An ideal dye for intraocular use should effectively stain the target tissue while being easy to apply and remove. Additionally, it should not have any adverse effects resulting from prolonged contact with the retinal tissue. Recently, concerns have been raised about the safety of some vital dyes during surgical procedures as they may cross the internal limiting membrane and deposit on the retina. In this study, we aimed to investigate whether commercially available vital dyes, VIEW-ILM® and TWIN® (AL.CHI.MI.A. S.r.l., Ponte San Nicolò, Padova, Italy), have the potential to cross the internal limiting membrane during vitreoretinal surgery and deposit on the retina. Furthermore, we evaluated their safety in vitro and in vivo.
A human-like pars plana vitrectomy was performed on porcine eyes ex vivo, with VIEW-ILM® or TWIN® used to stain the internal limiting membrane either with or without subsequent internal limiting membrane peeling. The two dyes were then extracted from retinal punches with or without internal limiting membrane, and quantified using high performance liquid chromatography. Safety was evaluated through in vitro cytotoxicity tests and in vivo skin sensitization and irritation tests according to ISO standards.
High performance liquid chromatography analyses demonstrated that VIEW-ILM® and TWIN® effectively stained the internal limiting membrane without crossing the membrane. No residual dyes were found in the retinal layers after internal limiting membrane removal. Furthermore, both in vitro and in vivo safety tests confirmed the absence of cytotoxicity, skin sensitization, and irritation.
The results of this study support the safety and efficacy of VIEW-ILM® and TWIN® for internal limiting membrane staining. The experimental protocol described in this study could be utilized to gain a comprehensive understanding of the characteristics of vital dyes.

BACKGROUND: Macular hole (MH) can severely impair central vision. Although it can be treated with vitrectomy surgery, avoiding recurrence and improving visual acuity are still priorities to be addressed. This study aims to reveal the trends and hotspots about MH.
METHODS: The Web of Science Core Collection (WOSCC) was used to perform a bibliometric analysis investigating trends of MH research from 2002 to 2021. We evaluated the details of associated regions, institutions, authors, and journals. To construct and overlay network visualizations, VOSviewer software was used.
RESULTS: In total, 1518 publications were collected. Our analysis showed that MH research is becoming increasingly relevant, with Japan achieving the largest number of publications (291), largest number of citations (7745 in total), and highest h-index value (48). Retina published the most publications on this topic, totaling more than the next two journals combined. An analysis of keyword co-occurrence was evaluated, highlighting several novel keywords of interest, such as flap technique, transplantation, epiretinal proliferation (EP), foveal microstructure, and retinal sensitivity.
CONCLUSIONS: Details on MH research were uncovered by comprehensively analyzing the global trends and hotspots over the past two decades, presenting valuable information for future MH research. Japan, the USA, and China hold leading positions in research on this topic. Amendable surgical methods are a potential focus for improving prognosis.

Dissociated optic nerve fiber layer (DONFL) appearance is characterized by dimpling of the fundus when observed after vitrectomy with the internal limiting membrane (ILM) peeling in macular diseases. However, the cause of DONFL remains largely unknown. Optical coherence tomography (OCT) findings have indicated that the nerve fiber layer (NFL) and ganglion cells are likely to have been damaged in patients with DONFL appearance. Since DONFL appearance occurs at a certain postoperative period, it is unlikely to be retinal damage directly caused by ILM peeling because apoptosis occurs at a certain period after tissue damage and/or injury. However, it may be due to ILM peeling-induced apoptosis in the retinal tissue. Anoikis is a type of apoptosis that occurs in anchorage-dependent cells upon detachment of those cells from the surrounding extracellular matrix (i.e., the loss of cell anchorage). The anoikis-related proteins βA3/A1 crystallin and E-cadherin are reportedly expressed in retinal ganglion cells. Thus, we theorize that one possible cause of DONFL appearance is ILM peeling-induced anoikis in retinal ganglion cells.