Characterization of the effects of retinal pigment epithelium-conditioned media on porcine and aged human retina.

BACKGROUND: Retinal pigment epithelium (RPE) cells produce neurotrophic factors that rescue photoreceptors from degeneration. Previously, we showed that conditioned medium (CM) from fetal vs adult RPE cells resulted in significantly better porcine retinal preservation, and possessed significantly higher levels of hepatocyte growth factor (HGF) and pigment epithelium-derived factor (PEDF). This study aimed to further describe the effects of human fetal RPE-CM on porcine and aged human retina, and to characterize its effects biochemically. METHODS: RPE-CM was harvested from passage-2 fetal RPE, 7 days after passage, 24-hours after exposure to basal medium. After culture in RPE-CM, porcine retinal morphology was assessed with confocal microscopy. The effects of RPE-CM on porcine and aged human retina survival were assessed by cytotoxicity and apoptosis biochemical assays. To characterize RPE-CM biochemically, effects of heating, digesting with proteinase-K, dilution, concentration, and fractionation were tested. Recombinant proteins and neutralizing antibodies were used to identify proteins that might contribute to the salutary effects of RPE-CM on porcine retina. RESULTS: Culturing porcine retina in RPE-CM significantly preserved outer nuclear layer width and the number of nuclei in cross-section, and significantly decreased photoreceptor axon retraction. RPE-CM decreased porcine retinal death by 17-34 % (p<0.05) compared to basal medium. Human retina from age-related macular degeneration (AMD) and non-AMD donors responded similarly after culture in RPE-CM. Heating, proteinase-K digestion, and dilution significantly diminished RPE-CM-mediated preservation of porcine retina, whereas concentrating RPE-CM significantly enhanced its preservation of porcine retina. Molecular cut filtration identified retina-preserving activity in the 3-100 kDa filtrate. PEDF or HGF at 90 % receptor occupancy significantly improved retinal preservation over 48 h of culture compared to basal medium. Neutralizing PEDF in RPE-CM decreased its ability to reduce retinal apoptosis by 23-27 % (p<0.05). CONCLUSION: RPE-CM reduced biochemically and histologically measured degeneration in porcine retinae. This effect was concentration-dependent, and can be attributed to a protein component(s) in a 3-100 kDa molecular cut fraction. Human retina (including non-AMD and AMD Caucasian and non-AMD African-American) responds to culture in RPE-CM similarly to porcine retina. Receptor occupancy calculations and retinal viability data indicate that PEDF may be one of the components that contribute to retina preservation by RPE-CM.

Artificial vision.

A number treatment options are emerging for patients with retinal degenerative disease, including gene therapy, trophic factor therapy, visual cycle inhibitors (e.g., for patients with Stargardt disease and allied conditions), and cell transplantation. A radically different approach, which will augment but not replace these options, is termed neural prosthetics ("artificial vision"). Although rewiring of inner retinal circuits and inner retinal neuronal degeneration occur in association with photoreceptor degeneration in retinitis pigmentosa (RP), it is possible to create visually useful percepts by stimulating retinal ganglion cells electrically. This fact has lead to the development of techniques to induce photosensitivity in cells that are not light sensitive normally as well as to the development of the bionic retina. Advances in artificial vision continue at a robust pace. These advances are based on the use of molecular engineering and nanotechnology to render cells light-sensitive, to target ion channels to the appropriate cell type (e.g., bipolar cell) and/or cell region (e.g., dendritic tree vs. soma), and on sophisticated image processing algorithms that take advantage of our knowledge of signal processing in the retina. Combined with advances in gene therapy, pathway-based therapy, and cell-based therapy, "artificial vision" technologies create a powerful armamentarium with which ophthalmologists will be able to treat blindness in patients who have a variety of degenerative retinal diseases.

Epiretinal membrane removal in diabetic eyes: comparison of viscodissection with conventional methods of membrane peeling.

AIMS: To compare conventional methods of epiretinal membrane peeling with viscodissection. METHODS: 154 eyes with proliferative diabetic retinopathy (PDR) that underwent pars plana vitrectomy with membrane dissection (89 traditional, 65 viscodissection) were studied retrospectively. Incidence of retinal breaks (RBs), length of time under anaesthesia, postoperative intraocular pressure, retinal reattachment rate, and final visual acuity (VA) were measured. RESULTS: To compare cases of similar complexity, a "complexity score" was defined. The average complexity score for cases done with and without viscodissection was 4.7 and 3.2, respectively. The mean frequency of RBs in eyes undergoing viscodissection was 0.43 (SD 0.5) v 0.14 (0.35) RBs/eye without viscodissection. In complex cases, the frequency of posterior/peripheral RBs was 0.31 (0.47)/0.13 (0.34) RBs/eye, respectively, with viscodissection v 0.12 (0.33)/0.23 (0.43) RBs/eye without viscodissection. None of these differences were statistically significant. The average preoperative/postoperative VA (logMAR) in the viscodissection cohort was 1.7/1.3 (range 0.3 to >1.9/0.1 to >1.9) v 1.4/1 (range 0.48 to >1.9/0.1 to >1.9) in the non-viscodissection cohort, among eyes with 6 months of follow up. Anaesthesia duration was significantly shorter for cases done without viscodissection (p=0.03), but cases done with viscodissection were significantly more complex than cases done without viscodissection (p<0.0001). CONCLUSION: Viscodissection appears to be a safe and effective alternative technique in eyes with PDR. Owing to the retrospective nature of the study, additional studies are warranted.

Vitrectomy with silicone oil infusion in severe diabetic retinopathy.

AIMS: To determine the results of pars plana vitrectomy (PPV) and silicone oil infusion (SOI) in severe proliferative diabetic retinopathy (PDR). METHODS: The records of 23 eyes (21 patients: 12 males, nine females) with PDR who had undergone PPV and SOI were reviewed retrospectively. RESULTS: Average follow up was 5.4 months (range 1-25). Surgical indications were tractional retinal detachment (TRD) (17.4%), traction-rhegmatogenous retinal detachment (TRRD) (8.7%), TRD with vitreous haemorrhage (VH) (48%), TRD with neovascular glaucoma (NVG) (8.6%), TRD with fibrinoid syndrome (FS) (17.3%). With one operation, the retinal reattachment rate was 17/23 (74%). Among these 23 eyes, 11 (48%) had previously failed vitrectomy, and the retina was attached in 8/11 (73%) with a single procedure. With additional surgery employing PPV and SOI, the final reattachment rate was 20/23 (87%). The only cases with intraocular pressure <5 mm Hg had retinal detachment. Postoperative visual acuity (VA) improved in 10 eyes (44%), was unchanged in three (12%), and decreased in 10 eyes (44%). CONCLUSION: SO tamponade is useful in severely diseased eyes with PDR, even in the presence of rubeosis iridis (RI) and NVG, FS, or in cases with previously failed vitrectomy, especially in the presence of RI.

Use of viscodissection and silicone oil in vitrectomy for severe diabetic retinopathy.

Improvement in surgical techniques has led to improved anatomic and functional success rates following surgery for severe complications of proliferative diabetic retinopathy (PDR). We compared the anatomic and functional outcomes of surgery in a non-randomized, consecutive case series of patients with severe PDR. We found that viscodissection using Healon provides outcomes comparable to conventional pick and scissors dissection. We also found that adjunctive use of silicone oil can salvage selected cases with particularly severe manifestations of PDR (e.g., the fibrinoid syndrome). With proper selection of patients and techniques, the anatomic success rate can exceed 80% even in the most severe cases. The goal of this paper is to show the applicability of using viscodissection and silicone oil infusion during vitrectomy in eyes with severe PDR.

Early attachment of uncultured retinal pigment epithelium from aged donors onto Bruch's membrane explants.

Retinal pigment epithelium (RPE) transplantation might replace cells lost as a consequence of choroidal neovascular membrane excision in patients with age-related macular degeneration (AMD). Autologous transplantation of RPE cells harvested from a peripheral biopsy may overcome problems of immune rejection. To study the feasibility of autologous RPE cell transplantation, the authors examined the attachment of freshly harvested RPE cells from aged donors onto Bruch's membrane explants, debrided to (1) remove or (2) preserve the RPE basement membrane. Human retinal pigment epithelial sheets were harvested from adult donor eyes (N = 12, mean age 79.00 +/- 9.40 years) and, following incubation in collagenase, were mechanically fragmented into microaggregates. Microaggregates (approximately 120 000 cells) were seeded onto the paired explants (7 mm diameter) and incubated for 20 min, 1, 4, or 24 hr at 37 degrees C. The percent coverage of the debrided surface by microaggregates was determined by sampling the center of the explants with scanning electron microscopy. RPE microaggregate attachment to Bruch's membrane was significantly greater at all time points analysed in samples with intact basement membrane versus those with an exposed inner collagenous layer. Coverage of debridements retaining intact RPE basement membrane was 1.83 +/- 1.10% at 20 min, 3.54 +/- 2.14% at 1 hr, and 8.68 +/- 2.63% at 4 hr. Coverage of debridements lacking basement membrane was 0.10 +/- 0.04% at 20 min, 0.39 +/- 0.25% at 1 hr, and 0.63 +/- 0.42% at 4 hr. Based on their morphologic appearance, many cells were dying as early as 1 hr following seeding. To increase surface coverage, the authors seeded four times the above number of cells and incubated the specimens for 1 hr. Coverage on explants lacking RPE basement membrane showed no increase in the number of cells attached to the inner collagenous layer. There was a significant approximately three-fold increase in the number of cells attached in the presence of basement membrane. These results indicate that if RPE cells from aged human donors are used for transplantation, some modification of the Bruch's membrane surface or the cells must be considered for cell attachment and eventual cell survival.

Short-term study of allogeneic retinal pigment epithelium transplants onto debrided Bruch's membrane.

PURPOSE: To investigate the survival and behavior of retinal pigment epithelium (RPE) microaggregates transplanted onto hydraulically debrided Bruch's membrane and to compare results of using three different vehicles for cell delivery. METHODS: RPE microaggregates obtained from male cats were transplanted onto the tapetal area of female cats after native RPE was debrided. For the control, one of three vehicles was introduced into the debridements. Each transplant or control specimen was analyzed histologically and immunohistochemically. Transplanted male RPE cells were identified by in situ labeling of the cat Y chromosome. RESULTS: Histologically, significant numbers of condensed, darkly stained RPE nuclei were observed in all transplants compared with few TUNEL-positive RPE cells. Cellular retinaldehyde-binding protein was present up to day 7 in all RPE cells in transplants. In both transplant and control specimens, the antibody against the Ki-67 nuclear antigen labeled some RPE cells at day 3. TUNEL-positive outer nuclear layer nuclei were most frequently observed at day 1, but were much less frequent at 7 days in both transplant and control specimens. CONCLUSIONS: Transplanted RPE appeared to retain at least some markers of differentiation up to 7 days after surgery. Some proliferation of transplanted RPE cells was also seen. Apoptotic cell death of transplanted RPE, as judged by TUNEL staining was observed rarely. RPE transplants imposed no adverse effect on the overlying retina. RPE survival appeared to be similar with each of the three vehicles for cell delivery.

Anterior segment ischemia and retinal detachment after vertical rectus muscle surgery.

PURPOSE: The authors describe the clinical course of a woman who developed two complications following vertical strabismus repair: anterior segment ischemia (ASI) and retinal detachment. METHODS: A 62 year-old woman is described. She presented with new onset proptosis and left hypertropia with significant diplopia in all fields of gaze. This presentation, her 15 year history of thyroid disease, and preoperative computed tomography (CT) of the orbits were consistent with Graves' ophthalmopathy. Vertical strabismus repair was carried out by recessing the left superior rectus muscle and resecting the left inferior rectus muscle. RESULTS: The diplopia was eliminated. The patient developed significant postoperative ASI and iatrogenic rhegmatogenous retinal detachment in the left eye due to unsuspected globe perforation. She was treated with systemic corticosteroids and radial scleral buckling. CONCLUSIONS: Severe ASI following strabismus surgery is a well recognized complication, with age, thyroid ophthalmopathy, and manipulation of the vertical rectus muscles as risk factors. The retinal detachment soon after strabismus surgery was difficult to detect, possibly due to diminished visualization of the posterior segment as a result of ASI.

Morphologic preservation and variability of human donor retina.

PURPOSE: To facilitate studies of human retina and utilization of human retinal tissue for treatment of retinal diseases, we studied morphologic preservation in postmortem human retina. METHODS: Morphology of retinas from thirty-one human eyes was examined using light and electron microscopy. The inner and outer retina, rod and cone photoreceptor cells, and central and peripheral retina were compared with regard to morphologic preservation. Possible factors affecting survival were analyzed. RESULTS: The earliest postmortem change was vacuolation of the nerve fiber layer within a few hours postmortem, followed by vacuolation and cytoplasmic swelling of the inner retina. As compared with the inner retina, outer retinal structure was better preserved, i.e., the photoreceptor cells maintained better morphology. Rod cell morphology was better preserved than cone cell morphology, with good preservation of the rod outer segment disc membranes and the inner segment mitochondrial membranes. Thus, well-preserved rod photoreceptor cells were evident in specimens at least 48-hours postmortem. Peripheral retina was better preserved than the central retina including the fovea and perifovea. Factors affecting anatomical integrity included the total time postmortem and, more importantly, the time between death and enucleation. Other factors, including age and sex, did not appear to affect morphological preservation in the present study. CONCLUSIONS: Human retina postmortem remained morphologically intact for a relatively long period of time, with differential preservation among different geographic areas and cell types. This morphologic evidence is consistent with previous findings of functional preservation (e.g. , photoresponses) in such tissue. This study may shed some light on understanding of human retina and its utilization for retinal transplantation.

Mutilating facial sarcoidosis.

We report a patient with massive facial sarcoidosis. While skin involvement is a common manifestation of sarcoidosis, it is unusual to see it in the dramatic form of cutaneous tumors with mutilation of the central face. There are few reports of tumoral cutaneous sarcoidosis like that of our patient.

Central retinal vein occlusion: report of two familial cases.

The authors report a 46-year-old father and 17-year-old son who each presented with unilateral central retinal vein occlusion (CRVO) and bilateral abnormalities of retinal vascular perfusion. The son presented with a nonperfused CRVO in the left eye, developed traction-rhegmatogenous retinal detachment treated with vitreous surgery, and developed prolonged arteriovenous filling in the retina of the fellow eye. The father presented with progressive CRVO in the right eye, developed choroido-vitreal neovascularization following laser treatment to create a chorioretinal anastomosis, underwent vitrectomy for retinal detachment and vitreous hemorrhage in that eye, and developed prolonged arm-eye and retinal arteriovenous circulation times in the fellow eye. An extensive evaluation (including hematological studies and imaging of the major vessels of the neck) failed to reveal a predisposing cause in either patient although echocardiography disclosed a mitral valve thrombus in the father. After institution of coumadin therapy, the circulatory parameters in the fellow eye of each patient improved.

Central retinal vein occlusion: review of management.

Central retinal vein occlusion is usually a disease of the elderly and is often associated with systemic vascular disease, e.g., hypertension, diabetes mellitus, arteriosclerotic vascular disease. Younger patients, especially those less than 45 years of age, with retinal vein occlusion should be evaluated carefully for the possibility of an underlying thrombotic tendency. The authors describe the ocular manifestations, pathogenesis, associated conditions, patient evaluation, and treatment of patients with central retinal vein occlusion.

Drusen in age-related macular degeneration: pathogenesis, natural course, and laser photocoagulation-induced regression.

Drusen are subretinal pigment epithelial deposits that are characteristic of but not uniquely associated with age-related macular degeneration (AMD). Age-related macular degeneration is associated with two types of drusen that have different clinical appearances and different prognoses. Hard drusen appear as small, punctate, yellow nodules and can precede the development of atrophic AMD. Areolar atrophy of the retinal pigment epithelium (RPE), choriocapillaris, and outer retina develop as the drusen disappear, but drusen can regress without evidence of atrophy. Soft drusen appear as large (usually larger than 63 microm in diameter), pale yellow or grayish-white, dome-shaped elevations that can resemble localized serous RPE detachments. They tend to precede the development of clinically evident RPE detachments and choroidal neovascularization. Drusen characteristics correlated with progression to exudative maculopathy include drusen number (five or more), drusen size (larger than 63 microm in diameter), and confluence of drusen. Focal hyperpigmentation in the macula and systemic hypertension also are associated with an increased risk of developing choroidal new vessels (CNVs). Large drusen are usually a sign of diffuse thickening of Bruch's membrane with basal linear deposit, a vesicular material that probably arises from the RPE, constitutes a diffusion barrier to water-soluble constituents in the plasma, results in lipidization of Bruch's membrane, and creates a potential cleavage plane between the RPE basement membrane and the inner collagenous layer of Bruch's membrane through which CNVs can grow. Disappearance of drusen spontaneously and in areas adjacent to laser photocoagulation scars was first noted by Gass (Gass JD: Arch Ophthalmol 90:206-217, 1973; Trans Am Acad Ophthalmol Otolaryngol 75:580-608, 1971). Subsequent reports have confirmed these observations. Photocoagulation-induced drusen regression might prevent patients with drusen from developing exudative maculopathy. The mechanism for spontaneous drusen regression probably involves RPE atrophy. The mechanism for photocoagulation-induced drusen regression is unknown. If photocoagulation-induced drusen regression is anatomically similar to atrophy-associated drusen regression, then the former will be associated with dissolution of basal linear deposit and a residuum of basal laminar deposit. Sarks and coworkers (Sarks JP, Sarks SH, Killingsworth MC: Eye 11:515-522, 1997) proposed that this in turn will eliminate the potential cleavage plane between the RPE basement membrane and inner collagenous layer of Bruch's membrane through which CNVs grow, thus retarding the growth of CNVs.

Abnormal collagen fibrils in nanophthalmos: a clinical and histologic study.

PURPOSE: To report the successful treatment of choroidal detachment in a patient with nanophthalmos and to report histopathologic findings in this patient's sclera. METHODS: Choroidal detachment, secondary angle closure, and nanophthalmos were diagnosed using biomicroscopy, indirect ophthalmoscopy, and echography. Full-thickness sclerectomies in four quadrants were made on the right eye. Sclerae from these sclerectomies were studied ultrastructurally. RESULTS: Best-corrected visual acuity improved to RE, 20/60 from 20/100 preoperatively; the anterior chamber deepened, and the choroidal detachment resolved. Histopathologic studies of each of the three scleral layers disclosed abnormal collagen fibrils that were frayed, split, and contained lightly stained cores. CONCLUSION: New findings include the identification of collagen with lightly stained centers and identification of differences in collagen morphology in different areas of the sclera in a nanophthalmic eye.

Diagnosis of phacoanaphylactic endophthalmitis by fine needle aspiration biopsy.

Diagnosis of phacoanaphylactic endophthalmitis (or lens induced uveitis), a rare autoimmune disease, is difficult due to variable clinical presentation. We sought to diagnose a case based on the cytopathology of the anterior chamber aspirate. This is a case report of spontaneous phacoanaphylactic endophthalmitis in a 79-year-old woman with no history of eye trauma or surgery. After clinical examination, diagnostic anterior chamber paracentesis was performed. Cytologic examination of the aspirate revealed polymorphonuclear leukocytes, histiocytes, and plasma cells surrounding amorphous lens material. A mature cataract was removed subsequently, and the eye has remained free of inflammation postoperatively. As the clinical diagnosis of phacoanaphylactic endophthalmitis is often difficult, cytopathology of an anterior chamber aspiration specimen may be useful in diagnosing this rare, treatable condition. As far as we know, this is the first case report of the diagnosis of phacoanaphylactic endophthalmitis solely by anterior chamber fine needle aspiration biopsy.