Tyrosine triple mutated AAV2-BDNF gene therapy in an inner retinal injury model induced by intravitreal injection of N-methyl-D-aspartate (NMDA)

Abstract

Purpose: Glaucoma is a group of chronic optic neuropathies characterized by the degeneration of retinal ganglion cells (RGCs) and their axons, and they ultimately cause blindness. Because neuroprotection using neurotrophic factors against RGC loss has been proven a beneficial strategy, extensive attempts have been made to perform gene transfer of neurotrophic proteins. This study used the inner retinal injury mouse model to evaluate the neuroprotective effect of tyrosine triple mutated and self-complementary adeno-associated virus (AAV) encoding brain-derived neurotrophic factor (BDNF; tm-scAAV2-BDNF).

Methods: C57BL/6J mice were intravitreally injected with 1 μl of tm-scAAV2-BDNF and its control AAV at a titer of 6.6 E+13 genome copies/ml. Three weeks later, 1 μl of 2 mM N-methyl-D-aspartate (NMDA) was administered in the same way as the viral injection. Six days after the NMDA injection, we assessed the dark-adapted electroretinography (ERG). Mice were sacrificed at one week after the NMDA injection, followed by RNA quantification, protein detection, and histopathological analysis.

Results: The RNA expression of BDNF in retinas treated with tm-scAAV2-BDNF was about 300-fold higher than that of its control AAV. Meanwhile, the expression of recombinant BDNF protein increased in retinas treated with tm-scAAV2-BDNF. In addition, histological analysis revealed that tm-scAAV2-BDNF prevented thinning of the inner retina. Furthermore, b-wave amplitudes of the tm-scAAV2-BDNF group were significantly higher than those of the control vector group. Histopathological and electrophysiological evaluations showed that tm-scAAV2-BDNF treatment offered significant protection against NMDA toxicity.

Conclusions: Results showed that tm-scAAV2-BDNF-treated retinas were resistant to NMDA injury, while retinas treated with the control AAV exhibited histopathological and functional changes after the administration of NMDA. These results suggest that tm-scAAV2-BDNF is potentially effective against inner retinal injury, including normal tension glaucoma.

Figure 1

Retinal GFP and BDNF expression after intravitreal injection of tm-scAAV2-GFP and tm-scAAV2-BDNF. A: Fluorescence microscopic images of cross sections treated with tm-scAAV2-GFP and NMDA. Green: GFP, Blue: DAPI. Scale bar represents 50 μm. B: Relative RNA expression levels of BDNF analyzed by the comparative threshold cycle method. GAPDH was used to normalize gene expressions (n = 6 in each group, *p<0.05 for a two-tailed Mann–Whitney U test). C: Protein expression levels of BDNF after intravitreal injection of tm-scAAV2-BDNF, as analyzed by ELISA (n = 4 in each group, *p<0.05 for a two-tailed Mann–Whitney U test).

Figure 2

Histological analysis. A: Exemplary images of hematoxylin and eosin-stained cross sections obtained from the buffer-treated (left), control vector-NMDA-treated (middle), and therapeutic vector-NMDA-treated (right) groups. Images were captured at a distance of 1,000 μm from the ONH. Scale bar represents 50 μm. GCL: ganglion cell layer, IPL: inner plexiform layer, INL: inner nuclear layer. B: Comparison of inner retinal thickness at individual points that were 250 µm from the ONH in the three groups (buffer only, n = 6; control vector-NMDA, n = 6; therapeutic vector-NMDA, n = 9, *p<0.05 in SNK test).

Figure 3

Immunofluorescence analysis on cross sections. A: Exemplary images of cross sections obtained from the buffer-treated (left), control vector-NMDA-treated (middle), and therapeutic vector-NMDA-treated (right) groups. Red: Brn3a, Blue: DAPI. Images were captured at a distance of 1,000 µm from the ONH. Scale bar represents 100 μm. B: Total number of Brn3a-positive cells in GCL from one ora serrata to the other (n = 3 in each group, *p<0.05 in SNK test).

Figure 4

ERG. A: Representative ERG waveforms from the buffer-treated (left), control vector-NMDA-treated (middle), and therapeutic vector-NMDA-treated (right) groups. B: Statistical analysis of the a- and b-waves of each group (buffer only, n = 4; control vector-NMDA, n = 8; therapeutic vector-NMDA, n = 12, *p<0.05 and **p<0.01 in SNK test).

Figure 5

Immunofluorescence analysis of cross sections. A: Exemplary images of cross sections obtained from the buffer-treated (left), control vector-NMDA-treated (middle), and therapeutic vector-NMDA-treated (right) groups. Red: GFAP, Blue: DAPI. Images were captured at a distance of 1,000 µm from the ONH. Scale bar represents 100 μm. B: Quantification of GFAP-positive area in the three groups (buffer only, n = 4; control vector-NMDA, n = 3; therapeutic vector-NMDA, n = 3, *p<0.05 and **p<0.01 in SNK test).