Toxicity profiles of subretinal indocyanine green, Brilliant Blue G, and triamcinolone acetonide: a comparative study.

BACKGROUND: This study introduces a novel porcine model to examine the histopathological and electrophysiological consequences of retinotoxicity exerted by dyes commonly used for internal limiting membrane (ILM) staining. METHODS: Indocyanine green (ICG) 0.5 mg/ml, Brilliant Blue G (BBG) 0.25 mg/ml and triamcinolone acetonide (TA) 13 mg/ml was injected subretinally in 12 vitrectomized pig eyes. At 6 weeks, retinas were examined by multifocal electroretinography (mfERG), ophthalmoscopy, fluorescein angiograpy, histopathology, and apoptosis assay. RESULTS: mfERG responses were significantly lower in ICG-injected eyes than in healthy fellow eyes (p = 0.039). The ratio between injected eyes and healthy fellow eyes was lower in the ICG group than in the BBG (p = 0.009) and TA group (p = 0.025). No difference between BBG and TA existed. All retinas were reattached, and fluorescein angiographies showed a window defect corresponding to the injected areas but no blood-retina barrier break-down. Histopathology confirmed damage to the outer retina after ICG, but not after BBG and TA. No apoptosis was found at 6 weeks. CONCLUSIONS: Subretinal ICG induces histological and functional damage to the retina, suggesting that ICG should be used with caution in macular hole surgery, where subretinal migration can occur. In contrast, BBG and TA appear safe after subretinal injection.

Pharmacokinetics of intravitreal glial cell line-derived neurotrophic factor: experimental studies in pigs.

The purpose of this study was to establish the intravitreal (ITV) pharmacokinetics of glial cell line-derived neurotrophic factor (GDNF) and observe possible complications after ITV injection. Twenty Danish landrace pigs and 34 eyes were included in the study; 30 were injected with 100 ng of GDNF, two controls were injected without GDNF, and two received no injection. At post-injection time points of 1, 2, 3, 6 hours (h), 1, 2, 4 or 7 days (d) eyes were enucleated and the ITV concentration of GDNF (cGDNF) was determined by enzyme-linked immunosorbent assay, and activity was tested using a retinal ganglion cell line (RGC5) bioassay. Indirect ophthalmoscopy, intraocular pressure assessment, and fundus photography were performed before enucleation. There was initial variability in the cGDNF, but after 24h GDNF was cleared in a monoexponential fashion with a half-life of 37 h (CL 33-43 h). Therapeutic concentrations were present for 15 d (CL 13-18d) when an extrapolation was done. GDNF-injected vitreous samples stimulated increased survival of RGC5s at 24h post-delivery (p=0.002) compared with no-GDNF vitreous controls. This effect was independent of intraocular incubation time when cGDNF was normalized to 5 ng/ml. A semi-logarithmic dose-response curve showed linearity between 0.1 and 10 ng/ml. None of the eyes showed any signs of inflammation or other complications. A single ITV GDNF injection of 100 ng leads to therapeutic levels for 15 days in the porcine eye. The GDNF was stable in the intraocular environment and no adverse events were observed. GDNF might therefore play a role in the future treatment of acute retinal damage.

[Visual loss under silicone oil]. / Visusminderung unter Silikon.

Silicone oil is used as intravitreal tamponading agent in surgery for rhegmatogenous retinal detachment (RRD) cases complicated with proliferative vitreoretinopathy (PVR). Recently, a number of case series have appeared where profound central visual loss has been found in eyes after uncomplicated vitrectomy with silicone tamponade for RRD in eyes with seemingly good visual potential. Several reports have demonstrated the migration of silicone oil droplets into the retina and the optic nerve, others the widespread loss of myelinated optic nerve fibres. These reports are reviewed, and it is concluded that caution is warranted when silicone oil is used in eyes with good visual potential. Finally the additional danger of central visual loss should be taken into consideration when deciding to use silicone oil or gas as intravitreal tamponade.

Calcium-independent phospholipase A2 regulates retinal pigment epithelium proliferation and may be important in the pathogenesis of retinal diseases.

Calcium-independent phospholipase A2, group VIA (iPLA2-VIA) is involved in cell proliferation. This study aimed to evaluate the role of iPLA2-VIA in retinal pigment epithelium (RPE) cell proliferation and in retinal diseases involving RPE proliferation. A human RPE cell line (ARPE-19) was used to explore this role in vitro. Proliferating ARPE-19 cells had increased expression and activity of iPLA2-VIA. iPLA2-VIA was found in the nuclei of proliferating ARPE-19 cells, whereas in confluent ARPE-19 cells, with limited proliferation, iPLA2-VIA was primarily found in the cytosol. Inhibition of iPLA2-VIA decreased the rate of proliferation, whereas over expression of iPLA2-VIA increased the rate of proliferation. Using an experimental porcine model of RPE proliferation we demonstrated significant nuclear upregulation of iPLA2-VIA in proliferating RPE cells in vivo. We furthermore evaluated the expression of iPLA2-VIA in proliferative vitreoretinopathy (PVR). PVR membranes revealed nuclear expression of iPLA2-VIA in the RPE cells which had migrated and participated in the formation of the membranes. Overall, the present results point to an important role of iPLA2-VIA in the regulation of RPE proliferation suggesting that iPLA2-VIA may be considered as a possible pharmaceutical target in retinal diseases involving RPE proliferation and migration.

Glaucoma detection with damato multifixation campimetry online.

PurposeTo evaluate Damato Multifixation Campimetry Online (DMCO), a free-of-charge internet-based visual field test. DMCO exists in three versions: DMCO BASIC, DMCO STANDARD, and DMCO ADVANCED. The main focus was (i) to investigate the sensitivity and the specificity of the existing DMCO versions in the detection of glaucomatous visual field loss and (ii) to define and evaluate algorithms for the interpretation of DMCO results.MethodsThe study design was an evaluation of a diagnostic test and included 97 individuals performing DMCO and white-on-white perimetry. Interpretation algorithms were devised to define abnormality, and these were evaluated using the Glaucoma Staging System as gold standard. Receiver operating characteristic (ROC) curves and area under the ROC (AUC) were calculated.ResultsAUCs from 15 algorithms ranged from 0.79 to 0.90. The most promising algorithm combined results from two successive DMCO STANDARD tests. The sensitivity was highly dependent on the severity of glaucoma. Hence, for eyes with mild, moderate, advanced, and severe glaucoma, the DMCO test demonstrated a sensitivity of 11.8, 71.4, 100, and 100%, respectively. The specificity was as high as 98.1%. Median duration per eye to complete the DMCO STANDARD test was 86 s for the control group and 125 s in participants with glaucoma.ConclusionsDMCO shows promise as a free-of-charge online tool to identify glaucomatous visual field defects in a preselected population. Ongoing studies are evaluating the use of DMCO in a nonselected population.

Quantitative vitreous fluorophotometry applying a mathematical model of the eye.

A slit-lamp fluorophotometric method is presented that permits calculation of a blood-retinal barrier permeability to fluorescein (P) and a diffusion coefficient for fluorescein in the vitreous body (D). The calculations are performed by relating the time course of the free--not protein bound--fluorescein concentration in the bloodstream with the fluorescein concentration profile in the vitreous body. The combination is performed automatically on a computer by applying a simplified mathematical model of the eye. P refers to the area of the barrier of the model eye. In a group of six normal persons, the mean P was (1.1 +/- 0.4) X 10(-7) cm/sec (mean +/- SD), while in six diabetic patients with background retinopathy and macular edema the mean P was (7.1 +/- 3.8 ) X 10(-7) cm/sec. The mean D was (7.4 +/- 3.4) X 10(-6) cm2/sec in the normal group and (9.6 +/- 2.0) X 10(-6) cm2/sec in diabetic patients, corresponding as a first approximation to free diffusion in water. Model calculations show that knowing the fluorescein concentration in the bloodstream is considerably significant for the calculation of the permeability, contributing factors up to 50%. For the low-permeation situation, subtraction of the preinjection scan contributes a factor of 50% for both permeability and diffusion coefficient. The exact placement in the vitreous body of the concentration profile, by applying a formalism that transforms slit-lamp movement to intraocular distance, contributes a factor of 20% on the diffusion coefficient. The permeability obtained with the model can be calculated as the ratio between area of vitreous and plasma fluorescein concentration curves within 20%. Active transport of fluorescein across the blood-retinal barrier in the direction of vitreous to blood does not seem to be significant within the first 2 hr after fluorescein injection.

Lactate transport in freshly isolated human fetal retinal pigment epithelium.

PURPOSE: To study transport mechanisms for small monocarboxylic acids in the apical and basolateral membranes of freshly isolated, human fetal retinal pigment epithelium. METHODS: The epithelium was mounted in a small Ussing chamber that allowed separate perfusion of both the apical and basal compartments and simultaneous measurements of intracellular pH, transepithelial potential, and tissue resistance. Intracellular pH was measured using a pH-sensitive dye, 2',7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein. RESULTS: When 10-100 mM lactate or pyruvate was added to the apical bath the cells acidified by 0.10-0.25 pH units. There were no differences between the initial rates of intracellular acidification produced by L-lactate and D-lactate. These rates could be described as Michaelis-Menten functions of the concentrations of lactate and pyruvate. The Km values were 42 +/- 12 mM for L-lactate and 34 +/- 8 mM for pyruvate. The rates of acidification caused by 50 mM L-lactate were reversibly reduced by 44% or 35% after apical administration of probenecid (2 mM) or alpha-cyano-4-hydroxycinnamate (2 mM), and irreversibly reduced by 78% after apical administration of the sulfhydryl-reagent mersalyl acid (2 mM). The intracellular acidifications caused by apical pyruvate (50 mM) were completely and reversibly inhibited by 50 mM apical L-lactate. Addition of 50 to 100 mM lactate to the basal bath caused intracellular alkalinizations, which could be inhibited by Na+ removal in the basal bath or by 2 mM alpha-cyano-4-hydroxycinnamate in the apical bath.

Optic nerve oxygen tension in pigs and the effect of carbonic anhydrase inhibitors.

PURPOSE: To evaluate how the oxygen tension of the optic nerve (ONP(O)2) is affected by the administration of the carbonic anhydrase inhibitors dorzolamide and acetazolamide and by alterations in oxygen and carbon dioxide in the breathing mixture. METHODS: Polarographic oxygen electrodes were placed in the vitreous humor immediately over the optic disc in 20 anesthetized pigs. Blood gasses and cardiovascular physiology were monitored. ONP(O)2 was recorded continuously with breathing gasses of 21% O2-79% N2, 100% O2, 20% O2-80% N2, and 5.19% CO2-19.9%, O2-74.9% N2. Acetazolamide (15-1000 mg) and dorzolamide (6-1000 mg) were administered intravenously. RESULTS: The mean (+/- SD) ONP(O)2 was found to be 24.1+/-11.6 mm Hg when the pigs were breathing room air and 50.7+/-29.3 mm Hg when they were breathing 100% O2 (n = 15; P < 0.001). In response to breathing 5.19% CO2, ONP(O)2 changed from 20.8+/-5.6 mm Hg (with 20.0% O2) to 28.9+/-3.6 mm Hg (n = 4; P < 0.001). Intravenous injections of 500 mg dorzolamide increased ONP(O)2 from 16.4+/-6.1 mm Hg to 26.9+/-12.2 mm Hg, or 52.5%+/-21.2% (n = 5; P = 0.017). A dose-dependent effect on ONP(O)2 was seen with intravenous dorzolamide doses of 1000, 500, 250, 125, 63, 27, 15, and 6 mg. Intravenous injections of 500 mg acetazolamide increased ONP(O)2 from 23.6+/-9.5 mm Hg to 30.9+/-10.0 mm Hg (n = 6; P < 0.001), and a dose-dependent effect was seen with doses of 1000, 500, 250, 125, 31, and 15 mg. CONCLUSIONS: ONP(O)2 is significantly increased by the carbonic anhydrase inhibition of dorzolamide and acetazolamide, and the effect is dose dependent. These data demonstrate for the first time a direct effect of carbonic anhydrase inhibitors on ONP(O)2.

Human retinal pigment epithelial cell-induced apoptosis in activated T cells.

PURPOSE: The immune privilege of the eye has been thought to be dependent on physical barriers and absence of lymphatic vessels. However, the immune privilege may also involve active immunologic processes, as recent studies have indicated. The purpose of the present study was to investigate whether human retinal pigment epithelial (RPE) cells can induce apoptosis in activated T cells. METHODS: Fas ligand (FasL) expression was detected by flow cytometry and immunohistochemistry. Cultured RPE cells were cocultured with T-cell lines and peripheral blood lymphocytes for 6 hours to 2 days. Induction of apoptosis was detected by 7-amino-actinomycin D and annexin V staining. RESULTS: Retinal pigment epithelial cells expressed FasL and induced apoptosis in activated Fas+ T cells. Blocking of Fas-FasL interaction with antibody strongly inhibited RPE-mediated T-cell apoptosis. Retinal pigment epithelial cells induced apoptosis in several activated T-cell populations and T-cell lines, including T-cell antigen receptor (TCR)-CD3-negative T-cell lines. In contrast, RPE cells induced little or no apoptosis in resting peripheral T cells. Major histocompatibility complex (MHC) class II monoclonal antibodies, which block alloactivation, had no inhibitory effect on RPE-mediated T-cell apoptotic responses in MHC class II-specific CD4+ T-cell lines. CONCLUSIONS: Retinal pigment epithelial cells express FasL and induce TCR-independent apoptosis in activated human T cells through Fas-FasL interaction. Retinal pigment epithelial cells may constitute an immunologic functional barrier against potentially harmful T cells.

The copper T380A intrauterine device in women with type II diabetes mellitus.

OBJECTIVE: To evaluate the safety and efficacy of the copper T380A intrauterine device (IUD) in women with type II, non-insulin-dependent diabetes mellitus. METHODS: From June 1988, 176 women with type II diabetes, in whom the copper T380A IUD was inserted, were followed prospectively until: study closure (June 1993); termination for pregnancy, expulsion, or removal for medical or personal reasons; or termination for loss to follow-up. RESULTS: Sixteen women never returned after initial insertion, leaving 160 women who were followed a total of 3066 months, with 117 continuing follow-up after their reexamination visit 6-12 weeks after insertion. None developed acute salpingitis. The overall removal rates per 100 woman-years were as follows: for pregnancy, 1.57; for expulsion, 1.96; for discontinuation because of medical reasons (including pain and bleeding), 4.31; and for personal reasons, 3.91. The continuation rate at the end of 3 years after insertion was 70%. CONCLUSION: The copper T380A IUD appears to be safe and effective in women with type II diabetes when standard criteria for IUD insertion are followed.

Optic nerve oxygen tension: the effects of timolol and dorzolamide.

BACKGROUND/AIMS: The authors have previously reported that carbonic anhydrase inhibitors such as acetazolamide and dorzolamide raise optic nerve oxygen tension (ONPO(2)) in pigs. The purpose of the present study was to investigate whether timolol, which belongs to another group of glaucoma drugs called beta blockers, has a similar effect. In addition, the effect of dorzolamide and timolol in combination was studied. METHODS: Polarographic oxygen electrodes were placed transvitreally over the optic disc in anaesthetised pigs and ONPO(2) was recorded continually. Drugs were administered intravenously either as 100 mg timolol followed by 500 mg dorzolamide (n = 5), 500 mg dorzolamide followed by 100 mg timolol (n = 5), or 100 mg timolol and 500 mg dorzolamide given simultaneously (n = 5). Arterial blood pressure, blood gasses, and heart rate were recorded. RESULTS: ONPO(2) was unaffected by administration of 100 mg timolol as an intravenous injection (n = 5). Administration of 500 mg dorzolamide by itself significantly increased ONPO(2) from 2.96 (SD 0.62) kPa to 3.69 (SD 0.88) kPa (n = 4, p = 0.035). The dorzolamide induced ONPO(2) increase was not significantly different from the ONPO(2) increases were seen when dorzolamide was administered simultaneous with (n = 5) or 35 minutes (n = 5) after 100 mg timolol. CONCLUSION: Systemic administration of timolol does not affect the optic nerve oxygen tension despite its lowering effect on the intraocular pressure. Additionally, timolol does not affect the ONPO(2) increasing effect of dorzolamide.

Optic nerve oxygen tension: effects of intraocular pressure and dorzolamide.

AIM: To investigate the influence of acute changes in intraocular pressure on the oxygen tension in the vicinity of the optic nerve head under control conditions and after intravenous administration of 500 mg of the carbonic anhydrase inhibitor dorzolamide. METHODS: Domestic pigs were used as experimental animals. Oxygen tension was measured by means of a polarographic electrode in the vitreous 0.5 mm anterior to the optic disc. This entity is called the optic nerve oxygen tension. Intraocular pressure was controlled by a hypodermic needle inserted into the anterior chamber and connected to a saline reservoir. RESULTS: When the intraocular pressure was clamped at 20 cm H2O optic nerve oxygen tension was 20 (5) mm Hg (n=8). Intravenous administration of dorzolamide caused an increase in optic nerve oxygen tension of 43 (8)% (n=6). Both before and after administration of dorzolamide optic nerve oxygen tension was unaffected by changes in intraocular pressure, as long as this pressure remained below 60 cm H2O. At intraocular pressures of 60 cm H(2)O and below, dorzolamide significantly increased optic nerve oxygen tension. CONCLUSION: Intravenous administration of 500 mg dorzolamide increases the oxygen tension at the optic nerve head during acute increases in intraocular pressure.

Indomethacin lowers optic nerve oxygen tension and reduces the effect of carbonic anhydrase inhibition and carbon dioxide breathing.

BACKGROUND/AIMS: Prostaglandins are important in blood flow regulation. Carbon dioxide (CO(2)) breathing and carbonic anhydrase inhibition increase the oxygen tension in the retina and optic nerve. To study the mechanism of this effect and the role of cyclo-oxygenase in the regulation of optic nerve oxygen tension (ONPO(2)), the authors investigated how indomethacin affects ONPO(2) and the ONPO(2) increases caused by CO(2) breathing and carbonic anhydrase inhibition in the pig. METHODS: Optic nerve oxygen tension was measured in 11 pigs with a polarographic oxygen electrode. The tip of the electrode was placed 0.5 mm above the optic disc. The effects of indomethacin, CO(2) breathing (3%) before and after indomethacin treatment, and carbonic anhydrase inhibition with or without indomethacin treatment were investigated. RESULTS: Administration of 300 mg indomethacin decreased optic nerve oxygen tension significantly. Carbonic anhydrase inhibition and CO(2) breathing increased ONPO(2) significantly. After indomethacin had been given, the rise in ONPO(2) caused by CO(2) breathing and carbonic anhydrase inhibition was significantly reduced. CONCLUSION: Systemic administration of indomethacin decreases the optic nerve oxygen tension; this is probably the result of decreased blood flow through vasoconstriction of vessels in the optic nerve. Additionally, indomethacin diminishes the ONPO(2) increasing effect of CO(2) breathing and carbonic anhydrase inhibition, thus affecting the reactivity of vessels in the optic nerve.

Bcl-2, Bax, and c-Fos expression correlates to RPE cell apoptosis induced by UV-light and daunorubicin.

PURPOSE: The aim of this study was to determine the role of Bcl-2, Bcl-X L, Bax, and c-Fos in regulation of apoptosis, induced by ultraviolet-light A (UV-A) and daunorubicin (DNR), in retinal pigment epithelium (RPE) cells grown on bovine extracellular matrix (ECM)-coated or uncoated plastic dishes. METHODS: Apoptosis in confluent RPE cells cultured on ECM-coated or uncoated dishes was induced by UV-A or DNR. Apoptosis was detected by 7-amino-actinomycin D labeling followed by flow cytometry and by terminal deoxy-transferase mediated X-dUTP nick end labeling (TUNEL). Cellular expression of Bcl-2, Bcl-X L, Bax, and c-Fos was determined by the use of antibodies and flow cytometry, Western blot analysis, and immunocytochemical staining. RESULTS: Both UV-A and DNR induce apoptosis in human RPE cells in vitro. Human fetal RPE cells grown on ECM-coated dishes were significantly more resistant to UV-A or DNR induced apoptosis than cells grown on uncoated dishes. RPE cells grown on ECM-coated dishes expressed higher Bcl-2 levels and lower Bax levels compared to cells grown on uncoated dishes. However, Bcl-X L and c-Fos levels were comparable in the two cultures. After UV-A or DNR treatment, Bcl-2, Bcl-X L, Bax, and c-Fos levels were differently regulated in cells grown on ECM-coated dishes compared to cells grown on uncoated dishes. CONCLUSION: A significant protection against apoptosis of RPE cells grown on ECM compared to cells grown on uncoated plastic dishes was found after exposure to UV-A or DNR. This protection was found to be proportionally correlated to the anti-apoptotic protein Bcl-2 and inversely correlated to the expression of Bax. Furthermore a sustained induction and expression of c-Fos was found to correlate to a higher percentage of apoptotic cells of RPE cells grown on plastic. These findings demonstrate that ECM is of great importance for RPE cell survival during noxious stimuli and points out the essential role for a healthy Bruch's Membrane (BM) for RPE survival.

[Retinal detachment. The vitrectomy technique]. / Nethindeløsning. Opereret med corpus vitreum-teknik.

One hundred and ten eyes from 110 patients who in 1992 had undergone vitrectomy for regmatogenous retinal detachment were included in a retrospective study. Two year follow-up was obtained for 97 eyes. At the end of follow-up 45% of the eyes had complete anatomical success. Fifty-five percent had attachment of the macula. Anatomical success after a single operation was obtained in 33%. A visual acuity of more than 0.05 was obtained for 35% of the eyes at the end of follow-up. Eighty patients had a follow-up interview; of these 56% stated that the advantages of the operation outweighed the disadvantages.

[Retinal detachment. The scleral buckling technique]. / Nethindeløsning. Opereret med ekstern teknik.

Eighty-four eyes from 83 patients who in 1992 had undergone scleral buckling surgery for rhegmatogenous retinal detachment were included in a retrospective study. Two year follow-up was obtained for 72 eyes. At the end of follow-up 93% of the eyes had complete anatomical success. A visual acuity of 0.5 or more was obtained for 45% of the eyes at the end of follow-up. Sixty-three patients stated that the advantages of the operation outweighed the disadvantages.

[Age related macular degeneration. A widespread disease]. / Aldersrelateret maculadegeneration. En folkesygdom.

Age-related macular degeneration, AMD, is the commonest cause of legal blindness in the industrialised world. Epidemiological data suggest that in Denmark more than 80,000 persons suffer impaired vision in at least one eye, because of AMD. More than 4000 are legally blind owing to this disease. AMD has two major phenotypes: wet and dry. Most severe visual losses are caused by wet AMD, where new blood vessels form under the macula. A new treatment of this condition is now available. Photodynamic therapy with verteporfin has been investigated in a double-blind, randomised clinical trial with more than 600 patients. This study has been scrutinised by a Cochrane review, which has recommended criteria for the treatment. For eyes meeting these inclusion criteria, photodynamic therapy can reduce the occurrence of moderate and severe visual loss over a two-year period by more than 60%. It is estimated that around 1000 eyes in Denmark will meet the inclusion criteria for photodynamic therapy.

Photoreceptor Differentiation following Transplantation of Allogeneic Retinal Progenitor Cells to the Dystrophic Rhodopsin Pro347Leu Transgenic Pig.

Purpose. Transplantation of stem, progenitor, or precursor cells has resulted in photoreceptor replacement and evidence of functional efficacy in rodent models of retinal degeneration. Ongoing work has been directed toward the replication of these results in a large animal model, namely, the pig. Methods. Retinal progenitor cells were derived from the neural retina of GFP-transgenic pigs and transplanted to the subretinal space of rhodopsin Pro347Leu-transgenic allorecipients, in the early stage of the degeneration and the absence of immune suppression. Results. Results confirm the survival of allogeneic porcine RPCs without immune suppression in the setting of photoreceptor dystrophy. The expression of multiple photoreceptor markers by grafted cells included the rod outer segment-specific marker ROM-1. Further evidence of photoreceptor differentiation included the presence of numerous photoreceptor rosettes within GFP-positive grafts, indicative of the development of cellular polarity and self-assembly into rudiments of outer retinal tissue. Conclusion. Together, these data support the tolerance of RPCs as allografts and demonstrate the high level of rod photoreceptor development that can be obtained from cultured RPCs following transplantation. Strategies for further progress in this area, together with possible functional implications, are discussed.

The influence of brightness on functional assessment by mferg: a study on scaffolds used in retinal cell transplantation in pigs.

To determine the effect of membrane brightness on multifocal electroretinograms (mfERGs), we implanted poly lactic-co-glycolic acid (PLGA) membranes in the subretinal space of 11 porcine eyes. We compared membranes with their native shiny white color with membranes that were stained with a blue dye (Brilliant Blue). Histological and electrophysiological evaluation of the overlying retina was carried out 6 weeks after implantation. Histologically, both white and blue membranes degraded in a spongiform manner leaving a disrupted outer retina with no preserved photoreceptor segments. Multifocal ERG revealed the white membranes to have a significantly higher P1-amplitude ratio than the blue (P = 0.027), and a correlation between brightness ratio and P1-amplitude ratio was found (r = 0.762). Based on our findings, we conclude that bright subretinal objects can produce normal mfERG amplitude ratios even when the adjacent photoreceptors are missing. Functional assessment with mfERG in scaffold implant studies should therefore be evaluated with care.

Subretinal implantation of electrospun, short nanowire, and smooth poly(ε-caprolactone) scaffolds to the subretinal space of porcine eyes.

Biodegradable scaffolds play an important adjunct role in transplantation of retinal progenitor cells (RPCs) to the subretinal space. Poly(ε-Caprolactone) (PCL) scaffolds with different modifications were subretinally implanted in 28 porcine eyes and evaluated by multifocal electroretinography (mfERG) and histology after 6 weeks of observation. PCL Short Nanowire, PCL Electrospun, and PCL Smooth scaffolds were well tolerated in the subretinal space in pigs and caused no inflammation and limited tissue disruption. PCL Short Nanowire had an average rate of preserved overlying outer retina 17% higher than PCL Electrospun and 25% higher than PCL Smooth. Furthermore, PCL Short Nanowire was found to have the most suitable degree of stiffness for surgical delivery to the subretinal space. The membrane-induced photoreceptor damage could be shown on mfERG, but the reductions in P1 amplitude were only significant for the PCL Smooth. We conclude that of the tested scaffolds, PCL Short Nanowire is the best candidate for subretinal implantation.