Carnosic acid slows photoreceptor degeneration in the Pde6b(rd10) mouse model of retinitis pigmentosa

Abstract

The photoreceptor cell death associated with the various genetic forms of retinitis pigmentosa (RP) is currently untreatable and leads to partial or complete vision loss. Carnosic acid (CA) upregulates endogenous antioxidant enzymes and has proven neuroprotective in studies of neurodegenerative models affecting the brain. In this study, we examined the potential effect of CA on photoreceptor death in the Pde6b(rd10) mouse model of RP. Our data shows that CA provided morphological and functional preservation of photoreceptors. CA appears to exert its neuroprotective effects through inhibition of oxidative stress and endoplasmic reticulum stress.

Figure 1. ERG results obtained from rd10 mice treated with vehicle (n = 12 at P21; n = 5 at P28) and CA (n = 10 at P21; n = 8 at P28), and from wild-type (WT) mice treated with vehicle (n = 4 at P21) and CA (n = 4 at P21).

(a) Typical dark-adapted ERG waveforms at P21. (b) Typical light-adapted ERG waveforms at P21. (c) Luminance-response curves of dark-adapted ERG a-wave at P21 and P28. (d) Luminance-response curves of dark-adapted ERG b-wave at P21 and P28. (e) Luminance-response curves of light-adapted ERG b-wave at P21 and P28. The error bars indicate standard errors. Under both dark-adapted and light-adapted conditions, the ERG a- and b-wave amplitudes were significantly higher in CA treated group than in the vehicle group at P21 (all p < 0.01) and P28 (all p < 0.05), while the difference was not significant in the WT groups (all p > 0.05).

Figure 2. CA treatment reduced retinal cell death in rd10 mice at P21.

(a) Representative images (20×) of TUNEL assay in rd10 mice and WT mice treated with vehicle or CA. The red spots indicate the TUNEL-positive cells. Scale bar indicates 50 μm. (b) Average number of dead cells in retinas of rd10 mice and WT mice. Data was expressed as mean ± SD (n = 3). **p < 0.01 vs. vehicle group.

Figure 3. CA treatment ameliorated the reduction of ONL thickness in rd10 mice.

(a) Representative images (40×) of retinal histology from rd10 mice treated with vehicle or CA. Scale bar indicates 30  μm. (b) Ratio of ONL to INL thickness. Data was expressed as mean ± SD in vehicle group (n = 4 at P21, n = 6 at P28) and CA group (n = 6 at P21 and n = 6 at P28). **p < 0.01 vs. vehicle group.

Figure 4. CA treatment enhanced Nrf2 expression in rd10 retina at P21.

a. Representative images of western blot for total Nrf2 and nuclear Nrf2. b. Average protein expression of total Nrf2 and nuclear Nrf2. n = 3 in each group. The error bars indicate standard deviations. *p < 0.05 vs. vehicle group.

Figure 5. CA treatment downregulated the expression of some protein markers of oxidative stress in rd10 retina at P21.

a. Representative images (20x) of retinal immunohistochemistry for p-JNK (red). Scale bar indicates 50 µm. b. Representative images of western blot of JNK, p-JNK, p38 and p-p38. c. Average protein expression of JNK, p-JNK, p38 and p-p38. n = 3 in each group. The error bars indicate standard deviations. *p < 0.05, **p < 0.01 vs. vehicle group.

Figure 6. CA treatment downregulated the expression of some markers of ER stress in rd10 retina at P21. CA treatment inhibits the expression of some markers of ER stress.

a. Representative images (20x) of retinal immunohistochemistry for GRP78/BiP (red). Scale bar indicates 50 μm. b. Representative images of western blot for GRP78/BiP, p-IRE1α, ATF4 and ATF6. c. Average protein expression of GRP78/BiP, p-IRE1α, ATF4 and ATF6 tested by western blot. n = 3 in each group. The error bars indicate standard deviations. *p < 0.05, **p < 0.01 vs. vehicle group.

Figure 7. CA treatment upregulated SIRT1 expression and downregulated p-p65 expression in rd10 retina at P21. CA treatment upregulated SIRT1 expression and downregulated p-p65 expression.

a. Representative images (20x) of retinal immunohistochemistry for SIRT1 (red). Scale bar indicates 50 μm. b. Representative images of western blot for SIRT1, p65 and p-p65. c. Average protein expression of SIRT1, p65 and p-p65 tested by western blot. n = 3 in each group. The error bars indicate standard deviations. *p < 0.05 vs. vehicle group.