Oroxylin A promotes retinal ganglion cell survival in a rat optic nerve crush model

Abstract

Purpose: To investigate the effect of oroxylin A on the survival of retinal ganglion cells (RGC) and the activation of microglial cells in a rat optic nerve (ON) crush model.

Methods: Oroxylin A (15mg/Kg in 0.2ml phosphate-buffered saline) or phosphate-buffered saline (PBS control) was immediately administered after ON crush once by subcutaneous injection. Rats were euthanized at 2 weeks after the crush injury. The density of RGC was counted by retrograde labeling with FluoroGold and immunostaining of retina flat mounts for Brn3a. Electrophysiological visual function was assessed by flash visual evoked potentials (FVEP). TUNEL assay, immunoblotting analysis of glial fibrillary acidic protein (GFAP), inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in the retinas, and immunohistochemistry of GFAP in the retinas and ED1 in the ON were evaluated.

Results: Two weeks after the insult, the oroxylin A-treated group had significantly higher FG labeled cells and Brn3a+ cells suggesting preserved RGC density in the central and mid-peripheral retinas compared with those of the PBS-treated group. FVEP measurements showed a significantly better preserved latency of the P1 wave in the ON-crushed, oroxylin A-treated rats than the ON-crushed, PBS treated rats. TUNEL assays showed fewer TUNEL positive cells in the ON-crushed, oroxylin A-treated rats. The number of ED1 positive cells was reduced at the lesion site of the optic nerve in the ON-crushed, oroxylin A-treated group. Increased GFAP expression in the retina was reduced greatly in ON-crushed, oroxylin A-treated group. Furthermore, administration of oroxylin A significantly attenuated ON crush insult-induced iNOS and COX-2 expression in the retinas.

Conclusions: These results demonstrated that oroxylin A hasss neuroprotective effects on RGC survival with preserved visual function and a decrease in microglial infiltration in the ONs after ON crush injury.

Fig 1. Improvement in RGC density in the retinas treated oroxylin A two weeks after ON crush.

(A, F) RGC densities in the sham group were 2577 ± 383/mm² and 1550 ± 325/mm², respectively. Two weeks after ON crush, the densities of RGCs in the central retina of the (B) PBS-treated group and (C) oroxylin A-treated group were 510 ± 158/mm² and 1352 ±476/mm² respectively, and (E, F) in mid-peripheral retina were 439 ±139/mm² and 960 ± 465/mm² respectively, showing a significant preservative effect by oroxylin A (n = 6 in each group; p<0.05) *p<0.05.

Fig 2. Retina flat mounts with Brn-3a validated RGC survival in Oroxylin-A treated group.

Representative image of Brn-3a+ cells in the central retina (A-C) and mid-peripheral retina (D-F) in each group. The oroxylin-A treated group shows significantly higher Brn-3a+ cells compared to PBS treated group in the (G) central (894 ±367/mm² vs 475± 203/mm²) and(H) mid-peripheral retina (800± 199/mm² vs 474 ± 254/mm²) respectively. n = 6 in each group; ***p<0.001.

Fig 3. Improvement in the latency of the P1 wave in FVEPs after oroxylin A treatment.

A. Representative flash VEP tracings at 2 weeks after ON crush. B. The latency of the P1 wave was 85 ± 15 ms, 154 ± 31 ms and 100 ± 12 ms in the sham, PBS-treated and oroxylin A-treated rats, respective Oroxylin A-treated group had shorter P1 latency than the PBS-treated group. (n = 6 in each group, p<0.05). *p<0.05.

Fig 4. Assays of TUNEL revealed a decreased number of apoptotic cell after oroxylin A treatment.

The upper column was reprehensive of the TUNEL in the retinas among the three groups. The lower column illustrates that there were 1.2 ± 0.9 positive cells/HPF in the RGC layers of retina in the sham-operated rats, 11.0 ± 3.5 positive cells/HPF in the PBS-treated group and 4.0 ± 2.4 positive cells/HPG in the oroxylin A-treated rats (n = 6 in each group). *p<0.05, ***p<0.001. Scale bar: 50μM. GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; ONL, outer nuclear layer; OPL, outer plexiform layer.

Fig 5. Less infiltration of ED1 in ONs treated with oroxylin A 2 weeks after ON crush.

The upper column was representative of ED1 staining in the longitudinal sections of ON. The lower column indicates that the ED1 positive cells/HPF in the sham group, PBS-treated group and oroxylin A-treated group were 4.1 ± 2.2, 72.0 ± 23.3 and 33.5 ± 16.3, respectively. n = 6 in each group. *p<0.05, ***p<0.001.

Fig 6. Oroxylin A attenuated retinal gliosis at 2 weeks after ON crush.

(A) GFAP (astrocytes and Muller cells) immunoreactivity in retinal sections. Effects of oroxylin A on the suppression of GFAP level in the retina at 2 weeks after ON crush. (B) Western blotting showing the expression levels of GFAP in the retina. In the bar graph, the expression level of GFAP is expressed as a ratio to GAPDH expression Values for sham-operated retinas were set to 1. Results represent the means ± S.D for three independent experiments. *p<0.05. **p<0.01. Scale bar: 50μm.

Fig 7. Reducing the expression level of iNOS and COX-2 in the retinas treated with oroxylin A after ON crush.

(A) Effects of oroxylin A on suppression of iNOS and COX-2 in the retina at 2 weeks after ON crush. (B.C) Quantitative analysis of (A). In the bar graph, the expression level of iNOS and COX-2 are expressed as a ratio to GAPDH expression Values for sham-operated retinas were set to 1. Results represent the means ± S.D for three independent experiments. *p<0.05.