实验性视网膜脱离及复位后视网膜色素上皮细胞抗增殖性细胞核抗原的表达

目的 观察视网膜脱离和复位过程中抗增殖性细胞核抗原(proiferating cell nuclear antigen, PCNA)表达的状况，以评价其在接触抑制机制中的意义。 方法 对72只猫眼行晶状体 囊外摘除和玻璃体切除手术。3周后，利用微穿刺技术制作视网膜脱离和复位动物模型，在不同时间取眼球并制成组织切片。采用免疫组织化学方法，观察视网膜脱离后视网膜色素上皮(retinal pigment-epithelium, RPE)细胞PCNA的表达。光镜下确定PCNA阳性的RPE细胞并量化增殖反应。采用方差分析(ANOVA)方法 ，对脱离组的脱离区和非脱离区及复位组复位区RPE细胞层PCNA的表达水平进行比较。 结果 在视网膜脱离后24 h，脱离组脱离区视网膜RPE细胞层出现PCNA阳性细胞 ，5～6 d达到高峰，20 d后逐渐回落。在复位组复位区，PCNA标记的RPE细胞明显少于脱离组脱离区。脱离组非脱离区视网膜很少有PCNA标记的RPE细胞。3组PCNA标记的RPE细胞数比较，差异有显著性的意义。 结论 视网膜脱离诱导RPE细胞增殖，而视网膜复位后这种增殖受到抑制。表明视网膜复位过程中，接触抑制的机制在发挥作用。（中华眼底病杂志，2003，19：20-23）     

实验性视网膜脱离的病理改变及细胞增生

目的:探讨实验性视网膜脱离的病理变化和细胞增生。方法:成年白化病兔32眼在间接检眼镜直视下,先抽取玻璃体液0.5mL软化眼球,用50nm玻璃微管在下方6:00赤道部部位刺入视网膜造成裂孔并注射生理盐水0.5mL于视网膜下间隙。术后0.5,1,3,6,24h;3,7d和14d摘除眼球作组织学观察并以免疫组织化学的方法检测视网膜下细胞增生。结果:32只兔眼术后均发生视网膜脱离,视网膜脱离范围3:00～9:00(髓腺下方视网膜均脱离)。自发性复位的时间在3～14d,组织学切片显示,3d可见视网膜色素上皮细胞积聚团,7dRPE细胞增生肥大呈巨噬细胞样改变,14dRPE多层化改变,并见巨噬细胞样RPE来源于单层的RPE,游离RPE层后,吸附于脱离的光感受器细胞外节;3d增生细胞核抗原即表达阳性,增生细胞表达角蛋白阳性。结论:在此模型中,RPE细胞表现出细胞增生,巨噬细胞样细胞也来源于RPE。     

Retinal reattachment of the primate macula. Photoreceptor recovery after short-term detachment

The macula of the neural retina from 12 adult rhesus monkeys (Macaca mulatta) was detached from the overlying retinal pigment epithelium (RPE) by subretinal injection of a balanced salt solution. Seven days later, the two layers were reapposed by draining fluid from the vitreous cavity and replacing it with a 3:1 mixture of sulphur hexafluride gas and air. Animals were sacrificed at 1 hr, 2 days and 7 days after detachment, and at periods ranging from 3 to 14 days after reattachment. At 2-7 days prior to sacrifice, some eyes received an intravitreal injection of 3H-L-fucose. The eyes were then fixed for light and electron microscopy (EM), and tissue sections were processed for autoradiography (ARG) or immunocytochemistry. During the 7-day detachment interval, rod outer segments (ROSs) and cone outer segments (COSs) degenerated, but inner segments remained intact and the rest of the retina appeared normal. The apical RPE surface dedifferentiated during the detachment interval. At 3 days after reattachment, a regrowth of rudimentary ROSs and COSs had occurred, but the disc stacking was clearly abnormal. ROSs and COSs both showed an increase in length and a tendency to return to their normal configurations with increasing time after reattachment. ROSs and COSs regained approximately 40% of their normal lengths after a 2-week reattachment period; however, persistent outer segment abnormalities were frequently found in otherwise well regenerated areas. Autoradiographic results confirmed that new disc members were synthesized subsequent to reattachment. Newly synthesized rod disc membranes were uniformly labeled using antibodies to bovine opsin. Regenerating outer segments interdigitated with newly formed apical RPE processes, and radiolabeled phagosomes were identified within the RPE cytoplasm by 1 week after reattachment. Proliferation of the RPE cell layer was identified at some locations in all animals, and was strongly correlated with a lack of underlying outer segment regeneration. Because of the short detachment interval, and the absence of underlying pathology or trauma, the recovery process described here probably represents an example of optimum recovery after retinal reattachment.     

Retinal detachment in the cat: the pigment epithelial-photoreceptor interface

Twenty-six cat retinae were surgically detached by injecting fluid into the subretinal space (SRS). The retinae were then studied by light and electron microscopy at detachment intervals ranging from 1/2 hr to 14 months. Degenerative and proliferative changes occur at the retinal pigment epithelial (RPE)-photoreceptor interface very soon after detachment, and the severity of these changes depends upon both the duration and height of the detachment. The specialized apical RPE processes that ensheath the outer segments are replaced by a uniform fringe of short, undifferentiated processes. The apical RPE surface becomes mounded, and this mounding becomes more pronounced at longer detachment durations. Labeling experiments with 3H-thymidine showed that some cat RPE cells enter a phase of stimulated DNA synthesis 12-24 hrs after detachment; RPE mitotic figures are first apparent 48 hrs after detachment. In the cat, discrete regions of proliferated RPE cells usually appear in one of several configurations. A number of different cell types, including polymorphonuclear neutrophils, monocytes at various maturational stages, photoreceptor cells, Müller cells, and RPE cells, appear in the expanded SRS of detached retinae. Rod and cone outer segments degenerate rapidly and become membrane bound sacs by 3 days postdetachment; the assembly of new outer segment membrane apparently does not stop completely even at moderately long detachment intervals (ie, 2 months). Degenerative changes in the inner segments do not take place with the same rapidity as those in the outer segments. The changes that occur at the RPE-photoreceptor interface are rapid, progressive, and sometimes irreversible events that have significant implications for photoreceptor recovery following retinal reattachment surgery.     

Glial fibrillary acidic protein increases in Müller cells after retinal detachment

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.     

N-cadherin expression in a rat model of retinal detachment and reattachment

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.     

Docosahexaenoic acid increases in frog retinal pigment epithelium following rod photoreceptor shedding.

The vertebrate retina conserves docosahexaenoic acid (22:6n-3) during n-3 fatty acid deficiency. The mechanism of conservation is not known, although recycling of this fatty acid between the retinal pigment epithelium (RPE) and retina is one possibility. We examined the role of the RPE in conservation of 22:6n-3 by quantitating the fatty acids and phospholipid molecular species (PLMS) in frog RPE before and after light-stimulated shedding of rod outer segments (ROS). RPE cells were dissociated with brush agitation and purified by a discontinuous ficoll density gradient. One hour after the light-induced shedding of ROS, the phagocytosed ROS tip and opsin content of RPE had increased. Simultaneously, the levels of 22:6n-3 and 22:6(n-3)-containing PLMS were increased in the RPE. Within 8 hr following the shedding event, 22:6n-3 in the RPE had returned to the dark level. These findings indicate that the phagocytosed ROS tips contain 22:6n-3 and that the RPE metabolizes these ROS tips and eliminates 22: 6n-3 from the cell. Thus, the RPE is intimately involved in the metabolism of 22: 6n-3 in the retina. The recycling of 22: 6n-3 from the RPE to the retina is a possible means of conserving this important fatty acid in the retina.     

Tritiated uridine labeling of the retina: changes after retinal detachment.

As part of a study designed to examine the response of photoreceptor cells to outer segment injury (retinal detachment), the pattern of RNA labeling ([3H]uridine incorporation) has been determined in detached cat retinas. Retinas were experimentally detached from the adjacent cellular layer (the retinal pigment epithelium:RPE) by injecting fluid into the extracellular space between the retina and RPE. Twenty-four hours before the animals were killed they received intravitreal injections of [3H]uridine. Autoradiograms were prepared from plastic sections 1.0 micron thick taken from detached retinal regions and, because the detachments do not encompass all of the retina, from nearby attached retinal regions. Twenty-four hours after retinal detachment there is a decrease in labeling intensity of the photoreceptors and Müller's glia in the region of detachment (compared to cells in nearby attached regions). Seventy-two hours after retinal separation, the same result is obtained in the photoreceptors, but labeling intensity is greatly increased in both the nuclei and cytoplasm of Müller's glia. The decrease in [3H]uridine labeling of the photoreceptors correlates with a decreased staining intensity of the cytoplasm and ultrastructural signs of necrosis. The striking change in the pattern and intensity of labeling of the Müller cells precedes extensive hypertrophy of these cells and the appearance within their cytoplasm of numerous 10-nm diameter filaments. Two weeks, and also 1 month, after detachment the pattern and labeling levels are similar to those observed 1 day after retinal separation. These data suggest a highly localized change in metabolism because the change in RNA labeling is restricted to the region of detached retina.     

Distribution of p27(KIP1), cyclin D1, and proliferating cell nuclear antigen after retinal detachment

Purpose To examine the expression of the p27(KIP1), cyclin D1, and proliferating cell nuclear antigen (PCNA) in the retina and retinal pigment epithelium (RPE) after retinal detachment.Methods Normal eyes and eyes at 2 or 4 days after retinal detachment with the C57B16 mouse were analyzed by immunocytochemistry using anti-p27(KIP1), anti-cyclin D1, and anti-proliferating cell nuclear antigen (PCNA) antibodies as well as anti-glutamate synthetase (GS) antibody.Results The p27(KIP1) positive nuclei were distributed in the inner nuclear layer (INL) and the RPE of the normal mice eye. In the INL, p27(KIP1) was detected in the middle sublayer, where the nuclei of glutamate synthetase positive Müller cells were situated. In contrast, cyclin D1 was not detected either in the retina or in the RPE. At 2 and 4 days after the retinal detachment, RPE cells under the detached retina were negative for p27(KIP1) and positive for cyclin D1 and PCNA. In the INL of the detached retina, p27(KIP1) was detected after 2 days, but was not detected after 4 days. In contrast, PCNA was not detected in the INL after 2 days, but was detected after 4 days. Cyclin D1 was detected in the middle sublayer of the INL at both 2 and 4 days after the retinal detachment.Conclusion These results suggested that degradation of p27(KIP1) and expression of cyclin D1 was involved in the proliferation of the Müller cells as well as RPE cells after retinal detachment.     

Opsin distribution and protein incorporation in photoreceptors after experimental retinal detachment.

The distribution of opsin was examined immunocytochemically after experimental retinal detachment in adult cats. Retinal detachments were produced by injecting fluid between the retinal pigment epithelium and neural retina. One to 60 days later the animals were killed. Tissue areas from detached and attached retinal regions from the eye with the detached retina, as well as normal (control) retinas, were processed for post-embedding light and electron microscopic immunocytochemistry. In normal and attached retinal regions, anti-opsin labeled the outer segments and Golgi apparatus most heavily, although the entire photoreceptor plasma membrane was labeled at a low level. Beginning at 2 days after retinal detachment, immunolabeling increased in the photoreceptor inner segment, cell body and synaptic terminal plasma membranes. This pattern of anti-opsin labeling continued at all intervals up through the 60-day detachment time-point. Injection of radiolabeled amino acid in detachments from 1 to 30 days show that radiolabeled protein is still transported to the truncated outer segments of the photoreceptor cells. In addition, these outer segment disks label with anti-opsin. These data imply that opsin continues to be transported and incorporated into the outer segments of photoreceptors showing severe degeneration as a result of long-term detachment from the RPE.     

Studies of experimentally induced retinal degeneration: 2. Early morphological changes produced by lipid peroxides in the albino rabbit

When pure, synthetic lipid hydroperoxides (LHP) were injected into the vitreous body of albino rabbits, electrical activity was decreased in all components of the electroretinogram (ERG) in a progressive and time-related manner. In the early phase morphological changes occurred at the interface between the photoreceptor outer segments and the retinal pigment epithelium (RPE) in response to LHP. These findings commenced within a few hours after injection and continued during the following 2-3 weeks, when the ERG was completely extinguished. Two days after injection, the rod outer segments (ROS) were swollen and damage of the RPE apical villi was observed. This initial event was followed by additional changes in the RPE which embraced swelling, accumulation of residual bodies, complete loss of ROS and enlargement and disruption of Bruch's membrane. The precursors of residual bodies appeared to result from focal, peroxidative damage to ROS discs which apparently rendered these materials undegradable by the RPE. As ROS degeneration continued, the RPE showed hypertrophy and modification. These studies provided evidence for a sequential destruction of the neural retina and RPE during oxidative damage involving lipid peroxidation. The mechanism appeared to differ from that produced by other toxic compounds or those which resulted from vitamin E or A deficiency. This new model system is thought to be useful in 1) explaining differences in susceptibility of inner ROS disks versus other membranes, 2) determining how the RPE metabolizes abnormal ROS, 3) studying RPE reactivity following trauma and/or retinal detachment, and 4) determining factors which produce degeneration of Bruch's membrane.     

Morphological recovery in the reattached retina

After experimental retinal detachment in the cat, a number of morphological changes take place in retinal and RPE cells. Following reattachment, the ultrastructural relationship between the photoreceptors and the RPE is re-established, but it does not return to the predetachment state even after short detachment episodes coupled with prolonged recovery periods. All of the reattached retinae show some degree of abnormality, ranging from subtle changes in photoreceptor ultrastructure to dramatic degenerative effects in the outer retina. Abrupt transitions in morphology from one reattached area to an adjacent area are not unusual. Photoreceptor recovery varies widely between animals, and between adjacent regions within the same retina. Ensheathment of outer segments by RPE apical processes is abnormal. In some reattached areas rod outer segment dimensions and disc structure are near normal as is the displacement rate of rod outer segment discs. In others, especially in areas of RPE or Müller cell proliferation and hypertrophy, the outer segments are shortened or absent completely, and there is a reduction of cell bodies in the outer nuclear layer. In some retinae, recovery in cones is inferior to that in rods. At short detachment durations (less than 1 wk) morphological recovery in the reattached retina is optimal while at long intervals (greater than 1 month) recovery is poor. The changes at the photoreceptor-RPE interface identified in the reattached cat retina probably have adverse effects on visual recovery when they occur within the human macula.     

Cytochalasin D inhibits L-glutamate-induced disc shedding without altering L-glutamate-induced increase in adhesiveness

Excitatory amino acid-stimulated disc shedding is correlated with the appearance of microfilament-rich ensheathing processes of the retinal pigment epithelium (RPE) and increased apparent adhesiveness between photoreceptors and RPE in explanted eyecups of Xenopus laevis. We have compared the time course of disc shedding and increased retinal adherence during L-glutamate treatment. Increased adherence was measured on the basis of the tendency of the apical RPE domains to partition with isolated neural retinas. In medium supplemented with L-glutamate (12 mM) or kainate (100 microM), a glutamate analog, the time course of increased partitioning of melanin pigment-rich cell fragments which contain ensheathing processes differs, even though the kinetics of induced disc shedding is the same in either case. Co-treatment with cytochalasin D (5 microM) completely blocks L-glutamate-induced disc shedding, as well as formation of microfilament-rich ensheathing processes, even though it has little effect upon apparent adhesiveness. The virtually complete dissociation of the effects of L-glutamate on disc shedding from that on increased adhesiveness of photoreceptors to RPE suggests that increased retinal adherence and pseudopod formation may be unrelated causally.     

Retinal pigment epithelial cells play a central role in the conservation of docosahexaenoic acid by photoreceptor cells after shedding and phagocytosis.

The involvement of retinal pigment epithelial (RPE) cells in the recycling of docosahexaenoic acid (DHA), from phagocytized disc membranes back to the retina, was studied in frogs subsequent to injection of [3H]DHA via the dorsal lymph sac. Rod outer segments (ROS) gradually accumulated [3H]DHA as a dense, heavily labeled region that arrived at the distal tips by 28 days post-injection. Autoradiographic analysis at the time of maximal shedding and phagocytosis (1-2 hr after the onset of light) showed diffusely (before 28 days) and heavily (after 28 days) labeled phagosomes in RPE cells. Biochemical analysis of the [3H]DHA-containing lipids of discs that contribute to the labeling of RPE cells after phagocytosis was also performed. Between 27 and 34 days, when 12% of retinal [3H]DHA-lipids present in disc membranes are phagocytized by RPE cells, total retinal labeling remained unchanged. Taken together, these data suggest that the [3H]DHA of the densely labeled region of the ROS was recycled back to the photoreceptor cells only after it had reached the RPE cells following 28 days post-injection. We conclude that, following daily phagocytosis of ROS tips, RPE cells play a central role in the conservation and redelivery of ROS-derived DHA back to photoreceptor cells through the interphotoreceptor matrix.     

Redistribution of Mn++-dependent pyrimidine 5'-nucleotidase (MDPNase) activity during shedding and phagocytosis

Ultrastructural cytochemistry was used to localize a previously undescribed retinal enzyme activity, ie, "manganese-dependent pyrimidine 5'-nucleotidase" (MDPNase) activity in retinas from rats raised in cyclic light and killed at various times in the lighting cycle. In retinas fixed during the last 3 hr of darkness, at the beginning of the shedding period, heavy cytochemical staining was observed over the extracellular surfaces of the apical processes of the retinal pigment epithelium (RPE) cells. The adjacent distal ends of the rod outer segments (ROS), ensheathed by the apical processes, were also stained along their surfaces. During the first 3 hr after light onset, at the time of maximal shedding and phagocytic activity, the tips of the ROS and ROS phagosomes were heavily labeled with reaction product distributed throughout the interdisc spaces. The RPE apical processes, previously heavily stained, were at this time weakly reactive relative to the ROS tips and phagosomes. After the shedding peak (4-8 hr after light onset), the tips of the ROS were no longer labeled with reaction product, and the apical processes were unreactive. The proximal portions of the ROS were weakly stained throughout the lighting cycle. The observed patterns of redistribution of MDPNase activity before, during, and after the shedding peak suggest that the presence of the enzyme in the ROS tips may be correlated with shedding. Changes in the staining of the RPE apical processes in relation to cyclic light further suggest that this enzyme may be transferred from the apical processes to the ROS tips prior to shedding.     

Retinal detachment in the cat: the outer nuclear and outer plexiform layers

The retinae of cats were surgically detached for 1/2 hr to 14 months, and the outer nuclear (ONL) and outer plexiform layers (OPL) were studied by light and electron microscopy. The longer the duration or the greater the height of detachment the more likely was the occurrence of cell death. Histologic signs of degeneration were present 1 hr after detachment. The number of photoreceptor nuclei in the ONL decreased significantly by 1 month. Loss of cells in the ONL occurred by necrosis and by the migration of photoreceptor cell bodies into the subretinal space. The OPL degenerated by the necrosis of cell processes and synaptic terminals and by the retraction of the synaptic terminals. By 2 weeks most synaptic terminals were necrotic or in the process of retracting. Photoreceptor synaptic contact with second order neurons was diminished by 30 days and was essentially absent by 50 days. Müller cells proliferated and hypertrophied; their nuclei and cell processes filled the intraretinal spaces left by the degenerating photoreceptors. In addition, Müller cells protruded into the subretinal space and formed multiple layers of cell bodies and processes between the retina and retinal pigment epithelium. By 14 months these subretinal Müller cell processes covered the entire detached retina, and appeared morphologically like an astroglial scar. Similar changes in human retinal detachments may significantly influence the degree of visual recovery after retinal reattachment, especially in retinae detached for more than a few days.     

急性中心性浆液性脉络膜视网膜病变的频域光相干断层扫描特征

目的 观察急性中心性浆液性脉络膜视网膜病变(CSC)频域光相干断层扫描(FD-OCT)的图像特征.方法 对比分析31例单眼急性CSC患者的FD-OCT以及荧光素眼底血管造影(FFA)检查资料.观察患眼FFA检查发现的渗漏点视网膜色素上皮(RPE)层、视网膜脱离区域以及对侧无症状眼RPE层的FD-OCT图像特征.结果 31只患眼中,FFA检查共发现荧光渗漏点34个.其中,RPE层面18个,占52.9%.荧光渗漏点在FD-OCT图像中表现为色素上皮脱离(PED)者17只眼,占54.8%;8只眼8个渗漏点表现为RPE层突起,分别占患眼及渗漏点的25.8%、23.5%;4只眼的6个渗漏点表现为RPE层不规则,分别占患眼及渗漏点的12.9%、17.6%;2只眼2个渗漏点表现为RPE层无明显异常,分别占患眼及渗漏点的6.5%、5.9%.其中,7只眼7个渗漏点见RPE层缺损,分别占患眼及渗漏点的22.6%、20.6%.所有患眼均可检测到后极部浆液性视网膜脱离以及光感受器内外节(IS/OS)层强反光带的断裂或消失.24只眼可见外节(0S)层厚度不均,占77.4%;10只眼可见外节层剥脱样改变,占32.3%;8只眼可见视网膜神经上皮外层以及RPE表面的颗粒样强反光点,占25.8%.0S层厚度不均的24只眼中,14只眼可见下垂征.31只无症状对侧眼中,11只眼存在RPE异常,占35.5%.其中,2只眼为PED,占无症状对侧眼的6.5%;1只跟为RPE层突起,占无症状对侧眼的3.2%;8只眼表现为RPE层不规则,占无症状对侧眼的25.8%.结论 急性CSC患眼渗漏点处RPE层的FD-OCT图像特征为PED、RPE层突起、RPE层不规则、RPE层无明显异常.视网膜脱离区域FD-OCT图像特征为浆液性视网膜脱离、IS/OS层断裂或消失、OS层厚度不均、OS层剥脱样改变、视网膜神经上皮外层以及RPE表面颗粒样强反光点.     

Expression pattern of erythropoietin and erythropoietin receptor in experimental model of retinal detachment

PURPOSE: Erythropoietin (EPO), known for its role in erythroid cell differentiation, has been suggested to have a neuroprotective effect on retinal neurons. The aim of the current study was to investigate the expression pattern of EPO and erythropoietin receptor (EPOR) in the detached retina of a rat model of retinal detachment (RD). METHODS: Forty-eight albino Sprague-Dawley rats were randomized into normal control group (n = 6) and RD model group (n = 42). The rat RD model was established by slow injection of 1.4% sodium hyaluronate into the upper half of the subretinal space, in which the upper half of the neurosensory retina was detached from the underlying retinal pigment epithelium (RPE). The expression of EPO and EPOR mRNA/protein was determined at 1, 3, 6, 12, 24, 48, and 72 hr after sodium hyaluronate injection by semiquantitative RT-PCR and Western blot analysis, respectively. Meanwhile, the distributions of EPO and EPOR were examined by immunohistochemistry. RESULTS: EPO and EPOR mRNA levels increased gradually after sodium hyaluronate injection and peaked 48 hr later. EPO mRNA level became significantly higher than that in the normal control group at 12 hr after injection, and EPOR mRNA level became significantly higher at 6 hr (p < 0.05). Moreover, the protein levels of EPO and EPOR presented a similar tendency to the mRNA level and became significantly higher than those in normal control group at 3 hr after injection (p < 0.05). Immunohistochemical results showed that both EPO and EPOR were strongly expressed all over the neurosensory retina 48 hr after injection, but EPOR was not found in the rod outer segment. Only weak expression of EPO and EPOR was noticed in the retina of normal control group. CONCLUSIONS: The levels of EPO and EPOR in detached retina increase with the duration of retinal detachment and reach their peaks at 48 hr; most layers of detached neurosensory retina can express EPO and EPOR. It is indicated that the EPO/EPOR system might play an important role in protection of retinal neurons during RD; supplementation with exogenous EPO might promote the survival of retinal neurons.     

Facilitation of artificial retinal detachment for macular translocation surgery tested in rabbit

PURPOSE. For macular translocation surgery, the native attached retina has to be detached either locally or completely. Although different surgical techniques are used, there is a general search for supporting procedures that facilitate and accelerate the retinal detachment. METHODS. Pars plana vitrectomies were performed in pigmented rabbits. In all experimental groups, a local retinal detachment was created by infusing the test solution with a thin glass micropipette attached to a glass syringe. In control animals a standard balanced salt solution was used at room temperature, in combination with a standard vitrectomy light source. In two test groups, a calcium- and magnesium-free solution was used for the vitrectomy, under illumination by a standard light source in group I (solution at room temperature) and group II (solution heated up to body temperature). In group III the rabbits were dark-adapted for half an hour, and then, during surgery, a red filter was used in front of the light source (standard balanced salt solution at room temperature). After the rabbits were killed at the end of surgery, the adherence of the retinal pigment epithelium (RPE) to the neural retina in the detachment area was quantified microscopically, and the morphologic integrity of the detached retinal tissue was examined by light and electron microscopy. No electrophysiology was performed. RESULTS. In all four groups, it was possible to detach the retina. The maximum adherence of the RPE cells to the neural retina was observed in the control group. Virtually no decrease in adherence was found in test group II (36 degrees C solution without calcium and magnesium), whereas a significant decrease was seen in both group I (calcium- and magnesium-free solution at room temperature) and group III (dark adaptation-red light technique; standard balanced salt solution at room temperature). In none of the experimental groups was any obvious damage of the retinal structure observed, even after exposure to the test solutions for 60 minutes. CONCLUSIONS. Both dark adaptation (red illumination) and the use of a calcium chloride- and magnesium chloride-free solution (at room temperature) can facilitate retinal detachment in macular translocation surgery. Both techniques are proposed as a gentle support for the operation, because they protect an intact RPE cell layer and do not cause retinal damage at the ultrastructural level.     

Rhodopsin regeneration in the normal and in the detached/replaced retina of the skate.

The bleaching and regeneration of rhodopsin in the skate retina was studied by means of fundus reflectometry, both in the normal eyecup preparation and after the retina had been detached and then replaced on the surface of the pigment epithelium (RPE). After bleaching virtually all the rhodopsin in the retinal test area of the normal eyecup, more than 90% of the photopigment was reformed after about 2 hr in darkness; over most of this time course, rhodopsin density rose linearly at a rate of 0.875% min-1 with a half-time of 55 min. Detaching the retina from its pigment epithelium resulted in a number of abnormalities, both structural and functional. Histological examination of the detached/replaced (D/R) retina showed striking alterations in the structural integrity of the RPE cells at their interface with the neural retina. The cells appeared vacuolated and misshapen, and the apical processes of the RPE, which normally ensheath the receptor outer segments, were shredded and free of their association with the visual cells. These morphological changes, as well as dilution of the IRBP content of the subretinal space caused by separation of the tissues, appear to be the main factors contributing to the functional abnormalities in rhodopsin kinetics. But despite these abnormalities and the persistent detachment, the rate of regeneration and the amount of rhodopsin reformed after bleaching were reduced by less than 50% of their normal values. The fact that a significant fraction of the bleached rhodopsin was regenerated under these conditions indicates that 11-cis retinal formed in the RPE was able to traverse a much greater than normal subretinal space to reach the opsin-bearing photoreceptor membranes.