We showed that ILM insertion combined with NGF injection was effective at early period of postoperation for patients with large idiopathic MHs (minimum diameter > 400 μm). To the best of our knowledge, this is the first time that NGF has been used as a neuroprotective agent during MH treatment. Both surgical techniques produced excellent anatomical and functional outcomes throughout the entire follow-up time, without ILM sheet dislocation. OCT revealed the significant reductions in the sizes of EZ and ELM defects. The improvement of BCVA in the NGF group at 3rd month postoperation was better than that of the insertion-only group, as was photoreceptor layer re-establishment.
ILM peeling always aids the anatomical closure of MHs21,22, but the closure rate for large MHs after the first surgery with ILM peeling is not very high and usually results in poorer visual acuity. Moreover, large MHs always tend to be V- or W-shaped, or flat-to-open (a flat MH lacking retinal pigment epithelium) after closure, and are usually associated with persistent photoreceptor loss, retinal pigment epithelium defects, and foveal tissue loss, leading to a poor visual recovery11,23. Several treatment modifications have been tested, including an inverted ILM flap technique10 and autologous ILM transplantation24. The inverted flap may not attach to the hole margin either during or after surgery, as the flap loss using OCT scan after the surgery could be observed. While free ILM fragment transplantation may afford only a small degree of clearance of liquid and result in instability during gas-liquid exchange. However, all the above therapeutic strategy has low closure rate, insufficient photoreceptor layer re-establishment and consequently poorer visual acuity. In 2016, Andrew et al. described a modified technique that place the ILM directly into the MH, in any direction and a viscoelastic cap is used to improve flap retention25. Lai et al. also used blood clots to stabilize and seal ILM flaps within MHs during air-fluid exchange26. But the toxic and uncertain effects of these additive on Müller cells and retinal pigment epithelium (RPE) cells remain unclear. Here, we inserted the inverted flap into the MH to stabilize the flap and injected NGF to improve healing process and we found that patients in both groups exhibited complete anatomical MH closure, from 1 to 6 months after surgery; the closure rates were thus 100%, implying NGF might be a synergetic protective factor during ILM insertion for the treatment of MHs.
ILM peeling allows residual Müller cells to reach the bare area of an MH, serving as a basement membrane regulating cell growth and as a scaffold for glial cell proliferation10,27,28. However, proliferation of activated glial cells triggers scarring; ILM fragments transplanted into holes may impede approximation of the neurosensory retina and the re-arranged photoreceptor cells20. We observed prominent foveal glial tissues on OCT scans of some eyes in both groups; although the MHs had closed, the BCVAs of these patients did not exhibit marked improvements. Park et al. indicated that no eye in an ILM insertion group exhibit complete recovery of the photoreceptor layers (the EZ and ELM)20. In this study, although only one eye of the control group and two of the NGF group exhibited complete recovery of ELM, and one of each group exhibited complete recovery of EZ integrity, the residual EZ and ELM defect sizes were significantly lower in the NGF group, and greater BCVA improvement and a shorter recovery period were observed. It is confirmed that a U-shaped closure is most common after use of the inverted-flap technique and is associated with better functional prognosis than other types of closure23. In our data, U-shaped foveae were observed in five of nine (55.6%) eyes in the NGF group and three of nine (33.3%) in the insertion group. We found no significant between-group difference in foveal configuration. Interestingly, the foveal configuration might be less important in terms of functional prognosis than hitherto thought, because several eyes in the NGF group with V-shaped or on the other hand not very good U-shaped foveae exhibited excellent EZ and ELM recoveries and good postoperative BCVAs (Figure 4, other OCT scans data not show). Thus, NGF might aid photoreceptor recovery and improve visual acuity, although the detailed mechanisms require further elucidation.
NGF is a classical neuroprotective factor produced by Müller cells and plays a critical role in retinal neovascularization18. NGF can reduce photoreceptor apoptosis after retinal detachment injury and protect Müller cells, relieving rat retinal gliosis by modulating the Trk-A signaling pathway29. NGF also supports the existence of retinal ganglion cells (RGCs) and photoreceptors, which directly inhibits degeneration, and NGF stimulates additional neurotrophic factor expression from Müller cells, which indirectly enhances photoreceptor survival30,31. Intravitreal NGF injection has been tested in the rat model of retinitis pigmentosa of the Royal College of Surgeons32. Liu and Ying et al. had suggested that 35 retinitis pigmentosa patients were treated with repeat intravitreal injections of NGF (30 µg/0.15 mL) to protect the RPE after safety evaluation in rats and rabbits33 and no toxic reactions or side-effects were evident. In this study, we employed the same NGF concentration, and found no toxic effects or abnormal tissue reactions during the entire follow-up. Our previous in vitro research also revealed that NGF was not toxic to Müller cells34.
The limitations of our study included the small sample size and the relatively short follow-up period. The patients were limited because of the lack of C3F8 which was necessary for the MH surgery in our study. We had to stop our investigation when the C3F8 ran out. C3F8 production was banned in our country and C3F8 produced by foreign manufacturers had not been approved by NMPA. We sincerely hope that the production of C3F8 could be restored to clinical use as soon as possible. Our study was a very preliminary study and randomized controlled clinical studies involving a larger number of patients and longer follow-ups were required to evaluate the anatomical and functional outcomes of the central macular fovea and the safety of NGF. However, our findings suggested that NGF could be used to treat large MHs.
In conclusion, we found that ILM sheet insertion into a MH combined with NGF injection had the potential to improve visual function in patients with large iFTMH in the short time for the initial surgical treatment. NGF produced better recovery of the photoreceptor layers and, consequently, might have superior postoperative visual acuity. Therefore, NGF could be valuable during initial treatment of large MHs.