Low-dose chloroquine mediated neuroprotection against n-methyl-d-aspartate induced excitotoxicity in adult mice_Chinese Journal of Ocular Fundus Diseases

Authors：

Liu Yifan ,  Shen Yin

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

Corresponding author：

Shen Yin, Email: yinshen@whu.edu.cn

Keywords：

Chloroquine; Retinal ganglion cells; N-methylaspartate; Toxic actions; Animal experimentation

DOI：

10.3760/cma.j.cn511434-20190903-00275

Video：

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Objective To investigate the protective effects of different concentrations of chloroquine on RGC in n-methyl-d-aspartate (NMDA) injured mice and its possible mechanisms.Methods Fifty-four healthy male C57/BL6 mice were randomly divided into three groups, 18 in each group. The mice in low-dose chloroquine group were intraperitoneally injected with chloroquine solution at a dose of 10 mg/kg daily. Mice in high-dose chloroquine group were intraperitoneally injected with chloroquine solution at a dose of 100 mg/kg, and the mice in control group were intraperitoneally injected with the same volume of PBS. NMDA intravitreal injection was performed 2 days after intraperitoneal injection, 5 nmoles NMDA was injected into the left eye, and the same volume of PBS was injected into the right eye as a control. The RGC staining of retinal plaques were performed 7 days after NMDA injection, and the number of alive RGC was calculated. The visual acuity and electroretinogram were used to evaluate the electrophysiological functions of RGC at 9 and 10 days after modeling. Real-time quantitative PCR and retinal frozen sections and glial fibrillary acidic protein (GFAP) immunofluorescence staining were performed 11 days after NMDA injection to evaluate the glial activation of the retina. The density, visual acuity, and the amplitude of PhNR-wave of RGC between groups were compared by one-way analysis of variance.Results At 7 days after NMDA injection, the density of RGC in retinal patch of low-dose chloroquine group was significantly higher than that of intraperitoneal injection of PBS control group (F=54.41, P＜0.01). The density of RGC in retinal patch of high-dose chloroquine group was lower than that of control group (F=1.18, P＞0.05). The visual acuity was higher than control group, and the difference was statistically significant (F=9.10, P＜0.05). The amplitude of PhNR-wave was significantly higher in low-dose chloroquine group than that of the control group (F=17.60, P＜0.01). The mRNA level of inflammatory factor and GFAP positive signal was also significantly lower than that of the control group (F=23.66, P＜0.05). The amplitude of PhNR-wave, the expression of GFAP (F=110.20, P＜0.01) and the mRNA level of inflammatory factors (F=167.60, 17.78; P＜0.01) in the high-dose chloroquine group were higher than the other two groups, and the differences were statistically significant.Conclusions In NMDA injury retinal model, low-dose chloroquine significantly increased the survival and physiological function of RGC, and the mechanism may be related to the inhibition of glial activation and inflammatory response. High-dose of chloroquine would aggravate the apoptosis of RGC.

Citation： Liu Yifan, Shen Yin. Low-dose chloroquine mediated neuroprotection against n-methyl-d-aspartate induced excitotoxicity in adult mice. Chinese Journal of Ocular Fundus Diseases, 2020, 36(4): 295-301. doi: 10.3760/cma.j.cn511434-20190903-00275 Copy

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2.	Uhlemann AC, Krishna S. Antimalarial multi-drug resistance in Asia: mechanisms and assessment[J]. Curr Top Microbiol Immunol, 2005, 295: 39-53.
3.	Yuyama K, Yamamoto N, Yanagisawa K. Chloroquine-induced endocytic pathway abnormalities: cellular model of GM1 ganglioside-induced Abeta fibrillogenesis in Alzheimer's disease[J]. FEBS Lett, 2006, 580(30): 6972-6976. DOI: 10.1016/j.febslet.2006.11.072.
4.	Caughlin S, Hepburn J, Liu Q, et al. Chloroquine restores ganglioside homeostasis and improves pathological and behavioral outcomes post-stroke in the rat[J]. Mol Neurobiol, 2019, 56(5): 3552-3562. DOI: 10.1007/s12035-018-1317-0.
5.	Biraboneye AC, Madonna S, Laras Y, et al. Potential neuroprotective drugs in cerebral ischemia: new saturated and polyunsaturated lipids coupled to hydrophilic moieties: synthesis and biological activity[J]. J Med Chem, 2009, 52(14): 4358-4369. DOI: 10.1021/jm900227u.
6.	Gaynes BI, Torczynski E, Varro Z, et al. Retinal toxicity of chloroquine hydrochloride administered by intraperitoneal injection[J]. J Appl Toxicol, 2008, 28(7): 895-900. DOI: 10.1002/jat.1353.
7.	Costedoat-Chalumeau N, Dunogue B, Leroux G, et al. A critical review of the effects of hydroxychloroquine and chloroquine on the eye[J]. Clin Rev Allergy Immunol, 2015, 49(3): 317-326. DOI: 10.1007/s12016-015-8469-8.
8.	Bonanomi MT, Dantas NC, Medeiros FA. Retinal nerve fibre layer thickness measurements in patients using chloroquine[J]. Clin Exp Ophthalmol, 2006, 34(2): 130-136. DOI: 10.1111/j.1442-9071.2006.01167.x.
9.	Li X, Fei J, Lei Z, et al. Chloroquine impairs visual transduction via modulation of acid sensing ion channel 1a[J]. Toxicol Lett, 2014, 228(3): 200-206. DOI: 10.1016/j.toxlet.2014.05.008.
10.	刘诗亮, 陈媛媛, 胡单萍, 等. N-甲基-D-天冬氨酸诱导的正常眼压青光眼小鼠模型的实验研究[J]. 武汉大学学报(医学版), 2014, 35(5): 710-715.Liu SL, Chen YY, Hu SP, et al. Experimental study of normal-tension glaucoma mouse model induced by N-methyl-D-aspartate[J]. Medical Journal of Wuhan University, 2014, 35(5): 710-715.
11.	Galindo-Romero C, Valiente-Soriano FJ, Jimenez-Lopez M, et al. Effect of brain-derived neurotrophic factor on mouse axotomized retinal ganglion cells and phagocytic microglia[J]. Invest Ophthalmol Vis Sci, 2013, 54(2): 974-985. DOI: 10.1167/iovs.12-11207.
12.	Prusky GT, Alam NM, Douglas RM. Enhancement of vision by monocular deprivation in adult mice[J]. J Neurosci,, 2006, 26(45): 11554-11561. DOI: 10.1523/JNEUROSCI.3396-06.2006.
13.	Connor KM, Krah NM, Dennison RJ, et al. Quantification of oxygen-induced retinopathy in the mouse: a model of vessel loss, vessel regrowth and pathological angiogenesis[J]. Nat Protoc, 2009, 4(11): 1565-1573. DOI: 10.1038/nprot.2009.187.
14.	Savarino A, Lucia MB, Giordano F, et al. Risks and benefits of chloroquine use in anticancer strategies[J]. Lancet Oncol, 2006, 7(10): 792-793. DOI: 10.1016/S1470-2045(06)70875-0.
15.	Easterbrook M. Ocular effects and safety of antimalarial agents[J]. Am J Med, 1988, 85(4A): 23-29. DOI: 10.1016/0002-9343(88)90358-0.
16.	Lyons JS, Severns ML. Detection of early hydroxychloroquine retinal toxicity enhanced by ring ratio analysis of multifocal electroretinography[J]. Am J Ophthalmol, 2007, 143(5): 801-809. DOI: 10.1016/j.ajo.2006.12.042.
17.	Zhao C, Lu S, Tajouri N, et al. In vivo confocal laser scanning microscopy of corneal nerves in leprosy[J]. Arch Ophthalmol, 2008, 126(2): 282-284. DOI: 10.1001/archophthalmol.2007.67.
18.	Hirata Y, Yamamoto H, Atta MS, et al. Chloroquine inhibits glutamate-induced death of a neuronal cell line by reducing reactive oxygen species through sigma-1 receptor[J]. J Neurochem, 2011, 119(4): 839-847. DOI: 10.1111/j.1471-4159.2011.07464.x.
19.	Lam TT, Abler AS, Kwong JM, et al. N-methyl-D-aspartate (NMDA)--induced apoptosis in rat retina[J]. Invest Ophthalmol Vis Sci, 1999, 40(10): 2391-2397.
20.	Schluter A, Aksan B, Fioravanti R, et al. Histone deacetylases contribute to excitotoxicity- triggered degeneration of retinal ganglion cells in vivo[J]. Mol Neurobiol, 2019, 56(12): 8018-8034. DOI: 10.1007/s12035-019-01658-x.
21.	Lambuk L, Iezhitsa I, Agarwal R, et al. Antiapoptotic effect of taurine against NMDA-induced retinal excitotoxicity in rats[J]. Neurotoxicology, 2019, 70: 62-71. DOI: 10.1016/j.neuro.2018.10.009.
22.	Bosco A, Breen KT, Anderson SR, et al. Glial coverage in the optic nerve expands in proportion to optic axon loss in chronic mouse glaucoma[J]. Exp Eye Res, 2016, 150: 34-43. DOI: 10.1016/j.exer.2016.01.014.
23.	Bringmann A, Pannicke T, Grosche J, et al. Muller cells in the healthy and diseased retina[J]. Prog Retin Eye Res, 2006, 25(4): 397-424. DOI: 10.1016/j.preteyeres.2006.05.003.
24.	Tezel G, Chauhan BC, LeBlanc RP, et al. Immunohistochemical assessment of the glial mitogen-activated protein kinase activation in glaucoma[J]. Invest Ophthalmol Vis Sci, 2003, 44(7): 3025-3033. DOI: 10.1167/iovs.02-1136.
25.	Lam TT, Kwong JM, Tso MO. Early glial responses after acute elevated intraocular pressure in rats[J]. Invest Ophthalmol Vis Sci, 2003, 44(2): 638-645. DOI: 10.1167/iovs.02-0255.
26.	Lin M, Chen Y, Jin J, et al. Ischaemia-induced retinal neovascularisation and diabetic retinopathy in mice with conditional knockout of hypoxia-inducible factor-1 in retinal Muller cells[J]. Diabetologia, 2011, 54(6): 1554-1566. DOI: 10.1007/s00125-011-2081-0.
27.	Shivakumar S, Panigrahi T, Shetty R, et al. Chloroquine protects human corneal epithelial cells from desiccation stress induced inflammation without altering the autophagy flux[J/OL]. Biomed Res Int, 2018, 2018: 7627329[2018-11-01].https://doi.org/10.1155/2018/7627329. DOI:10.1155/2018/7627329.
28.	Jain V, Ravindran E, Dhingra NK. Differential expression of Brn3 transcription factors in intrinsically photosensitive retinal ganglion cells in mouse[J]. J Comp Neurol, 2012, 520(4): 742-755. DOI: 10.1002/cne.22765.
29.	Viswanathan S, Frishman LJ, Robson JG, et al. The photopic negative response of the flash electroretinogram in primary open angle glaucoma[J]. Invest Ophthalmol Vis Sci, 2001, 42(2): 514-522.
30.	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.
31.	Chrysostomou V, Crowston JG. The photopic negative response of the mouse electroretinogram: reduction by acute elevation of intraocular pressure[J]. Invest Ophthalmol Vis Sci, 2013, 54(7): 4691-4697. DOI: 10.1167/iovs.13-12415.
32.	Burroughs SL, Kaja S, Koulen P. Quantification of deficits in spatial visual function of mouse models for glaucoma[J]. Invest Ophthalmol Vis Sci, 2011, 52(6): 3654-3659. DOI: 10.1167/iovs.10-7106.

1. Seki M, Lipton SA. Targeting excitotoxic/free radical signaling pathways for therapeutic intervention in glaucoma[J]. Prog Brain Res, 2008, 173: 495-510. DOI: 10.1016/S0079-6123(08)01134-5.
2. Uhlemann AC, Krishna S. Antimalarial multi-drug resistance in Asia: mechanisms and assessment[J]. Curr Top Microbiol Immunol, 2005, 295: 39-53.
3. Yuyama K, Yamamoto N, Yanagisawa K. Chloroquine-induced endocytic pathway abnormalities: cellular model of GM1 ganglioside-induced Abeta fibrillogenesis in Alzheimer's disease[J]. FEBS Lett, 2006, 580(30): 6972-6976. DOI: 10.1016/j.febslet.2006.11.072.
4. Caughlin S, Hepburn J, Liu Q, et al. Chloroquine restores ganglioside homeostasis and improves pathological and behavioral outcomes post-stroke in the rat[J]. Mol Neurobiol, 2019, 56(5): 3552-3562. DOI: 10.1007/s12035-018-1317-0.
5. Biraboneye AC, Madonna S, Laras Y, et al. Potential neuroprotective drugs in cerebral ischemia: new saturated and polyunsaturated lipids coupled to hydrophilic moieties: synthesis and biological activity[J]. J Med Chem, 2009, 52(14): 4358-4369. DOI: 10.1021/jm900227u.
6. Gaynes BI, Torczynski E, Varro Z, et al. Retinal toxicity of chloroquine hydrochloride administered by intraperitoneal injection[J]. J Appl Toxicol, 2008, 28(7): 895-900. DOI: 10.1002/jat.1353.
7. Costedoat-Chalumeau N, Dunogue B, Leroux G, et al. A critical review of the effects of hydroxychloroquine and chloroquine on the eye[J]. Clin Rev Allergy Immunol, 2015, 49(3): 317-326. DOI: 10.1007/s12016-015-8469-8.
8. Bonanomi MT, Dantas NC, Medeiros FA. Retinal nerve fibre layer thickness measurements in patients using chloroquine[J]. Clin Exp Ophthalmol, 2006, 34(2): 130-136. DOI: 10.1111/j.1442-9071.2006.01167.x.
9. Li X, Fei J, Lei Z, et al. Chloroquine impairs visual transduction via modulation of acid sensing ion channel 1a[J]. Toxicol Lett, 2014, 228(3): 200-206. DOI: 10.1016/j.toxlet.2014.05.008.
10. 刘诗亮, 陈媛媛, 胡单萍, 等. N-甲基-D-天冬氨酸诱导的正常眼压青光眼小鼠模型的实验研究[J]. 武汉大学学报(医学版), 2014, 35(5): 710-715.Liu SL, Chen YY, Hu SP, et al. Experimental study of normal-tension glaucoma mouse model induced by N-methyl-D-aspartate[J]. Medical Journal of Wuhan University, 2014, 35(5): 710-715.
11. Galindo-Romero C, Valiente-Soriano FJ, Jimenez-Lopez M, et al. Effect of brain-derived neurotrophic factor on mouse axotomized retinal ganglion cells and phagocytic microglia[J]. Invest Ophthalmol Vis Sci, 2013, 54(2): 974-985. DOI: 10.1167/iovs.12-11207.
12. Prusky GT, Alam NM, Douglas RM. Enhancement of vision by monocular deprivation in adult mice[J]. J Neurosci,, 2006, 26(45): 11554-11561. DOI: 10.1523/JNEUROSCI.3396-06.2006.
13. Connor KM, Krah NM, Dennison RJ, et al. Quantification of oxygen-induced retinopathy in the mouse: a model of vessel loss, vessel regrowth and pathological angiogenesis[J]. Nat Protoc, 2009, 4(11): 1565-1573. DOI: 10.1038/nprot.2009.187.
14. Savarino A, Lucia MB, Giordano F, et al. Risks and benefits of chloroquine use in anticancer strategies[J]. Lancet Oncol, 2006, 7(10): 792-793. DOI: 10.1016/S1470-2045(06)70875-0.
15. Easterbrook M. Ocular effects and safety of antimalarial agents[J]. Am J Med, 1988, 85(4A): 23-29. DOI: 10.1016/0002-9343(88)90358-0.
16. Lyons JS, Severns ML. Detection of early hydroxychloroquine retinal toxicity enhanced by ring ratio analysis of multifocal electroretinography[J]. Am J Ophthalmol, 2007, 143(5): 801-809. DOI: 10.1016/j.ajo.2006.12.042.
17. Zhao C, Lu S, Tajouri N, et al. In vivo confocal laser scanning microscopy of corneal nerves in leprosy[J]. Arch Ophthalmol, 2008, 126(2): 282-284. DOI: 10.1001/archophthalmol.2007.67.
18. Hirata Y, Yamamoto H, Atta MS, et al. Chloroquine inhibits glutamate-induced death of a neuronal cell line by reducing reactive oxygen species through sigma-1 receptor[J]. J Neurochem, 2011, 119(4): 839-847. DOI: 10.1111/j.1471-4159.2011.07464.x.
19. Lam TT, Abler AS, Kwong JM, et al. N-methyl-D-aspartate (NMDA)--induced apoptosis in rat retina[J]. Invest Ophthalmol Vis Sci, 1999, 40(10): 2391-2397.
20. Schluter A, Aksan B, Fioravanti R, et al. Histone deacetylases contribute to excitotoxicity- triggered degeneration of retinal ganglion cells in vivo[J]. Mol Neurobiol, 2019, 56(12): 8018-8034. DOI: 10.1007/s12035-019-01658-x.
21. Lambuk L, Iezhitsa I, Agarwal R, et al. Antiapoptotic effect of taurine against NMDA-induced retinal excitotoxicity in rats[J]. Neurotoxicology, 2019, 70: 62-71. DOI: 10.1016/j.neuro.2018.10.009.
22. Bosco A, Breen KT, Anderson SR, et al. Glial coverage in the optic nerve expands in proportion to optic axon loss in chronic mouse glaucoma[J]. Exp Eye Res, 2016, 150: 34-43. DOI: 10.1016/j.exer.2016.01.014.
23. Bringmann A, Pannicke T, Grosche J, et al. Muller cells in the healthy and diseased retina[J]. Prog Retin Eye Res, 2006, 25(4): 397-424. DOI: 10.1016/j.preteyeres.2006.05.003.
24. Tezel G, Chauhan BC, LeBlanc RP, et al. Immunohistochemical assessment of the glial mitogen-activated protein kinase activation in glaucoma[J]. Invest Ophthalmol Vis Sci, 2003, 44(7): 3025-3033. DOI: 10.1167/iovs.02-1136.
25. Lam TT, Kwong JM, Tso MO. Early glial responses after acute elevated intraocular pressure in rats[J]. Invest Ophthalmol Vis Sci, 2003, 44(2): 638-645. DOI: 10.1167/iovs.02-0255.
26. Lin M, Chen Y, Jin J, et al. Ischaemia-induced retinal neovascularisation and diabetic retinopathy in mice with conditional knockout of hypoxia-inducible factor-1 in retinal Muller cells[J]. Diabetologia, 2011, 54(6): 1554-1566. DOI: 10.1007/s00125-011-2081-0.
27. Shivakumar S, Panigrahi T, Shetty R, et al. Chloroquine protects human corneal epithelial cells from desiccation stress induced inflammation without altering the autophagy flux[J/OL]. Biomed Res Int, 2018, 2018: 7627329[2018-11-01].https://doi.org/10.1155/2018/7627329. DOI:10.1155/2018/7627329.
28. Jain V, Ravindran E, Dhingra NK. Differential expression of Brn3 transcription factors in intrinsically photosensitive retinal ganglion cells in mouse[J]. J Comp Neurol, 2012, 520(4): 742-755. DOI: 10.1002/cne.22765.
29. Viswanathan S, Frishman LJ, Robson JG, et al. The photopic negative response of the flash electroretinogram in primary open angle glaucoma[J]. Invest Ophthalmol Vis Sci, 2001, 42(2): 514-522.
30. 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.
31. Chrysostomou V, Crowston JG. The photopic negative response of the mouse electroretinogram: reduction by acute elevation of intraocular pressure[J]. Invest Ophthalmol Vis Sci, 2013, 54(7): 4691-4697. DOI: 10.1167/iovs.13-12415.
32. Burroughs SL, Kaja S, Koulen P. Quantification of deficits in spatial visual function of mouse models for glaucoma[J]. Invest Ophthalmol Vis Sci, 2011, 52(6): 3654-3659. DOI: 10.1167/iovs.10-7106.

Chinese Journal of Ocular Fundus Diseases

Low-dose chloroquine mediated neuroprotection against n-methyl-d-aspartate induced excitotoxicity in adult mice

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