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Immunohistochemical localization of basic fibroblast growth factor in mature and developing retinas of normal and RCS rats. 总被引:5,自引:0,他引:5
Basic fibroblast growth factor (bFGF) delays photoreceptor degeneration when injected intraocularly in Royal College of Surgeons (RCS) rats with inherited retinal dystrophy. In the present study, we have determined the localization of endogenous bFGF in retinas of normal and RCS rats during the normal developmental period (postnatal days 0-20) and the period of photoreceptor degeneration in RCS rats (days 20-90). bFGF was localized immunohistochemically by indirect immunoperoxidase using two different polyclonal antibodies and one monoclonal antibody against bFGF. bFGF was present in retinas as early as birth, and remained through adult age. Controls using either PBS, non-immune IgG or antibody preabsorbed with bFGF peptide were devoid of label. In normal rats between the ages of birth and postnatal day (P) 4, bFGF was found in developing ganglion cells, superficial blood vessels, some of the innermost cells in the neuroblastic layer, developing horizontal cells, and retinal pigment epithelial (RPE) cells. Between P0 and P4, the intensity of staining increased significantly in horizontal cells. From P6-P10, some cells in the inner nuclear layer remained positive, but horizontal cell staining became less intense in the central retina. The superficial vessels, ganglion cells and RPE cells also remained positive for bFGF. At P20-25, when the retina was essentially mature, bFGF was found in RPE cells, most cells of the ganglion cell layer, and many cells of the inner nuclear layer, but horizontal cells and blood vessels showed a lower concentration of bFGF than they did at younger ages. At P45 and older, blood vessels no longer showed bFGF immunoreactivity. The staining pattern in RCS rats was indistinguishable from that for normal rats at all ages examined. These results show that bFGF is present in the developing and adult rat retina in some neural cells, in addition to vessels and RPE cells. The transient elevated expression of bFGF immunoreactivity in developing horizontal cells and blood vessels suggests a possible role for this growth factor in retinal development. In addition, if RCS retinas possess any difference in bFGF localization or concentration compared to normal retinas, it must be too small to detect by immunohistochemical means, or at least with the reagents used. 相似文献
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背景人类和哺乳动物视觉发育关键期内视网膜处于发育阶段,其各个亚层的正确排列以及细胞的准确定位是视觉信息正常传递的基础之一。频域光学相关断层扫描(SD—OCT)可活体测量并连续观察大鼠视网膜厚度的变化。目的了解视觉发育关键期内SD大鼠视网膜厚度的变化规律。方法选择新生SD大鼠30只,其中10只于出生后14、18、21、24、42d进行双眼眼底SD—OCT扫描,对视网膜厚度以及部分亚层包括内界膜(ILM)至内丛状层(IPL)、内核层(INL)、外核层(ONL)至视网膜色素上皮(RPE)层的厚度进行生物测量。另取20只SD大鼠,分别于14、18、21、24、42d各处死4只大鼠并摘除双眼,制作石蜡切片行苏木精一伊红染色,对视网膜厚度以及部分亚层结构进行测量。并对两种方法测量数据的相关性进行评价。结果大鼠视网膜CirrusHD—OCT图像由内向外可显示视网膜IPL、INL、外丛状层(OPL)、ONL、光感受器内节/外节(IS/OS)层以及RPE层等结构。比较各个时间点的视网膜厚度、ILM至IPL厚度、INL厚度以及ONL至RPE层厚度,差异均有统计学意义(F=15.425,P=0.000;F=3.973,P=0.007;F=17.529,P:0.000;F=7.038,P=0.000)。随大鼠日龄的增加,视网膜厚度、INL厚度及ONL至RPE层厚度均呈递减趋势。测量视网膜组织切片各对应层次厚度,并与OCT测量结果进行线性回归分析,结果表明两种方法测量的视网膜厚度、INL厚度以及ONL至RPE层厚度均呈正相关(r=0.794,P=0.000;r=0.784,P=0.000;r=0.681,P=0.000)。结论CirrusHD—OCT可以清晰地显示并测量大鼠视网膜各主要层次,且与组织学测量具有较好的相关性。视觉发育关键期内大鼠视网膜逐渐变薄,OCT测量主要表现为INL厚度和ONL至RPE层厚度减少。 相似文献
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Tsumura T Murata A Yamaguchi F Sugimoto K Hasegawa E Hatase O Nairn AC Tokuda M 《Vision research》1999,39(19):3165-3173
Ca2+/calmodulin-dependent protein kinase I (CaM-kinase I) in rat retina was analyzed by immunohistochemical analysis, Western blot analysis and kinase activity assay. Western blot analysis revealed two immunoreactive bands similar to those detected in the brain. Developmental studies revealed that CaM-kinase I expression increased in accordance with postnatal development. Expression of CaM-kinase I in the retinas of rats raised in the complete darkness markedly decreased. CaM-kinase I activity assay supported these findings. Synapsin I was shown to be a possible intrinsic substrate of CaM-kinase I in rat retina. These results elucidated that CaM-kinase I is expressed in the retina and may play an important role in the retinal functions and that the expression of CaM-kinase I is regulated by light stimulation. 相似文献
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Immunocytochemical localization of interphotoreceptor retinoid-binding protein in developing normal and RCS rat retinas 总被引:3,自引:0,他引:3
A J Eisenfeld A H Bunt-Milam J C Saari 《Investigative ophthalmology & visual science》1985,26(5):775-778
Interphotoreceptor retinoid-binding protein (IRBP) was localized immunocytochemically in developing normal and RCS rat retinas. IRBP was present in normal and RCS neural retinas on the day after birth (postnatal day 2, P2) to P8 in the space between the neuroblastic layer and the retinal pigment epithelium (RPE). The presence of IRBP prior to the development of outer segments (OS) suggests that OS formation is not linked temporally with IRBP secretion. On P10, staining was confined to the interphotoreceptor space with an intense band of label adjacent to the RPE. This staining pattern persisted in normal rats throughout development and until P18 in RCS rats. On P18, anti-IRBP staining in the RCS was spread evenly throughout the OS layer with no intense band of label adjacent to the RPE and after P18, there was decreased staining with anti-IRBP. On P45 and later, no staining of the RCS retina was found with anti-IRBP. Immunoblots of normal and RCS retinas corroborated the results from immunocytochemical staining. These findings suggest that IRBP may be synthesized in the photoreceptors, but is not abnormal in amount or distribution prior to onset of retinal degeneration in the RCS rat. 相似文献
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Van Den Berghe L Sainton K Gogat K Marchant D Dufour E Bonnel S Gadin S Menasche M Abitbol M 《Investigative ophthalmology & visual science》2004,45(5):1297-1305
PURPOSE: To identify proteins secreted by the retinal pigment epithelium (RPE) and to analyze their cellular distribution in normal and pathologic rat retinas at various stages of eye development. METHODS: A cDNA library was constructed with RNA isolated from porcine RPE sheets and screened by using the yeast signal sequence trap system. In situ hybridization, immunohistochemistry, and semiquantitative RT-PCR analysis were performed on rat retinas. RESULTS: The cDNA encoding prosaposin was isolated. This is the first time this gene has been shown to be expressed in the retina. Prosaposin mRNA was detected in the rat RPE cell monolayer and in ganglion cells 14, 21, and 45 days after birth. The amount of prosaposin mRNA increased between days 14 and 45 after birth in normal retinas (rdy+), but not in the pathologic retinas (rdy-) of RCS rats. CONCLUSIONS: Several techniques were used to determine the localization of prosaposin in rat retinas. The increase in the amount of prosaposin mRNA in normal retinas coincided with the maturation of photoreceptor cells and the beginning of the phagocytosis process. In addition, the RCS rdy- RPE cells, characterized by the abrogation of the ingestion phase of the photoreceptor outer segments, are deficient in prosaposin expression. 相似文献
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Neuronal migration and glial remodeling in degenerating retinas of aged rats and in nonneovascular AMD 总被引:3,自引:0,他引:3
PURPOSE: To demonstrate structural and immunocytochemical changes associated with light-induced degeneration in albino rat retinas and human AMD retinas. METHODS: Retinas from Wistar rats aged 3, 6, or 10 months were examined by immunocytochemistry, with antibodies to neuronal and glial markers. Results were compared with human nonneovascular AMD retinas. RESULTS: In aging rat retinas, many photoreceptors were lost in response to normal ambient light exposure. Photoreceptor loss was preceded by loss of RPE cells. Müller cells extended processes through gaps in Bruch's membrane, into the choroid. immunolabeling for gamma-aminobutyric acid (GABA), the glycine transporter Glyt-1, and the rod bipolar cell marker PKC revealed the presence of numerous neuronal somata and processes that appeared to have migrated into the choroidal region. Processes of presumptive ganglion cells remodeled and stratified in the choroid, where strong labeling for synaptic vesicle antigens was present. Myelination of retinal ganglion cell axons was also observed, especially in the peripheral retina. In AMD retinas, glial rearrangement and displacement of neurons suggestive of their migration were also observed. CONCLUSIONS: In response to loss of RPE and photoreceptor cells, adult retinal neurons migrate out of the retina along remodeled processes of Müller cells. The presence of synaptic vesicle antigens suggests the formation of new synapses between migrating neurons. The myelination is probably due to the ingress of Schwann cells from the sclera. The presence of some similar changes in human AMD retinas suggests that these findings are of broad significance for determining the likely events in transplantation of neurons in the human retina and elsewhere. 相似文献
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Agarwal N Martin E Krishnamoorthy RR Landers R Wen R Krueger S Kapin MA Collier RJ 《Experimental eye research》2002,74(4):445-453
Betaxolol (racemic), a beta-adrenoceptor antagonist that is used to lower intraocular pressure in the treatment of glaucoma, has been shown to protect inner retina cells from various insults. To determine if such protection could be afforded to retinal photoreceptors and retinal pigment epithelial cells (RPE), levobetaxolol (S-betaxolol) was evaluated in a photic-induced retinopathy model. Rats were dosed (IP) with vehicle or levobetaxolol (10 and 20 mg kg(-1)) 48, 24 and 0 hr prior to exposure for 6 hr to fluorescent blue light. The electroretinogram (ERG) and retinal morphology were assessed after a 3 week recovery period. Evaluation of the ERG demonstrated significant protection of retinal function in levobetaxolol (20 mg kg(-1))-dosed rats compared to vehicle-dosed rats. Similarly, the RPE and outer nuclear layer were significantly thicker in levobetaxolol (20 mg kg(-1))-dosed rats compared to vehicle-dosed rats. To elucidate potential mechanism(s) of the neuroprotective activity of levobetaxolol, bFGF and CNTF mRNA levels in normal rat retinas were evaluated 12 hr after a single i.p. injection. Northern blot analysis of levobetaxolol treated retinas demonstrated a 10-fold up-regulation of bFGF and a two-fold up-regulation of CNTF mRNA levels, trophic factors that have been shown to inhibit retinal degeneration in a number of species. These studies suggest that levobetaxolol can be used as a novel neuroprotective agent to ameliorate retinopathy. 相似文献
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We have examined the presence and distribution of integrin and fibronectin in the retinas of 21-day chick embryos and adult rats, with particular emphasis on the question of localization in the retinal pigment epithelium (RPE). Isolated sheets of RPE solubilized and separated by gel electrophoresis contain integrin, as indicated by immunoblotting with polyclonal and monoclonal antibodies to the complex. By the same technique, antibodies to fibronectin reacted with a single protein in the isolated RPE. In both chick and rat, integrin and fibronectin were localized by indirect immunofluorescence exclusively to the basement membrane of the RPE, the choriocapillaris and the retinal-vitreal border. When isolated RPE cells from chick retinas were examined, integrin was seen to be present along the basolateral surfaces of the cells as well. Similarly, in the intact rat retina, staining for integrin could be seen along the lateral surfaces of some of the RPE cells. Neither integrin nor fibronectin were present along the apical surfaces of the RPE in either rat or chick. The close similarity between the location of integrin and fibronectin supports the idea that the RPE adheres to the basal lamina at least in part via integrin-fibronectin linkages. A clear implication of our results is that the adhesion between RPE and retina requires a different set of linkage proteins. 相似文献
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The immunolocalization of 70 kD stress protein (SP70) was investigated in the retinal tissues of normal Sprague-Dawley (SD) rat and that of the Royal College of Surgeons (RCS) rat with inherited retinal dystrophy. From postnatal day 2 to 15, SP70 was present in the maturing retinal tissues of both rat strains. In the RCS rat retina of postnatal day 22, at the onset of retinal degeneration, SP70 was expressed in the retinal pigment epithelium (RPE). At postnatal day 40, immunostaining for SP70 was considerably reduced in the degenerating RCS retina. In the RCS retina at postnatal day 90, immunostaining for SP70 was completely lost except for the ganglion cells and the inner plexiform layers. These results suggested that, at the onset of retinal degeneration, the RCS retina may have a state of metabolic stress, which induced SP70 expression in the RPE. At the end stage of retinal degeneration, the immunostaining for SP70 was lost, suggesting the lack of production of SP70 in the degenerated retinal tissue. 相似文献
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In the neural retina, glial cells control formation of ionic gradients by mediating transmembrane water fluxes through aquaporin (AQP) water channels. Retinal content and immunolocalization of two water channels, AQP1 and AQP4, in the diabetic rat retinas during high-salt loading were examined in this study. Diabetes was induced by an intraperitoneal injection of streptozotocin. Diabetic and control animals were observed after varying lengths of exposure to normal- and high-salt conditions. Ultrathin sections of retinal tissue, stained with uranyl acetate and lead citrate, were photographed using a transmission electron microscope (TEM). Retinal wholemounts were immunostained with AQP1 and AQP4 antibody to detect the immunolocalization changes by confocal microscopy. AQP1 and AQP4 content were evaluated by Western blot analysis. In the retinas of high-salt loading diabetic animals, obviously increased intracellular edema was observed by TEM in ganglion cells and mitochondrial swelling was observed in glial cells. Immunolocalization of AQP1 increased from the posterior to peripheral retina. Western blot results indicated that a high-salt diet may cause increased retinal content of AQP4 and may exacerbate increased retinal content of AQP1 caused by diabetic retinopathy. High-salt loading may increase neural retinal edema in rats with diabetic retinopathy, and altered glial cell mediated water transport via AQP channels in the retina may play an important role in the neural retinal edema formation and resolution. 相似文献
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Sánchez MC Barcelona PF Luna JD Ortiz SG Juarez PC Riera CM Chiabrando GA 《Experimental eye research》2006,83(6):1378-1385
The low-density lipoprotein receptor-related protein-1 (LRP-1) is a high-molecular weight receptor of the LDL receptor gene family. Its ability to bind and internalize both proteinases and proteinase-inhibitor complexes from the extracellular space suggests that it has a major role in modulating uncontrolled retinal cell proliferation. In order to test this assumption, we investigated the expression of LRP-1 and receptor-associated ligands in a rat model of oxygen-induced retinal neovascularization. Wistar albino rats were placed into incubators at birth and exposed to an atmosphere alternating between 50% and 10% of oxygen every 24 h. After 14 days, the animals were allowed to recover in room air and sacrificed at postnatal day 20 (P20). The protein expression of LRP-1 and alpha2-macroglobulin (alpha2M) in the retina from unexposed and hyperoxia-exposed rats was investigated by Western blot. The localization of LRP-1 after neovascularization was assessed by immunohistochemical staining. The activity of metalloproteinases (MMPs) was determined by zymography. Histological analysis was done to quantitate the neovascular response in these animals. Western blot analysis showed that LRP-1 was expressed, along with alpha2M, in the retina of rats with oxygen-induced neovascularization at P20. By immunohistochemical analysis, positive staining for LRP-1 appeared in cells extending from the inner limiting membrane (ILM) to the outer limiting membrane (OLM). The cells of the retina that expressed LRP-1 were identified by immunofluorescence as Müller cells. Zymographic analysis demonstrated increased activity of MMP-2 and MMP-9 under neovascular conditions. This is the first demonstration of the involvement of LRP-1 in retinal neovascularization. In retinas of rats with oxygen-induced neovascularization, the expression of LRP-1 and alpha2M was increased along with an enhanced activity of MMPs, suggesting that LRP-1 expression may play a role in modulating retinal neovascularization by regulating proteolytic activity. 相似文献
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Wu BX Moiseyev G Chen Y Rohrer B Crouch RK Ma JX 《Investigative ophthalmology & visual science》2004,45(11):3857-3862
PURPOSE: To investigate the expression of RDH10, an all-trans retinol dehydrogenase identified in the retinal pigment epithelium (RPE), in retinal Muller cells. METHODS: The RDH10 protein levels in mouse eyecups and bovine tissues were examined by Western blot analysis using a polyclonal antibody against RDH10. The cellular localization in the retina was determined by immunohistochemistry. Expression of RDH10 in rMC-1, a cell line derived from rat Muller cells, was determined by RT-PCR and Western blot analysis. All-trans retinol dehydrogenase activity assays were performed using lysates from rMC-1 cells. The generation of all-trans retinal from tritiated all-trans retinol was analyzed by HPLC. RESULTS: RDH10, retinal G protein-coupled receptor (RGR), and RPE65 all had higher expression levels in the eyecups of BALB/c than in C57Bl/6 mice. In addition to the RPE, RDH10 was also detected at lower levels in the retina and liver. Immunohistochemistry showed that RDH10 was localized in Muller cells in retinal sections. RDH10 was detected in rMC-1 cells, at both the RNA and protein levels. The rat RDH10 cDNA containing the full-length coding region was cloned from rMC-1 cells. The rat RDH10 cDNA encodes a protein of 341 amino acids and shares 99% sequence identity with human, bovine, and mouse RDH10 at the amino acid level. In rMC-1 cells, all-trans retinol dehydrogenase activity was detected in the microsomal fraction. NADP was shown to be the preferred cofactor, which is identical with the cofactor preference of the recombinant RDH10. CONCLUSIONS: RDH10 was expressed in retinal Muller cells, in addition to the RPE. RDH10 generates all-trans retinal, which is the substrate for the photoisomerase RGR in Muller cells. 相似文献
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P A Erickson S K Fisher C J Guérin D H Anderson D D Kaska 《Experimental eye research》1987,44(1):37-48
Retinal detachment, separation of the neural retina from the retinal pigment epithelium (RPE), initiates a series of changes in the eye which result in loss of vision if the retina is not rapidly reattached to the RPE. Many of the complex effects of this separation on the cell biology of the retina have yet to be determined. We report here a change in the amount and location of a specific cytoskeletal protein, glial fibrillary acidic protein (GFAP), within Müller cells after retinal detachment. Cat neural retina and RPE were separated by injecting fluid into the extracellular space between the retina and RPE. Normal retinas and retinas detached for 30 days were fixed and embedded for conventional electron microscopy or immunocytochemistry, or homogenized and processed by SDS-PAGE for immunoblot analysis with anti-GFAP. In normal retinas and in attached retinal regions of eyes with retinal detachment, GFAP was detected only in the end feet of the Müller cells as 10 nm diameter filaments and as a diffuse component over the cytoplasm. By contrast, in regions where the retina was detached from the RPE, GFAP was localized throughout the Müller cells in both of these forms. Immunoblots revealed a significant increase in anti-GFAP labeling of a 51,000 MW band from the detached retina. 相似文献
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PURPOSE: To observe the changes in N-cadherin expression in the retina after experimental retinal detachment (RD) and reattachment in the rat and to explore the role N-cadherin might play after RD. METHODS: Forty rat retinas were detached by transscleral injection of 1.4% sodium hyaluronate into the subretinal space. The eyes were enucleated at different time intervals (n = 5), followed by fixation, embedding, and sectioning. The differences in N-cadherin expression in the normal retina, detached retina, and spontaneously reattached retina were determined. Furthermore, an N-cadherin antagonist was injected in combination with 1.4% sodium hyaluronate into the subretinal space in another 10 eyes, in an attempt to demonstrate the role N-cadherin plays after RD. RESULTS: N-cadherin was not expressed in the RPE layer of the normal rat retina. After RD, intense immunolabeling of N-cadherin was seen in the RPE cells, the photoreceptors, and the outer limiting membrane (OLM). An increasing number of cytokeratin (CK)-positive cells likely to be RPE cells was found attached to the outer surface of the detached neural retina. Where the retina was reattached, the N-cadherin immunolabeling rapidly decreased. In eyes treated with an N-cadherin antagonist, the retinas appeared thinner than that in eyes without treatment, and the photoreceptor nuclei showed significantly loss. Moreover, CK-positive cells attached to the outer surface of the detached retina were markedly fewer in number. CONCLUSIONS: Increased expression of N-cadherin in the RPE cells, the photoreceptor cells, and the OLM of the retina after RD may contribute to RPE cell migration and photoreceptor survival. These changes could be reversed by retinal reattachment. 相似文献
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Enhanced expression of adhesion molecules of the retinal vascular endothelium in spontaneous diabetic rats 总被引:12,自引:0,他引:12
Otsuka Long-Evans Tokushima Fatty (OLETF) rats are a spontaneously diabetic strain with clinical features resembling those of human noninsulin-dependent diabetes mellitus. These rats show increased leukocyte entrapment in the retina. The present study was designed to investigate the immunolocalization of intercellular adhesion molecules-1 (ICAM-1) and P-selectin in the retinas of OLETF rats. Four 72-week-old male OLETF rats and 4 age- and sex-matched Long-Evans Tokushima Otsuka (LETO) control rats were used. Western blot analysis and immunohistochemical study were performed using an anti-P-selectin monoclonal antibody (mAb) and an anti-ICAM-1 mAb. Western blot analysis showed increased expression of both ICAM-1 and P-selectin in the retinas of OLETF rats. Immunohistochemically, OLETF rats expressed greater amounts of ICAM-1 and P-selectin in the retinal vascular endothelium than did LETO rats. These findings demonstrated the upregulation of ICAM-1 and P-selectin in the retina of OLETF rats. The enhanced expression of these adhesion molecules might participate in the pathogenesis of early diabetic microangiopathy in the retina. 相似文献
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We have studied permeability of retinal pigment epithelial (RPE) cell junctions in Royal College of Surgeons rats with inherited retinal degeneration, and their genetic controls, using the horseradish peroxidase and lanthanum nitrate electron microscope tracer techniques. We find that early in the dystrophic process, at two postnatal weeks in the pink-eyed retina and three postanatal weeks in the black-eyed retina, RPE cell tight junctions form a barrier to extracellular tracer. However, at three postnatal weeks in the pink-eyed retina, at about the same time that degenerating photoreceptor nuclei begin to appear, RPE cell tight junctions become permeable. The permeability increase occurs later in the black-eyed strain, but by six postnatal weeks junctions are permeable in both strains. By 72 postnatal days, when most photoreceptor nuclei have disappeared, many RPE cells are abnormal in shape, with an elongated and flattened appearance, and some appear to have lost their junctions entirely. In the horseradish peroxidase experiments, many pinocytotic vesicles filled with reaction product were observed in the dystrophic RPE after the junctional breakdown. This suggests that an increase in transcellular transport may also occur in the dystrophic RPE. 相似文献