N-Methyl-D-Aspartate (NMDA)-induced Apoptosis in Rabbit Retina

Yong Wook Park; Sun Hwa Chae; Ji-Woong Lee; Oh Ju Kwon; Jae Pil Shin; Chang Jin Jeon; Si Yeol Kim

doi:10.3341/jkos.2008.49.7.1146

Journal List > J Korean Ophthalmol Soc > v.49(7) > 1008025

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Wook Park, Chae, Lee, Kwon, Shin, Jeon, and Kim: N-Methyl-D-Aspartate (NMDA)-induced Apoptosis in Rabbit Retina

Original Article

J Korean Ophthalmol Soc 2008;49(7):1146-1153.

Published online: 7 September 2008

DOI: https://doi.org/10.3341/jkos.2008.49.7.1146

N-Methyl-D-Aspartate (NMDA)-induced Apoptosis in Rabbit Retina

Yong Wook Park, M.D.¹, Sun Hwa Chae, M.D.¹, Ji-Woong Lee, M.D.¹, Oh Ju Kwon, Ph.D.², Jae Pil Shin, M.D.^1,, Chang Jin Jeon, Ph.D.³, Si Yeol Kim, M.D.¹

Department of Ophthalmology, Kyungpook National University, College of Medicine, Daegu, Korea

Department of Ophthalmic Optics, Busan College of Information Technology, Pusan, Korea

Department of Biology, Kyungpook National University, College of Natural Science, Daegu, Korea

Address reprint requests to Jae Pil Shin, M.D. Department of Ophthalmology, Kyungpook National University Hospital #50 Samduk 2-ga, Jung-gu, Daegu 700-721, Korea Tel: 82-53-420-5817, Fax: 82-53-426-6552, E-mail: jpshin@hitel.net

Received 18 March 2008 Accepted 14 June 2007

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Purpose

To evaluate the involvement of apoptosis in N-methyl-D-aspartate (NMDA)-induced excitotoxicity in the rabbit retina.

Methods

After intravitreal injection of 680 and 2,000 nmoles of NMDA in rabbit eyes, the eyes were enucleated at 2, 16, and 60 hours and 1 and 2 weeks. The apoptotic cell death was determined with TdT-mediated biotin-dUTP nick end labeling (TUNEL) stain, and immunohistochemical stains of Bcl-2, Bax, and caspase-3 were performed.

Results

TUNEL showed increased labeling scattered in the ganglion cell layer and inner nuclear layer from 16 to 60 hours. The number of TUNEL-positive nuclei decreased at 60 hours, and none was observed at 2 hours, 1 week, and 2 weeks. More TUNEL-positive nuclei were seen with injection of 2,000 nmoles compared to 680 nmoles. Bcl-2, Bax, and caspase-3 were seen histologically as early as 2 hours in the ganglion cell layer and inner nuclear layer; there was no stained nuclei with the TUNEL stain. At 2 hours after intravitreal NMDA injection, Bcl-2, Bax, and caspase-3 were also stained in Müller cells.

Conclusions

This study showed that apoptosis is involved in NMDA-induced excitotoxicity in the rabbit retina. Bcl-2, Bax, and caspase-3 may play important roles in modulating the apoptosis in NMDA-induced excitotoxicity, and Müller cells are involved in the apoptotic pathway.

Keywords: Apoptosis, Bax, Bcl-2, Caspase-3, NMDA

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Figure 1.

TUNEL stain of the retinas at 2 (A), 16 (D), 60 (G) hours after 680 nmoles of intravitreal NMDA injection and at 2 (B), 16 (E), 60 (H) hours after 2,000 nmoles of intravitreal NMDA injection and at 2 (C), 16 (F), 60 (I) hours after PBS injection (×400). A, B: No noticeable labeled nuclei are seen in the ganglion cell layer (GCL) and the inner nuclear layer (INL). (D, E, H) Scattered and intensely labeled nuclei are seen in the GCL and INL. G: No labeled nuclei are seen at 60 hours after 680 nmoles of NMDA injection. (C, F, I) No noticeable nuclei are stained after intravitreal PBS injection. GCL=ganglion cell layer; IPL=inner plexiform layer; INL=inner nuclear layer; OPL= outer plexiform layer; ONL=outer nuclear layer.

Figure 2.

Bcl-2 immunohistochemical staining of the retinas at 2 (A), 16 (C), 60 (E) hours after 680 nmoles of intravitreal NMDA injection and at 2 (B), 16 (D), 60 (F) hours after 2,000 nmoles of intravitreal NMDA injection (×400). A, B: Bcl-2 are stained in the ganglion cell layer and Müller cell (arrow). C, D, E, F: Bcl-2 are stained strongly in the GCL and INL. GCL=ganglion cell layer; IPL=inner plexiform layer; INL=inner nuclear layer; OPL= outer plexiform layer; ONL=outer nuclear layer.

Figure 3.

Bax immunohistochemical staining of the retinas at 2 (A), 16 (C), 60 (E) hours after 680 nmoles of intravitreal NMDA injection and at 2 (B), 16 (D), 60 (F) hours after 2,000 nmoles of intravitreal NMDA injection (×400). (A) Bax are stained in the ganglion cell layer and Müller cell (arrow). (B, C, D, E, F) Bax are stained in the GCL and INL. GCL=ganglion cell layer; IPL=inner plexiform layer; INL=inner nuclear layer; OPL=outer plexiform layer; ONL=outer nuclear layer.

Figure 4.

Caspase-3 immunohistochemical staining of retinas at 2 (A), 16 (C), 60 (E) hours after 680 nmoles of intravitreal NMDA injection and at 2 (B), 16 (D), 60 (F) hours after 2,000 nmoles of intravitreal NMDA injection (×400). (A) Caspase-3 are stained in the ganglion cell and Müller cell (arrow). (B) Caspase-3 are stained in the ganglion cell layer. (C, D, E, F) Caspase-3 are stained strongly in the GCL and INL. GCL=ganglion cell layer; IPL=inner plexiform layer; INL=inner nuclear layer; OPL=outer plexiform layer; ONL=outer nuclear layer.

Table 1.

Result of TUNEL-positive cell count in NMDA-induced apoptosis of rabbit retinas after different concentration of NMDA injection

Cell count	2 hours	16 hours	60 hours	1 week	2 weeks
680 nmole	0	8.33±2.08*	0^†	0	0
2,000 nmole	0	23.33±7.09*	7.67±2.08^†	0	·0

^* † p<0.05 by Mann-Whitney U-test.

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