Dose-dependent effects of NMDA on retinal and optic nerve morphology in rats

Abstract

Aim: To investigate dose-dependent effects of N-methyl-D-aspartate (NMDA) on retinal and optic nerve morphology in rats.

Methods: Sprague Dawley rats, 180-250 g in weight were divided into four groups. Groups 1, 2, 3 and 4 were intravitreally administered with vehicle and NMDA at the doses 80, 160 and 320 nmol respectively. Seven days after injection, rats were euthanized, and their eyes were taken for optic nerve toluidine blue and retinal hematoxylin and eosin stainings. The TUNEL assay was done for detecting apoptotic cells.

Results: All groups treated with NMDA showed significantly reduced ganglion cell layer (GCL) thickness within inner retina, as compared to control group. Group NMDA 160 nmol showed a significantly greater GCL thickness than the group NMDA 320 nmol. Administration of NMDA also resulted in a dose-dependent decrease in the number of nuclei both per 100 µm GCL length and per 100 µm² of GCL. Intravitreal NMDA injection caused dose-dependent damage to the optic nerve. The degeneration of nerve fibres with increased clearing of cytoplasm was observed more prominently as the NMDA dose increased. In accordance with the results of retinal morphometry analysis and optic nerve grading, TUNEL staining demonstrated NMDA-induced excitotoxic retinal injury in a dose-dependent manner.

Conclusion: Our results demonstrate dose-dependent effects of NMDA on retinal and optic nerve morphology in rats that may be attributed to differences in the severity of excitotoxicity and oxidative stress. Our results also suggest that care should be taken while making dose selections experimentally so that the choice might best uphold study objectives.

Keywords: N-methyl-D-aspartate; dose-dependent effects; excitotoxicity; glaucoma; optic nerve; retina.

Figure 1. Representative microphotographs of rat retinal sections stained with H&E after administering different doses of NMDA

A: The PBS group, normal retinal histology; B-D: The NMDA 80, 160 and 320 nmol groups, respectively. NMDA injection, particularly at higher doses, caused severe degenerative changes, with an initial stage of massive cellular swelling, and subsequently, retinal cells loss. Scale bar represents 100 µm. GCL: Ganglion cell layer; IPL: Inner plexiform layer.

Figure 2. Quantitative morphometric analysis of inner retinal changes in response to various doses of NMDA

A: Thickness of GCL within inner retina (%); B: No. of ganglion cells nuclei per 100 µm GCL length; C: No. of ganglion cells nuclei per 100 µm² of GCL; D: No. of nuclei per 100 µm² of inner retina. Group 1 was injected with PBS (vehicle), group 2 was injected with 80 nmol NMDA, group 3 was injected with 160 nmol NMDA, and group 4 was injected with 320 nmol NMDA. Inner retina=GCL+IPL. n=10, ^aP<0.05 vs PBS, ^bP<0.01 vs PBS, ^cP<0.001 vs PBS, ^dP<0.001 vs NMDA 80 nmol, and ^eP<0.01 vs NMDA 160 nmol.

Figure 3. Representative microphotographs of optic nerve semithin sections stained with toluidine blue

A: The PBS group, normal optic nerve histology; B-D: Groups of NMDA 80, 160 and 320 nmol, respectively; B: Comparable distribution of glial cells to the control group with slight degeneration of nerve fibers; C: Denser glial cells with degeneration of nerve fibers; D: Presence of glial cells and extensive vacuolation indicates progressive degenerative changes occupying the entire section. Scale bar represents 50 µm. Asterisk indicates nerve fiber degeneration; Triangle symbol indicates vacuolation.

Figure 4. Quantitative estimation of the effect of intravitreal administration of different doses of NMDA on optic nerve morphology

PBS group served as the control. n=6, ^aP<0.05 vs PBS, ^bP<0.001 vs PBS, ^cP<0.05 vs NMDA 80 nmol.

Figure 5. Representative TUNEL stained microphotographs of retinal sections

A: PBS group, no apoptotic signal was detected in control group; B-D: Groups of NMDA 80, 160 and 320 nmol, respectively; B: Few detection of apoptosis ganglion cells; C: Several apoptotic signals were detected; D: A multiple presence of apoptotic signals was observed. Scale bar represents 100 µm; arrows indicate the presence of apoptotic cells. GCL: Ganglion cell layer; IPL: Inner plexiform layer.

Figure 6. Quantitative estimation of the retinal cell apoptosis in response to various doses of NMDA, as measured by TUNEL staining

PBS served as the control. n=6, ^aP<0.01 vs PBS, ^bP<0.001 vs PBS, ^cP<0.01 vs NMDA 80 nmol, ^dP<0.01 vs NMDA 160 nmol.