Peripheral and central effects of NADPH oxidase inhibitor, mitoapocynin, in a rat model of diisopropylfluorophosphate (DFP) toxicity

Abstract

Organophosphates (OP) are highly toxic chemical nerve agents that have been used in chemical warfare. Currently, there are no effective medical countermeasures (MCMs) that mitigate the chronic effects of OP exposure. Oxidative stress is a key mechanism underlying OP-induced cell death and inflammation in the peripheral and central nervous systems and is not mitigated by the available MCMs. NADPH oxidase (NOX) is one of the leading producers of reactive oxygen species (ROS) following status epilepticus (SE). In this study, we tested the efficacy of the mitochondrial-targeted NOX inhibitor, mitoapocynin (MPO) (10 mg/kg, oral), in a rat diisopropylfluorophosphate (DFP) model of OP toxicity. In DFP-exposed animals, MPO decreased oxidative stress markers nitrite, ROS, and GSSG in the serum. Additionally, MPO significantly reduced proinflammatory cytokines IL-1β, IL-6, and TNF-α post-DFP exposure. There was a significant increase in GP91^phox, a NOX2 subunit, in the brains of DFP-exposed animals 1-week post-challenge. However, MPO treatment did not affect NOX2 expression in the brain. Neurodegeneration (NeuN and FJB) and gliosis [microglia (IBA1 and CD68), and astroglia (GFAP and C3)] quantification revealed a significant increase in neurodegeneration and gliosis after DFP-exposure. A marginal reduction in microglial cells and C3 colocalization with GFAP in DFP + MPO was observed. The MPO dosing regimen used in this study at 10 mg/kg did not affect microglial CD68 expression, astroglial count, or neurodegeneration. MPO reduced DFP-induced oxidative stress and inflammation markers in the serum but only marginally mitigated the effects in the brain. Dose optimization studies are required to determine the effective dose of MPO to mitigate DFP-induced changes in the brain.

Keywords: GP91phox; diisopropylfluorophosphate (DFP); gliosis; oxidative stress; proinflammatory cytokines.

FIGURE 1

A summary of experimental design and SE quantification. (A) Schematic illustration of the experimental timeline of DFP administration and MPO treatment. (B,C) SE severity by seizure stage over time and SE severity score- the time (in minutes) spent in CS (≥stage 3) during the 60 min post DFP exposure in-mixed sex cohort (B) and between males and females (C). There was no significant difference in SE severity between vehicle and MPO treated animals (B, mixed effects analysis) or between males and females (C, unpaired t-test). n = 12 per group. Data presented as mean ± SEM.

FIGURE 2

Oxidative stress markers in the serum. Comparison between control, PBS + MPO, DFP + VEH, and DFP + MPO for nitrite (μM) using a Griess assay, superoxide anion levels (RFU) using a reactive oxygen species (ROS) assay, and oxidized glutathione (μM) using a GSSG assay. Compared to the control, there was a significant increase in nitrite and GSSG in DFP + VEH animals, but not in DFP + MPO was observed. DFP + VEH ROS concentration was high (p = 0.68) compared to all other groups. Kruskal–Wallis test for nitrite and ROS; and one-way ANOVA for GSSG. n = 8 per group. *Represents the DFP effect compared to control and PBS + MPO. #Represents effects of MPO in DFP compared to DFP + VEH. *p < 0.05, #p < 0.05. Data presented as mean ± SEM.

FIGURE 3

Serum cytokine and chemokine assays. MPO significantly reduced DFP-induced proinflammatory cytokines IL-1β, IL-6, TNF-α, IL-10, and MCP1 levels did not alter in any groups. Kruskal–Wallis test for IL-1β and IL-6 and one-way ANOVA for IL-10, TNF-α, and MCP1. n = 8 per group. *Represents the DFP effect compared to control and PBS + MPO. #Represents effects of MPO in DFP compared to DFP + VEH, *p < 0.05, #p < 0.05. Data presented as mean ± SEM.

FIGURE 4

Oxidative stress markers in the brain 1-week post-DFP. (A) Representative images of the piriform cortex (PC) showing GP91^phox (green), IBA1-positive microglial cells (red), and DAPI (white), a nuclear stain. The overlay images exemplify colocalization. (B) GP91^phox and IBA1 colocalized-microglial cells quantification from CA1, CA3, DG, amygdala (AMY), and PC regions increased in DFP groups compared to non-DFP groups. MPO did not have an effect. DFP + MPO Gp91^phox-positive microglia were significantly higher than control animals in all regions quantified. Kruskal–Wallis test. (C,E) The overall group effect of MPO on GP91^phox-IBA1 colocalization and IBA1 positive microglia cells per field in CA1, CA3, DG, Amy, and PC are shown. Horizontal bars indicate a significant difference between control vs. DFP + VEH and PBS + MPO vs. DFP + MPO. Overall, MPO, did not have an effect on GP91^phox IBA1 colocalization. Microglia absolute count was reduced, though it was not significant. Two-way ANOVA mixed effects analysis. (D) IBA1-positive microglia was not affected by treatment with MPO. In each region, DFP significantly increased the quantity of IBA1-positive cells. There were significantly more IBA1-positive cells in the CA3 and DG regions of DFP + MPO compared to PBS + MPO animals which further demonstrates the effect of DFP. One-way ANOVA. n = 8 control groups, n = 12 DFP groups. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 5

Microgliosis 1-week post-DFP. (A) Representative images of CD68 positive (green) microglia (IBA1, red) with DAPI (white) in the dentate gyrus (DG) from controls and DFP-exposed animals treated with vehicle or MPO. (B) Quantification of CD68 + microglia from CA1, CA3, DG, amygdala (AMY), and piriform cortex (PC). CD68 expression in DFP groups was upregulated in all these regions compared to controls. Kruskal–Wallis test. (C) Bars indicate a significant difference in group effect between control vs. DFP + VEH and PBS + MPO vs. DFP + MPO. Two-way ANOVA mixed effect analysis. n = 8 in non-DFP groups, n = 12 in DFP groups. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 6

Astrogliosis 1-week post-DFP. (A) Representative IHC images of C3 positive (green) astroglia (red) in the amygdala (AMY). (B) There was an increase in C3 GFAP-positive cells in CA1, CA3, DG, Amy, and PC in DFP-exposed animals compared to respective controls. Kruskal–Wallis test. (C) In grouped hippocampal and extrahippocampal regions, there was a significant upregulation of C3 + GFAP colocalization in DFP + VEH and DFP + MPO compared to control and PBS + MPO groups. Two-way ANOVA mixed effect analysis. (D) As determined by the Kruskal–Wallis test (CA1, CA3, and AMY) and one-way ANOVA (DG and PC), DFP nor MPO had an effect on absolute GFAP-positive cell counts in the individual brain regions except for the PC, where there was a significant increase in PBS + MPO animals. (E) In contrast, there was a significant difference in the brain as a whole between control vs. DFP + VEH and PBS + MPO vs. DFP + MPO determined by two-way ANOVA mixed effect analysis. MPO had no effect on DFP-induced astrogliosis (n = 8 in non-DFP groups, n = 12 in DFP groups). (F) An example of a glial scar, identified by astrocytes (green) lining clusters of microglia (red), and reduced neuronal cells (white) at the core of the scar. (G) The number of animals with glial scars in DFP + VEH and DFP + MPO were compared. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 7

Neurodegeneration 1-week post-DFP. (A) Representative images of neurodegeneration demonstrated by FJB-positive (green) NeuN (red) in the amygdala (AMY) of each treatment group. (B,C) Cell quantification of FJB and NeuN colocalization in individual and grouped hippocampal (CA1, CA3, and DG), and extrahippocampal (AMY and PC) regions. FJB-positive NeuN cells significantly increased in both DFP + VEH and DFP + MPO groups. One-way ANOVA (CA1, CA3, DG, and AMY), Kruskal–Wallis (PC), and two-way ANOVA mixed effect analysis (grouped regions). (D,E) The number of NeuN + cells per field in individual and grouped regions CA1, CA3, DG, AMY, and PC. (D) Individual regions were analyzed by one-way ANOVA (CA1, CA3, DG, and AMY) and Kruskal–Wallis (PC). In the DG, AMY, and PC, there was significant neuronal loss in DFP + MPO as compared to PBS + MPO animals. In the DG, PBS + MPO-treated animals had significantly more neurons than controls. (E) A two-way ANOVA mixed effect analysis was used to evaluate the grouped regions. Bars indicate a significant difference between control vs. DFP + VEH and PBS + MPO vs. DFP + MPO. (F) The percentage of FJB + neurons in CA1 compared to CA3 in DFP + VEH and DFP + MPO animals. No significant differences were observed between groups. n = 8 in non-DFP groups, n = 12 in DFP groups. *p < 0.05, **p < 0.01, ***p < 0.001.