Charles A. Garcia

In vitro degradation of thin poly(DL-lactic-co-glycolic acid) films

This study was designed to investigate the in vitro degradation of thin poly(DL-lactic-co-glycolic acid) (PLGA) films for applications in retinal pigment epithelium transplantation and guided tissue regeneration. PLGA films of copolymer ratios of 75:25 and 50:50 were manufactured with thickness levels of 10 microm (thin) and 100 microm (thick). Degradation of the films occurred during sample processing, and thin films with a higher surface area to volume ratio degraded faster. Sample weight loss, molecular weight loss, dimensional, and morphological changes were analyzed over a 10-week period of degradation in 0.2 M of phosphate-buffered saline (PBS), pH 7.4, at 37 degrees C. All PLGA films degraded by heterogeneous bulk degradation. Sample weights remained relatively constant for the first several weeks and then decreased dramatically. The molecular weights of PLGA films decreased immediately upon placement in PBS and continued to decrease throughout the time course. PLGA 50:50 films degraded faster than 75:25 films due to their higher content of hydrophilic glycolic units. The results also demonstrated that thick films degrade faster than corresponding thin films with the same composition. This was attributed to the greater extent of the autocatalytic effect, which further was confirmed by heterogeneous gel permeation chromatograms. These studies suggest that the degradation rate of thin films can be engineered by varying film thicknesses.

Retinitis Pigmentosa: A Symposium on Terminology and Methods of Examination

This report represents a summary of opinions expressed at a meeting of specialists interested in retinitis pigmentosa (RP) and allied diseases, at which an attempt was made to define some minimum guidelines for ocular evaluation of these disorders. The term RP would be reserved for a group of hereditary disorders that diffusely involve photoreceptor and pigment epithelial function, and should not be used when a secondary cause is suspected. RP may be classified by genetic type (single cases without known affected relatives should be termed isolated or simplex), by the topography of retinal involvement, and by the severity of disease (to identify subtypes with mild or localized disease). Patients should have at least one comprehensive examination that conforms to basic standards, preferable early in the course of the disease. The visual field examination should use both a small and a large test light. Electroretinographic testing should (1) use a full-field stimulus, and (2) routinely document three independent responses (cone, rod, and mixed cone-rod). Patients should be identifiable for future study or therapeutic trials. They should be counseled about the disease and followed regularly. No specific therapy exists at present for most of these diseases, but optical and night vision aids are available. Sunglasses for outdoor use are recommended until more is known about whether long-term exposure to bright sunlight alters the course of these diseases.

Retinal pigment epithelium cells cultured on synthetic biodegradable polymers

Alterations in the normal structure or functions of retinal pigment epithelium (RPE) can result in a number of ocular diseases. Implantation of RPE cells cultured on thin, biodegradable polymer films may provide a means of transplanting an organized sheet of RPE cells with distinct apical/basal characteristics for the restoration of normal RPE function. We have investigated the interactions of human RPE cells with different biodegradable polymer films to assess their suitability as substrates for RPE culture. Four biodegradable polymers were used: low molecular weight (MW) 50:50 poly(DL-lactic-co-glycolic acid) (PLGA); high MW 50:50 PLGA; 75:25 PLGA; and poly(L-lactic acid) (PLLA). Polymer film substrates were manufactured using a solvent casting technique. Human fetal RPE cells (10-16 weeks gestational) were plated on the polymer substrates and the cultures assessed with respect to cell attachment and proliferation. Histological and immunohistochemical studies were performed on the cells after 8 days in culture. RPE cells attached to all the polymers studied after 8 h in culture. After 8 h, 80.2 +/- 9.5% and 82.3 +/- 7.9% of the plated cells were attached to substrates of high MW 50:50 PLGA and 75:25 PLGA, respectively. The cells proliferated on all substrates, and there was about a threefold increase in cell number over the 8-day culture period on all the polymers studied. Immunohistochemistry after 8 days in culture demonstrated RPE cells labeled with a distinct reaction product for cytokeratin in the cell cytoplasm. All the polymers studied were suitable for RPE culture; however, high MW 50:50 PLGA and 75:25 PLGA proved to be the best in terms of manufacturing properties, cell attachment, and proliferation. These polymers can provide a suitable substrate for RPE cell culture and hold promise for the subretinal implantation of organized sheets of RPE cells.

Biodegradation and tissue reaction to intravitreous biodegradable poly(D,L-lactic-co-glycolic)acid microspheres

We studied the biodegradation of and the tissue reaction to microspheres of 50:50 poly(D,L-lactic-co-glycolic)acid (PLGA) (viscosity-average MW: 3000 d), injected intravitreous in rabbits. These microspheres are under investigation as injectable devices for intravitreous sustained drug delivery. The rate of intravitreous degradation of PLGA microspheres has not been well documented in the literature. Twenty two pigmented rabbits underwent gas vitrectomy in one eye: 19 eyes received 2.5 mg of PLGA microspheres in 1 ml of balanced salt solution (BSS) and 3 control eyes received 1 ml of BSS only. Slit lamp exam and indirect ophthalmoscopy were performed periodically from day 1 to 6 months after surgery. The eyes were enucleated and studied by light microscopy and immunohistochemistry at various time points. The electroretinogram (ERG) was recorded in a subgroup of rabbits before injection and after 1 and 6 months. The amount of microspheres in the vitreous cavity progressively decreased. At 6 months microspheres were found in 1/4 rabbits at indirect ophthalmoscopy and in 4/4 rabbits histopathologically. A mild localized, non progressive foreign body reaction was observed. The cell reaction was composed mostly of vimentin and glial fibrillary acidic protein positive cells which probably represent glial cells and fibroblasts. The choroid and retina were normal. The ERG showed no abnormalities. No clinical inflammatory signs were observed 4 days postoperatively and thereafter.

Retinal lesions as clues to disseminated bacterial and candidal infections frequency, natural history, and etiology

Invasive bacterial and candidal infections are known to involve the retina, but the natural history of the retinal lesions and the utility of ophthalmologic consultation in the critical care setting as a diagnostic tool are not well understood. We 1) performed weekly funduscopic examinations on 77 medical and surgical patients in intensive care units (ICUs), 2) analyzed results of serial ocular examinations in 180 non-neutropenic patients with candidemia, and 3) reviewed the English literature on the association of retinal lesions with disseminated bacterial or candidal infection (DBCI).We found that 15 (19%) of the ICU patients had retinal lesions consistent with DBCI. Of these 15, 1 had clearly sepsis-related retinal lesions, while 13 (87%) had 1 or more systemic disease that could have explained their retinal findings (6 diabetic retinopathy; 2 human immunodeficiency virus infection/acquired immunodeficiency syndrome (HIV/AIDS) retinopathy; 2 hypertensive retinopathy; 1 hemolytic uremic syndrome, and 1 leukemia). Multivariate analysis revealed that systemic disease (odds ratio 8.37, 95% confidence intervals: 3.24–21.56) independently correlated with the presence of retinal lesions while DBCI, trauma, hyperalimentation, and transfusion of blood products were not independently predictive in any analysis.Twenty of the 180 (15%) candidemic patients had retinal lesions. Two (1%) had classic 3-dimensional white lesions with vitreal extension, and 5 (2.7%) had chorioretinal lesions without vitreal haziness. Notably, 10% of patients had superficial retinal hemorrhages and/or cotton wool spots that could have been due to either candidemia or a systemic disease (diabetes, hypertension, renal failure, closed head trauma). Concurrent bacteremia occurred in 3 of the 27 patients with eye lesions. Retinal lesions resolved in a mean of 33 days. None of the patients had symptoms at the time of the retinal finding.We found 3 studies that prospectively assessed retinal lesions in bacteremic patients. The frequency of retinal lesions in these series varied from 12% to 26%, with the most common lesions being cotton wool spots followed by superficial retinal hemorrhages. White-centered hemorrhages were seen in about 15% ± 2 of bacteremic patients. Five studies prospectively evaluated candidemic patients for Candida endophthalmitis. These studies observed rates from 0% to 78% for lesions consistent with candidal endophthalmitis. Most studies performed recently found that nonspecific lesions such as cotton wool spots or superficial retinal hemorrhages occurred with a frequency of 11% to 20%. The availability of less toxic antifungal agents, more frequent use of empirical therapy, and the trend to early treatment may be altering the frequency of this complication.Observation of a classic 3-dimensional retina-based vitreal inflammatory process is virtually diagnostic of endogenous endophthalmitis due to Candida spp., but such lesions are relatively uncommon. Conversely, nonspecific lesions that could be due to bacterial or candidal endophthalmitis (cotton wool spots, retinal hemorrhages, and Roth spots) are seen frequently. These lesions are most often due to an underlying systemic disease rather than an infection. Serial examinations provide the best evidence that a given lesion is due to an intercurrent infection. The current low rate of vitreal extension of retinal process appears to be due to the high rate of empirical or therapeutic use of antifungal agents in high-risk patient groups. Ophthalmoscopy should be performed in patients with known candidemia. However, ophthalmoscopic examination seems to have little value in assisting with the discovery of occult disseminated candidiasis or bacterial infection.

Mortality of Mexican Americans With NIDDM: Retinopathy and other predictors in Starr County, Texas

Objective— To determine the rate and risk factors of mortality in a cohort of Mexican Americans with NIDDM. Research Design and Methods— A cohort of 353 Mexican Americans with NIDDM were identified between 1981 and 1986. All individuals underwent extensive evaluations that included physical, historical, ophthalmological, and laboratory assessments. This cohort was followed prospectively for a mean of 8 yr. Follow-up included mortality surveillance, death certificate extraction, and a combination of annual and intermediate examinations. Results— The cohort experienced 67 mortality events. One-third of all deaths were premature <65 yr of age) and most often were attributed to diseases of the heart (60.0%). In no case was diabetes listed as the cause of death, although it was listed as a contributing cause in 25.5% of cases. Men had a higher mortality rate than women. In both sexes, baseline retinopathy was identified as an important predictor of subsequent mortality. Mortality was significantly elevated in those with nonproliferative retinopathy and even further elevated in those with proliferative disease (relative risks of ≥ 4 for proliferative disease). Conclusions— Mexican Americans with NIDDM are experiencing premature and excessive mortality compared with the general population. The results clearly link microvascular complications with macrovascular disease, but this link is not explained by a more untoward profile of traditional cardiovascular risk factors. Retinopathy appears to serve as an important monitor of the progression of diabetes and when identified would warrant aggressive action to inhibit or slow the processes leading to subsequent mortality.

Manufacture and characterization of poly(α-hydroxy ester) thin films as temporary substrates for retinal pigment epithelium cells

Abstract For many disorders of the retinal pigment epithelium (RPE) for which there are no effective treatments, transplantation of RPE cells may provide a viable means of restoring function. Using a solvent casting technique, we have manufactured thin films of poly( l -lactic acid) and poly( dl -lactic-co-glycolic acid) 75:25 and 50:50. Non-porous, flexible films with controlled thickness as thin as 12 ± 3 μm and reproducible surface morphologies and flexural properties were produced. Fetal human RPE cells were found to attach to these substrates when cultured in vitro . The films made using this technique may provide a means of transplanting allogeneic RPE cells as a therapy for a number of ocular diseases related to RPE dysfunction.

RETINAL PIGMENT EPITHELIUM CELL CULTURE ON THIN BIODEGRADABLE POLY(DL-LACTIC-CO-GLYCOLIC ACID) FILMS

Thin films of 50:50 and 75:25 poly(DL-lactic-co-glycolic acid) (PLGA) were manufactured with a controlled thickness of less than 10 microm. The effect of PLGA copolymer ratio on in vitro cell attachment, proliferation, morphology, and tight junction formation was evaluated using a human D407 retinal pigment epithelium (RPE) cell line. Almost complete cell attachment was achieved on both PLGA films after 8 h of cell seeding, which was comparable to that on tissue culture polystyrene (TCPS) controls. The initial cell seeding density affected attachment, and the optimal value for 50:50 PLGA was 25000 cells cm(-2). After 7 days of in vitro culture, cell density on 50:50 and 75:25 PLGA films increased 45 and 40 folds, respectively, and a 34-fold increase was observed on TCPS. The RPE cells cultured on PLGA films at confluence had a characteristic cobblestone morphology. Confluent RPE cells also developed normal tight junctions in vitro which were concentrated mainly at the apical surfaces of cell-cell junctions. These results demonstrated that thin biodegradable PLGA films can provide suitable substrates for human RPE cell culture, and may serve as temporary carriers for subretinal implantation of organized sheets of RPE.

The Pattern Electroretinogram in Diabetes

In 27 normal subjects and 64 insulin-dependent diabetic patients, we evaluated the pattern electroretinogram, which may reflect the neural activity of the spatially sensitive retinal ganglion cells. The amplitude of the pattern electroretinogram was reduced in diabetic patients who had no observable retinal changes. The amplitude was further reduced with increasing retinopathy. The pattern electroretinogram amplitude change was a more sensitive indicator of retinal change among the diabetic subpopulations than the statistically significant changes in latency. A significant correlation between pattern electroretinogram amplitude and the duration of diabetes were found in diabetic patients with either no observable retinopathy or minimal background retinopathy. The pattern electroretinogram may be useful as a quantitative, dependent variable to establish and monitor short-term metabolic and physiologic changes in diabetic patients.

Proton beam irradiation of subfoveal choroidal neovascularisation in age-related macular degeneration

Purpose To assess the safety and potential toxicity of proton beam radiation in the treatment of subfoveal choroidal neovascular membrane (CNVM) due to age-related manner degeneration (ARMD) in a prospective, non-randomised study.Methods Forty-eight eyes of 46 consecutive patients with subfoveal CNVM due to ARMD, not amenable to laser photocoagulation, were treated prospectively with a single proton beam exposure. Two dose regimens were evaluated: 8 CGE (Cobalt Gray Equivalent) and 14 CGE. Patients were followed for an average of 22.1 months after proton beam treatment.Results At the 12 month follow-up, 44% of eyes in the 8 CGE group and 75% of the eyes in the 14 CGE group had stabilised or improved visual acuity. Complex size in the 8 CGE group as measured on standard fluorescein angiography (FA), decreased or had no change initially but showed less effect over time, while the eyes treated with 14 CGE maintained decreased leakage over the follow-up period of 12 months. However, 11 eyes in the 14 CGE group experienced radiation retinopathy, with the onset between 3 and 30 months. Seven of these 11 eyes have demonstrated some visual loss but only 1 eye developed severe visual loss at 15 months after proton treatment.Conclusions To date, 14 CGE has suggested a favourable influence on visual function and growth inhibition of CNVM. Proton beam irradiation appears to inhibit CNVM growth. The 14 CGE dose regimen appears to have a longer effect of CNVM growth than does 8 CGE, with overall stabilisation of visual function and growth inhibition. Radiation retinopathy has developed over time, but severe visual loss has been limited. On the basis of the incidence of radiation retinopathy, adjustments in the total radiation dosage and/ or fractionation of the dosage should be considered.