Diet-incorporated saracatinib, a Src tyrosine kinase inhibitor, counteracts diisopropylfluorophosphate (DFP)-induced chronic neurotoxicity in the rat model

Abstract

Acute exposure to diisopropylfluorophosphate (DFP), an organophosphate nerve agent, induces status epilepticus (SE) and epileptogenesis despite treatment with countermeasures immediately after exposure. Epileptogenesis is characterized by increased Src family tyrosine kinases (SFK), nitrooxidative stress, reactive gliosis, and neurodegeneration in the early stage, followed by spontaneous seizures at a later stage. We hypothesized that treating with saracatinib (SAR), an SFK inhibitor, would mitigate the early markers of epileptogenesis. Therefore, in this study, we investigated the effects of SAR in the diet (10-20 mg/kg/day, high-dose or 5-10 mg/kg/day, low-dose) fed for four weeks post-DFP. SAR-in-diet significantly mitigated DFP-induced nitrooxidative stress markers (nitrite, GSH/GSSG) and pro-inflammatory cytokines/chemokine (TNF-α, IL-17, IL-6, IL-18, IL-1α, MCP-1) in serum and cerebrospinal fluid. DFP-exposed rats exhibited increased reactive microglia (IBA1 +CD68) and reactive astrocytes (GFAP+C3) in extrahippocampal and thalamic regions. Both SAR dosing regimens significantly reduced reactive astrogliosis across several brain regions in both sexes, while the low-dose SAR-in-diet significantly reduced both microgliosis and reactive microgliosis in the laterodorsal thalamic (LDT) nucleus in males. Sex-specific effects of SAR at high-dose were observed in astrogliosis in females in the dentate gyrus, subiculum, amygdala, and LDT. Both SAR dosing regimens significantly reduced neurodegeneration (FJB-positive neurons) in males in the centromedian thalamic nucleus. Pearson correlation analyses revealed strong associations between key epileptogenic markers and SAR-in-diet treatment. These findings underscore the complex relationship between the early markers of epileptogenesis and the disease-modifying potential of SAR-in-diet. Additionally, the SAR-in-diet treatment approach is translational and reduces handling stress in animals in long-term studies.

Keywords: Diisopropylfluorophosphate (DFP) model; Key proinflammatory cytokines; Neuroinflammation; Nitro-oxidative stress; Organophosphate nerve agents; Saracatinib (AZD0530).

Fig. 1.

Experimental design illustrating DFP exposure, interventions, and the timeline of various endpoints.

Fig. 2.

Body weight progression and SE severity. (A) There was no significant difference in the body weight progression between vehicle and SAR-treated LD and HD groups post-DFP challenge. (B-C) There were no significant differences in the SE severity between the vehicle and SAR treated groups. (D-F) There was no significant difference in the latency and SE severity between males and females. (A) Mixed-effect analysis, n = 12/group for mixed sex cohort and 6/sex; (B) One-way ANOVA (Kruskal-Wallis test), n = 12/group; (C) Mixed-effect analysis, n = 12/group; (D) unpaired t-test, n = 18/sex; (E) Mann-Whitney test, n = 18/sex; (F) Mixed-effect analysis, n = 18/sex.

Fig. 3.

Comparison and correlation analysis of serum SAR concentrations and consumption rates in animals fed with varying SAR-in-diet concentrations. (A) Daily SAR rate received in diet (mg/kg). (B-C) There was a significant correlation between the SAR rate consumed (mg/kg) and serum SAR concentration (ng/mL). (C) Pearson correlation analysis.

Fig. 4.

Nitrite and GSH/GSSG levels. SAR-in-diet significantly reduced the nitrite levels (A) and increased the GSH/GSSG ratio in DFP-exposed rats (B). (A, B) Two-way ANOVA with Tukey’s multiple comparisons test, n = 9–10/group for mixed sex cohort or 4–5/sex, ***p < 0.001, ****p < 0.0001 vs control; #p < 0.05, ##p < 0.01, ####p < 0.0001 vs DFP+Veh; ӿp < 0.05 vs DFP+SAR (LD). No sex differences were observed, and the data were pooled together.

Fig. 5.

The effects of DFP and vehicle or SAR treatment in the serum (A) and CSF (B) at 4 weeks post-exposure. The key proinflammatory cytokines and chemokine TNF-α, IL-17, IL-6, IL-18, IL-1α, and MCP-1 in the serum (A) and CSF (B) were significantly reduced SAR treatment. (A, B) Two-way ANOVA with Tukey’s multiple comparisons test, n = 8–10/group for mixed sex cohort or 4–5/sex. **p < 0.01, ***p < 0.001, ****p < 0.0001 vs control; ##p < 0.01; ###p < 0.001, ####p < 0.0001 vs DFP+Veh. No sex differences were observed, and the data were pooled together.

Fig. 6.

Microgliosis. (A) Representative photomicrographs of the brain sections from male rats stained for IBA1 (red-microglia), CD68 (green-reactive microglia), and DAPI (blue-nuclear stain); (B) Representative higher magnification images of reactive/homeostatic microglia. Scale bar 100 μm. Microgliosis (C-E) and reactive microgliosis (F-H). The overall effect (C, F), the regional difference in mixed-sex (D, G), and the difference in males and females in the LDT region of the brain (E, H). (C, F) Mixed-effects analysis with Tukey’s multiple comparisons test; (D, E and G, H) Region-wise two-way ANOVA with Tukey’s multiple comparisons test. For all, n = 9–10/group for mixed sex cohort or 4–5/ sex. *p < 0.05, **p < 0.01, ***p < 0.001 vs control; #p < 0.05 vs DFP+Veh; ӿӿp < 0.01 vs DFP+SAR (LD). DG-dentate gyrus; CA-cornu ammonis; AMY-amygdala; PC-piriform cortex; LDT-laterodorsal thalamic nucleus, MDT-mediodorsal thalamic nucleus, and CMT-centromedian thalamic nucleus. Sex difference was observed, so the graph was plotted separately for males and females in the region where the difference was detected.

Fig. 7.

Astrogliosis. (A) Representative photomicrographs of the brain sections of rats stained for GFAP (red-astrocyte), C3 (green-reactive astrocyte), and DAPI (blue-nuclear stain); (B) Representative higher magnification images of reactive/homeostatic astrocytes. Scale bar 100 μm. Astrogliosis (C-F) and reactive astrogliosis (G-H). The overall effect (C, G), the regional difference in mixed-sex (D, H), and differences in males (E) and females (F) are shown for astrogliosis. (C, G) Two-way ANOVA with Tukey’s multiple comparisons tests; (D, E, F, and H) Region-wise two-way ANOVA with Tukey’s multiple comparisons tests. For all, n = 9–10/group for mixed sex cohort or 4–5/ sex. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs control; #p < 0.05, ##p < 0.01, ###p < 0.001, ####p < 0.0001 vs DFP+Veh; ӿӿp < 0.01 vs DFP+SAR (LD). DG-dentate gyrus; CA-cornu ammonis; AMY-amygdala; PC-piriform cortex; LDT-laterodorsal thalamic nucleus, MDT-mediodorsal thalamic nucleus, and CMT-centromedian thalamic nucleus. Sex difference was observed, so the graph was plotted separately for males and females in the region where the difference was detected.

Fig. 8.

SAR-in-diet significantly rescued SE-induced neurodegeneration. Immunohistochemistry (IHC) of NeuN (red-labeled cells) combined with FJB staining (green-labeled cells, with co-localized cells appearing yellow), along with representative images from the CMT region (A). (B) Representative higher magnification images showing FJB positive neurons. The overall effect (C), the regional difference in the mixed-sex cohort (D), and the sex difference in the CMT region of the thalamus (E). (C) Two-way ANOVA with Tukey’s multiple comparisons tests; (D, E) Region-wise two-way ANOVA with Tukey’s multiple comparisons tests. For all, n = 9–10/group for mixed sex cohort or 4–5/ sex. *p < 0.05, ***p < 0.001, ****p < 0.0001 vs control; ###p < 0.001, ####p < 0.0001 vs DFP+Veh. DG-dentate gyrus; CA-cornu ammonis; AMY-amygdala; PC-piriform cortex; LDT-laterodorsal thalamic nucleus, MDT-mediodorsal thalamic nucleus, and CMT-centromedian thalamic nucleus. Sex difference was observed, so the graph was plotted separately for males and females in the region where the difference was detected.

Fig. 9.

Pearson correlation showed a strong significant correlation with different parameters. Heatmap summarizing Pearson correlation matrices between SE severity, various assays and IHC outcomes. Black crosses (X) indicate non-significant correlations, with r values in each box. n = 10 control, 10 DFP+Veh, 10 DFP+SAR (LD), 9 DFP+SAR (HD).