Novel therapeutics for treating organophosphate-induced status epilepticus co-morbidities, based on changes in calcium homeostasis

Abstract

Organophosphate (OP) chemicals include pesticides such as parathion, and nerve gases such as sarin and soman and are considered major chemical threat agents. Acute OP exposure is associated with a cholinergic crisis and status epilepticus (SE). It is also known that the survivors of OP toxicity exhibit neurobehavioral deficits such as mood changes, depression, and memory impairment, and acquired epilepsy. Our research has focused on addressing the need to develop effective therapeutic agents that could be administered even after prolonged seizures and would prevent or lessen the chronic morbidity associated with OP-SE survival. We have developed rat survival models of OP pesticide metabolite paraoxon (POX) and nerve agent sarin surrogate diisopropyl fluorophosphate (DFP) induced SE that are being used to screen for medical countermeasures against an OP attack. Our research has focused on studying neuronal calcium (Ca²⁺) homeostatic mechanisms for identifying mechanisms and therapeutics for the expression of neurological morbidities associated with OP-SE survival. We have observed development of a "Ca²⁺ plateau" characterized by sustained elevations in neuronal Ca²⁺ levels in OP-SE surviving rats that coincided with the appearance of OP-SE chronic morbidities. These Ca²⁺ elevations had their origin in Ca²⁺ release from the intracellular stores such that blockade with antagonists like dantrolene, carisbamate, and levetiracetam lowered OP-SE mediated Ca²⁺ plateau and afforded significant neuroprotection. Since the Ca²⁺ plateau lasts for a prolonged period, our studies suggest that blocking it after the control of SE may represent a unique target for development of novel countermeasures to prevent long term Ca²⁺ mediated OP-SE neuropsychiatric comorbidities such as depression, anxiety, and acquired epilepsy (AE).

Keywords: Calcium; Carisbamate; Cell death; Cognitive deficits; DFP; Dantrolene; Depression; Levetiracetam; Paraoxon; Status epilepticus.

Figure 1.. Paradigm for POX and DFP induced SE survival models

At time 0, male Sprague-Dawley rats (weight- 280-310g) are injected with OP agent POX or DFP. These agents are prepared fresh in ice-cold PBS and kept on ice before their sub-cutaneous administration. One-minute later atropine sulfate and 2-PAM prepared in saline are injected intramuscularly. Rats start to show progression of cholinergic symptoms typified by vacuous chewing movement, salivation, Straub tail response, intense tremors and ultimately continues seizures (SE). From the appearance of first Stage-4 SE, rats are allowed to seize for 1-h at which point midazolam is injected to stop the SE. Surviving rats are injected with saline and sucrose-milk solution to aid their recovery. Test countermeasures are administered immediately after midazolam intervention. OP-SE survival, Ca²⁺ estimations, and neuropathology are assessed during the first few weeks. Development of chronic morbidities are assessed at latent timepoints beginning at 3-months post OP-SE survival (models originally published in refs: and 26).

Figure 2.. Neuronal injury following POX induced SE

A-D. Representative photomicrographs of Fluoro-Jade C (FJC) staining in the dentate gyrus-hilus region, parietal cortex, amygdala, and thalamus 2 days after POX SE. Scale bars, 200 μm. E. Quantitative analyses of FJC labeling. FJC positive cells indicative of neuronal injury were observed in hilus, thalamus and cortex of POX rats 48-h after SE termination. Control rats did not exhibit any FJC labeling. (*p<0.05 compared to control, t-test, n= 4-6 rats). (Data originally published in ref: 25).

Figure 3.. Development of chronic behavioral morbidities following POX induced SE

Approximately 3-months following POX SE, surviving rats were tested on various behavioral assays for assessing symptoms of depression and memory impairments. A. Forced Swim Test (FST): Static image of a rat in FST cylinder. Graph shows increased immobility time in POX SE compared to control rats during the FST, indicative of behavioral despair. B. Sucrose Preference Test (SPT): Rats have a free-choice of consuming regular water or 1% sucrose water for a 24-h period. Graph shows decreased sucrose consumption in POX SE rats compared to control rats on the SPT indicative of anhedonia. C. Elevated Plus Maze (EPM): Images shows EPM paradigm. The vertical bars are open arms and horizontal bars are closed arms. Movement of rats traced in Red is shown in EPM over a 5-min test. An almost solid Red line can be seen for closed-arm activity for POX rats. Adjoining graph shows quantitation indicative of enhanced anxiety in POX SE rats as characterized by significantly less time spent in the open arm of the EPM. D. Novel Object Recognition (NOR): In this paradigm, in phase-I rats are allowed to explore an arena with two-identical objects for a 3-min period. After 1-h delay, rats are re-introduced in the same arena now containing one novel object. Time spent by the rats exploring the two objects is recorded over a 2-min period. Adjoining graph shows quantitation of a NOR test suggestive of an impaired recognition memory in POX SE rats as displayed significantly less time spent exploring the novel object. All data expressed as mean ± SEM, *p<0.05, t-test, n= 8 rats. (Data originally published in ref: 43).

Figure 4.. Development of the Ca²⁺ plateau following OP-SE

A. Pseudo-color ratiometric images of representative acutely isolated hippocampal neurons from control, POX and DFP treated SE rats. Control neurons had bluish color that corresponds to lower Fura-2 ratio while OP-SE neurons had orange-red color that corresponds to higher Fura-2 ratio. B. Representative Ca²⁺ imaging study providing quantitation of[Ca²⁺]_i in 6 acutely isolated neurons obtained from control and SE rats 24-h after POX SE injury. C. Hippocampal CA1 [Ca²⁺]_i from age-matched control (white bar) and POX rats were isolated 1-h and 1, 7 and 30 days after SE (black bars). [Ca²⁺]_i in POX-SE rats was significantly higher than control values at all the time-points and did not return to base-line even at 30-days post SE (Ca²⁺ plateau). (Data originally published in refs: and 26).

Figure 5.. Mechanisms for the generation and maintenance of Ca²⁺ plateau following OP-SE

A. Quantitating the effects of pharmacological agents on the Ca²⁺ plateau. No effect of the application of MK-801 to 4 acutely isolated neurons manifesting the Ca²⁺ plateau 24h after SE. Lowering of Ca²⁺ plateau following application of dantrolene to 4 acutely isolated neurons manifesting the Ca²⁺ plateau 24h after SE. B. Hippocampal CA1 [Ca²⁺]_i from control rats (white bar), DFP rats (black bar), and DFP + MK-801 (grey bars) were isolated 1-h after SE. MK-801 pretreatment (4 mg/kg, i.p.) prevented the elevations in [Ca²⁺]_i that occur following DFP induced SE. However, MK-801 treatment 1-h after DFP-induced SE did not significantly affect DFP-SE induced [Ca²⁺]_i elevations. C. Hippocampal CA1 [Ca²⁺]_i from control rats (white bar), POX rats (black bars), and POX + drugs (shaded bars) were isolated 48-h after SE. Drugs were administered at 1, 6, and 18-h post termination of SE with midazolam. [Ca²⁺]_i in neurons isolated from POX-SE rats treated with either dantrolene (DANT, 50 μM bath-application to isolated neurons) or levetiracetam (LEV, 50 mg/kg, i.p.) or carisbamate (CRB, 90 mg/kg, i.p.) were significantly lower than POX SE rats (no drugs) values at the respective time point. All data represented as mean ± SEM. *p<0.05 (Data originally published in refs: and 26).

Figure 6.. Blockade of the Ca²⁺ plateau is neuroprotective following POX-SE

A. Representative photomicrographs of Fluoro-Jade C (FJC) staining in the dentate gyrus-hilus region, parietal cortex, amygdala, and thalamus of a POX rat 2 days after POX SE, and POX + dantrolene (10 mg/kg, i.m.), and POX + carisbamate (90 mg/kg, i.m.) treated rats. Scale bars, 200 μm. Dantrolene or carisbamate were administered 60-mins post POX SE and resulted in a minimum of approximately 60% reduction in FJC labeling across all the regions analyzed. B. Quantitative analyses of FJC labeling. Control rats did not exhibit any FJC labeling. FJC positive cells indicative of neuronal injury were observed in hilus, amygdala, thalamus and cortex of POX rats 48-h after SE termination. Rats treated with dantrolene or carisbamate showed significantly less FJC labeling in these brain regions (*p<0.05 compared to control, t-test, n= 6 rats). Dantrolene was obtained from Sigma Chemical Company and was dissolved in saline. Warm water sonication for about 10 min was needed to get the drug in solution. Carisbamate was provided by Johnson & Johnson (Pharmaceutical Research & Development L.L.C., Titusville, NJ, U.S.A.) and was dissolved in sterile water made slightly basic with sodium hydroxide. Levetiracetam was obtained in injectable form (Hospira Company). (Data originally published in ref: 33).