S-Nitrosylation of HDAC2 and the acetylation of histone H3 in the rat retina by IPRG001.

<p>(A) S-Nitrosylation of HDAC2 1 day after intraocular administration of IPRG001. NO scavenger, c-PTIO or nNOS inhibitor, ETPI was treated 1 h before IPRG001 treatment. Biotinylated proteins were mixed with avidin beads, eluted and analyzed by western blotting with anti-HDAC2 antibody. **P<0.01 vs control. ⁺P<0.01 vs IPRG001 treatment alone (n = 3). (B) Levels of acetylated histone H3 (AcH3) after treatment of IPRG001. *P<0.05, **P<0.01 vs control (n = 3). (C) nNOS dependent histone H3 acetylation by IPRG001. ETPI was treated 1 h before IPRG001. *P<0.01 vs control. ⁺P<0.01 vs IPRG001 treatment alone (n = 3). (D–I) Immunohistochemistry of AcH3 and NeuN in rat RGCs. Levels of AcH3 were increased 1 day after IPRG001 treatment (G–I) compared to vehicle control (D–F). (D and G) Immunoreactivity of AcH3. (E and H) NeuN staining. (F and I) Merged images. Scale = 20 µm.</p

Upregulation of RARβ expression in RGCs after IPRG001 treatment.

<p>(A) Western blot analysis of RARβ protein expression increased at 1 day after treatment of IPRG001. *P<0.01 vs control (n = 3). (B) Levels of RARβ expression by IPRG001 with or without optic nerve injury. *P<0.01 vs control (n = 3). (C–H) RARβ and NeuN expression in the retina after treatment of IPRG001. (C and F) Immunoreactivity of RARβ protein was increased in RGCs 1 day after treatment with IPRG001 (F) compared to vehicle control (C). Scale = 50 µm. (D and G) NeuN staining of C and F. (E and H) Merged images. (I) nNOS/NO-dependent expression of RARβ by IPRG001. *P<0.05, **P<0.01 vs control, ⁺P<0.01 vs IPRG001 treatment alone (n = 3).</p

IPRG001 activates NADPHd staining and nNOS protein expression in the rat retina.

<p>(A, B) NADPHd staining in the retina increased at 1 day post-treatment with 100 pmol/eye of IPRG001 (B) as compared to vehicle control (A). Scale = 100 µm. (C) Western blot analysis of nNOS protein after treatment of IPRG001. *P<0.01 vs control (n = 3). (D) Levels of nNOS expression by IPRG001 with or without optic nerve injury. *P<0.01 vs control (n = 3). (E–J) nNOS and NeuN expression in the retina after treatment of IPRG001. (E and F) Immunoreactivity of nNOS protein was increased in RGCs at 1 day after IPRG001 treatment (F) compared to vehicle control (E). Scale = 50 µm. (G and H) NeuN staining of E and F. (I and J) Merged images.</p

No effect of RARβ expression on RGCs survival induced by IPRG001.

<p>(A-D) Surviving RGCs stained by TUJ1 in whole mount retina. (A) Vehicle control. Scale = 50 µm. (B) 10 days after injury. (C) Injury plus IPRG001. (D) Injury plus IPRG001 with RARβ-specific siRNA. (E) Quantification of surviving RGCs after nerve injury with or without siRNA for RARβ. *P<0.01 vs control, ⁺P<0.01 vs injury (n = 8).</p

Correlation between axon outgrowth and RARβ expression in rat retina after birth.

<p>(A) P1 retinal explant, (B) P14 retinal explant, (C) P60 retinal explant, Scale = 100 µm. (D) Quantification of axon outgrowth from rat retinal explants at various days after birth. Y axis shows % of explants with neurites longer than 200 µm. *P<0.01 vs P1 retina. (n = 3, 5 rats of each stage). (E) Quantification of RARβ expression in rat retina during development. *P<0.05, **P<0.01 vs P1 (n = 3). (F–N) RARβ and NeuN expression in the rat retina during development. (F, I, L) RARβ expression at P1 (F), P14 (I) and P60 (L). Scale = 50 µm. (G, J, M) NeuN-positive RGCs at P1 (G), P14 (J), and P60 (M). (H, K, N) Merged images of each day at P1 (H), P14 (K), and P60 (N). (O) P1 retina treated with scrambled siRNA (P) siRNA for RARβ in P1. Scale = 100 µm. (Q) siRNA of RARβ suppressed expression of RARβ and neurite outgrowth in P1 retina. *P<0.01 vs vehicle control (Con) and scrambled siRNA(Scr) (RARβ expression), ⁺P<0.01 vs Con and Scr (neurite outgrowth), (n = 3, 5 rats of each treatment).</p

IPRG001-induced rat optic nerve regeneration under RARβ-dependent conditions <i>in vivo</i>.

<p>(A, C, E, G) Longitudinal sections of the adult rat optic nerve showing GAP-43 positive axons extending over the injury site (asterisks) after 2 weeks after optic nerve injury. (B, D, F, H) Enlarged image of the area enclosed within the white box of A, C, E, G. Scale = 50 µm. (A) Vehicle control, Scale = 250 µm. (C) IPRG001, (E) IPRG001 plus siRNA for RARβ. (G) IPRG001 plus c-PTIO (500 pmol). (I) Quantification of axonal regrowth at two indicated proximal points from the injury site (250 µm and 500 µm). **P<0.01 vs control, ⁺P<0.01 vs injury plus IPRG001 (n = 8, 6 rats per each group).</p

Data_Sheet_1_Relationship between tinnitus and olfactory dysfunction: audiovisual, olfactory, and medical examinations.xlsx

IntroductionSensory dysfunctions and cognitive impairments are related to each other. Although a relationship between tinnitus and subjective olfactory dysfunction has been reported, there have been no reports investigating the relationship between tinnitus and olfactory test results.MethodsTo investigate the relationship between tinnitus and olfactory test results, we conducted sensory tests, including hearing and visual examinations. The subjects included 510 community-dwelling individuals (295 women and 215 men) who attended a health checkup in Yakumo, Japan. The age of the subjects ranged from 40 to 91 years (mean ± standard deviation, 63.8 ± 9.9 years). The participants completed a self-reported questionnaire on subjective tinnitus, olfactory function, and hearing function, as well as their lifestyle. The health checkup included smell, hearing, vision, and blood examinations.ResultsAfter adjusting for age and sex, the presence of tinnitus was significantly associated with subjective olfactory dysfunction, poor olfactory test results, hearing deterioration, vertigo, and headache. Additionally, high serum calcium levels and a low albumin/globulin ratio were significantly associated with low physical activity and nutrition. Women scored higher than men in olfactory and hearing examinations, but there was no gender difference in vision examinations.ConclusionSubjective smell dysfunction and poor smell test results were significantly associated with tinnitus complaints. Hearing and vision were associated even after adjusting for age and sex. These findings suggest that evaluating the mutual relationships among sensory organs is important when evaluating the influence of sensory dysfunctions on cognitive function.</p