Ophthalmic Surgery, Lasers and Imaging Retina

Case Report 

Dedifferentiation of Retinal Pigment Epithelium in a Patient With Chronic Retinal Detachment

Mert Simsek, MD, FICO; Serdar Ozates, MD; Bayram Gulpamuk, MD; Berrin Buyukeren, MD; Mehmet Yasin Teke, MD

Abstract

A 34-year-old woman presented with vision loss in her right eye, which had persisted for approximately 12 months. Funduscopy showed horseshoe retinal tear at the 8-o'clock position and retinal detachment in inferior, nasal, and temporal quadrants, with a pigmented demarcation line in the right eye. Diffuse punctate accumulations were noted on the detached retinal surface and in the vitreous. Fundus autofluorescence imaging detected diffuse punctate accumulations, which produced hyperautofluorescence spots. Cytological examination of a vitreous sample detected CD68-positive and cytokeratin- and CD45-negative macrophages. These findings indicate that epithelial-to-mesenchymal transition occurred in the retinal pigment epithelium of our patient.

[Ophthalmic Surg Lasers Imaging Retina. 2018;49:716–720.]

Abstract

A 34-year-old woman presented with vision loss in her right eye, which had persisted for approximately 12 months. Funduscopy showed horseshoe retinal tear at the 8-o'clock position and retinal detachment in inferior, nasal, and temporal quadrants, with a pigmented demarcation line in the right eye. Diffuse punctate accumulations were noted on the detached retinal surface and in the vitreous. Fundus autofluorescence imaging detected diffuse punctate accumulations, which produced hyperautofluorescence spots. Cytological examination of a vitreous sample detected CD68-positive and cytokeratin- and CD45-negative macrophages. These findings indicate that epithelial-to-mesenchymal transition occurred in the retinal pigment epithelium of our patient.

[Ophthalmic Surg Lasers Imaging Retina. 2018;49:716–720.]

Introduction

The retinal pigment epithelium (RPE) is a single layer of polygonal cells present below the sensorial retina that plays various roles in maintaining the optimal function and health of the retina, especially photoreceptor layer. The RPE performs different functions, including storage of metabolites, external blood-retinal barrier function, nutrient transport, phagocytosis of degraded outer segment materials, and vitamin A storage.1 In order to function properly, RPE cells need to be highly differentiated.

Fundus autofluorescence (FAF) is a rapid, noninvasive, and reproducible fundus imaging modality for detecting lipofuscin, the predominant fluorophore in the RPE. RPE alteration, pigment accumulation, lipofuscinopathies (Stargardt and Best diseases), and various retinal pathologies (age-related macular degeneration, central serous chorioretinopathy, and chloroquine retinopathy) cause changes in autofluorescence patterns.2

In this study, we examined funduscopy findings and assessed RPE transdifferentiation detected by the cytological examination of a pathological sample obtained from a patient who presented with retinal detachment, which had persisted for approximately 12 months. Moreover, we discussed the reason underlying diffuse punctate hyperautofluorescence observed in the vitreous during FAF imaging.

Case Report

A 34-year-old female patient presented with impaired vision in her right eye that had persisted for nearly 12 months. In her examination, best-corrected visual acuity was at hand motion level in the right eye and was 10/10 in the left eye. Intraocular pressure, as measured through applanation, was 10 mm Hg and 15 mm Hg in the right and left eyes, respectively.

Biomicroscopy detected normal anterior segment findings for both the eyes. Dilated funduscopy of the right eye with a three-mirror ocular lens revealed a horseshoe retinal tear at the 8-o'clock position and retinal detachment in inferior, nasal, and temporal quadrants, including the macula; however, no anomaly was detected in superior retinal quadrant. A pigmented demarcation line caused by the long-standing detachment separated the detached retinal quadrant from the normal retina (Figure 1). Dilated funduscopy of the left eye yielded normal findings.

Demarcation line at digital color fundus photography.

Figure 1.

Demarcation line at digital color fundus photography.

Diffuse punctate accumulations were noted on the detached retinal surface and in the vitreous. FAF imaging detected diffuse punctate accumulations, which demonstrated hyperautofluorescence (Figures 2 and 3). Optic coherence tomography also revealed these accumulations in the vitreous and on the retinal surface (Figure 4).

Hyperautofluorescence spots at vitreous and retinal surface.

Figure 2.

Hyperautofluorescence spots at vitreous and retinal surface.

Hyperautofluorescence spots at vitreous and retinal surface.

Figure 3.

Hyperautofluorescence spots at vitreous and retinal surface.

Hyperautofluorescence spots at vitreous and retinal surface.

Figure 4.

Hyperautofluorescence spots at vitreous and retinal surface.

Pars plana vitrectomy (PPV) and intravitreal tamponade were planned in the right eye to provide its anatomical integrity. The patient was operated, and a vitreous sample was obtained without turning on the infusion fluid for performing cytological examination. PPV was performed, and the retina was mobilized. Endolaser was applied to the inferior half of retina and the retinal tear at the 8-o'clock position. The operation was ended by injecting perfluoropropane (C3F8) for tamponade. Control examination performed after 3 weeks showed C3F8 absorption and retinal attachment.

Vitreous aspiration was performed using a 27-gauge needle attached to a 1-mL syringe inserted through the pars plana at 3.5 mm from the limbus and directed toward the optic nerve to slowly aspirate 0.2 mL vitreous sample before performing vitrectomy and without turning on the infusion fluid. The sample was transported to Hacettepe University in a sterile container.

Cytological examination of the vitreous sample detected CD68-positive and cytokeratin (CK)-negative macrophages. Moreover, most macrophages were stained with HMB-45 and few macrophages were stained with Prussian blue (Figure 5).

40× magnification, staining in vitreous samples. (A) CD68, (B) Pan-CK, (C) HMB45, and (D) Prussian Blue.

Figure 5.

40× magnification, staining in vitreous samples. (A) CD68, (B) Pan-CK, (C) HMB45, and (D) Prussian Blue.

Discussion

Retinal inflammation, trauma, aging, and detachment disrupt the physiological structure and normal functions of the RPE. RPE dysfunction causes a disturbance in the normal retinal function and loss of optimal retinal micro-environment. During retinal detachment, RPE cells become round, lose their microvilli, detach from basal membrane, aggregate, and form clusters.3

RPE cells are post-mitotic and differentiated cells. During retinal injury, growth factors such as FGF, TGF-β, and HGF; inflammatory mediators; and cytokines induce metaplasia, thus disrupting the normal structure of differentiated RPE cells.4 This is called dedifferentiation, transdifferentiation, or epithelial-to-mesenchymal transition. During the process of dedifferentiation, RPE cells lose various intracellular and cell surface proteins and pigments. Moreover, their cytoskeletal protein expressions are altered, giving them a mobile character. The resulting physiologically active macrophagic or fibroblastic RPE cells can replicate and move to the retinal surface by passing through the retinal break. It is known that transforming RPE cells are responsible for the development of proliferative vitreoretinopathy and epiretinal membrane.5

Despite being functionally different, dedifferentiated RPE cells retain some cellular markers. One of the best-characterized markers of differentiated RPE cells is the family of cytoskeletal proteins known as CK.6 CK-7, CK-8, CK-18, and CK-19 are the most-studied markers. CK-8 and CK-18 expressions are often downregulated, and CK-7 expression is often upregulated in dedifferentiated RPE cells. CK-19 is associated with the mobility of RPE cells. However, some dedifferentiated RPE cells do not express any of the examined CK proteins. Some dedifferentiated RPE cells also express CD68, a transmembrane protein that is highly expressed by bone marrow-derived macrophages.7 However, CD68 is not expressed by differentiated RPE cells of the healthy eyes.

Cytological examination of the vitreous sample obtained from our patient detected pan CK-negative and CD68-positive cells, suggesting that cell deposits observed on the retinal surface and in the vitreous contained macrophages. These macrophages were suggested to originate from differentiated RPE cells during chronic retinal detachment through transdifferentiation in response to existing inflammatory mediators and cytokines by acquiring macrophagic features similar to those of bone marrow-derived macrophages.

CD45 is co-expressed along with CD68 in bone marrow-derived macrophages.8 It is an important surface marker enabling us to determine whether a macrophage has originated from bone marrow or not. Cytological examination of our case's vitreous sample showed CD45 was negative. CD68 positivity along with CD45 negativity ruled out the possibility that these macrophages arrived secondary to possible previous vitreous hemorrhage caused by retinal detachment.

Boulton et al. showed that dedifferentiated RPE cells can synthesize melanin and show increased tyrosinase activity in tissue culture.9 Intracytoplasmic examination of the vitreous sample indicated that most cells showed HMB45 (a melanocyte marker) positivity and few cells showed Prussian blue (a hemosiderin marker) positivity.

Lipofuscin, a fluorophore accumulating in lysosomes after the degradation of the external segment of photoreceptors, is the main source of autofluorescence.2 However, there are also other fluorophores besides lipofuscin. Connective tissue elements such as collagen and elastin, pigments such as hemosiderin, and accumulations such as drusen induce hyper-autofluorescence in FAF imaging.10,11 In our patient, diffuse punctate hyperautofluorescence detected by FAF imaging was suggested to be caused by hemosiderin present in macrophages.

Hemorrhage results in hemoglobin degradation and hemosiderin production, which induces hyperautofluorescence in FAF imaging. The origin of hemosiderin in macrophages detected in our patient was unclear because the patient had no bleeding at presentation or during diagnosis. However, the patient had long-standing retinal detachment, which was diagnosed in the late period. Therefore, hemosiderin detected in the macrophages of our patient may be produced by bleeding that occurred in the past.

In conclusion, we observed that retinal inflammation due to chronic retinal detachment stimulated the dedifferentiation of post-mitotic RPE cells. Moreover, we detected hemosiderin-laden macrophages that were produced as a result of metaplasia and functioned similar to bone marrow-derived macrophages. Furthermore, hemosiderin caused the hyperautofluorescence detected by FAF imaging.

Our results indicate that determination of the exact time of RPE transition and visualization of hemosiderin-laden macrophages entering the anterior chamber fluid from the vitreous by FAF imaging may help in determining the approximate time of retinal detachment. Particularly, this will considerably help in determining the time of retinal detachment in cases where the retina cannot be visualized directly (because of corneal opacities, mature cataract, intravitreal hemorrhage, etc.) and in predicting postoperative prognosis.

References

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Authors

From the Department of Ophthalmology, Sorgun State Hospital, Yozgat, Turkey (MS); the Department of Ophthalmology, Dr. Sami Ulus Maternity and Children's Health and Diseases Training and Research Hospital, Ankara, Turkey (SO); the Department of Ophthalmology, Ulucanlar Eye Training and Research Hospital, Ankara, Turkey (BG, MYT); and the Department of Pathology, Hacettepe University Faculty of Medicine, Ankara, Turkey (BB).

The authors report no relevant financial disclosures.

Address correspondence to Mert Simsek, MD, FICO, Sorgun State Hospital, Ahmetefendi Mahallesi, Şehit Cemal Şimşek Caddesi, No: 37, 66700, Sorgun, Yozgat, Turkey; email: mertsimsek86@gmail.com.

Received: January 09, 2018
Accepted: August 03, 2018

10.3928/23258160-20180831-11

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