Calcium overload is associated with lipofuscin formation in human retinal pigment epithelial cells fed with photoreceptor outer segments.

PURPOSE: To investigate the role of Ca²(+) in lipofuscin formation in human retinal pigment epithelial (RPE) cells that phagocytize bovine photoreceptor outer segments (POSs). METHODS: Cultured human RPE cells fed with 2 × 107per l bovine POS were treated with flunarizine, an antagonist of Ca²(+) channel, or/and centrophenoxine, a lipofuscin scavenger. The Ca²(+) changes and lipofuscin formation were measured with fluoresence dye Fluo-3/AM ester, laser scanning confocal microscopy (LSCM) and flow cytometry (FCM). The activity of RPE cells was measured by methyl thiazolyl tetrazolium (MTT) assay and argyrophilic nucleolar organizer regions (AgNORs) assay. RESULTS: The Ca²(+) fluorescence intensity (CFI) of RPE cells fed with POS was significantly increased compared with the controls (165.36 ± 29.92 U). It reached a peak with 777.33 ± 63.86 U (P<0.01) at 12 h, and then decreased but still maintained a high level of 316.90 ± 36.07 U (P<0.01) for 4 days. Flunarizine and centrophenoxine significantly decreased the Ca²(+) overload to 227.18 ± 14.00 U at 12 h and 211.06 ± 20.45 U at 4 days. FCM confirmed these changes. The drugs also showed an inhibitory effect on the lipofuscin formation. The proliferation rate of the cells fed with POS increased significantly. Both drugs had inhibitory effects on the activity of the cultured cells. This tendency was confirmed by AgNORs assay. CONCLUSIONS: The Ca²(+) inflow initiated lipofuscin accumulation in RPE cells fed with POS. Flunarizine and centrophenoxine can decrease Ca²(+) overload and lipofuscin formation in RPE cells, accompanied by maintaining cellular vitality.

Strengthening tight junctions of retinal microvascular endothelial cells by pericytes under normoxia and hypoxia involving angiopoietin-1 signal way.

PURPOSE: To determine the effects of pericytes and angiopoietin-1 on the expression of occludin and zonula occludens-1 (ZO-1) in retinal endothelial cells (ECs) under both normoxic and hypoxic conditions. METHODS: Rat primary retinal microvascular ECs were cultured under normoxia or hypoxia in either absence or presence of pericytes conditioned medium (PCM). PCM was pretreated with or without angiopoietin-1 neutralizing antibody. Immuofluorescent staining, Western blot and RT-PCR were used to detect the alterations of occludin and ZO-1 expression. RESULTS: Under normoxia, PCM strengthened occludin and ZO-1 immunofluorescent staining at cytomembrane as well as increased their expression at both protein and mRNA level. When pretreated with angiopoietin-1 neutralizing antibody, occludin upregulation induced by PCM was significantly blocked at protein level (62%) and mRNA level (34%). Under hypoxia, the continuity of occludin and ZO-1 staining at cell boundaries was disrupted consistent with a decrease of their protein level by 31 and 27%, respectively. Also occludin and ZO-1 mRNA level decreased by 46 and 57%, respectively. PCM was observed to partially increase expression of occludin at protein and mRNA level. Angiopoietin-1 antibody slightly inhibited (16%) PCM induced occludin mRNA increase under hypoxia. CONCLUSION: Pericytes improved the integrity of endothelial barrier through inducing occludin and ZO-1 expression at protein and mRNA level under normoxia. Under hypoxia, pericytes could partially reverse occludin decrease. These protecting effects of pericytes on endothelial barrier were at least in part mediated by angiopoietin-1.

Decreased chaperone activity of alpha-crystallin in selenite cataract may result from selenite-induced aggregation.

PURPOSE: To investigate the role of chaperone activity of alpha-crystallin in selenite-induced cataract formation. METHODS: Selenite cataract was induced in Sprague-Dawley rats by five subcutaneous injections of sodium selenite over a 20-day period starting at 8-10 days postpartum. alpha-Crystallin was separated from the rat lenses by size-exclusion chromatography. Bovine alpha(L)-crystallin and beta(L)-crystallin were isolated for studies in vitro, and for the chaperone assays. The protective effects of both alpha(H)- and alpha(L)-crystallin were measured spectrophotometrically in four different assay procedures including the thermally induced aggregation of catalase and beta(L)-crystallin, and the fructation- and heat-induced inactivation of catalase. The bovine alpha(L)-crystallin was incubated with different concentrations of sodium selenite for 72 h and then its chaperone activity against heat-induced beta(L)-crystallin aggregation was assayed. The aggregation of selenite-treated alpha(L)-crystallin was analysed by molecular sieve high-performance liquid chromatography (HPLC). RESULTS: The protection of alpha(H)-crystallin was less than that of alpha(L)-crystallin in both normal and cataractous lenses. The chaperone activities of both alpha(H)- and alpha(L)-crystallin in selenite cataract were decreased compared with normal lenses. The protection provided by both alpha(H)-crystallin and alpha(L)-crystallin against the thermal aggregation of catalase was much greater than their protection against thermally and chemically induced inactivation. HPLC analysis demonstrated aggregation of alpha-crystallin by sodium selenite after 24 h incubation in a dose-dependent fashion. CONCLUSION: The chaperone activity of alpha-crystallin presented parallel patterns of activity with different methods, further supporting the view that the different assays measure essentially the same property. The decreased chaperone activity of alpha-crystallin in selenite cataract may result from selenite-induced aggregation.

Migration of retinal pigment epithelial cells in vitro modulated by monocyte chemotactic protein-1: enhancement and inhibition.

BACKGROUND: The migration of retinal pigment epithelial (RPE) cells is an initial step in the development of proliferative vitreoretinopathy (PVR). This in vitro study was carried out to investigate the effects of monocyte chemotactic protein-1 (MCP-1) on the migration and proliferation of RPE cells. METHODS: We used an in vitro wound healing model in which a small area of a confluent monolayer of human RPE (HRPE) cells was denuded with a razor blade. The cultures were subsequently incubated with MCP-1, IL-1beta, TNF-alpha, or combinations thereof. Neutralizing IgG1 of antihuman MCP-1, dexamethasone (DEX) or daunorubicin were also added to the cultures to test their inhibitory effects on migration of RPE cells. HRPE migration was measured as the number of cells that entered the denuded area. The effect of MCP-1 on proliferation of HRPE cells was examined by MTT assay. RESULTS: MCP-1 stimulated HRPE cell migration in a dose-dependent manner. IL-1beta or TNF-alpha slightly stimulated HRPE cell migration, but adding anti-MCP- IgG1 significantly reduced this effect. MCP-1-induced migration could be inhibited by DEX but not by daunorubicin. MCP-1 did not show a significant effect on HRPE cell proliferation. CONCLUSION: MCP-1 stimulates HRPE cell migration, suggesting that this chemokine regulates the development of PVR at the initial stage. The migration of HRPE cells induced by IL-1beta and TNF-alpha may be associated with the MCP-1 that HRPE cells secretes under the stimulation of these two cytokines. The knowledge that MCP-1-induced migration of HRPE cells is inhibited by DEX may be useful in devising an effective treatment for PVR.

Prevention of experimental proliferative vitreoretinopathy with daunomycin and triamcinolone based on the time course of the disease.

BACKGROUND: Our previous experiments showed a limited effect of treatment with daunomycin when given at the inflammatory phase of the development of proliferative vitreoretinopathy (PVR) induced by macrophages in rabbits. In the present study, we tested the efficacy of daunomycin when given at the proliferative phase and combined with triamcinolone given separately at the inflammatory phase in the same model. METHODS: Four groups of rabbits, 16 animals in each, respectively received 5 microg daunomycin on day 6; 1 mg triamcinolone immediately after macrophage injection; 1 mg triamcinolone immediately and 5 microg daunomycin on day 6 (combined drugs); and 0.1 ml saline (controls). Ophthalmoscopy and 3H-thymidine autoradiography were use to evaluate the effects of drugs on traction retinal detachments and cellular proliferation in the vitreous and on the retina. RESULTS: Retinal detachment occurred in 33.3%, 16.1%, 8.3% and 83.3% (P<0.01) of the eyes treated with daunomycin, triamcinolone, combined drugs, and the controls, respectively. Autoradiography revealed significantly decreased numbers of labelled nuclei on days 7 and 14 in daunomycin-treated eyes compared with controls. Significantly decreased numbers of inflammatory cells and labelled cells were noted in eyes treated with triamcinolone and combined drugs. CONCLUSION: Daunomycin given at the proliferative phase, and combined with triamcinolone given at the inflammatory phase of PVR, can be more effective in preventing PVR development than daunomycin given at the inflammatory phase.

Temporary vitreous gas tamponade by perfluoromethylcyclopentane.

Using perfluoromethylcyclopentane (FMCP; US patent no. 5,441,989, granted 1995) we have developed a new vitreous gas tamponade in a rabbit model that allows complete filling of the vitreous cavity without vitrectomy and without a significant increase in intraocular pressure. In humans this procedure would allow the blockage of inferior and posterior retinal holes without special positioning of the patient. Perfluoromethylcyclopentane (FMCP), a liquid perfluorocarbon with a boiling point slightly above body temperature, is injected in minute volumes into the vitreous cavity, where it vaporizes, thereby filling a gas volume approximately 500 times its liquid volume. FMCP was injected into the midvitreous in six rabbits (six eyes). After 2-3 days a complete gas tamponade was achieved in three eyes. Two eyes showed 75-90% filling, and one eye was filled only 50% with gas. Intraocular pressure was highest in the completely filled eyes, ranging from 26.6 to 38.8 mmHg. In all eyes the maximum expansion of the gas bubble lasted 2 weeks. One eye developed a retinal detachment. All eyes showed transient subcapsular cataracts. The results of this study showed that intravitreal injection of FMCP, a new perfluorocarbon liquid, results in a complete gas tamponade of the vitreous cavity which lasts 2 weeks without severe intraocular pressure rise and without vitrectomy. This procedure will be especially useful for eyes that have retinal detachment from inferior or posterior retinal holes. Injection of a conventional gas such as SF6 or C3F8 usually does not block retinal holes in inferior or posterior locations without tedious positioning and risk of (transient) glaucoma. Since the mechanism of transition of FMCP from liquid to gas in the vitreous is poorly understood, we are currently studying FMCP vaporization in an in vitro eye model.

[Vitrectomy combined with posterior chamber intraocular lens implantation in complicated ocular injuries].

Posterior chamber intraocular lens implantation combined with lensectomy-vitrectomy and/or removal of intraocular foreign bodies in one operation was performed on 12 patients with ocular injuries involving both anterior and posterior segments. There were 6 cases with intravitreal or intraretinal foreign bodies, 3 cases with lens dislocation in the vitreous by blunt trauma and 3 cases with vitreous hemorrhage and cataract. Pre-operatively, the visual acuities of all patients ranged from light perception to 0.02, while postoperatively, their visual acuities were 0.5-1.5 in 8 cases, 0.3 in 2 cases, finger counting and 0.05 in other 2 cases which were related to optic nerve atrophy and retinal vein occlusion, respectively. The results suggest that the combined surgery benefit the fast visual rehabilitation in selected young patients with unilateral ocular injuries.

[Factors affecting the visual prognosis after extraction of congenital cataract with IOL implantation].

Multivariated analysis of corrected visual acuities after extraction of congenital cataract with IOL implantation in 24 patients (41 eyes), including 14 children (22 eyes) under 12 years of age, revealed that 6 factors significantly affected the postoperative results, namely, the pattern of lenticular opacity, monocularity, strabismus, nystagmus, after-cataract formation and its time of onset. Therefore, operation as early as possible is recommended for cases with these factors and IOL implantation considered, with discretion, in children for whom other forms of visual aids are not available.

[Triamcinolone acetonide in the prevention of experimental proliferative vitreoretinopathy].

Macrophages were used to induce an experimental model of proliferative vitreoretinopathy (PVR) for the evaluation of drug efficacy of triamcinolone acetonide in the prevention of PVR. After injection of macrophages into the rabbit vitreous, 1 mg of triamcinolone and 0.1 ml saline, respectively, were also injected into the vitreous of the treated group and the control group. Afterwards, on the 28th day, retinal detachment developed in 77% of the eyes in the control group and in 13% of the eyes in the treated group (n = 30, P < 0.01). The time of triamcinolone being cleared up from the vitreous was 35-63 days (average 45.5 days). Electroretinogram and transmission electron microscopic examinations demonstrated that up to 4 mg of triamcinolone was nontoxic to the retina. The results suggest that during inflammatory stage, the use of triamcinolone effectively and safely prevent the development of PVR.

[Primary implantation of posterior chamber intraocular lenses in eyes with defective posterior capsule].

Primary implantation of posterior chamber intraocular lens (PC IOL) was performed in 70 eyes with defective posterior capsule following extracapsular cataract extraction. The visual acuity was 0.5 or better at 3 months and 12 months postoperative in 97.1% and 88.6% of the cases respectively. Complications were rare. The authors put forth the following principles for such cases: (1) injection of air or Healon to maintain a deep anterior chamber: (2) the insertion or rotation of the IOL should be away from the area of capsular rupture; (3) the long axis of the IOL should cross the meridian of the posterior capsule rupture: (4) the residual anterior capsule may be utilized to support the IOL; (5) complete clearance of any vitreous in the anterior chamber; and (6) performance of at least 2 peripheral iridectomies to guard against pupillary block. This technique is applicable in eyes with peripheral posterior or capsule defect of less than 120 degrees or with central rupture of less than 4mm.

[The operative use of perfluorodecalin in severe proliferative vitreoretinopathy].

Perfluorocarbon liquids can be used as an operative hydrodynamic tool during vitreous surgery. The high specific gravity exerts a pressure on the retina to open up the retinal funnel, to displace the subretinal fluid and to stabilize the posterior retina for membrane peeling, endo-laser photocoagulation, and to release anterior traction. The authors reviewed 27 patients of a variety of severe proliferative vitreo-retinopathies (PVR) treated with the perfluorocarbon procedure, including idiopathic PVR in 9 cases, traumatic PVR in 7 cases, aphakic or pseudophakic PVR in 5 cases, intraocular inflammatory PVR in 4 cases and other forms in 2 cases. The success rate was 81.5% and the final visual acuity ranged from 0.05 to 0.5 in 50.0% of the patients that attained retinal reattachment. The retina failed to reattach in patients with rupture of the eyeball or advanced proliferative diabetic retinopathy. The surgical techniques and management of operative complications were discussed.

Repair of outer blood-retinal barrier after severe ocular blunt trauma in rabbits.

Retinal contusion is a leading cause of visual loss in ocular blunt trauma. However, its pathogenesis remains controversial. We established a rabbit model of severe retinal contusion with energy of about 2.87 J. Typical retinal edema and sometimes subretinal hemorrhage reproducibly occurred at the posterior pole after injury. These subsided 1 week later with depigmentation in the lesion. Histopathological examination revealed severe damage of the outer layer of retina, for example, disruption of retinal pigment epithelium (RPE) and photoreceptors. Electroretinography showed a decrease in the b wave by 38-47% in amplitudes (P < 0.01) during the first 3 days and then returned, although not to normal level. To investigate the damage and repair of blood-retinal barrier (BRB), 5 ml 2% lanthanum solution (La) was injected via the common carotid artery 1-2 min before enucleation. La diffused in the interphotoreceptor space through the damaged junctions of RPE 1 h-3 days after injury. La also reached the nuclei level of photoreceptors up to 14 days after injury. Although a glial scar with scattered RPE cells attached to Bruch's membrane in the severely damage area, no La diffusion was found in the retina 4 weeks after trauma. These results showed incomplete repair of outer BRB after severe blunt trauma.

Corticosteroids and daunomycin in the prevention of experimental proliferative vitreoretinopathy induced by macrophages.

An experimental model of proliferative vitreoretinopathy (PVR) induced by macrophages simulates a special form of wound healing process in the eye and mimics the development of PVR from its initial stage. We used this model for the evaluation of drug efficacy in the prevention of PVR. One mg triamcinolone acetonide (TA), 10 micrograms daunomycin-liposome (DL), 5 micrograms free daunomycin (FD) and 0.1 ml saline or empty liposomes (as controls) were injected into the vitreous in four groups of animals (30 or 40 rabbit eyes each) after macrophage injection. Retinal detachment developed in 77.5% of the control eyes on day 28, compared to 13.3% of the TA-treated eyes (P < 0.01), to 33.3% of the eyes treated with DL (P < 0.01), and 50% of the FD-treated eyes (P < 0.05). TA cleared up from the vitreous within 35-63 days (average 45.5 days). The half-time of FD clearance was 145.5 min. Although DL declined rapidly during the first 2 days, there was an average of 0.64 microgram/ml daunomycin in the vitreous on day 14. Transmission electron microscopy showed that FD at a dosage of over 5 micrograms or DL over 20 micrograms was toxic to the retina and that up to 4 mg TA was nontoxic. These results suggest that steroids such as TA, given at the inflammatory stage, can effectively and safely prevent the development of PVR, and that encapsulation in liposomes of cytotoxic agents such as daunomycin can enhance drug efficacy and reduce toxicity. The time course of initiation and development of PVR is important in the selection of particular drugs.

Ultrastructures of the glial epiretinal membrane induced by activated macrophages.

A rabbit model of glial epiretinal membrane was established following the injection of activated macrophages into the vitreous. The membrane was composed entirely of cells with glial characteristics, ie, abundant intermediate filaments, microvilli, junctional complexes and basement membranes. The extracellular matrix of the mature membranes contained collagen fibrils of 10 to 15 and 20 to 25 nm in diameter. Fusiform densities were seen adjacent to the cell membrane and cells with indented nuclei were found in thick membranes. These observations demonstrate that glial cells in epiretinal membranes may synthesize collagen and possess myofibroblast-like properties.

[Ultrastructures of the glial epiretinal membrane].

Rabbit models of glial epiretinal membrane were established following the injection of activated macrophages into the vitreous. The membrane was composed of cells with glial characteristics, i.e., abundant intermediate filaments, microvilli, junctional complexes, and basement membranes. The extracellular matrix of mature membranes contained collagen fibrils 10 to 15 and 20 to 25 nm in diameter. Fusiform densities were seen adjacent to the cell membrane, and cells with indented nuclei were found in thick epiretinal membranes. These observations demonstrate that glial cells in the epiretinal membrane synthesize collagen and may have properties like myofibroblasts.

Fibrovascular proliferation and retinal detachment after intravitreal injection of activated macrophages in the rabbit eye.

Injection of activated macrophages into the posterior vitreous of the rabbit induced vigorous fibrovascular proliferation over the optic disk and medullary rays, as demonstrated by 3H-thymidine autoradiography. One week after injection, endothelial cells and pericytes of the capillaries near the inner surface of the optic disk and rays were labeled; fibroblast-like cells, which were also labeled, migrated and formed vitreous strands. By the second week after injection, the fibrovascular tissue proliferated most actively, and traction medullary ray detachment and peripapillary retinal fold formation were observed. The cellular proliferation was accompanied by inflammatory cell infiltration. Glial cells within the optic disk, as well as retinal pigment epithelial cells beneath the detached retina, were labeled by 3H-thymidine. These results demonstrate that the fibrovascular proliferation originates from the vessel complex of the optic disk and medullary rays in this experimental model of retinal detachment.

Glial epiretinal membranes and contraction. Immunohistochemical and morphological studies.

It has been suggested that glial cells do not contribute substantially to the contractile forces generated by epiretinal membranes. We have established a rabbit model in which epiretinal membranes form on the inferior peripheral retina after the injection of activated macrophages into the vitreous. By two months, the membranes were extensive but without evidence of traction. At four months, however, full-thickness retinal folds were present beneath the thick epiretinal membrane. A homogeneous glial cell composition was suggested by light microscopic examination of serial sections through several membranes. Immunohistochemical staining with anti-glial fibrillary acidic protein and antivimentin and immunoelectron microscopy confirmed that these thick epiretinal membranes were composed entirely of glial cells, which may cause mild traction on the retina; this traction is associated with cell alignment and the tissue bridges connecting the membrane and the retina. The fusiform densities and indented nuclei suggested that the glial cells within the membrane may possess some characteristics of myofibroblasts.

Liquefaction of rabbit vitreous by ferrous ions.

Intravitreal injection of 0.7 mumol of ferrous chloride in 0.1 ml into the rabbit eye resulted in liquefaction of the vitreous gel and condensation of vitreous collagen fibrils within two weeks; injection of 0.1 mumol did not cause any obvious vitreous degeneration, although retina damage was noted in the posterior pole. Macrophages migrated at the vitreoretinal interface and local posterior vitreous separation was observed after the injection of ferrous solution. This suggests that the least amount of ferrous ions necessary to cause liquefaction of the rabbit vitreous is in the range of 16.8 to 39.2 micrograms of elemental iron, a concentration of 0.3 to 0.7 mM in the vitreous. Since 0.1 ml of blood contains approximately 50 micrograms of iron, it is possible, at least theoretically, that the iron released by hemoglobin following vitreous hemorrhage could induce liquefaction of the vitreous.

Posterior vitreous separation and retinal detachment induced by macrophages.

Macrophages, which migrate into the vitreous in conditions such as vitreous hemorrhage and penetrating ocular injury, may contribute to the development of intravitreous cellular proliferation and posterior vitreous separation. To investigate this possibility, activated macrophages were harvested from the peritoneal cavity and injected into the vitreous of rabbits. As early as 8 days after macrophage injection, posterior vitreous separation and glial epiretinal membrane formation began to occur. Two weeks after injection, vitreous strands that approached the optic disc and medullary rays were evident; fibroblasts proliferated over the disc or rays and induced retinal detachment. These findings support the hypothesis that macrophages in the vitreous may, in part, mediate cellular proliferation and posterior vitreous separation.