Miyake-Apple view of inner side of sclerotomy during microincision vitrectomy surgery.

PURPOSE: To examine the inner surface of the sclerotomy during microincision vitrectomy surgery by Miyake-Apple view. METHODS: The anterior half of porcine eyes was attached to a transparent acrylic plate with cyanoacrylate glue. Then, either a 23-gauge or a 25-gauge trocar-cannula was inserted through the sclera obliquely. The inner surface of the entrance site was observed posteriorly by Miyake-Apple view. These images were compared with the endoscopic view of two patients who underwent vitreous surgery for an epiretinal membrane. RESULTS: When the trocar-cannula was inserted obliquely, the Miyake-Apple view showed that the ciliary epithelium at the sclerotomy site was stretched. When the trocar-cannula was inserted vertically, the ciliary epithelium was folded, and the folds remained even after the trocar was removed. Vitreous strands were seen incarcerated into the sclerotomy site. In human eyes, a folding of the ciliary epithelium was not clearly seen with the endoscopic view but the incarcerated vitreous was seen. CONCLUSION: The Miyake-Apple view provided a precise, in vivo, observation of the inner surface of the entry site. It disclosed the morphological stress on the ciliary epithelium by the sclerotomy.

Retinal pigment epithelium (RPE)-choroid graft translocation in the treatment of an RPE tear: preliminary results.

AIM: To investigate whether retinal pigment epithelium (RPE)-choroid translocation would be a suitable treatment for RPE tears, which have a poor prognosis and are encountered more often since the introduction of anti-(vascular endothelial growth factor (VEGF)) therapy for exudative age-related macular degeneration (AMD). METHODS: Prospective interventional case series of six eyes of six patients with AMD with an RPE tear treated with an RPE-choroid translocation. The RPE tear occurred in a vascularised pigment epithelium detachment in four patients and after treatment in the other two. Preoperative and postoperative evaluation included ETDRS visual acuity (VA) and fixation testing. The follow-up period ranged from 6 months to 2 years. RESULTS: The mean preoperative VA was 20/160 (range 20/400-20/80). The mean VA at the last examination after surgery was 20/80 (range 1/60-20/50). One of the six patients had a preoperative VA of >/=20/80, and four had a VA of 20/80 or better at their last examination. Foveal fixation on the graft was present in five of the six eyes up to the last examination. CONCLUSION: These preliminary data show that an RPE-choroid translocation may be a treatment option for patients with an RPE tear.

Investigation of selective retina treatment (SRT) by means of 8 ns laser pulses in a rabbit model.

BACKGROUND: It has been shown that selective retina treatment (SRT) using a train of 1.7 microseconds laser pulses allows selective damage of the retinal pigment epithelium (RPE) while sparing the adjacent photoreceptors and thus avoiding laser scotoma. It was the purpose of this work to investigate SRT laser effects with Q-switched pulses of only 8 nanoseconds in duration by evaluating the angiographic and ophthalmoscopic damage thresholds and the damage range by histology in a rabbit model. MATERIALS AND METHODS: A flash lamp pumped frequency doubled (532 nm) Nd:YAG laser with 8 nanoseconds pulse duration was used. In total 210 laser lesions, each calculated to be 102 microm in diameter on retina, were applied through a slit lamp onto the fundus of six eyes of Chinchilla Bastard rabbits. The rabbits were irradiated with increasing energies with single pulses and a train of 10 laser pulses at 10 Hz. After treatment fundus photography and angiography were performed to determine the damage thresholds (ED(50)-probability of RPE cell damage and neurosensory retinal damage) as well as the safety range between both thresholds (ratio of angiographic ED(86) vs. ophthalmoscopic ED(14)). Selected histology was taken for single and repetitive pulse lesions after treatment. RESULTS: Angiographic and ophthalmoscopic ED(50)-thresholds decreased with increasing number of pulses. For single pulse application ophthalmoscopic and angiographic ED(50) were determined to 365 and 144 mJ/cm(2), respectively. Regarding 10 pulses 266 and 72 mJ/cm(2) were found. No retinal hemorrhages or disruptions were observed for both sets of parameters. The therapeutic window between angiographic and ophthalmoscopic threshold revealed a factor of 3.1 for single pulses and 2.3 for repetitive pulse irradiation. The safety range respectively had a factor of 0.8 (single pulses) and 1.7 (10 pulses). Histologic examination of laser lesions with single and repetitive pulses at radiant exposures within the therapeutic window-292 and 213 mJ/cm(2) respectively-revealed damaged RPE, intact Bruch's membrane and choriocapillaries. Photoreceptors were partly spared but also damaged to various extents. CONCLUSIONS: Short laser pulses of 8 nanoseconds pulse duration can damage the RPE without retinal hemorrhage or disruption. Selective damage of the RPE without affecting the photoreceptors can only rarely be achieved due to the small safety range. Thus, so far microsecond laser pulses for SRT seems favorable compared to nanosecond pulses in order to prevent unintentional photoreceptor damage.

Retinal pigment epithelial tears after intravitreal bevacizumab injection for predominantly classic choroidal neovascularization.

PURPOSE: To detect retinal pigment epithelium (RPE) tears in predominantly classic choroidal neovascularization (CNV) secondary to age-related macular degeneration (AMD) treated with intravitreal bevacizumab injections. METHODS: Forty consecutive patients with predominantly classic CNV secondary to AMD were treated with 1.25 mg of intravitreal bevacizumab. Patients were evaluated with visual acuity (VA) measured with Early Treatment Diabetic Retinopathy Study charts, optical coherence tomography, and fluorescein angiography. RESULTS: Three patients developed a RPE tear after the first injection. The first patient had been treated with verteporfin therapy and VA remained unchanged. In the other two cases the CNV was naive and VA improved since the foveal center was not involved by the tear and macular edema was reduced. CONCLUSIONS: RPE tears can occur following intravitreal bevacizumab injections in patients with predominantly classic CNV although VA is not always affected.

Influence of pulse duration and pulse number in selective RPE laser treatment.

BACKGROUND AND OBJECTIVES: The therapeutic effect of laser treatment for macular diseases is related to the damage to the retinal pigment epithelium (RPE) and the subsequent restoration of the defect due to RPE proliferation. In contrast to conventional laser treatment, it is possible to damage the RPE selectively and to spare the photoreceptors by using repetitive microsecond laser pulses. It was the aim of the study to investigate the influence of pulse duration and number of pulses on angiographically and ophthalmoscopically visible retinal damage thresholds in order to optimize treatment modalities. STUDY DESIGN/MATERIALS AND METHODS: In total, 625 laser lesions with various parameters were applied to the retina in 11 eyes of 6 Chinchilla breed rabbits using an experimental laser system (Nd:YLF at 527 nm). Pulse duration (1.7 microseconds and 200 nanoseconds) and number of pulses (100, 10, and 1 pulses) were varied at a constant repetition rate of 100 Hz. Damage thresholds were determined in terms of ophthalmoscopic and fluorescein angiographic visibility, and the therapeutic window (TW; angiographic ED(50) vs. ophthalmoscopic ED(50)) as well as the safety range (SR; angiographic ED(84) vs. ophthalmoscopic ED(16)) between both thresholds were calculated. Selected laser lesions were evaluated by histology. RESULTS: Generally, the ED(50) radiant exposure for angiographic visibility decreases for shorter laser pulses and with an increase in the number of pulses. The TW for both pulse durations (1.7 microseconds and 200 nanoseconds) was wider with 100 pulses than with single pulses. The widest TW was found for 100 pulses at 200 nanoseconds pulse duration (5.9-fold above the angiographic threshold), and the smallest TW with a factor of 1.6 was found for 1.7 microseconds single pulses. In terms of SR, only irradiation with 100 pulses at 200 nanoseconds pulse duration was associated with a ratio >2. Independently of pulse duration, histological examination of laser sites 1 hour after irradiation revealed widely intact photoreceptors, while the underlying RPE was damaged. CONCLUSIONS: Pulse duration and number of pulses have a significant influence on RPE damage thresholds and consecutively on TW and SR. Because fundus pigmentation in humans may vary intra- and interindividually by a factor of 2, a large TW and ideally also a large SR should be ensured in a clinical treatment context. In rabbits, the safety range with 200 nanoseconds pulses is higher than with the pulse duration of 1.7 microseconds currently in clinical use. These findings suggest the need for clinical pilot studies to prove whether these results can be transposed to the situation in humans.

Targeting of the retinal pigment epithelium (RPE) by means of a rapidly scanned continuous wave (CW) laser beam.

BACKGROUND AND OBJECTIVES: Selective treatment of the retinal pigment epithelium (RPE) by repetitively applying green micros-laser pulses is a new method for retinal diseases associated with a degradation of the RPE, which spares the neural retina. We investigated an alternative approach to realize repetitive micros-laser exposure by rapidly scanning a continuous wave (CW)-laser beam across the RPE. STUDY DESIGN/MATERIALS AND METHODS: An Ar(+) laser beam (514 nm) with a diameter of 18.75 microm was repetitively scanned across porcine RPE samples in vitro providing an irradiation time of 1.6 micros per point on the central scan axis. RPE cell damage was investigated by means of the fluorescence viability assay Calcein-AM. RESULTS: The ED(50) cell damage is 305 mJ/cm(2) when applying 10 scans with a repetition rate of 500 Hz. The threshold decreases with the number of scans, a saturation was found at 135 mJ/cm(2) with more than 500 exposures applied. The depth of focus in beam direction is 350 microm, defined by an increase of the threshold radiant exposure by 20%. CONCLUSIONS: Targeting of pigmented cells with high local resolution has been proved with a laser-scanning device. Looking ahead selective RPE-treatment, the adaptation of a laser-scanning device on a slit-lamp or into a modified retina angiograph seems to be an attractive alternative to the pulsed micros laser device.

Effects of hypoxia on retinal pigmented epithelium cells: protection by antioxidants.

Age-related macular degeneration and other eye diseases, such as diabetic retinopathy, are probably linked to the effects of oxygen radicals derived from light or metabolic reactions. We have investigated the effects of hypoxia on bovine retinal pigmented epithelial cells (RPE) and the response of these cells to two antioxidants that have previously shown a beneficial action against free radical-linked senescent involution. The main results of the study were as follows: (i) Hypoxia induced apoptotic damage on RPE cells, with LDH leakage and ATP reduction; (ii) both vitamin C (VC) and N-acetyl-cysteine (NAC) treatment protected against hypoxia-induced apoptosis, with less DNA fragmentation. In our opinion, these findings justify further experimental and clinical work to investigate the role of hypoxia in the mechanisms of age-related RPE injury and death as well as the potential of antioxidant administration to prevent or delay retinal degenerative processes caused by oxygen-dependent pathophysiological conditions.

Threshold determinations for selective retinal pigment epithelium damage with repetitive pulsed microsecond laser systems in rabbits.

BACKGROUND AND OBJECTIVE: In both clinical and animal studies, it has been shown that repetitive short laser pulses can cause selective retinal pigment epithelium damage (RPE) with sparing of photoreceptors. Our purpose was to determine the ophthalmoscopic and angiographic damage thresholds as a function of pulse durations by using different pulsed laser systems to optimize treatment modalities. MATERIALS AND METHODS: Chinchilla-breed rabbits were narcotized and placed in a special holding system. Laser lesions were applied using a commercial laser slit lamp, contact lens, and irradiation with a frequency-doubled Nd:YLF laser (wave-length: 527 nm; repetition rate: 500 Hz; number of pulses: 100; pulse duration: 5 micros, 1.7 micros, 200 ns) and an argon-ion laser (514 nm, 500 Hz, 100 pulses, 5 micros and 200 ms). In all eyes, spots with different energies were placed into the regio macularis with a diameter of 102 microm (tophat profile). After treatment, fundus photography and fluorescein angiography were performed and radiant exposure for ED50 damage determined. Speckle measurements at the fiber tips were performed to determine intensity peaks in the beam profile. RESULTS: Using the Nd:YLF laser system, the ophthalmoscopic ED50 threshold energies were 25.4 microJ (5 micros), 32 microJ (1.7 micros), and 30 microJ (200 ns). The angiographic ED50 thresholds were 13.4 microJ (5 micros), 9.2 microJ (1.7 micros), and 6.7 microJ (200 ns). With the argon laser, the angiographic threshold for 5 micros pulses was 5.5 microJ. The ophthalmoscopic threshold could not be determined because of a lack of power; however, it was > 12 microJ. For 200 ms, the ED50 radiant exposures were 20.4 mW ophthalmoscopically and 19.2 mW angiographically. Speckle factors were found to be 1.225 for the Nd:YLF and 3.180 for the argon laser. Thus, the maximal ED50 -threshold radiant exposures for the Nd:YLF were calculated to be 362 mJ/cM2 (5 micros), 478 mJ/cm2 (1.7 micros), and 438 mJ/cm2 (200 ns) ophthalmoscopically. Angiographically, the thresholds were 189 mJ/cm2 (5 micros), 143 mJ/cm2 (1.7 micros), and 97 mJ/cm2 (200 ns). For the argon laser, the maximal ED50 radiant exposure threshold was 170 mJ/cm2 angiographically. CONCLUSION: The gap between the angiographic and the ophthalmoscopic thresholds for the 200 ns regime (4.5 times above angiographic ED50) was wider than for the 1.7 micros regime (3.3 times above the angiographic ED50). This would suggest the appropriate treatment would be 200 ns pulses. However, histologies have yet to prove that nonvisible mechanical effects increase with shorter pulse durations and could reduce the "therapeutic window." When comparing the thresholds with 5 micros pulses from the argon and Nd:YLF laser, it demonstrates that intensity modulations in the beam profile must be considered.

Uptake dynamics and retinal tolerance of phosphorothioate oligonucleotide and its direct delivery into the site of choroidal neovascularization through subretinal administration in the rat.

This study aimed to investigate uptake dynamics and retinal tolerance of phosphorothioate oligonucleotides (PS-oligos) following subretinal injection. A fluorescent-labeled PS-oligo (FL-oligo) with random sequence was administered into the subretinal space of rat by transsclera-choroid-retinal pigment epithelium (RPE) injection at doses of 0.129, 1.29, and 12.9 microg in 2.0 microl solution. The uptake dynamics were evaluated by fundus fluorescent photography in real time and by fluorescence microscopy using flat mounts and cryosections. Immunophenotyping for CD4+, CD8+ cytotoxic lymphocytes, and CD68+ macrophages was performed to assess cellular infiltration in the retina. In addition, the FL-oligo was injected subretinally in a rat model of choroidal neovascularization (CNV) for direct delivery into the site of CNV. Subretinal administration of FL-oligo resulted in both dose-dependent and time-dependent distribution in the retina, where it accessed the RPE and all layers of the neuroretina. CD4+, CD8+ cytotoxic lymphocytes, and CD68+ macrophages were observed at the site of needle penetration. However, in areas far from the injection site where the FL-oligo appeared strongly, cellular infiltration was absent, and the retinal morphology was preserved very well. The FL-oligo was successfully delivered into the site of intense laser photocoagulation. It was predominantly localized to the RPE, macrophages, and some choroid cells and remained detectable for at least 56 days after injection. Our results demonstrate for the first time that subretinal injection efficiently introduced PS-oligo into the RPE and neuroretina with an acceptable level of safety. Subretinal administration of antiangiogenic oligonucleotides may hold great potential for the treatment of CNV.

Induction of interleukin-8 in human retinal pigment epithelial cells after denuding injury.

AIM: To determine interleukin 8 (IL-8) and monocyte chemoattractant protein 1 (MCP-1) expression in response to mechanical injury in human retinal pigment epithelial (HRPE) cells. METHODS: Enzyme linked immunosorbent assay (ELISA) was performed to determine IL-8 and MCP-1 secretion by HRPE cells after mechanical denudation. IL-8 and MCP-1 mRNA expression by HRPE cells was assessed using semiquantitative RT-PCR. The effects of immunosuppressive drugs, dexamethasone (DEX) and cyclosporin A (CSA), as well as immunosuppressive cytokines, interleukin 4 (IL-4), interleukin 10 (IL-10), and interleukin 13 (IL-13), on chemokine expression in HRPE cells after denuding injury were analysed. RESULTS: Mechanical injury induced HRPE IL-8 mRNA and IL-8 secretion. Although MCP-1 mRNA was enhanced slightly after denuding injury, MCP-1 secretion was not increased. DEX and CSA inhibited HRPE chemokine expression after injury. IL-4 and IL-13 enhanced IL-8 and MCP-1 production by HRPE cells after injury while IL-10 had no effect. CONCLUSIONS: These results suggest that IL-8 may be involved in retinal inflammatory responses to injury and that DEX and/or CSA treatment may help control the inflammatory components of retinal diseases such as proliferative vitreoretinopathy.

Gene expression profile of ARPE-19 during repair of the monolayer.

BACKGROUND: The purpose of this study was to identify the profile of gene expression during retinal pigment epithelial (RPE) wound repair. METHODS: ARPE-19 cells derived from a human RPE cell line were grown for 4 weeks and injured by creating multiple concentric wounds. Unwounded cultures served as controls. During the proliferative phase of wound repair, total RNA was extracted from control and wounded cultures, and a [32P]dATP-labeled cDNA probe was synthesized and hybridized to Atlas Arrays (Clontech, Palo Alto, Calif.) containing 588 cDNAs. The autoradiograms obtained were then analyzed using the Molecular Dynamics software program. Semiquantitative PCR was carried out to confirm up-regulation of four genes associated with wound repair. ELISA was performed to quantitate the secreted MCP-1. RESULTS: In wounded cultures prominent up-regulation (greater than fivefold) was seen for genes encoding DNA synthesis and DNA repair proteins. A greater than threefold increase was seen for genes encoding mitogen-activated protein kinase, CD44, MCP-1 (monocyte chemotactic protein), thymosin beta-10, and HDGF (hepatoma-derived growth factor), among others. Genes encoding tumor suppressors were downregulated three- to five-fold in the wounded compared with the unwounded cultures. Semiquantitative PCR confirmed up-regulation of transcripts for thymosin beta-10, HDGF, CD44, and MCP-1. ELISA showed a 20% increase in secreted MCP-1. CONCLUSIONS: Gene array analysis revealed a differentiation program that included increased expression of genes involved in wound repair (adhesion molecules, cytokines, signal transducers), along with increased MCP-1 secretion. The RPE may be an early participant in the inflammatory response that occurs with proliferative vitreoretinopathy.

Lesäo ocular em epidermodisplasia verruciforme / Ocular lesion in epidermodysplasia verruciformis

Descriçäo de um caso de epidermodisplasia verruciforme com acometimento ocular. A tumoraçäo corneana existente foi ressecada. Näo houve recidivas mesmo três anos após a intervençäo. Estudo histopatológico é apresentado. Ao que sabemos, trata-se do primeiro caso onde é descrita a associaçäo entre tal displasia e carcinoma espinocelular córneo-conjuntival in situ

Scanning and transmission electron microscopic findings during RPE wound healing in vivo.

OBJECTIVE: To examine the scanning (SEM) and transmission (TEM) electron microscopic features of an in vivo rabbit model of retinal pigment epithelial (RPE) wound healing. METHODS: Hydraulic debridement of the RPE was performed in one eye of each of 35 pigmented rabbits using a pars plana vitrectomy approach. Five of the 35 eyes were examined by either SEM or TEM on each of the following postoperative days: 0, 2, 4, 7, 14, 28 and 56. RESULTS: TEM revealed that hydraulic RPE debridement results in only focal damage to the RPE basement membrane portion of Bruch's membrane and that this damage is repaired by day 7 without ultrastructural sequelae. SEM and TEM disclosed that the RPE cells at the margin of the debrided bed become flattened and enlarged and evolve a cytoskeletal reorganization with altered apical-basal polarity consistent with the development of a migrating phenotype. This is followed by gradual restoration to a more normal stationary RPE phenotype after initial closure (reepithelialization) of the RPE defect on day 7. RPE hyperplasia also occurs and may contribute to this repair process. Tight junctions are re-established among the apical surfaces of monolayered and multilayered RPE cells by day 7, coinciding with the restoration of the blood outer retinal barrier. CONCLUSION: Hydraulic debridement of the RPE in vivo is a useful investigational model that provides important insight into the pathogenesis of outer retinal disorders and their treatment with such techniques as submacular surgery or RPE transplantation.

Laser induced bubble formation in the retina.

BACKGROUND AND OBJECTIVE: The immediate thermodynamic effects of absorption of a laser pulse in the retina are theoretically studied to understand underlying physical damage mechanisms at threshold fluences. Damage is most likely to occur at threshold levels in the retinal pigment epithelium due to the strong absorption by the melanosomes. METHODS: The retinal pigment epithelium is modeled as an aqueous environment with absorption occurring at small spherical sites with absorption coefficients representative of melanosomes. For laser pulse durations of less than 10(-6) seconds, heat conduction is negligible during energy deposition and the resulting large energy density in the melanosomes will cause vaporization of the medium immediately surrounding a melanosome. RESULTS: We developed expressions for calculating the size of bubbles produced as a function of pulse characteristics and melanosome properties. We show that for pulse durations between 10(-6) and 10(-9) seconds, bubble formation will occur for laser fluences that are smaller than those required to cause Arrhenius-type thermal damage. CONCLUSION: Bubble formation is likely to be the mechanism of threshold damage to the retina for laser pulses durations in the time regime between 10(-6) and 10(-9) seconds.