Mechanisms Underlying Neuroprotection by the NSAID Mefenamic Acid in an Experimental Model of Stroke

Abstract

Stroke is a devastating neurological event with limited treatment opportunities. Recent advances in understanding the underlying pathogenesis of cerebral ischemia support the involvement of multiple biochemical pathways in the development of the ischemic damage. Fenamates are classical non-steroidal anti-inflammatory drugs but they are also highly subunit-selective modulators of GABA_A receptors, activators of I_KS potassium channels and antagonists of non-selective cation channels and the NLRP3 inflammosome. In the present study we investigated the effect of mefenamic acid (MFA) in a rodent model of ischemic stroke and then addressed the underlying pharmacological mechanisms in vitro for its actions in vivo. The efficacy of MFA in reducing ischemic damage was evaluated in adult male Wistar rats subjected to a 2-h middle cerebral artery occlusion. Intracerebroventricular (ICV) infusion of MFA (0.5 or 1 mg/kg) for 24 h, significantly reduced the infarct volume and the total ischemic brain damage. In vitro, the fenamates, MFA, meclofenamic acid, niflumic acid, and flufenamic acid each reduced glutamate-evoked excitotoxicity in cultured embryonic rat hippocampal neurons supporting the idea that this is a drug class action. In contrast the non-fenamate NSAIDs, ibuprofen and indomethacin did not reduce excitotoxicity in vitro indicating that neuroprotection by MFA was not dependent upon anti-inflammatory actions. Co-application of MFA (100 μM) with either of the GABA_A antagonists picrotoxin (100 μM) or bicuculline (10 μM) or the potassium channel blocker tetraethylammonium (30 mM) did not prevent neuroprotection with MFA, suggesting that the actions of MFA also do not depend on GABA_A receptor modulation or potassium channel activation. These new findings indicate that fenamates may be valuable in the adjunctive treatment of ischemic stroke.

Keywords: MCAO; excitotoxicity; fenamates; ischemic stroke; neuroprotection.

FIGURE 1

The experimental protocol and timeline for the middle cerebral artery occlusion (MCAO) experiments. Top left is a schematic dorsal view of rat brain and middle cerebral artery and its major branches supplying blood to the temporoparietal cortex. The top right illustrates a ventral view of the intraluminal filament occluding the left internal carotid artery beyond the bifurcation of the left middle cerebral artery to induce MCAO. The lower panel shows the timeline for drug treatment, MCAO, reperfusion and post-MCAO evaluation.

FIGURE 2

Mefenamic acid (MFA) reduces brain damage following MCAO. Left inset shows a schematic of the rat brain with the diagonal lines representing the seven 2 mm thick coronal brain sections obtained from each animal. The top right shows a coronal section 0.0 mm from bregma in a control MCAO animal. The contralateral (non-injured) hemisphere appears red due to the reaction of viable cells with the TTC stain (red). The infarct, in the core of the ipsilateral (ischemic) hemisphere in the temporal cortex, appears white due to the absence of live cells. The penumbra in the striatum appears pink due to a mixed population of both live and dead cells. The lower panels show TTC stained serial sections (rostral to caudal is left to right) from a control (vehicle) and MFA (1 mg/kg) treated animal. Note the large infarcted cortical areas in the vehicle treated animal.

FIGURE 3

Mefenamic acid and sodium salicylate are neuroprotective in the rodent transient MCAO model. The histograms show (A) penumbra volume, (B) infarct volume, (C) total ischemic damage (TID) and (D) edema volume in animals treated with either MFA (0.5 or 1 mg/kg), sodium salicylate (1 mg/kg) or vehicle control. Drugs were infused into the left lateral cerebral ventricle by osmotic mini pump for 24 h beginning 1 h prior to MCAO. MFA significantly reduced infarct, edema, and TID; salicylate also reduced infarct and TID but the penumbra volume was not reduced in any of the treatment groups. Values are expressed as mean ± SEM for n = 8 in each group. ^∗p ≤ 0.05; ^∗∗p ≤ 0.01.

FIGURE 4

Excitotoxity was reduced by fenamate NSAIDs in vitro: (top) phase contrast photomicrographs of hippocampal neurons 9 days in culture untreated and cells after exposure to glutamate (Glu, 5 μM), glutamate plus MFA (100 μM) or glutamate plus MK-801 (10 μM). (Bottom) Shows histogram summaries of similar experiments conducted with mefenamic acid, flufenamic acid, niflumic acid or meclofenamic acid on glutamate induced cell death. Fenamates or MK-801 were co-incubated during glutamate exposure and immediately after exposure for 24 h. Cell death was assayed 24 h post-exposure and significantly reduced (^∗∗p ≤ 0.01) by treatment with all the fenamates at 100 μM. Niflumic acid and meclofenamic acid, at 10 μM, also significantly reduced cell death by 54% (^∗p ≤ 0.05) and 56% (^∗∗p ≤ 0.01) respectively. Note that MK-801 even reduced LDH (cell death) below the untreated cells. The scale bar is 40 microns at 300x magnification.

FIGURE 5

Impact of Cyclooxygenase inhibition on glutamate-evoked neurotoxicity: hippocampal neurons 9 days in culture were exposed to glutamate (5 μM) in the absence (control) or presence of either sodium salicylate (SAL), indomethacin (INDO), or ibuprofen (IBU) (each at 10 and 100 μM). Drugs were co-incubated during glutamate exposure and immediately after exposure for 24 h. Cell death was measured 24 h post-exposure using Cytotox-96. Sodium salicylate significantly reduced cell death by 56% (^∗∗p ≤ 0.01) and 65% (^∗∗p ≤ 0.01) at 10 and 100 μM, respectively. However, neither indomethacin nor ibuprofen reduced glutamate-induced cell death. The NMDA channel blocker, MK-801 (10 μM) reduced cell death by 98% (^∗∗p ≤ 0.01).

FIGURE 6

Impact of GABA_A receptor and ion channel inhibition on the neuroprotective effects of MFA in vitro: hippocampal neurons cultured for 9 days were exposed to glutamate (5 μM) and test drugs by co-incubating during exposure and immediately after exposure for 24 h. Cell death was quantified using Cytotox-96 at 24 h post-exposure. (Top) Treatment with MFA (100 μM), chlordiazepoxide (CDZ, 100 μM), or sodium pentobarbital (Pento, 100 μM) reduced glutamate induced cell death by 62, 74, and 81% (^∗∗p ≤ 0.01) respectively. Picrotoxin (PTX, 100 μM) abolished the neuroprotective effect of CDZ and sodium pentobarbital on glutamate-evoked cell death but did not significantly alter the action of MFA. Picrotoxin (100 μM) alone did not reduce glutamate evoked cell death. (Bottom) MFA, CDZ or pentobarbital (all tested at 100 μM) reduced glutamate induced cell death by 62, 74, and 81% (^∗∗p ≤ 0.01) respectively. Bicuculline (BIC, 10 μM) abolished the effect of pentobarbital and significantly reduced the neuroprotective effect of CDZ on glutamate induced cell death, but failed to alter the effect of MFA. Bicuculline (10 μM) alone did not reduce glutamate evoked cell death ^∗p < 0.05.

FIGURE 7

Involvement of K channels in the neuroprotective effects of MFA in vitro: hippocampal neurons in culture for 9 days were exposed to glutamate (5 μM). Drugs were co-incubated during glutamate exposure and immediately after for 24 h. Cell death was quantified using the Cytotox-96 24 h later. Treatment with MFA (100 μM) or nicorandil (NIC, 100 μM) reduced glutamate mediated cell death by 65 and 60% (^∗∗p ≤ 0.01) respectively. Tetraethylammonium (TEA, 30 mM) alone did not significantly alter the level of glutamate induced cell death. Co-application of TEA (30 mM) completely abolished the neuroprotective effect of nicorandil (100 μM) but co-incubation of TEA (30 mM) with MFA (100 μM) did not significantly affect the neuroprotective actions of this fenamate NSAID.