Intravitreal Administration of rhNGF Enhances Regenerative Processes in a Zebrafish Model of Retinal Degeneration.

Pasquale Cocchiaro,Marcello Allegretti,Laura Brandolini,Vincenzo Di Donato,Rodolfo Mastropasqua,Davide Rubbini,Andrea Aramini,Flavio Mantelli

doi:10.3389/fphar.2022.822359

Abstract

Nerve growth factor (NGF) is the best characterized neurotrophin, and it is known to play an important role in ocular homeostasis. Here, we demonstrated the expression of NGF receptors in adult zebrafish retina and optimized a light-induced retina degeneration (LID) zebrafish model that mimics human cone-rod disorders, demonstrating that intravitreal (IV) administration of rhNGF can boost zebrafish retinal regeneration in this model. Adult zebrafish retinae exposed to 60 h of light irradiation (60 h LID) displayed evident reduction of outer nuclear layer (ONL) thickness and cell number with presence of apoptotic cells. Retinal histologic evaluation at different time points showed that IV therapeutic injection of rhNGF resulted in an increase of ONL thickness and cell number at late time points after damage (14 and 21 days post injury), ultimately accelerating retinal tissue recovery by driving retinal cell proliferation. At a molecular level, rhNGF activated the ERK1/2 pathway and enhanced the regenerative potential of Müller glia gfap- and vim-expressing cells by stimulating at early time points the expression of the photoreceptor regeneration factor Drgal1-L2. Our results demonstrate the highly conserved nature of NGF canonical pathway in zebrafish and thus support the use of zebrafish models for testing new compounds with potential retinal regenerative properties. Moreover, the pro-regenerative effects of IV-injected NGF that we observed pave the way to further studies aimed at evaluating its effects also in mammals, in order to expedite the development of novel rhNGF-based therapeutic approaches for ophthalmological disorders.

Highlights

A recent study confirmed the expression of ngfrb and ntrk1 genes in sensory neurons during zebrafish early development (Hahn et al, 2020), the expression of the two Nerve growth factor (NGF) receptors in adult zebrafish retina has not been reported yet
In order to evaluate the potential regenerative effect of rhNGF administration in adult zebrafish retinal degeneration model, we first assessed the expression of NGF receptors in adult zebrafish retina
At 36 hpi, an upregulation of drgal1-L2 transcription was observed in both injected and untreated eyes compared to the basal expression level (2 folds change); at 72 hpi, a drastic threefold increase was detected in rhNGF-injected retinal tissues compared to the untreated ones. These results suggest that activation of NGF pathway by injection of exogenous growth factor might enhance the regenerative potential of the tissue, and this effect is possibly mediated by a rhNGF-induced proliferation of Müller glia (MG) cell populations in the early post-injury time points, which would result in an increased size in the pool of MG in later time points

Summary

Introduction

Inherited, acquired, or iatrogenic, the retinal conditions are complex and multi-factorial diseases characterized by progressive bilateral degeneration of the rod and cone photoreceptors (Narayan et al, 2016; Al-Zamil and Yassin 2017). NGF is the best characterized member of the neurotrophin family and has been widely described as a critical neuronal survival factor It plays a crucial role in regulating the homeostasis of ocular tissues (Aloe et al, 2012), where it is expressed with its receptors TrkA and p75 (Garcia et al, 2017): TrkA has high affinity and selectivity for NGF binding and can trigger different signaling pathways related to cell survival, proliferation, and differentiation, which involve the activation of ERK, PI3K, and PLC-γ (Wang et al, 2014); on the contrary, p75 has low affinity for NGF and high affinity for proNGF and is implicated in cell death mediation (Coassin et al, 2008). Increasing lines of evidence have shown that NGF can be crucial for the treatment of blinding diseases, such as retinal degenerations, and several studies have already described the protective effect of NGF administration in experimental models of retinitis pigmentosa (RP) (Rocco et al, 2015) and age-related macular degeneration (AMD) (Lambiase et al, 2009)

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Conclusion