Differential effects of non-steroidal anti-inflammatory drugs on mitochondrial dysfunction during oxidative stress

Brain Research, 462 (1988) 275-285 275 Elsevier

BRE 13990

Differential effects of non-steroidal anti-inflammatory drugs on seizures produced by pilocarpine in rats

C. I k o n o m i d o u - T u r s k i 1'* , E . A . C a v a l h e i r o 2, L. Turski 1 , Z . A . B o r t o l o t t o 2, Z. K l e i n r o k 1, L.S . C a l d e r a z z o - F i l h o 2 and W . A . Tur sk i 1

1Department of Pharrnacology, Medical School, Lublin (Poland) and 2 Escola Paulista de Medicina, Department of Neurology and Neurosurgery, Laboratory of Experimental Neurology, Sao Paulo, S.P. (Brazil)

(Accepted 3 May 1988)

Key words: Pilocarpine; Non-steroidal anti-inflammatory drugs; Seizure; Epilepsy; Brain damage

The muscarinic cholinergic agonist pilocarpine induces in rats seizures and status epilepticus followed by widespread damage to the forebrain. The present study was designed to investigate the effect of 5 non-steroidal anti-inflammatory drugs, sodium salicylate, phenylbutazone, indomethacin, ibuprofen and mefenamic acid, on seizures produced by pilocarpine. Pretreatment of rats with sodium salicylate, EDs0 103 mg/kg (60-174), and phenylbutazone, 59 mg/kg (50-70) converted the non-convulsant dose of pilocarpine, 200 mg/kg, to a convulsant one. Indomethacin, 1-10 mg/kg, and ibuprofen, 10-100 mg/kg, failed to modulate seizures produced by pilocarpine. Mefenamic acid, 26 (22-30) mg/kg, prevented seizures and protected rats from seizure-related brain damage induced by pilocarpine, 380 mg/kg. These results indicate that non-steroidal anti-inflammatory drugs differentially modulate the threshold for pilocarpine-induced seizures.

INTRODUCTION

The salicylates and non-steroidal anti-inflammato-

ry drugs belong to the most widely used therapeutics in human medicine 33. Their therapeutic indications

extend from pain medication to treatment and pro-

phylaxis of cardiovascular diseases and the therapy of inflammatory states, rheumatoid arthritis and de- generative disorders 33. The mechanism responsible

for pharmacological actions of anti-inflammatory

drugs involves interference with prostaglandin metabolism 11,33.

Attention to the role of prostaglandins in the central nervous system increased after the finding of

Hor ton in 1964 that intracerebroventricular or intra- venous infusions of prostaglandins of the E series

produce stupor and prolonged sedation in cats and chicks 19. There is some uncertainty over the physio- logical role of prostaglandins in the mammalian

brain, mainly because concentrations of prostanoids and their precursor, an unsaturated fatty acid arachi- donate, are very low under normal conditions 7'14' 15,23. However, experimental evidence shows that hy-

poxia and convulsions trigger the release of arachido- nate from membrane phospholipids 44 and accelerate

its metabolism via the cyclooxygenase pathway to prostaglandins 3"5"6"15"17"44. A marked rise in free fatty

acid content 3'23 and increased synthesis of prosta-

glandins in the brain is seen during chemically and electrically induced convulsions in rodents 3"5'7'16, 28,32,34. More detailed studies demonstrated a posi-

tive correlation between the duration of seizures and prostanoid formation in brain regions primarily in- volved in the epileptic process 3A3'29'3°. A decreased

formation of prostaglandins in spontaneously convulsing gerbils 31 gave rise to the assumption that in-

creased synthesis of prostanoids in the brain might serve as an endogenous anticonvulsant mechanism.

* Present address: Department of Psychiatry, Washington University School of Medicine, 4940 Audubon Avenue, St. Louis, MO 63110, U.S.A.

Correspondence: L. Turski. Present address: Department of Neuropsychopharmacology, Schering AG, Sellerstr. 6-8, D-1000 Berlin 65, F.R.G.

0006-8993/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)

276

In fact, inhibition of brain prostaglandin formation by non-steroidal anti-inflammatory drugs increases the susceptibility of experimental animals to seizures 1<31"34. Furthermore, certain anti-inflammatory

drugs produce electrographic changes consistent with CNS excitation 42 and can, themselves, be epi- leptogenic in high doses 2"42"45.

The present study was therefore designed to investigate the effect of the 5 most commonly prescribed non-steroidal anti-inflammatory drugs, sodium salicylate, phenylbutazone, indomethacin, ibuprofen and mefenamic acid, on the convulsant action of pilocarpine in rats. The pilocarpine model has been found to be useful for studying preventive and therapeutic approaches to therapy-resistant forms of epilepsy 37,4°.

Some of these data have been communicated to the Ninth Congress of the Polish Pharmacological So- ciety in Lublin 36.

MATERIALS AND METHODS

Animals The experimental subjects were adult male Wistar

rats, 250-280 g in weight. The rats were housed individually and maintained on a standard light-dark cycle with ad libitum access to chow pellets and wa- ter. The assignment of rats to experimental groups and the determination of subsequent time of behavioral and electroencephalographic (EEG) testing for different treatment groups was random. The behavioral observations and monitoring of the EEG took place between 08.00 and 20.00 h. Experimental groups consisted of 4-12 animals.

Drugs Pilocarpine hydrochloride, methylscopolamine ni-

trate, sodium salicylate, indomethacin, ibuprofen, mefenamic acid and phenylbutazone were obtained from Sigma (St. Louis, MO, U.S.A.). The drugs were dissolved in saline or suspended in a 3% solu- tion of Tween 80 (Loba, Vienna, Austria) and administered i.p. or s.c. in a volume of 0.5 ml/100 g body wt.

Pilocarpine was administered i.p. in doses of 200 and 380 mg/kg. Methylscopolamine, 1 mg/kg, was administered s.c. 30 min prior to the injection of either dose of pilocarpine.

Sodium salicylate was administered in doses of 50, 100,200 and 300 mg/kg, phenylbutazone in doses o f 25, 50, 75 and 100 mg/kg. Indomethacin was given in doses of 1, 5 and 10 mg/kg, while ibuprofen in doses of 10, 25, 50 and 100 mg/kg. Mefenamic acid was administered in doses of 10, 20, 30 and 40 mg/kg.

Sodium salicylate and phenylbutazone were administered 45 min prior to the injection of pilocarpine, 200 mg/kg. Mefenamic acid was administered 45 min prior to pilocarpine, 380 mg/kg. Indomethacin

and ibuprofen were given 45 min prior to either 200 or 380 mg/kg pilocarpine. The timing interval chosen for non-steroidal anti-inflammatory drugs is related to their maximal efficacy in suppressing seizures in other experimental models of epilepsy 34'42. The dose

of the non-steroidal anti-inflammatory drug required

to trigger or block the seizure response in 50% of rats (EDs0 effective dose) given 200 and 380 mg/kg of pilocarpine, respectively, was determined by computer analysis of the data obtained from experiments with 3 or 4 different dosages. The criterion used to indicate convulsive response was status epilepticus defined as continuous seizures (stage IV/V according to Ra- cine 27) persisting for a period of at least 30 min before

spontaneous termination. The incidence of seizure response (probit transformed percentages) was plotted vs log dose of either drug tested. The EDs0 and the confidence limits were estimated by fitting the data by linear regression analysis 22.

Surgery and electrophysiological procedures For depth recordings, bipolar twisted electrodes

(tip diameter 100 /~m, interelectrode distance 500 /~m) were stereotaxically positioned in the dorsal hippocampus (AP 4.0, L +2.6, V +1.7) and amygdala (AP 4.6, L +4.0, V-3 .6) 2t under sodium pentobarbital (Nembutal, Ceva, Neuilly-sur-Seine, France; 50 mg/kg i.p.) anesthesia and anchored to the skull with dental acrylic. Surface recordings were led from jew- eller screws positioned bilaterally over the occipital cortex. An additional screw placed in the frontal si- nus served as a reference (indifferent) electrode. Sig- nals under investigation were amplified by a Beck- man model RM polygraph (time constant 0.03 s, high cut off filter 15 Hz). EEG recordings and behavioral observations were carried out in a plexiglass compartment (30 x 30 x 45 cm). Before the monitoring of the EEG, animals were individually placed in the

recording compartment and allowed 30 min for habit- uation to the recording set-up. The baseline EEG recordings were made for 30 min and the animals received an i.p. injection of anti-inflammatory drugs or solvent. Subsequently, the rats received pilocarpine, 200 or 380 mg/kg i.p. EEG recordings were made continuously and behavior noted for periods ranging from 4 to 8 h following the injection of pilocarpine. Additional recordings were made between 10-12, 20-24, 36-48 h and 5-10 days. The correct location of implanted electrodes was confirmed histologically in Cresyl violet-stained serial sections.

Morphological techniques The brains were processed for morphological anal-

ysis by light microscopy 24-72 h, 5-14 days and 21-31 days after the administration of pilocarpine and anti-inflammatory drugs. The details for histolo- gical processing are described elsewhere 2°'36. Brief- ly, the rats were anesthetized with an overdose of sodium pentobarbital and perfused with fixative con- taining 10% acetic acid, 10% formaldehyde and 80% methanol. The brains were fixed in situ at 4 °C for 24 h, then removed and processed for paraffin embed- ding. Subsequently, serial sections of the entire brain were cut coronally at 10 pm, with every 10th section being mounted on a glass slide and stained with Cre- syl violet or according to the Fink and Heimer tech- nique 1°.

RESULTS

Behavior Pilocarpine. Pilocarpine, 200 mg/kg (n = 6), ren-

dered animals akinetic immediately following the injection. Mild tremor of the head, occasional myoclo- nic movements or head bobbing, scratching and teeth chattering comprised the behavior for up to 1-2 h following the injection. This activity subsided within 30-60 min and behavior was indistinguishable from that in saline-treated control rats (n = 4). None of the rats subjected to pilocarpine, 200 mg/kg, developed seizures or status epilepticus.

Piiocarpine, 380 mg/kg (n = 5), induced akinesia, ataxic lurching, gustatory automatisms and head tremor immediately following the injection. After 20-30 min this behavior progressed to limbic seizures with rearing, forelimb clonus, salivation, masticatory

277

jaw movements and falling. The seizures commenced after 32.5 _+ 5 min (n = 5), recurred every 2-5 min, and led to status epilepticus at 50-60 min. Two animals died in the course of seizures elicited by pilocarpine, 380 mg/kg (2/5).

Sodium salicylate. Sodium salicylate increased the severity of seizures produced by pilocarpine, 200 mg/kg, in a dose-dependent manner. The EDs0 for sodium salicylate as a factor triggering seizures in rats treated with pilocarpine, 200 mg/kg, was 103 mg/kg (61-174; n -- 28). Two out of 6 animals receiving sodium salicylate, 50 mg/kg, and pilocarpine, 200 mg/kg, developed limbic seizures and status epilepticus (2/6). Four out of 6 rats treated with sodium salicylate, 100 mg/kg, and pilocarpine, 200 mg/kg, showed seizures and status epilepticus (4/6). Appli- cation of sodium salicylate, 200 mg/kg, led to limbic seizures and status epilepticus in 5 out of 10 rats (5/10) treated with pilocarpine, 200 mg/kg. Three animals died in the course of seizures (3/10). In animals pretreated with sodium salicylate, 300 mg/kg (n = 6), pilocarpine, 200 mg/kg, elicited limbic seizures and severe status epilepticus in 5 out of 6 rats, which led to the death of 3 rats (3/6). Sodium salicylate alone failed to induce either akinesia or muscle relaxation in doses of less than 300 mg/kg.

Phenylbutazone. Phenylbutazone increased the susceptibility of rats to seizures produced by pilocarpine, 200 mg/kg, with an EDs0 of 59 mg/kg (50-70; n = 24). Although none of 4 rats receiving phenylbutazone, 25 mg/kg (0/4), developed limbic seizures, two out of 6 animals pretreated with 50 mg/kg displayed convulsions and status epilepticus (2/6) following pilocarpine, 200. The status epilepticus led to the death of one animal (1/6). The treatment with phenylbutazone, 75 mg/kg, and pilocarpine, 200 mg/kg, led to limbic seizures and status epilepticus in 5 out of 7 rats (5/7). In this treatment group status epilepticus led to the death of 4 ani~nals (4/7). With phenylbutazone, 100 mg/kg, and pilocarpine, 200 mg/kg, all 6 rats developed status epilepticus (6/6). Four rats from this treatment group died in the course of protracted seizures (4/6). Phenylbutazone, 100 mg/kg, slightly re- duced the muscle tone in rats.

Indomethacin. Indomethacin did not trigger seizures in rats subjected to subconvulsant dose of pilocarpine, 200 mg/kg. None of the rats given indomethacin, 1 (n = 4), 5 (n = 4) and 10 mg/kg (n = 7), and

278

pilocarpine, 200 mg/kg, developed limbic seizures or status epilepticus. Indomethacin, 1 (n = 4), 5 (n = 7) and 10 mg/kg (n = 12), had also no effect on the severity of seizures and the lethal toxicity of pilocarpine, 380 mg/kg. All rats receiving indomethacin and pilocarpine, 380 mg/kg (n = 23), displayed limbic seizures and status epilepticus after a short latency of 20-35 min. Seven animals (7/23) died in the course of seizures between 2 and 4 h following the injection of pilocarpine. The remaining 16 rats displayed a full pattern of convulsions culminating in status epilepticus. Indomethacin, 5 and 10 mg/kg, did not affect ex- ploratory activity in rats. Neither loss of righting re- flex nor muscle relaxation was detected following the treatment with indomethacin, 5 and 10 mg/kg.

lbuprofen. Ibuprofen did not trigger convulsions in rats treated with pilocarpine 200 mg/kg. The rats re-

ceiving ibuprofen in doses of 10 (n = 4), 25 (n = 4), 50 (n = 8) and 100 mg/kg (n -- 4), and pilocarpine, 200 mg/kg, did not develop convulsions usually elicited by pilocarpine, 380 mg/kg, in drug naive rats 39'4°.

Ibuprofen, 50 mg/kg (n = 5) and 100 mg/kg (n = 6), also conferred no protection against seizures induced by pilocarpine, 380 mg/kg. The time course, spec- trum and quality of behavioral alterations did not differ from those monitored following application of pilocarpine, 380 mg/kg, alone. The lethal toxicity of pilocarpine, 380 mg/kg, in ibuprofen treated rats did not differ from that in drug-naive rats. The high doses of ibuprofen, 50 and 100 mg/kg, resulted in slight akinesia.

Mefenamic acid. Mefenamic acid had a marked suppressant action upon the convulsions induced by pilocarpine, 380 mg/kg. The EDs0 for mefenamic

a b 5rain Ilfte~ NSL C I I rain d 311 rain

C A 1 _ . : - , - - r ~ . . . . . . - % = : . : . . . . : . . : • . = : L ~ . . ~ ~ • . - ~ . , . ~ . ~ . . , : ; ~ ' = . : = ~ - t : ~ : . . . . : - • :" , , ~ , . , ~ " J - : - t . " . " " " t ' : . " ~ .

e )rain |f l l~ PILO f IO rain g 21 rain h 32 rain HPC - _ . . . . ,,_ . . . . .-~_ . . . . . ; .=!. ,_,~_:,. ,_., ,a~...~,.k., . . .~_. I I i , , , ~J. IJ.I..LLr L..,Jj LlJJ.

Fig. ]. The electrographic activity elicited by pilocarpine (PILO) in rats subjected to the treatment with sodium salicylate (NSL). NSL, 300 mg/kg, was administered i.p. 45 rain prior to injection of PILO, 200 mg/kg, a: pre-drug control recordings, b-d: electrographic correlates 5-38 min after the injection of NSL. e: significant 0-rhythm in the hippocampus and low-voltage fast activity in the AMG and CX are registered 3 min after PILO. f: high-voltage spiking is registered in the hippocampus, while low voltage activity dominates amygdala and cortex, g,h: the spiking activity becomes synchronized in all three recordings within 20-30 rain. i: the first electrographic seizure 53 rain after the injection of PILO. The waxing of the seizure is faster in the hippocampus and amygdala relative to cortex, j: the elecffographic seizures of variable duration recur every 3- ]0 min and progress to the status epilepticus within 1-2 h. k-m: the eiectrographic activity recorded 2-4 h after administration of PILO during status epilepticus, n: high voltage spiking with progressive depression of the background activity is registered 9-12 h after PILO. o: high-voltage spiking and depressed background activity characterize the EEO 16-24 h following the injection of PILO. Triangles ( ~ ) represent wet shakes. AMG, amygdala: CX, cortex; HPC, hippocampus.

279

acid against seizures produced by pilocarpine, 380 mg/kg, was 26 mg/kg (22-30; n = 22). Mefenamic acid, 10 mg/kg, did not affect seizures and lethal toxicity produced by pilocarpine, 380 mg/kg. All 5 rats in this group exhibited severe convulsions and status epilepticus (5/5), which led to the death of two animals (2/5). Mefenamic acid, 20 mg/kg, protected one out of 4 rats against iimbic seizures and status epilepticus (3/4), while 30 mg/kg conferred protection of 3 out of 6 rats against status epilepticus induced by pilocarpine, 380 mg/kg (3/6). Mefenamic acid, 40 mg/kg, protected 7 out of 7 rats against seizures elicited by pilocarpine, 380 mg/kg (0/7). Two animals in this group developed single episodes of seizures, which did not proceed to status epilepticus. The lethal toxicity of pilocarpine, 380 mg/kg, was abolished by mefenamic acid, 40 mg/kg. With mefenamic acid, 30 and 40 mg/kg, animals were sedated and showed a lowered muscle tone.

Electroencephalography Pilocarpine. Pilocarpine, 200 mg/kg (n = 4), pro-

duced significant theta (0 rhythm in the hippocampus and low-voltage fast activity in the cortex and amygdala. Isolated spikes or bursts of polyspiking sporadi- cally occurred in the hippocampus 1-2 h after the administration of the drug. The EEG was indistinguishable from background pattern 2-4 h after the application of pilocarpine, 200 mg/kg.

The injection of pilocarpine, 380 mg/kg (n = 4), induced a significant 0-rhythm, which superseded the background activity in the hippocampus, whereas low-voltage fast activity occurred in the cortex and amygdala. This activity progressed to high-voltage fast activity with spiking in the hippocampus. The spiking activity rapidly spread to amygdala and cortex, became well synchronized and evolved into electrographic seizures. The seizures recurred every 3-10 min and led to status epilepticus 40-60 min af-

a b 10 min after PBZ c 32 min d 5 min after PILO HPC ~'~ ~ ~ - ~ , ~ - , ~ e ~ ~ . v ~

e 12 rain | 23min g38 rain I;1 l HPC

4

h 47 min I 52 min

i~"," ,%111~ ( ~ I tII~I~]Ttl 1I , , ' ~ ' l I~ ~ I lilll llll ~'~ i

HPC

Is

Fig. 2. The electrographic activity elicited by pilocarpine (PILO) in rats subjected to the treatment with phenylbutazone (PBZ). PBZ, 100 mg/kg, was administered i.p. 45 min prior to injection of PILO, 200 mg/kg, a: pre-drug control recordings, b,c: electrographic correlates 10 and 32 min following the injection of PBZ. d: a significant 0-rhythm represents the characteristic pattern of the EEG registered 2-10 min following the injection of PILO. e: high-voltage spiking is initially recorded in the hippocampus, whereas the activity in amygdala and cortex becomes faster, f: the spiking activity spreads to the amygdala and cortex within 20-25 min post-injection, and becomes well synchronized in all records, g: the first electrographic seizure registered 38 min after the injection of PILO. Spiking activity originating in the hippocampus precedes the evolution of the seizure, h,i: the seizures recur every 3-10 min and progress to status epilepticus within 1-2 h after PILO. j: the paroxysmal activity registered during status epilepticus 1 h after injection of PILO. k: the activity pattern registered 4 h after injection of PILO. By 12-24 h, the EEG progressively normalizes, the paroxysmal activity gradually abates and the background activity becomes depressed. The EEG returns to the pre-drug patterns after a delay of 3-7 days. AMG, amygdala; CX, cortex; HPC, hippocampus.

280

ter the injection of pilocarpine, 380 mg/kg. This pattern of EEG activity lasted for several hours (6-16 h) and then the seizure activity gradually abated. The EEG usually returned to the pre-drug pattern within 3-5 days.

Sodium salicylate. Sodium salicylate, 300 mg/kg (n = 6), did not change the electrographic activity in hippocampus, amygdala and cortex (Fig. lb-d). Fig. 1 demonstrates the electrographic alterations in rats pretreated with sodium salicylate, 300 mg/kg, and subjected to the action of pilocarpine, 200 mg/kg. High-voltage spiking and fast activity rapidly ap- peared (up to 10 min) in the hippocampus (Fig. If), whereas low-voltage fast activity dominated amygdaloid and cortical recordings (Fig. le,f). The spiking and bursts of polyspiking spread to amygdala (Fig. lg) and cortex (Fig. lh). The seizures commenced 30-60 min following the injection of pilocarpine, 200 mg/kg (Fig. li). Wet dog shakes were frequently ob- served during the waning phase of the seizures (Fig. li). The ictal periods with well developed seizures recurred every 3-10 min and led to status epilepticus after 1-2 h (Fig. lk). This activity pattern lasted for several hours (6-12 h) and then gradually abated during 6-24 h (Fig. ll-n). The EEG remained de-

pressed within 24-48 h post-injection (Fig. lo) and slowly returned to the pre-drug patterns within 72-96 h.

Phenylbutazone. Phenylbutazone, 100 mg/kg (n = 4), transiently increased the voltage but did not change the morphology of the EEG in all recordings (Fig. 2b-d). The electrographic activity following the injection of pilocarpine, 200 mg/kg, in rats pretreated with phenylbutazone, 100 mg/kg, was charac-

terized by rapid buildup of spiking activity (Fig. 2f), which spread to amygdala and cortex (Fig. 2f) and progressed to seizures within 20-40 min (Fig. 2g). The seizures led to status epilepticus after a latency of 30-60 min (Fig. 2j). Status epilepticus lasted for up to 6-10 h (Fig. 2k). The EEG gradually normal- ized within 24-48 h and was indistinguishable from the pre-drug pattern after 3-5 days.

Indomethacin. Indomethacin modified neither the EEG nor EEG changes produced by pilocarpine, 200 (n = 5) or 380 mg/kg (n = 7). After the injection of pilocarpine, 200 mg/kg, in rats pretreated with indomethacin, 10 mg/kg, the quality of electrographic alterations did not differ from that registered in drug- naive rats subjected to the action of this dose of pilocarpine. Administration of pilocarpine, 380 mg/kg, in

a b 8 min after MEF c 30 min

d 5min after PILO e 13rain f 32 rain

g 46min h lh i 2h

AMG

Fig. 3. The electrographic activity elicited by pilocarpine (PILO) in rats subjected to the treatment with mefenamic acid (MEF). MEF, 40 mg/kg, was administered i.p. 45 min prior to injection of PILO, 380 mg/kg, a: pre-drug control recordings, b,c: the electrographic correlates 8 and 30 min after the injection of MEF. d: high-voltage fast activity replaces theta rhythm in the hippocampus and dominates amygdaloid and cortical records 5 min after PILO. e,f: isolated spikes and bursts of polyspikes are transiently registered in the hippocampus and amygdala 10-30 min following the injection of PILO, whereas cortical records remain fast. g,h: high voltage fast activity alternates with 0-rhythm, while bursts of polyspikes are registered in amygdala for up to 1 h after PILO. i: 1-2 h after PILO EEG normalizes in all recordings (a). AMG, amygdala; CX, cortex; HPC, hippocampus.

rats receiving indomethacin, 10 mg/kg, resulted in a sequential development of severe seizures and status epilepticus. In animals, which survived status epilepticus, the seizure activity gradually abated during 6-24 h and the EEG returned to the pre-drug pattern within 3-7 days, although isolated spikes were registered up to 10 days after the administration of pilocarpine.

Ibuprofen. Ibuprofen, 100 mg/kg (n = 9), depressed the electrographic activity in all recordings shortly after the injection. High voltage slow activity and slow waves prevailed in the EEG for up to 2-4 h. The pattern of electrographic activity induced by piiocarpine, 200 mg/kg (n = 4), in rats pretreated with ibuprofen, 100 mg/kg, did not differ from that registered in drug-naive rats. Ibuprofen, 100 mg/kg (n = 5), did not change the susceptibility of rats to seizures produced by pilocarpine, 380 mg/kg. In such rats, the 0-rhythm in the hippocampus was replaced with high- voltage fast activity and spiking, which rapidly spread to amygdala and cortex. This activity gradually progressed to electrographic seizures and status epilepticus. The quality and timing of electrographic changes did not differ from those registered in drug- naive rats subjected to the convulsant action of pilocarpine, 380 mg/kg.

Mefenamic acid. After the injection of mefenamic acid, 40 mg/kg (n = 7), the low-voltage fast activity superseded background activity in the hippocampus, amygdala and cortex (Fig. 3b,c). This pattern of electrographic activity, registered in all recordings for up to 1 h following the injection of mefenamic acid, 40 mg/kg, was interrupted by isolated spikes. Pilocar- pine, 380 mg/kg, induced a reappearance of the 0- rhythm in the hippocampus (Fig. 3d) and generated high-voltage fast activity in the amygdala and cortex (Fig. 3d) in rats pretreated with mefenamic acid, 40 mg/kg (n = 7). The 0-rhythm alternated with periods of high-voltage fast activity and spiking during 30 min to 1 h (Fig. 3e-g). The amygdala recordings showed high-voltage fast activity and isolated spikes or bursts of polyspiking (Fig. 3g,h). The cortical recordings displayed minor changes limited to the appearance of short-lasting periods of fast activity. This type of transient alterations disappeared within 2-3 h (Fig. 3h,i). The EEG became indistinguishable from pre- drug activity after 1-2 h (Fig. 3i).

281

Neuropathology Pilocarpine. Pilocarpine, 200 mg/kg (n = 4), did

not induce detectable damage to the forebrain in rats. The brains of rats subjected to the convulsant action of pilocarpine, 380 mg/kg (n = 3), showed a characteristic damage pattern involving the hippocampus, thalamus, amygdaloid complex, piriform and entorhinal cortex, neocortex and substantia nigra. The detailed topography and extent of the damage to the rat forebrain following seizures elicited by pilocarpine, 380 mg/kg, have been thoroughly described elsewhere 39.4°.

Sodium salicylate. Extensive damage to the hippocampus, thalamus, amygdala, piriform and entorhinal cortex, neocortex and substantia nigra was found in brains of rats, which received sodium salicylate and pilocarpine, 200 mg/kg (Fig. 4A,H). The topography of this damage resembled that produced by convulsant dose of pilocarpine, 380 mg/kg. Two out of 6 rats receiving sodium salicylate, 50 mg/kg, showed a widespread damage to the brain. With sodium salicylate, 100 mg/kg, 4 out of 6 rats developed damage to the forebrain, while two rats which survived the treatment with sodium salicylate, 200 mg/kg, and pilocarpine, 200 mg/kg, underwent morphological injury. Two rats receiving sodium salicylate, 300 mg/kg, and pilocarpine, 200 mg/kg, which survived status epilepticus also showed severe de- structions in the dorso-lateral parts of the caudate- putamen and one animal displayed cell loss in the glo- bus pallidus. The rats was greater treatment groups pine, 200 mg/kg.

extent of the damage in these two relative to that detected in other with sodium salicylate and pilocar-

Phenylbutazone. Phenylbutazone increased the susceptibility of rats to the brain damaging action of pilocarpine (Fig. 4C,G). Four rats, which received phenylbutazone, 25 mg/kg, and pilocarpine, 200 mg/kg, but did not develop seizures showed no pa- thology in their brains. With phenylbutazone, 50 mg/kg, one out of two rats, which survived status epilepticus developed damage to the forebrain. The extent of the damage in this animal was limited to the thalamus, CA1 subfield of the hippocampus, amygdala, piriform cortex and substantia nigra. The damage to the neocortex and entorhinal cortex was less relative to drug-naive rats subjected to convulsant action of pilocarpine, 380 mg/kg. The damage to the

b~

forebrain in rats, which survived the treatment with

phenylbutazone, 75 mg/kg (n = 1) or 100 mg/kg (n = 2), and pilocarpine, 200 mg/kg, was widespread

and more severe relative to rats subjected to moder-

ate doses of the drug.

Indomethacin and ibuprofen. Neither indometha-

cin, 10 mg/kg (n = 3), nor ibuprofen, 100 mg/kg (n =

2), affected the development of the damage to the

forebrain induced by pilocarpine, 380 mg/kg (Fig. 4E). Both drugs had no effect on the susceptibility of

rats to the brain damaging action of pilocarpine, 200

mg/kg, as two rats receiving indomethacin, 10 mg/kg,

and two rats receiving ibuprofen, 100 mg/kg, and pi- Iocarpine, 200 mg/kg, developed no morphological

alterations indicative of cell injury (Fig. 4F).

Mefenarnic acid. Mefenamic acid had a marked

suppressant effect upon the morphological sequelae of seizures produced by pilocarpine, 380 mg/kg (Fig.

4B,D). None out of 7 rats pretreated with mefenamic

acid, 40 mg/kg, and pilocarpine, 380 mg/kg, devel-

oped morphological alterations in the brain. After

mefenamic acid, 30 mg/kg, 3 out of 6 animals showed a protection against neuronal degeneration. One animal in this group died in the course of seizures, while

two remaining rats showed an attenuated pattern of damage confined to the piriform cortex, hippocam-

pus and thalamus. Mefenamic acid, 20 mg/kg, protected one out of 4 rats against seizures and brain

damage induced by pilocarpine, 380 mg/kg, while 10

mg/kg provided no protection against cell death in

the forebrain of 3 animals.

283

DISCUSSION

This study demonstrates that sodium salicylate' and

phenylbutazone increase the susceptibility of rats to seizures induced by pilocarpine. Indomethacin and

ibuprofen have no influence on the convulsant action

of pilocarpine, whereas mefenamic acid prevents pi-

locarpine-induced convulsions and protects against

epileptic brain damage.

The non-steroidal anti-inflammatory drugs have a

variety of actions including blockade of adenosine uptake, interference with Ca 2÷ uptake, inhibition of

prostaglandin inactivating enzymes, competition for

receptor sites with specific prostaglandins and inhibition of prostaglandin synthetase 12. The seizure-mod-

ulating action of non-steroidal anti-inflammatory

drugs is interpreted in terms of inhibition of brain prostaglandin synthesis 35. The lowering of the sei-

zure threshold for pilocarpine by sodium salicylate

and phenylbutazone might also be due to inhibition

of brain prostanoid synthesis. This proposal, however, offers no explanation for the failure of indome-

thacin and ibuprofen to modulate the threshold for pilocarpine-induced seizures. Indomethacin and ibu-

profen are actually more efficient inhibitors of prostanoid formation than salicylates ~'12"34. In the pentyl-

enetetrazol seizure model in mice indomethacin and

ibuprofen enhance the lethal toxicity and indomethacin accelerates the onset of convulsions 34. Indome-

thacin and ibuprofen also block adenosine uptake in

rat brain synaptosomes, whereas phenylbutazone is

Fig. 4. The damage to the forebrain induced by pilocarpine in rats subjected to the action of non-steroidal anti-inflammatory drugs. Survival times: 72 h to 21 days. Cresyl violet or Fink-Heimer stain, A: a breakdown of the morphological structure of the CA 1 subfield of the dorsal hippocampus of a rat pretreated with sodium salicylate, 300 mg/kg, and subsequently given pilocarpine, 200 mg/kg. The neuronal population of the pyramidal layer is severely damaged. Shrunken neurons are surrottnded by dilated spaces. Survival time: 10 days. x 196. B: no sign of neuronal degeneration is visible in the CA3 subfield of the dorsal hippocampus in a rat pretreated with mefenamic acid, 40 mg/kg, prior to pilocarpine, 380 mg/kg. Survival time: 5 days. x 196. C: advanced necrosis with neuronal depopula- tion and disruption of the neuropil in the lateral thalamus of the rat treated with phenylbutazone, 100 mg/kg, and pilocarpine, 200 mg/kg. Survival time: 3 days. × 196. D: mefenamic acid, 40 mg/kg, protects lateral thalamus from morphological sequelae of seizures induced by pilocarpine, 380 mg/kg. This photomicrograph demonstrates unchanged morphology of lateral thalamus, Survival time: 21 days. x 196. E: extensive disruption of the cytoarchitecture of the pyriform cortex of the rat treated with indomethacin, 10 mg/kg, and pilocarpine, 380 mg/kg. Disintegration of the neuropil and edema are evident throughout all layers of the pyriform cortex. Survival time: 7 days. × 196. F: a normal morphology of the pyriform cortex in a rat treated with ibuprofen, 100 mg/kg, and pilocarpine, 200 mg/kg. No sign of neuronal injury is seen throughout the pyriform cortex. Survival time: 5 days. × 196. G: a grave breakdown of the morphological structure of substantia nigra pars reticulata after treatment with phenylbutazone, 100 mg/kg, and pilocarpine, 200 mg/kg. Survival time: 3 days. × 196. H: extensive neuronal loss and edema in the basolateral amygdaloid nucleus in the rat treated with sodium salicylate, 300 mg/kg, and pilocarpine, 200 mg/kg, Nearly total neuronal depletion and destruction of the neuropil is present throughout the entire basolateral nucleus. Survival time: 5 days. x 196.

284

ineffective in this respect 26'43. Adenos ine and related

purines have potent depressant effects on central and

per ipheral neurons in mammals 26 and possess anti-

convulsant proper t ies against a wide range of chemi-

cally-induced seizures 9. 2-Chloroadenosine, an

adenosine receptor agonist, has been shown to pre-

vent the occurrence of convulsions and brain damage

in rats t rea ted with pi locarpine 38. Thus, the various

actions of indomethacin and ibuprofen, both on pros-

taglandin synthesis 35 and adenosine uptake 26 may in-

terfere with their effect on the seizure threshold for

pi locarpine.

The most intriguing finding of this study is the dem-

onstrat ion of an anticonvulsant effect of mefenamic

acid. This compound repor tedly produces CNS exci-

tation 42 and in high doses clonic/tonic seizures in ro-

dents 42 and humans 2'45. Al though controversy sur-

rounds the exact mechanism of the anticonvuisant ac-

tion of mefenamic acid in the pi locarpine model , the

actions of fenamates on prostaglandin metabol ism

and adenosine uptake may in some way aid the un-

derstanding of how this drug modulates the seizure

threshold. The fenamates antagonize the action of

prostaglandins in guinea pig and human al imentary 4

and bronchial muscle s , and inhibit adenosine uptake

in brain synaptosomal preparat ions 26. However , the

fenamates affect the uptake of adenosine less pro-

foundly than indomethacin and ibuprofen 26. Mefe-

namic acid is also a weaker inhibitor of pros tanoid

formation in comparison to indomethacin 12'24. The

demonstra t ion of anticonvulsant action of mefenam-

ic acid in pi locarpine model forms and interest ing

parallel to observations on seizure-protective effects

of mefenamic acid in rats subjected to systemic or in-

tracortical administrat ion of penicillin 41. In this prep-

aration mefenamic acid delayed the onset of seizures

and decreased the number of convulsions 41.

The present research demonstra tes that sodium sa-

licylate and phenylbutazone, at doses which corre-

spond to those used for ant i - inf lammatory medica-

tion in humans 33, facilitate the convulsant action of

pi locarpine in rats. Since the involvement of cholin-

ergic mechanisms in the generat ion of limbic seizures

in exper imental animals and man has recently been proposed 1s'25'39"4°, these results may indicate the

need for careful monitoring of the dosage of sodium

salicylate and phenylbutazone in the t rea tment of

chronic polyarthri t is or cardiovascular and degenera-

tive diseases in patients suffering from epilepsy.

ACKNOWLEDGEMENTS

This research was suppor ted by project grants

from the Polish Academy of Sciences, F A P E S P ,

CNPq and F I N E P (Brasil).

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