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Induction of chronic epileptiform activity in the rat by an inhibitor of cholesterol synthesis, U18666A
Brain Research, 150 (1978) 343-351 © Elsevier/North-HollandBiomedicalPress
343
INDUCTION OF CHRONIC EPILEPTIFORM ACTIVITY IN THE RAT BY AN INHIBITOR OF CHOLESTEROL SYNTHESIS, U18666A
GEORGE G. BIERKAMPER* and RICHARD J. CENEDELLA Department of Pharmacology, West Virginia University, Morgantown, W.Va. and Department of Biochemistry, Kirksville College of Osteopathic Medicine, Kirksville, Mo. 63501 (U.S.A.)
(Accepted November 10th, 1977)
SUMMARY Earlier work in our and other laboratories suggest that alteration of brain lipids, primarily sterols, could be a precondition for the development of epileptiform activity. The present study further tests this hypothesis by attempting to produce chronic epileptiform activity in the rat by a drug which impairs biosynthesis &cerebral cholesterol. Starting one day after birth, weekly injection of the rat with U18666A, 3-fl(2-diethylaminoethoxy)androst-5-en-17-one hydrochloride (10 mg/kg, s.c.), produced a reduced seizure threshold to flurothyl ether and a recurrent, spontaneous seizure state by the sixth and tenth weeks of life, respectively. These conditions were not seen if treatment was delayed until rats were about 4 weeks old. The seizure pattern, as seen by continuous ECoG and EMG recordings, consisted of a 3-5 sec burst of high voltage spiking and corresponding increases in muscle activity. However, major motor seizures were not produced. The total episode lasted 10-15 sec. Seizure frequency ranged from 5 to 21 per day. U 18666A decreases cholesterol synthesis,presumably by inhibiting enzymatic reduction of desmosterol to cholesterol. After the first two weeks of treatment, cerebral cortical cholesterol levels decreased to about 50 ~o of control cortical levels. However, the concentration of cerebral total sterols did not change because desmosterol levels reciprocally increased. In spite of continued drug dosage, cholesterol and desmosterol levels of treated rats approached those of controls by 8 weeks of age. These observations, plus finding that a seizure-prone state did not develop in rats when the onset of drug treatment was postponed until about 4 weeks of age, suggest that alterations of brain sterols early in development of the mammalian brain can result in development of a chronic, epileptiform condition later in life.
* Present Address: Department of EnvironmentalHealth Sciences, The Johns Hopkins University School of Hygieneand Public Health, 615 N, WolfeSt., Baltimore, Md. 21205, U.S.A.
344 INTRODUCTION A relationship between lipids, especially sterols, and the etiology or expression of epilepsy is suggested by several lines of investigation. Elevation of serum cholesterol levels decrease convulsive seizures in epileptic monkeys 11. Diets rich in cholesterol decrease the susceptibility of mice and rats to pentylenetetrazole-induced seizures 1. Evidence for a direct relationship between alterations of cerebral cortical sterols and cobalt-induced epilepsy has recently been obtained from studies in our laboratory 2,3. This model of chronic epilepsy or epileptiform activity is produced by insertion of cobalt-metal slivers into the cerebral cortex 5. We observed that the cortical concentration of unesterified (free) cholesterol decreased and the concentration of esterified cholesterol greatly increased in rats implanted with epileptogenic metals (cobalt and nickel) but not in those implanted with non-epileptogenic metals (copper and stainless steel) 2. These changes were observed to precede or parallel the first appearance of epileptiform activity as measured by electrocorticography. In view of an apparent relationship between cerebral cortical sterols and this experimental epilepsy, we examined the possibility that an inhibitor of biosynthesis of cerebral cholesterol might induce chronic epileptiform activity in the rat. The present paper reports the successful attempt to induce chronic epileptiform activity in the rat with the drug UI8666A (3-fl(2-diethylaminoethoxy)androst-5-en-17-one hydrochloride). Cerebral lipid changes produced by U 18666A are also described. MATERIALS AND METHODS
Treatment of rats Beginning one day after birth, Sprague-Dawley rats (Hilltop Lab Animals, Scottdale, Pa.) were injected (s.c.) every fourth or seventh day with either 10 mg/kg of U18666A dissolved in olive oil or with plain olive oil (controls). These doses of U18666A were usually continued for 8-12 weeks and were well tolerated by the rat, as judged by normal gross appearance and normal weight gain. Flurothyl ether seizure threshold testing The sensitivity of control and treated rats to induction of convulsions by fluorothyl ether (Airco, Murray Hill, N.J.) was tested by a modification of the method of Truitt et al. 17. Briefly, a rat was placed into a two-gallon desiccator jar, 2 ml of a solution of flurothyl (10% in 95 % ethanol, v/v) was injected into a gauze sponge in the base of the chamber. The time elapsed to development of clonic seizure (with loss of righting reflex) after sealing the chamber was used as the measure of brain excitability to this chemical. Eleetrocorticography Treated and age-matched control rats were anesthetized with Innovar (McNeil Labs, Fort Washington, Pa.), and permanent electrodes (stainless steel screws) were placed over the left and right frontal and parietal cortices for bipolar electrocortico-
345 graphy (ECoG) recording as described by Colasanti et alA All electrodes were connected to a headpiece module, which was held in place with dental acrylic. Rats were placed into individual monitoring cages, where the headpieces were connected through a 6-conductor cable to a mercury swivel-junction box at the top of the cage. E C o G and E M G activities were recorded continuously for 7-10 days using a 4-channel polygraph (Grass model 5D). U18666A was not given during the periods of recording. Animals had free access to food and water and were on a 12 h light/dark cycle. Isolation and quantification o f cerebral lipids
Total lipids from the cerebral cortex of treated and age-matched control rats were isolated at 1, 14-15, 30 and 56 days of age. Rats were sacrificed by decapitation, brains quickly removed and blotted dry. Samples of cortex (100-200 mg), free of white matter, were removed with a scalpel and frozen within 2.5 min of decapitation. The frozen cortical samples were weighed and directly homogenized in 20 vols of chloroform-methanol (C :M, 2:1, v/v) using a Tekmar (Polytron-like) homogenizer. Total lipids were recovered and subsequently fractioned by thin-layer chromatography on silica gel G into various classes as described before 4. The silica gel areas corresponding to free cholesterol (sterols) and phospholipids were separately recovered. Total free sterols, extracted from the silica gel with chloroform, were separated and quantified on a Packard Becker 417 gas chromatograph, using the column and operating conditions described by Vela and Acevedo TM. Sterol identity was confirmed by a comparison to standards. Total phospholipids were extracted from the silica gel with methanol and quantitated by the colorimetric method of Raheja et a1.14. In some cases sterol esters were measured following saponification as we described earlier z. Measurements of cerebral lipids focused on the cortex rather than whole brain, since electrical activity of the brain which is recorded at the head's surface is thought to originate from the cortex 10. The influence of dosage frequency with U18666A upon the concentration of cerebral sterols was examined by giving U18666A at either 10 mg/kg (s.c.) every fourth day or 10 mg/kg (s.c.) every seventh day. The more extensive data was collected with animals dosed every fourth day.
TABLE I Flurothyl seizure thresholds o f rats treated with U18666A Treatment*
n
Body weight (gm)
Time to clonus** (sec)
Control U18666A
6 6
85 4- 3 79 4- 6
599 4- 57 384 4- 29***
* Beginning one day after birth, rats received U18666A (10 mg/kg, s.c.) once weekly for 4 weeks. Controls received plain olive oil. At 6 weeks of age male rals were tested for seizure thresholds. ** Seizure susceptibility was assessed by the modified Flurothyl inhalation method of Truitt et allL Values are means 4- S.E.M. *** P(t) < 0.01 (Student's 't' test).
346
Fig. 1. Typical recordings of ECoG and EMG activity from UI8666A treated rats. The tracings are simultaneous high speed recordings (25 mm/sec) from two different rats. Stereotypic seizure patterns are seen for both animals. The EMG activity for rat-I is not integrated; whereas, that for rat-2 is integrated. Both rats were recorded in the awake state. The animals had been treated from one day after birth with 10 mg/kg (s.c.) of U18666A every 7th day for 10 weeks. Bipolar recordings were made across the right frontal (RF) and left parietal (LP) electrodes. EMG activity was recorded bilaterally from the temporalis muscles.
RES U LTS
Flurothyl seizure threshold tests Weekly injection of U 18666A (10 mg/kg, s.c.) into neonatal rats for 4 consecutive weeks caused a reduction in the seizure threshold of male rats to the convulsant flurothyl as compared with age-matched controls when both groups were tested at 6 weeks of age. The rats treated with UI8666A convulsed approximately 3.5 min (about 35 ~o) earlier than control animals (Table I). In contrast, if onset of the treatment period (10 mg U18666A/kg, s.c., once weekly for 6 weeks) was delayed until the rats were 4 weeks old, no change was seen in the seizure threshold of treated rats. Injection of only the mothers (10 mg/kg/week, s.c.) during either the last two weeks of pregnancy or during the first 4 weeks of nursing had no effect on seizure thresholds to flurothyl of mothers or pups.
Electrocorticography Figs. 1 and 2 show typical recordings of E C o G and E M G activity from rats in the awake state which had been treated with U18666A (10 mg/kg, s.c.) once weekly for 10 weeks. Treatment was started one day after birth. The stereotypic seizure pattern consisted of 3-5 sec bursts of high voltage spiking and a corresponding increase in integrated muscle activity. Behaviorally, the rats assumed a 'frozen' posture during the high voltage burst; this was usually followed by rapid licking and chewing movements and brief rhythmic twitching o f the forelimbs. During this time the rat appeared disoriented, and did not show a startle reflex to loud noises. This total opisode lasted 10-15 sec. None of the U18666A-treated rats were ever observed to have a generalized seizure, nor did they lose their righting reflex. In fact, without the E C o G recordings
347
Fig. 2. Typical slow speed recordings (0.5 mm/sec) of the ECoG and EMG activity from a control and a UI8666A treated rat. The conditions of treatment and the recording methods are identical to those described for Fig. 1. Control rats received plain olive oil. S, stereotypic seizure pattern recorded at slow speed.
it is doubtful that the presence ofepileptiform activity would have been suspected, since no violent m o t o r activity was observed. Recurrent, spontaneous seizure episodes continued to occur in rats which were observed 3 months after termination of the initial 10-12-week drug treatment regimen. By continuous 24 h E C o G and E M G recording, the mean frequency of these stereotypic seizure episodes was obtained for a 7-day period in 12 U18666A-treated rats and in 4 control rats (Table II). Seizure frequency for the U18666A-treatedmale rats ranged from about 5 to 21 seizures per day, with an average of about 10 stereotypic seizures per day. No day-to-night variations were noted in seizure frequency; sleep-awake cycle variations in seizure frequency were not examined. These results represent the frequency of only stereotypic seizure patterns. Some animals treated with U18666A generated frequent spike-wave complexes without the 3-5 sec high voltage bursts. Of the three U18666A-treated female rats examined, only one showed such seizure bursts. Another female exhibited occasional spike-wave complexes without a single sustained high voltage burst, and a third female showed no abnormal E C o G activity. None of the control rats generated any epileptiform activity during the 7-10 days that they were recorded.
348 TABLE II Frequency of seizures observed over a 7-day period in U18666A-treated rats Rat number
1
2 3 4 5 6 7 8 9 10 11 12 Controls
Sex
Mean number of seizures/day ± S.E.M.*
M
0.8 7.6 q- 0.6 10.3 ± 1.1 20.8 ± 2.5 5.8 d: 0.6 15.1 ± 0.9 14.4 ± 1.3 5.3 ~ 0.8 4.8 ± 1.0 ** *** 1.4 ± 0.5 **
M M M M M M M M F F F (3 m, 1 f)
10.6 ±
* Mean seizure frequency i S.E.M. measured by continuous 24 h monitoring of the ECoG over seven consecutive days. ** No epileptogenic activity. *** Frequent spike-wave complexes.
Cerebral c o r t e x lipids
Measurement of cerebral cortical sterols from U 18666A-treated rats (10 mg/kg, s.c., every fourth day) over the first 8 weeks after birth revealed that by two weeks of age cortical cholesterol levels decreased to about 50 ~ of the concentration found in the cortex of age-matched control rats (Fig. 3). Total cortical sterol concentrations were not decreased because demosterol levels reciprocally increased. In spite of continued dosage with U18666A, after two weeks of treatment cholesterol and desmosterol levels of treated rats began to approach those of controls and by the eighth week much of the earlier differences in cortical sterol composition between the two groups had disappeared. However, at the eighth week there was some indication that the total sterol level in cortex from treated rats was slightly less than that from controls. Also, within limits, the frequency of treatment with the 10 mg/kg dose of U18666A apparently had little effect upon the measured cortical lipids. For example, at two weeks o f age desmosterol comprised 52.4 ± 0.8 ~o (n = 10) of the total cortical sterols of rats treated every fourth day; whereas, it comprised 54.4 i 1.3 ~ (n ---- 12) of those treated every seventh day. Fumagalli et al. 7 similarly observed that the alterations of brain sterols in the neonatal rat produced by 20,25-diazacholesterol were largely independent of the amount or frequency of the drug dose. No significant differences were found in the cortical concentrations of total phospholipids between control and treated rats at any of the times examined. Cerebral cortical levels of esterified cholesterol were significantly lower in treated as compared with control rats after both 14 and 30 days of treatment but not after 56 days (data not shown). Unexpectedly, little or no desmosterol was present in the total sterol esters isolated from cortex of treated or control rats. No differences
349 II
CONTROL CHOLESTEROL........ TREATED
I0
t
TRE~..y,,.'"""
...........
CONTROL
DESMOSTEROL. . . . . . . . . . .
9
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/
.,,:[
8 X b.l F-a0 c.)
~
7
6
l.U t.;, -.. ._I 0 n-" hl I---
s o ~ 'S
its
Total Sterol (mg / g)
sf
~f
,'r,
.,s sS so
/
rr
a:
Z
/s
Age
i"
5
/
/
/
4
(Days~ 1 14
s
s" /
3
7.32 + 0.12 + 0 17
*56 "'.,
Treated
2,65 +_ 0.10 6.04 + 0.11 6.05 +_ 0.15
30
,i,~ s
Control
10 89
7.45+-0-07 10.25+-0.16
P(t) of diff, between control and treated
0.02.
.%
2 E •.
I
I
.
.
I
I
5
I0
•
I I 14 15
.
.
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.
I
.
.
.
.
.
-,-,-•
' ~ . L ..............J........... l ........... J _ _ = . - - ~ .......... L.a 30 56
DAYS OF AGE
Fig. 3. Effect of treatment of the rats with U 18666A upon cerebral cortex concentrations of cholesterol and desmosterol. Beginning one day after birth, rats received either 10 mg/kg (s.c.) of U18666A dissolved in olive oil every 4th day for up to eight weeks or plain olive oil (controls). Individual litters of control and treated rats were sacrificed at 1, 14-15, 30, and 56 days of age. Point are the mean 4S.E.M. (bars) of 10-12 rats at each time except day one where n = 6 (cortices from two rats were pooled per sample). The differences in concentrations of cholesterol and desmosterol between control and treated rats at each time are all statistically significant, P(t) < 0.001. were observed between the response of treated male and female rats to the lipid altering effects of U18666A. DISCUSSION U18666A lowers cortical levels of cholesterol and reciprocally increases those of desmosterol through a presumed inhibition of the enzymatic reduction of desmosterol to cholesterol 13. U18666A was chosen for the present study because Jurgelski et al. 8 reported that treatment of the juvenile opossum with this drug for 15-25 weeks resulted in the induction of a chronic somatosensory epilepsy characterized by major motor seizures. However, no epileptiform activity was reported by Winer et al. 19 to occur in rats given U18666A orally for 4 weeks at 15 mg/kg/day beginning at 3 weeks of age. In contrast, we observed chronic epileptiform activity in rats, particularly male rats, treated for 10-12 weeks with this drug when treatment was initiated one day after birth. Spontaneous seizure activity continued months after termination of drug treatment. Apparently there is a critical period in development of the rat during which U18666A can affect sterol metabolism in the brain. Our studies indicate that this period is probably the first two weeks after birth, since following two weeks of treatment cortical levels
350 of desmosterol sharply decreased and cholesterol increased in spite of continued drug dosage. Also, if the onset of drug treatment was delayed until the rats were about 4 weeks old, evidence for seizure development was not obtained. Scott and Barber 1~ similarly observed that if triparanol, another inhibitor of enzymatic reduction of desmosterol, was given twice weekly to mice for 12 weeks (the first dose given one day after birth) the per cent content of desmosterol in brain increased only until day 17 and then decreased to zero per cent by the seventh week. These results suggest that the transport of U18666A and triparanol into the brain decreases after about two weeks of age. The present studies indicated that U18666A affects the critical period of rapid development of the brain which takes place during the first 3 weeks of the rat's neonatal life 9A2 ; a period during which the brain is particularly vulnerable to metabolic insult ",9. The reduced concentration of cholesterol in the brain of U18666A treated rats during this important early phase of development might result in abnormal myelination or in formation of abnormal neuronal or glial cell membranes. Although cortical membranes formed subsequent to this period of depressed cholesterol and elevated desmosterol levels would be normal, a population of permanently altered structures could predispose the animal to epileptiform activity. In conclusion, our observations support the theory that derangement of brain sterols during periods of rapid development and growth of the brain can cause permanent structural or functional alterations which could lead to a chronic epileptic condition. The present studies also provide a new experimental model of chronic epileptiform activity in the mammal. This model apparently has a unique feature of involving frequent spontaneous seizures that are detectable by electroencephalography and that do not involve major increases of motor activity. In this regard, the epileptiform activity appears to share similarities with pure petit-mal discharges in children and adolescents 15 ACKNOWLEDGEMENT This project was supported by a grant from the Epilepsy Foundation of America.
REFERENCES 1 Alexander, G. J. and Kopeloff, L. M., Induced hypercholest©remia and decreased susceptibihty to seizures in experimental animals, Exp. NeuroL, 32 (197t) 134-140. 2 Bierkamper, G. G., Craig, C. R. and Cenedella, R. J., Cerebral cortical sterol changes in cobaltinduced epilepsy, Fed. Prec., 35 (1976) 544. 3 Cenedella, R. J. and Craig, C. R., Changes in cerebral cortical lipids in cobalt-induced epilepsy, J. Neurochem., 21 (1973) 743-748. 4 Ccnedella, R. J., Gaili, C. and Paoletti, R., Brain free fatty acid levels in rats sacrificed by decapitation versus focused microwave irradiation, Lipids, 10 (1975) 290-293. 5 Colasanti, B. K., Hartman, E. R. and Craig, C. R., Eleetrocorticogram and behavioral correlates during the development of chronic cobalt experimental epilepsy in the rat, Epilepsia (Basel), 15 (1974) 361-373. 6 Dobbing, J., Vulnerable periods in developing brain. In A. N. Davison and J. Dobbing (Eds,), Applied Neurochemistry, Blackweil, Oxford, 1968, pp. 287-316.
351 7 Fumagalli, R., Smith, M. E., Urna, G. and Paoletti, R., The effect of hypocholesterolemic agents on myelinogenesis, J. Neurochem., 16 (1969) 1329-1339. 8 Jurgelski, W., Jr., Hudson, P. M. and Vogel, F. S., Induction of a chronic somatosensory epilepsy in the opossum (Didelphia Virginiana Kerr) with an inhibitor of cholesterol biosynthesis, Brain Research, 64 (1973) 466--471. 9 Kabara, J. J., A critical review of brain cholesterol metabolism. Prog. Brain Res., 40 (1973) 363-382. 10 Klemn, W. R. (Ed.), Animal Electroencephalography, Academic Press, New York, 1969, pp. 12-15. 11 Kopeloff, L. M. and Alexander, G. J., Serum cholesetrol in monkeys with chronic epileptic foci, Life Sci., 10 (1971) 869-876. 12 Norton, W. T. and Poduslo, S. E., Myelination in rat brain: changes in myelin composition during brain maturation, J. Neurochem., 21 (1973) 759-773. 13 Phillips, W. A. and Avigan, J., Inhibition of cholesterol biosynthesis in the rat by 3fl-(2 diethylaminoethoxy) androst-5-en-17-one, hydrochloride, Proc. Soc. exp. Biol. (N.Y.), 112 (1963) 233-236. 14 Raheja, R. K., Kaur, C., Sinhg, A. and Bhatia, I. S., New colorimetric method for the quantitative estimation of phospholipids without acid digestion, J. Lipid Res., 14 (1973) 695-697. 15 Sadove, M. S., Becka, D. and Gibbs, F. A., (Eds.) Electroencephalography for Anesthesiologists and Surgeons, J. B. Lippincott, Philadelphia, 1967, pp. 69-70. 16 Scott, T. G. and Barber, V. C., An enzyme histochemical and biochemical study of the effect of an inhibitor of cholesterol synthesis on myelinatingmouse brain, J. Neurochem., 11 (1964)423-429. 17 Truitt, E. G., Jr., Ebersberger, E. M. and Ling, A. S. C., Measurement of brain excitability by use of hexafluorodiethyl ether (Indoklon), J. Pharmacol. Exp. Ther., 129 (1971) 33-38. 18 Vela, B. A. and Acevedo, H. F., Determination of urinary cholesterol by gas-liquid chromatography, Steroids, 14 (1969) 499-516. 19 Winer, N., Klachko, D. M., Baer, R. D., Langley, P. L. and Burns, T. W., Myotonic response induced by inhibitors of cholesterol biosynthesis, Science, 153 (1966) 312-313.
343
INDUCTION OF CHRONIC EPILEPTIFORM ACTIVITY IN THE RAT BY AN INHIBITOR OF CHOLESTEROL SYNTHESIS, U18666A
GEORGE G. BIERKAMPER* and RICHARD J. CENEDELLA Department of Pharmacology, West Virginia University, Morgantown, W.Va. and Department of Biochemistry, Kirksville College of Osteopathic Medicine, Kirksville, Mo. 63501 (U.S.A.)
(Accepted November 10th, 1977)
SUMMARY Earlier work in our and other laboratories suggest that alteration of brain lipids, primarily sterols, could be a precondition for the development of epileptiform activity. The present study further tests this hypothesis by attempting to produce chronic epileptiform activity in the rat by a drug which impairs biosynthesis &cerebral cholesterol. Starting one day after birth, weekly injection of the rat with U18666A, 3-fl(2-diethylaminoethoxy)androst-5-en-17-one hydrochloride (10 mg/kg, s.c.), produced a reduced seizure threshold to flurothyl ether and a recurrent, spontaneous seizure state by the sixth and tenth weeks of life, respectively. These conditions were not seen if treatment was delayed until rats were about 4 weeks old. The seizure pattern, as seen by continuous ECoG and EMG recordings, consisted of a 3-5 sec burst of high voltage spiking and corresponding increases in muscle activity. However, major motor seizures were not produced. The total episode lasted 10-15 sec. Seizure frequency ranged from 5 to 21 per day. U 18666A decreases cholesterol synthesis,presumably by inhibiting enzymatic reduction of desmosterol to cholesterol. After the first two weeks of treatment, cerebral cortical cholesterol levels decreased to about 50 ~o of control cortical levels. However, the concentration of cerebral total sterols did not change because desmosterol levels reciprocally increased. In spite of continued drug dosage, cholesterol and desmosterol levels of treated rats approached those of controls by 8 weeks of age. These observations, plus finding that a seizure-prone state did not develop in rats when the onset of drug treatment was postponed until about 4 weeks of age, suggest that alterations of brain sterols early in development of the mammalian brain can result in development of a chronic, epileptiform condition later in life.
* Present Address: Department of EnvironmentalHealth Sciences, The Johns Hopkins University School of Hygieneand Public Health, 615 N, WolfeSt., Baltimore, Md. 21205, U.S.A.
344 INTRODUCTION A relationship between lipids, especially sterols, and the etiology or expression of epilepsy is suggested by several lines of investigation. Elevation of serum cholesterol levels decrease convulsive seizures in epileptic monkeys 11. Diets rich in cholesterol decrease the susceptibility of mice and rats to pentylenetetrazole-induced seizures 1. Evidence for a direct relationship between alterations of cerebral cortical sterols and cobalt-induced epilepsy has recently been obtained from studies in our laboratory 2,3. This model of chronic epilepsy or epileptiform activity is produced by insertion of cobalt-metal slivers into the cerebral cortex 5. We observed that the cortical concentration of unesterified (free) cholesterol decreased and the concentration of esterified cholesterol greatly increased in rats implanted with epileptogenic metals (cobalt and nickel) but not in those implanted with non-epileptogenic metals (copper and stainless steel) 2. These changes were observed to precede or parallel the first appearance of epileptiform activity as measured by electrocorticography. In view of an apparent relationship between cerebral cortical sterols and this experimental epilepsy, we examined the possibility that an inhibitor of biosynthesis of cerebral cholesterol might induce chronic epileptiform activity in the rat. The present paper reports the successful attempt to induce chronic epileptiform activity in the rat with the drug UI8666A (3-fl(2-diethylaminoethoxy)androst-5-en-17-one hydrochloride). Cerebral lipid changes produced by U 18666A are also described. MATERIALS AND METHODS
Treatment of rats Beginning one day after birth, Sprague-Dawley rats (Hilltop Lab Animals, Scottdale, Pa.) were injected (s.c.) every fourth or seventh day with either 10 mg/kg of U18666A dissolved in olive oil or with plain olive oil (controls). These doses of U18666A were usually continued for 8-12 weeks and were well tolerated by the rat, as judged by normal gross appearance and normal weight gain. Flurothyl ether seizure threshold testing The sensitivity of control and treated rats to induction of convulsions by fluorothyl ether (Airco, Murray Hill, N.J.) was tested by a modification of the method of Truitt et al. 17. Briefly, a rat was placed into a two-gallon desiccator jar, 2 ml of a solution of flurothyl (10% in 95 % ethanol, v/v) was injected into a gauze sponge in the base of the chamber. The time elapsed to development of clonic seizure (with loss of righting reflex) after sealing the chamber was used as the measure of brain excitability to this chemical. Eleetrocorticography Treated and age-matched control rats were anesthetized with Innovar (McNeil Labs, Fort Washington, Pa.), and permanent electrodes (stainless steel screws) were placed over the left and right frontal and parietal cortices for bipolar electrocortico-
345 graphy (ECoG) recording as described by Colasanti et alA All electrodes were connected to a headpiece module, which was held in place with dental acrylic. Rats were placed into individual monitoring cages, where the headpieces were connected through a 6-conductor cable to a mercury swivel-junction box at the top of the cage. E C o G and E M G activities were recorded continuously for 7-10 days using a 4-channel polygraph (Grass model 5D). U18666A was not given during the periods of recording. Animals had free access to food and water and were on a 12 h light/dark cycle. Isolation and quantification o f cerebral lipids
Total lipids from the cerebral cortex of treated and age-matched control rats were isolated at 1, 14-15, 30 and 56 days of age. Rats were sacrificed by decapitation, brains quickly removed and blotted dry. Samples of cortex (100-200 mg), free of white matter, were removed with a scalpel and frozen within 2.5 min of decapitation. The frozen cortical samples were weighed and directly homogenized in 20 vols of chloroform-methanol (C :M, 2:1, v/v) using a Tekmar (Polytron-like) homogenizer. Total lipids were recovered and subsequently fractioned by thin-layer chromatography on silica gel G into various classes as described before 4. The silica gel areas corresponding to free cholesterol (sterols) and phospholipids were separately recovered. Total free sterols, extracted from the silica gel with chloroform, were separated and quantified on a Packard Becker 417 gas chromatograph, using the column and operating conditions described by Vela and Acevedo TM. Sterol identity was confirmed by a comparison to standards. Total phospholipids were extracted from the silica gel with methanol and quantitated by the colorimetric method of Raheja et a1.14. In some cases sterol esters were measured following saponification as we described earlier z. Measurements of cerebral lipids focused on the cortex rather than whole brain, since electrical activity of the brain which is recorded at the head's surface is thought to originate from the cortex 10. The influence of dosage frequency with U18666A upon the concentration of cerebral sterols was examined by giving U18666A at either 10 mg/kg (s.c.) every fourth day or 10 mg/kg (s.c.) every seventh day. The more extensive data was collected with animals dosed every fourth day.
TABLE I Flurothyl seizure thresholds o f rats treated with U18666A Treatment*
n
Body weight (gm)
Time to clonus** (sec)
Control U18666A
6 6
85 4- 3 79 4- 6
599 4- 57 384 4- 29***
* Beginning one day after birth, rats received U18666A (10 mg/kg, s.c.) once weekly for 4 weeks. Controls received plain olive oil. At 6 weeks of age male rals were tested for seizure thresholds. ** Seizure susceptibility was assessed by the modified Flurothyl inhalation method of Truitt et allL Values are means 4- S.E.M. *** P(t) < 0.01 (Student's 't' test).
346
Fig. 1. Typical recordings of ECoG and EMG activity from UI8666A treated rats. The tracings are simultaneous high speed recordings (25 mm/sec) from two different rats. Stereotypic seizure patterns are seen for both animals. The EMG activity for rat-I is not integrated; whereas, that for rat-2 is integrated. Both rats were recorded in the awake state. The animals had been treated from one day after birth with 10 mg/kg (s.c.) of U18666A every 7th day for 10 weeks. Bipolar recordings were made across the right frontal (RF) and left parietal (LP) electrodes. EMG activity was recorded bilaterally from the temporalis muscles.
RES U LTS
Flurothyl seizure threshold tests Weekly injection of U 18666A (10 mg/kg, s.c.) into neonatal rats for 4 consecutive weeks caused a reduction in the seizure threshold of male rats to the convulsant flurothyl as compared with age-matched controls when both groups were tested at 6 weeks of age. The rats treated with UI8666A convulsed approximately 3.5 min (about 35 ~o) earlier than control animals (Table I). In contrast, if onset of the treatment period (10 mg U18666A/kg, s.c., once weekly for 6 weeks) was delayed until the rats were 4 weeks old, no change was seen in the seizure threshold of treated rats. Injection of only the mothers (10 mg/kg/week, s.c.) during either the last two weeks of pregnancy or during the first 4 weeks of nursing had no effect on seizure thresholds to flurothyl of mothers or pups.
Electrocorticography Figs. 1 and 2 show typical recordings of E C o G and E M G activity from rats in the awake state which had been treated with U18666A (10 mg/kg, s.c.) once weekly for 10 weeks. Treatment was started one day after birth. The stereotypic seizure pattern consisted of 3-5 sec bursts of high voltage spiking and a corresponding increase in integrated muscle activity. Behaviorally, the rats assumed a 'frozen' posture during the high voltage burst; this was usually followed by rapid licking and chewing movements and brief rhythmic twitching o f the forelimbs. During this time the rat appeared disoriented, and did not show a startle reflex to loud noises. This total opisode lasted 10-15 sec. None of the U18666A-treated rats were ever observed to have a generalized seizure, nor did they lose their righting reflex. In fact, without the E C o G recordings
347
Fig. 2. Typical slow speed recordings (0.5 mm/sec) of the ECoG and EMG activity from a control and a UI8666A treated rat. The conditions of treatment and the recording methods are identical to those described for Fig. 1. Control rats received plain olive oil. S, stereotypic seizure pattern recorded at slow speed.
it is doubtful that the presence ofepileptiform activity would have been suspected, since no violent m o t o r activity was observed. Recurrent, spontaneous seizure episodes continued to occur in rats which were observed 3 months after termination of the initial 10-12-week drug treatment regimen. By continuous 24 h E C o G and E M G recording, the mean frequency of these stereotypic seizure episodes was obtained for a 7-day period in 12 U18666A-treated rats and in 4 control rats (Table II). Seizure frequency for the U18666A-treatedmale rats ranged from about 5 to 21 seizures per day, with an average of about 10 stereotypic seizures per day. No day-to-night variations were noted in seizure frequency; sleep-awake cycle variations in seizure frequency were not examined. These results represent the frequency of only stereotypic seizure patterns. Some animals treated with U18666A generated frequent spike-wave complexes without the 3-5 sec high voltage bursts. Of the three U18666A-treated female rats examined, only one showed such seizure bursts. Another female exhibited occasional spike-wave complexes without a single sustained high voltage burst, and a third female showed no abnormal E C o G activity. None of the control rats generated any epileptiform activity during the 7-10 days that they were recorded.
348 TABLE II Frequency of seizures observed over a 7-day period in U18666A-treated rats Rat number
1
2 3 4 5 6 7 8 9 10 11 12 Controls
Sex
Mean number of seizures/day ± S.E.M.*
M
0.8 7.6 q- 0.6 10.3 ± 1.1 20.8 ± 2.5 5.8 d: 0.6 15.1 ± 0.9 14.4 ± 1.3 5.3 ~ 0.8 4.8 ± 1.0 ** *** 1.4 ± 0.5 **
M M M M M M M M F F F (3 m, 1 f)
10.6 ±
* Mean seizure frequency i S.E.M. measured by continuous 24 h monitoring of the ECoG over seven consecutive days. ** No epileptogenic activity. *** Frequent spike-wave complexes.
Cerebral c o r t e x lipids
Measurement of cerebral cortical sterols from U 18666A-treated rats (10 mg/kg, s.c., every fourth day) over the first 8 weeks after birth revealed that by two weeks of age cortical cholesterol levels decreased to about 50 ~ of the concentration found in the cortex of age-matched control rats (Fig. 3). Total cortical sterol concentrations were not decreased because demosterol levels reciprocally increased. In spite of continued dosage with U18666A, after two weeks of treatment cholesterol and desmosterol levels of treated rats began to approach those of controls and by the eighth week much of the earlier differences in cortical sterol composition between the two groups had disappeared. However, at the eighth week there was some indication that the total sterol level in cortex from treated rats was slightly less than that from controls. Also, within limits, the frequency of treatment with the 10 mg/kg dose of U18666A apparently had little effect upon the measured cortical lipids. For example, at two weeks o f age desmosterol comprised 52.4 ± 0.8 ~o (n = 10) of the total cortical sterols of rats treated every fourth day; whereas, it comprised 54.4 i 1.3 ~ (n ---- 12) of those treated every seventh day. Fumagalli et al. 7 similarly observed that the alterations of brain sterols in the neonatal rat produced by 20,25-diazacholesterol were largely independent of the amount or frequency of the drug dose. No significant differences were found in the cortical concentrations of total phospholipids between control and treated rats at any of the times examined. Cerebral cortical levels of esterified cholesterol were significantly lower in treated as compared with control rats after both 14 and 30 days of treatment but not after 56 days (data not shown). Unexpectedly, little or no desmosterol was present in the total sterol esters isolated from cortex of treated or control rats. No differences
349 II
CONTROL CHOLESTEROL........ TREATED
I0
t
TRE~..y,,.'"""
...........
CONTROL
DESMOSTEROL. . . . . . . . . . .
9
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/
.,,:[
8 X b.l F-a0 c.)
~
7
6
l.U t.;, -.. ._I 0 n-" hl I---
s o ~ 'S
its
Total Sterol (mg / g)
sf
~f
,'r,
.,s sS so
/
rr
a:
Z
/s
Age
i"
5
/
/
/
4
(Days~ 1 14
s
s" /
3
7.32 + 0.12 + 0 17
*56 "'.,
Treated
2,65 +_ 0.10 6.04 + 0.11 6.05 +_ 0.15
30
,i,~ s
Control
10 89
7.45+-0-07 10.25+-0.16
P(t) of diff, between control and treated
0.02.
.%
2 E •.
I
I
.
.
I
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5
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•
I I 14 15
.
.
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.
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.
.
.
.
.
-,-,-•
' ~ . L ..............J........... l ........... J _ _ = . - - ~ .......... L.a 30 56
DAYS OF AGE
Fig. 3. Effect of treatment of the rats with U 18666A upon cerebral cortex concentrations of cholesterol and desmosterol. Beginning one day after birth, rats received either 10 mg/kg (s.c.) of U18666A dissolved in olive oil every 4th day for up to eight weeks or plain olive oil (controls). Individual litters of control and treated rats were sacrificed at 1, 14-15, 30, and 56 days of age. Point are the mean 4S.E.M. (bars) of 10-12 rats at each time except day one where n = 6 (cortices from two rats were pooled per sample). The differences in concentrations of cholesterol and desmosterol between control and treated rats at each time are all statistically significant, P(t) < 0.001. were observed between the response of treated male and female rats to the lipid altering effects of U18666A. DISCUSSION U18666A lowers cortical levels of cholesterol and reciprocally increases those of desmosterol through a presumed inhibition of the enzymatic reduction of desmosterol to cholesterol 13. U18666A was chosen for the present study because Jurgelski et al. 8 reported that treatment of the juvenile opossum with this drug for 15-25 weeks resulted in the induction of a chronic somatosensory epilepsy characterized by major motor seizures. However, no epileptiform activity was reported by Winer et al. 19 to occur in rats given U18666A orally for 4 weeks at 15 mg/kg/day beginning at 3 weeks of age. In contrast, we observed chronic epileptiform activity in rats, particularly male rats, treated for 10-12 weeks with this drug when treatment was initiated one day after birth. Spontaneous seizure activity continued months after termination of drug treatment. Apparently there is a critical period in development of the rat during which U18666A can affect sterol metabolism in the brain. Our studies indicate that this period is probably the first two weeks after birth, since following two weeks of treatment cortical levels
350 of desmosterol sharply decreased and cholesterol increased in spite of continued drug dosage. Also, if the onset of drug treatment was delayed until the rats were about 4 weeks old, evidence for seizure development was not obtained. Scott and Barber 1~ similarly observed that if triparanol, another inhibitor of enzymatic reduction of desmosterol, was given twice weekly to mice for 12 weeks (the first dose given one day after birth) the per cent content of desmosterol in brain increased only until day 17 and then decreased to zero per cent by the seventh week. These results suggest that the transport of U18666A and triparanol into the brain decreases after about two weeks of age. The present studies indicated that U18666A affects the critical period of rapid development of the brain which takes place during the first 3 weeks of the rat's neonatal life 9A2 ; a period during which the brain is particularly vulnerable to metabolic insult ",9. The reduced concentration of cholesterol in the brain of U18666A treated rats during this important early phase of development might result in abnormal myelination or in formation of abnormal neuronal or glial cell membranes. Although cortical membranes formed subsequent to this period of depressed cholesterol and elevated desmosterol levels would be normal, a population of permanently altered structures could predispose the animal to epileptiform activity. In conclusion, our observations support the theory that derangement of brain sterols during periods of rapid development and growth of the brain can cause permanent structural or functional alterations which could lead to a chronic epileptic condition. The present studies also provide a new experimental model of chronic epileptiform activity in the mammal. This model apparently has a unique feature of involving frequent spontaneous seizures that are detectable by electroencephalography and that do not involve major increases of motor activity. In this regard, the epileptiform activity appears to share similarities with pure petit-mal discharges in children and adolescents 15 ACKNOWLEDGEMENT This project was supported by a grant from the Epilepsy Foundation of America.
REFERENCES 1 Alexander, G. J. and Kopeloff, L. M., Induced hypercholest©remia and decreased susceptibihty to seizures in experimental animals, Exp. NeuroL, 32 (197t) 134-140. 2 Bierkamper, G. G., Craig, C. R. and Cenedella, R. J., Cerebral cortical sterol changes in cobaltinduced epilepsy, Fed. Prec., 35 (1976) 544. 3 Cenedella, R. J. and Craig, C. R., Changes in cerebral cortical lipids in cobalt-induced epilepsy, J. Neurochem., 21 (1973) 743-748. 4 Ccnedella, R. J., Gaili, C. and Paoletti, R., Brain free fatty acid levels in rats sacrificed by decapitation versus focused microwave irradiation, Lipids, 10 (1975) 290-293. 5 Colasanti, B. K., Hartman, E. R. and Craig, C. R., Eleetrocorticogram and behavioral correlates during the development of chronic cobalt experimental epilepsy in the rat, Epilepsia (Basel), 15 (1974) 361-373. 6 Dobbing, J., Vulnerable periods in developing brain. In A. N. Davison and J. Dobbing (Eds,), Applied Neurochemistry, Blackweil, Oxford, 1968, pp. 287-316.
351 7 Fumagalli, R., Smith, M. E., Urna, G. and Paoletti, R., The effect of hypocholesterolemic agents on myelinogenesis, J. Neurochem., 16 (1969) 1329-1339. 8 Jurgelski, W., Jr., Hudson, P. M. and Vogel, F. S., Induction of a chronic somatosensory epilepsy in the opossum (Didelphia Virginiana Kerr) with an inhibitor of cholesterol biosynthesis, Brain Research, 64 (1973) 466--471. 9 Kabara, J. J., A critical review of brain cholesterol metabolism. Prog. Brain Res., 40 (1973) 363-382. 10 Klemn, W. R. (Ed.), Animal Electroencephalography, Academic Press, New York, 1969, pp. 12-15. 11 Kopeloff, L. M. and Alexander, G. J., Serum cholesetrol in monkeys with chronic epileptic foci, Life Sci., 10 (1971) 869-876. 12 Norton, W. T. and Poduslo, S. E., Myelination in rat brain: changes in myelin composition during brain maturation, J. Neurochem., 21 (1973) 759-773. 13 Phillips, W. A. and Avigan, J., Inhibition of cholesterol biosynthesis in the rat by 3fl-(2 diethylaminoethoxy) androst-5-en-17-one, hydrochloride, Proc. Soc. exp. Biol. (N.Y.), 112 (1963) 233-236. 14 Raheja, R. K., Kaur, C., Sinhg, A. and Bhatia, I. S., New colorimetric method for the quantitative estimation of phospholipids without acid digestion, J. Lipid Res., 14 (1973) 695-697. 15 Sadove, M. S., Becka, D. and Gibbs, F. A., (Eds.) Electroencephalography for Anesthesiologists and Surgeons, J. B. Lippincott, Philadelphia, 1967, pp. 69-70. 16 Scott, T. G. and Barber, V. C., An enzyme histochemical and biochemical study of the effect of an inhibitor of cholesterol synthesis on myelinatingmouse brain, J. Neurochem., 11 (1964)423-429. 17 Truitt, E. G., Jr., Ebersberger, E. M. and Ling, A. S. C., Measurement of brain excitability by use of hexafluorodiethyl ether (Indoklon), J. Pharmacol. Exp. Ther., 129 (1971) 33-38. 18 Vela, B. A. and Acevedo, H. F., Determination of urinary cholesterol by gas-liquid chromatography, Steroids, 14 (1969) 499-516. 19 Winer, N., Klachko, D. M., Baer, R. D., Langley, P. L. and Burns, T. W., Myotonic response induced by inhibitors of cholesterol biosynthesis, Science, 153 (1966) 312-313.