In vivo effects of single or combined topical neuroprotective and regenerative agents on degeneration of retinal ganglion cells in rat optic nerve crush model

Yuta Kitamura,Shuichi Yamamoto,Takayuki Baba,Guzel Bikbova,Toshiyuki Oshitari

doi:10.1038/s41598-018-36473-2

Yuta Kitamura, Shuichi Yamamoto + Show 3 more

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https://doi.org/10.1038/s41598-018-36473-2

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Abstract

To determine the effectiveness of a single or a combination of topical neurotrophic factors (NFs) in protecting retinal ganglion cells (RGCs) in the rat optic nerve crush (ONC) model, the left ONC was performed to induce the death of the RGCs in adult Sprague-Dawley rats. The NFs studied were tauroursodeoxycholic acid (TUDCA), citicoline, neurotrophin-4 (NT-4), combined TUDCA/citicoline (Doublet-1), combined TUDCA/NT-4 (Doublet-2), combined TUDCA/citicoline/NT-4 (Triplet), and PBS. After 2 weeks, the number of RGCs was determined by Brn3a immunostaining. The optic nerves were immunostained for anti-Growth Associated Protein-43(GAP-43) and -200kD neurofilament heavy antibody to study optic nerve regeneration. Two weeks after the ONC, the densities of RGCs in all treated eyes were significantly higher than that of the PBS treated eyes. In the Triplet group, the number of RGC axons after ONC was significantly higher than that in all of the single treatment groups and the number of TUNEL positive cells was significantly reduced and the number of GAP-43 immunopositive axons was significantly greater than those in the PBS group. Neovascularization was observed only in the Doublet-1 group. We conclude that the combination of the three NFs was the most effective way to protect RGCs after the ONC.

Highlights

There is an accumulation of glycated proteins, lipids, and nucleic acid with increasing age which results from the elevated blood glucose
To assess the neuroprotective effects of the topical neurotrophic factors, we determined the number of surviving retinal ganglion cells (RGCs) two weeks after the optic nerve was crushed
This was done by immunostaining the RGCs with Brn3a, a member of the POU-domain transcription factors which is expressed in RGC

Summary

Introduction

There is an accumulation of glycated proteins, lipids, and nucleic acid with increasing age which results from the elevated blood glucose. We determined earlier that neuroprotective and regenerative factors such as neurotrophin-4 (NT-4)[4,7], citicoline[8], tauroursodeoxycholic acid (TUDCA)[5], and their combinations enhanced the regeneration of neurites in AGEs-exposed retinas in vitro[9]. These findings suggested that they were axon-protective agents that could be used to treat neuronal diseases associated with AGEs accumulation. The results indicated that citicoline eyedrops can enhance the visual function of patients with glaucoma without adverse side effects[12,13] Another recent study evaluated the neuroprotective effect of topical citicoline in a mouse model of diabetic retinopathy[14]. The mechanisms of the RGC death after optic nerve injuries[17,18,19,20] are, in part, common with those in our culture system or in degenerating neurons[2,4,5,6,9] of human diabetic retinas[3,21]

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