Time-Dependent Changes of Cell Proliferation After Laser Photocoagulation in Mouse Chorioretinal Tissue

Lee, Si Hyung; Kim, Hoon Dong; Park, Yeo Jin; Ohn, Young-Hoon; Park, Tae Kwann

doi:10.1167/iovs.14-16112

Cited by 9 publications

(9 citation statements)

References 28 publications

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“…In our previous experiment, RPE proliferative activity following cwPC was elevated during the first week, and cell apoptosis was prominent during the first 24 hours. 19 Because we also expected to observe obviously TUNEL-positive cells after 24 hours of SRT-like cwPC lesions in the previous study, the TUNEL assay was performed 24 hours after SRT application to evaluate the pattern of cell death. Cell death following SRT was not identified in the neural retina, and the distribution of cell death was limited to the RPE layer.…”

Section: Discussionmentioning

confidence: 99%

“…The periodic IP EdU injection methods described in our previous paper were modified for this study. 19 The IP EdU injection schedule from the time of SRT is presented in Figure 1. The mice received periodic IP injections at the following intervals: 0 to 3 hours (n ¼ 5), 3 to 12 hours (n ¼ 5), 12 to 24 hours (n ¼ 5), 1 to 3 days (n ¼ 5), 3 to 7 days (n ¼ 5), and 7 to 14 days (n ¼ 5) after SRT.…”

Section: Intraperitoneal 5-ethynyl-2 0 -Deoxyuridine Injectionmentioning

confidence: 99%

“…19 Briefly, commercially available Click-iT Imaging Kits (Invitrogen, Carlsbad, CA, USA) were used for EdU assays after periodic IP EdU injections following SRT, and the manufacturer's protocols were followed. The tissue prepara- …”

Section: Edu Assaymentioning

confidence: 99%

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Retinal Pigment Epithelium Responses to Selective Retina Therapy in Mouse Eyes

Kim

Jang

Lee

et al. 2016

Invest. Ophthalmol. Vis. Sci.

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Citation: Kim HD, Jang SY, Lee SH, et al. Retinal pigment epithelium responses to selective retina therapy in mouse eyes. Invest Ophthalmol Vis Sci. 2016;57:3486-3495. DOI:10.1167/iovs.16-19508 PURPOSE. To investigate the characteristics of retinal pigment epithelium (RPE) and retinal damage induced by selective retina therapy (SRT) in mice, and to elucidate longitudinal changes in RPE cells.METHODS. C57BL/6J mice received SRT and continuous-wave laser photocoagulation (cwPC). The cell death pattern was evaluated using TUNEL assay, and proliferative potential of the RPE cells was evaluated using 5-ethynyl-2 0 -dexoyuridine (EdU) assay. To investigate the cell-cell integrity of RPE cells, b-catenin staining was performed. The number and hexagonality of RPE cells in the SRT-treated area were estimated using a Voronoi diagram with time periods of 3 hours to 14 days. Antibodies to microphthalmia-associated transcription factor (MiTF) and orthodenticle homeobox 2 (Otx2) were used to confirm the specific characteristics of RPE cells in the SRT-treated area.RESULTS. The number of TUNEL-positive cells located in the neural retina was significantly lower in lesions treated with SRT compared to those treated with cwPC. EdU-positive RPE cells were first detected 3 to 12 hours after SRT, and increased until 3 to 7 days after SRT. bcatenin staining showed that hexagonality was compromised and subsequently, RPE cells expanded in size within the targeted location. The number of RPE cells in SRT lesions decreased gradually until 12 hours after SRT and recovered by 14 days. Upregulated expression of MiTF and Otx2 was observed for 2 weeks in the SRT lesions.CONCLUSIONS. Selective retina therapy seems to induce selective RPE damage without collateral thermal injury in the neural retina. Furthermore, SRT-treated lesions recovered by proliferation of RPE cells that were present in the treated lesions and by expansion of adjacent RPE cells.Keywords: microphthalmia-associated transcription factor (MiTF), orthodenticle homeobox 2 (Otx2), retinal pigment epithelium, selective retina therapy L aser photocoagulation is a mainstay for the treatment of several retinal conditions associated with retinal pigment epithelium (RPE) dysfunction. 1-4 Continuous-wave laser photocoagulation (cwPC) has been used in the treatment of various macular diseases, such as diabetic macular edema (DME), agerelated macular degeneration, and central serous chorioretinopathy (CSC), over several decades.2-4 Light energy from cwPC is converted into heat in the melanosomes of RPE cells. By heat diffusion, this thermal energy not only damages the RPE but also results in collateral damage of surrounding components, particularly overlying photoreceptors, Bruch's membrane, and choriocapillaries. [5][6][7][8] This is a desired effect because it induces scar formation, which prevents further retinal detachment in retinal holes, and damages photoreceptors during panretinal photocoagulation, which is thought to preserve the central macula in diabetic retinopathy. Howev...

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Section: Discussionmentioning

confidence: 99%

Section: Intraperitoneal 5-ethynyl-2 0 -Deoxyuridine Injectionmentioning

confidence: 99%

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Retinal Pigment Epithelium Responses to Selective Retina Therapy in Mouse Eyes

Kim

Jang

Lee

et al. 2016

Invest. Ophthalmol. Vis. Sci.

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“…RPE cells of the adult murine eye are largely mitotically quiescent, 33 providing no such easy access to the nucleus. Injury to the retina, however, has been shown to induce RPE cell proliferation through the process of epithelial-to-mesenchymal transition (EMT); 34 one such injury is laser-induced photocoagulation of murine RPE 35, 36. In order to determine whether transfection of RPE in vivo by PEG-SS-POD/pCAGLuc nanoparticles could be enhanced by induction of RPE cell division by laser-induced photocoagulation, we injected C57BL/6J mice in the subretinal space with PEG-SS-POD/pCAGLuc nanoparticles either without laser treatment or immediately following laser treatment.…”

Section: Resultsmentioning

confidence: 99%

Reducible PEG-POD/DNA Nanoparticles for Gene Transfer In Vitro and In Vivo: Application in a Mouse Model of Age-Related Macular Degeneration

Dasari

Cashman

Kumar‐Singh

2017

Molecular Therapy - Nucleic Acids

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Non-viral gene delivery systems are being developed to address limitations of viral gene delivery. Many of these non-viral systems are modeled on the properties of viruses including cell surface binding, endocytosis, endosomal escape, and nuclear targeting. Most non-viral gene transfer systems exhibit little correlation between in vitro and in vivo efficiency, hampering a systematic approach to their development. Previously, we have described a 3.5 kDa peptide (peptide for ocular delivery [POD]) that targets cell surface sialic acid. When functionalized with polyethylene glycol (PEG) via a sulfhydryl group on the N-terminal cysteine of POD, PEG-POD could compact plasmid DNA, forming 120- to 180-nm homogeneous nanoparticles. PEG-POD enabled modest gene transfer and rescue of retinal degeneration in vivo. Systematic investigation of different stages of gene transfer by PEG-POD nanoparticles was hampered by their inability to deliver genes in vitro. Herein, we describe functionalization of POD with PEG using a reducible orthopyridyl disulfide bond. These reducible nanoparticles enabled gene transfer in vitro while retaining their in vivo gene transfer properties. These reducible PEG-POD nanoparticles were utilized to deliver human FLT1 to the retina in vivo, achieving a 50% reduction in choroidal neovascularization in a murine model of age-related macular degeneration.

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“…1,2 In this experiment, we used a shorter duration (0.02 sec) than previously performed, and were able to minimize the photothermal damage of photoreceptor and the RPE cells. After 28 days of injection, immunostaining with b-catenin, a wellknown protein involved in both cell-cell adhesion and canonical Wnt signaling, 3 clearly showed that short-pulse laser resulted in robust EGFP expression of not only RPE cells in the laser-treated site but also cells nearby the lesion for all capsid types (Fig.…”

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confidence: 99%

Laser Photocoagulation Induces Transduction of Retinal Pigment Epithelial Cells by Intravitreally Administered Adeno-Associated Viral Vectors

Lee

Kong

Lyu

et al. 2015

Human Gene Therapy Methods

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Figure 1. Short-pulse laser pretreatment induced transduction of adeno-associated viral (AAV) vector into retinal pigment epithelial (RPE) cells. Shown are representative confocal microscopic RPE wholemount images (200·) of the control (laser-untreated/vector-uninjected, laser-treated/vector-uninjected, and laser-untreated/vector-injected; A-E) and scAAV type 2-, 5-, and 8-treated eyes after laser pretreatment (F-H). Morphological changes of RPE cells were also observed at the lasered region. AAV5 vector (G) transduced fewer RPE cells compared with AAV2 (F) and AAV8 (H). Several RPE cells adjacent to the laser site were also transduced by AAV vectors. Laser-untreated/vector-uninjected and laser-treated/vector-uninjected control eyes did not show any EGFP expression (A and E), while laser-untreated/vector-injected eyes demonstrated few of sporadic EGFP expression in the RPE cells. The wholemount was processed with b-catenin (red) immunostaining to show the intact cell-to-cell junction. Some of the RPE cells showed cytoplasmic accumulation of b-catenin, indicating the activation of canonical Wnt signaling pathway. *Center of laser-treated region. The scale bars represent 100 lm.

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Time-Dependent Changes of Cell Proliferation After Laser Photocoagulation in Mouse Chorioretinal Tissue

Cited by 9 publications

References 28 publications

Retinal Pigment Epithelium Responses to Selective Retina Therapy in Mouse Eyes

Retinal Pigment Epithelium Responses to Selective Retina Therapy in Mouse Eyes

Reducible PEG-POD/DNA Nanoparticles for Gene Transfer In Vitro and In Vivo: Application in a Mouse Model of Age-Related Macular Degeneration

Laser Photocoagulation Induces Transduction of Retinal Pigment Epithelial Cells by Intravitreally Administered Adeno-Associated Viral Vectors

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