1400 W, a selective inducible nitric oxide synthase inhibitor, mitigates early neuroinflammation and nitrooxidative stress in diisopropylfluorophosphate-induced short-term neurotoxicity rat model

Abstract

Organophosphate nerve agent (OPNA) exposure induces acute and long-term neurological deficits. OPNA exposure at sub-lethal concentrations induces irreversible inhibition of acetylcholinesterase and cholinergic toxidrome and develops status epilepticus (SE). Persistent seizures have been associated with increased production of ROS/RNS, neuroinflammation, and neurodegeneration. A total of 1400W is a novel small molecule, which irreversibly inhibits inducible nitric oxide synthase (iNOS) and has been shown to effectively reduce ROS/RNS generation. In this study, we investigated the effects of 1400W treatment for a week or two weeks at 10 mg/kg or 15 mg/kg per day in the rat diisopropylfluorophosphate (DFP) model. 1400W significantly reduced the number of microglia, astroglia, and NeuN+FJB positive cells compared to the vehicle in different regions of the brain. 1400W also significantly reduced nitrooxidative stress markers and proinflammatory cytokines in the serum. However, neither of the two concentrations of 1400W for two weeks of treatment had any significant effect on epileptiform spike rate and spontaneous seizures during the treatment period in mixed sex cohorts, males, or females. No significant sex differences were found in response to DFP exposure or 1400W treatment. In conclusion, 1400W treatment at 15 mg/kg per day for two weeks was more effective in significantly reducing DFP-induced nitrooxidative stress, neuroinflammatory and neurodegenerative changes.

Keywords: 1400W; cytokines; epilepsy; iNOS; neurodegeneration; neuroinflammation; nitrooxidative stress; organophosphate nerve agent.

Figure 1

Experimental design for non-telemetry (A) and telemetry groups (B) illustrating the DFP exposure, intervention, and the endpoints.

Figure 2

SE severity quantification during the 60 min post DFP in non-telemetry and telemetry groups. There were no significant differences in the SE severity between the vehicle and 1400W treated groups in both sexes. One-way ANOVA with Tukey’s post-hoc for multiple comparisons (A,B; n = 11–12), (C,D; n = 16–19), (E,F; n = 10–13), and (G,H; n = 16–18).

Figure 3

The effects of 1400W treatment for a week on DFP-induced astrogliosis and microgliosis. Representative images of GFAP (green, for astrocytes), IBA1 (gray, for microglia), and NeuN (red, for neurons) positive cells representing Amygdala (AMY), CA1, CA3, Dentate gyrus (DG), Piriform Cortex (PC), Subiculum (SUB) regions of the brains from control, vehicle+1400W, DFP + vehicle, DFP + 1400W (10 and 15 mg/kg) groups (A). The representative enlarged images for each group and the brain regions are shown in the adjacent columns, scale bar = 50 μM. The mixed effects model revealed a significant increase in GFAP+ve cells in brain as a whole in DFP + vehicle group and 1400W at both 10 and 15 mg/kg significantly reduced the GFAP positive cells (B). Two-way ANOVA showed an increase in GFAP positive cells within different regions of the brain following DFP exposure and 1400W reduced the DFP-induced astrogliosis (C). The mixed effects model revealed a significant increase in microgliosis in DFP + vehicle group but 1400W at either of the concentration had no significant effect. However, two-way ANOVA analysis of individual brain regions showed a significant reduction of microgliosis at 15 mg/kg in CA1, CA3 and SUB regions (D,E), n = 8–12. Repeated measure Two-way ANOVA (mixed effects) with Tukey’s post-hoc for multiple comparisons. * Represents DFP effect compared to vehicle (control); # represents the 1400W effect compared to DFP + vehicle group, * represents DFP effect compared to the vehicle (control) and # represents 1400W effect compared to DFP + vehicle group. *p < 0.05, **p < 0.01, ***p < 0.001, #p < 0.05, ##p < 0.01, ###p < 0.001.

Figure 4

The effects of 1400W treatment for 2 weeks on DFP-induced astrogliosis and microgliosis. Representative images of GFAP (green, for astrocytes) and IBA1 (gray, for microglia) positive cells representing Amygdala (AMY), CA1, CA3, Dentate gyrus (DG), Piriform Cortex (PC), Subiculum (SUB) regions of the brains from control, vehicle+1400W, DFP + vehicle, DFP + 1400W (10 and 15 mg/kg) groups (A). The representative enlarged images for each group and the brain regions are shown in the adjacent columns, scale bar = 50 μM. The mixed effects model revealed a significant increase in both GFAP+ve and IBA1 + ve cells in brain as a whole in the DFP + vehicle group. 1400W at both 10 and 15 mg/kg significantly reduced astrogliosis and microgliosis (B,D). Two-way ANOVA showed an increase in GFAP positive cells within different regions of the brain following DFP exposure. 1400W at both concentrations reduced the DFP-induced astrogliosis in all brain regions. However, at 10 mg microgliosis was not reduced in the DG and PC (C,E), n = 6–12. Repeated measure Two-way ANOVA (mixed effects) with Tukey’s post-hoc for multiple comparisons. * Represents DFP effect compared to the vehicle (control); # represents the 1400W effect compared to DFP + vehicle group * represents DFP effect compared to vehicle (control) and # represents 1400W effect compared to DFP + vehicle group. *p < 0.05, **p < 0.01, ***p < 0.001, #p < 0.05, ##p < 0.01, ###p < 0.001.

Figure 5

The effects of 1400W treatment for a week on DFP-induced neurodegeneration. Representative images of NeuN (red, neurons) and FJB (green) positive cells from Amygdala (AMY), CA1, CA3, Dentate gyrus (DG), Piriform Cortex (PC), Subiculum (SUB) regions are shown. Co-localized (orange) cells represent degenerating neurons. Sections were counterstained with DAPI (blue) to represent all nuclei, scale bar = 50 μM (A). Mixed effects model revealed a significant increase in degenerating neurons in brain as a whole in DFP + vehicle group and 1400W at 15 mg/kg significantly reduced the FJB positive neurons (B).The two-way ANOVA analysis showed an increase in FJB positive neurons within different regions of the brain following DFP treatment and 1400W treatment significantly reduced FJB positive neurons at 15 mg/kg in DG, PC and SUB (C). n = 7–12, Repeated measure Two-way ANOVA (mixed effects) with Tukey’s post-hoc for multiple comparisons. * Represents DFP effect compared to the vehicle (control); # represents the 1400W effect compared to DFP + vehicle group * represents DFP effect compared to vehicle (control) and # represents 1400W effect compared to DFP + vehicle group. *p < 0.05, **p < 0.01, ***p < 0.001, #p < 0.05, ##p < 0.01, ###p < 0.001.

Figure 6

The effects of 1400W treatment for 2 weeks on neurodegeneration. Representative images of NeuN (red, for neurons) and FJB (green) positive cells from Amygdala (AMY), CA1, CA3, Dentate gyrus (DG), Piriform Cortex (PC), Subiculum (SUB) regions are shown. Co-localized (orange) cells represent degenerating neurons. Sections were counterstained with DAPI (blue) to represent all nuclei, scale bar = 50 μM (A). Mixed effects model revealed a significant increase in degenerating neurons in brain as a whole in DFP + vehicle group at 15 days post-exposure (B).The two-way ANOVA analysis showed an increase in FJB positive neurons within different regions of the brain following DFP exposure and 1400W treatment significantly reduced the DFP induced increase in FJB positive neurons at 10 mg/kg in AMY, CA3 and DG, while 15 mg/kg reduced in all regions except the PC (C), n = 7–12. Repeated measure Two-way ANOVA (mixed effects) with Tukey’s post-hoc for multiple comparisons. * Represents DFP effect compared to vehicle (control); # represents the 1400W effect compared to DFP + vehicle group * represents DFP effect compared to vehicle (control) and # represents 1400W effect compared to DFP + vehicle group. *p < 0.05, **p < 0.01, ***p < 0.001, #p < 0.05, ##p < 0.01, ###p < 0.001.

Figure 7

The effects of 1400W on the DFP-induced serum pro-inflammatory cytokines. At 8-day post DFP, 1400 W at 10 mg/kg significantly reduced the DFP induced IL-1β and TNF-α but not MCP-1, while 1400W at 15 mg/kg significantly reduced DFP induced IL-1β, IL-6, TNF-α and MCP-1 (A). At 15-day post DFP, 1400 W both at (10 mg/kg and 15 mg/kg) significantly reduced the upregulated cytokine levels following DFP exposure (B), n = 8, one-way ANOVA with Tukey’s post-hoc for multiple comparisons; *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 8

The effects of DFP and 1400W on serum glutathione profiles. At 8-day, 1400W (10 mg/kg or 15 mg/kg) had no significant effect on serum GSH and GSSG levels (A). At 15-day post DFP, 1400 W (15 mg/kg) significantly reduced GSSG levels and also increased the GSH/GSSG ratio in DFP exposed rats (B), n = 8, one-way ANOVA with Tukey’s post-hoc for multiple comparisons; *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 9

The effects of DFP and 1400W on serum nitro-oxidative stress markers. At 8-day post DFP, 1400 W (15 mg/kg) significantly reduced serum levels of nitrite and ROS levels in DFP exposed rats (A,B). At 15-day post DFP, 1400 W (15 mg/kg) significantly reduced serum nitrite in DFP exposed rats (C), n = 8, one-way ANOVA with Tukey’s post-hoc for multiple comparisons; *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 10

The effects of DFP and 1400W on epileptiform spikes and SRS during the 15 days post-exposure. Epileptiform spikes were compared in mixed sex cohorts and between males and females (A–D). There were no significant differences in the epileptiform spike rate between any treatment groups in mixed sex cohorts (A,B) or between males and females (C,D). Electrographic seizures were also compared in mixed sex cohorts and between males and females (E–H). There were also no significant differences in the electrographic seizures (SRS) between any treatment groups in mixed sex cohorts (E,F) or between males and females (G,H). One-way ANOVA with Tukey’s post-hoc for multiple comparisons. (A,B; n = 10–13), (C,D; n = 16–18), (E,F, n = 10–13), (G,H; n = 16–18).