The present results establish that soman leads to large increases in expression of COX-2 protein. The greatest increase in COX-2 was observed within subregions of the hippocampus (i.e., dentate gyrus, CA1 and CA3). Significant increases in COX-2 were also observed in the basolateral amygdala, piriform cortex and ventral thalamus. COX-1 expression was not altered by soman. The soman-induced increase in COX-2 was time-dependent and developed at a relatively slow rate by comparison to the very rapid onset of hyper-cholinergic (0-5 min) and hyper-glutamatergic (5-40 min) conditions known to occur after soman exposure (Shih and McDonough, 1997
). Changes in COX-2 levels were very slight for 12 hr after soman treatment and increased to maximal levels over the course of 24-48 hours. COX-2 levels declined somewhat from 48 hours to 7 days, the last time-point sampled presently, but remained significantly elevated nonetheless. Immunoblotting studies confirmed soman-induced increases in COX-2 with increases as large as 15-fold by comparison to controls. The lack of effect of soman on COX-1 levels was also confirmed by immunoblotting.
These results are significant for several important reasons. First, COX-2 is the initial enzyme in the biosynthesis of PGEs. The PGEs participate in inflammatory processes throughout the body (Kuehl and Egan, 1980
). In brain, PGEs have excitatory effects on neurons (Bezzi et al., 1998
; Nishihara et al., 1995
; Sekiyama et al., 1995
) and they have been implicated in numerous seizure conditions (Cole-Edwards and Bazan, 2005
) to include roles for both initiation and propagation (Takemiya et al., 2006
). Second, soman-induced increases in COX-2 occur in brain regions known to be damaged by NAs (Baille et al., 2005
; Collombet et al., 2005
; Collombet et al., 2008
; McDonough et al., 1989
; McDonough et al., 1999
; Shih and McDonough, 1999
). Prior results linking soman-induced neuropathology to seizure intensity and duration (Chapman et al., 2006
) make it all the more interesting that increases in COX-2 protein are positively correlated with seizure intensity after soman exposure. This result also confirms that seizure activity per se triggers COX-2 induction and not soman (i.e., some soman-treated rats do not develop seizures and show very low levels of COX-2). Third, increases in COX-2 develop rather slowly after soman, a compound that causes physiological distress with an extremely rapid onset and which can lead to death within minutes. The identification of increased COX-2 as a late-occurring effect of soman exposure has important implications for the treatment of NA poisoning (see below).
The present results are consistent with prior studies implicating inflammatory processes in the action of NAs. Recent microarray analyses showed increases in COX-2 mRNA that reached a maximum of about 7-fold between 9-12 hours after soman administration (Dillman et al., 2009
). Our studies also recapitulate others showing that NAs increase brain PGEs to their highest levels 24-48 hours post-exposure (Chapman et al., 2006
). The present results expand these findings substantially by establishing that soman effects on COX-2 gene expression extend to increased translation of COX-2 protein, and they provide the biochemical basis for increased PGE production. Our results are also unique in that they identify the cell-type in which COX-2 is increased by soman. Double-labeling immunohistochemical studies showed clearly that COX-2-positive cells in soman treated rats were co-labeled with NeuN, establishing their identity as neurons. COX-2 immunoreactivity was not observed in activated microglia or in astrocytes. Considering only the hippocampus, the morphology of COX-2 containing cells in soman treated rats suggests their identity as pyramidal and granular neurons. Although gliosis is increased by NAs (Baille et al., 2005
; Collombet et al., 2005
; Collombet et al., 2008
; Grauer et al., 2008
; Zimmer et al., 1997
), glial activation does not include elevated COX-2 expression and it appears that microglial and astrocyte participation in NA-induced neuropathology involves inflammatory and excitatory mechanisms that could act to enhance COX-2 mediated effects that occur only in neurons.
Significant strides have been made recently in developing therapies for NA intoxication. Current treatment involves a four-pronged approach (i.e., carbamate cholinesterase inhibitor, atropine, 2-PAM Cl and diazepam) aimed at counteracting the immediate effects of NA (Berry and Davies, 1970
; Dirnhuber et al., 1979
; Moore et al., 1995). This therapy is very effective in increasing survival after NA exposure but it does not always prevent seizures and neuronal damage. A number of modifications in this treatment strategy have been made to optimize seizure control (McDonough et al., 2009
; Shih et al., 2007
) because the neuropathology associated with NA exposure is linked to seizure intensity and duration (Chapman et al., 2006
). It is evident that several different pathological systems are recruited after NA exposure. The early cholinergic overload leads to seizures that can be controlled by anti-muscarinic drugs. A second, later phase of increased excitatory (glutamate/NMDA) transmission follows and seizures occurring in this phase are responsive to benzodiazepines. However, limits in the duration of their anti-seizure activity or delays in their application after NA exposure can lead to the re-emergence of seizures and the risk of additional brain damage.
It has become clear that the development of neuronal damage after exposure to NA can be quite protracted. In animal models of NA poisoning, a progressive neuronal degeneration in hippocampus (Collombet et al., 2006
) and amygdala (Collombet et al., 2008
) continues for 60-90 days after exposure. Behavioral deficits reflective of the brain region damaged are also seen chronically to include altered cognition in the case of hippocampal damage (Filliat et al., 2007
; Grauer et al., 2008
) and increased emotional behavior (e.g., conditioned and unconditioned fear) in the case of damage to the amygdala (Coubard et al., 2008
). In humans exposed to NA (victims of the Tokyo sarin attack (Ohbu et al., 1997
)) emotional and cognitive changes have been seen up to 7 years post-exposure (Miyaki et al., 2005
) and may be related to persistent structural changes in brain caused by a single exposure to sarin (Yamasue et al., 2007
). In light of these findings, the present results suggest the possibility that COX-2 expression is triggered as a late-occurring response to soman-induced seizures, and the associated increase in the production of PGEs could play a role in neuronal degeneration well after early cholinergic and glutamatergic overloads. Seizures arising in response to a PGE overload will not likely respond to anticholinergics and benzodiazepines. Therefore, some consideration should be given to the use of COX-2 selective inhibitors (NSAIDs) to prevent or minimize neuronal damage that occurs through non-cholinergic and nonglutamatergic mechanisms.