Downregulation of heat shock protein B8 protects retinal ganglion cell after optic nerve injury in mice_Chinese Journal of Ocular Fundus Diseases

Authors：

Xie Feijia , He Tao , Yang Ning , Yang Jiayi , Hua Dihao , Luo Jinyuan , Li Zongyuan ,  Xing Yiqiao

Eye Center, Renmin Hospital of Wuhan University, Wuhan 430060, China;

Corresponding author：

Xing Yiqiao, Email: Yiqiao_xing57@whu.edu.cn

Keywords：

Heat-shock proteins; Optic nerve injuries; Retinal Ganglion Cells; Electrooculography; Animal experimentation

DOI：

10.3760/cma.j.cn511434-20200609-00271

Video：

Export PDF Favorites Scan Get Citation

Abstract Full text Figures/Tables Video References Cited by

Objective To 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).Methods Adeno-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.Results Compared 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).Conclusions Expression 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.

Citation： Xie Feijia, He Tao, Yang Ning, Yang Jiayi, Hua Dihao, Luo Jinyuan, Li Zongyuan, Xing Yiqiao. Downregulation of heat shock protein B8 protects retinal ganglion cell after optic nerve injury in mice. Chinese Journal of Ocular Fundus Diseases, 2021, 37(4): 298-306. doi: 10.3760/cma.j.cn511434-20200609-00271 Copy

1.	Guy WM, Soparkar CN, Alford EL, et al. Traumatic optic neuropathy and second optic nerve injuries[J]. JAMA Ophthalmol, 2014, 132(5): 567-571. DOI: 10.1001/jamaophthalmol.2014.82.
2.	Kumaran AM, Sundar G, Chye LT. Traumatic optic neuropathy: a review[J]. Craniomaxillofac Trauma Reconstr, 2015, 8(1): 31-41. DOI: 10.1055/s-0034-1393734.
3.	Tang Z, Zhang S, Lee C, et al. An optic nerve crush injury murine model to study retinal ganglion cell survival [J/OL]. J Vis Exp, 2011(50): 2685[2011-04-25]. https://doi.org/10.3791/2685. DOI: 10.3791/2685.
4.	郑幼平, 吴小桃, 田原, 等. 银杏内酯B对视神经钳夹伤后大鼠视网膜神经节细胞的保护作用[J]. 中国医学创新, 2015, 12(21): 13-16. DOI: 10.3969/j.issn.1674-4985.2015.21.005.Zheng YP, Wu XT, Tian Y, et al. Protective effect of ginkgolide B on retinal ganglion cells after crush of intra orbital optic nerve in rats[J]. Medical Innovation of China, 2015, 12(21): 13-16. DOI: 10.3969/j.issn.1674-4985.2015.21.005.
5.	于荣国, 韩金栋, 颜华, 等. 活化的巨噬细胞在外伤性视神经损伤修复中的作用[J]. 中华眼底病杂志, 2011, 27(1): 70-73. DOI: 10.3760/cma.j.issn.1005-1015.2011.01.016.Yu RG, Han JD, Yan H, et al. The role of activated macrophages in the repair of traumatic optic nerve injury[J]. Chin J Ocul Fundus Dis, 2011, 27(1): 70-73. DOI: 10.3760/cma.j.issn.1005-1015.2011.01.016.
6.	Li F, Xiao H, Hu Z, et al. Exploring the multifaceted roles of heat shock protein B8 (HSPB8) in diseases[J]. Eur J Cell Biol, 2018, 97(3): 216-229. DOI: 10.1016/j.ejcb.2018.03.003.
7.	Carra S, Seguin SJ, Lambert H, et al. HspB8 chaperone activity toward poly(Q)-containing proteins depends on its association with Bag3, a stimulator of macroautophagy[J]. J Biol Chem, 2008, 283(3): 1437-1444. DOI: 10.1074/jbc.M706304200.
8.	Li F, Tan J, Zhou F, et al. Heat shock protein B8 (HSPB8) reduces oxygen-glucose deprivation/reperfusion injury via the induction of mitophagy[J]. Cell Physiol Biochem, 2018, 48(4): 1492-1504. DOI: 10.1159/000492259.
9.	Fontaine JM, Sun X, Hoppe AD, et al. Abnormal small heat shock protein interactions involving neuropathy-associated HSP22 (HSPB8) mutants[J]. FASEB J, 2006, 20(12): 2168-2170. DOI: 10.1096/fj.06-5911fje.
10.	Acunzo J, Katsogiannou M, Rocchi P. Small heat shock proteins HSP27 (HspB1), alphaB-crystallin (HspB5) and HSP22 (HspB8) as regulators of cell death[J]. Int J Biochem Cell Biol, 2012, 44(10): 1622-1631. DOI: 10.1016/j.biocel.2012.04.002.
11.	Luo J, He T, Yang J, et al. SIRT1 is required for the neuroprotection of resveratrol on retinal ganglion cells after retinal ischemia-reperfusion injury in mice[J]. Graefe's Arch Clin Exp Ophthalmol, 2020, 258(2): 335-344. DOI: 10.1007/s00417-019-04580-z.
12.	Cameron EG, Xia X, Galvao J, et al. Optic nerve crush in mice to study retinal ganglion cell survival and regeneration [J/OL]. Bio Protoc, 2020, 10(6): e3559[2020-03-20]. https://doi.org/10.21769/BioProtoc.3559. DOI: 10.21769/BioProtoc.3559.
13.	May CA, Lutjen-Drecoll E. Morphology of the murine optic nerve[J]. Invest Ophthalmol Vis Sci, 2002, 43(7): 2206-2212.
14.	Prusky GT, Alam NM, Beekman S, et al. Rapid quantification of adult and developing mouse spatial vision using a virtual optomotor system[J]. Invest Ophthalmol Vis Sci, 2004, 45(12): 4611-4616. DOI: 10.1167/iovs.04-0541.
15.	Li B, Barnes GE, Holt WF. The decline of the photopic negative response (PhNR) in the rat after optic nerve transection[J]. Doc Ophthalmol, 2005, 111(1): 23-31. DOI: 10.1007/s10633-005-2629-8.
16.	Casson RJ, Chidlow G, Wood JP, et al. The effect of retinal ganglion cell injury on light-induced photoreceptor degeneration[J]. Invest Ophthalmol Vis Sci, 2004, 45(2): 685-693. DOI: 10.1167/iovs.03-0674.
17.	Osborne NN, Perry VH. Effect of neonatal optic nerve transection on some classes of amacrine cells in the rat retina[J]. Brain Res, 1985, 343(2): 230-235. DOI: 10.1016/0006-8993(85)90739-5.
18.	Dijk F, Kamphuis W. An immunocytochemical study on specific amacrine cell subpopulations in the rat retina after ischemia[J]. Brain Res, 2004, 1026(2): 205-217. DOI: 10.1016/j.brainres.2004.08.014.
19.	Coffey PJ, Girman S, Wang SM, et al. Long-term preservation of cortically dependent visual function in RCS rats by transplantation[J]. Nat Neurosci, 2002, 5(1): 53-56. DOI: 10.1038/nn782.
20.	Kretschmer F, Tariq M, Chatila W, et al. Comparison of optomotor and optokinetic reflexes in mice[J]. J Neurophysiol, 2017, 118(1): 300-316. DOI: 10.1152/jn.00055.2017.
21.	方晏红, 张学东, 雷博. 视网膜电图震荡电位的研究进展及其在糖尿病视网膜病变中的应用[J]. 中华眼底病杂志, 2013, 29(1): 106-108. DOI: 10.3760/cma.j.issn.1005-1015.2013.01.032.Fang YH, Zhang XD, Lei B. Progress of electroretinogram oscillatory potential and its application in diabetic retinopathy[J]. Chin J Ocul Fundus Dis, 2013, 29(1): 106-108. DOI: 10.3760/cma.j.issn.1005-1015.2013.01.032.
22.	Bui BV, Fortune B. Ganglion cell contributions to the rat full-field electroretinogram[J]. J Physiol, 2004, 555(Pt 1): 153-173. DOI: 10.1113/jphysiol.2003.052738.
23.	Alarcon-Martinez L, Aviles-Trigueros M, Galindo-Romero C, et al. ERG changes in albino and pigmented mice after optic nerve transection[J]. Vision Res, 2010, 50(21): 2176-2187. DOI: 10.1016/j.visres.2010.08.014.
24.	Zhang ZZ, Gong YY, Shi YH, et al. Valproate promotes survival of retinal ganglion cells in a rat model of optic nerve crush[J]. Neuroscience, 2012, 224: 282-293. DOI: 10.1016/j.neuroscience.2012.07.056.
25.	Rey-Funes M, Larrayoz IM, Contartese DS, et al. Hypothermia prevents retinal damage -generated by optic nerve trauma in the rat [J/OL]. Sci Rep, 2017, 7(1): 6966[2017-07-31]. https://doi.org/10.1038/s41598-017-07294-6. DOI: 10.1038/s41598-017-07294-6.
26.	Gargini C, Belfiore MS, Bisti S, et al. The impact of basic fibroblast growth factor on photoreceptor function and morphology[J]. Invest Ophthalmol Vis Sci, 1999, 40(9): 2088-2099.
27.	Alarcon-Martinez L, de la Villa P, Aviles-Trigueros M, et al. Short and long term axotomy-induced ERG changes in albino and pigmented rats[J]. Mol Vis, 2009, 15: 2373-2383.
28.	Grinblat GA, Khan RS, Dine K, et al. RGC neuroprotection following optic nerve trauma mediated by intranasal delivery of amnion cell secretome[J]. Invest Ophthalmol Vis Sci, 2018, 59(6): 2470-2477. DOI: 10.1167/iovs.18-24096.
29.	Depre C, Hase M, Gaussin V, et al. H11 kinase is a novel mediator of myocardial hypertrophy in vivo[J]. Circ Res, 2002, 91(11): 1007-1014. DOI: 10.1161/01.res.0000044380.54893.4b.
30.	Knoferle J, Koch JC, Ostendorf T, et al. Mechanisms of acute axonal degeneration in the optic nerve in vivo[J]. Proc Natl Acad Sci USA, 2010, 107(13): 6064-6069. DOI: 10.1073/pnas.0909794107.
31.	Kang LH, Zhang S, Jiang S, et al. Activation of autophagy in the retina after optic nerve crush injury in rats[J]. Int J Ophthalmol, 2019, 12(9): 1395-1401. DOI: 10.18240/ijo.2019.09.04.
32.	Klimek C, Jahnke R, Wordehoff J, et al. The Hippo network kinase STK38 contributes to protein homeostasis by inhibiting BAG3-mediated autophagy[J]. Biochim Biophys Acta Mol Cell Res, 2019, 1866(10): 1556-1566. DOI: 10.1016/j.bbamcr.2019.07.007.

1. Guy WM, Soparkar CN, Alford EL, et al. Traumatic optic neuropathy and second optic nerve injuries[J]. JAMA Ophthalmol, 2014, 132(5): 567-571. DOI: 10.1001/jamaophthalmol.2014.82.
2. Kumaran AM, Sundar G, Chye LT. Traumatic optic neuropathy: a review[J]. Craniomaxillofac Trauma Reconstr, 2015, 8(1): 31-41. DOI: 10.1055/s-0034-1393734.
3. Tang Z, Zhang S, Lee C, et al. An optic nerve crush injury murine model to study retinal ganglion cell survival [J/OL]. J Vis Exp, 2011(50): 2685[2011-04-25]. https://doi.org/10.3791/2685. DOI: 10.3791/2685.
4. 郑幼平, 吴小桃, 田原, 等. 银杏内酯B对视神经钳夹伤后大鼠视网膜神经节细胞的保护作用[J]. 中国医学创新, 2015, 12(21): 13-16. DOI: 10.3969/j.issn.1674-4985.2015.21.005.Zheng YP, Wu XT, Tian Y, et al. Protective effect of ginkgolide B on retinal ganglion cells after crush of intra orbital optic nerve in rats[J]. Medical Innovation of China, 2015, 12(21): 13-16. DOI: 10.3969/j.issn.1674-4985.2015.21.005.
5. 于荣国, 韩金栋, 颜华, 等. 活化的巨噬细胞在外伤性视神经损伤修复中的作用[J]. 中华眼底病杂志, 2011, 27(1): 70-73. DOI: 10.3760/cma.j.issn.1005-1015.2011.01.016.Yu RG, Han JD, Yan H, et al. The role of activated macrophages in the repair of traumatic optic nerve injury[J]. Chin J Ocul Fundus Dis, 2011, 27(1): 70-73. DOI: 10.3760/cma.j.issn.1005-1015.2011.01.016.
6. Li F, Xiao H, Hu Z, et al. Exploring the multifaceted roles of heat shock protein B8 (HSPB8) in diseases[J]. Eur J Cell Biol, 2018, 97(3): 216-229. DOI: 10.1016/j.ejcb.2018.03.003.
7. Carra S, Seguin SJ, Lambert H, et al. HspB8 chaperone activity toward poly(Q)-containing proteins depends on its association with Bag3, a stimulator of macroautophagy[J]. J Biol Chem, 2008, 283(3): 1437-1444. DOI: 10.1074/jbc.M706304200.
8. Li F, Tan J, Zhou F, et al. Heat shock protein B8 (HSPB8) reduces oxygen-glucose deprivation/reperfusion injury via the induction of mitophagy[J]. Cell Physiol Biochem, 2018, 48(4): 1492-1504. DOI: 10.1159/000492259.
9. Fontaine JM, Sun X, Hoppe AD, et al. Abnormal small heat shock protein interactions involving neuropathy-associated HSP22 (HSPB8) mutants[J]. FASEB J, 2006, 20(12): 2168-2170. DOI: 10.1096/fj.06-5911fje.
10. Acunzo J, Katsogiannou M, Rocchi P. Small heat shock proteins HSP27 (HspB1), alphaB-crystallin (HspB5) and HSP22 (HspB8) as regulators of cell death[J]. Int J Biochem Cell Biol, 2012, 44(10): 1622-1631. DOI: 10.1016/j.biocel.2012.04.002.
11. Luo J, He T, Yang J, et al. SIRT1 is required for the neuroprotection of resveratrol on retinal ganglion cells after retinal ischemia-reperfusion injury in mice[J]. Graefe's Arch Clin Exp Ophthalmol, 2020, 258(2): 335-344. DOI: 10.1007/s00417-019-04580-z.
12. Cameron EG, Xia X, Galvao J, et al. Optic nerve crush in mice to study retinal ganglion cell survival and regeneration [J/OL]. Bio Protoc, 2020, 10(6): e3559[2020-03-20]. https://doi.org/10.21769/BioProtoc.3559. DOI: 10.21769/BioProtoc.3559.
13. May CA, Lutjen-Drecoll E. Morphology of the murine optic nerve[J]. Invest Ophthalmol Vis Sci, 2002, 43(7): 2206-2212.
14. Prusky GT, Alam NM, Beekman S, et al. Rapid quantification of adult and developing mouse spatial vision using a virtual optomotor system[J]. Invest Ophthalmol Vis Sci, 2004, 45(12): 4611-4616. DOI: 10.1167/iovs.04-0541.
15. Li B, Barnes GE, Holt WF. The decline of the photopic negative response (PhNR) in the rat after optic nerve transection[J]. Doc Ophthalmol, 2005, 111(1): 23-31. DOI: 10.1007/s10633-005-2629-8.
16. Casson RJ, Chidlow G, Wood JP, et al. The effect of retinal ganglion cell injury on light-induced photoreceptor degeneration[J]. Invest Ophthalmol Vis Sci, 2004, 45(2): 685-693. DOI: 10.1167/iovs.03-0674.
17. Osborne NN, Perry VH. Effect of neonatal optic nerve transection on some classes of amacrine cells in the rat retina[J]. Brain Res, 1985, 343(2): 230-235. DOI: 10.1016/0006-8993(85)90739-5.
18. Dijk F, Kamphuis W. An immunocytochemical study on specific amacrine cell subpopulations in the rat retina after ischemia[J]. Brain Res, 2004, 1026(2): 205-217. DOI: 10.1016/j.brainres.2004.08.014.
19. Coffey PJ, Girman S, Wang SM, et al. Long-term preservation of cortically dependent visual function in RCS rats by transplantation[J]. Nat Neurosci, 2002, 5(1): 53-56. DOI: 10.1038/nn782.
20. Kretschmer F, Tariq M, Chatila W, et al. Comparison of optomotor and optokinetic reflexes in mice[J]. J Neurophysiol, 2017, 118(1): 300-316. DOI: 10.1152/jn.00055.2017.
21. 方晏红, 张学东, 雷博. 视网膜电图震荡电位的研究进展及其在糖尿病视网膜病变中的应用[J]. 中华眼底病杂志, 2013, 29(1): 106-108. DOI: 10.3760/cma.j.issn.1005-1015.2013.01.032.Fang YH, Zhang XD, Lei B. Progress of electroretinogram oscillatory potential and its application in diabetic retinopathy[J]. Chin J Ocul Fundus Dis, 2013, 29(1): 106-108. DOI: 10.3760/cma.j.issn.1005-1015.2013.01.032.
22. Bui BV, Fortune B. Ganglion cell contributions to the rat full-field electroretinogram[J]. J Physiol, 2004, 555(Pt 1): 153-173. DOI: 10.1113/jphysiol.2003.052738.
23. Alarcon-Martinez L, Aviles-Trigueros M, Galindo-Romero C, et al. ERG changes in albino and pigmented mice after optic nerve transection[J]. Vision Res, 2010, 50(21): 2176-2187. DOI: 10.1016/j.visres.2010.08.014.
24. Zhang ZZ, Gong YY, Shi YH, et al. Valproate promotes survival of retinal ganglion cells in a rat model of optic nerve crush[J]. Neuroscience, 2012, 224: 282-293. DOI: 10.1016/j.neuroscience.2012.07.056.
25. Rey-Funes M, Larrayoz IM, Contartese DS, et al. Hypothermia prevents retinal damage -generated by optic nerve trauma in the rat [J/OL]. Sci Rep, 2017, 7(1): 6966[2017-07-31]. https://doi.org/10.1038/s41598-017-07294-6. DOI: 10.1038/s41598-017-07294-6.
26. Gargini C, Belfiore MS, Bisti S, et al. The impact of basic fibroblast growth factor on photoreceptor function and morphology[J]. Invest Ophthalmol Vis Sci, 1999, 40(9): 2088-2099.
27. Alarcon-Martinez L, de la Villa P, Aviles-Trigueros M, et al. Short and long term axotomy-induced ERG changes in albino and pigmented rats[J]. Mol Vis, 2009, 15: 2373-2383.
28. Grinblat GA, Khan RS, Dine K, et al. RGC neuroprotection following optic nerve trauma mediated by intranasal delivery of amnion cell secretome[J]. Invest Ophthalmol Vis Sci, 2018, 59(6): 2470-2477. DOI: 10.1167/iovs.18-24096.
29. Depre C, Hase M, Gaussin V, et al. H11 kinase is a novel mediator of myocardial hypertrophy in vivo[J]. Circ Res, 2002, 91(11): 1007-1014. DOI: 10.1161/01.res.0000044380.54893.4b.
30. Knoferle J, Koch JC, Ostendorf T, et al. Mechanisms of acute axonal degeneration in the optic nerve in vivo[J]. Proc Natl Acad Sci USA, 2010, 107(13): 6064-6069. DOI: 10.1073/pnas.0909794107.
31. Kang LH, Zhang S, Jiang S, et al. Activation of autophagy in the retina after optic nerve crush injury in rats[J]. Int J Ophthalmol, 2019, 12(9): 1395-1401. DOI: 10.18240/ijo.2019.09.04.
32. Klimek C, Jahnke R, Wordehoff J, et al. The Hippo network kinase STK38 contributes to protein homeostasis by inhibiting BAG3-mediated autophagy[J]. Biochim Biophys Acta Mol Cell Res, 2019, 1866(10): 1556-1566. DOI: 10.1016/j.bbamcr.2019.07.007.

Chinese Journal of Ocular Fundus Diseases

Downregulation of heat shock protein B8 protects retinal ganglion cell after optic nerve injury in mice

Abstract Full text Figures/Tables Video References Cited by

Previous Article

Next Article

Format

Content