Acute organophosphate (OP) toxicity poses a significant threat to both military and civilian personnel as it can lead to a variety of cholinergic symptoms including the development of status epilepticus (SE). Depending on its severity, SE can lead to a spectrum of neurological changes including neuroinflammation and neurodegeneration. In this study, we determined the impact of SE severity and duration on disease promoting parameters such as gliosis and neurodegeneration and the efficacy of a disease modifier, saracatinib (AZD0530), a Src/Fyn tyrosine kinase inhibitor. Animals were exposed to 4 mg/kg diisopropylfluorophosphate (DFP, s.c.) followed by medical countermeasures. We had five experimental groups: controls (no DFP), animals with no continuous convulsive seizures (CS), animals with ∼20-min continuous CS, 31-60-min continuous CS, and > 60-min continuous CS. These groups were then assessed for astrogliosis, microgliosis, and neurodegeneration 8 days after DFP exposure. The 31-60-min and > 60-min groups, but not ∼20-min group, had significantly upregulated gliosis and neurodegeneration in the hippocampus compared to controls. In the piriform cortex and amygdala, however, all three continuous CS groups had significant upregulation in both gliosis and neurodegeneration. In a separate cohort of animals that had ∼20 and > 60-min of continuous CS, we administered saracatinib for 7 days beginning three hours after DFP. There was bodyweight loss and mortality irrespective of the initial SE severity and duration. However, in survived animals, saracatinib prevented spontaneous recurrent seizures (SRS) during the first week in both severity groups. In the ∼20-min CS group, compared to the vehicle, saracatinib significantly reduced neurodegeneration in the piriform cortex and amygdala. There were no significant differences in the measured parameters between the naïve control and saracatinib on its own (without DFP) groups. Overall, this study demonstrates the differential effects of the initial SE severity and duration on the localization of gliosis and neurodegeneration. We have also demonstrated the disease-modifying potential of saracatinib. However, its’ dosing regimen should be optimized based on initial severity and duration of CS during SE to maximize therapeutic effects and minimize toxicity in the DFP model as well as in other OP models such as soman.

Diisopropylfluorophosphate (DFP), an organophosphate nerve agent (OPNA), exposure causes status epilepticus (SE) and epileptogenesis. In this study, we tested the protective effects of saracatinib (AZD0530), a Src kinase inhibitor, in mixed-sex or male-only Sprague Dawley rats exposed to 4–5 mg/kg DFP followed by 2 mg/kg atropine and 25 mg/kg 2-pralidoxime. Midazolam (3 mg/kg) was given to the mixed-sex cohort (1 h post-DFP) and male-only cohort (~30 min post-DFP). Saracatinib (20 mg/kg, oral, daily for 7 days) or vehicle was given two hours later and euthanized eight days or ten weeks post-DFP. Brain immunohistochemistry (IHC) showed increased microgliosis, astrogliosis, and neurodegeneration in DFP-treated animals. In the 10-week post-DFP male-only group, there were no significant differences between groups in the novel object recognition, Morris water maze, rotarod, or forced swim test. Brain IHC revealed significant mitigation by saracatinib in contrast to vehicle-treated DFP animals in microgliosis, astrogliosis, neurodegeneration, and nitro-oxidative stressors, such as inducible nitric oxide synthase, GP91phox, and 3-Nitrotyrosine. These findings suggest the protective effects of saracatinib on brain pathology seem to depend on the initial SE severity. Further studies on dose optimization, including extended treatment regimen depending on the SE severity, are required to determine its disease-modifying potential in OPNA models.

Glial scars have been observed following stab lesions in the spinal cord and brain but not observed and characterized in chemoconvulsant-induced epilepsy models. Epilepsy is a disorder characterized by spontaneous recurrent seizures and can be modeled in rodents. Diisopropylfluorophosphate (DFP) exposure, like other real-world organophosphate nerve agents (OPNAs) used in chemical warfare scenarios, can lead to the development of status epilepticus (SE). We have previously demonstrated that DFP-induced SE promotes epileptogenesis which is characterized by the development of spontaneous recurrent seizures (SRS), gliosis, and neurodegeneration. In this study, we report classical glial scars developed in the piriform cortex, but not in the hippocampus, by 8 days post-exposure. We challenged both male and female rats with 4–5 mg/kg DFP (s.c.) followed immediately by 2 mg/kg atropine sulfate (i.m.) and 25 mg/kg pralidoxime (i.m.) and one hour later by midazolam (i.m). Glial scars were present in the piriform cortex/amygdala region in 73% of the DFP treated animals. No scars were found in controls. Scars were characterized by a massive clustering of reactive microglia surrounded by hypertrophic reactive astrocytes. The core of the scars was filled with a significant increase of IBA1 and CD68 positive cells and a significant reduction in NeuN positive cells compared to the periphery of the scars. There was a significantly higher density of reactive GFAP, complement 3 (C3), and inducible nitric oxide synthase (iNOS) positive cells at the periphery of the scar compared to similar areas in controls. We found a significant increase in chondroitin sulfate proteoglycans (CS-56) in the periphery of the scars compared to a similar region in control brains. However, there was no change in TGF-β1 or TGF-β2 positive cells in or around the scars in DFP-exposed animals compared to controls. In contrast to stab-induced scars, we did not find fibroblasts (Thy1.1) in the scar core or periphery. There were sex differences with respect to the density of iNOS, CD68, NeuN, GFAP, C3 and CS-56 positive cells. This is the first report of cortical glial scars in rodents with systemic chemoconvulsant-induced SE. Further investigation could help to elucidate the mechanisms of scar development and mitigation strategies.