Original Article
Downregulation of heat shock protein B8 protects retinal ganglion cell after optic nerve injury in mice
Xie Feijia, He Tao, Yang Ning, Yang Jiayi, Hua Dihao, Luo Jinyuan, Li Zongyuan, Xing Yiqiao
Published 2021-04-25
Cite as Chin J Ocul Fundus Dis, 2021, 37(4): 298-306. DOI: 10.3760/cma.j.cn511434-20200609-00271
Abstract
ObjectiveTo investigate the effect of heat shock protein B8 (HspB8) downregulation on retinal ganglion cell (RGC) and retinal function in the mice model of optic nerve injury (ONC).
MethodsAdeno-Associated Virus (AAV) 2 AAV2-shHspB8-GFP was constructed to knockdown HspB8. 66 adult male C57/BL6 mice were randomly divided into the control group, the ONC group, the AAV2-shHspB8 group, the ONC+AAV2-shHspB8 group, and the ONC+AAV2-GFP group. There were 10, 20, 16, 10 and 10 mice respectively, and both eyes were used as experimental eyes. Western blot was used to evaluate the expression of HspB8 on day 3 and 7 after ONC. By GFP immunofluorescence staining, the efficacy of AAV2-shHspB8-GFP transfer was accessed. Moreover, it was possible to identify functional and RGC survival differences between groups by optomotor response (OMR), dark adapted full-field flash electroretinogram (ff-ERG), oscillatory potentials (OPs), photopic negative response (PhNR) and retinal flat-mount RGC counting 5 days after ONC. Comparisons between two groups were made using Mann-Whitney U test, unpaired t-test, unpaired t-test with Welch’s correction, one-way ANOVA, and Bonferroni t test.
ResultsCompared with the control group, the expression of HSPB8 protein in the retina of mice in ONC3 group was significantly increased, and the difference was statistically significant (F=43.63, P<0.01). Compared with the control group, the ONC group showed obviously lower visual acuity (P<0.01), lower a-wave, b-wave, OPs, PhNR amplitude, longer b-wave latency (P<0.05), and the survival rates of RGC in ONC3 group, ONC5 group and ONC7 group decreased in a time-dependent manner(F=384.90, P<0.01). Transfection of AAV2 efficiency was highest on 4 weeks after IVT. Besides, there was no significant differences between the control group and the AAV2-shHspB8 group on visual acuity, ff-ERG, OPs, PhNR and RGC survival (P>0.05). In comparison of the control group, we found that RGC survival of the ONC5+AAV2-shHspB8 group was significantly elevated (F=10.62, P<0.01).
ConclusionsExpression of HspB8 on the retina can be induced by ONC. The investigation of RGC counting, visual acuity, and ff-ERG revealed that optic nerve injury destructed functionality of mice retina and resulted to RGC death ultimately. The Most crucial finding of this research is that HspB8 knockdown had a neuroprotective effect in RGC after ONC.
Key words:
Heat-shock proteins; Optic nerve injuries; Retinal Ganglion Cells; Electrooculography; Animal experimentation
Contributor Information
Xie Feijia
Eye Center, Renmin Hospital of Wuhan University, Wuhan 430060, China
He Tao
Eye Center, Renmin Hospital of Wuhan University, Wuhan 430060, China
Yang Ning
Eye Center, Renmin Hospital of Wuhan University, Wuhan 430060, China
Yang Jiayi
Eye Center, Renmin Hospital of Wuhan University, Wuhan 430060, China
Hua Dihao
Eye Center, Renmin Hospital of Wuhan University, Wuhan 430060, China
Luo Jinyuan
Eye Center, Renmin Hospital of Wuhan University, Wuhan 430060, China
Li Zongyuan
Eye Center, Renmin Hospital of Wuhan University, Wuhan 430060, China
Xing Yiqiao
Eye Center, Renmin Hospital of Wuhan University, Wuhan 430060, China
