Assessment of pharmacokinetics, safety, and neuroprotective efficacy of an adjunct intramuscular verapamil therapy in a rat model of organophosphate DFP-induced status epilepticus

Assessment of pharmacokinetics, safety, and neuroprotective efficacy of an adjunct intramuscular verapamil therapy in a rat model of organophosphate DFP-induced status epilepticus | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Assessment of pharmacokinetics, safety, and neuroprotective efficacy of an adjunct intramuscular verapamil therapy in a rat model of organophosphate DFP-induced status epilepticus Yam Nath Paudel , Robert E. Blair , Elisa Hawkins , Matthew S. Halquist , Melissa Morgan , Jason Funderburk , Daniel Calvano , Jennifer Koblinski , Hope Richard , View ORCID Profile Laxmikant S. Deshpande doi: https://doi.org/10.1101/2025.06.25.661332 Yam Nath Paudel 1 Departments of Neurology, School of Medicine, Virginia Commonwealth University , Richmond, VA 23298, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Robert E. Blair 1 Departments of Neurology, School of Medicine, Virginia Commonwealth University , Richmond, VA 23298, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Elisa Hawkins 1 Departments of Neurology, School of Medicine, Virginia Commonwealth University , Richmond, VA 23298, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Matthew S. Halquist 2 Departments of Pharmaceutics, Bioanalytical Shared Resource Laboratory, School of Pharmacy, Virginia Commonwealth University , Richmond, VA 23298, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Melissa Morgan 2 Departments of Pharmaceutics, Bioanalytical Shared Resource Laboratory, School of Pharmacy, Virginia Commonwealth University , Richmond, VA 23298, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Jason Funderburk 2 Departments of Pharmaceutics, Bioanalytical Shared Resource Laboratory, School of Pharmacy, Virginia Commonwealth University , Richmond, VA 23298, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Daniel Calvano 3 Departments of Pathology, School of Medicine , Tissue and Data Acquisition and Analysis Core, VCU Massey Comprehensive Cancer Center, Virginia Commonwealth University , Richmond, VA 23298, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Jennifer Koblinski 3 Departments of Pathology, School of Medicine , Tissue and Data Acquisition and Analysis Core, VCU Massey Comprehensive Cancer Center, Virginia Commonwealth University , Richmond, VA 23298, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Hope Richard 3 Departments of Pathology, School of Medicine , Tissue and Data Acquisition and Analysis Core, VCU Massey Comprehensive Cancer Center, Virginia Commonwealth University , Richmond, VA 23298, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Laxmikant S. Deshpande 1 Departments of Neurology, School of Medicine, Virginia Commonwealth University , Richmond, VA 23298, USA 4 Departments of Pharmacology and Toxicology, School of Medicine, Virginia Commonwealth University , Richmond, VA 23298, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Laxmikant S. Deshpande For correspondence: Laxmikant.Deshpande{at}vcuhealth.org Abstract Full Text Info/History Metrics Preview PDF Abstract Lethal organophosphate (OP) exposure leads to status epilepticus (SE), which, despite standard-of-care therapy, is associated with acute mortality and long-term morbidities. Neuronal injury and inflammation are reported following OP-SE, and drugs targeted at these processes have produced beneficial outcomes. Verapamil (VPM) is a calcium-channel blocker used as an antihypertensive drug. It exhibits neuroprotective and anti-inflammatory actions in experimental models of CNS injuries. Here, we investigated the feasibility of an adjunctive intramuscular (i.m.) VPM therapy in OP Diisopropyl Fluorophosphate (DFP)-induced SE. We also investigated the safety and toxicity of i.m. VPM and compared its pharmacokinetic (PK) profile to oral (p.o.) administration. Rats were injected with DFP (4 mg/kg, s.c.). One minute later, atropine (0.5 mg/kg, i.m.) and 2-PAM (25 mg/kg, i.m.) were injected, and at 1-hour post-SE, midazolam (1.78 mg/kg, i.m.) was administered. Rats were then treated with VPM (10 mg/kg, i.m, 3 days). Histological analysis was conducted to assess neuronal injury and injection-site pathology. In a separate group of rats, PK studies were conducted on blood and brain homogenates treated once with saline or VPM (10 mg/kg, p.o. or i.m.). Our data demonstrated that following DFP-SE, i.m. VPM achieved higher blood and brain levels and exhibited a favorable PK profile compared to p.o. route. VPM therapy did not cause significant muscle pathology and produced a robust neuroprotective response. Our studies provide evidence that the i.m. route is an effective method for delivering VPM following SE producing significant neuroprotective outcomes compared to treatment with the standard-of-care alone in OP-SE. Introduction Status Epilepticus (SE) is a clinical emergency associated with high mortality and significant neurological morbidities 1 – 3 . There are several causes for SE, including stroke, traumatic brain injury, withdrawal from anti-seizure medications, alcohol withdrawal, and exposure to certain toxic chemicals 4 . One such category of chemicals that could lead to the rapid induction of SE is the organophosphate (OP) compounds 5 , 6 . These compounds are diverse in their applicability and include commonly used pesticides, industrial chemicals, and additives to jet fuels 7 . OP compounds also include lethal chemical warfare nerve agents (CWNA) 8 . OP exposure can thus occur accidentally, occupationally, domestically, or during war/ terrorism-related scenarios 9 – 12 . Furthermore, intentional use of OP compounds for self-harm is also unfortunately common in developing nations 13 , 14 . OP compounds are inhibitors of the enzyme acetylcholinesterase, leading to accumulation of acetylcholine at the synapse 8 , 15 , 16 . Depending on the OP dose and the level of AChE inhibition, symptoms of OP exposure range from salivation, lacrimation, and headaches to tremors, muscle fasciculations, and seizures. These symptoms could further evolve into unremitting seizures (status epilepticus, SE), respiratory difficulties, and eventually death if prompt medical care is not provided 17 , 18 . Emergency treatment for OP intoxication includes respiratory support and treatment with a three-drug regimen standard of care (SOC) recommended by the Food and Drug Administration (FDA) to manage hypercholinergic signs. These drugs include atropine (a muscarinic receptor antagonist), pralidoxime (an AChE reactivator), and midazolam (a benzodiazepine to manage seizures) 19 , 20 . These emergency procedures have significantly reduced the acute mortality associated with OP exposures. However, despite these treatments, survival from OP exposure is associated with chronic neurological co-morbidities, including memory impairments, mood disorders, and even spontaneous recurrent seizures (SRS). Thus, effective treatments are needed to control not only the acute mortality but also lower the incidences of chronic morbidity associated with OP exposures 21 , 22 . Cellular injury and neurodegeneration are key processes underlying the neurological consequences of OP toxicity 15 , 23 – 25 . Our lab has studied neuronal calcium homeostatic mechanisms in rat models of OP toxicity 26 . We reported protracted elevations in hippocampal calcium levels using the paraoxon (POX) 6 and diisopropyl fluorophosphate (DFP) 5 models of OP-induced SE. Calcium-induced calcium release mechanisms were found to mediate these chronic elevations in calcium. Indeed, treatment with ryanodine receptor (RyR) and inositol-tris-phosphate (IP3R) antagonists such as dantrolene and levetiracetam not only lowered the OP-SE-induced neuronal calcium levels but also produced a neuroprotective effect 27 . Ketamine, which prevents calcium entry by blocking the N-methyl-D-aspartate receptors (NMDAR), has also been neuroprotective in some OP-SE models 28 , 29 . Neuroinflammation has also been reported in OP-SE, and pharmacological treatment aimed at suppressing neuroinflammatory processes is being investigated to improve neurological outcomes following OP intoxication 30 – 35 . Verapamil (VPM) is a phenylalkylamine calcium channel blocker approved by the Food and Drug Administration (FDA) in the 1980s for the management of high blood pressure as well as for treating angina and cardiac arrhythmias 36 . The non-FDA-approved indications for VPM include its use for treating cluster headaches 37 , acute coronary syndrome 38 , and cerebral vasospasm 39 . The drug is typically administered orally, and an intravenous formulation is available. The usual dose range is 120 to 360 mg/day given orally and up to 10 mg parenterally 40 . VPM has a long human safety record with minimal side effects and drug-drug interactions 41 . Recent findings regarding the molecular actions of VPM, in addition to calcium-channel inhibition, have demonstrated a potent neuroprotective and anti-inflammatory action, raising the possibility that VPM could also be an effective OP countermeasure drug. For example, VPM increased neuronal survival and memory in a mouse stroke model 42 . VPM (20 mg/kg, i.p.) also prevented neuronal damage and rescued memory following severe hypoglycemia in rats 43 . VPM (10 mg/kg, i.p.) is also reported to decrease apoptosis in a rat model of ischemia/ reperfusion injury 44 . In a cellular model of Alzheimer’s Disease, destabilization of calcium homeostasis and enhanced glutamate excitotoxicity were noted, which were attenuated by VPM 45 . Similarly, VPM was reported to be neuroprotective in a cellular model of dopaminergic neurotoxicity 46 . VPM also rescued motor neurons by reducing endoplasmic reticulum stress in a mouse model of ALS 47 . Together, these findings point toward a neuroprotective profile for VPM, providing a rationale for testing VPM as a potential therapy to attenuate OP-SE toxicities. In this study, using a rat model of DFP-induced SE 5 , we compared the pharmacokinetics of VPM when administered orally (p.o.) or intramuscularly (i.m.). The i.m. route was selected given the necessity for ease of administration during an OP-based mass casualty scenario. VPM is also a substrate for P-glycoprotein (P-gp), a transmembrane protein that moves drugs out of the cell 48 , 49 . Therefore, we also measured brain levels under both SE and no-SE conditions to investigate the brain penetrance and availability of VPM. We also investigated the neuroprotective efficacy of adjunctive i.m. VPM therapy in various brain regions following DFP-induced SE when co-administered with the standard FDA-approved three-drug regimen of atropine, 2-PAM, and midazolam indicated for OP intoxication. Muscle pathology at the injection site was also evaluated to investigate the safety of repeatedly administering i.m. injections. VPM therapy. Finally, a stability analysis of our VPM formulation was also conducted. Materials and Methods Drugs and Chemicals DFP (purity > 90%) was obtained from Chem Service Inc. (West Chester, PA). Verapamil hydrochloride, atropine sulfate, and pralidoxime chloride (2-PAM) were obtained from Millipore-Sigma (St. Louis, MO). Midazolam vials (5 mg/mL) were obtained from Med-Vet International (Mettawa, IL). To prepare a working DFP solution (4 mg/mL), the desired quantity of DFP was removed using a Hamilton syringe and added to a glass bottle containing ice-cold phosphate-buffered saline (PBS), and then gently vortexed. The bottle was kept on ice, and syringes were drawn and kept on ice until the time of subcutaneous (s.c.) injections. Time on ice (between dilution in PBS and injection) was never more than ten minutes. Verapamil, Atropine sulfate, and 2-PAM were dissolved in saline (SAL, 0.9% NaCl) and then sterile-filtered. Animals All animal use procedures were per the National Institute of Health Guide for the Care and Use of Laboratory Animals and approved by Virginia Commonwealth University’s Institutional Animal Care and Use Committee. Male and Female Sprague-Dawley rats were obtained from Envigo (Indianapolis, IN) at 9 weeks of age. Animals were housed two per cage at 20-22°C with a 12 Light: 12 Dark hour cycle (lights on 0600-01800 h) and free access to food and water. Induction of DFP-SE As shown in Figure 1 , in accordance with our previously published protocol for inducing DFP-induced SE 5 , 50 , separate cohorts of rats were injected with DFP (4 mg/kg, s.c.). One minute later, atropine (0.5 mg/kg, i.m.) and 2-PAM (25 mg/kg, i.m.) were injected to permit survival and progression into SE. DFP exposure produced signs of progressive cholinergic hyperstimulation culminating in continuous seizures. We utilized the Racine criteria for assessing SE severity 51 , 52 . The criteria for the behavioral seizure score were: 0 - behavioral arrest, hair raising, excitement, and rapid respiration; 1 - mouth movement of lips/tongue, vibrissae movement, and salivation; 2 – head "bobbing"/clonus; 3 – forelimb clonus; 4 – clonic rearing, and 5 – clonic rearing with loss of postural control. A Racine score of 4-5 was used as the criterion to mark the onset of SE, approximately 5-10 minutes post-DFP injection. Our previous studies have shown that this Racine score correlates with the tonic-clonic phase of SE and is accompanied by an EEG frequency of >6 Hz, which meets the clinical criteria of SE 5 , 50 . At 1-h post-SE onset, midazolam (1.78 mg/kg, i.m.) was administered to suppress SE. Midazolam administration was delayed by 1 h post-DFP-SE onset to simulate a practical therapeutic window for first responders or hospital admission following mass casualty events 5 , 53 , 54 . These doses were determined using human equivalents calculated using a human-to-rat dose translation equation 55 , 56 . Control rats were age-matched, no-SE condition that received either SAL or VPM. Download figure Open in new tab Figure 1: Timeline for induction of DFP-SE and VPM interventions post SE At time 0, Sprague-Dawley rats (both male and female) were injected with the OP agent DFP (4 mg/kg, s.c.). One minute later, atropine sulfate (AtSO 4 ; 0.5 mg/kg, i.m.) and 2-PAM (25 mg/kg, i.m.) were injected. SE onset was approximately 10 min. Rats were allowed to seize for 1 h, at which point midazolam (1.78 mg/kg, i.m.) was injected. For PK studies, VPM (10 mg/kg) was administered once via the p.o. or i.m. route, and blood and brain samples were collected at various time points up to 24 hours. For histological studies, rats were injected with saline or VPM (10 mg/kg, i.m., b.i.d. ) for three consecutive days. Mortality was assessed daily till the fourth day, when the rats were euthanized and perfused for brain and muscle collection. Verapamil treatment Rats that met the SE severity criteria (Racine 4-5) and received midazolam at 1-h post-DFP-SE onset were randomized into two treatment groups: those receiving saline (SAL) or VPM. Previous studies indicated that VPM could be administered i.p. in rodent seizure models at doses up to 40 mg/kg 57 – 59 . A pilot study with two doses of VPM (10 and 20 mg/kg, i.m.) at 1 h after DFP-SE was conducted in rats (n=5). Rats safely tolerated both doses in these pilot studies. For this study, we chose a 10 mg/kg twice-daily treatment regimen as this dose was closer to the human-equivalent dose 56 and would allow for a stable plasma level given a t 1/2 of 5 h in humans 60 , 61 . Thus, VPM was administered concomitantly with the first dose of midazolam at 60 minutes post-DFP-SE onset and subsequently twice daily for up to 72 hours, for a total of six VPM doses. The control group was given i.m. SAL injections for the same duration and frequency. For i.m. administration, VPM or SAL was administered in the quadriceps muscle using a 28-gauge needle. The injection volume was 0.1mL/kg. Injection sites were alternated between the lateral and contralateral sides for the twice-daily dosing protocol. Manifestations of distress and discomfort (such as limping, licking the injection site, and other ambulation-related issues) were subjectively graded as: none, mild, moderate, and severe. Mild discomfort was noted on the last day of i.m. injections, which quickly resolved without any intervention. No ambulatory deficits were observed in these rats. Muscle pathology was conducted as described below. Pharmacokinetic analyses For the pharmacokinetics study, rats were dosed once with SAL or VPM (10 mg/kg). Blood samples were obtained in duplicate from 3-4 rats at each of the 10 time points for the two routes of administration (i.m. and p.o.) following saline or DFP administration. Following anesthesia, rats were euthanized and blood samples were collected through cardiac ventricles at 0, 5 min, 30 min, and at 1, 2, 4, 6, 12, 18, and 24 h following VPM administration. The blood was placed in an EDTA microcuvette and frozen at -80 °C until analyzed. Concurrently with blood collection, the brain was rapidly dissected, and the cortex was removed and snap frozen in isopentane (-15 ° C) and stored at -80 ° C until use. VPM levels were measured in whole blood and cortex using liquid chromatography tandem mass spectrometry (LC-MS/MS). The linear range was 0.05-1200 ng/mL for VPM. Sample preparation 50 µL of rat whole blood was analyzed for VPM levels using a protein precipitation extraction method. Briefly, each aliquot was transferred to a 1.5 mL micro-centrifuge tube, and 500 µL of extraction solvent consisting of 70% methanol and 30% water was added, following the 50 µL of internal standard solution (AT-d 7 and LV-d 6 at a concentration of 500 ng/mL). Sample tubes were vortexed for 1 min and sonicated for 30 min. Samples were then centrifuged for 10 min at 13.2g and transferred to an amber vial. VPM was analyzed using LC-MS/MS with multiple reaction monitoring (MRM) transitions on an AB Sciex 6500+ Qtrap (Sciex, USA) with an LC-20-AD (Shimadzu, USA) UPLC system. Chromatographic separation was performed on a Zorbax Eclipse Plus C18 Column (2.1 X 100mm, I.D. 3.5 µm, Agilent Technologies, USA). The column temperature was ambient; the flow rate was 1.0 mL/min, and the injection volume was 1 µL. The mobile phase A consisted of 0.2% formic acid in water, and mobile phase B in methanol. Isocratic conditions were used at acetonitrile (95%) and 0.2% formic acid (5% ) in reagent water. Data were analyzed with Analyst 1.6 Quantitation Wizard in accordance with the Bioanalytical Laboratories Standard Operating Procedures. Perfusion and tissue collection At 96-h post DFP-SE, a separate group of rats underwent perfusion and fixation procedures to obtain brain and muscle tissue. Following induction of deep anesthesia with ketamine/xylazine (75 mg/ 7.5 mg/kg, i.p.), rats underwent transcardial perfusion with isotonic saline and then perfused with 250 ml of 4% paraformaldehyde in a 0.1 M phosphate buffer (pH 7.4). Brains and quadricep muscles were removed and allowed further fixation for 24 h in buffered 4% paraformaldehyde at 4 ° C. Brains were then cryoprotected in 30% sucrose in 0.1 M phosphate buffer (pH 7.4) for 3 days at 4 ° C. Brains were then embedded in OCT by snap freezing in isopentane (-15° C) and stored at -80°C until use. Post-fixed muscle was transferred to a 70% ethanol solution and processed as described below. Fluoro-Jade staining Coronal brain sections were prepared using a Leica CM3050S cryostat (Leica Biosystems, Deer Park, IL) and adhered to glass slides (Superfrost Plus; Fisher Scientific, Pittsburgh, PA) and stored at -80 ° C until use. Briefly, 20 μm-thick sections were collected between (-1.8 mm to -4.56 mm Bregma, based on rat anatomic atlas 62 , 63 ). The sectioning spans the entire dorsal hippocampal formation and includes the somatosensory cortex, thalamus, amygdala, and piriform cortex. Each slide contained sections representing all experimental groups (age-matched control, DFP-SAL, DFP-VPM). Every 20th section through the rat hippocampus is selected from at least six animals for labeling and staining 27 , 53 , 54 , 64 – 66 . Slides were dried in a desiccant chamber at 55 °C for 30 min prior to staining. Slides were first incubated in a solution of 1% NaOH in 80% ethanol for 5 min, followed by hydration in 70% ethanol and then ddH 2 O for 2 min each. Slides were then incubated in a 0.06% KMnO 4 solution for 10 min, followed by washing in ddH 2 O for 2 min. Slides were then stained in a 0.0004% Fluoro-Jade C (FJC) solution in 0.1% acetic acid for 20 minutes. Stained slides underwent 3× washes in ddH 2 O for 2 min each and then dried in a desiccant chamber at 55 °C for 30 min. Stained slides were then cleared with xylene for 5 min and cover-slipped with DPX mounting agent. FJC-stained sections were viewed under 20X magnification on an Olympus IX-70 microscope under brightfield or fluorescein/FITC filter settings, and 16-bit grayscale digital images were acquired with a digital CCD camera (ORCA-ER; Hamamatsu Corp) using MetaMorph7.8 (Molecular Devices, San Jose, CA). All microscope/camera settings for FJC image acquisition were constant throughout. Grayscale images were analyzed using ImageJ 1.53 (NIH). Specific staining was measured using auto-threshold segmentation (Yen method) of selected regions of interest and represented as cell counts/mm 2 (analyze particles module) 27 , 65 , 66 . Assessment of muscle pathology To evaluate the safety of i.m. VPM administrations, quadricep muscle specimens were obtained at 1 day and 3 months post the last i.m. injection of the VPM treatment regimen. To conserve animal numbers, muscle specimens for acute toxicity assessment were obtained from the same rats that were utilized for dissecting the brain for FJC staining. A separate cohort of rats was utilized for the 3-month time point. These time points were selected to assess whether repeated VPM injections caused any muscle injury at the site of injection, either acutely or chronically. Muscle specimens were obtained as described in the perfusion section above. Muscle samples were embedded in paraffin, and 5-micron-thick sections were obtained. These sections were then stained with Hematoxylin and Eosin (H&E) for histopathological analysis. Automated H&E staining was performed with the Dako CoverStainer (Agilent). A standard four-point inflammation scale was used for scoring 67 , 68 as described in Table 1 . The score for each site was the average of the scores for each of the three tissue blocks. View this table: View inline View popup Download powerpoint Table 1: Inflammation scale for assessment of injection site muscle pathology Stability analysis of VPM formulation VPM formulation (10 mg/mL in sterile saline) in multiple glass bottles was stored at 25°C ± 2°C and 60 ± 5% Relative Humidity. At the respective time points (Day 0, 90, 203), three bottles were randomly chosen, and the stability of the VPM formulation was assessed under ICH QA1 (R2) guidelines [Stability Testing of New Drug Substances and Products] and using a similar USP VPM injection monograph (USP29-NF24). Similar to the USP29-NF24 VPM monograph, a system suitability solution was prepared at 1.9 mg/mL of USP Verapamil Hydrochloride RS and 1.5 mg/mL of USP Verapamil Related Compound B RS in mobile phase (see Table 2 ). The VPM standard solution (2.5 mg/mL) was also prepared in the mobile phase. Sample Preparation (Verapamil HCL Injection Sample): Dilute sample to a working solution of 2.5 mg/L in mobile phase. Dilute working solution to 0.5 mg/L in the mobile phase. Impurity Standard solution: 2.5 mg/mL of USP Verapamil Hydrochloride RS, and 7.5 µg/mL each of USP Verapamil Related Compound A RS, USP Verapamil Related Compound E RS, and USP Verapamil Related Compound F RS in Mobile phase. Filter the samples and standards using a syringe filter into 1 mL autosampler tubes. View this table: View inline View popup Download powerpoint Table 2: HPLC Conditions for VPM concentration determination Experimental rigor and Data analysis Male and Female rats were housed in separate rooms. All the drugs were prepared fresh daily and belonged to the same respective batch to minimize experimental variability due to possible minor differences between drug lots. DFP-SE induction protocol and VPM treatment were carried out on separate days for male and female rats. Rats were randomized, ignoring sex and the estrous cycle stages. Experimental groups were blinded until the data were analyzed. The study excluded animals that did not present with SE onset or failed to achieve a Racine score of 4 or higher. Two independent observers assessed the severity of SE. Data analysis was performed using GraphPad Prism 10 software. Data represented as mean ± SD. Mixed-effect analysis utilized a two-way ANOVA. Multiple t-tests were employed as appropriate. A p-value ≤ 0.05 was considered to indicate statistical significance. For pharmacokinetic analysis, model-independent methods were used to estimate the pharmacokinetic parameters of VPM in rat blood samples. The area under the curve (AUC) was determined utilizing the parameters programmed in Microsoft Excel. AUC from time zero to the last sampling time was calculated using the linear trapezoidal rule. The AUC from the last sampling time was extrapolated to infinity by dividing the last measured plasma concentration by the terminal elimination rate constant. Cmax and Tmax were obtained directly from the concentration-time curves. The AUC data are expressed as mean ± SD and compared using a One-Way Analysis of Variance (ANOVA) followed by a post-hoc Tukey test with a significance set at p<.05. Results Pharmacokinetic studies after i.m. and p.o. treatment with VPM VPM blood levels were measured in control rats (no DFP-SE) at various time intervals after administration by i.m. and p.o. routes. Table 3 presents data on the pharmacokinetics (PK) of VPM treatment in control rats. In both control male and female rats, increased bioavailability (AUC) and higher drug concentrations in plasma (Cmax) were observed when VPM was administered i.m. compared to p.o. The time to peak VPM level (Tmax) was delayed following i.m. dosing under control conditions. View this table: View inline View popup Download powerpoint Table 3. Summary of i.m. to p.o. pharmacokinetics for Verapamil in control rats. Next, we investigated the PK profile of i.m. VPM administered after DFP-SE. As shown in Table 4 , non-compartmental analysis revealed comparable PK values for various PK parameters in male and female rats. However, compared to the control (no-DFP SE) condition, an even higher Cmax was obtained after DFP-SE. Furthermore, the time to Tmax after i.m. VPM administration in DFP-SE was decreased compared to control, no-SE rats, and was similar to the values obtained in non-SE conditions upon p.o. administration. View this table: View inline View popup Download powerpoint Table 4. Summary of i.m. pharmacokinetics for verapamil in Control and DFP-SE rats Figure 2 shows a comparison of plasma concentration (ng/mL) versus time profiles for VPM in male and female rats for the two dosing routes. Mixed-sex analysis indicated that the i.m. route provided significantly higher and sustained blood VPM levels in both male and female rats ( Fig. 2A ). Significantly higher VPM levels were reached within 30 minutes of i.m. injection and remained significantly elevated at all subsequently measured time points compared to p.o. dosing in both the male ( Fig. 2B ) and the female ( Fig. 2C ) rats (n=3-4 rats/sex/ timepoint, multiple t-test for each time point, *p<0.05). No significant differences in VPM levels were noted within the sexes for i.m. or the p.o. routes ( Fig. 2D, E ). Download figure Open in new tab Figure 2: Blood levels of VPM following p.o. and i.m. administration Comparison of the PK profile of VPM (10 mg/kg) following p.o. and i.m. administration in control rats. Levels of VPM were analyzed using LC-MS/ MS in blood specimens collected from control, no-SE male and female rats at time points ranging from 0 min to 1440 min (24 h). A. significantly higher and sustained levels of VPM were noted after i.m. compared to p.o. dosing. B, C. significantly higher VPM levels were achieved in both male and female rats. D, E. No sex differences were noted for VPM levels following i.m. or p.o. administration. (Each data point represents the mean ± SD of concentration values (ng/ml) from 3 rats. *p<.05, Tukey post-hoc analysis was used to compare concentrations). Brain levels of VPM Upon i.m. administration, VPM was detected in the brains of male and female rats under control and DFP-SE conditions ( Table 3 ). Mixed-sex analysis revealed differences in VPM brain levels under the control and DFP-SE conditions. Following DFP-SE, male rats attained significantly higher VPM concentrations as early as 30 minutes, and this level was sustained for up to 1 hour. Subsequently, male rats continued to exhibit higher VPM concentrations for up to 4 hours. Beyond this time point, the VPM concentration in male rats began to decline while remaining stable in female rats. Significantly higher VPM levels were observed in female rats at the 6-hour time point. At the 18-hour time point, VPM was undetectable in male rats but measurable in female rats ( Figure 3 ; n=3-4 rats/timepoint/group, *p<.05, two-way ANOVA, Bonferroni’s multiple comparison test). Download figure Open in new tab Figure 3: Brain levels of VPM following i.m. administration VPM (10 mg/kg, i.m.) was administered once to control and DFP-SE male and female rats, and the brain was dissected at various time points ranging from 0 to 24 hours. The concentration of cortical VPM was then estimated using LC-MS/ MS. A. Mixed-sex analysis showed higher levels of brain VPM in both control and DFP-SE conditions. B-C. Higher levels of VPM were observed under the DFP-SE condition compared to the no-SE condition in both male (B) and female (C) rats. D-E. Male rats showed higher VPM levels, which peaked earlier than those in female rats under both control (D) and DFP-SE conditions (E). Significant differences at each time point are indicated (n=3-4 rats/timepoint/group, *p<.05, two-way ANOVA, Bonferroni’s multiple comparison test). Muscle safety studies after i.m. treatment with VPM As illustrated in Figure 4A , one day after the last i.m. injection, the pathological scores for quadricep muscle inflammation were not significantly different between DFP-SE rats treated with SAL and those treated with VPM. As illustrated in Figure 4B , three months post-injections, the muscle pathology score for the DFP-SE rats treated with SAL control did not differ significantly from the VPM tissue. No gender-specific differences were noted in inflammation scores between SAL and VPM treatment at both the acute and chronic time points (n=4 rats/group, two-way ANOVA, *p<.05). Download figure Open in new tab Figure 4: Acute and chronic muscle pathology following i.m. VPM therapy The data represent the muscle damage score for the i.m. VPM therapy protocol (twice-daily for 3 days) when evaluated 1 day and 3 months after the end of the injections regimen in the following groups: DFP + SAL (blue) and DFP + VPM (green) in male and female rats. No significant sex or treatment differences in the inflammation scores were noted (A) acutely or (B) chronically (data represented as mean inflammation score ± SD; n=4 rats/group, two-way ANOVA, *p<.05). Assessment of the neuroprotective efficacy of i.m. VPM Injured brain cells undergoing neurodegeneration in DFP-SE rats exhibited bright green fluorescence in FJC-stained sections ( Figure 5A ). Sex-specific spatial differences in DFP-induced neuronal injury were observed ( Figure 5B-E ). In the male rats, significantly more FJC+ cells were found in the CA1, DG, and PC regions compared to female rats ( Figure 5E ; Two-way ANOVA, post-hoc Tukey test, *p<0.05, n=6-8 rats per group). Download figure Open in new tab Download figure Open in new tab Figure 5: Neuroprotective effects of VPM. A. Extensive FJC+ staining indicative of dying neurons is seen in multiple brain regions from DFP-SE rats, which is markedly reduced in VPM-treated DFP rats (scale 200 μM). B. A significant decrease in FJC+ cell counts was seen across the brain regions measured in VPM-treated DFP rats compared to SAL-treated DFP rats. C-D. Regional differences in VPM’s neuroprotective efficacy were noted between male and female rats following DFP-SE. E. Male rats were more sensitive to injury following DFP-SE across the CA1, DG, and PC regions. F. No sex differences were noted for the neuroprotective actions of VPM across all the brain regions quantified. (n= 8 per group, *p<.05, two-way ANOVA, Sidak multiple comparison test). Mixed-sex analysis also revealed that significantly more FJC+ cells were present in the CA1, CA3, and DG regions of the hippocampus, along with the PC, in DFP rats treated with SAL compared to those treated with VPM ( Figure 5B-D ; Two-way ANOVA, post-hoc Tukey test, *p<0.05, n= 6-8 rats per group). No sex differences in the neuroprotective effects of VPM were observed ( Figure 5F ). No FJC+ cells were found in brain sections from control rats. ( Figure 5A ) Stability of VPM formulation Stability studies based on the USP monograph for VPM injection demonstrated that our VPM injectable formulation (10 mg/mL in sterile saline) remained within 90-110% of the labeled claim at all tested time points (0, 90, and 203 days), indicating minimal degradation of VPM in our formulation even after 6 months post-constitution ( Table 6 , n= 3 bottles assayed at each time point). Discussion The results from our study demonstrate that i.m. VPM can achieve higher blood and brain levels and faster kinetics compared to p.o. administration in both male and female rats. Our VPM formulation had a long shelf life and underwent slight degradation even at 200 days after constitution. Adjunctive treatment regimen consisting of i.m. VPM (10 mg/kg) administered along with the OP-SE standard-of-care therapy, starting 1 hour after DFP-SE onset and continuing for three days, was found to be safe and did not cause significant muscle pathology either acutely or chronically. Finally, i.m. VPM therapy significantly reduced neuronal injury in multiple brain regions following DFP-SE in both male and female rats. Since this neuroprotective adjunct treatment will initially require on-field administration, an i.m. route of administration is desirable as it provides speed for rapid intervention during a mass exposure event or battlefield-related forward care situations 69 . VPM is commonly administered p.o. We therefore first compared the PK profile of VPM in whole blood when administered p.o. and i.m. to control, no-SE rats ( Table 3 ). Non-compartmental analyses revealed that VPM, when administered via the i.m. route, produced significantly higher drug levels and exhibited greater bioavailability than when administered p.o. in both male and female rats. In contrast, time to peak VPM level (Tmax) was delayed following i.m. dosing under control conditions. However, following DFP-SE, a higher Cmax was obtained compared to the Cmax following i.m. administration in control, no-SE conditions. Interestingly, the duration to Tmax obtained after i.m. administration in DFP-SE was shorter than the Tmax obtained after i.m. administration in no-SE rats and was comparable to the values obtained in no-SE conditions upon p.o. administration. Overall, these results indicated that the i.m. route provided significantly higher VPM levels, achieved stable therapeutic blood levels, and demonstrated a favorable PK profile, suggesting it could be an effective route to deliver VPM in OP-SE ( Table 4 ; Figure 2 ). The blood-brain barrier (BBB) is a diffusion barrier that regulates substances entering the brain. In addition, P-glycoprotein (P-gp) is a transporter protein that protects the brain from xenobiotics. These mechanisms play a role in limiting the entry and levels of drugs in the brain. In a rat model of ischemia-induced CNS injury, the level of VPM penetrance in the brain was reported to be dependent on BBB breakdown and also regulated by P-gp 70 . Thus, we studied VPM brain levels following DFP-induced SE. It has been previously reported that following DFP-SE, the BBB becomes leaky and increased permeability is noticed as early as 6 h following SE and persists for up to 7 days 71 , 72 . Furthermore, VPM is reported to bind and inhibit P-gp directly. In our studies, under both control and DFP-SE conditions, VPM was measurable in the brain upon i.m. administration ( Table 5 ; Figure 3 ). Higher VPM brain levels were observed in both male and female rats following DFP-SE compared to the control condition. Overall, there was a trend toward greater and quicker VPM brain accumulation in males compared to females. However, a statistical significance was not noted. Interestingly, VPM levels were sustained longer in female rats than male rats under both SE and no-SE conditions. These differences in VPM brain kinetics could partially explain the sex differences seen in the neuroprotective efficacy of VPM. It may also warrant optimization of dose and frequency of administration between the two sexes. Future studies in our lab are planned to investigate these sex differences. View this table: View inline View popup Download powerpoint Table 5. Brain levels of VPM (ng/g) in Control and DFP-SE rats Significant neuronal damage has been reported following OP-SE 5 , 6 , 30 , 33 , 64 , 73 . Brain regions that show the most severe damage include the hippocampal regions (CA1, CA3, DG), as well as cortical regions such as the parietal cortex and the piriform cortex, along with nuclear regions in the amygdala and the thalamus. Injury to these critical structures also damages the brain circuitry and compromises the functionality housed in these brain regions. In agreement with these findings, widespread neuronal damage to structures in the limbic system and cortical areas was noted following DFP-SE ( Figure 5 ). Sex-specific and regional differences in vulnerability to neuronal damage following DFP-SE have been previously reported. For example, one study reported that FJB+ cells were significantly higher in the dentate gyrus but not in the cortical region in adult female rats compared to adult male rats 74 . Interestingly, male juvenile rats exhibited greater cell injury across all brain regions following DFP-SE compared to female juvenile rats 75 . In our study, FJC+ cells were significantly higher in male rats in the CA1, DG, and PC regions. In contrast, female rats exhibited greater neuronal injury in the CA3 region following DFP-SE. VPM intervention had a significant neuroprotective action in the CA1 and PC regions for both sexes. However, VPM was protective in the DG region in only male rats. In the CA3 regions, VPM provided some neuroprotection, as evidenced by a lower number of FJC+ cells in both sexes following DFP-SE; however, significance could not be achieved. It is worth noting that male rats exhibited significantly higher VPM brain levels following DFP-SE, but the drug persisted longer in females. Whether these PK-related differences contribute to the sex-related differences in neuronal injury outcomes following DFP-SE will be addressed through investigations of VPM dose and duration in a future study. We also investigated the pathological effects of repeated i.m. administrations of VPM by conducting muscle histology at the injection site 67 , 68 . Our studies demonstrated that the i.m. treatment regimen with VPM did not produce any overt inflammation or other pathology at the injection sites at both acute and chronic time points after the end of the injection period. No sex differences were noted in muscle pathology following i.m. VPM administrations ( Figure 4 ). While a few clusters of scattered infiltrates indicative of mild inflammation were noted at acute time points, the pathology scores between DFP rats treated with SAL or VPM were not different. At 3 months after the last VPM injection, the muscle pathology revealed some minimal infiltrates in some rats, while the majority had pathology scores of 0, indicating the absence of inflammation. These studies establish the safety of i.m. VPM and demonstrates that this route of administration may be an effective method to deliver VPM post-SE. Our VPM formulation had an extended shelf life and showed little degradation even after 200 days following constitution ( Table 6 ). The stability and ease of VPM constitution for i.m. administration will afford benefits for stockpiling VPM formulations in the event it is further optimized as an OP countermeasure. View this table: View inline View popup Download powerpoint Table 6. Stability of VPM formulation Our study had some limitations. First, we did not assess electrographic SE and instead assessed SE severity on the Racine scale. We have previously shown that rats displaying a Racine score of 4 and above reliably exhibit electrographic EEG patterns akin to SE 5 , 50 . Additionally, other studies have shown that rats from mixed-sex cohorts housed in the same room yielded reproducible SE severity in both sexes and both EEG telemetry (surgery) and non-telemetry (non-surgery) groups 76 . Second, we did not quantify neuroinflammatory changes in this study. The goal of this study was to validate the safety and neuroprotective efficacy of a VPM formulation that could be administered i.m. after a reasonable delay following DFP-SE. Studies are underway in our laboratory to investigate the anti-inflammatory effects of VPM and its underlying molecular mechanism. In conclusion, the data from this study demonstrate that adjunct treatment with VPM could provide effective countermeasures for extending neuroprotective efficacy and improving long-term neurological outcomes after OP intoxication. Acknowledgement This work was supported by the Countermeasures Against Chemical Threats (CounterACT) Program, the National Institutes of Health (NIH) Office of the Director (OD), and the National Institute of Neurological Disorders and Stroke (NINDS) [Grant Number-UG3 NS133630]. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the federal government. Services in support of the research project were generated by the VCU Massey Comprehensive Cancer Center Tissue and Data Acquisition and Analysis Shared Resource, supported, in part, with funding from NIH-NCI Cancer Center Support Grant P30 CA016059. Funder Information Declared Countermeasures Against Chemical Threats (CounterACT) Program, the National Institutes of Health (NIH) Office of the Director (OD), and the National Institute of Neurological Disorders and Stroke (NINDS) , UG3 NS133630 NIH-NCI Cancer Center Support Grant , P30 CA016059 References 1. ↵ Helmstaedter C . Cognitive outcome of status epilepticus in adults . Epilepsia . 2007 ; 48 Suppl 8 : 85 – 90 . Epub 2008/03/11. PubMed PMID: 18330010 . OpenUrl CrossRef PubMed 2. ↵ Betjemann JP , Lowenstein DH . Status epilepticus in adults . Lancet Neurol . 2015 ; 14 ( 6 ): 615 – 24 . Epub 20150420. doi: 10.1016/S1474-4422(15)00042-3 . PubMed PMID: 25908090 . OpenUrl CrossRef PubMed 3. ↵ Kamppi L , Kamppi A , Strzelczyk A . Mortality and morbidity of status epilepticus over the long term . Epilepsy Behav . 2024 ; 158 : 109918 . Epub 20240713. doi: 10.1016/j.yebeh.2024.109918 . PubMed PMID: 39003945 . OpenUrl CrossRef PubMed 4. ↵ Trinka E , Hofler J , Zerbs A . Causes of status epilepticus . Epilepsia . 2012 ; 53 Suppl 4 : 127 – 38 . Epub 2012/09/14. doi: 10.1111/j.1528-1167.2012.03622.x . PubMed PMID: 22946730 . OpenUrl CrossRef PubMed 5. ↵ Deshpande LS , Carter DS , Blair RE , DeLorenzo RJ . Development of a prolonged calcium plateau in hippocampal neurons in rats surviving status epilepticus induced by the organophosphate diisopropylfluorophosphate . Toxicol Sci . 2010 ; 116 ( 2 ): 623 – 31 . Epub 2010/05/26. doi: kfq157 [pii] 10.1093/toxsci/kfq157 . PubMed PMID: 20498005 ; PMCID: PMC2905411 . OpenUrl CrossRef PubMed Web of Science 6. ↵ Deshpande LS , Carter DS , Phillips KF , Blair RE , DeLorenzo RJ . Development of status epilepticus, sustained calcium elevations and neuronal injury in a rat survival model of lethal paraoxon intoxication . Neurotoxicology . 2014 ; 44C : 17 – 26 . Epub 2014/05/03. doi: 10.1016/j.neuro.2014.04.006 . PubMed PMID: 24785379 . OpenUrl CrossRef PubMed 7. ↵ Costa LG . Organophosphorus Compounds at 80: Some Old and New Issues . Toxicol Sci . 2018 ; 162 ( 1 ): 24 – 35 . Epub 2017/12/12. doi: 10.1093/toxsci/kfx266 . PubMed PMID: 29228398 ; PMCID: PMC6693380 . OpenUrl CrossRef PubMed 8. ↵ Bajgar J . Organophosphates/nerve agent poisoning: mechanism of action, diagnosis, prophylaxis, and treatment . Adv Clin Chem . 2004 ; 38 : 151 – 216 . Epub 2004/11/04. PubMed PMID: 15521192 . OpenUrl CrossRef PubMed Web of Science 9. ↵ Sugiyama A , Matsuoka T , Sakamune K , Akita T , Makita R , Kimura S , Kuroiwa Y , Nagao M , Tanaka J . The Tokyo subway sarin attack has long-term effects on survivors: A 10-year study started 5 years after the terrorist incident . PLoS One . 2020 ; 15 ( 6 ): e0234967 . Epub 20200623. doi: 10.1371/journal.pone.0234967 . PubMed PMID: 32574198 ; PMCID: PMC7310687 . OpenUrl CrossRef PubMed 10. Anger WK , Farahat FM , Lein PJ , Lasarev MR , Olson JR , Farahat TM , Rohlman DS . Magnitude of behavioral deficits varies with job-related chlorpyrifos exposure levels among Egyptian pesticide workers . Neurotoxicology . 2020 ; 77 : 216 – 30 . Epub 20200130. doi: 10.1016/j.neuro.2020.01.012 . PubMed PMID: 32006538 ; PMCID: PMC7085493 . OpenUrl CrossRef PubMed 11. Jaga K , Dharmani C . Sources of exposure to and public health implications of organophosphate pesticides . Rev Panam Salud Publica . 2003 ; 14 ( 3 ): 171 – 85 . doi: 10.1590/s1020-49892003000800004 . PubMed PMID: 14653904 . OpenUrl CrossRef PubMed 12. ↵ White RF , Steele L , O’Callaghan JP , Sullivan K , Binns JH , Golomb BA , Bloom FE , Bunker JA , Crawford F , Graves JC , Hardie A , Klimas N , Knox M , Meggs WJ , Melling J , Philbert MA , Grashow R . Recent research on Gulf War illness and other health problems in veterans of the 1991 Gulf War: Effects of toxicant exposures during deployment . Cortex; a journal devoted to the study of the nervous system and behavior . 2016 ; 74 : 449 – 75 . Epub 2015/10/24. doi: 10.1016/j.cortex.2015.08.02210.1016/j.cortex.2015.08.022 . Epub 2015 Sep 25. PubMed PMID: 26493934 ; PMCID: 4724528 . OpenUrl CrossRef PubMed 13. ↵ Ajdacic-Gross V , Weiss MG , Ring M , Hepp U , Bopp M , Gutzwiller F , Rossler W . Methods of suicide: international suicide patterns derived from the WHO mortality database . Bulletin of the World Health Organization . 2008 ; 86 ( 9 ): 726 – 32 . Epub 2008/09/18. PubMed PMID: 18797649 ; PMCID: 2649482 . OpenUrl CrossRef PubMed Web of Science 14. ↵ Mohanraj R , Kumar S , Manikandan S , Kannaiyan V , Vijayakumar L . A public health initiative for reducing access to pesticides as a means to committing suicide: findings from a qualitative study . International review of psychiatry . 2014 ; 26 ( 4 ): 445 – 52 . Epub 2014/08/20. doi: 10.3109/09540261.2014.924094 . PubMed PMID: 25137110 . OpenUrl CrossRef PubMed 15. ↵ Tsai YH , Lein PJ . Mechanisms of organophosphate neurotoxicity . Curr Opin Toxicol . 2021 ; 26 : 49 – 60 . Epub 2021/07/27. doi: 10.1016/j.cotox.2021.04.002 . PubMed PMID: 34308007 ; PMCID: PMC8302047 . OpenUrl CrossRef PubMed 16. ↵ Aroniadou-Anderjaska V , Figueiredo TH , de Araujo Furtado M , Pidoplichko VI , Braga MFM . Mechanisms of Organophosphate Toxicity and the Role of Acetylcholinesterase Inhibition . Toxics . 2023 ; 11 ( 10 ). Epub 20231018. doi: 10.3390/toxics11100866 . PubMed PMID: 37888716 ; PMCID: PMC10611379 . OpenUrl CrossRef PubMed 17. ↵ Tattersall J . Seizure activity post organophosphate exposure . Front Biosci (Landmark Ed ). 2009 ; 14 ( 10 ): 3688 – 711 . Epub 20090101. doi: 10.2741/3481 . PubMed PMID: 19273303 . OpenUrl CrossRef PubMed 18. ↵ Chuang CS , Yang KW , Yen CM , Lin CL , Kao CH . Risk of Seizures in Patients with Organophosphate Poisoning: A Nationwide Population-Based Study . Int J Environ Res Public Health . 2019 ; 16 ( 17 ). Epub 20190829. doi: 10.3390/ijerph16173147 . PubMed PMID: 31470499 ; PMCID: PMC6747140 . OpenUrl CrossRef PubMed 19. ↵ Eddleston M , Buckley NA , Eyer P , Dawson AH . Management of acute organophosphorus pesticide poisoning . Lancet . 2008 ; 371 (9612): 597 -607. Epub 2007/08/21. doi: S0140-6736(07)61202-1 [pii] 10.1016/S0140-6736(07)61202-1 . PubMed PMID: 17706760 . OpenUrl CrossRef PubMed Web of Science 20. ↵ Nerve Agents - Emergency Department/Hospital Management [Internet] 2013 . Available from: http://chemm.nlm.nih.gov/na_hospital_mmg.htm#top . 21. ↵ Jett DA , Laney JW . Civilian research on chemical medical countermeasures . Toxicol Mech Methods . 2021 ; 31 ( 4 ): 242 – 3 . Epub 20191002. doi: 10.1080/15376516.2019.1669250 . PubMed PMID: 31575312 . OpenUrl CrossRef PubMed 22. ↵ Jett DA , Spriggs SM . Translational research on chemical nerve agents . Neurobiol Dis . 2020 ; 133 : 104335 . Epub 20181120. doi: 10.1016/j.nbd.2018.11.020 . PubMed PMID: 30468862 . OpenUrl CrossRef PubMed 23. ↵ Flannery BM , Bruun DA , Rowland DJ , Banks CN , Austin AT , Kukis DL , Li Y , Ford BD , Tancredi DJ , Silverman JL , Cherry SR , Lein PJ . Persistent neuroinflammation and cognitive impairment in a rat model of acute diisopropylfluorophosphate intoxication . J Neuroinflammation . 2016 ; 13 ( 1 ): 267 . Epub 20161012. doi: 10.1186/s12974-016-0744-y . PubMed PMID: 27733171 ; PMCID: PMC5062885 . OpenUrl CrossRef PubMed 24. Naughton SX , Terry AV , Jr . . Neurotoxicity in acute and repeated organophosphate exposure . Toxicology . 2018 ; 408 : 101 – 12 . Epub 2018/08/26. doi: 10.1016/j.tox.2018.08.011 . PubMed PMID: 30144465 ; PMCID: PMC6839762 . OpenUrl CrossRef PubMed 25. ↵ Voorhees JR , Rohlman DS , Lein PJ , Pieper AA . Neurotoxicity in Preclinical Models of Occupational Exposure to Organophosphorus Compounds . Front Neurosci . 2016 ; 10 : 590 . Epub 2017/02/06. doi: 10.3389/fnins.2016.00590 . PubMed PMID: 28149268 ; PMCID: PMC5241311 . OpenUrl CrossRef PubMed 26. ↵ Deshpande LS , DeLorenzo RJ . Novel therapeutics for treating organophosphate-induced status epilepticus co-morbidities, based on changes in calcium homeostasis . Neurobiol Dis . 2020 ; 133 : 104418 . Epub 20190312. doi: 10.1016/j.nbd.2019.03.006 . PubMed PMID: 30872159 ; PMCID: PMC6742584 . OpenUrl CrossRef PubMed 27. ↵ Deshpande LS , Blair RE , Huang BA , Phillips KF , DeLorenzo RJ . Pharmacological blockade of the calcium plateau provides neuroprotection following organophosphate paraoxon induced status epilepticus in rats . Neurotoxicol Teratol . 2016 ; 56 : 81 – 6 . Epub 2016/05/26. doi: 10.1016/j.ntt.2016.05.002 . PubMed PMID: 27224207 ; PMCID: 4935577 . OpenUrl CrossRef PubMed 28. ↵ Lewine JD , Weber W , Gigliotti A , McDonald JD , Doyle-Eisele M , Bangera N , Paulson K , Magcalas C , Hamilton DA , Garcia E , Raulli R , Laney J . Addition of ketamine to standard-of-care countermeasures for acute organophosphate poisoning improves neurobiological outcomes . Neurotoxicology . 2018 ; 69 : 37 – 46 . Epub 20180830. doi: 10.1016/j.neuro.2018.08.011 . PubMed PMID: 30172622 . OpenUrl CrossRef PubMed 29. ↵ Lazar S , Neufeld-Cohen A , Egoz I , Baranes S , Gez R , Glick P , Cohen M , Gutman H , Chapman S , Gore A . Efficacy of a combined anti-seizure treatment against cholinergic established status epilepticus following a sarin nerve agent insult in rats . Toxicol Appl Pharmacol . 2024 ; 484 : 116870 . Epub 20240221. doi: 10.1016/j.taap.2024.116870 . PubMed PMID: 38395364 . OpenUrl CrossRef PubMed 30. ↵ Gage M , Putra M , Wachter L , Dishman K , Gard M , Gomez-Estrada C , Thippeswamy T . Saracatinib, a Src Tyrosine Kinase Inhibitor, as a Disease Modifier in the Rat DFP Model: Sex Differences, Neurobehavior, Gliosis, Neurodegeneration, and Nitro-Oxidative Stress . Antioxidants (Basel ). 2021 ; 11 ( 1 ). Epub 20211228. doi: 10.3390/antiox11010061 . PubMed PMID: 35052568 ; PMCID: PMC8773289 . OpenUrl CrossRef PubMed 31. Pearson-Smith JN , Patel M . Antioxidant drug therapy as a neuroprotective countermeasure of nerve agent toxicity . Neurobiol Dis . 2020 ; 133 : 104457 . Epub 20190425. doi: 10.1016/j.nbd.2019.04.013 . PubMed PMID: 31028872 ; PMCID: PMC7721294 . OpenUrl CrossRef PubMed 32. Rojas A , Ganesh T , Lelutiu N , Gueorguieva P , Dingledine R . Inhibition of the prostaglandin EP2 receptor is neuroprotective and accelerates functional recovery in a rat model of organophosphorus induced status epilepticus . Neuropharmacology . 2015 ; 93 : 15 – 27 . Epub 20150203. doi: 10.1016/j.neuropharm.2015.01.017 . PubMed PMID: 25656476 ; PMCID: PMC4387070 . OpenUrl CrossRef PubMed 33. ↵ S SV , Massey N , Meyer C , Wang C , Thippeswamy T . Diet-incorporated saracatinib, a Src tyrosine kinase inhibitor, counteracts diisopropylfluorophosphate (DFP)-induced chronic neurotoxicity in the rat model . Biomed Pharmacother . 2025 ; 189 : 118234 . Epub 20250612. doi: 10.1016/j.biopha.2025.118234 . PubMed PMID: 40513390 . OpenUrl CrossRef PubMed 34. Ramakrishnan S , Singh T , Chen A , Wu X , Reddy DS . Neurosteroid mitigation of developmental neurological dysfunction, long-term epileptic biomarkers, chronic neuroinflammation, and neurodegeneration in a pediatric rat model of organophosphate exposure . J Pharmacol Exp Ther . 2025 ; 392 ( 5 ): 103555 . Epub 20250319. doi: 10.1016/j.jpet.2025.103555 . PubMed PMID: 40273512 . OpenUrl CrossRef PubMed 35. ↵ Shah EJ , Grunwald WC , Jr. , Garrett TL , Brown TL , Cool DR . Sarin-Induced Neuroinflammation in Mouse Brain Is Attenuated by the Caspase Inhibitor Q-VD-OPh . J Pharmacol Exp Ther . 2024 ; 388 ( 2 ): 367 – 75 . Epub 20240117. doi: 10.1124/jpet.123.001820 . PubMed PMID: 37918856 ; PMCID: PMC10801781 . OpenUrl Abstract / FREE Full Text 36. ↵ Kangilaski J , McBride G . Calcium blockers for heart disease: two approved, more to come . JAMA . 1982 ; 247 ( 14 ): 1911 – 3 , 7. doi: 10.1001/jama.247.14.1911 . PubMed PMID: 6121064 . OpenUrl CrossRef PubMed 37. ↵ Weaver-Agostoni J. Cluster headache . Am Fam Physician . 2013 ; 88 ( 2 ): 122 - 8 . PubMed PMID: 23939643 . OpenUrl PubMed 38. ↵ Amsterdam EA , Wenger NK , Brindis RG , Casey DE , Jr. , Ganiats TG , Holmes DR , Jr. , Jaffe AS , Jneid H , Kelly RF , Kontos MC , Levine GN , Liebson PR , Mukherjee D , Peterson ED , Sabatine MS , Smalling RW , Zieman SJ . 2014 AHA/ACC Guideline for the Management of Patients with Non-ST-Elevation Acute Coronary Syndromes: a report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines . J Am Coll Cardiol . 2014 ; 64 (24):e139-e228. Epub 20140923. doi: 10.1016/j.jacc.2014.09.017 . PubMed PMID: 25260718 . OpenUrl CrossRef PubMed 39. ↵ Jun P , Ko NU , English JD , Dowd CF , Halbach VV , Higashida RT , Lawton MT , Hetts SW . Endovascular treatment of medically refractory cerebral vasospasm following aneurysmal subarachnoid hemorrhage . AJNR Am J Neuroradiol . 2010 ; 31 ( 10 ): 1911 – 6 . Epub 20100708. doi: 10.3174/ajnr.A2183 . PubMed PMID: 20616179 ; PMCID: PMC4067264 . OpenUrl Abstract / FREE Full Text 40. ↵ Whelton PK , Carey RM , Aronow WS , Casey DE , Jr. , Collins KJ , Dennison Himmelfarb C , DePalma SM , Gidding S , Jamerson KA , Jones DW , MacLaughlin EJ , Muntner P , Ovbiagele B , Smith SC , Jr. , Spencer CC , Stafford RS , Taler SJ , Thomas RJ , Williams KA , Sr. , Williamson JD , Wright JT , Jr . . 2017 ACC/AHA/AAPA/ABC/ACPM/AGS/APhA/ASH/ASPC/NMA/PCNA Guideline for the Prevention, Detection, Evaluation, and Management of High Blood Pressure in Adults: A Report of the American College of Cardiology/American Heart Association Task Force on Clinical Practice Guidelines . Hypertension . 2018 ; 71 ( 6 ): e13 - e115 . Epub 20171113. doi: 10.1161/HYP.0000000000000065 . PubMed PMID: 29133356 . OpenUrl CrossRef PubMed 41. ↵ Fahie S , Cassagnol M. Verapamil. StatPearls . Treasure Island (FL) 2022 . 42. ↵ Maniskas ME , Roberts JM , Aron I , Fraser JF , Bix GJ. Stroke neuroprotection revisited: Intra-arterial verapamil is profoundly neuroprotective in experimental acute ischemic stroke . J Cereb Blood Flow Metab . 2016 ; 36 ( 4 ): 721 – 30 . Epub 20151002. doi: 10.1177/0271678X15608395 . PubMed PMID: 26661189 ; PMCID: PMC4821022 . OpenUrl CrossRef PubMed 43. ↵ Jackson DA , Michael T , Vieira de Abreu A , Agrawal R , Bortolato M , Fisher SJ . Prevention of Severe Hypoglycemia-Induced Brain Damage and Cognitive Impairment With Verapamil . Diabetes . 2018 ; 67 ( 10 ): 2107 – 12 . Epub 20180503. doi: 10.2337/db18-0008 . PubMed PMID: 29724724 ; PMCID: PMC6152340 . OpenUrl Abstract / FREE Full Text 44. ↵ Jangholi E , Sharifi ZN , Hoseinian M , Zarrindast MR , Rahimi HR , Mowla A , Aryan H , Javidi MA , Parsa Y , Ghaffarpasand F , Yadollah-Damavandi S , Arani HZ , Shahi F , Movassaghi S . Verapamil Inhibits Mitochondria-Induced Reactive Oxygen Species and Dependent Apoptosis Pathways in Cerebral Transient Global Ischemia/Reperfusion . Oxid Med Cell Longev . 2020 ; 2020 : 5872645 . Epub 20201017. doi: 10.1155/2020/5872645 . PubMed PMID: 33133347 ; PMCID: PMC7591985 . OpenUrl CrossRef PubMed 45. ↵ Kim HS , Park CH , Cha SH , Lee JH , Lee S , Kim Y , Rah JC , Jeong SJ , Suh YH . Carboxyl-terminal fragment of Alzheimer’s APP destabilizes calcium homeostasis and renders neuronal cells vulnerable to excitotoxicity . FASEB J . 2000 ; 14 ( 11 ): 1508 – 17 . doi: 10.1096/fj.14.11.1508 . PubMed PMID: 10928985 . OpenUrl CrossRef PubMed Web of Science 46. ↵ Liu Y , Lo YC , Qian L , Crews FT , Wilson B , Chen HL , Wu HM , Chen SH , Wei K , Lu RB , Ali S , Hong JS . Verapamil protects dopaminergic neuron damage through a novel anti-inflammatory mechanism by inhibition of microglial activation . Neuropharmacology . 2011 ; 60 ( 2-3 ): 373 – 80 . Epub 20101013. doi: 10.1016/j.neuropharm.2010.10.002 . PubMed PMID: 20950631 ; PMCID: PMC3014428 . OpenUrl CrossRef PubMed 47. ↵ Zhang X , Chen S , Lu K , Wang F , Deng J , Xu Z , Wang X , Zhou Q , Le W , Zhao Y . Verapamil Ameliorates Motor Neuron Degeneration and Improves Lifespan in the SOD1(G93A) Mouse Model of ALS by Enhancing Autophagic Flux . Aging Dis . 2019 ; 10 ( 6 ): 1159 – 73 . Epub 20191201. doi: 10.14336/AD.2019.0228 . PubMed PMID: 31788329 ; PMCID: PMC6844595 . OpenUrl CrossRef PubMed 48. ↵ Summers MA , Moore JL , McAuley JW . Use of verapamil as a potential P-glycoprotein inhibitor in a patient with refractory epilepsy . The Annals of pharmacotherapy . 2004 ; 38 ( 10 ): 1631 – 4 . Epub 20040824. doi: 10.1345/aph.1E068 . PubMed PMID: 15328394 . OpenUrl CrossRef PubMed 49. ↵ Ledwitch KV , Gibbs ME , Barnes RW , Roberts AG . Cooperativity between verapamil and ATP bound to the efflux transporter P-glycoprotein . Biochem Pharmacol . 2016 ; 118 : 96 – 108 . Epub 20160813. doi: 10.1016/j.bcp.2016.08.013 . PubMed PMID: 27531061 ; PMCID: PMC5341695 . OpenUrl CrossRef PubMed 50. ↵ Blair RE , Hawkins E , Pinchbeck LR , DeLorenzo RJ , Deshpande LS . Chronic Epilepsy and Mossy Fiber Sprouting Following Organophosphate-Induced Status Epilepticus in Rats . J Pharmacol Exp Ther . 2024 ; 388 ( 2 ): 325 – 32 . Epub 20240117. doi: 10.1124/jpet.123.001739 . PubMed PMID: 37643794 . OpenUrl Abstract / FREE Full Text 51. ↵ Racine RJ . Modification of seizure activity by electrical stimulation. I. After-discharge threshold . ElectroencephalogrClinNeurophysiol . 1972 ; 32 ( 3 ): 269 – 79 . OpenUrl CrossRef PubMed Web of Science 52. ↵ Racine RJ . Modification of seizure activity by electrical stimulation. II. Motor seizure . Electroencephalogr Clin Neurophysiol . 1972 ; 32 ( 3 ): 281 – 94 . Epub 1972/03/01. PubMed PMID: 4110397 . OpenUrl CrossRef PubMed Web of Science 53. ↵ Reddy DS , Zaayman M , Kuruba R , Wu X . Comparative profile of refractory status epilepticus models following exposure of cholinergic agents pilocarpine, DFP, and soman . Neuropharmacology . 2021 ; 191 : 108571 . Epub 20210418. doi: 10.1016/j.neuropharm.2021.108571 . PubMed PMID: 33878303 ; PMCID: PMC8217316 . OpenUrl CrossRef PubMed 54. ↵ Guignet M , Dhakal K , Flannery BM , Hobson BA , Zolkowska D , Dhir A , Bruun DA , Li S , Wahab A , Harvey DJ , Silverman JL , Rogawski MA , Lein PJ . Persistent behavior deficits, neuroinflammation, and oxidative stress in a rat model of acute organophosphate intoxication . Neurobiol Dis . 2020 ; 133 : 104431 . Epub 20190321. doi: 10.1016/j.nbd.2019.03.019 . PubMed PMID: 30905768 ; PMCID: PMC6754818 . OpenUrl CrossRef PubMed 55. ↵ Nair AB , Jacob S . A simple practice guide for dose conversion between animals and human . J Basic Clin Pharm . 2016 ; 7 ( 2 ): 27 – 31 . doi: 10.4103/0976-0105.177703 . PubMed PMID: 27057123 ; PMCID: PMC4804402 . OpenUrl CrossRef PubMed 56. ↵ Reagan-Shaw S , Nihal M , Ahmad N . Dose translation from animal to human studies revisited . FASEB J . 2008 ; 22 ( 3 ): 659 – 61 . Epub 2007/10/19. doi: fj.07-9574LSF [pii] 10.1096/fj.07-9574LSF . PubMed PMID: 17942826 . OpenUrl CrossRef PubMed Web of Science 57. ↵ Wurpel JN , Iyer SN . Calcium channel blockers verapamil and nimodipine inhibit kindling in adult and immature rats . Epilepsia . 1994 ; 35 ( 2 ): 443 – 9 . doi: 10.1111/j.1528-1157.1994.tb02458.x . PubMed PMID: 8156971 . OpenUrl CrossRef PubMed Web of Science 58. George B , Kulkarni SK , Mathur R . Motor and electrographic response of refractory experimental status epilepticus in rats and effect of calcium channel blockers . Indian J Exp Biol . 2000 ; 38 ( 2 ): 105 – 12 . PubMed PMID: 11218825 . OpenUrl PubMed 59. ↵ Vezzani A , Wu HQ , Stasi MA , Angelico P , Samanin R . Effect of various calcium channel blockers on three different models of limbic seizures in rats . Neuropharmacology . 1988 ; 27 ( 5 ): 451 – 8 . doi: 10.1016/0028-3908(88)90126-8 . PubMed PMID: 3393267 . OpenUrl CrossRef PubMed 60. ↵ Follath F , Ha HR , Schutz E , Buhler F . Pharmacokinetics of conventional and slow-release verapamil . Br J Clin Pharmacol . 1986 ; 21 Suppl 2 : 149S – 53S . doi: 10.1111/j.1365-2125.1986.tb02864.x . PubMed PMID: 3756058 ; PMCID: PMC1400738 . OpenUrl CrossRef PubMed 61. ↵ McAllister RG , Jr ., Kirsten EB . The pharmacology of verapamil. IV. Kinetic and dynamic effects after single intravenous and oral doses . Clin Pharmacol Ther . 1982 ; 31 ( 4 ): 418 – 26 . doi: 10.1038/clpt.1982.54 . PubMed PMID: 7060323 . OpenUrl CrossRef PubMed Web of Science 62. ↵ Paxinos G , Watson C . The rat brain in stereotaxic coordinates: hard cover edition : Elsevier ; 2006 . 63. ↵ Paxinos G , Watson CR , Emson PC . AChE-stained horizontal sections of the rat brain in stereotaxic coordinates . J Neurosci Methods . 1980 ; 3 ( 2 ): 129 – 49 . doi: 10.1016/0165-0270(80)90021-7 . PubMed PMID: 6110810 . OpenUrl CrossRef PubMed Web of Science 64. ↵ Wu X , Kuruba R , Reddy DS . Midazolam-Resistant Seizures and Brain Injury after Acute Intoxication of Diisopropylfluorophosphate, an Organophosphate Pesticide and Surrogate for Nerve Agents . J Pharmacol Exp Ther . 2018 ; 367 ( 2 ): 302 – 21 . Epub 2018/08/18. doi: 10.1124/jpet.117.247106 . PubMed PMID: 30115757 ; PMCID: PMC6193253 . OpenUrl Abstract / FREE Full Text 65. ↵ Rojas A , McCarren HS , Wang J , Wang W , Abreu-Melon J , Wang S , McDonough JH , Dingledine R . Comparison of neuropathology in rats following status epilepticus induced by diisopropylfluorophosphate and soman . Neurotoxicology . 2021 ; 83 : 14 – 27 . Epub 20201219. doi: 10.1016/j.neuro.2020.12.010 . PubMed PMID: 33352274 ; PMCID: PMC7987879 . OpenUrl CrossRef PubMed 66. ↵ Rojas A , Abreu-Melon J , Wang S , Dingledine R . Time-dependent neuropathology in rats following organophosphate-induced status epilepticus . Neurotoxicology . 2022 ; 91 : 45 – 59 . Epub 20220429. doi: 10.1016/j.neuro.2022.04.010 . PubMed PMID: 35500718 . OpenUrl CrossRef PubMed 67. ↵ Patterson EE , Goel V , Cloyd JC , O’Brien TD , Fisher JE , Dunn AW , Leppik IE . Intramuscular, intravenous and oral levetiracetam in dogs: safety and pharmacokinetics . Journal of veterinary pharmacology and therapeutics . 2008 ; 31 ( 3 ): 253 – 8 . Epub 2008/05/13. doi: 10.1111/j.1365-2885.2008.00948.x . PubMed PMID: 18471147 . OpenUrl CrossRef PubMed 68. ↵ Deshpande LS , Blair RE , Halquist M , Kosmider L , DeLorenzo RJ . Intramuscular atenolol and levetiracetam reduce mortality in a rat model of paraoxon-induced status epilepticus . Ann N Y Acad Sci . 2020 ; 1480 ( 1 ): 219 – 32 . Epub 20200922. doi: 10.1111/nyas.14500 . PubMed PMID: 32961584 ; PMCID: PMC7708441 . OpenUrl CrossRef PubMed 69. ↵ Jett DA . Finding new cures for neurological disorders: a possible fringe benefit of biodefense research? Sci Transl Med . 2010 ; 2 ( 23 ): 23p s12. Epub 2010/04/09. doi: 10.1126/scitranslmed.3000752 . PubMed PMID: 20375001 . OpenUrl CrossRef PubMed 70. ↵ Fang W , Lv P , Geng X , Shang E , Yang Q , Sha L , Li Y . Penetration of verapamil across blood brain barrier following cerebral ischemia depending on both paracellular pathway and P-glycoprotein transportation . Neurochem Int . 2013 ; 62 ( 1 ): 23 – 30 . Epub 20121108. doi: 10.1016/j.neuint.2012.10.012 . PubMed PMID: 23142723 . OpenUrl CrossRef PubMed 71. ↵ Bernardino PN , Hobson BA , Huddleston SL , Andrew PM , MacMahon JA , Saito NH , Porter VA , Bruun DA , Harvey DJ , Garbow JR , Gelli A , Chaudhari AJ , Lein PJ . Time- and region-dependent blood-brain barrier impairment in a rat model of organophosphate-induced status epilepticus . Neurobiol Dis . 2023 ; 187 : 106316 . Epub 20231004. doi: 10.1016/j.nbd.2023.106316 . PubMed PMID: 37797902 ; PMCID: PMC11000668 . OpenUrl CrossRef PubMed 72. ↵ Bernardino PN , Luo AS , Andrew PM , Unkel CM , Gonzalez MI , Gelli A , Lein PJ . Evidence Implicating Blood-Brain Barrier Impairment in the Pathogenesis of Acquired Epilepsy following Acute Organophosphate Intoxication . J Pharmacol Exp Ther . 2024 ; 388 ( 2 ): 301 – 12 . Epub 20240117. doi: 10.1124/jpet.123.001836 . PubMed PMID: 37827702 ; PMCID: PMC10801776 . OpenUrl Abstract / FREE Full Text 73. ↵ Li Y , Lein PJ , Liu C , Bruun DA , Tewolde T , Ford G , Ford BD . Spatiotemporal pattern of neuronal injury induced by DFP in rats: a model for delayed neuronal cell death following acute OP intoxication . Toxicol Appl Pharmacol . 2011 ; 253 ( 3 ): 261 – 9 . Epub 2011/04/26. doi: S0041-008X(11)00130-X [pii] 10.1016/j.taap.2011.03.026 . PubMed PMID: 21513723 ; PMCID: PMC3108263 . OpenUrl CrossRef PubMed 74. ↵ Singh T , Ramakrishnan S , Wu X , Reddy DS . Sex Differences in Organophosphate Model of Benzodiazepine-Refractory Status Epilepticus and Neuronal Damage . J Pharmacol Exp Ther . 2024 ; 388 ( 2 ): 313 – 24 . Epub 20240117. doi: 10.1124/jpet.123.001747 . PubMed PMID: 37770202 ; PMCID: PMC10801723 . OpenUrl Abstract / FREE Full Text 75. ↵ Gonzalez EA , Calsbeek JJ , Tsai YH , Tang MY , Andrew P , Vu J , Berg EL , Saito NH , Harvey DJ , Supasai S , Gurkoff GG , Silverman JL , Lein PJ . Sex-specific acute and chronic neurotoxicity of acute diisopropylfluorophosphate (DFP)-intoxication in juvenile Sprague-Dawley rats . Curr Res Toxicol . 2021 ; 2 : 341 – 56 . Epub 20210914. doi: 10.1016/j.crtox.2021.09.002 . PubMed PMID: 34622217 ; PMCID: PMC8484742 . OpenUrl CrossRef PubMed 76. ↵ Rao NS , Meyer C , Vasanthi SS , Massey N , Samidurai M , Gage M , Putra M , Almanza AN , Wachter L , Thippeswamy T . DFP-Induced Status Epilepticus Severity in Mixed-Sex Cohorts of Adult Rats Housed in the Same Room: Behavioral and EEG Comparisons . Front Cell Dev Biol . 2022 ; 10 : 895092 . Epub 20220510. doi: 10.3389/fcell.2022.895092 . PubMed PMID: 35620057 ; PMCID: PMC9127803 . OpenUrl CrossRef PubMed View the discussion thread. 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