**1. Introduction**

The retina is a multilayered light-sensing neural tissue located at the back of the eye [1]. The outer retina is composed of photoreceptors, which convert light into electrical signals and transmit them to the inner neurons and to the ganglion cells in the inner retina. The ganglion cell layer is also composed of Muller glia, whose foot processes are embedded into a thin transparent layer in the basal lamina of the inner retina called the inner limiting membrane (ILM). The ILM forms a boundary between the retina and the underlying vitreous [2–4].

Epiretinal membrane (ERM), a fibrocellular tissues forms on the surface of ILM, mainly on the macula. ERMs can be either: (a) idiopathic or (b) associated with ocular inflammatory diseases such as diabetic retinopathy and retinal detachment [5,6]. ERMs distort the underlying ILM and alter retinal morphology and function. Survival and proliferation of the cells present on the ERM also depends upon the oxygen level. Ischemic conditions lead to accumulation/activation of the transcription factor, hypoxia-inducible factor (HIF-1 α), which causes secretion of vasoactive cytokines vascular leakage and macular edema [7]. Growth factors such as hepatocyte growth factor (HGF), heparin-binding epidermal growth factor (HB-EGF), and epidermal growth factor (EGF) others and other inflammatory molecules released due to any retinal insults also induce the migration of retinal cells like RPE to the vitreous to proliferate and induce the retinal membrane formation.

Currently, ILM removal along with ERM peeling is considered the gold standard treatment and results in minimal recurrence of the ERMs. However, the extent of successful restoration of vision varies as ERM peel can lead to retinal breaks and perturb outer retina function. Therefore, ILM peeling requires a decision on the inevitability of ERM removal only for selected needful patients.

We propose that understanding the cellular composition and gene expression signature of the ERMs will assist in designing strategies to tackle ERM formation with minimal e ffects on the ILM or avoid ERM peeling in patient where it may play a beneficial role, such as in preventing macular hole formation [8,9]. Previous remarkable studies have identified hyalocytes, glial cells, retinal pigment epithelial cells, fibrocytes and myofibroblast along with non-cellular component like fibronectin, and actin [10,11]. However, a comprehensive analysis of the di fferent components and a gene expression profile related to oxidative stress and pro-inflammatory signaling associated genes have not been investigated.

In the present study, we carried out a comparative histological, ultrastructural and gene expression analysis of fibrocellular membranes associated with inflammatory conditions of the eye. Our results expand our understanding on the role of activated microglia in ERMs and sugges<sup>t</sup> that immunomodulation by targeting microglia could be a potential therapy for a better clinical managemen<sup>t</sup> of the condition.

#### **2. Materials and Methods**

#### *2.1. Enrollment of Subjects*

Initially, ILM specimens (*n* = 30) were collected during vitreo-retinal surgery from June 2016 to September 2017 at the L.V. Prasad Eye Institute by a single vitreoretinal surgeon (Jay Chhablani) and were used for Hematoxylin and Eosin (H&E), transmission electron microscopy (TEM) and immunohistochemistry (IHC)-based evaluations. Additional membranes (*n* = 15, 3 in MH, 8 in PDR and 4 in RD) were collected from the additional ORs of two other vitreo-retinal surgeons (Mudit Tyagi and Rajeev Reddy Pappuru) for the targeted gene expression profiling. This prospective study was approved (Ethic Ref No. LEC 02-14-029) by the Institutional Review Board (IRB) of L.V. Prasad Eye Institute, Hyderabad, India (LVPEI) and adhered to the tests of the Declaration of Helsinki. A prior informed consent was obtained from each study subject. All subjects underwent a comprehensive ophthalmic examination with Snellen's visual acuity, slit lamp examination, intraocular pressure measurement using applanation tonometry and dilated fundus examination. All eyes except eyes with RRD underwent preoperative spectral domain optical coherence tomography (OCT) Cirrus

High Definition-OCT (Carl Zeiss Meditec, Dublin, CA, USA). Five-line high definition raster scan was performed.

The membranes removed as part of the routine surgical managemen<sup>t</sup> were collected from di fferent pathological conditions. Subjects with the diagnosis of vitreoretinal interface disorders including idiopathic macular hole (MH), proliferative diabetic retinopathy (PDR) and rhegmatogenous retinal detachment (RD) who underwent pars plana vitrectomy (PPV) with Brilliant blue-G (BBG)—assisted inner limiting membrane peeling (ILM peeling) were included in the study.

## *2.2. Surgical Details*

Surgical procedure included 23- or 25-gauge vitrectomy. After induction of posterior vitreous detachment, vitrectomy was completed. Epiretinal membranes were removed using end-gripping forceps. For ILM staining, 0.02% brilliant blue was used under air infusion. After one minute of staining, ILM at the macular area was removed using ILM forceps. In eyes undergoing macular hole repair, C3F8 gas (12–15%) was used as tamponade. Cataract surgery was performed during the follow-up in cases where it was needed with standard phacoemulsification procedure, and posterior chamber intraocular lens was implanted. In cases with rhematogenous RD, brilliant blue staining of ILM was performed under fluid with infusion canula o ff.
