Neuroprotection Against NMDA-Induced Retinal Damage by Philanthotoxin-343 Involves Reduced Nitrosative Stress

Abstract

N-methyl-D-aspartate receptor (NMDAR) overstimulation is known to mediate neurodegeneration, and hence represents a relevant therapeutic target for neurodegenerative disorders including glaucoma. This study examined the neuroprotective effects of philanthotoxin (PhTX)-343 against NMDA-induced retinal injury in rats. Male Sprague Dawley rats were divided into three groups; group 1 received phosphate buffer saline as the negative control, group 2 was injected with NMDA (160 nM) to induce retinal excitotoxic injury, and group 3 was pre-treated with PhTX-343 (160 nM) 24 h before NMDA exposure. All treatments were given intravitreally and bilaterally. Seven days post-treatment, rats were subjected to visual behaviour assessments using open field and colour recognition tests. Rats were then euthanized, and the retinas were harvested and subjected to haematoxylin and eosin (H&E) staining for morphometric analysis and 3-nitrotyrosine (3-NT) ELISA protocol as the nitrosative stress biomarker. PhTX-343 treatment prior to NMDA exposure improved the ability of rats to recognize visual cues and preserved visual functions (i.e., recognition of objects with different colours). Morphological examination of retinal tissues showed that the fractional ganglion cell layer thickness within the inner retina (IR) in the PhTX-343 treated group was greater by 1.28-fold as compared to NMDA-treated rats (p < 0.05) and was comparable to control rats (p > 0.05). Additionally, the number of retinal cell nuclei/100 μm² in IR for the PhTX-343-treated group was greater by 1.82-fold compared to NMDA-treated rats (p < 0.05) and was comparable to control group (p > 0.05). PhTX-343 also reduced the retinal 3-NT levels by 1.74-fold compared to NMDA-treated rats (p < 0.05). In conclusion, PhTX-343 pretreatment protects against NMDA-induced retinal morphological changes and visual impairment by suppressing nitrosative stress as reflected by the reduced retinal 3-NT level.

Keywords: N-methyl-D-aspartate receptor; N-methyl-D-aspartate receptor overstimulation; excitotoxicity; glaucoma; neurodegeneration; philanthotoxin-343; retinal ganglion cell; visual behavioural analysis.

FIGURE 1

Overall study design of the project. PBS, phosphate buffer saline; NMDA, N-methyl-D-aspartate; PhTX-343, philanthotoxin-343; 3-NT, 3-nitrotyrosine.

FIGURE 2

Experimental design and placement of objects for colour object recognition test in open field arena. The experimental design has three phases; habituation stage (trial 1) with empty open arena, familiarization phase (trials 2 and 3) with two blue colour objects (OB1; and OB2;) in the arena, and colour replacement phase (trial 4) where one blue object (OB1;) is replaced with a green object (OG;). Rats were placed at the centre of the arena alone and allowed to explore freely, the rat’s movements were recorded using Any-maze software. OB1, object blue 1; OB2, object blue 2, OG, object green.

FIGURE 3

Effect of PhTX-343 on NMDA-induced changes towards exploratory and locomotor activities of rats in open field test. Bar graphs show (A) Total distance travelled, (B) Total immobile time, and (C) Total immobile episodes. Representative track plots for each group are shown in panel (D) PBS group, (E) NMDA group, and (F) PhTX-343 pre-treatment prior to NMDA exposure. *p < 0.05 vs PBS; #p < 0.05 vs NMDA. PBS, phosphate buffer saline; NMDA, N-methyl-D-aspartate; PhTX-343, philanthotoxin-343.

FIGURE 4

Effect of PhTX-343 on NMDA-induced changes in colour object recognition in rats (A) Bar graph showing the total number of approaches of rats at the colour objects; during familiarization stage. The open field arena contained two blue objects (OB1; and OB2;), while during the colour replacement phase, one blue object (OB1;) was replaced with a green object (OG;). Representative track plots during familiarization stage for each group are shown in panel (B) PBS group, (C) NMDA group, and (D) PhTX-343 pre-treatment prior to NMDA exposure. Representative track plots during colour replacement phase for each group are shown in panel (E) PBS group, (F) NMDA group and (G) PhTX-343 pre-treatment group. *p < 0.05 vs PBS; #p < 0.05 vs NMDA. PBS, phosphate buffer saline; NMDA, N-methyl-D-aspartate; PhTX-343, philanthotoxin-343.

FIGURE 5

Effect of PhTX-343 on NMDA-induced changes in retinal morphology (×20 magnification) in rats. Photomicrographs (scale bar 100 µm) show H&E-stained retinal sections 7 days after intravitreal treatment for (A) PBS (control group), (B) NMDA-induced group and (C) Pre-treatment with PhTX-343, 24 h prior to NMDA administration. Morphometric quantification are presented as bar graphs (D) Fractional GCL thickness (%) within IR, and (E) Number of retinal cell nuclei per 100 µm² of IR. The NMDA group demonstrated significant GCL thinning and lower retinal cell nuclei density compared to the PBS and PhTX-343 groups. Both analysis showed comparable results between the PhTX-343 and PBS groups. *p < 0.05 vs control; ^# p < 0.05 vs NMDA. PBS, phosphate buffer saline; NMDA, N-methyl-D-aspartate; PhTX-343, philanthotoxin-343, GCL, ganglion cell layer, IR, inner retina, IPL, inner plexiform layer.

FIGURE 6

Effect of PhTX-343 7-days pretreatment on NMDA-induced retinal nitrosative stress in rats quantified by retinal 3-nitrotyrosine (3-NT) ELISA. The NMDA group had significantly higher 3-NT content compared to rats treated with PBS and pretreated with PhTX-343 prior to NMDA exposure. The 3-NT content for PBS and PhTX-343 groups were comparable. *p < 0.05 vs PBS; ^# p < 0.05 vs NMDA. PBS, phosphate buffer saline; NMDA, N-methyl-D-aspartate; PhTX-343, philanthotoxin-343.