Co-administration of subtherapeutic diazepam enhances neuroprotective effect of COX-2 inhibitor, NS-398, after lithium pilocarpine-induced status epilepticus
Introduction
Status epilepticus (SE) is a neurologic emergency that requires prompt intervention to prevent long-term morbidity and mortality. Both human clinical studies and rodent models of SE indicate that prolonged seizure activity causes neuronal death via several pathways, and rapid treatment of SE reduces this injury and subsequent morbidity (Lowenstein and Alldredge, 1998, Alldredge et al., 2001, Klitgaard et al., 2002, Morimoto et al., 2004). Benzodiazepines, such as diazepam and lorazepam, are widely used as first-line agents to terminate seizure activity, but unfortunately, SE becomes progressively more refractory to these agents after 30–40 min (Walton and Treiman, 1988, Jones et al., 2002, Naylor et al., 2005). Thus, developing therapies for treating SE that more effectively terminate later stage seizure activity, enhance neuroprotection, or suppress the long-term consequences of SE is an important goal.
Inflammatory mediators contribute to the negative consequences of SE (Ravizza et al., 2011, Vezzani et al., 2012). The cyclooxygenase-2 (COX-2) pathway, in particular, has been studied in many animal models of SE and reportedly affects neuronal excitability (Chen and Bazan, 2005, Zhang et al., 2008), susceptibility to neuronal death (Ho et al., 1998, Kawaguchi et al., 2005), and influx of several antiepileptic drugs across the blood–brain barrier (Bauer et al., 2008, Schlichtiger et al., 2010, van Vliet et al., 2010), all of which may influence the severity of SE. COX-2 is an inflammatory enzyme involved in prostaglandin synthesis that is expressed under basal conditions in excitatory neurons from the cortex, hippocampus, hypothalamus and amygdala (Yamagata et al., 1993, Adams et al., 1996, Kaufmann et al., 1996). Increased synaptic activity associated with seizures markedly amplifies the expression of COX-2 within 30 min from the beginning of seizures, with the peak effect estimated to be about 24 h in different animal models of seizures (Voutsinos-Porche et al., 2004, Kawaguchi et al., 2005, Jung et al., 2006, Takemiya et al., 2006). COX-2 knockout mice showed decreased incidence of after-discharges, reduced after-discharge duration, and delayed induction of convulsive seizures compared to control mice after rapid kindling, suggesting that COX-2 facilitates the recurrence of seizures (Takemiya et al., 2003). The COX-2 pathway has also been implicated in regulating the transport of several antiepileptic drugs across the blood–brain barrier (Schlichtiger et al., 2010, van Vliet et al., 2010). Thus hypothetically, COX-2 inhibitors could directly suppress SE, facilitate transport of antiepileptic drugs to brain tissue, and reduce subsequent damage to neurons. However, when attempts to study the effect of COX-2 inhibition on the severity of SE and resultant neuronal damage using primarily behavioral observation of seizure activity, blocking of the COX-2 pathway has given mixed results, with some studies showing neuroprotection (Kunz and Oliw, 2001b, Jung et al., 2006, Takemiya et al., 2006, Bauer et al., 2008, Polascheck et al., 2010), and others showing no neuroprotection (Holtman et al., 2009, Holtman et al., 2010, Pekcec et al., 2009) or even exacerbation of neuronal death (Baik et al., 1999, Kim et al., 2008).
The aim of the present study was to investigate the effect of COX-2 inhibition, both alone and in the presence of diazepam, on the severity of SE and on neuronal damage. Brain electrical activity was monitored continuously for 24 h with subdural electroencephalogram (EEG) electrodes, thus allowing analysis of the effect of COX-2 and diazepam on the electrographic SE, not just behavioral seizure activity. Neuronal damage in the hippocampus was assessed by FluoroJade B staining of brain sections obtained 24 h after induction of SE. We hypothesized that inhibition of COX-2 decreases the duration and/or intensity of electrographic SE, possibly through decreased pharmacoresistance to benzodiazepines, with resultant neuroprotection.
Section snippets
Experimental animals
Adult male Sprague–Dawley rats (Charles River, USA) weighing 250–400 g were kept under 12-h light/dark conditions with free access to food and water. All procedures were approved by the University of Utah Animal Care and Use Committee.
Electrode implantation
The animals were anesthetized with isoflurane (2%) and placed in a stereotaxic device. Bipolar electrodes (MS333-3-B, Plastics One, Roanoke, VA, USA) were used for subdural recordings. Two holes (500 μm) were drilled on the right side of the midline under the
Lithium pilocarpine with and without COX-2 inhibition and/or diazepam evokes prolonged SE
Treatment with lithium-pilocarpine evoked prolonged and sustained SE lasting several hours (Fig. 2). The first convulsive seizure, chosen as time point zero for further treatments, was observed clinically as either Racine stage 4 (rearing) or 5 (rearing with falling). Within a few minutes, all animals progressed to Racine stage 5 behavior, and had been in sustained SE when treatment was given at 30 min. During the 24 h video EEG monitoring period, the mortality rate was: 29% and 36% in vehicle
Discussion
Three key results were obtained in this study: (1) Treatment with the selective COX-2 inhibitor NS-398—with or without diazepam—did not have a significant effect on the duration or intensity of electrographic SE. (2) COX-2 inhibition significantly decreased neuronal damage in the CA3 and hilus in the dorsal hippocampus. (3) This effect was greatly enhanced when NS-398 was associated with diazepam, and neuroprotection was obtained throughout the entire hippocampus.
Conclusions
Inhibition of COX-2 activity 30 min after pilocarpine-induced SE with a repeat dose at 6 h leads to moderate neuroprotection, but does not significantly affect duration or intensity of electrographic SE. The neuroprotective effect is greatly enhanced when COX-2 inhibitor is associated with diazepam, indicating that COX-2 inhibitors may have potential therapeutic applications in association with benzodiazepines, and may improve clinical outcomes following SE.
Acknowledgments
This research was supported by the Counter-ACT Program, National Institutes of Health Office of the Director (NIH OD), and the National Institute of Neurological Disorders and Stroke (NINDS), Grants No. N01-NS-4-2359 (CT, WP, and FED) and K08-NS-070957 (JE). We gratefully acknowledge the technical assistance of Katie Ricks and Spencer Clark.
References (54)
- et al.
Prostaglandin E2 protects cultured cortical neurons against N-methyl-d-aspartate receptor-mediated glutamate cytotoxicity
Brain Res
(1994) - et al.
Cyclooxygenase-2 selective inhibitors aggravate kainic acid induced seizure and neuronal cell death in the hippocampus
Brain Res
(1999) - et al.
Temporal profile of neuronal injury following pilocarpine or kainic acid-induced status epilepticus
Epilepsy Res
(2000) - et al.
Effect of cyclooxygenase inhibitors on pentylenetetrazol (PTZ)-induced convulsions: possible mechanism of action
Prog Neuropsychopharmacol Biol Psychiatry
(2006) - et al.
Lithium pilocarpine-induced status epilepticus in postnatal day 20 rats results in greater neuronal injury in ventral versus dorsal hippocampus
Neuroscience
(2011) The temporal evolution of neuronal damage from pilocarpine-induced status epilepticus
Brain Res
(1996)- et al.
Post-treatment, but not pre-treatment, with the selective cyclooxygenase-2 inhibitor celecoxib markedly enhances functional recovery from kainic acid-induced neurodegeneration
Neuroscience
(2004) - et al.
Induction of cyclooxygenase (COX)-2 but not COX-1 gene expression in apoptotic cell death
J Neuroimmunol
(1998) - et al.
Cox-2 inhibition can lead to adverse effects in a rat model for temporal lobe epilepsy
Epilepsy Res
(2010) - et al.
Effects of SC58236, a selective COX-2 inhibitor, on epileptogenesis and spontaneous seizures in a rat model for temporal lobe epilepsy
Epilepsy Res
(2009)