Neurotoxicology 74 (2019) 19–27 Contents lists available at ScienceDirect Neurotoxicology journal homepage: www.elsevier.com/locate/neuro Full Length Article Time dependent dual effect of anti-inflammatory treatments on sarin- induced brain inflammation: Suggested role of prostaglandins T ⁎ Shira Chapman, Ettie Grauer , Rellie Gez, Inbal Egoz, Shlomi Lazar Israel Institute for Biological Research, Israel ARTICLE INFO ABSTRACT Keywords: A common consequence of exposure to organophosphate nerve agents is the centrally mediated seizure activity Sarin that appears even after conventional treatment with atropine and oximes. We have previously demonstrated a Organophosphates major inflammatory response with subsequent brain damage which was correlated with the duration of the fl Anti-in ammatory sarin-induced seizures (Chapman et al., 2006). In the present work seizures were induced by the nerve agent Prostaglandins sarin (1.2 LD50) insufficiently treated 1 min later by atropine and trimedoxime bromide (TA), with additional Convulsions midazolam treatment either 5 or 30 min after continuous seizure activity. The efficacy of both steroidal and nonsteroidal anti-inflammatory drugs (NSAIDs), as well as other drugs that were reported as beneficial in neuroprotection, were evaluated for their contribution as adjunct treatment against sarin induced seizures and the ensuing inflammatory brain damage. Results show that both steroids and NSAIDs were harmful when ad- ministered during convulsions, and steroids were at best ineffective if administered at their termination. However, if administered at termination of convulsions, the NSAID ibuprofen, the selective COX 2 inhibitor nimesulide and the PLA2 inhibitor quinacrine were partially effective in reducing brain inflammatory markers. Administration of exogenous analogs of prostaglandins (PGE2) immediately following sarin-induced convulsions was found to have a beneficial effect in reducing brain inflammatory markers measured at 24 h and one week post sarin exposure. These findings support the hypothesis that elevated levels of PGE2 have a beneficial role immediately following sarin induced seizures, and that early inhibition of PGE2 production by both steroids and NSAID is contraindicative. 1. Introduction mRNA of the pro-inflammatory markers IL-1β, TNFα, and IL-6 within 6 h following exposure (Svensson et al., 2001; Williams et al., 2003). The nerve agent sarin is a highly toxic, irreversible organopho- However, the effects of anti-inflammatory treatments following nerve sphate (OP) cholinesterase (ChE) inhibitor used in the terror attacks in agents have not been previously reported. In various models of seizures Japan (Okumura et al., 2003) and, more recently, in the civil war in and brain injury there are conflicting reports as to the efficacy of dif- Syria (Eisenkraft et al., 2014; Rosman et al., 2014). Sarin exposure ferent anti-inflammatory treatments. While some report ameliorating results in a dose dependent hyper-secretion, fasciculation, tremor, effects of steroids in reducing lipid peroxidation and providing neuro- convulsions, respiratory failure and death (Munro et al., 1994). In an- protection (Hall, 1993), others were skeptical of their beneficial effect imals, OP intoxication was shown to induce a dose-dependent, wide (Gomes et al., 2005). Steroids were helpful in reducing vasogenic spread and specific brain damage (Kadar et al., 1995; Lazar et al., edema (Sztriha et al., 1986), and in inhibiting COX-2 and PGE2 pro- 2016), the severity of which was found to correlate with the extent and duction following seizures (Ciceri et al., 2002). In contrast, there are duration of convulsions (McDonough and Shih, 1997; McDonough reports of aggravation of seizures following steroid treatment (Duffy et al., 1999, 2000; Chapman et al., 2006; de Araujo Furtado et al., et al., 2014; Supko and Johnston, 1994; Lee et al., 1989) which was also 2010). We have previously demonstrated a specific, time dependent seen in cell cultures (Semba et al., 1996). The same conflicting effects inflammatory response following sarin-induced seizures (Chapman have been reported for non-steroidal anti-inflammatory drugs (NA- et al., 2006, 2015, Lazar et al., 2016). Exposure to the nerve agent SIDs). While neuroprotective effects of ibuprofen and indomethacin soman also resulted in a microglia activation (Zimmer et al., 1997), have been reported following cerebral ischemia (Iwata et al., 2010; induction of COX-2 (Angoa-Pérez et al., 2010) and an increase in the Girgis et al., 2013; Lopes et al., 2016; Tutak et al., 2005), exacerbation ⁎ Corresponding author. E-mail address: [email protected] (E. Grauer). https://doi.org/10.1016/j.neuro.2019.05.006 Received 7 March 2019; Received in revised form 12 May 2019; Accepted 12 May 2019 Available online 13 May 2019 0161-813X/ © 2019 Elsevier B.V. All rights reserved. S. Chapman, et al. Neurotoxicology 74 (2019) 19–27 of seizures following NSAID have also been documented (Régnier et al., 2.4. Inflammation markers 2010; Auriel et al., 2014). The documentation of the effect of the COX2 inhibitors nimesulide and rofecoxib on seizures have been pre- 2.4.1. Prostaglandine (PGE2) dominantly positive. Both are reported as protective either by elevation PGE2 was assayed according to SIGMA protocol for PGE2 antibodies of seizure threshold (Akula et al., 2008), or by neuroprotection (Wang (p 5164). Each sample tube contained 25–100 μl of brain homogenate et al., 2012). The present study evaluated the efficacy of anti-in- and a reaction mixture containing 0.5 ml PGE2 antibodies (sigma) and flammatory treatments, as adjunct therapy to the standard treatment of 100 μlof[3H]- PGE2 (Amersham, UK) was added. OP poisoning, in ameliorating inflammatory markers and the ensuing Cytokines were assayed by DuoSet ELISA development systems for brain injury that resulted from sarin-induced prolong convulsions. The rats, TNFα (DY510), IL-1β (DY501) and IL-6 (DY506), R&D Systems, role of prostaglandins was delineated as timing of COX inhibition was according to the protocols provided with the kits. found to be crucial for the efficacy of treatments. 2.4.2. TSPO The glial cell marker 18kDTranslocater protein (TSPO, formerly 2. Method termed PBR) was assayed using labeled PK-11195 ([3 H]PK-11195, specific activity 83.5 Ci/mmol, purchased from Perkin Elmer, USA). 2.1. Animals Binding of [3 H]PK-11195 to rat brain membranes was performed with minor modifications as previously described (Benevides et al., 2001). All procedures involving animals were in accordance with the Guide Briefly, reaction mixture (in 12 × 75 test tubes), at a final volume of for the Care and Use of Laboratory Animals, National Academy Press, 1 ml, contained 300 μl Tris−HCl buffer (50 mM, pH 7.4), 100 μl[3H] Washington, DC, 2011, and were approved by the IIBR animal care and PK-11195 solution (1 nM), 100 μl PK-11195 solution (10 μM final con- – use committee. Male albino Sprague-Dawley rats weighing 280 300 g centration for nonspecific binding) or buffer and 500 μl membrane were purchased from Charles River (England). Animals were housed on suspension (diluted 1:10 from the initial brain homogenate). Non- bedding in plastic cages, 3 per cage, in a controlled environment with a specific binding amounted to 5–15% of total ligand bound. Tubes were constant temperature of 21 ± 2 °C and 12 h light/dark cycle, lights on incubated for 1 h at 0–4 °C and the reaction terminated by rapid fil- at 6 a.m.. Food pellets and water were available ad lib. Animals were tration over GF/B filters. Radioactivity was assessed using Packard li- allowed at least 5 days of acclimatization before onset of experiment. quid scintillation analyzer (1600 TR). (Chapman et al., 2015) 2.5. Histological staining and scoring 2.2. Materials Brains were rapidly removed from the skull, immersed in 4% neu- 2.2.1. Sarin tral buffered paraformaldehyde (pH = 7.0; Gadot, Israel) at 4 °C, and (isopropyl methylphosphono-fluoridate) was supplied by the processed routinely for paraffin embedding. Serial 7μm-thick sections Department of Organic Chemistry at the Israel Institute for Biological were cut in the coronal plane, mounted on positively charged plus Research (IIBR). Sarin (at least 96% pure, based on 1H and 31 P NMR) slides and air dried overnight. Next, sections were deparaffinized [xy- was dissolved in propylene glycol and kept frozen. For intramuscularly lene(2 x 5 min), 100% ethanol (2 x 3 min), 95% ethanol (3 min), 80% (i.m.) injections, fresh dilute solutions in saline were prepared for each ethanol (3 min) and distilled water for 2 min] and rehydrated through experiment. 100 and 95% alcohol to distilled water. Slides were stained with he- matoxylin and eosin (H & E) for light microscopy examination. 2.2.2. Drugs The severity of brain damage was semi-quantitatively scored ac- fi Atropine sulfate, trimedoxime bromide (TMB4), Nimesulide, cording to a modi ed histological scoring scale previously described Quinacrine Ibuprofen and Misoprostol (Sigma-Aldrich). Midazolam (Bloch-Shilderman et al., 2005). The score is based on the typical solution (Dormicum; Hoffman-LaRoche, 5 mg/ml), Solu-Medrol neuropathology following OP poisoning (Kadar et al., 1995) and in- (Methylprednisolone, 125 mg/2 ml) Dinoprostone (Prostine E2, 10 mg/ cludes the hippocampus, piriform and thalamus. For each of these brain – ml) and Iloprost (Ilomedine 0.1 mg/ml) purchased from a local phar- areas the score ranged from 0 (intact brain), 1- for minor changes, 2 – macy. Atropine and TMB4 were dissolved in saline and administered moderate changes, 3 severe damage. Maximum score per brain was i.m. Nimesulide, ibuprofen, quinacrine and misoprostol were dissolved therefore-9 in DMSO and administered intraperitoneally (i.p.), Methylprednisolone, dinoprostone (Prostin) and iloprost (Ilomedin) were administered i.p. in 2.6. Experimental outline their provided solutions. All the treatments were administered in neu- roprotective doses determined based on published literature. Solutions 2.6.1. Exposure and initial treatment μ of atropine (20 mg/ml) and TMB4 (30 mg/ml) were prepared in sterile Rats were exposed to sarin by i.m.