Lowering the calcium concentration in the subretinal space in vivo loosens retinal adhesion.

Retinal adhesiveness in vitro is reduced by lowering the external calcium (Ca2+) concentration. The effects of lowering subretinal Ca2+ concentration in living rabbit eyes was investigated by making experimental retinal detachments (blebs) filled with Ca(2+)-free disodium edetate solution. Unlike blebs made with Hanks' solution, these low-Ca2+ blebs enlarged progressively after they were formed, and they were surrounded by a wide whitish halo. This halo region had weak adhesion (shown by the rapid spread of fluorescein solution into the halo and by the measurement of local adhesiveness after enucleation). The retinal pigment epithelial microvilli in the halo appeared stretched toward the center of the blebs as if there had been retinal traction or movement. Measurements of retinal adhesiveness in vivo showed it to be decreased to about 30% of normal by use of this solution.     

Measurement of retinal adhesive force in the in vivo rabbit eye

A new method to evaluate the retinal adhesive force, which was defined as the force needed to achieve adhesive failure per unit length, in the in vivo rabbit eye was tested. A small dome-shaped retinal detachment (bleb) was made in the posterior pole by injecting balanced salt solution into the subretinal space. A separate measuring micropipette with a tip diameter of 5 microns was inserted into the subretinal space. The subretinal pressure was measured directly with the resistance servonulling method as the solution was injected repeatedly into the subretinal space. According to Laplace's law, the retinal adhesive force per unit length in normal Dutch rabbits was calculated to be (1.8 +/- 0.2) X 10(2) dyn/cm (n = 87). This value was about five times larger than that reported previously with in vitro peeling methods. The new method will resolve several problems encountered in the previous methods and will allow for the measurement of the retinal adhesive force more physiologically and precisely.     

Light-induced migration of retinal microglia into the subretinal space.

PURPOSE: To explore the effects of light exposure and deprivation on the distribution and function of microglia in the subretinal space of mice. METHODS: Using a monoclonal antibody, 5D4, that identifies resting, ramified microglia, the distribution and density of microglia in the retina, and the subretinal space were determined by confocal microscopy and by immunohistochemistry of cryopreserved sections of eyes of albino and pigmented mice exposed to diverse levels of light, ranging from complete darkness to intense brightness. Axotomized retinal ganglion cells were retrograde labeled by fluorescent tracer to determine whether the marker colocalizes to 5D4+ cells. Electron microscopy was used to evaluate microglia for evidence of phagocytosis. RESULTS: 5D4+ microglia in pigmented eyes were limited to the inner retinal layers, but in albino eyes 5D4+ cells were found in the outer retinal layers and subretinal space as well. The subretinal space of eyes of albino mice raised from birth in complete darkness contained few 5D4+ cells, but exposure to light caused the rapid accumulation of 5D4+ cells at this site. 5D4+ cell density in the subretinal space correlated directly with intensity of ambient light. Retrograde labeling of axotomized ganglion cells resulted in 5D4+ cells in the subretinal space that contained the retrograde label. Subretinal microglia contained phagocytized rod outer segment discs. On intense light exposure, 5D4+ cells adopted an active morphology, but failed to express class II major histocompatibility complex (MHC) molecules. CONCLUSIONS: Light exposure induced retinal microglia migration into the subretinal space in albino mice. Subretinal microglia appeared to augment through phagocytosis the capacity of pigment epithelium to take up the photoreceptor debris of light toxicity. The unexpected presence of these cells in the subretinal space raises questions concerning their potential contribution to immune privilege in this space and to the fate of retinal transplants.     

Photothermal, cryogenic, and diathermic effects of retinal adhesive force in vivo.

Argon laser photocoagulation, cryoretinopexy, and diathermy of moderate intensity is applied to 140 rabbit eyes. Retinal adhesive force is measured from 1 day to 6 months following the treatment, using the authors' new in vivo method. A small retinal detachment is induced within the area surrounded by the burns. At the moment when the detachment starts to expand beyond the burns, subretinal pressure is measured using a resistance servo-nulling method. Retinal adhesive force is then calculated according to Laplace's law and compared with that of untreated eyes. Both photocoagulation and diathermy enhanced adhesiveness within 24 hours to 128% and 122%, respectively, of normal levels. Cryoretinopexy reduced the retinal adhesiveness during the first week, but afterwards generated as much adhesiveness as the other two methods. Beyond 6 months, under the conditions described here by the authors, the adhesive force was stronger after diathermy (279%) than after cryoretinopexy (214%) or laser photocoagulation (220%).     

Effects of indocyanine green injection on the retinal surface and into the subretinal space in rabbits

PURPOSE: To evaluate the effects of indocyanine green (ICG) injection on the retinal surface and into the subretinal space of rabbit eyes. METHODS: Twenty-two Dutch-belted rabbits underwent two-port vitrectomy followed by injection of ICG (5 mg/mL) on the retinal surface and into the subretinal space. Balanced salt solution (BSS) was also injected subretinally. The locations where ICG was delivered (both epiretinal and subretinal) were exposed to light from an endoilluminator for 7 minutes. The animals were examined at 1, 7, and 14 days after surgery. The eyes were studied by fluorescein angiography as well as light and electron microscopy. RESULTS: No damage was observed after epiretinal ICG injection, but subretinal ICG injection resulted in damage to the outer nuclear layer, photoreceptor inner and outer segments, and retinal pigment epithelium. This damage was more severe with longer follow-up. Control experiments without ICG, in which balanced salt solution was injected into the subretinal space or light was delivered on the epiretinal surface, demonstrated only damage to the photoreceptor outer segments. CONCLUSION: Subretinal delivery of ICG (5 mg/mL) in rabbits induces retinal pigment epithelium, photoreceptor inner and outer segment, and outer nuclear layer damage. These mechanisms of damage may explain the retinal pigment epithelium changes that are sometimes seen after ICG-assisted internal limiting membrane peeling in humans.     

Surgical treatment of retinal detachment with and without puncture of the subretinal space

Proposed are indications for a drainage of the subretinal space on the basis of results of 345 retinal detachment operations with and without evacuation of the subretinal fluid. These indications are dependent on the recognition of the so called prognostical factors unfavourable for the surgical treatment of retinal detachment.     

Shifting subretinal fluid in rhegmatogenous retinal detachment.

In a consecutive series of 470 cases of rhegmatogenous retinal detachment 25 (5%) were found to have shifting subretinal fluid (SRF) at the preoperative examination. The study showed that the association between SRF and rhegmatogenous retinal detachment is unusual but not rare. Shifting SRF was most often associated with aphakic and longstanding retinal detachment, and found in cases in which the retinal holes were small.     

Pathologic response of the retinal pigment epithelium. The effect of mucopolysaccharide in the subretinal space to phagocytosis. Part 5

Previously we reported that retinal pigment epithelial cells (RPE) showed different phagocytotic activity according to the charge characteristics of the surface of polystyrene particles which were injected into the subretinal space of albino rats. In this study we examined whether subretinal mucopolysaccharide controls RPE phagocytosis or not, using positive or negative charged particles (3 microns in diameter). The technique of ruthenium red staining, a cationic dye, was employed with special efforts to detect the relationship between acid mucopolysaccharide and particles at the electron microscopic level. Six hours after injection of the positive charged particles, the ruthenium red staining revealed fine granular electron-dense materials, coating the surfaces of many small substances surrounding polystyrene particles which were not yet phagocytized by RPE. On the other hand, there was no staining of negative ones which were already phagocytized by RPE. At 24 hours, no staining was observed on the surface of either particles. These findings suggest that an anionic property of mucopolysaccharide in the subretinal space controls the phagocytosis of RPE.     

视网膜下腔免疫赦免研究进展

视网膜下腔是免疫赦免部位,能诱导免疫偏离,产生免疫耐受。血-视网膜屏障的隔离作用、TGF-β和IFN等免疫因子的调节作用;CD95L、可溶性因子、Galectin-Ⅰ和TRAIL等所介导的RPE对T淋巴细胞的调节作用均参与了视网膜下腔的免疫赦免机制。RPE、小胶质细胞以及视网膜微血管内皮细胞等相关APC均参与了移植物的免疫排斥反应过程。视网膜移植免疫排斥反应过程中,免疫抑制剂的使用和免疫耐受状态的产生可以有效降低术后免疫排斥反应的发生。     

Transplantation of embryonic retina to the subretinal space in rabbits.

Embryonic rabbit retina can be transplanted to the subretinal space of adult rabbit with a new method, which gives a high rate of successful short-term transplants. Embryonic (stage E 15) neural retina cells were injected through an incision just behind the sclerocorneal border with a thin (inner diameter 0.15-0.4 mm, outer diameter 0.3-0.5 mm) plastic tube attached to a specially designed instrument, by which the length of the protruding plastic tip could be controlled. The retina was penetrated from the vitreous side and the donor tissue was injected into the subretinal space. The cells survived in the host for at least 5 months, although the long-term survival rate tended to decrease. The transplanted cells matured and differentiated, forming an approximation of the layered, retinal structure with some anomalies (e.g. rosettes). The subretinal location offers an interesting and convenient way of studying the development of retinal cell transplants in rabbits. Large transplants can be produced, and the risk for failures due to erroneous vitreous placement is small.     

Immunolocalization of N-acetylgalactosaminylphosphotransferase in the adult retina and subretinal space.

The cell surface N-acetylgalactosaminylphosphotransferase (GalNAcPTase) modulates N-cadherin-mediated adhesion among embryonic chick retinal cells (Balsamo et al., 1990). We are investigating the potential role of this transferase in modulating adhesive interactions in the adult retina. Using a previously characterized monoclonal anti-GalNAcPTase, we have used immunohistochemical and immunoblot techniques to localize and characterize the transferase in the retinas of the post metamorphic frog (Xenopus laevis), adult cow, and adult cynomolgus macaque. On frozen sections, anti-GalNAcPTase specifically labels the outer segments of photoreceptors in all species. Immunolabel appears at the surface of, or between rod outer segments in all species. The nerve fiber layer also shows high immunoreactivity in all species. Monkey cone outer segments are also highly immunoreactive. Photoreceptor inner segments are clearly immunoreactive in the cow retina. Immunoblots of purified cow rod outer segments show immunolabeled bands near 220 kDa, which is the molecular mass of the GalNAcPTase used as immunogen. Purified Xenopus rod outer segments are not immunoreactive on blots, while soluble interphotoreceptor matrix (IPM) shows immunoreactive bands principally at 113-130, and 166 kDa. Cow soluble IPM shows immunoreactivity at 180 kDa. Based on these findings, we propose that the GalNAcPTase, or a fragment thereof, is a component of the IPM, and perhaps of the photoreceptor outer segment as well.     

A new method to evaluate retinal adhesive force in living rabbit eye

A new method to evaluate the retinal adhesive force in rabbit eye in vivo is described. A small dome-shaped retinal detachment (bleb) was made in the posterior pole fundus of Dutch rabbit by injecting balanced salt solution into the subretinal space through a micropipette. The transmural pressure was directly measured with a micropressure measuring system, utilizing the resistance servo-nulling methods when the bleb started to extend again. According to Laplace's law, the retinal adhesive force of normal Dutch rabbits was calculated to be (1.8 +/- 0.2) x 10(2) dyn/cm (n = 84). This value was about 5 times larger than that previously obtained by peeling methods in vitro. Our newly developed method might resolve several problems encountered in the previous methods and allow for the measurement of the retinal adhesive force more physiologically and precisely.     

A tissue-engineered approach towards retinal repair: Scaffolds for cell transplantation to the subretinal space

Background  

Several mechanisms of retina degeneration result in the deterioration of the outer retina and can lead to blindness. Currently, with the exception of anti-angiogenic treatments for wet age-related macular degeneration, there are no treatments that can restore lost vision. There is evidence that photoreceptors and embryonic retinal tissue, transplanted to the subretinal space, can form new synapses with surviving host neurons. However, these transplants have yet to result in a clinical treatment for retinal degeneration.     

Toxicity of endogenous peroxynitrite and effects of puerarin on transplanted retinal pigment epithelial sheets in the subretinal space in mice

AIM: To evaluate the toxicity of endogeneous peroxynitrite on transplanted retinal pigment epithelial (RPE) sheets and the effect of puerarin on their survival in the C57BL/6 mice after RPE sheets have been transplanted into SD rats' subretinal space . METHODS: C57BL/6 mice eyes were used to culture RPE cells. Ninety-six SD rats were involved in the experiment. They were divided into control（block control） , streptozotocin(STZ, negative control), untransplanted RPE (positive control) and transplanted RPE groups respectively. Diabetes was induced in SD rats by intra-peritoneal STZ injection in the latter three groups. Saline was injected into the subretinal space of 24 SD rats in the untransplanted RPE group and primary RPE sheets were injected into the subretinal space of 24 SD rats in the transplanted RPE group. Puerarin (45mg/kg) was administrated into both untransplanted RPE and transplanted RPE groups of diabetic rats through intra- peritoneal injection route after RPE sheets transplantation. At 20,40,60 days after surgery,Western blotting analysis, DNA ladder and RT-PCR were used for determining the differences in expression of nitrotyrosine (NT, the foot print of peroxynitrite ), apoptosis and iNOS mRNA in the control, STZ, untransplanted RPE and transplanted RPE groups respectively. HE staining was used for determining the RPE survival in the subretinal space of the transplanted RPE group. RESULTS: Apoptosis and expression of NT and iNOS mRNA were observed in STZ, untransplanted RPE and transplanted RPE groups, but were delayed in untransplanted RPE and transplanted RPE groups in a time-dependent manner compared with control and STZ groups (P＜0.01）. There were no differences between the two groups (P>0.01). NT, DNA ladder, iNOS mRNA were down-regulated, which were associated with the decrease of expression of peroxynitrite. Numerous pigmented cells emerged and increased in number in the subretinal space during the 60-day observation period after transplantation. On day 20, heavily pigmented cells were visible at the transplant site; On day 40, monolayer and multilayered transplant was visible in the subretinal space; On day 60, heavily pigmented monolayer and multilayered transplants with round apical profile were present along Bruch's membrane. CONCLUSION: Puerarin increased the 60-day survival of C57BL/6 mice RPE xenografts in the SD rats' subretinal space, which may be related to its direct inhibition of apoptosis of RPE cells and antagnism of damage of peroxynitrite to RPE cells.     

The effect of subretinal viscoelastics on the porcine retinal function

The functional consequence of long-term retinal detachment in the porcine model is examined by multifocal electroretinography (mfERG). Retinal detachment (RD) in humans leaves permanent visual impairment, despite anatomical successful reattachment surgery. To improve treatment, adjuvant pharmaceutical therapy is needed, and can only be tested in a suitable animal model. The porcine model is promising and the mfERG is well validated in this model. RD was induced in 18 pigs by vitrectomy and healon injection of various concentrations. Preoperatively and 6 weeks postoperatively eight animals were examined by mfERG. The major component P1 was analyzed statistically. Indirect ophthalmoscopy and bilateral color fundus photography (FP) were performed. Selected animals underwent high-resolution optical coherence tomography (OCT). Examination by ophthalmoscopy and FP showed that the RDs remained detached for the 6 weeks of follow-up. The P1 amplitude of the mfERG did not differ significantly between the detached areas, the surrounding attached areas, and the healthy eye (p = 0.25). Similarly, P1 implicit time did not differ between the areas (p = 0.85). The lack of functional consequences of long-term RD makes the porcine model unsuitable for examining adjuvant pharmaceutical RD treatment. Future studies should focus on foveated primates.     

视网膜下间隙移植免疫排斥反应的实验观察

探讨视网膜下间隙的免疫学特点,方法ＤＢＡ／２鼠视网膜加或不加免疫佐剂、人外周血单个核细胞移植到Ｃ５ＹＢＬ／６或ＢＡＬＢ／Ｃ鼠的视多膜下间隙。２周后行眼组织病理学检查。结果视网的植物存活,无炎性细胞浸润。人ＰＢＭＣ移植发生免疫排斥反应。结论视网膜下间隙移植,发生一定限度的免疫赦免。