Subretinal Implantation of RPE on a Carrier in Minipigs: Guidelines for Preoperative Preparations, Surgical Techniques, and Postoperative Care.

Degenerative disorders of the retina (including age-related macular degeneration), which originate primarily at or within the retinal pigmented epithelial (RPE) layer, lead to a progressive disorganization of the retinal anatomy and the deterioration of visual function. The substitution of damaged RPE cells (RPEs) with in vitro cultured RPE cells using a subretinal cell carrier has shown potential for re-establishing the anatomical structure of the outer retinal layers and is, therefore, being further studied. Here, we present the principles of a surgical technique that allows for the effective subretinal transplantation of a cell carrier with cultivated RPEs into minipigs. The surgeries were performed under general anesthesia and included a standard lens-sparing three-port pars plana vitrectomy (PPV), subretinal application of a balanced salt solution (BSS), a 2.7 mm retinotomy, implantation of a nanofibrous cell carrier into the subretinal space through an additional 3.0 mm sclerotomy, fluid-air exchange (FAX), silicone oil tamponade, and closure of all the sclerotomies. This surgical approach was used in 29 surgeries (18 animals) over the past 8 years with a success rate of 93.1%. Anatomic verification of the surgical placement was carried out using in vivo fundus imaging (fundus photography and optical coherence tomography). The recommended surgical steps for the subretinal implantation of RPEs on a carrier in minipig eyes can be used in future preclinical studies using large-eye animal models.

Determination of Micropulse Modes with Targeted Damage to the Retinal Pigment Epithelium Using Computer Modeling for the Development of Selective Individual Micropulse Retinal Therapy.

PURPOSE: When using a serial laser system for selective impact on the retinal pigment epithelium (RPE), there is a challenge to determine the optimal range of micropulse parameters which result in targeted damage to the RPE. This study proposes a computer model that has identified the optimal parameters to be applied. METHODS: This study was conducted on 18 patients who were diagnosed with acute central serous chorioretinopathy and transparent optical media, aged 35 to 46 years old, and type 2 and 3 on the Fitzpatrick scale. Testing of the micropulse mode was performed on the Navilas 577s laser system; 864 spots were analyzed in total. Considering the probability of damage visualization at different laser power, the computer simulation of tissue heating and protein denaturation was performed to determine the micropulse modes which resulted in selective damage to the RPE. RESULTS: The computer model parameter ΔE = 3.34 × 105 J/mol was determined from fitting the model predictions to the autofluorescence test results. The micropulse modes with a micropulse duration of 50-100 µs, duty cycle 2.4-4.8%, 10 ms-pulse envelope (5 micropulses), and spot diameter of 100 µm have efficiency and selectivity above 67% and correspond to the optimal therapeutic window for targeted RPE damage at a certain power. Increasing the micropulse duration, number of micropulses, and duty cycle leads to a decrease in the selective effect on the RPE and higher damage to adjacent tissues. CONCLUSION: The concepts of efficiency and selectivity have been introduced to quantify the amount of damage caused. The optimal range of micropulse parameters which result in effective and selective damage on the RPE has been determined for the Navilas 577s laser system. The proposed method can be used for any other serial laser system. A comparison of the different micropulse modes, as well as the CW modes, has been performed.

Changes in entropy on polarized-sensitive optical coherence tomography images after therapeutic subthreshold micropulse laser for diabetic macular edema: A pilot study.

PURPOSE: To investigate the dynamics of the healing process after therapeutic subthreshold micropulse laser (SMPL) for diabetic macular edema (DME) using polarization-sensitive optical coherence tomography (PS-OCT). METHODS: Patients with treatment-native or previously-treated DME were prospectively imaged using PS-OCT at baseline, 1, 2, 3, and 6 months. The following outcomes were evaluated: changes in the entropy value per unit area (pixel2) in the retinal pigment epithelium (RPE) on the B-scan image; changes in the entropy value in each stratified layer (retina, RPE, choroid) based on the ETDRS grid circle overlaid with en face entropy mapping, not only the whole ETDRS grid area but also a sector irradiated by the SMPL; and the relationship between edema reduction and entropy changes. RESULTS: A total of 11 eyes of 11 consecutive DME patients were enrolled. No visible signs of SMPL treatment were detected on PS-OCT images. The entropy value per unit area (pixel2) in the RPE tended to decrease at 3 and 6 months from baseline (35.8 ± 17.0 vs 26.1 ± 9.8, P = 0.14; vs 28.2 ± 18.3, P = 0.14). Based on the en face entropy mapping, the overall entropy value did not change in each layer in the whole ETDRS grid; however, decrease of entropy in the RPE was observed at 2, 3, and 6 months post-treatment within the SMPL-irradiated sectors (P < 0.01, each). There was a positive correlation between the change rate of retinal thickness and that of entropy in the RPE within the SMPL-irradiated sector at 6 months (r2 = 0.19, P = 0.039). CONCLUSION: Entropy measured using PS-OCT may be a new parameter that facilitates objective monitoring of SMPL-induced functional changes in the RPE that could not previously be assessed directly. This may contribute to a more promising therapeutic evaluation of DME. CLINICAL TRIAL: This clinical study was registered in UMIN-CTR (ID: UMIN000042420).

A novel quantitative analysis method for idiopathic epiretinal membrane.

PURPOSE: To introduce a novel method to quantitively analyse in three dimensions traction forces in a vast area of the ocular posterior pole. METHODS: Retrospective analysis of 14 eyes who underwent peeling surgery for idiopathic, symptomatic and progressive epiretinal membrane. The technique measures the shift in position of vascular crossings after surgery from a fixed point, which is the retinal pigmented epithelium. This shift is defined as the relaxation index (RI) and represents a measure of the postoperative movement of the retina due to released traction after surgery. RESULTS: Best-corrected visual acuity was significantly better than baseline at all follow ups while the RI had its maximum value at baseline. Moreover, we found a significant correlation between best-corrected visual acuity at 6 months and RI at baseline. CONCLUSION: While all previous published methods focused on bi-dimensional changes observed in a small region, this study introduces a three-dimensional assessment of tractional forces. Future integration of RI into built-in processing software will allow systematic three-dimensional measurement of intraretinal traction.

Investigation of the therapeutic mechanism of subthreshold micropulse laser irradiation in retina.

PURPOSE: Subthreshold micropulse laser irradiation has been used for the treatment of retinal edema; however, there are few reports about the mechanism of its therapeutic effect. In this study, we compared threshold short pulse and subthreshold micropulse laser irradiation in mice and investigated their mechanism. METHODS: Nine to 12-week-old male C57BL/6J mice were used in this study. After general anesthesia, threshold short pulse or subthreshold micropulse laser irradiation was performed on the right eye using IQ577. Enucleation was performed 24 h after the laser irradiation, and histological and gene expression analyses were carried out. RESULTS: Coagulation spots and atrophy of the retinal pigment epithelium were observed after threshold short pulse laser irradiation but not after subthreshold micropulse laser irradiation. Twenty-four hours after laser, aquaporin (AQP) 1, 2, 7, and 11 levels were significantly elevated by 1.7- to 3-fold in the threshold short pulse laser group compared with non-treated control group. AQP 3 was increased significantly and prominently by 100-fold. VEGF-A and VEGFR2 were upregulated 1.5- and 2.3-fold, respectively. In the subthreshold micropulse laser group, AQP 3 was increased by 6-fold compared with the non-treated control group. Angiopoietin-1 and the adrenomedullin (AM) receptor CLR were decreased by 0.6-fold and 0.5-fold, respectively. CONCLUSION: Threshold short pulse laser irradiation caused retinal damage and prominent changes in the expression of various genes. Contrarily, subthreshold micropulse laser irradiation did not induce retinal damage; it upregulated AQP 3, which might have improved retinal edema by drainage of subretinal fluid.

Selective retina therapy monitoring by speckle variance optical coherence tomography for dosimetry control.

SIGNIFICANCE: Selective retina therapy (SRT) selectively targets the retinal pigment epithelium (RPE) and reduces negative side effects by avoiding thermal damages of the adjacent photoreceptors, the neural retina, and the choroid. However, the selection of proper laser energy for the SRT is challenging because of ophthalmoscopically invisible lesions in the RPE and different melanin concentrations among patients or even regions within an eye. AIM: We propose and demonstrate SRT monitoring based on speckle variance optical coherence tomography (svOCT) for dosimetry control. APPROACH: M-scans, time-resolved sequence of A-scans, of ex vivo bovine retina irradiated by 1.7-µs duration laser pulses were obtained by a swept-source OCT. SvOCT images were calculated as interframe intensity variance of the sequence. Spatial and temporal temperature distributions in the retina were numerically calculated in a 2-D retinal model using COMSOL Multiphysics. Microscopic images of treated spots were obtained before and after removing the upper neural retinal layer to assess the damage in both RPE and neural layers. RESULTS: SvOCT images show abrupt speckle variance changes when the retina is irradiated by laser pulses. The svOCT intensities averaged in RPE and photoreceptor layers along the axial direction show sharp peaks corresponding to each laser pulse, and the peak values were proportional to the laser pulse energy. The calculated temperatures in the neural retina layer and RPE were linearly fitted to the svOCT peak values, and the temperature of each lesion was estimated based on the fitting. The estimated temperatures matched well with previously reported results. CONCLUSION: We found a reliable correlation between the svOCT peak values and the degree of retinal lesion formation, which can be used for selecting proper laser energy during SRT.

Surgical Approaches for Cell Therapeutics Delivery to the Retinal Pigment Epithelium and Retina.

Developing successful surgical strategies to deliver cell therapeutics to the back of the eye is an essential pillar to success for stem cell-based applications in blinding retinal diseases. Within this chapter, we have attempted to gather all key considerations during preclinical animal trials.Guidance is provided for choices on animal models, options for immunosuppression, as well as anesthesia. Subsequently we cover surgical strategies for RPE graft delivery, both as suspension as well as in monolayers in small rodents, rabbits, pigs, and nonhuman primate. A detailed account is given in particular on animal variations in vitrectomy and subretinal surgery, which requires a considerable learning curve, when transiting from human to animal. In turn, however, many essential subretinal implantation techniques in large-eyed animals are directly transferrable to human clinical trial protocols.A dedicated subchapter on photoreceptor replacement provides insights on preparation of suspension as well as sheet grafts, to subsequently outline the basics of subretinal delivery via both the transscleral and transvitreal route. In closing, a future outlook on vision restoration through retinal cell-based therapeutics is presented.

Intraoperative Drainage of a Bullous Serous Pigment Epithelial Detachment.

BACKGROUND AND OBJECTIVE: To describe a novel, simple technique for surgically draining a bullous serous pigment epithelial detachment (PED). PATIENTS AND METHODS: Pars plana vitrectomy was performed with confirmed elevation of the hyaloid face. Proportional diathermy allowed stepwise entry into the PED superotemporally through an initially small, needle-point focus while providing control of any potential bleeding. Thick fluid was aspirated with a soft-tipped cannula, fluid-air exchange was performed, and intravitreal bevacizumab was injected before removing the cannulas. RESULTS: The PED was successfully completely drained intraoperatively and remained flat at 1 week postoperatively. However, the draining site ultimately closed, and continued exudation from choroidal neovascularization led to recurrent PED and eventual nonhemorrhagic retinal pigment epithelial tear despite aggressive treatment with aflibercept and photodynamic therapy. The early visual acuity benefit may relate to resolution of hyperopic shift. CONCLUSION: Serous PED can be surgically reduced without hemorrhagic complications, but long-term success depends upon control of the underlying choroidal neovascularization. [Ophthalmic Surg Lasers Imaging Retina. 2019;50:510-513.].

Clinical and molecular markers in retinal detachment-From hyperreflective points to stem cells and inflammation.

PURPOSE: Retinal detachment (RD) is one of the most frequently diagnosed ophthalmologic conditions requiring prompt surgical intervention. Combination of proper surgical technique and new diagnostic markers, both clinical and molecular, can help improve the diagnosis and prognosis of RD treatment. METHODS: 12 patients with rhegmatogenous RD (rRD) were included into the study after obtaining patient consent and Regional Ethical Approval (average age: 58.1 ± 17.4 years). OCT was performed before and after 23G vitrectomy for RD. Pure subretinal fluid (SRF) was collected during surgery and analyzed by protein array profiling on a panel of 105 inflammatory cytokines (Human XL Cytokine Array), while the effect of SRF upon human macrophages-driven phagocytosis of apoptotic retinal pigment epithelial (RPE) cells ex vivo was quantified by flow cytometry. Immunohistochemistry (IHC) of retinectomized tissue due to PVR caused by RD was performed to determine presence of markers for microglial cells (CD34), macrophages and activated microglia (CD68), regulator of the immune response to infection (NFkB), progenitor and stem cell marker (Sox2), pluripotency marker (Oct4) and intermediate filament markers (GFAP and Nestin). RESULTS: OCT of fresh RD patients contained pre-operatively hyper reflective points (HRPs) at the detached neuroretina border and proximal to the RPE layer-their size and number decreased following successful reattachment surgery. IHC of the retinectomized tissue from detached retina due to severe PVR showed presence of cell conglomerates at the detached neuroretina border which were positive for CD68, NFkB, Sox2 and GFAP, less positive for CD47 and Nestin and negative for Oct4 and CD34. The SRF contained at least 37 cytokines with higher, and 4 cytokine with lower concentration compared to that in vitreous from non-RD pathology; when used as conditional medium to human macrophages ex vivo, the SRF doubled their capacity for engulfing dying RPEs. CONCLUSIONS: Fresh RD can be hallmarked by presence of HRPs at the detached neuroretina border on OCT; the HRPs decrease in size and number after successful reattachment surgery, and likely resemble the macrophage conglomerates seen by IHC. The neuroretina in RD contains progenitor/stem-like cells and signs of inflammatory reaction, while the SRF contains inflammatory cytokines and other factors which increase the ability of professional phagocytes to engulf dying RPE, or for that matter, other dying cells in the retina.

Retinal pigment epithelial tears.

A retinal pigment epithelial (RPE) tear is a well-known complication of retinal pigment epithelial detachments (PED) and may cause a significant visual impairment. The most common cause is a vascularized PED in patients with exudative age-related macular degeneration (AMD). The development of diagnostic imaging techniques brings us closer to the etiology and pathophysiological mechanisms of this entity, offering us new strategies for treatment and follow-up. The advent of intravitreal antiangiogenic treatment (anti-VEGF) has led to an increase in the number of reported cases of RPE tears, which are an important vision-limiting factor during treatment. However, RPE tears may occur spontaneously or as a consequence of thermal laser treatment, photodynamic therapy or anti-VEGF therapy. It is accepted that the mechanism of RPE tears is multifactorial. The optimization of the functional outcome of this complication has been described with continuous treatment with antiangiogenic drugs. The goal of the present review is to evaluate the incidence, risk factors and treatment of RPE tears.

Motion-invariant SRT treatment detection from direct M-scan OCT imaging.

PURPOSE: Selective retina therapy (SRT) is a laser treatment targeting specific posterior retinal layers. It is focused on inducing damage to the retinal pigment epithelium (RPE), while sparing other retinal tissue compared to traditional photocoagulation. However, the targeted RPE layer is invisible with most imaging modalities and induced SRT lesions cannot be monitored. In this work, imaging scans acquired from an experimental setup that couples the SRT laser beam with an optical coherence tomography (OCT) beam are analyzed in order to evaluate the treatment as they occur. METHODS: We isolated a small part of the time-resolved scan corresponding to the end of the treatment, for which we have microscopic evidence of the SRT outcome. We then use a convolutional neural network to correspond each scan to the treatment result. We explore which aspects of the scan convey more valuable information for a robust therapy evaluation. By only using this adequately small part, we can achieve an online estimation, while being resilient to eye movement. RESULTS: The available dataset consists of time- resolved OCT scans of 98 ex vivo porcine eyes, treated with different energy levels. The proposed method yields high performance in the task of predicting whether the applied energy was adequate for SRT treatment, by focusing on the immediate OCT signal acquired during treatment time. CONCLUSIONS: We propose a strategy toward online noninvasive SRT treatment assessment, able to provide a satisfying evaluation of a treatment status, that therefore could be used for the planning of the treatment continuation.

Differential autophagic effects of vital dyes in retinal pigment epithelial ARPE-19 and photoreceptor 661W cells.

Indocyanine green (ICG) and brilliant blue G (BBG) are commonly used vital dyes to remove internal limiting membrane (ILM) in vitreoretinal surgery. The vital dyes have shown cytotoxic effects in ocular cells. Autophagy is a stress responsive pathway for either protecting cells or promoting cell death. However, the role of autophagy in ocular cells in response to the vital dyes remains unknown. In this study, we found that ICG and BBG reduced cell viability in both human retinal pigment epithelial ARPE-19 and mouse photoreceptor 661W cells. ICG and BBG induced lipidated GFP-LC3-II and LC3-II in ARPE-19 and 661W cells. Combination treatment with the autophagy inhibitor chloroquine indicated that ICG and BBG reduced autophagic flux in ARPE-19 cells, whereas the vital dyes induced autophagic flux in 661W cells. Moreover, genetic and pharmacological ablation of autophagy enhanced vital dyes-induced cytotoxicity in ocular cells. Dietary supplements, including resveratrol, lutein, and CoQ10, induced autophagy and diminished the cytotoxic effects of ICG and BBG in ocular cells. These results suggest that autophagy may protect ARPE-19 and 661W cells from vital dyes-induced damage.

Early Subretinal Allograft Rejection Is Characterized by Innate Immune Activity.

Successful subretinal transplantation is limited by considerable early graft loss despite pharmacological suppression of adaptive immunity. We postulated that early innate immune activity is a dominant factor in determining graft survival and chose a nonimmunosuppressed mouse model of retinal pigment epithelial (RPE) cell transplantation to explore this. Expression of almost all measured cytokines by DH01 RPE cells increased significantly following graft preparation, and the neutrophil chemoattractant KC/GRO/CINC was most significantly increased. Subretinal allografts of DH01 cells (C57BL/10 origin) into healthy, nonimmunosuppressed C57BL/6 murine eyes were harvested and fixed at 1, 3, 7, and 28 days postoperatively and subsequently cryosectioned and stained. Graft cells were detected using SV40 large T antigen (SV40T) immunolabeling and apoptosis/necrosis by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL). Sections were also immunolabeled for macrophage (CD11b and F4/80), neutrophil (Gr1 Ly-6G), and T-lymphocyte (CD3-ɛ) infiltration. Images captured with an Olympus FV1000 confocal microscope were analyzed using the Imaris software. The proportion of the subretinal bolus comprising graft cells (SV40T+) was significantly (p < 0.001) reduced between postoperative day (POD) 3 (90 ± 4%) and POD 7 (20 ± 7%). CD11b+, F4/80+, and Gr1 Ly-6G+ cells increased significantly (p < 0.05) from POD 1 and predominated over SV40T+ cells by POD 7. Colabeling confocal microscopic analysis demonstrated graft engulfment by neutrophils and macrophages at POD 7, and reconstruction of z-stacked confocal images confirmed SV40T inside Gr1 Ly-6G+ cells. Expression of CD3-ɛ was low and did not differ significantly between time points. By POD 28, no graft cells were detectable and few inflammatory cells remained. These studies reveal, for the first time, a critical role for innate immune mechanisms early in subretinal graft rejection. The future success of subretinal transplantation will require more emphasis on techniques to limit innate immune-mediated graft loss, rather than focusing exclusively on suppression of the adaptive immune response.

Surgical treatment in combined hamartoma of the retina and retinal pigment epithelium. / Tratamiento quirúrgico en un hamartoma combinado de retina y epitelio pigmentario.

CASE REPORT: The case is presented of a 39 year-old man with a combined hamartoma of the retina and retinal pigment epithelium, who experienced progressive visual loss and worsening of metamorphopsia. The patient underwent vitrectomy and epiretinal component peeling, with improvement in visual acuity, metamorphopsia, and retinal architecture, assessed by optical coherence tomography. DISCUSSION: Selected patients with combined hamartomas of the retina and retinal pigment epithelium may benefit from surgical management.

Morphologic changes in the retina after selective retina therapy.

PURPOSE: To investigate structural changes in the retina by histologic evaluation and in vivo spectral domain optical coherence tomography (SD-OCT) following selective retina therapy (SRT) controlled by optical feedback techniques (OFT). METHODS: SRT was applied to 12 eyes of Dutch Belted rabbits. Retinal changes were assessed based on fundus photography, fluorescein angiography (FAG), SD-OCT, light microscopy, transmission electron microscopy (TEM), and scanning electron microscopy (SEM) at each of the following time points: 1 h, and 1, 3, 7, 14 and 28 days after SRT. BrdU (5'-bromo-2'-deoxy-uridine) incorporation assay was also conducted to evaluate potential proliferation of RPE cells. RESULTS: SRT lesions at1 h after SRT were ophthalmoscopically invisible. FAG showed leakage in areas corresponding to SRT lesions, and hyperfluorescence disappeared after 7 days. SD-OCT showed that decreased reflectivity corresponding to RPE damage was restored to normal over time in SRT lesions. Histologic analysis revealed that the damage in SRT lesions was primarily limited to the retinal pigment epithelium (RPE) and the outer segments of the photoreceptors. SEM and TEM showed RPE cell migration by day 3 after SRT, and restoration of the RPE monolayer with microvilli by 1 week after SRT. At 14 and 28 days, ultrastructures of the RPE, including the microvilli and tight junctions, were completely restored. The outer segments of the photoreceptors also recovered without sequelae. Interdigitation between the RPE and photoreceptors was observed. BrdU incorporation assay revealed proliferation of RPE on day 3 after SRT, and peak proliferation was observed on day 7 after SRT. CONCLUSION: Based on multimodal imaging and histologic assessment, our findings demonstrate that SRT with OFT could selectively target the RPE without damaging the neurosensory retina. Therefore, the use of SRT with OFT opens the door to the possibility of clinical trials of well-defined invisible and nondestructive retina therapy, especially for macular disease.

Thermofusion of the retina with the RPE to seal tears during retinal detachment repair.

PURPOSE: To develop an animal model to test the hypothesis that immediate adhesion of the retina to the choroid (retinopexy) can be created by elimination of the water separating the retina from the retinal pigment epithelium (RPE) prior to photocoagulation. The retina and RPE are hydrophobic lipoprotein structures separated intraoperatively by a thin layer of fluid despite surgical drainage. If the RPE and retina are contacting, heating should create a unified local coagulum and achieve instantaneous fusing of the retina and RPE, thus sealing the subretinal space around the retinal tear. The surgical technique and histological findings in a rabbit model of rhegmatogenous retinal detachment (RRD) are reported here. METHOD: Nine Dutch-belted, pigmented rabbits underwent vitrectomy with lensectomy, creation of localised retinal detachment by subretinal injection of balanced salt solution (BSS), enlargement of the hole and fluid-gas exchange to "re-attach" the retina. Dehydration of the retina surrounding the hole was achieved by an airstream from a flute needle. A laser (810 nm) was applied in long pulses to achieve a mild retinal reaction around the hole in the dehydrated adjacent retina. The BSS irrigation was resumed. Eyes were then enucleated and the treated retina examined histologically. RESULTS: The dehydrated and lasered retinal tear margin demonstrated fusion of the retina with the RPE/choroid. The non-dehydrated adjacent areas showed thermal tissue changes in the retina, RPE/choroid and adjacent sclera but remained separated by persistent subretinal fluid and no fusion or unified coagulum developed. CONCLUSION: Immediate laser-induced thermal fusion of the retina with the RPE at the margin of a retinal tear can be achieved by removing the subretinal fluid prior to photocoagulation. The integrated coagulum seals the tear margin preventing further fluid entering the subretinal space, thus correcting the cause of RRD. This method may facilitate RRD repair without buckling or internal tamponade.

An easy, rapid method to isolate RPE cell protein from the mouse eye.

The retinal pigment epithelium (RPE) is essential for maintaining the health of the neural retina. RPE cell dysfunction plays a critical role in many common blinding diseases including age-related macular degeneration (AMD), diabetic retinopathy, retinal dystrophies. Mouse models of ocular disease are commonly used to study these blinding diseases. Since isolating the RPE from the choroid has been challenging, most techniques separate the RPE from the retina, but not the choroid. As a result, the protein signature actually represents a heterogeneous population of cells that may not accurately represent the RPE response. Herein, we describe a method for separating proteins from the RPE that is free from retinal and choroidal contamination. After removing the anterior segment and retina from enucleated mouse eyes, protein from the RPE was extracted separately from the choroid by incubating the posterior eyecup with a protein lysis buffer for 10 min. Western blot analysis identified RPE65, an RPE specific protein in the RPE lysates, but not in choroidal lysates. The RPE lysates were devoid of rhodopsin and collagen VI, which are abundant in the retina and choroid, respectively. This technique will be very helpful for measuring the protein signal from the RPE without retinal or choroidal contamination.