘｄｉｅｓｙｎｔｈａｓｅ ,ＮＯＳ)的催化下分解为Ｌ 瓜氨酸和ＮＯ ,ＮＯ通过提高鸟苷酸环化酶 (ｃＧＭＰ)的水平而发挥生物效应[3] 。在大鼠视网膜内ＮＯＳ主要存在于无长突细胞中[4 ] 。我们通过观察 6 0ｄ的皇家外科学院鼠 (ＲｏｙａｌＣｏｌｌｅｇｅｏｆＳｕｒｇｅｏｕＲａｔ,ＲＣＳ)在接受视细胞移植后重建视网膜中含ＮＯＳ的无长突细胞的变化 ,以及和Ｗｉｓｔａｒ鼠、ＲＣＳ手术对…     

视细胞移植术后RCS鼠视网膜 γ-氨基丁酸的分布观察

目的通过纯视细胞移植, 观察重建视网膜神经传导通 路中抑制性神经递质γ氨基丁酸(γ-Aminobutyric Acid,GABA)的分布和变化。方法取同龄Wistar/皇家外科学院鼠(royal college of surgeon rat,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2周取RCS鼠手术眼，手术对照眼，RCS鼠对照眼，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。测量内丛状层(inner plexiform layer,IPL)和节细胞层(ganglion cell layer,GCL)的光密度，计数无长突细胞的数量， 方差分析组间差异,配对t检验RCS鼠手术眼组内差异。结果在视细胞移植术后2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鼠重建视网膜中内丛状层GABA免疫反应阳性 的神经纤维光密度测量值差异有显著性的意义 P < 0．001，而与Wi star鼠眼比较无明显区别。和手术对照眼，RCS对照眼，Wistar鼠组比，GABA能无长突细胞 (amacrine cell)明显增多(P<0．05)。节细胞层GABA阳性免 疫反应光密度测量值和上述几组比变化不明显(P>0．05)。接受移植的RCS鼠各部位的无长突细胞在移植部二侧和对侧赤道，后极部之间的差异无显著性 的意义( P = 0．3436)。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抑制性神经递质GABA的分布接近正常，无长突细胞反应性增生。(中华眼底病杂志,2001,17:128-131)     

视网膜色素上皮移植细胞超微结构的观察

目的 观察视网膜色素上皮移植细胞的超微结构,为临床应用提供实验基础。方法 取有色兔眼球后段,去除视网膜的神经上皮层,获得视网膜色素上皮细胞作为移植的供体细胞。以１０只无色兔为移植的受体,内路法将供体色素上皮细胞移植到视网膜下腔,电镜观察移植细胞的超微结构。结果 移植的１０只眼,细胞成功移植到视网膜下腔,可见移植细胞粘附于受体的Ｂｒｕｃｈ膜并形成基底内褶,细胞间紧密连接,细胞内可见吞噬体。结论 内路     

视网膜视细胞的成片移植

目的 探索用准分子激光切削技术制备视网膜单层细胞植片,经内入路视网膜下腔的单层视细胞成片移植。方法 用准分子激光对大鼠视网膜进行切削,制取单层视细胞植片,此后,按内入路手术方法进行了兔视网膜下腔的异种移植。结果 切削后所得视细胞植片由单层视细胞组成,结构完整,包括外丛状层、外核层和外节层;视细胞植片经明胶包埋后被准确植入宿主视网膜下腔中,移植术后第1,2天宿主观视网膜未能复位,呈脱离状态,移植物没能与视网膜色素上皮层相贴;移植后10天,宿主视网膜复位,视细胞移植片平铺于宿主视网膜下腔中,植片视细胞外节也宿主视网膜色素上皮层相贴;移植后10天,宿主视网膜复位,视细胞移植片平铺于宿主视网膜下腔中,植片视细胞外节与宿主视网膜色素上皮层相贴,未见明显免疫排异现象。结论 准分子激光制备单层视细胞植片方法简单、可行;初步观察到内入路单层视细胞成片移植后,视细胞植片能够在宿主视网膜下腔中以正常生理位置存活;视网膜下腔为理想的视网膜移植的受位。     

Hereditary retinal degeneration and photoreceptor apoptosis

目的研究遗传性视网膜变性的发生机制。方法对生后不同鼠龄ＲＣＳ（ＲｏｙａｌＣｏｌｅｇｅｏｆＳｕｒｇｅｏｎｓ）和ＳＤ（Ｓｐｒａｇｕｅ－Ｄａｗｌｅｙ）大鼠的视网膜进行光镜观察、计算机自动图象分析和凋亡细胞的ＴＵＮＥＬ（ＴｄＴ－ｍｅｄｉａｔｅｄｂｉｏｔｉｎ－ｄＵＴＰｎｉｃｋ－ｅｎｄｌａｂｅｌ－ｉｎｇ）检测。结果与同龄ＳＤ大鼠相比，ＲＣＳ大鼠出生后１５ｄ，视细胞（ｐｈｏｔｏｒｅｃｅｐｔｏｒ，ＰＲＣ）外节膜盘堆积，以后相继出现内节排列紊乱、消失，细胞核固缩，从生后３０ｄ到６０ｄ，ＰＲＣ迅速消失。从出生后４０ｄ，视网膜色素上皮（ｒｅｔｉｎａｌｐｉｇｍｅｎｔｅｐｉｔｈｅｌｉｕｍ，ＲＰＥ）细胞出现增殖和排列紊乱。１００ｄ时，ＲＰＥ层出现新生血管。ＴＵＮＥＬ检测，ＲＣＳ大鼠视网膜ＰＲＣ阳性数目从２５ｄ起开始逐渐增加，３５ｄ时达高峰。结论ＲＣＳ大鼠的视网膜变性以ＰＲＣ凋亡为主，ＲＰＥ紊乱和视网膜新生血管是视网膜变性的晚期病变。     

同化培养液双界面法培养纯化的视网膜神经节细胞

视网膜神经节细胞 (ｒｅｔｉｎａｌｇａｎｇｌｉｏｎｃｅｌｌｓ ,ＲＧＣｓ)具有高度分化性 ,生后一般不再进行有丝分裂 ,ＲＧＣｓ体外培养属于原代培养 ;ＲＧＣｓ体外混合培养多能存活 4周 ,而纯化培养大多只能存活 4 8ｈ。我们采用星形胶质细胞同化培养液双界面法培养纯化的ＲＧＣｓ存活时间≥ 2周 ,为纯化ＲＧＣｓ提供较好的实验模型。一、材料和方法1 乳鼠脑源性单层星型胶质细胞的培养[1] :取生后 1～5ｄ的Ｓｐｒａｇｕｅ Ｄａｗｌｅｙ(ＳＤ)乳鼠 (清洁级 ,华中科技大学同济医学院动物实验中心提供 ) 5只 ,断头处死 ,取额顶叶脑组织置于…     

Limitation of anatomical integration between subretinal transplants and the host retina

PURPOSE: In previous studies of subretinal transplantation in rabbits, the host photoreceptor layer seemed to prevent the bridging of neuronal fibers between the graft and the host retina. The current study was undertaken to determine whether the same phenomenon occurs in transplants to the subretinal space of the vascularized retina of rats. Bridging of fibers was examined in transplants to animals of different genetic backgrounds (normal versus dystrophic rats), of different ages, and after different survival times. METHODS: Sprague-Dawley (SD) rat retinal tissue from embryonic day (E)18 was subretinally grafted to adult (60-day-old) normal SD rats, to RCS rats (32 and 73 days old), and to adult (60-day-old) transgenic P23H rats. After various survival times (28-183 days), transplanted retinas were processed for routine histology and immunocytochemistry. Antibodies against calbindin, neuronal nitric oxide synthase (NOS), and protein kinase C (PKC) were used to identify specific retinal cell types and their processes. RESULTS: The shape and position of the immunoreactive cell bodies indicated that the expected neuronal populations were labeled within the grafts and in the host retina. Labeled neuronal processes were also observed. In each case, NOS-, calbindin-, and PKC-immunolabeled fibers formed bridges between the graft and the host tissues. However, regardless of the extent of host photoreceptor cell loss, the age of the recipient, or the genetic background, bridging fibers were observed only in areas where the host photoreceptor layer was discontinuous or completely missing. CONCLUSIONS: The present study demonstrates that the host photoreceptor layer plays a role in limiting graft-host anatomical integration.     

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)     

Subretinal transplantation of bone marrow mesenchymal stem cells delays retinal degeneration in the RCS rat model of retinal degeneration

Because there is no effective treatment for this retinal degeneration, potential application of cell-based therapy has attracted considerable attention. Several investigations support that bone marrow mesenchymal stem cells (MSCs) can be used for a broad spectrum of indications. Bone marrow MSCs exert their therapeutic effect in part by secreting trophic factors to promote cell survival. The current study investigates whether bone marrow MSCs secrete factor(s) to promote photoreceptor cell survival and whether subretinal transplantation of bone marrow MSCs promotes photoreceptor survival in a retinal degeneration model using Royal College of Surgeons (RCS) rats. In vitro, using mouse retinal cell culture, it was demonstrated that the conditioned medium of the MSCs delays photoreceptor cell apoptosis, suggesting that the secreted factor(s) from the MSCs promote photoreceptor cell survival. In vivo, the MSCs were injected into the subretinal space of the RCS rats and histological analysis, real-time RT-PCR and electrophysiological analysis demonstrated that the subretinal transplantation of MSCs delays retinal degeneration and preserves retinal function in the RCS rats. These results suggest that MSC is a useful cell source for cell-replacement therapy for some forms of retinal degeneration.     

益气明目丸对视网膜色素变性大鼠视网膜Bax、Caspase-3表达的影响

目的　观察益气明目丸对视网膜色素变性大鼠视网膜中Bax、Caspase-3表达的影响。方法　24只RCS大鼠分为3组，每组8只，雌雄各半，其中，空白组:RCS（rdy+/+，p+/+）大鼠灌胃生理盐水；模型组:RCS（rdy-/-，p-/-）大鼠灌胃生理盐水；益气明目丸组:RCS（rdy-/-，p-/-）大鼠灌胃益气明目丸悬浊溶液。灌胃30 d后，HE染色观察各组视网膜各层结构，免疫组织化学染色法检测各组视网膜组织中Bax、Caspase-3的平均光密度值，Western blot法测定各组视网膜组织中Bax、Caspase-3蛋白的相对表达量。结果　HE染色结果显示，益气明目丸组大鼠视网膜厚度明显高于模型组，感光细胞核数目也较模型组增多，外丛状层、外核层、视杆视锥层较模型组结构更清晰。免疫组织化学染色检测结果显示，益气明目丸组Bax、Caspase-3平均光密度值（0.133±0.030、0.069±0.024）均较模型组(0.211±0.036、0.119±0.027)减少（均为P＜0.01）。Western blot检测结果显示，益气明目丸组Bax蛋白、Caspase-3蛋白相对表达量（0.374±0.051、0.628±0.045）均明显低于模型组(0.768±0.061、0.802±0.074)，差异均有统计学意义（均为P＜0.01）。结论　益气明目丸对视网膜色素变性大鼠视网膜的超微结构具有保护作用；益气明目丸通过抑制视网膜上Bax、Caspase-3的表达减轻视网膜感光细胞的凋亡，达到保护视细胞的目的。     

Photoreceptor rescue in the RCS rat without pigment epithelium transplantation

Transplantation of normal retinal pigment epithelium (RPE) to the subretinal space has been reported to rescue photoreceptors in the RCS rat. Moreover, the rescue effect was surprisingly large considering the relatively small number of RPE cells transplanted. The reason for this widespread rescue of photoreceptors is not known, nor is the mechanism for outer segment phagocytosis in photoreceptors not apposed to the transplanted RPE cells. This suggests that the rescue effect may not be solely mediated by the transplanted cells. We therefore wished to test whether the transplantation surgery itself might contribute to the rescue of RCS photoreceptors. For these control experiments, we performed the surgery on juvenile RCS rats as described by others for the transplantation of RPE but instead of injecting RPE, we injected saline. We sacrificed the RCS control operates two months following surgery. In the area of the surgery (superior retinal quadrant) the outer nuclear layer (ONL) was up to 8-10 photoreceptor cells thick, while at the extreme inferior margin of the retina the ONL was almost eliminated. To investigate the role of temporary retinal detachment in photoreceptor rescue we repeated the above experiment using our trans-corneal approach to the subretinal space. This procedure results in a large temporary retinal detachment and little or no damage to the choroid and sclera.(ABSTRACT TRUNCATED AT 250 WORDS)     

Visual response properties of retinal ganglion cells in the royal college of surgeons dystrophic rat

PURPOSE: Alterations in retinal ganglion cell response patterns were profiled in dystrophic Royal College of Surgeons (hereafter RCS) rats over the first 100 postnatal days as a baseline for retinal rescue and vision restoration strategies. This method enabled the evaluation of the extent to which postreceptoral neuronal attributes in degenerating retinas mirror inferred declines in photoreceptor function. METHODS: Single-unit responses from large retinal ganglion cells were recorded from age-matched dystrophic RCS (RCS-rdy(-)) and congenic RCS-p(+) (hereafter wild-type or wt) rats were recorded in vitro under visual control. Cells were profiled with conventional spatial and flux stimulus modulations. RESULTS: Ganglion cell single unit and population attributes alter slowly over the course of photoreceptor degeneration in dystrophic RCS rats, with significant decreases in apparent receptive field size, contrast sensitivity, and threshold sensitivity detected by the first month of life. Spatial frequency tuning and contrast responses were extremely weak by postnatal day (P)76, paralleled by a progressive decline in signal-to-noise (S-N) ratio to roughly unity by postnatal day (P)107. This decline was only a simple loss of responsivity, as background firing rates increased substantially over time. Whereas wt retinas were dominated by ON-center cells (15/23 cells), dystrophic animals were dominated by OFF-center cells by P47 (24/27 cells). CONCLUSIONS: The first definitive signs of degeneration in dystrophic RCS rats are parallel decreases in ganglion cell threshold sensitivity and receptive field size, followed by deterioration in spatial summation. Arguably, these changes can be qualitatively explained as photoreceptor signaling losses. However, the apparent shift in population profile from ON- to OFF-center ganglion cells long before loss of the b-wave at P90 implies that a reactive mechanism such as bipolar cell rewiring and/or transformation of neuronal phenotypes occur during the early phase of photoreceptor stress, before rod and cone death.     

Functional and structural characteristics of photoreceptor cells rescued in RPE-cell grafted retinas of RCS dystrophic rats

In this study retinas of pink-eyed 26 day-old RCS dystrophic rats were injected with normal, melanotic retinal pigment epithelial (RPE) cells into the subretinal space. The areas containing underlying photoreceptor cells were subsequently investigated by electron microscopy at 60 days and light microscopy immunocytochemistry at 60 and 120 days. Retinal regions containing viable RPE-cell grafts exhibited melanin-contained RPE cell transplants attached to Bruch's membrane and containing shed rod outer segments (ROS). Also, the ROS of these photoreceptor cells exhibited a normal-appearing structural relationship with apical membrane projections of these RPE cells. The debris zone, prominent in retinas of 60 day-old RCS dystrophic rats, was much reduced in RPE-cell grafted regions. By at least 3 months after RPE transplantation, immunostaining for Na+,K+-ATPase was demonstrated along inner segments (IS) of photoreceptor cells in grafted regions, as was also shown in control retinas, but not in non-grafted regions. Opsin immunostaining in RPE-cell grafted regions and in control retinas was detected along ROS and rod inner segments (RIS) and at the periphery of cell bodies in the outer nuclear layer. This study has shown that transplanted normal RPE cells in retinas of RCS dystrophic rats appear to have normal functional and structural characteristics at least three months after transplantation. Furthermore, the rescued PRC's exhibited a normal distribution of and immunostaining density for the membrane-bound enzyme Na+,K+-ATPase and the photo-pigment opsin.     

Retinal transplants restore visually evoked responses in rats with photoreceptor degeneration

PURPOSE: To assess whether transplantation of intact sheets of fetal retina with retinal pigment epithelium (RPE) into a retina with photoreceptor degeneration restores visually evoked responses. METHODS: Sheets of fetal retina with RPE were transplanted into the subretinal space of Royal College of Surgeons (RCS) rats at 37 to 69 days of age. Sixty-three days to 10 months after transplantation, multiunit visual responses were recorded in the superior colliculus (SC) of transplanted rats, age-matched untransplanted rats, and rats with sham surgery. RESULTS: In 19 of 29 RCS rats with transplants, visually evoked responses were recorded from and restricted to a small area of the SC that corresponds topographically to the portion of the retina in which the transplant was placed. Outside of this area, no visual responses were evoked. Visually evoked responses were never recorded in age-matched, nontransplanted RCS rats. Visually evoked responses were recorded in 6 of 13 RCS rats with sham surgery, but these responses were significantly different from responses in rats with transplants. CONCLUSIONS: These results demonstrate that this transplantation technique restores visually evoked responses in the brain. Although the underlying mechanism is unknown, we propose that the central visual response results from increased synaptic efficacy within the host retina. If it can be established that functional connections between the transplant and the host retina produce the effect, then it would indicate that the technique could be explored as a therapeutic strategy in some diseases of retinal degeneration.     

Retinal degeneration is delayed by tissue factor pathway inhibitor-2 in RCS rats and a sodium-iodate-induced model in rabbits

PURPOSE: To investigate the in vivo effects of tissue factor pathway inhibitor 2 (TFPI-2), which stimulates proliferation of retinal pigment epithelial cells, but not the proliferation of fibroblast and vascular endothelial cells in vitro, on retinal degeneration using a sodium-iodate (SI)-induced model in rabbits and Royal Collage of Surgeons (RCS) rats. METHODS: 79 microg of recombinant TFPI-2 (rTFPI-2) or vehicle alone was injected intravitreously to 18 eyes of 12 pigmented rabbits a day after 20 mg/kg of SI was intravenously administered. Retinal function was assessed 4, 7, 14, and 21 days after the injection by analysing amplitudes of the c-wave of a bright flash electroretinogram. Additionally, 10 microg of rTFPI-2 or vehicle alone was injected intravitreously to 11 eyes of RCS rats at both 3 and 4 weeks old, then the retina was examined histologically at 5 weeks old. RESULTS: The rTFPI-2-treated eyes in rabbits showed a significantly less decrease in the relative amplitude of the c-wave than control eyes on days 4 and 7. The thickness of the outer nuclear layer was significantly thicker and the vacuole in the photoreceptor layer was less frequently observed in the rTFPI-2-treated RCS rats than the controls. CONCLUSIONS: Intravitreal injection of TFPI-2 rescues SI-induced retinal degeneration in rabbits and naturally occurring retinal degeneration in RCS rats at least partly. These results may suggest that this compound can be utilized in the treatment of retinal degeneration.