97
Brain Research, 553 (1991) 97-104 1991 Elsevier Science Publishers B.V. 0006-8993/91/$03.50 ADONIS 000689939116747X
BRES 16747
Motor and electroencephalographic response of refractory experimental status epilepticus in rats to treatment with MK-801, diazepam, or MK-801 plus diazepam Nancy Y. Walton and David M. Treiman Neurology and Research Services, Department of Veterans Affairs Medical Center, West Los Angeles, CA (U.S.A.) and Department of Neurology, UCLA School of Medicine, Los Angeles, CA (U.S.A.)
(Accepted 29 January 1991) Key words: Status epilepticus; Seizure; Lithium; Pilocarpine; MK-801; Diazepam; Electroencephalogram; Frequency analysis
Pharmacologic control of refractory status epilepticus has been little-studied in experimental models. In this experiment, rats in status epilepticus induced by lithium and pilocarpine were treated with MK-801 alone, diazepam alone or MK-801 plus diazepam, with treatment begun at a time when this model of status is refractory to anticonvulsant drugs. EEG samples were digitized before and for two hours after treatment, and the digitized samples subjected to computerized frequency analysis. MK-801 plus diazepam halted all manifestations of status epilepticus. Although neither MK-801 alone nor diazepam alone stopped the ongoing electrographie status epileptieus, both drugs diminished motor seizures and total EEG power. MK-801 treatment prevented the progression of changes in EEG pattern which normally occurs in this model of status epilepticus, while diazepam did not. MK-801, with and without diazepam, allowed the rats to survive the episode of status epilepticus, but rats treated with MK-801 alone required several days to recover completely, while the MK-801 plus diazepam rats appeared normal the next day. MK-801 may be a useful agent for treatment of human refractory status epilepticus, because of its neuroprotective action as well as its ability to potentiate GABAergic drugs.
INTRODUCTION Sequential injections of lithium and pilocarpine (separated by 20-24 h) reliably induce status epilepticus in rats which lasts from 3 - 6 h and is universally fatal 7,1°,14. Status epilepticus can be prevented if antiepileptic drugs are given before pilocarpine and status can be aborted if treatment is given shortly after the onset of seizure activity 12. If treatment is delayed until the status is well established, the seizures are refractory to treatment 12. We have described a predictable sequence of E E G patterns which occurs in generalized convulsive status epilepticus in humans as well as experimental models of status epilepticus in the rat (including lithium/pilocarpine) 17. Examples of these patterns, recorded from a single rat during an episode of status epilepticus induced by lithium and pilocarpine, are shown in Fig. 1. The transition of lithium/pilocarpine status epilepticus from responsive to refractory can be monitored by the E E G pattern seen at the time treatment is given. In an earlier experiment, we reported that 20 mg/kg diazepam controlled status in 100% of rats treated when the E E G
displayed discrete electrographic seizures (the initial status pattern) and 50% of rats treated when the E E G displayed merging seizures (the second pattern), but in only 17% of rats treated when the E E G displayed high-amplitude, continuous, rapid spiking or periodic epileptiform discharges (the third and fourth patterns) 2°. Lithiurn/pilocarpine status m a y provide a model for studying the basic mechanisms responsible for refractory status epilepticus in humans and evaluating new drugs with potential for treating this condition. We describe here experiments using MK-801, both alone and followed by diazepam, as a treatment for status epilepticus induced by lithium and pilocarpine which has progressed to the refractory stage. MK-801 is a noncompetitive N M D A antagonist which acts at the PCP site on the open cation channel associated with the N M D A receptor 5. O r m a n d y et al. x3 reported that MK-801 blocked the development of lithium/pilocarpine status epilepticus when it was given prior to pilocarpine; when given after status epilepticus was established, MK-801 had little effect on the ongoing electrographic activity, but did increase the 24 h survival rate. Fariello et al. 4
Correspondence: N.Y. Walton, Department of Neurology, UCLA School of Medicine, 710 Westwood Plaza (C-128), Los Angeles, CA 90024, U.S.A.
98 reported that while MK-801 protected against kainic acid neurotoxicity, the electrographic manifestations of kainate-induced status epilepticus were actually worsened by MK-801 pretreatment. Neuroprotective effects have also been described when MK-801 is given prior to onset of status epilepticus
induced by intrahippocampal quinolinic acid is. In this experiment, MK-801 and/or diazepam were administered after status epilepticus induced by lithium and pilocarpine was well established. Effects produced by either drug alone or the two drugs in combination on ongoing motor and electrographic manifestations of
~ ~ !
'1 '
/I
150M.V I
ii~
i
,~
II
'l
1~
i
I
15C NL
I second
I
!'
I
,~ '
t
I~
Isec
o.
e
,jr.
IJ
41
!
, t
~
t~
"i
~oo~v~ C.
f
'
*
.
I
!
/
:
H "~
i '~
',,I
I,'V
I'
II
"
I second
d
350/~Vl
Isec
Fig. 1. The predictable sequence of EEG patterns seen in generalized convulsive status epilepticus in humans as well as in experimental models of status epilepticus (SE) in the rat 17. Each of these examples is taken from the same rat, in which status epilepticus was induced by injections of lithium and pilocarpine. The time and sensitivity calibrations are noted for each EEG sample. Recordings are from epidural screw electrodes with channel 1 being fight frontal to parietal, channel 2 left frontal to parietal, channel 3 fight to left frontal, and channel 4 fight to left parietal. a: baseline EEG, recorded just prior to injection of piiocarpine, b: end of discrete electrographic seizure, 3 rain after onset of SE. c: merging seizures, 5 rain after onset of SE. d: continuous, high-amplitude, rapid spiking, 20 rain after onset of SE and 12 min after the onset of continuous spiking pattern, e: periodic epileptiform discharges (PEDs) on a relatively fiat background, 118 rain after onset of SE and 3 min after the first 10 second episode of PEDs.
99 status e p i l e p t i c u s w e r e e v a l u a t e d .
TABLE I
Drug regimens studied MATERIALS AND METHODS Adult, male Sprague-Dawley rats, weighing 175-200 g at the time of surgery (Bantin and Kingman) were used as subjects in this experiment. Rats were housed singly after surgery in plexiglas cages with contact bedding. Food and water were available ad lib throughout. Room temperature was maintained at 22 °C and a 24-h diurnal lighting schedule was controlled automatically, with lights on from 06.00 to 18.00 each day. Epidural recording electrodes were placed surgically under ketamine/xylazine anesthesia as has been described elsewhere 2°. One week postoperative recovery was allowed prior to experimentation. Lithium chloride was purchased from Sigma Chemicals, Inc, and dissolved at 0.9% for injection in sterile water. Pilocarpine was purchased from Sigma Chemicals and dissolved for injection in normal saline (100 mg/ml). MK-801 was purchased from Research Biochemicals Inc. and reconstituted in ethanol (50 mg/ml), then diluted to 2 mg/ml with 30% polyethylene glycol 400. Diazepam was purchased from Elkius-Sirm as a 5 mg/ml solution in a vehicle containing 40% propylene glycol, 10% ethanol and 1.5% benzyl alcohol. Vehicle solutions to match both MK-801 and diazepam were also prepared. An EEG sample was obtained prior to the pilocarpine injection and EEG was then monitored continuously. In order to obtain EEG samples for frequency analysis, the output from the EEG machine amplifiers was transmitted by cable to an IBM AT-compatible microcomputer via a LabMaster DMA analog-to-digital converter (Techmar). The system was calibrated using a 50/~V 16 Hz sine wave signal. Sixty-four second samples of artifact-free EEG were analyzed for each rat at the following times: baseline, after 10 min continuous spiking (just prior to the first treatment injection), after 30 min continuous spiking (10 rain after the second treatment injection), then every 30 rain thereafter. Frequency and Coherence Topography System software (Neurolmages, Inc.) was used to digitize and analyze the EEG. Data were sampled at 512 points per channel per second. The program analyzed the data into frequency spectra with a 4 Hz bandwidth covering the range from 2 to 58 Hz, based on a fast Fourier transform algorithm. The frequency ranges used for statistical analysis (2-6 Hz, 6-18 Hz, 18-58 Hz) were established by factor analysis of results from earlier experiments which showed that changes in power distribution in these ranges could account for 70% of the total variance seen. EEG power is a concept which has been described in various complex mathematical terms. The fast Fourier transform used in spectral analysis partitions the complex EEG waves into theoretical sine wave components with different frequencies. The power associated with ranges, or bins, of these frequencies can then be thought of as the voltage-time area covered by the sine waves. Power is thus a function both of how many sine wave cycles of various frequencies occur and their peak amplitudes. Because there is much debate as to what absolute unit of power is appropriate for EEG analysis 19, power in this study is expressed as a multiple of the power seen at baseline in the same animal. Status epilepticus was induced by injection of 3 mmol/kg lithium chloride i.p., followed in 20-24 h by s.c. injection of 30 mg/kg pilocarpine. Six rats were treated with each of the drug regimens shown in Table I, with the first injection given when the EEG had displayed a pattern of continuous, high-amplitude rapid spiking for 10 min. The second injection was given 10 min after the first. These rats were observed for 2 h after treatment. An additional 4 rats each were treated with 2 mg/kg MK-801, alone and with 20 mg/kg diazepam, observed continuously for at least 8 h, then rechecked the following morning (about 12 h later). Two rats each were treated with the MK-801 plus diazepam combination, but with treatment initiation postponed until either 30 or 60 min continuous spiking on EEG. These animals were observed for 2 h after treatment.
First injections were given at 1 ml/kg b. wt. Second injections were given at 4 ml/kg b. wt. n = 6 per group.
Group
First injection
Second injection
Vehicle
vehicle for MK-801 2 mg/kg MK-801
vehicle for diazepam vehicle for diazepam 20 mg/kg diazepam 20 mg/kg diazepam
MK-801 Diazepam MK 801 plus diazepam
vehicle for MK-801 2 mg/kg MK-801
In order to rule out the possibility that observed differences might have resulted from a pharmacokinetic interaction of MK-801 with diazepam, 6 rats were treated during status epilepticus with either vehicle plus diazepam or MK-801 plus diazepam, exactly as has already been described. These rats were decapitated 10 min after diazepam injection and the brains rapidly removed and frozen. Six non-seizing control rats were also treated with vehicle plus diazepam or MK-801 plus diazepam and decapitated 10 rain after the diazepam injection. Cortical diazepam concentrations were assayed using a modification of a method developed in this laboratory for Iorazepam 6. The solid-phase extraction step described for the Iorazepam assay was not used because too much diazepam was lost in this step and the run time was extended to 30 rain. Statistical analysis was done by two-way ANOVA. Treatment group (see Table I) served as one factor, while time from onset of continuous spiking served as the second factor (repeated measures on the same subjects were used for this factor). Individual comparisons were made using the Tukey HSD procedure. Statistics were calculated by computer using the SYSTAT program (Systat), with alpha set at 0.01.
RESULTS D a t a will b e p r e s e n t e d as a m e a n + s.d. unless n o t e d o t h e r w i s e . O n s e t o f status e p i l e p t i c u s , o p e r a t i o n a l l y d e f i n e d as t h e a p p e a r a n c e o f cortical spiking o n E E G , o c c u r r e d 21.5 _+ 7.5 m i n a f t e r i n j e c t i o n o f p i l o c a r p i n e ( r a n g e = 1 1 - 4 6 m i n ) . T h e E E G p a t t e r n o f discrete e l e c t r o g r a p h i c seizures lasted 6.0 + 2.6 m i n ( r a n g e = 2 - 1 3 m i n ) , f o l l o w e d by 6.6 + 3.8 m i n o f m e r g i n g seizures ( r a n g e = 2 - 2 2 m i n ) p r i o r to t h e o n s e t of c o n t i n u o u s spiking. O t h e r e x p e r i m e n t e r s 1°,12,13 h a v e r e p o r t e d treatm e n t i n j e c t i o n t i m e s in t e r m s o f m i n u t e s since i n j e c t i o n o f p i l o c a r p i n e . T h e i n t e r v a l f r o m p i l o c a r p i n e i n j e c t i o n to i n j e c t i o n of first t r e a t m e n t d r u g was 44.2 + 12.6 min ( r a n g e = 3 3 - 7 3 m i n ) in t h e s e rats. A n i m a l s t r e a t e d with t w o v e h i c l e i n j e c t i o n s c o n t i n u e d to h a v e m o t o r seizures consisting o f c o n t i n u o u s clonic j e r k i n g of the h e a d , plus f o r e l i m b c l o n u s visible w h e n e v e r t h e l i m b was n o t b e a r i n g w e i g h t . T h e rats t e n d e d to walk in circles, r a r e l y s t a n d i n g still. T h e y did n o t lose m o t o r t o n e o r t h e ability to m a i n t a i n an u p r i g h t p o s t u r e d u r i n g t h e 2-h o b s e r v a t i o n p e r i o d e x c e p t for b r i e f falls at
100 I
:I
I
I,
I
!
'. '
,,,
~
'
i ,l!
I !~~,
I,!~
'i~l;'A 4
V,
'
~
'~
J'
i
~
,i~r}' 1' }]'?lq'y\
',W~'J,
i,;~, i i l i
lO00~v[ I second
3~
700/~.V[~ I second
I
,
J
'
r
a W~#,~.d~,rW~,..~.%.4%~\,.~h,t~,Q~¢C~..~%4~ ,:%~,,.~v~%~,~,,,~,,.s
I! I I ' l J ! !
II
I
[ I ~,'
I
I I,'
II
;
'
,!''.~
,''
, I;
lO00~V I 2b
I second
3~
ZOo~vl I second
-!:: .'"tr, ~'~ ~
~
,'
~ . . ~ . \ ~ * '~
't' ~, .~,,,,.,~r',,,il ~'r,l ii,"f"T 4~'(/'t"],:,t(¢,. .g
,' /.4',i,,:)!4
a,
I
J
'i I
It
~.
7oo,,vl__ Isecond
Fig. 2. Examples of EEGs recorded 10, 30 and 120 min from the onset of continuous spiking in rats treated with vehicle only. All samples are from the same animal as in Fig. 1. Montage is like those in Fig. 1, and time and amplitude calibrations are given for each sample, a: 10 min from the onset of continuous spiking, b: 30 min from the onset of continuous spiking, c: 120 min from the onset of continuous spiking.
the onset of status and near the end of the observation period, when motor seizure symptoms were most pronounced. Fig. 2 shows examples of E E G s from these vehicle-treated rats. Either MK-801+vehicle or vehicle+diazepam decreased visible motor seizure activity. MK-801-treated rats assumed a prone position by 20 min after injection, with muscular rigidity as is typical of this class of drugs. No convulsive movements of any kind were visible in these rats. E E G examples from an MK-801-treated rat are shown in Fig. 3. Diazepam-treated rats became limp and unresponsive within 5 min of injection, but subtle convulsive movements consisting of small clonic jerks of the facial muscles and/or whiskers which occurred in synchrony with epileptiform discharges on the E E G could still be seen in these animals. More overt convulsive movements returned by I h after diazepam injection, and two animals attempted to walk around 90 min after diazepam injection. Fig. 4 shows examples of E E G s from a diazepam-treated rat. Electrographic manifestations of
3~
500~,v[__ I second
Fig. 3. Examples of EEGs recorded 10, 30 and 120 rnin after the
onset of continuous spiking in rats treated with MK-80I. All samples are from the same rat. Montage is like those in Fig. 1 and the time and amplitude calibrations are given for each sample, a: 10 min from the onset of continuous spiking, before injection of MK-801. b: 30 rain from the onset of continuous spiking, 20 min after injection of MK-801. c: 120 min from the onset of continuous spiking, 110 min after injection of MK-801. status epilepticus continued in all animals treated with either MK-801 or diazepam alone, with an obvious decrease in the overall amplitude of epileptiform discharges. However, while the E E G ' s of diazepam-treated rats all eventually developed periodic epileptiform discharges (see Fig. 1 for example), this pattern was never seen in the E E G s of MK-801-treated rats. The 4 rats treated with MK-801 alone and allowed to survive were alive the following day, with about 20% loss of body weight, but eating and drinking normally. These animals had recovered completely by 3 days after status induction. We had found in an earlier experiment that diazepam-treated rats do not survive 24 h, as has been reported for untreated animals in this model 1244. In contrast, rats treated with MK-801 followed by diazepam ceased all movements except respiration within 5 min of the diazepam injection. The E E G converted to a low-amplitude, slow pattern at the time motor seizures
101
stopped, and did not change noticeably for several hours (see Fig. 5). Two of the rats did have brief episodes of intermittent low-amplitude spike activity for 5-10 rain after the initial cessation of status, before the record converted permanently to a postictal pattern. The 4 animals treated with this drug combination and allowed to survive were alive the next day, with no loss of body weight and apparently normal behavior. The MK-801 plus diazepam regimen did not control status epilepticus when the treatment was delayed until 30 or 60 min of continuous spiking. Rats treated at these later times all stopped having motor seizures, but the electrographic manifestations of status epilepticus were unaffected. There were no statistically significant differences between cortical diazepam concentrations in the brains of rats receiving vehicle plus diazepam compared to those receiving MK-801 plus diazepam, either in the animals treated during status epilepticus (vehicle plus diazepam = 8.8 + 3.5 ~g/g; MK-801 plus diazepam = 10.4 + 3.8/~g/g) or the non-seizing controls (vehicle plus diazepam = 5.7
+ 1.8 #g/g); MK-801 plus diazepam = 5.7 _+ 2.0 #g/g). Computerized frequency analysis of the EEG seen with these different t r e a t m e n t regimens r e v e a l e d distinct changes induced by each drug. The analysis of variance results are summarized in Table II. All rats had massive increases in total E E G power (compared to that seen prior to injection of pilocarpine) at 10 rain after the onset of continuous spiking, before any treatment had been given. Total power at this time was 84.6 + 35.15 times baseline power, and no statistically significant differences were found among the 4 treatment groups. Total power in vehicle-treated rats remained at this level at 30 and 60 min after the onset of continuous spiking, then declined 36% at 90 min and another 36% from 90-120 min. Total power decreased significantly after treatment with either MK-801 or diazepam alone, to around 40 times baseline power, and remained at this level from 30-120 min. In contrast, when rats were treated with MK-801 plus
I
z :
'
,
,t
i
i
i'
'l
I
~
.~.~J.:~,4.--J,~;,'~,~'.,,~+,'~,q.#,r'+,;~
"i' " ~ '
+';"ii~"~"
~",il'
I
~
'/
:,
'
~
'
't.~~'+;,'..-J~i-!,i;,~-' '.;..J',~.~' ,' -+. ;-.,'
",'"'l " " i
h
"'i
"'
i'/':"
~t
i
50
t
~ ~
'
lOOOVJ
1
I second
40
o/t,./,.,a
,
.j,
!.r"., l'/-;L ~".,',',:-','".,~'~- ~i..',.'~ '.',~,~ "' ~, e : L,,'., '. i
l
®
~1
I~ ¢ c ~ I
i
i
it
'
'
I
5., ,
2oo~vl
,
,ooo~vI Isec~nd
'--
5c
•
,I
',~
'
"~
I
!
Isec0nd
Fig. 5, Examples of E E O s recorded 10, 30 and 120 min since the
4¢
~ooo~vl Iseco~d
Fig. 4. Examples of EEGs recorded 10, 30 and 120 min after the onset of continuous spiking from rats treated with diazepam only. All samples are from the same animal. Montage is the same as those in Fig. 1 and time and amplitude calibrations are given for each sample, a: 10 min from onset of continuous spiking, before injection of diazepam, b: 30 min from onset of continuous spiking, 10 rain after injection of diazepam, c: 120 min from onset of continuous spiking, 100 min after injection of diazepam.
onset of continuous spiking in rats treated with MK-801 plus diazepam. All samples are from the same rat. Montage is like those in Fig. 1 and the time and ampfitude calibrations are given for each sample, a: 10 rain from onset of continuous spiking, before injection of either MK-801 or diazepam, b: 30 rain from onset of continuous spiking, 20 rain after injection of MK-801 and 10 min after injection of diazepam. Motor seizures stopped 8 min before this recording, c: 120 min from onset of continuous spiking, 110 rain after injection of MK-801 and 100 rain after injection of diazepam. No motor seizures nor electrographic epileptiform activity occurred during the previous 108 min.
i
102 d i a z e p a m , total p o w e r d r o p p e d to less than that seen at baseline, and r e m a i n e d at this level during the period from 30-120 rain. Total p o w e r d a t a for each of the 4 t r e a t m e n t groups is shown in Fig. 6. The percentage of total p o w e r in the 2 - 6 Hz bin was 21.6 + 3.28 just prior to any t r e a t m e n t , without statistically significant differences among the t r e a t m e n t groups. This percentage was not significantly changed at 30 min after the onset of continuous spiking in vehiclet r e a t e d rats, but rose to over 45% at 60 min. F u r t h e r non-significant increases were seen at 90 and 120 min, to just o v e r 50%. Rats treated with diazepam alone had a linear increase in percentage of p o w e r in the 2 - 6 Hz range, from 27% at 30 min to 41% at 120 min. Rats which received either MK-801 alone or MK-801 followed by d i a z e p a m showed a d r o p in percentage of p o w e r in the 2 - 6 H z bin, to about 15% of the total, at 30 min. This percentage, in MK-801-alone-treated rats, then rose to 27% at 90 min and was unchanged at 120 min. Rats t r e a t e d with MK-801 followed by diazepam had 30% of total E E G p o w e r in the 2 - 6 Hz bin from 60-120 min. The percentage of p o w e r occurring at frequencies from 2 - 6 Hz for all t r e a t m e n t groups is presented in Fig. 7. Percentage of p o w e r in bins covering the range from 6 - 1 8 Hz was a r o u n d 25% just prior to treatment, without statistically significant differences among the treatment groups. Vehicle-treated rats had percentages ranging from 3 5 - 4 0 % at times from 30-120 rain. Rats treated with d i a z e p a m alone had 4 0 - 4 5 % of the total p o w e r occurring in this range at all points after treatment. MK-801-alone-treated rats increased the percentage of p o w e r in this range from 34% at 30 min to 44% at 120 min. W h e n rats were treated with both drugs, the percentage of p o w e r in the 6-18 Hz range did not change
80-
~
~
VEHICLE
x
tr tl.I
~ w IJJ
\\.\ 40-
-J
\\ ,\
20-
0
J
I 60
30
I 90
_ _
-
I 120
TIME (min) Fig. 6. Total EEG power, expressed as a multiple of the total power seen at baseline for each animal, for the four drug treatment groups used in this study. Time on the x-axis is since the onset of continuous spiking on EEG. The 10 minute time point is just prior to initiation of treatment. Each point is the mean + S.E.M. for 6 rats. Points marked with an * are different from the before-treatment point for that drug treatment group (ANOVA, P < 0.01).
significantly from 30 to 120 rain. Since total E E G p o w e r was so very low in these animals, total p o w e r in this frequency bin was also very low, c o m p a r e d to when the animals were seizing. Percentage of p o w e r at 6-18 Hz for all 4 t r e a t m e n t groups is shown in Fig. 8. The percentage of total p o w e r occurring at frequencies from 18-58 Hz was a r o u n d 45% just prior to t r e a t m e n t , without significant differences among the treatment groups. A t 30 min after the onset of continuous spiking, the rats treated with either vehicle or d i a z e p a m alone had about 35% of the total p o w e r falling in this very fast frequency bin, and the percentages of both groups d r o p p e d to 1 5 - 2 0 % by 120 min, with the diazepamtreated rats declining gradually while the vehicle-treated rats' percentage d r o p p e d at 60 min and r e m a i n e d unchanged to 120 min. In sharp contrast, rats receiving
TABLE II
N 50--
Summary of analysis of variance
~
,
VEH CILE ,
*
*
I 90
I 120
F values marked with an ** have associated probabilities less than 0.001, those marked with an * have associated probabilities less than 0.01
Variable
Effect (d])
F
Total EEG power
treatment group (3,20) time (4,80) interaction (12,80) treatment group (3,20) time (4,80) in teraction ( 12,80) treatment group (3,20) time (4,80) interaction (12,80) treatment group (3,20) time (4,80) interaction (12,80)
6.657* 50.512"* 5.345** 7.648"* 19.557'* 3.573 ** 9.397'* 2.793 1.613 27.596** 36.146"* 5.283 **
% power, 2-6 Hz % power 6-18 Hz % power 18-58 Hz
,,,
2010-
0
O
I 30
I 60
TIME (rain) Fig. 7. Percentage of total EEG power in bins covering frequencies from 2 to 6 Hz. Time on the x-axis is the same as in Fig. 2. Each point is the mean + S.E.M. for 6 rats. Points marked with an * are different from the pretreatment point for that group (ANOVA, P < 0.01).
103 50-
!~DZP ,OZP_.t • 40--
I VEHICLE
.~.tI' MK-801 n
30-_
20
0
I 30
_
. . . . . . . .
60I
I 90
I 120
TIME (min)
Fig. 8. Percentage of total EEG power in bins covering frequencies from 6-18 Hz. Otherwise, as Fig. 7.
MK-801 with or without diazepam had 50-55% of total E E G power in the 18-58 Hz range at 30 rain after the onset of continuous spiking. This percentage then dropped to 40% at 60 min before the two groups' values diverged. MK-801-alone-treated rats declined to 30% at 90 min and remained at this level, while rats treated with MK-801 plus diazepam maintained about 40% of total E E G power in this range at both the 90 and 120 min points. Percentage of power from 18--58 Hz for all four treatment groups is shown in Fig. 9. DISCUSSION MK-801 treatment clearly did not stop status epilepticus in this model unless it was followed by diazepam. The same dose of diazepam alone, given without MK-801 pretreatment, did not control status epilepticus in an earlier experiment 2°, when given at the same time as the MK-801 in this experiment, nor in this experiment when it was given at the same time as diazepam was given to the combination-treatment rats. Although these two
60-
N ,SO"I" U'~ 40I 3o-
UJ O~ 20O.
~ lo-
0
0
I
30
I
60
I
90
I
120
TIME (min)
Fig. 9. Percentage of total EEG power in bins covering frequencies from 18 to 58 Hz. Otherwise, as Fig. 7.
drugs act at different receptor sites in the brain, these data support the hypothesis that, by blocking activity at N M D A sites, MK-801 also modulates activity at the G A B A A receptor, where diazepam is known to bind. Others have presented evidence from in vitro studies which suggests that N M D A receptor activation produces a decline in GABAergic inhibition 1'3'8"9'16. Kapur and Lothman 11 have reported that MK-801 pretreatment prevented the loss of GABAergic inhibition which normally occurs following induction of recurrent seizures in urethane-anesthetized rats. In addition, recent reports describing a potentiation of general anesthetics by MK801 also lend support to this hypothesis 2,15. It is, of course, possible that the synergism seen when MK-801 and diazepam were given in combination was the result of a pharmacokinetic rather than a pharmacodynamic interaction. However, we found no difference between the cortical diazepam concentrations of rats given both drugs and those given only diazepam, when measurements were made 10 rain following diazepam injection to either rats in status epilepticus or non-seizing controls. The electrographic manifestations of status epilepticus in the rats treated only with diazepam were indistinguishable from those of the vehicle-treated rats by 120 min after the onset of continuous spiking (100 min after injection of diazepam). Total E E G power decreased by half shortly after treatment with diazepam, but by 120 min the vehicle-treated rats had declined to the same level, while the diazepam-treated rats did not change from 30-120 min. MK-801 produced a very different electrographic response from diazepam. Total E E G power dropped a little less, then gradually declined to the same level as the diazepam-treated rats. The distribution of power in the E E G of MK-801-treated rats was marked by less power in the 2-6 Hz bin and more power in the 18-58 Hz bins than was seen in the diazepam-treated group. The percentage of power at 6-18 Hz was the same at 30 min as it had been before treatment, then increased steadily to 120 min, as the percentage of power at 18-58 Hz declined. By visual inspection, these E E G s appeared to have an increased spike rate and the spikes were of shorter duration than before treatment with MK-801 (see Fig. 3). The question, of course, is why MK-801-alone-treated rats should survive the status in relatively good condition while diazepam-alone-treated animals do not, given that both treatments failed to halt the electrographic manifestations of status epilepticus. Fariello et al. 4 interpreted a similar finding when MK-801 was given prior to induction of status epilepticus with kainic acid to mean that the electrographic manifestations of status epilep-
104 ticus were worse, even though MK-801 allowed better survival rates. We believe this d a t a should be interpreted that the E E G s of MK-801-treated rats failed to progress through the sequence of E E G patterns which u n t r e a t e d and d i a z e p a m - t r e a t e d rats did, but rather continued at the stage of r a p i d continuous spiking. The maintenance of status epilepticus at this stage should be regarded as a positive t r e a t m e n t outcome. We never saw periodic epileptiform discharges in the E E G s of MK-801-treated animals, and the computerized analysis d e m o n s t r a t e d that the build-up in slow activity which occurred in the vehicle- or d i a z e p a m - t r e a t e d rats did not occur with MK-801 treatment. T h e increase in 2 - 6 Hz percentage of p o w e r as status epilepticus progresses may be the electrographic m a r k e r of neuronal injury. This m a y also provide a clue as to why MK-801 plus d i a z e p a m failed to stop status epilepticus when t r e a t m e n t initiation was delayed to 30 or 60 min after the onset of continuous spiking. These rats already had increasing percentages of p o w e r at 2 - 6 Hz at the time treatment was
started. It is unclear w h e t h e r the failure of the combination t r e a t m e n t represents d e c r e a s e d efficacy of MK801, d i a z e p a m or both drugs. A d d i t i o n a l studies of this p h e n o m e n o n are clearly required. Since formal neuropathological studies at different status durations have not been published on this m o d e l of status epilepticus, one can only speculate as to how extensive and severe the damage might be at any given time in the status episode. MK-801 followed by d i a z e p a m (or any o t h e r of the G A B A A receptor-acting anticonvulsants) has real potential for t r e a t m e n t of refractory status epilepticus in humans. The neuroprotective action of MK-801 would be a m a j o r advantage in these cases, which frequently take a very long time to control and which have a very high risk of death or p e r m a n e n t neurological sequelae.
REFERENCES
of lithium potentiation of pilocarpine-induced status epilepticus in rats, Exp. Neurol., 91 (!986) 471-480. 11 Kapur, J. and Lothman E.W., NMDA receptor activation mediates the loss of GABAergic inhibition induced by recurrent seizures, Epilepsy Res., 5 (1990) 103-111. 12 Morrisett, R.A., Jope, R.S. and Snead, O.C., Effects of drugs on the initiation and maintenance of status epilepticus induced by administration of pilocarpine to lithium-pretreated rats, Exp. Neurol., 97 (1986) 193-200. 13 Ormandy, G.C., Jope, R.S. and Snead, O.C., Anticonvulsant actions of MK-801 on the lithium-pilocarpine model of status epilepticus in rats, Exp. Neurol., 106 (1989) 172-180. 14 Persinger, M.A., Makarec, K. and Bradley, J.-C., Characteristics of limbic seizures evoked by peripheral injections of lithium and pilocarpine, Physiol. Behav., 44 (1988) 27-37. 15 Scheller, M.S., Zornow, M.H., Fleischer, J.E., Shearman, G.T. and Greber, T.E, The non-competitive N-methyI-D-aspartate receptor antagonist, MK-801, profoundly reduces volatile anesthetic requirements in rabbits, Neuropharmacology, 28 (1989) 677-681. 16 Stelzer, A., Slater, N.T. and Ten Bruggencate, G., Activation of NMDA receptors blocks GABAergic inhibition in an in vitro model of epilepsy, Nature, 326 (1987) 698-701. 17 Treiman, D.M., Walton, N.Y. and Kendrick, C., A progressive sequence of electroencephalographic changes during generalized convulsive status epilepticus, Epilepsy Res., 5 (1990) 49-60. 18 Vezzani, A., Serafini, R., Stasi, M.A., Caccia, S., Conti, I., Tridico, R.V. and Samanin, R., Kinetics of MK-801 and its effect on quinolinic acid-induced seizures and neurotoxicity in rats, J. Pharmacol. Exp. Ther., 249 (1989) 278-283. 19 Walter, D.O., On units and dimensions for reporting spectral intensities, Electroencephalogr. Clin. Neurophysiol., 24 (1968) 486-487. 20 Walton, N.Y. and Treiman, D.M., Response of status epilepticus induced by lithium and pilocarpine to treatment with diazepam, Exp. Neurol., 101 (1988) 267-275.
1 Baldino, E, Wolfson, B., Heinemann, U. and Gutnick, M.J., An N-methyI-D-aspartate (NMDA) receptor antagonist reduces bicuculline-induced depolarization shifts in neocortical explant cultures, Neurosci. Lett., 70 (1986) 101-105. 2 Daniel, L.C. The non-competitive N-methyl-D-aspartate antagonists, MK-801, phencyelidine and ketamine, increase the potency of general anesthetics, Pharmacol. Biochem. Behav., 36 (1990) 111-115. 3 Dingledine, R., Hymes, A. and King, G.L., Involvement of N-methyl-D-aspartate receptors in epileptiform bursting in the rat hippocampal slice, Z Neurophysiol., 380 (1986) 175-189. 4 Fariello, R.G., Golden, G.T., Smith, G.G. and Reyes, P.E, Potentiation of kainic acid epileptogenicity and sparing from neuronal damage by an NMDA receptor antagonist, Epilepsy Res., 3 (1989) 206-213. 5 Foster, A.C. and Wong, E.H.E, The novel anticonvulsant MK-801 binds to the activated state of the N-methyi-D-aspartate receptor in rat brain, Br. J. Pharmacol., 91 (1987) 403-409. 6 Gunawan, S., Walton, N.Y. and Treiman, D.M., Analysis of lorazepam in rat brain using liquid/liquid and solid-phase extraction in combination with high performance liquid chromatography, Biomed. Chromatogr., 4 (1990) 168-170. 7 Honehar, M.P., Olney, J.W. and Sherman, W.R., Systemic cholinergic agents induce seizures and brain damage in lithiumtreated rats, Science, 220 (1983) 323-325. 8 Horne, A.L., Harrison, N.L., Turner, J.D. and Simmons, M.A., Spontaneous paroxysmal activity induced by zero magnesium and bicuculline: suppression by NMDA antagonists and GABA mimetics, Eur. J. Pharmacol., 122 (1986) 221-238. 9 Jones, R.S.G., Epileptiform events induced by GABA antagonists in entorhinal cortical cells in vitro are partly mediated by N-methyl-D-aspartate receptors, Brain Research, 457 (1988) 113-121. 10 Jope, R.S., Morrisett, R.A. and Snead, O.C., Characterization
Acknowledgements. The technical assistance of Mr. James Kang in carrying out these experiments is gratefully acknowledged. This work was supported by a grant from the Department of Veterans Affairs. Portions of this work were presented at the 1990 meeting of the American Epilepsy Society.
Brain Research, 553 (1991) 97-104 1991 Elsevier Science Publishers B.V. 0006-8993/91/$03.50 ADONIS 000689939116747X
BRES 16747
Motor and electroencephalographic response of refractory experimental status epilepticus in rats to treatment with MK-801, diazepam, or MK-801 plus diazepam Nancy Y. Walton and David M. Treiman Neurology and Research Services, Department of Veterans Affairs Medical Center, West Los Angeles, CA (U.S.A.) and Department of Neurology, UCLA School of Medicine, Los Angeles, CA (U.S.A.)
(Accepted 29 January 1991) Key words: Status epilepticus; Seizure; Lithium; Pilocarpine; MK-801; Diazepam; Electroencephalogram; Frequency analysis
Pharmacologic control of refractory status epilepticus has been little-studied in experimental models. In this experiment, rats in status epilepticus induced by lithium and pilocarpine were treated with MK-801 alone, diazepam alone or MK-801 plus diazepam, with treatment begun at a time when this model of status is refractory to anticonvulsant drugs. EEG samples were digitized before and for two hours after treatment, and the digitized samples subjected to computerized frequency analysis. MK-801 plus diazepam halted all manifestations of status epilepticus. Although neither MK-801 alone nor diazepam alone stopped the ongoing electrographie status epileptieus, both drugs diminished motor seizures and total EEG power. MK-801 treatment prevented the progression of changes in EEG pattern which normally occurs in this model of status epilepticus, while diazepam did not. MK-801, with and without diazepam, allowed the rats to survive the episode of status epilepticus, but rats treated with MK-801 alone required several days to recover completely, while the MK-801 plus diazepam rats appeared normal the next day. MK-801 may be a useful agent for treatment of human refractory status epilepticus, because of its neuroprotective action as well as its ability to potentiate GABAergic drugs.
INTRODUCTION Sequential injections of lithium and pilocarpine (separated by 20-24 h) reliably induce status epilepticus in rats which lasts from 3 - 6 h and is universally fatal 7,1°,14. Status epilepticus can be prevented if antiepileptic drugs are given before pilocarpine and status can be aborted if treatment is given shortly after the onset of seizure activity 12. If treatment is delayed until the status is well established, the seizures are refractory to treatment 12. We have described a predictable sequence of E E G patterns which occurs in generalized convulsive status epilepticus in humans as well as experimental models of status epilepticus in the rat (including lithium/pilocarpine) 17. Examples of these patterns, recorded from a single rat during an episode of status epilepticus induced by lithium and pilocarpine, are shown in Fig. 1. The transition of lithium/pilocarpine status epilepticus from responsive to refractory can be monitored by the E E G pattern seen at the time treatment is given. In an earlier experiment, we reported that 20 mg/kg diazepam controlled status in 100% of rats treated when the E E G
displayed discrete electrographic seizures (the initial status pattern) and 50% of rats treated when the E E G displayed merging seizures (the second pattern), but in only 17% of rats treated when the E E G displayed high-amplitude, continuous, rapid spiking or periodic epileptiform discharges (the third and fourth patterns) 2°. Lithiurn/pilocarpine status m a y provide a model for studying the basic mechanisms responsible for refractory status epilepticus in humans and evaluating new drugs with potential for treating this condition. We describe here experiments using MK-801, both alone and followed by diazepam, as a treatment for status epilepticus induced by lithium and pilocarpine which has progressed to the refractory stage. MK-801 is a noncompetitive N M D A antagonist which acts at the PCP site on the open cation channel associated with the N M D A receptor 5. O r m a n d y et al. x3 reported that MK-801 blocked the development of lithium/pilocarpine status epilepticus when it was given prior to pilocarpine; when given after status epilepticus was established, MK-801 had little effect on the ongoing electrographic activity, but did increase the 24 h survival rate. Fariello et al. 4
Correspondence: N.Y. Walton, Department of Neurology, UCLA School of Medicine, 710 Westwood Plaza (C-128), Los Angeles, CA 90024, U.S.A.
98 reported that while MK-801 protected against kainic acid neurotoxicity, the electrographic manifestations of kainate-induced status epilepticus were actually worsened by MK-801 pretreatment. Neuroprotective effects have also been described when MK-801 is given prior to onset of status epilepticus
induced by intrahippocampal quinolinic acid is. In this experiment, MK-801 and/or diazepam were administered after status epilepticus induced by lithium and pilocarpine was well established. Effects produced by either drug alone or the two drugs in combination on ongoing motor and electrographic manifestations of
~ ~ !
'1 '
/I
150M.V I
ii~
i
,~
II
'l
1~
i
I
15C NL
I second
I
!'
I
,~ '
t
I~
Isec
o.
e
,jr.
IJ
41
!
, t
~
t~
"i
~oo~v~ C.
f
'
*
.
I
!
/
:
H "~
i '~
',,I
I,'V
I'
II
"
I second
d
350/~Vl
Isec
Fig. 1. The predictable sequence of EEG patterns seen in generalized convulsive status epilepticus in humans as well as in experimental models of status epilepticus (SE) in the rat 17. Each of these examples is taken from the same rat, in which status epilepticus was induced by injections of lithium and pilocarpine. The time and sensitivity calibrations are noted for each EEG sample. Recordings are from epidural screw electrodes with channel 1 being fight frontal to parietal, channel 2 left frontal to parietal, channel 3 fight to left frontal, and channel 4 fight to left parietal. a: baseline EEG, recorded just prior to injection of piiocarpine, b: end of discrete electrographic seizure, 3 rain after onset of SE. c: merging seizures, 5 rain after onset of SE. d: continuous, high-amplitude, rapid spiking, 20 rain after onset of SE and 12 min after the onset of continuous spiking pattern, e: periodic epileptiform discharges (PEDs) on a relatively fiat background, 118 rain after onset of SE and 3 min after the first 10 second episode of PEDs.
99 status e p i l e p t i c u s w e r e e v a l u a t e d .
TABLE I
Drug regimens studied MATERIALS AND METHODS Adult, male Sprague-Dawley rats, weighing 175-200 g at the time of surgery (Bantin and Kingman) were used as subjects in this experiment. Rats were housed singly after surgery in plexiglas cages with contact bedding. Food and water were available ad lib throughout. Room temperature was maintained at 22 °C and a 24-h diurnal lighting schedule was controlled automatically, with lights on from 06.00 to 18.00 each day. Epidural recording electrodes were placed surgically under ketamine/xylazine anesthesia as has been described elsewhere 2°. One week postoperative recovery was allowed prior to experimentation. Lithium chloride was purchased from Sigma Chemicals, Inc, and dissolved at 0.9% for injection in sterile water. Pilocarpine was purchased from Sigma Chemicals and dissolved for injection in normal saline (100 mg/ml). MK-801 was purchased from Research Biochemicals Inc. and reconstituted in ethanol (50 mg/ml), then diluted to 2 mg/ml with 30% polyethylene glycol 400. Diazepam was purchased from Elkius-Sirm as a 5 mg/ml solution in a vehicle containing 40% propylene glycol, 10% ethanol and 1.5% benzyl alcohol. Vehicle solutions to match both MK-801 and diazepam were also prepared. An EEG sample was obtained prior to the pilocarpine injection and EEG was then monitored continuously. In order to obtain EEG samples for frequency analysis, the output from the EEG machine amplifiers was transmitted by cable to an IBM AT-compatible microcomputer via a LabMaster DMA analog-to-digital converter (Techmar). The system was calibrated using a 50/~V 16 Hz sine wave signal. Sixty-four second samples of artifact-free EEG were analyzed for each rat at the following times: baseline, after 10 min continuous spiking (just prior to the first treatment injection), after 30 min continuous spiking (10 rain after the second treatment injection), then every 30 rain thereafter. Frequency and Coherence Topography System software (Neurolmages, Inc.) was used to digitize and analyze the EEG. Data were sampled at 512 points per channel per second. The program analyzed the data into frequency spectra with a 4 Hz bandwidth covering the range from 2 to 58 Hz, based on a fast Fourier transform algorithm. The frequency ranges used for statistical analysis (2-6 Hz, 6-18 Hz, 18-58 Hz) were established by factor analysis of results from earlier experiments which showed that changes in power distribution in these ranges could account for 70% of the total variance seen. EEG power is a concept which has been described in various complex mathematical terms. The fast Fourier transform used in spectral analysis partitions the complex EEG waves into theoretical sine wave components with different frequencies. The power associated with ranges, or bins, of these frequencies can then be thought of as the voltage-time area covered by the sine waves. Power is thus a function both of how many sine wave cycles of various frequencies occur and their peak amplitudes. Because there is much debate as to what absolute unit of power is appropriate for EEG analysis 19, power in this study is expressed as a multiple of the power seen at baseline in the same animal. Status epilepticus was induced by injection of 3 mmol/kg lithium chloride i.p., followed in 20-24 h by s.c. injection of 30 mg/kg pilocarpine. Six rats were treated with each of the drug regimens shown in Table I, with the first injection given when the EEG had displayed a pattern of continuous, high-amplitude rapid spiking for 10 min. The second injection was given 10 min after the first. These rats were observed for 2 h after treatment. An additional 4 rats each were treated with 2 mg/kg MK-801, alone and with 20 mg/kg diazepam, observed continuously for at least 8 h, then rechecked the following morning (about 12 h later). Two rats each were treated with the MK-801 plus diazepam combination, but with treatment initiation postponed until either 30 or 60 min continuous spiking on EEG. These animals were observed for 2 h after treatment.
First injections were given at 1 ml/kg b. wt. Second injections were given at 4 ml/kg b. wt. n = 6 per group.
Group
First injection
Second injection
Vehicle
vehicle for MK-801 2 mg/kg MK-801
vehicle for diazepam vehicle for diazepam 20 mg/kg diazepam 20 mg/kg diazepam
MK-801 Diazepam MK 801 plus diazepam
vehicle for MK-801 2 mg/kg MK-801
In order to rule out the possibility that observed differences might have resulted from a pharmacokinetic interaction of MK-801 with diazepam, 6 rats were treated during status epilepticus with either vehicle plus diazepam or MK-801 plus diazepam, exactly as has already been described. These rats were decapitated 10 min after diazepam injection and the brains rapidly removed and frozen. Six non-seizing control rats were also treated with vehicle plus diazepam or MK-801 plus diazepam and decapitated 10 rain after the diazepam injection. Cortical diazepam concentrations were assayed using a modification of a method developed in this laboratory for Iorazepam 6. The solid-phase extraction step described for the Iorazepam assay was not used because too much diazepam was lost in this step and the run time was extended to 30 rain. Statistical analysis was done by two-way ANOVA. Treatment group (see Table I) served as one factor, while time from onset of continuous spiking served as the second factor (repeated measures on the same subjects were used for this factor). Individual comparisons were made using the Tukey HSD procedure. Statistics were calculated by computer using the SYSTAT program (Systat), with alpha set at 0.01.
RESULTS D a t a will b e p r e s e n t e d as a m e a n + s.d. unless n o t e d o t h e r w i s e . O n s e t o f status e p i l e p t i c u s , o p e r a t i o n a l l y d e f i n e d as t h e a p p e a r a n c e o f cortical spiking o n E E G , o c c u r r e d 21.5 _+ 7.5 m i n a f t e r i n j e c t i o n o f p i l o c a r p i n e ( r a n g e = 1 1 - 4 6 m i n ) . T h e E E G p a t t e r n o f discrete e l e c t r o g r a p h i c seizures lasted 6.0 + 2.6 m i n ( r a n g e = 2 - 1 3 m i n ) , f o l l o w e d by 6.6 + 3.8 m i n o f m e r g i n g seizures ( r a n g e = 2 - 2 2 m i n ) p r i o r to t h e o n s e t of c o n t i n u o u s spiking. O t h e r e x p e r i m e n t e r s 1°,12,13 h a v e r e p o r t e d treatm e n t i n j e c t i o n t i m e s in t e r m s o f m i n u t e s since i n j e c t i o n o f p i l o c a r p i n e . T h e i n t e r v a l f r o m p i l o c a r p i n e i n j e c t i o n to i n j e c t i o n of first t r e a t m e n t d r u g was 44.2 + 12.6 min ( r a n g e = 3 3 - 7 3 m i n ) in t h e s e rats. A n i m a l s t r e a t e d with t w o v e h i c l e i n j e c t i o n s c o n t i n u e d to h a v e m o t o r seizures consisting o f c o n t i n u o u s clonic j e r k i n g of the h e a d , plus f o r e l i m b c l o n u s visible w h e n e v e r t h e l i m b was n o t b e a r i n g w e i g h t . T h e rats t e n d e d to walk in circles, r a r e l y s t a n d i n g still. T h e y did n o t lose m o t o r t o n e o r t h e ability to m a i n t a i n an u p r i g h t p o s t u r e d u r i n g t h e 2-h o b s e r v a t i o n p e r i o d e x c e p t for b r i e f falls at
100 I
:I
I
I,
I
!
'. '
,,,
~
'
i ,l!
I !~~,
I,!~
'i~l;'A 4
V,
'
~
'~
J'
i
~
,i~r}' 1' }]'?lq'y\
',W~'J,
i,;~, i i l i
lO00~v[ I second
3~
700/~.V[~ I second
I
,
J
'
r
a W~#,~.d~,rW~,..~.%.4%~\,.~h,t~,Q~¢C~..~%4~ ,:%~,,.~v~%~,~,,,~,,.s
I! I I ' l J ! !
II
I
[ I ~,'
I
I I,'
II
;
'
,!''.~
,''
, I;
lO00~V I 2b
I second
3~
ZOo~vl I second
-!:: .'"tr, ~'~ ~
~
,'
~ . . ~ . \ ~ * '~
't' ~, .~,,,,.,~r',,,il ~'r,l ii,"f"T 4~'(/'t"],:,t(¢,. .g
,' /.4',i,,:)!4
a,
I
J
'i I
It
~.
7oo,,vl__ Isecond
Fig. 2. Examples of EEGs recorded 10, 30 and 120 min from the onset of continuous spiking in rats treated with vehicle only. All samples are from the same animal as in Fig. 1. Montage is like those in Fig. 1, and time and amplitude calibrations are given for each sample, a: 10 min from the onset of continuous spiking, b: 30 min from the onset of continuous spiking, c: 120 min from the onset of continuous spiking.
the onset of status and near the end of the observation period, when motor seizure symptoms were most pronounced. Fig. 2 shows examples of E E G s from these vehicle-treated rats. Either MK-801+vehicle or vehicle+diazepam decreased visible motor seizure activity. MK-801-treated rats assumed a prone position by 20 min after injection, with muscular rigidity as is typical of this class of drugs. No convulsive movements of any kind were visible in these rats. E E G examples from an MK-801-treated rat are shown in Fig. 3. Diazepam-treated rats became limp and unresponsive within 5 min of injection, but subtle convulsive movements consisting of small clonic jerks of the facial muscles and/or whiskers which occurred in synchrony with epileptiform discharges on the E E G could still be seen in these animals. More overt convulsive movements returned by I h after diazepam injection, and two animals attempted to walk around 90 min after diazepam injection. Fig. 4 shows examples of E E G s from a diazepam-treated rat. Electrographic manifestations of
3~
500~,v[__ I second
Fig. 3. Examples of EEGs recorded 10, 30 and 120 rnin after the
onset of continuous spiking in rats treated with MK-80I. All samples are from the same rat. Montage is like those in Fig. 1 and the time and amplitude calibrations are given for each sample, a: 10 min from the onset of continuous spiking, before injection of MK-801. b: 30 rain from the onset of continuous spiking, 20 min after injection of MK-801. c: 120 min from the onset of continuous spiking, 110 min after injection of MK-801. status epilepticus continued in all animals treated with either MK-801 or diazepam alone, with an obvious decrease in the overall amplitude of epileptiform discharges. However, while the E E G ' s of diazepam-treated rats all eventually developed periodic epileptiform discharges (see Fig. 1 for example), this pattern was never seen in the E E G s of MK-801-treated rats. The 4 rats treated with MK-801 alone and allowed to survive were alive the following day, with about 20% loss of body weight, but eating and drinking normally. These animals had recovered completely by 3 days after status induction. We had found in an earlier experiment that diazepam-treated rats do not survive 24 h, as has been reported for untreated animals in this model 1244. In contrast, rats treated with MK-801 followed by diazepam ceased all movements except respiration within 5 min of the diazepam injection. The E E G converted to a low-amplitude, slow pattern at the time motor seizures
101
stopped, and did not change noticeably for several hours (see Fig. 5). Two of the rats did have brief episodes of intermittent low-amplitude spike activity for 5-10 rain after the initial cessation of status, before the record converted permanently to a postictal pattern. The 4 animals treated with this drug combination and allowed to survive were alive the next day, with no loss of body weight and apparently normal behavior. The MK-801 plus diazepam regimen did not control status epilepticus when the treatment was delayed until 30 or 60 min of continuous spiking. Rats treated at these later times all stopped having motor seizures, but the electrographic manifestations of status epilepticus were unaffected. There were no statistically significant differences between cortical diazepam concentrations in the brains of rats receiving vehicle plus diazepam compared to those receiving MK-801 plus diazepam, either in the animals treated during status epilepticus (vehicle plus diazepam = 8.8 + 3.5 ~g/g; MK-801 plus diazepam = 10.4 + 3.8/~g/g) or the non-seizing controls (vehicle plus diazepam = 5.7
+ 1.8 #g/g); MK-801 plus diazepam = 5.7 _+ 2.0 #g/g). Computerized frequency analysis of the EEG seen with these different t r e a t m e n t regimens r e v e a l e d distinct changes induced by each drug. The analysis of variance results are summarized in Table II. All rats had massive increases in total E E G power (compared to that seen prior to injection of pilocarpine) at 10 rain after the onset of continuous spiking, before any treatment had been given. Total power at this time was 84.6 + 35.15 times baseline power, and no statistically significant differences were found among the 4 treatment groups. Total power in vehicle-treated rats remained at this level at 30 and 60 min after the onset of continuous spiking, then declined 36% at 90 min and another 36% from 90-120 min. Total power decreased significantly after treatment with either MK-801 or diazepam alone, to around 40 times baseline power, and remained at this level from 30-120 min. In contrast, when rats were treated with MK-801 plus
I
z :
'
,
,t
i
i
i'
'l
I
~
.~.~J.:~,4.--J,~;,'~,~'.,,~+,'~,q.#,r'+,;~
"i' " ~ '
+';"ii~"~"
~",il'
I
~
'/
:,
'
~
'
't.~~'+;,'..-J~i-!,i;,~-' '.;..J',~.~' ,' -+. ;-.,'
",'"'l " " i
h
"'i
"'
i'/':"
~t
i
50
t
~ ~
'
lOOOVJ
1
I second
40
o/t,./,.,a
,
.j,
!.r"., l'/-;L ~".,',',:-','".,~'~- ~i..',.'~ '.',~,~ "' ~, e : L,,'., '. i
l
®
~1
I~ ¢ c ~ I
i
i
it
'
'
I
5., ,
2oo~vl
,
,ooo~vI Isec~nd
'--
5c
•
,I
',~
'
"~
I
!
Isec0nd
Fig. 5, Examples of E E O s recorded 10, 30 and 120 min since the
4¢
~ooo~vl Iseco~d
Fig. 4. Examples of EEGs recorded 10, 30 and 120 min after the onset of continuous spiking from rats treated with diazepam only. All samples are from the same animal. Montage is the same as those in Fig. 1 and time and amplitude calibrations are given for each sample, a: 10 min from onset of continuous spiking, before injection of diazepam, b: 30 min from onset of continuous spiking, 10 rain after injection of diazepam, c: 120 min from onset of continuous spiking, 100 min after injection of diazepam.
onset of continuous spiking in rats treated with MK-801 plus diazepam. All samples are from the same rat. Montage is like those in Fig. 1 and the time and ampfitude calibrations are given for each sample, a: 10 rain from onset of continuous spiking, before injection of either MK-801 or diazepam, b: 30 rain from onset of continuous spiking, 20 rain after injection of MK-801 and 10 min after injection of diazepam. Motor seizures stopped 8 min before this recording, c: 120 min from onset of continuous spiking, 110 rain after injection of MK-801 and 100 rain after injection of diazepam. No motor seizures nor electrographic epileptiform activity occurred during the previous 108 min.
i
102 d i a z e p a m , total p o w e r d r o p p e d to less than that seen at baseline, and r e m a i n e d at this level during the period from 30-120 rain. Total p o w e r d a t a for each of the 4 t r e a t m e n t groups is shown in Fig. 6. The percentage of total p o w e r in the 2 - 6 Hz bin was 21.6 + 3.28 just prior to any t r e a t m e n t , without statistically significant differences among the t r e a t m e n t groups. This percentage was not significantly changed at 30 min after the onset of continuous spiking in vehiclet r e a t e d rats, but rose to over 45% at 60 min. F u r t h e r non-significant increases were seen at 90 and 120 min, to just o v e r 50%. Rats treated with diazepam alone had a linear increase in percentage of p o w e r in the 2 - 6 Hz range, from 27% at 30 min to 41% at 120 min. Rats which received either MK-801 alone or MK-801 followed by d i a z e p a m showed a d r o p in percentage of p o w e r in the 2 - 6 H z bin, to about 15% of the total, at 30 min. This percentage, in MK-801-alone-treated rats, then rose to 27% at 90 min and was unchanged at 120 min. Rats t r e a t e d with MK-801 followed by diazepam had 30% of total E E G p o w e r in the 2 - 6 Hz bin from 60-120 min. The percentage of p o w e r occurring at frequencies from 2 - 6 Hz for all t r e a t m e n t groups is presented in Fig. 7. Percentage of p o w e r in bins covering the range from 6 - 1 8 Hz was a r o u n d 25% just prior to treatment, without statistically significant differences among the treatment groups. Vehicle-treated rats had percentages ranging from 3 5 - 4 0 % at times from 30-120 rain. Rats treated with d i a z e p a m alone had 4 0 - 4 5 % of the total p o w e r occurring in this range at all points after treatment. MK-801-alone-treated rats increased the percentage of p o w e r in this range from 34% at 30 min to 44% at 120 min. W h e n rats were treated with both drugs, the percentage of p o w e r in the 6-18 Hz range did not change
80-
~
~
VEHICLE
x
tr tl.I
~ w IJJ
\\.\ 40-
-J
\\ ,\
20-
0
J
I 60
30
I 90
_ _
-
I 120
TIME (min) Fig. 6. Total EEG power, expressed as a multiple of the total power seen at baseline for each animal, for the four drug treatment groups used in this study. Time on the x-axis is since the onset of continuous spiking on EEG. The 10 minute time point is just prior to initiation of treatment. Each point is the mean + S.E.M. for 6 rats. Points marked with an * are different from the before-treatment point for that drug treatment group (ANOVA, P < 0.01).
significantly from 30 to 120 rain. Since total E E G p o w e r was so very low in these animals, total p o w e r in this frequency bin was also very low, c o m p a r e d to when the animals were seizing. Percentage of p o w e r at 6-18 Hz for all 4 t r e a t m e n t groups is shown in Fig. 8. The percentage of total p o w e r occurring at frequencies from 18-58 Hz was a r o u n d 45% just prior to t r e a t m e n t , without significant differences among the treatment groups. A t 30 min after the onset of continuous spiking, the rats treated with either vehicle or d i a z e p a m alone had about 35% of the total p o w e r falling in this very fast frequency bin, and the percentages of both groups d r o p p e d to 1 5 - 2 0 % by 120 min, with the diazepamtreated rats declining gradually while the vehicle-treated rats' percentage d r o p p e d at 60 min and r e m a i n e d unchanged to 120 min. In sharp contrast, rats receiving
TABLE II
N 50--
Summary of analysis of variance
~
,
VEH CILE ,
*
*
I 90
I 120
F values marked with an ** have associated probabilities less than 0.001, those marked with an * have associated probabilities less than 0.01
Variable
Effect (d])
F
Total EEG power
treatment group (3,20) time (4,80) interaction (12,80) treatment group (3,20) time (4,80) in teraction ( 12,80) treatment group (3,20) time (4,80) interaction (12,80) treatment group (3,20) time (4,80) interaction (12,80)
6.657* 50.512"* 5.345** 7.648"* 19.557'* 3.573 ** 9.397'* 2.793 1.613 27.596** 36.146"* 5.283 **
% power, 2-6 Hz % power 6-18 Hz % power 18-58 Hz
,,,
2010-
0
O
I 30
I 60
TIME (rain) Fig. 7. Percentage of total EEG power in bins covering frequencies from 2 to 6 Hz. Time on the x-axis is the same as in Fig. 2. Each point is the mean + S.E.M. for 6 rats. Points marked with an * are different from the pretreatment point for that group (ANOVA, P < 0.01).
103 50-
!~DZP ,OZP_.t • 40--
I VEHICLE
.~.tI' MK-801 n
30-_
20
0
I 30
_
. . . . . . . .
60I
I 90
I 120
TIME (min)
Fig. 8. Percentage of total EEG power in bins covering frequencies from 6-18 Hz. Otherwise, as Fig. 7.
MK-801 with or without diazepam had 50-55% of total E E G power in the 18-58 Hz range at 30 rain after the onset of continuous spiking. This percentage then dropped to 40% at 60 min before the two groups' values diverged. MK-801-alone-treated rats declined to 30% at 90 min and remained at this level, while rats treated with MK-801 plus diazepam maintained about 40% of total E E G power in this range at both the 90 and 120 min points. Percentage of power from 18--58 Hz for all four treatment groups is shown in Fig. 9. DISCUSSION MK-801 treatment clearly did not stop status epilepticus in this model unless it was followed by diazepam. The same dose of diazepam alone, given without MK-801 pretreatment, did not control status epilepticus in an earlier experiment 2°, when given at the same time as the MK-801 in this experiment, nor in this experiment when it was given at the same time as diazepam was given to the combination-treatment rats. Although these two
60-
N ,SO"I" U'~ 40I 3o-
UJ O~ 20O.
~ lo-
0
0
I
30
I
60
I
90
I
120
TIME (min)
Fig. 9. Percentage of total EEG power in bins covering frequencies from 18 to 58 Hz. Otherwise, as Fig. 7.
drugs act at different receptor sites in the brain, these data support the hypothesis that, by blocking activity at N M D A sites, MK-801 also modulates activity at the G A B A A receptor, where diazepam is known to bind. Others have presented evidence from in vitro studies which suggests that N M D A receptor activation produces a decline in GABAergic inhibition 1'3'8"9'16. Kapur and Lothman 11 have reported that MK-801 pretreatment prevented the loss of GABAergic inhibition which normally occurs following induction of recurrent seizures in urethane-anesthetized rats. In addition, recent reports describing a potentiation of general anesthetics by MK801 also lend support to this hypothesis 2,15. It is, of course, possible that the synergism seen when MK-801 and diazepam were given in combination was the result of a pharmacokinetic rather than a pharmacodynamic interaction. However, we found no difference between the cortical diazepam concentrations of rats given both drugs and those given only diazepam, when measurements were made 10 rain following diazepam injection to either rats in status epilepticus or non-seizing controls. The electrographic manifestations of status epilepticus in the rats treated only with diazepam were indistinguishable from those of the vehicle-treated rats by 120 min after the onset of continuous spiking (100 min after injection of diazepam). Total E E G power decreased by half shortly after treatment with diazepam, but by 120 min the vehicle-treated rats had declined to the same level, while the diazepam-treated rats did not change from 30-120 min. MK-801 produced a very different electrographic response from diazepam. Total E E G power dropped a little less, then gradually declined to the same level as the diazepam-treated rats. The distribution of power in the E E G of MK-801-treated rats was marked by less power in the 2-6 Hz bin and more power in the 18-58 Hz bins than was seen in the diazepam-treated group. The percentage of power at 6-18 Hz was the same at 30 min as it had been before treatment, then increased steadily to 120 min, as the percentage of power at 18-58 Hz declined. By visual inspection, these E E G s appeared to have an increased spike rate and the spikes were of shorter duration than before treatment with MK-801 (see Fig. 3). The question, of course, is why MK-801-alone-treated rats should survive the status in relatively good condition while diazepam-alone-treated animals do not, given that both treatments failed to halt the electrographic manifestations of status epilepticus. Fariello et al. 4 interpreted a similar finding when MK-801 was given prior to induction of status epilepticus with kainic acid to mean that the electrographic manifestations of status epilep-
104 ticus were worse, even though MK-801 allowed better survival rates. We believe this d a t a should be interpreted that the E E G s of MK-801-treated rats failed to progress through the sequence of E E G patterns which u n t r e a t e d and d i a z e p a m - t r e a t e d rats did, but rather continued at the stage of r a p i d continuous spiking. The maintenance of status epilepticus at this stage should be regarded as a positive t r e a t m e n t outcome. We never saw periodic epileptiform discharges in the E E G s of MK-801-treated animals, and the computerized analysis d e m o n s t r a t e d that the build-up in slow activity which occurred in the vehicle- or d i a z e p a m - t r e a t e d rats did not occur with MK-801 treatment. T h e increase in 2 - 6 Hz percentage of p o w e r as status epilepticus progresses may be the electrographic m a r k e r of neuronal injury. This m a y also provide a clue as to why MK-801 plus d i a z e p a m failed to stop status epilepticus when t r e a t m e n t initiation was delayed to 30 or 60 min after the onset of continuous spiking. These rats already had increasing percentages of p o w e r at 2 - 6 Hz at the time treatment was
started. It is unclear w h e t h e r the failure of the combination t r e a t m e n t represents d e c r e a s e d efficacy of MK801, d i a z e p a m or both drugs. A d d i t i o n a l studies of this p h e n o m e n o n are clearly required. Since formal neuropathological studies at different status durations have not been published on this m o d e l of status epilepticus, one can only speculate as to how extensive and severe the damage might be at any given time in the status episode. MK-801 followed by d i a z e p a m (or any o t h e r of the G A B A A receptor-acting anticonvulsants) has real potential for t r e a t m e n t of refractory status epilepticus in humans. The neuroprotective action of MK-801 would be a m a j o r advantage in these cases, which frequently take a very long time to control and which have a very high risk of death or p e r m a n e n t neurological sequelae.
REFERENCES
of lithium potentiation of pilocarpine-induced status epilepticus in rats, Exp. Neurol., 91 (!986) 471-480. 11 Kapur, J. and Lothman E.W., NMDA receptor activation mediates the loss of GABAergic inhibition induced by recurrent seizures, Epilepsy Res., 5 (1990) 103-111. 12 Morrisett, R.A., Jope, R.S. and Snead, O.C., Effects of drugs on the initiation and maintenance of status epilepticus induced by administration of pilocarpine to lithium-pretreated rats, Exp. Neurol., 97 (1986) 193-200. 13 Ormandy, G.C., Jope, R.S. and Snead, O.C., Anticonvulsant actions of MK-801 on the lithium-pilocarpine model of status epilepticus in rats, Exp. Neurol., 106 (1989) 172-180. 14 Persinger, M.A., Makarec, K. and Bradley, J.-C., Characteristics of limbic seizures evoked by peripheral injections of lithium and pilocarpine, Physiol. Behav., 44 (1988) 27-37. 15 Scheller, M.S., Zornow, M.H., Fleischer, J.E., Shearman, G.T. and Greber, T.E, The non-competitive N-methyI-D-aspartate receptor antagonist, MK-801, profoundly reduces volatile anesthetic requirements in rabbits, Neuropharmacology, 28 (1989) 677-681. 16 Stelzer, A., Slater, N.T. and Ten Bruggencate, G., Activation of NMDA receptors blocks GABAergic inhibition in an in vitro model of epilepsy, Nature, 326 (1987) 698-701. 17 Treiman, D.M., Walton, N.Y. and Kendrick, C., A progressive sequence of electroencephalographic changes during generalized convulsive status epilepticus, Epilepsy Res., 5 (1990) 49-60. 18 Vezzani, A., Serafini, R., Stasi, M.A., Caccia, S., Conti, I., Tridico, R.V. and Samanin, R., Kinetics of MK-801 and its effect on quinolinic acid-induced seizures and neurotoxicity in rats, J. Pharmacol. Exp. Ther., 249 (1989) 278-283. 19 Walter, D.O., On units and dimensions for reporting spectral intensities, Electroencephalogr. Clin. Neurophysiol., 24 (1968) 486-487. 20 Walton, N.Y. and Treiman, D.M., Response of status epilepticus induced by lithium and pilocarpine to treatment with diazepam, Exp. Neurol., 101 (1988) 267-275.
1 Baldino, E, Wolfson, B., Heinemann, U. and Gutnick, M.J., An N-methyI-D-aspartate (NMDA) receptor antagonist reduces bicuculline-induced depolarization shifts in neocortical explant cultures, Neurosci. Lett., 70 (1986) 101-105. 2 Daniel, L.C. The non-competitive N-methyl-D-aspartate antagonists, MK-801, phencyelidine and ketamine, increase the potency of general anesthetics, Pharmacol. Biochem. Behav., 36 (1990) 111-115. 3 Dingledine, R., Hymes, A. and King, G.L., Involvement of N-methyl-D-aspartate receptors in epileptiform bursting in the rat hippocampal slice, Z Neurophysiol., 380 (1986) 175-189. 4 Fariello, R.G., Golden, G.T., Smith, G.G. and Reyes, P.E, Potentiation of kainic acid epileptogenicity and sparing from neuronal damage by an NMDA receptor antagonist, Epilepsy Res., 3 (1989) 206-213. 5 Foster, A.C. and Wong, E.H.E, The novel anticonvulsant MK-801 binds to the activated state of the N-methyi-D-aspartate receptor in rat brain, Br. J. Pharmacol., 91 (1987) 403-409. 6 Gunawan, S., Walton, N.Y. and Treiman, D.M., Analysis of lorazepam in rat brain using liquid/liquid and solid-phase extraction in combination with high performance liquid chromatography, Biomed. Chromatogr., 4 (1990) 168-170. 7 Honehar, M.P., Olney, J.W. and Sherman, W.R., Systemic cholinergic agents induce seizures and brain damage in lithiumtreated rats, Science, 220 (1983) 323-325. 8 Horne, A.L., Harrison, N.L., Turner, J.D. and Simmons, M.A., Spontaneous paroxysmal activity induced by zero magnesium and bicuculline: suppression by NMDA antagonists and GABA mimetics, Eur. J. Pharmacol., 122 (1986) 221-238. 9 Jones, R.S.G., Epileptiform events induced by GABA antagonists in entorhinal cortical cells in vitro are partly mediated by N-methyl-D-aspartate receptors, Brain Research, 457 (1988) 113-121. 10 Jope, R.S., Morrisett, R.A. and Snead, O.C., Characterization
Acknowledgements. The technical assistance of Mr. James Kang in carrying out these experiments is gratefully acknowledged. This work was supported by a grant from the Department of Veterans Affairs. Portions of this work were presented at the 1990 meeting of the American Epilepsy Society.
