Vitrectomy Before Intravitreal Injection of AAV2/2 Vector Promotes Efficient Transduction of Retinal Ganglion Cells in Dogs and Nonhuman Primates

AbstractPurposeTo determine if dietary administration of the dual ETA/ ETB receptor antagonist, macitentan, could protect retinal ganglion cells (RGCs) following endothelin-1 (ET-1) mediated vasoconstriction in Brown Norway rats.MethodsAdult male and female Brown Norway rats were either untreated or treated with macitentan (5 mg/kg body weight) once a day for 3 days followed by intravitreal injection of either 4 μl of 500 μM ET-1 (2 nmole/eye) or vehicle in one eye. Imaging of the retinal vasculature using fluorescein angiography was carried out at various time points, including, 5, 10, 15, 25 and 30 minutes. Following the imaging of the vasculature, rats were either treated with macitentan (5 mg/kg/body weight in dietary gels) or untreated (control gels without medication). Following treatments, rats were euthanized, retinal flat mounts were prepared, immunostained for RGC marker Brn3a, imaged and surviving RGCs were counted in a masked manner.ResultsVasoconstrictive effects following intravitreal ET-1 injection were greatly reduced in rats administered with macitentan in the diet prior to the ET-1 administration. ET-1 intravitreal injection produced a 31% loss of RGCs which was significantly reduced in macitentan-treated rats. Following ET-1 administration, GFAP immunostaining was increased in the ganglion cell layer as well as in the retrolaminar region, suggestive of astrocytic activation by ET-1 administration. RGC numbers in macitentan treated and ET-1 injected rats were similar to that observed in control retinas.ConclusionsET-1-mediated neurodegeneration could occur through both vascular and cellular mechanisms. The endothelin receptor antagonist, macitentan, has neuroprotective effects in retinas of Brown Norway rats that occurs through different mechanisms, including, enhancement of RGC survival and reduction ET-1 mediated vasoconstriction.

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Quantification of green fluorescent protein expression in mouse retinal ganglion cells following intravitreal injection of recombinant adeno-associated virus

Acta Ophthalmologica ◽

10.1111/j.1755-3768.2016.0537 ◽

2016 ◽

Vol 94 ◽

Author(s):

T. Khatib ◽

A. Osborne ◽

P. Widdowson ◽

K. Martin

Keyword(s):

Green Fluorescent Protein ◽

Protein Expression ◽

Intravitreal Injection ◽

Retinal Ganglion Cells ◽

Green Fluorescent Protein Expression ◽

Fluorescent Protein ◽

Ganglion Cells ◽

Retinal Ganglion ◽

Adeno Associated Virus ◽

Green Fluorescent

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The change of microglia numbers within the mice retina, optic nerve and chiasm following intravitreal AAV2-GFP injection

European Journal of Ophthalmology ◽

10.1177/11206721211049001 ◽

2021 ◽

pp. 112067212110490

Author(s):

Yuanfei Ji ◽

Bo Yu ◽

Yikui Zhang ◽

Wencan Wu

Keyword(s):

Optic Nerve ◽

Intravitreal Injection ◽

Retinal Ganglion Cells ◽

Ganglion Cells ◽

Optic Chiasm ◽

Optimal Concentration ◽

The Other ◽

Retinal Ganglion ◽

Sparse Labeling ◽

Microglial Morphology

Purpose To explore the optimized concentration of AAV2-GFP for sparse transfection of retinal ganglion cells (RGCs) and optic nerve (ON), and to examine the changes of microglial morphology and distribution in the retina, optic nerve and chiasm after injection. Methods We defined the optimal concentration of AAV2-GFP for sparse labeling of RGCs and axons in WT mice. We further explored the changes of microglial morphology and distribution in the retina, optic nerve and chiasm after intravitreal injection in CX3CR1+/GFP mice. Results 14 days after intravitreal injection of AAV2-GFP, live imaging of the retina showed that fundus fluorescence was very strong and dense at 2.16 × 1011 VG/retina, 2.16 × 1010 VG/retina, 2.16 × 109 VG/retina. RGCs were sparsely marked at a concentration 1:1000 (2.16 × 108 VG/retina) and fundus fluorescence was weak. The transfected RGCs and axons were unevenly distributed in the retina and significantly more RGCs were transfected near the injection site of AAV2-GFP compared to the other sites of the flat-mounted retina. Microglia density increased significantly in the retina and part of optic nerve, but not in the optic chiasm. The morphology of microglia was largely unchanged. Conclusions AAV2-GFP was highly efficient and the optimal concentration of sparsely labeled RGCs was 1:1000 (2.16 × 108 VG/retina). After intravitreal injection of AAV2-GFP, the number of microglia increased partly. The morphology of microglia was comparable.

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