Midazolam and isoflurane combination reduces late brain damage in the paraoxon-induced status epilepticus rat model

Journal Pre-proof Midazolam and isoflurane combination reduces late brain damage in the paraoxon-induced status epilepticus rat model Evyatar Swissa (Conceptualization) (Methodology) (Investigation) (Software) (Data curation) (Formal analysis) (Visualization) (Writing - original draft) (Writing - review and editing), Guy Bar-Klein (Conceptualization) (Methodology) (Investigation) (Data curation) (Validation) (Writing - original draft), Yonatan Serlin (Visualization) (Validation) (Writing - review and editing), Itai Weissberg (Formal analysis) (Visualization) (Writing - review and editing), Lyna Kamintsky (Software) (Data curation) (Formal analysis) (Visualization), Arik Eisenkraft (Conceptualization) (Methodology) (Resources) (Funding acquisition)Writingoriginal draft), Liran Statlender (Conceptualization) (Methodology), Shai Shrot (Conceptualization) (Methodology), Yossi Rosman (Conceptualization) (Methodology) (Funding acquisition), Ofer Prager (Visualization) (Validation) (Supervision) (Writing - review and editing), Alon Friedman (Supervision) (Project administration) (Funding acquisition) (Writing - original draft) (Writing - review and editing)

PII:

S0161-813X(20)30029-2

DOI:

https://doi.org/10.1016/j.neuro.2020.02.007

Reference:

NEUTOX 2592

To appear in:

Neurotoxicology

Received Date:

26 November 2019

Revised Date:

9 February 2020

Accepted Date:

17 February 2020

Please cite this article as: Swissa E, Bar-Klein G, Serlin Y, Weissberg I, Kamintsky L, Eisenkraft A, Statlender L, Shrot S, Rosman Y, Prager O, Friedman A, Midazolam and isoflurane combination reduces late brain damage in the paraoxon-induced status epilepticus rat model, Neurotoxicology (2020), doi: https://doi.org/10.1016/j.neuro.2020.02.007

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Midazolam and isoflurane combination reduces late brain damage in the paraoxoninduced status epilepticus rat model Evyatar Swissa1, Guy Bar-Klein2,3, Yonatan Serlin4, Itai Weissberg5, Lyna Kamintsky6, Arik Eisenkraft7, Liran Statlender8, Shai Shrot9,10, Yossi Rosman10, Ofer Prager1 and Alon Friedman1,6*

1

Departments of Brain and Cognitive Sciences, Physiology and Cell Biology, The Inter-

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Faculty Brain Science School, Zlotowski Center for Neuroscience, Ben-Gurion University of the Negev, Beer-Sheva, Israel 2

McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

Howard Hughes Medical Institute, Chevy Chase, MD, USA

4

Neurology Residency Training Program, McGill University, Montreal, QC, Canada

5

Internal Medicine Division, Soroka Medical Center, Beer-Sheva, Israel

6

Department of Medical Neuroscience, Dalhousie University, Halifax, Nova Scotia,

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3

Canada

The Institute for Research in Military Medicine, the Hebrew University, Jerusalem, and

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7

the IDF Medical Corps, Israel

Intensive Care Unit, Rabin Medical Center, Petah Tikva, Israel

9

Department of Radiology, Sheba Medical Center, Ramat-Gan, Israel Sackler School of medicine, Tel Aviv University, Tel Aviv, Israel

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10

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8

Correspondence to: Dr. Alon Friedman, Ben-Gurion University of the Negev, P.O.B. 653

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Beer-Sheva, 8410501 Israel, Email: [email protected]

Highlights 

Combination of anti-epileptics and midazolam promotes status epilepticus cessation

Midazolam plus valproate significantly shortened seizure duration and recurrence

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Status epilepticus duration was not associated with the degree of late brain injury



Isoflurane significantly reduced late brain injury compared to other agents tested

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

Abstract

Organophosphates (OPs) are widely used as pesticides and have been employed as warfare agents. OPs inhibit acetylcholinesterase, leading to over-stimulation of cholinergic

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synapses and can cause status epilepticus (SE). OPs poisoning can result in irreversible

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brain damage and death. Despite termination of SE, recurrent seizures and abnormal brain activity remain common sequelae often associated with long-term neural damage and

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cognitive dysfunction. Therefore, early treatment for prevention of seizures is of high interest. Using a rat model of paraoxon poisoning, we tested the efficacy of different

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neuroprotective and anti-epileptic drugs (AEDs) in suppressing early seizures and preventing brain damage. Electrocorticographic recordings were performed prior, during

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and after injection of 4.5 LD50 paraoxon, followed by injections of atropine and toxogonin (obidoxime) to prevent death. Thirty minutes later, rats were injected with midazolam

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alone or in combination with different AEDs (lorazepam, valproic acid, phenytoin) or neuroprotective drugs (losartan, isoflurane). Outcome measures included SE duration, early seizures frequency and epileptiform activity duration in the first 24-hours after poisoning. To assess delayed brain damage, we performed T2-weighted magnetic resonance imaging one month after poisoning. SE duration and the number of recurrent seizures were not affected by the addition of any of the drugs tested. Delayed brain injury

was most prominent in the septum, striatum, amygdala and piriform network. Only isoflurane anesthesia significantly reduced brain damage. We show that acute treatment with isoflurane, but not AEDs, reduces brain damage following SE. This may offer a new therapeutic approach for exposed individuals.

Keywords: Acetylcholinesterase, anesthesia, anti-epileptic drugs, organophosphates,

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recurrent seizures, status epilepticus

List of abbreviations

OPs, organophosphates; AChE, acetylcholinesterase; SE, status epilepticus; AEDs, antiepileptic drugs; BZDs, benzodiazepines; GABA, γ-aminobutyric acid; LD, lethal dose; IP,

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intraperitoneal; IM, intramuscular; ECoG, electrocorticography; ANN, artificial neural

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network; MID, midazolam; LOR, lorazepam; PHT, phenytoin; VPA, valproic acid; LOS, losartan; ISO, Isoflurane; ATOX, atropine + toxogonin; PBS, phosphate-buffered saline;

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PFA, paraformaldehyde; BBB, blood-brain barrier; MRI, magnetic resonance imaging; RS, recurrent seizures; T2w, T2 weighted; TR, repetition time; TE, excitation time; NEX,

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number of excitations; BH-FDR, Benjamini-Hochberg false discovery rate;

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1. Introduction

Organophosphates (OPs) are organic phosphorous compounds commonly used as pesticides (Marrs, 1993), and have also been used as chemical weapon nerve agents. Intentional use of OPs may result in severe injury or death to civilians and military personnel, as recently evident in Syria (Rosman et al., 2014), the United Kingdom (Chai et al., 2018) and elsewhere (Yanagisawa et al., 2006). OP pesticides are a frequent cause of

seizures following accidental poisoning, most often in developing countries (Costa, 2006; Marrs, 1993). OPs inhibit acetylcholinesterase (AChE), an enzyme that catalyzes acetylcholine (ACh) breakdown, leading to ACh accumulation and over-stimulation of cholinergic synapses (Antonijevic and Stojiljkovic, 2007). Acute exposure to high levels of OPs can lead to respiratory failure, loss of consciousness and seizures (Costa, 2006; Marrs, 1993). When seizures are prolonged (i.e. status epilepticus - SE), irreversible brain damage and death are common occurrences (Delgado-Escueta and Bajorek, 1982). SE is

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associated with cell loss, reactive gliosis and delayed neural dysfunction, causing severe neuro-psychiatric complications (Delgado-Escueta and Bajorek, 1982). SE has also been associated with cerebrovascular pathology and blood-brain barrier dysfunction (BBBD),

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manifested in the leakage of serum proteins into the brain neuropil (Carpentier et al., 1990; Friedman, 2011). Specifically, extravasation of the serum protein albumin has been shown activate

transforming

growth

factor

β

(TGF-β)

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to

signaling

in

astrocytes,

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neuroinflammation, extracellular matrix changes and excitatory synaptogenesis, resulting in abnormal network excitability (Bar-Klein et al., 2014a; David et al., 2009; Friedman, 2011; Weissberg et al., 2015). Although studied extensively (Carpentier et al., 1990; Costa,

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2006; Delgado-Escueta and Bajorek, 1982; Marrs, 1993), to date, there is no efficient treatment for the prevention of OP-induced SE and brain damage.

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The antidotal treatment for acute OPs poisoning consists of atropine, a peripheral

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muscarinic receptor antagonist, blocking ACh activity; oximes, which mediates dephosphorylation of the OP-AChE complex upon immediate administration, thus reactivating AChE; and if convulsions ensue - benzodiazepines (BZDs), γ-aminobutyric acid (GABA) receptors agonists, act by increasing synaptic inhibition to facilitate the termination of SE (Costa, 2006; Zilker, 2005). Midazolam is the most common BZD used for terminating OP-induced SE (Towne and DeLorenzo, 1999). We were able to

demonstrate midazolam efficacy in terminating paraoxon-induced SE (Bar-Klein et al., 2014b). However, despite SE termination, recurrent seizures and pathological epileptiform activity may continue for hours. Additionally, epilepsy developed in 50% of rats, and extensive brain damage was common (Bar-Klein et al., 2017; Shrot et al., 2014). While decline in the serum levels of BZDs (specifically midazolam, with elimination half-life <4 hours (Reves et al., 1985)) may explain seizure re-occurrence (Bar-Klein et al., 2014b), the rapid loss of functional postsynaptic GABAA receptors, partly due to subunit

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internalization, is a well-described cause of resistance to BZDs treatment during SE (Deeb et al., 2012; Naylor et al., 2005). Thus, additional therapeutic strategies to prevent delayed neuronal hyper-excitability and brain damage following SE are imperative.

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The anti-epileptic drugs (AEDs) phenytoin and valproic acid are currently the recommended treatment following initial BZD treatment (Shorvon and Ferlisi, 2011).

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Phenytoin is a blocker of voltage-gated sodium channels (Yaari et al., 1986), while valproic

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acid acts through multiple mechanisms, including enhancement of GABAergic inhibition and blockage of voltage-gated sodium and T-type calcium channels (Reddy and Kuruba, 2013).

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General anesthetics are known to suppress SE and epileptiform activity and are commonly used in cases of refractory SE, where other AEDs fails to terminate seizure activity

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(Mirsattari et al., 2004; Shorvon and Ferlisi, 2011). We recently showed that short and

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intermittent anesthesia with isoflurane has an anti-epileptogenic and neurovascular protective effect following SE (Bar-Klein et al., 2017, 2016). Treatment with losartan, an angiotensin II type 1 receptor antagonist, was shown to block brain TGF- signaling, reduce BBBD and prevent epileptogenesis after OP-induced SE in rats (Bar-Klein et al., 2017). The current study employs the paraoxon-induced SE model to test the efficacy of combinational treatment (i.e. midazolam together with another drug),

in terminating SE, preventing recurrent seizures, epileptiform activity, and the long-term brain damage. We combined early administration of midazolam with either long-acting AED (lorazepam, valproic acid or phenytoin), TGF-β signaling blocker (losartan), or inhalation anesthetic (isoflurane).

2. Materials and methods

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2.1 Materials Paraoxon-ethyl and atropine sulfate were purchased from Sigma-Aldrich (West Chester, PA, U.S.A.). Obidoxime chloride (Toxogonin) was obtained from Merck-Serono

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(Darmstadt, Germany). Midazolam was purchased from Rafa Laboratories (Jerusalem, Israel). Lorazepam (LOR) was purchased from Concept for Pharmacy (Kfar Saba, Israel),

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Phenytoin sodium (PHT) was purchased from Labesfal (Santiago de Besteiros, Portugal),

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and Valproate sodium (Orfiril, VPA) was purchased from Desitin Arzneimittel (Hamburg, Germany). Losartan potassium (Cozaar, LOS) was obtained from Teva Pharmaceutical

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Industries (Petah Tikva, Israel). Isoflurane (Terrel, ISO) was purchased from Piramal Critical care (Bethlehem, PA).

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2.2 Animal preparation

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All experimental procedures were approved by the Animal Care and Use Ethical Committee at the Ben-Gurion University of the Negev, Beer-Sheva, Israel. Fifty-three adult Sprague Dawley male rats (300-350 g; Envigo Ltd., Rehovot, Israel) were kept under a 12:12 h light and dark regimen and supplied with drinking water and food ad libitum. For electrode implantation, rats were deeply anesthetized with intraperitoneal (IP) injection of ketamine (75 mg/kg; Clorketam, Vetoquinol) and xylazine (5 mg/kg;

Sedaxylan, Eurovet Animal Health B.V) and were placed in a stereotactic frame. The skin was disinfected, anesthetized with local application of lidocaine, and a sagittal incision was made. Epidural electrodes were placed 3 mm caudal and ±2.5 mm lateral to bregma and a telemetric transmitter (CTA-F40 or CA-F40, Data Science International, St. Paul, MN) was implanted subcutaneously allowing continuous electrocorticographic (ECoG) recordings. 2.3 Paraoxon poisoning and treatment

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Animals were poisoned five days after surgery with intramuscular (IM) injection of paraoxon (1.45 mg/kg, equivalent to 4.5 LD50), followed by two successive IM injection of ATOX (atropine 3 mg/kg and toxogonin 20 mg/kg, equivalent to human dose), one and

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five minutes after exposure. Animals were then randomly assigned into six treatment groups. All animals were treated with midazolam (1 mg/kg, IM) 30 minutes following

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poisoning together with one of the following:

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AEDs: (i) lorazepam (LOR) (0.94 mg/kg, IP); (ii) valproic acid (VPA) (400 mg/kg, IP); (iii) phenytoin (PHT) (50 mg/kg, IP).

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Anti-inflammatory/anti-hypertensive: (iv) losartan (LOS) (60 mg/kg, IP). General inhalation anesthetic: (v) isoflurane anesthesia (ISO; administered in an anesthesia chamber (at the size of normal home cage containing clean bedding) at 2% with

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98% oxygen at 1l/h, 1-hour duration, induced at 6 hours after SE).

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Group (vi) received no further treatment and served as control (midazolam alone). 2.4 Electrocorticographic recordings and analysis ECoG recordings were initiated four days after implantation of electrodes, to allow the rats to fully recover from surgery. Recordings (1 kHz sampling rate) lasted up to 72 hours - 24 hours prior to poisoning (baseline) and a maximum of 48 hours after poisoning. Recordings

were analyzed off-line using an automated seizure detection algorithm, based on feature extraction and artificial neural network (ANN) clustering, as previously described (BarKlein et al., 2017, 2014, 2014b). ECoG analysis was performed blinded to treatment. Abnormal activity was classified into three patterns (Figure 1): (1) Early long-duration seizures which occurred within one minute of paraoxon injection (Figure 1A, referred below as 'status epilepticus’, SE). SE was characterized by consecutive distinct seizures (see below) and was considered to end when no ictal activity was recorded for at least 30

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min; (2) Distinct seizures were defined as continuous characteristic electrical activity automatically classified as a ‘seizure’ by ANN >1 and lasting >6 seconds (Figure 1B). Recurrent seizures were defined as seizures detected at least 30 min following the

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termination of the early SE; (3) Epileptiform activity – was defined as periods (>6 sec) of abnormal high-amplitude cortical activity with ANN output >0.7 and <1 (lower than the

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threshold for seizure definition) (Figure 1C).

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2.5 Magnetic Resonance Imaging

Brains were scanned ex-vivo using Aspect M2 MRI system (Aspect Imaging Technologies

intracardially

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Ltd., Lod, Israel). One month after poisoning, rats were deeply anesthetized and perfused

with

phosphate-buffered

saline

(PBS)

containing

4%

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paraformaldehyde (PFA). Brains were then removed and post-fixed in the same solution

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overnight at 4oc and were then transferred to storing solution containing 0.25% PFA in PBS. T2-weighted (T2w) fast spin echo sequence (TR/TE/NEX = 3,516/60/4; acquisition time, 93.3 minutes) was performed (30 mm field of view data matrix of 256 by 256, 0.12 mm in-plane resolution and slice thickness of 0.7 mm). Analysis was performed using inhouse Matlab scripts as described (Bar-Klein et al., 2017). Briefly, preprocessing was used to extract brain volume of interest and the creation of 3D brain objects which was registered

to a rat brain atlas. Automatic segmentation was next performed into anatomical brain regions. Extent of brain injury was assessed by calculating the percent volume with T2w abnormal signal in each of the 14 brain regions (comprising >91% of the scanned brain): midbrain, hindbrain, substantia nigra, septum, striatum, amygdala, corpus callosum, internal capsule, fimbria-fornix, cortex, hippocampus, pallidum, thalamus and piriform network (comprised of the olfactory structures, piriform cortex, dorsal endopiriform nucleus, and inferior olive).

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2.6 Statistical analysis

Differences between treatment groups in the duration of SE, number of seizures and MRI

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findings, were evaluated using either the Kruskal-Wallis test (multiple comparisons) or Mann-Whitney test, with Benjamini–Hochberg false discovery rate correction (BH FDR

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two-stage linear step-up procedure). SE termination and distinct seizures contingency were assessed using chi square test (χ2). K-means clustering was used to determine distribution

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of SE termination. Two-way analysis of variance (ANOVA) with repeated measure was used for analysis of the percentage of time characterized by pathological activity. Analyses

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3. Results

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were performed using GraphPad Prism 8 (GraphPad Software, La Jolla California USA).

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3.1 Survival

Overall, 10/53 animals died within the first 24 hours following poisoning, most (n=8, 15%) died within 30min of poisoning and before any additional anticonvulsant treatment was administered. Two additional rats died shortly after treatment with VPA. No significant differences in survival rates between treatment groups were found.

3.2 Midazolam resistance is prevalent after high-dose OP exposure Prolonged tonic-clonic convulsions (i.e. SE) were observed in all rats within one minute after the administration of paraoxon. Injection of midazolam (MID) at 30 minutes resulted in effective termination of the SE (i.e. complete cessation of ictal activity within one hour of MID injection) in 8 rats (57%, termed here as ‘responders’). In 6 rats (43%), no response to MID was noted and ictal ECoG activity was recorded for more than one hour after MID (hence ‘non responders’; SE duration: 33.7±2 vs. 137±16 min for responders and non-

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responders, respectively. P<0.001, Mann-Whitney; Figure 2A).

3.3 Addition of AED but not losartan or isoflurane ameliorates SE outcomes

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Administration of AED significantly increased the likelihood of immediate SE termination,

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and the percentage of non-responder animals (i.e. SE>1 hr.) dropped down from 43% in the MID group to 5% in the MID+AEDs groups (phenytoin, valproic acid, and lorazepam,

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n=18, p=0.0013, χ2). VPA rats had significantly shorter SE durations (n=6, avg. SE duration 31±1 min) compared to MID only (n=14, avg. SE duration 78±16 min, p=0.021,

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Kruskal-Wallis test with BH-FDR correction; Figure 2D). All rats treated with midazolam and losartan (LOS) were considered non-responders (n=5, avg. SE duration 91±6 min).

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MID+PHT and MID+VPA combinations had significantly shorter SEs compared with MID+LOS-treated rats (p=0.031, p=0.0015, respectively, Kruskal-Wallis test with BH-

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FDR correction; Figure 2D). We next counted the number of recurrent distinct seizures (Figure 2B, E) during the first 24 hours after poisoning (following termination of SE). Distinct seizures were detected in all groups, and were found in 8/14, 6/6, 4/6, 6/6, 5/5 and 6/6 respectively in MID, LOR, PHT, VPA, LOS or ISO groups, (p=0.16, χ2). Rats treated with a combination of MID and VPA or PHT had reduced number of recurrent seizures compared with MID only rats

(p=0.032, p=0.037, respectively, Kruskal-Wallis test with BH-FDR correction). We next measured the duration of the epileptiform activity (see Methods). Non-responders MID treated rats displayed abnormal ECoG for a significantly higher percentage of time compared to MID-responders during the first 24 hours following poisoning (p<0.001, Twoway ANOVA multiple comparisons, with BH-FDR correction; Figure 2C). A rapid decrease in pathological activity was noted in the responders groups (MID-responders (53%), VPA, PHT, LOR) within the first hour (30 min after administration of

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anticonvulsant treatment), to <10% of the time with abnormal activity. No significant differences were noted within the responders groups.

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3.4 Isoflurane anesthesia reduces brain damage

Using anatomical MRI images acquired one month following poisoning, we quantified the

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extent of brain injury (see Methods and (Bar-Klein et al., 2017)). Figure 3A shows T2 coronal sections of a representing brain from each treatment group. Above-threshold

averaged

total

brain

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hyperintense voxels are marked in red. No differences between the groups were found in volume

(MID=233.5±5.4,

n=6;

VPA=234.2±11.3,

n=4;

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LOR=216.4±18.7, n=4; ISO=256±6.4, n=5; LOS=243.5±13.2, n=6; volume units provided in mm3; Figure 3B). The striatum, amygdala and piriform network were the three sub-

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regions showing most injury one month after poisoning, when compared to sham controls.

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ISO-treated rats had significantly less damage in the striatum compared to MID, VPA and LOS treated rats (p=0.049, p<0.001, p=0.019, respectively, Kruskal-Wallis test). In the Piriform network, rats treated with ISO had significantly less damage compared to VPA treated rats (p=0.014, Kruskal-Wallis test). In the Amygdala, rats treated with MID alone or ISO had significantly less damage compared to VPA and LOR treated rats (p=0.036, p=0.017, p=0.024, p=0.012, respectively, Kruskal-Wallis test). Moreover, ISO treatment

significantly reduced the damage in the septum compared to VPA and LOS treatments (p=0.0091, p=0.015, respectively, Kruskal-Wallis test) (Figure 3B).

4. Discussion We examined several combinations of existing AEDs as well as the volatile anesthetic isoflurane, and the antihypertensive losartan, as potential treatments for aborting early postSE seizures and brain injury following high-dose (4.5LD50) paraoxon-induced SE. We have

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previously shown that with lower doses of paraoxon (1.4LD50) poisoning (Bar-Klein et al., 2014b; Shrot et al., 2014), a single injection of midazolam effectively aborts SE in all rats. In contrast, we show here that with higher levels of poisoning, 47% of the rats have

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persistent electrographic evidence of SE despite treatment with midazolam. To what extent the lack of response to midazolam is due to robust and prolonged ACh activity, is unclear.

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Non-cholinergic effects of OPs such as oxidative stress and free radical formation

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(Abdollahi et al., 2004), prolonged elevations of [Ca2+]i ‘Ca2+ plateau’ which may last up to 1 month following exposure (Deshpande et al., 2014), BZDs resistance due to down-

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regulation of GABAA (Deeb et al., 2012; Naylor et al., 2005) or other, not yet known mechanisms, may also play role. Importantly, adding either of the AEDs (VPA, PHT,

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LOR) in a therapeutic dose, increased significantly the likelihood of animals to respond to MID from 47 to 95% (p=0.0013). VPA treated rats had significantly shorter SEs compared

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to MID alone. Furthermore, VPA and PHT treated rats had significantly less recurrent seizures (RS) compared to MID alone. Although blocking SE has been shown to reduce brain injury (Deshpande et al., 2014; Fujikawa, 1996; Lowenstein and Alldredge, 1998; McDonough and Shih, 1997), it is also well established that RS, for up to 60 days following SE, can induce structural changes, neuroinflammation, and cellular injury (Bar-Klein et al., 2017; Pitkänen et al., 2002; van Vliet et al., 2014). Furthermore, it has been shown that RS

without SE can induce neuronal death and other neuropathologies (Navarro Mora et al., 2009). RS were frequent in our model, regardless of the treatment given, and were accompanied by epileptogenesis, which, as stated above, was characterized by an inflammatory

cascade

leading

to

pathological

neuronal

hyperexcitability

and

synaptogenesis. As previously described by our group (Bar-Klein et al., 2017, 2014a; Swissa et al., 2019; Weissberg et al., 2015), these processes may contribute to the development of epilepsy and delayed brain injury, subject to the extent and brain regions

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involved, compared to the acute phase following SE. The angiotensin- and TGFβ- signaling blocker, losartan was not effective in increasing response to MID during SE. Losartan aggravated the duration of epileptic activity, perhaps due to blood pressure lowering and

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reduced brain perfusion during this high energy-demanding state. Low dose, 1 hour long, isoflurane anesthesia did not reduce the number of seizures compared to other treatments,

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and seizures and abnormal epileptiform activity were observed shortly following

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anesthesia.

Interestingly, brain injury at 1-month after SE, did not follow the trend of the immediate

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ECoG data. We found no correlation between the duration of SE and/or early seizures and the extent of injury. Moreover, low-dose isoflurane administered in the current study –

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while did not block recurrent seizures, was effective, at least partially, in reducing structural brain lesions. The striatum, amygdala and piriform network are three sub-regions

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in which a late damage (1 month after paraoxon) has been previously demonstrated (BarKlein et al., 2017, 2016; Pitkänen et al., 2002). In particular, damage to the piriform network, has been associated with the development of epilepsy (Bar-Klein et al., 2017). While other studies were mainly concentrated on the acute brain damage after SE, and specifically the neuroprotective profile of isoflurane 30-120 min after paraoxon SE (Krishnan et al., 2017, 2016), our aim in this study was to quantify the late brain damage

at 1 month following SE, RS, and epileptogenesis. It is therefore suggested that ISO neuroprotective effect may arise from its anti-inflammatory properties, among other mechanisms. It is difficult to exclude that ISO neuroprotective effect was partly due to the drop in core body temperature, as previously shown in other injury models (Liu et al., 2017). Therefore, we measured the body temperature in an additional cohort of rats during 1 hour long (2% isoflurane) anesthesia. As expected, body temperature declined from 37.7±0.2 to 35.0±0.6 (mean difference 2.6°C±0.4 (n=5)). This change is modest compared

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to previously reported anesthesia-induced hypothermia in a mice stroke model (37.0±0.2°C to 26.8±0.2°C) (Liu et al., 2017). Other studies in rats showed that the therapeutic effect of hypothermia was achieved at body temperatures of 30-34°C in epileptic animals (Lundgren

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et al., 1994) and in a model of intracranial vessel occlusion (Zhao et al., 2007). Furthermore, the recently described anti-epileptogenic/neuroprotective effect of isoflurane

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in two independent SE models where core body temperature was controlled (Bar-Klein et

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al., 2016), hints that neuroprotection is mainly mediated by ISO itself. These results stress the importance of early mechanisms, independent of seizure activity, in the development of delayed brain injury. Moreover, this may also suggest that chronic administration, rather

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than a single acute dose, can mediate the neuro-protective, anti-inflammatory and antiepileptogenic effects of losartan previously described (Bar-Klein et al., 2017, 2014a). Our

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results emphasize the importance of developing early neuro-protective treatments, in

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addition to those currently used, aiming at stopping seizure activity. We showed that combined anti-epileptic treatments are effective in terminating SE, yet seizures may recur within 24 hours, as well as subsequent structural brain damage. Generalizability of our findings to SE from other causes rather than OPs is a limitation of this study. Future preclinical and clinical studies in different SE etiologies are required to test for the underlying mechanisms, dose-time-interval and detailed therapeutic protocol required to achieve

maximal neuroprotective effect of isoflurane with or without combined anti-seizure medications. CRediT_author_statement

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Evyatar Swissa: Conceptualization, Methodology, Investigation, Software, Data Curation, Formal analysis, Visualization, Writing- Original draft, reviewing and editing. Guy Bar-Klein: Conceptualization, Methodology, Investigation, Data Curation, Validation, Writing- Original draft. Yonatan Serlin: Visualization, Validation, WritingReviewing and Editing. Ofer Prager: Visualization, Validation, Supervision, WritingReviewing and Editing. Itai Weissberg: Formal analysis, Visualization, WritingReviewing and Editing. Lyna Kamintsky: Software, Data Curation, Formal analysis, Visualization. Arik Eisenkraft: Conceptualization, Methodology, Resources, Funding acquisition, Writing- Original draft. Liran Statlender: Conceptualization, Methodology. Shai Shrot: Conceptualization, Methodology. Yossi Rosman: Conceptualization, Methodology, Funding acquisition. Alon Friedman: Supervision, Project administration, Funding acquisition, Writing - Original draft. Review & Editing

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Declaration of Interest

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None of the authors has any conflict of interest to disclose. Declaration of interests

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The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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Acknowledgements

This study was supported by an unrestricted research grant from the Israeli Defense Forces

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(IDF) Medical Corps, Directorate of Defense Research & Development, Israeli Ministry of Defense (IMOD DDR&D), and the Nuclear Biological and Chemical (NBC) Protection Division at the Israeli Ministry of Defense, given to Dr. Alon Friedman.

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Figure legends

Figure 1: Typical ECoG patterns. Top panels: ECoG in black; mid panels: A 10 sec.

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middle section of the ECoG trace; bottom panels: ANN output in blue; horizontal line represents ANN threshold; examples extracted from a recording of a MID treated rat). A. Immediate SE evolved following IM paraoxon injection, and diminished after injection of

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midazolam (MID) (arrows in 1, 5 and 30 min represent treatment with ATOX and MID

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treatments, respectively); B. A distinct recurrent seizure; C. Pathological epileptiform discharges; and D. non-epileptiform baseline activity.

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Figure 2: Effect of treatment on SE length, recurrent seizures, and overall

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pathological activity. A. Duration (min) of status epilepticus. MID treatment at 30 minutes resulted in effective termination of the SE in 8 rats (57%, termed ‘responders’, blue circles).

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In 6 rats (43%), no response to MID was noted and ictal ECoG activity was recorded for more than one hour after MID (hence ‘non responders’, red circles). K-means clustering

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identified 2 clusters (SE duration: 33.7±2 vs. 137±16 min for responders and nonresponders, respectively. P=0.0007, Mann-Whitney). B. Number of distinct seizures during

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the first 24 hours after poisoning for the MID group. C. The percent of time in epileptiform state, averaged (±SEM) for each group and time frame, defined as the overall sum of high ANN outputs (>0.7), was used as a measure of total ictal and inter-ictal pathological activity. Non-responders MID treated rats (blue) displayed abnormal ECoG for a significantly higher percentage of time compared to MID-responders (red) up to 36 hours following (p<0.0001, Two-way ANOVA multiple comparisons, with BH-FDR correction).

D. Additional AED significantly increased the likelihood of immediate SE termination. Non-responder animals (i.e. SE>1 hr, k-means clustering) dropped down from 43% in the MID group to 5% in the AEDs groups (phenytoin, valproic acid, and lorazepam, n=18, p=0.0013, χ2). Addition of VPA significantly shortened SE duration compared to MID (p=0.021, Kruskal-Wallis test with BH-FDR correction). All rats treated with midazolam and losartan (LOS) were considered non-responders (n=5, avg. SE lengths 91±6 min) and had significantly longer SE compared to the VPA and PHT groups (p=0.0015, p=0.031,

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respectively, Kruskal-Wallis test with BH-FDR correction). E. Rats treated with VPA or PHT had reduced number of recurrent seizures compared with MID only rats (p=0.032,

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p=0.037, respectively, Kruskal-Wallis test with BH-FDR correction).

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Figure 3: T2w MRI for quantitative detection of pathological voxels. A. Apparent

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damage to brain tissue 1-month following poisoning. Top row: T2w coronal sections from an exemplary rat for each treatment, as well as non-poisoned animal (naïve); bottom row:

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corresponding sections with detected pathological voxels (incl. ventricles) masked red. B.

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Sub-region quantification of late brain damage. Bars showing the calculated percent volume of damaged tissue in each sub-region, averaged for each of the treatment groups, 1-month following poisoning. No significant differences in brain volume were noted among the groups (MID=233.5±5.4, n=6; VPA=234.2±11.3, n=4; LOR=216.4±18.7, n=4; ISO=256±6.4, n=5; LOS=243.5±13.2, n=6; volume units provided in mm3). Whole brain damage was significantly lower in rats treated with MID or ISO compared to VPA and

LOR groups (p=0.0057, p=0.0049, p=0.017, p=0.014, respectively, Kruskal-Wallis test). ISO-treated rats had significantly less damage in the striatum compared to MID, VPA and LOS treated rats (p=0.049, p<0.0001, p=0.019, respectively, Kruskal-Wallis test). In the Piriform network, rats treated with ISO had significantly less damage compared to VPA treated rats (p=0.014, Kruskal-Wallis test). In the Amygdala, rats treated with MID or ISO had significantly less damage compared to VPA and LOR treated rats (p=0.036, p=0.017, p=0.024, p=0.012, respectively, Kruskal-Wallis test). In the septum, ISO treatment

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significantly reduced the damage compared to VPA and LOS treatments (p=0.0091,

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p=0.015, respectively, Kruskal-Wallis test).