Protective effect of etomidate on cultured retinal ganglion cells with mechanical injury in vitro_Chinese Journal of Ocular Fundus Diseases

Authors：

Zhao Xuan ¹ , Bai Jue ² , You Siwei ³ , Cui Yuanyuan ¹ ,  Wu Mingmei ³

1. Department of Histology and Embryology, Faculty of Basic Medicine, Xi'an Medical University, Xi'an 710021, China;
2. Ophthalmic Hospital of Xi'an People’s Hospital (Xi'an Fourth Hospital), Xi'an 710004, China;
3. Department of Neurobiology, Air Force Medical University, Xi'an 710032, China;

Corresponding author：

Wu Mingmei, Email: wumm33@163.com

Keywords：

Etomidate; Retinal ganglion cells; Mechanical injury; Neuroprotection; In vitro culture

DOI：

10.3760/cma.j.cn511434-20211021-00595

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Objective To observe the protective effect of etomidate (ET) on cultured retinal ganglion cells (RGC) with mechanical injury in vitro. Methods New Sprague-Dawley rat RGC was cultured in vitro and identified by double immunofluorescent labeling of Thy1.1 and microtubule associated protein 2. The cultured primary cells were randomly divided into control group, RGC scratch group, ET low dose group (1 μmol/L), ET medium dose group (5 μmol/L) and ET high dose group (10 μmol/L). The RGC mechanical injury model was established by using iris knife to culture cells in RGC scratch group and ET group with different concentration. Seven days after modeling, the RGC survival rate of each group was detected by cell count Kit 8 proliferation assay. The apoptosis rate of RGC was detected by Annexin Ⅴ/propyl iodide double staining. Single factor analysis of variance was used to compare the groups. The pairwise comparison between groups was tested by the least significant difference method. Results The survival rates of RGC in RGC scratch group, ET low dose group, ET medium dose group and ET high dose group were (72.60±2.97)%, (73.73±1.14)%, (79.19±1.79)% and (83.88±0.94)%, respectively. The RGC apoptosis rates of control group, RGC scratch group, ET low dose group, ET medium dose group and ET high dose group were (5.08±0.17)%, (18.67±1.24)%, (17.96±0.74)%, (15.11± 0.56)% and (11.67±1.32)%, respectively. Comparison of RGC survival rate between groups: compared with RGC scratch group, the cell survival rate of ET low-dose group, ET medium-dose group and ET high-dose group was increased, and the difference between RGC scratch group and ET low-dose group was not statistically significant (P=0.728); the differences between RGC scratch group, ET medium dose group and ET high dose group were statistically significant (P＜0.001); the difference between ET medium dose group and ET high dose group was statistically significant (P=0.002). Comparison of apoptosis rate of RGC among groups: the apoptosis rate of RGC scratch group was significantly higher than that of control group, the difference was statistically significant (P＜0.001). Compared with RGC scratch group, the apoptosis rate of ET low-dose group, ET medium-dose group and ET high-dose group was decreased, and there was no statistical significance between RGC scratch group and ET low-dose group (P=0.869). The differences of apoptosis rate between RGC scratch group, ET medium dose group and ET high dose group were statistically significant (P＜0.05). The difference of apoptosis rate between ET medium dose group and ET high dose group was statistically significant (P=0.007). Conclusion ET has neuroprotective effect on RGC cultured in vitro with mechanical injury, and the protective effect increases with the increase of ET dose in a certain range.

Citation： Zhao Xuan, Bai Jue, You Siwei, Cui Yuanyuan, Wu Mingmei. Protective effect of etomidate on cultured retinal ganglion cells with mechanical injury in vitro. Chinese Journal of Ocular Fundus Diseases, 2023, 39(6): 489-493. doi: 10.3760/cma.j.cn511434-20211021-00595 Copy

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6.	Zhao X, Kuang F, You YY, et al. Etomidate affects the anti-oxidant pathway to protect retinal ganglion cells after optic nerve transection[J]. Neural Regen Res, 2019, 14(11): 2020-2024. DOI: 10.4103/1673-5374.259627.
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8.	Li R, Fan L, Ma F, et al. Effect of etomidate on the oxidative stress response and levels of inflammatory factors from ischemia-reperfusion injury after tibial fracture surgery[J]. Exp Ther Med, 2017, 13(3): 971-975. DOI: 10.3892/etm.2017.4037.
9.	Xie D, Li M, Yu K, et al. Etomidate alleviates cardiac dysfunction, fibrosis and oxidative stress in rats with myocardial ischemic reperfusion injury[J]. Ann Transl Med, 2020, 8(18): 1181. DOI: 10.21037/atm-20-6015.
10.	Jia L, Hao H, Wang C, et al. Etomidate attenuates hyperoxia-induced acute lung injury in mice by modulating the Nrf2/HO-1 signaling pathway[J]. Exp Ther Med, 2021, 22(1): 785. DOI: 10.3892/etm.2021.10217.
11.	Barnstable CJ, Drager UC. Thy-1 antigen: a ganglion cell specific marker in rodent retina[J]. Neuroscience, 1984, 11(4): 847-855. DOI: 10.1016/0306-4522(84)90195-7.
12.	Taschenberger H, Grantyn R. Several types of Ca²⁺ channels mediate glutamatergic synaptic responses to activation of single Thy-1-immunolabeled rat retinal ganglion neurons[J]. J Neurosci, 1995, 15(3 Pt 2): 2240-2254. DOI:10.1523/JNEUROSCI.15-03-02240.1995.
13.	Chung RS, Adlard PA, Dittmann J, et al. Neuron-glia communication: metallothionein expression is specifically up-regulated by astrocytes in response to neuronal injury[J]. J Neurochem, 2004, 88(2): 454-461. DOI: 10.1046/j.1471-4159.2003.02193.x.
14.	Kilic E, Weishaupt JH, Kilic U, et al. The superoxide dismutase1 (SOD1) G93A mutation does not promote neuronal injury after focal brain ischemia and optic nerve transection in mice[J]. Neuroscience, 2004, 128(2): 359-364. DOI: 10.1016/j.neuroscience.2004.06.064.
15.	Wang T, Cong R, Yang H, et al. Neutralization of BDNF attenuates the in vitro protective effects of olfactory ensheathing cell-conditioned medium on scratch-insulted retinal ganglion cells[J]. Cell Mol Neurobiol, 2011, 31(3): 357-364. DOI: 10.1007/s10571-010-9626-5.
16.	Daniels S, Roberts RJ. Post-synaptic inhibitory mechanisms of anaesthesia; glycine receptors[J]. Toxicol Lett, 1998, 100-101: 71-76. DOI: 10.1016/s0378-4274(98)00167-2.

1. Valk BI, Struys MMRF. Etomidate and its analogs: a review of pharmacokinetics and pharmacodynamics[J]. Clin Pharmacokinet, 2021, 60(10): 1253-1269. DOI: 10.1007/s40262-021-01038-6.
2. Watson JC, Drummond JC, Patel PM, et al. An assessment of the cerebral protective effects of etomidate in a model of incomplete forebrain ischemia in the rat[J]. Neurosurgery, 1992, 30(4): 540-544. DOI: 10.1227/00006123-199204000-00011.
3. Lee J, Kim D, Hong H, et al. Protective effect of etomidate on kainic acid-induced neurotoxicity in rat hippocampus[J]. Neurosci Lett, 2000, 286(3): 179-182. DOI: 10.1016/s0304-3940(00)01118-6.
4. Cayli SR, Ates O, Karadag N, et al. Neuroprotective effect of etomidate on functional recovery in experimental spinal cord injury[J]. Int J Dev Neurosci, 2006, 24(4): 233-239. DOI: 10.1016/j.ijdevneu.2006.04.003.
5. Xu ZX, Wu MM, Jiao XY, et al. Neuroprotective effect of etcmidate on axotomized retinal ganglion cells in adult rats[J]. Int Eye Sci, 2007, 7(4): 941-944. DOI: 10.3969/j.issn.1672-5123.2007.04.016.
6. Zhao X, Kuang F, You YY, et al. Etomidate affects the anti-oxidant pathway to protect retinal ganglion cells after optic nerve transection[J]. Neural Regen Res, 2019, 14(11): 2020-2024. DOI: 10.4103/1673-5374.259627.
7. Ates O, Yucel N, Cayli SR, et al. Neuroprotective effect of etomidate in the central nervous system of streptozotocin-induced diabetic rats[J]. Neurochem Res, 2006, 31(6): 777-783. DOI: 10.1007/s11064-006-9076-0.
8. Li R, Fan L, Ma F, et al. Effect of etomidate on the oxidative stress response and levels of inflammatory factors from ischemia-reperfusion injury after tibial fracture surgery[J]. Exp Ther Med, 2017, 13(3): 971-975. DOI: 10.3892/etm.2017.4037.
9. Xie D, Li M, Yu K, et al. Etomidate alleviates cardiac dysfunction, fibrosis and oxidative stress in rats with myocardial ischemic reperfusion injury[J]. Ann Transl Med, 2020, 8(18): 1181. DOI: 10.21037/atm-20-6015.
10. Jia L, Hao H, Wang C, et al. Etomidate attenuates hyperoxia-induced acute lung injury in mice by modulating the Nrf2/HO-1 signaling pathway[J]. Exp Ther Med, 2021, 22(1): 785. DOI: 10.3892/etm.2021.10217.
11. Barnstable CJ, Drager UC. Thy-1 antigen: a ganglion cell specific marker in rodent retina[J]. Neuroscience, 1984, 11(4): 847-855. DOI: 10.1016/0306-4522(84)90195-7.
12. Taschenberger H, Grantyn R. Several types of Ca²⁺ channels mediate glutamatergic synaptic responses to activation of single Thy-1-immunolabeled rat retinal ganglion neurons[J]. J Neurosci, 1995, 15(3 Pt 2): 2240-2254. DOI:10.1523/JNEUROSCI.15-03-02240.1995.
13. Chung RS, Adlard PA, Dittmann J, et al. Neuron-glia communication: metallothionein expression is specifically up-regulated by astrocytes in response to neuronal injury[J]. J Neurochem, 2004, 88(2): 454-461. DOI: 10.1046/j.1471-4159.2003.02193.x.
14. Kilic E, Weishaupt JH, Kilic U, et al. The superoxide dismutase1 (SOD1) G93A mutation does not promote neuronal injury after focal brain ischemia and optic nerve transection in mice[J]. Neuroscience, 2004, 128(2): 359-364. DOI: 10.1016/j.neuroscience.2004.06.064.
15. Wang T, Cong R, Yang H, et al. Neutralization of BDNF attenuates the in vitro protective effects of olfactory ensheathing cell-conditioned medium on scratch-insulted retinal ganglion cells[J]. Cell Mol Neurobiol, 2011, 31(3): 357-364. DOI: 10.1007/s10571-010-9626-5.
16. Daniels S, Roberts RJ. Post-synaptic inhibitory mechanisms of anaesthesia; glycine receptors[J]. Toxicol Lett, 1998, 100-101: 71-76. DOI: 10.1016/s0378-4274(98)00167-2.

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