Differential Impact of Severity and Duration of Status Epilepticus, Medical Countermeasures, and a Disease-Modifier, Saracatinib, on Brain Regions in the Rat Diisopropylfluorophosphate Model

Abstract

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.

Saracatinib, a Src Tyrosine Kinase Inhibitor, as a Disease Modifier in the Rat DFP Model: Sex Differences, Neurobehavior, Gliosis, Neurodegeneration, and Nitro-Oxidative Stress

“…We found FJB positive cells that were colocalized with NeuN as well as FJB positive cells without NeuN positive staining, as reported in our previous publication [ 70 ]. We counted both colocalized cells and FJB cells without NeuN in our assessment of neurodegeneration since NeuN was expected to downregulate in some neurons in response to stress [ 82 ].…”

“…Human studies have shown some adverse effects to SAR administration, such as fatigue, nausea, diarrhea, and headaches [ 67 , 86 ]. Notably, we did not observe any side effects in this study with 20 mg/kg daily dose, unlike our previous study (using 25 mg/kg twice a day for the first three days) in the DFP model, where SAR-treated animals had increased weight loss and mortality compared to VEH-treated animals [ 70 ]. Although SAR treatment did not significantly mitigate weight loss or morbidity measures (Irwin analysis), it appears that 20 mg/kg is more tolerable in these animals.…”

“…SAR was prepared once in 3–4 days and left stirring at room temperature to prevent precipitation. SAR formulation has been described in our previous publication [ 70 ].…”

“…We recently tested the short-term effects of SAR in animals that had about 20 min of convulsive seizures after DFP intoxication [ 70 ]. These animals did not develop SRS during the treatment period and had significantly reduced neurodegeneration compared to the VEH-treated group when tested 24 h after the last day of SAR treatment (i.e., 8 days post-DFP) [ 70 ]. Notably, the SAR-treated animals had significant weight loss compared to VEH controls.…”

“…Notably, the SAR-treated animals had significant weight loss compared to VEH controls. In that study, we administered 25 mg/kg SAR, beginning 4 h after DFP intoxication, twice daily at 12 h intervals during the first three days, followed by a single dose per day for the next four days [ 70 ]. In this study, we tested 20 mg/kg per day for 7 days to minimize toxicity and increase the efficacy of the drug.…”