Lovastatin exacerbates atypical absence seizures with only minimal effects on brain sterols

AY-9944 (AY) exacerbates chronic recurrent seizures in rats that are analogous to atypical absence epilepsy in humans. The mechanism by which AY affects the slow spike-and-wave discharges associated with these seizures is not known, but is thought to involve inhibition of cholesterol synthesis. We tested the hypothesis that seizures seen with AY are due to significant reduction in brain cholesterol and/or elevated brain 7-dehydrocholesterol by assessing whether three other cholesterol synthesis inhibitors mimic AY seizures in rats. Effects of AY on brain sterols and spike-and-wave discharge duration were compared with those of two other late-stage cholesterol inhibitors [BM 15.766 (BM) and U18666A (UA)] and to an HMG-CoA reductase (early-stage cholesterol) inhibitor, lovastatin. With BM or UA, prolongation of seizure duration and brain sterol changes was similar to that caused by AY. AY effects on both brain sterols and seizure duration were dose-related. Lovastatin, with or without concurrent AY, mimicked AY seizures but reduced brain cholesterol by <10% and did not significantly change brain 7-dehydrocholesterol.Either lovastatin has a different mechanism of action than these late-stage cholesterol inhibitors or the brain sterol changes are not directly responsible for seizures in this model. AY-9944 (AY) exacerbates chronic recurrent seizures in rats that are analogous to atypical absence epilepsy in humans. The mechanism by which AY affects the slow spike-and-wave discharges associated with these seizures is not known, but is thought to involve inhibition of cholesterol synthesis. We tested the hypothesis that seizures seen with AY are due to significant reduction in brain cholesterol and/or elevated brain 7-dehydrocholesterol by assessing whether three other cholesterol synthesis inhibitors mimic AY seizures in rats. Effects of AY on brain sterols and spike-and-wave discharge duration were compared with those of two other late-stage cholesterol inhibitors [BM 15.766 (BM) and U18666A (UA)] and to an HMG-CoA reductase (early-stage cholesterol) inhibitor, lovastatin. With BM or UA, prolongation of seizure duration and brain sterol changes was similar to that caused by AY. AY effects on both brain sterols and seizure duration were dose-related. Lovastatin, with or without concurrent AY, mimicked AY seizures but reduced brain cholesterol by <10% and did not significantly change brain 7-dehydrocholesterol. Either lovastatin has a different mechanism of action than these late-stage cholesterol inhibitors or the brain sterol changes are not directly responsible for seizures in this model. Atypical absence seizures are chronic and frequent in generalized epilepsies such as Lennox-Gastaut syndrome. They are characterized by spontaneous, bilaterally synchronous, slow spike-and-wave discharges (SWD) and are usually associated with intermittent impairment of consciousness and the ability to move during seizures (1Cortez M.A. McKerlie C. Snead 3rd O.C. A model of atypical absence seizures: EEG, pharmacology and developmental characterization.Neurology. 2001; 56: 341-349Crossref PubMed Scopus (87) Google Scholar). Unlike typical absence seizures, behavioral and electrocorticographic (ECoG) correlates of atypical absence seizures are not concordant in seizure onset and offset (2Cortez M.A. Cunnane S.C. Snead 3rd O.C. Brain sterols in the AY-9944 rat model of atypical absence seizures.Epilepsia. 2002; 43: 3-8Crossref PubMed Scopus (21) Google Scholar). During postnatal brain development, administration of the cholesterol synthesis inhibitor AY-9944 (AY) leads to permanent and recurrent atypical absence seizures in adult rats (1Cortez M.A. McKerlie C. Snead 3rd O.C. A model of atypical absence seizures: EEG, pharmacology and developmental characterization.Neurology. 2001; 56: 341-349Crossref PubMed Scopus (87) Google Scholar). This effect is clearest in Long-Evans rats, which are already susceptible to mild atypical absence seizures, but in which AY augments the seizure duration by 10- to 20-fold. Hence, we refer to this markedly heightened seizure activity as the “AY model” or as “AY seizures.” Seizure onset in the AY model occurs prepubertally (1Cortez M.A. McKerlie C. Snead 3rd O.C. A model of atypical absence seizures: EEG, pharmacology and developmental characterization.Neurology. 2001; 56: 341-349Crossref PubMed Scopus (87) Google Scholar). AY seizures in Long-Evans hooded rats and C3H mice (1Cortez M.A. McKerlie C. Snead 3rd O.C. A model of atypical absence seizures: EEG, pharmacology and developmental characterization.Neurology. 2001; 56: 341-349Crossref PubMed Scopus (87) Google Scholar, 2Cortez M.A. Cunnane S.C. Snead 3rd O.C. Brain sterols in the AY-9944 rat model of atypical absence seizures.Epilepsia. 2002; 43: 3-8Crossref PubMed Scopus (21) Google Scholar, 3Cortez M.A. Servanescu R.I. Snead III. O.C. The chronic model of atypical absence epilepsy induced by AY 9944 is reproducible in C3H mice: AFEEGRS data (Abstract).Can. J. Neurol. Sci. 2000; 27: S11PubMed Google Scholar, 4Dvornik D. Hill P. Effect of long-term administration of AY-9944, an inhibitor of 7-dehydrocholestrol Δ7-reductase, on serum and tissue lipids in the rat.J. Lipid Res. 1968; 9: 587-595Abstract Full Text PDF PubMed Google Scholar) mimic the human condition in most respects, including developmental ECoG correlates, behavior, and pharmacological profiles (1Cortez M.A. McKerlie C. Snead 3rd O.C. A model of atypical absence seizures: EEG, pharmacology and developmental characterization.Neurology. 2001; 56: 341-349Crossref PubMed Scopus (87) Google Scholar, 5Persad V. Cortez M.A. Snead 3rd O.C. A chronic model of atypical absence seizures: studies of developmental and gender sensitivity.Epilepsy Res. 2002; 48: 111-119Crossref PubMed Scopus (33) Google Scholar, 6Smith K.A. Bierkamper G.G. Paradoxical role of GABA in a chronic model of petit mal (absence)-like epilepsy in the rat.Eur. J. Pharmacol. 1990; 176: 45-55Crossref PubMed Scopus (50) Google Scholar, 7Smith K.A. Fisher R.S. The selective GABAB antagonist CGP-35348 blocks spike-wave burst in the cholesterol synthesis rat absence epilepsy model.Brain Res. 1996; 729: 147-150PubMed Google Scholar). Therefore, we have postulated that the AY model is a reproducible and valid model of atypical absence seizures. Early studies with AY have shown that it decreases cholesterol levels in all tissues studied (4Dvornik D. Hill P. Effect of long-term administration of AY-9944, an inhibitor of 7-dehydrocholestrol Δ7-reductase, on serum and tissue lipids in the rat.J. Lipid Res. 1968; 9: 587-595Abstract Full Text PDF PubMed Google Scholar, 8Xu G. Salen G. Shefer S. Ness G.C. Chen T.S. Zhao Z. Salen L. Tint G.S. Treatment of the cholesterol biosynthetic defect in Smith-Lemli-Opitz syndrome reproduced in rats by BM 15.766.Gastroenterology. 1995; 109: 1301-1307Abstract Full Text PDF PubMed Scopus (26) Google Scholar, 9Kolf-Clauw M. Chevy F. Siliart B. Wolf C. Mulliez N. Roux C. Cholesterol biosynthesis inhibited by BM15.766 induces holoprosencephaly in the rat.Teratology. 1997; 56: 188-200Crossref PubMed Scopus (44) Google Scholar, 10Gaoua W. Wolf C. Chevy F. Ilien F. Roux C. Cholesterol deficit but not accumulation of aberrant sterols is the major cause of the teratogenic activity in the Smith-Lemli-Opitz syndrome animal model.J. Lipid Res. 2000; 41: 637-646Abstract Full Text Full Text PDF PubMed Google Scholar, 11Liscum L. Klansek J.J. Niemann-Pick disease type C.Curr. Opin. Lipidol. 1998; 9: 131-135Crossref PubMed Scopus (96) Google Scholar). The decrease in cholesterol in the brains of AY-treated animals is associated with an elevation of the immediate cholesterol precursor, 7-dehydrocholesterol (2Cortez M.A. Cunnane S.C. Snead 3rd O.C. Brain sterols in the AY-9944 rat model of atypical absence seizures.Epilepsia. 2002; 43: 3-8Crossref PubMed Scopus (21) Google Scholar) (Fig. 1). We recently reported that decreased brain cholesterol is a transient effect of AY that reverses after AY injections are stopped, but that AY seizures are sustained long after the brain sterols return to normal (2Cortez M.A. Cunnane S.C. Snead 3rd O.C. Brain sterols in the AY-9944 rat model of atypical absence seizures.Epilepsia. 2002; 43: 3-8Crossref PubMed Scopus (21) Google Scholar). We reasoned that if the AY-induced brain sterol changes (i.e., lowered cholesterol and elevated 7-dehydrocholesterol) are involved in the pathogenesis of AY seizures, other late-stage cholesterol synthesis inhibitors affecting 7-dehydrocholesterol conversion to cholesterol should induce seizures similar to those seen in the AY model (1Cortez M.A. McKerlie C. Snead 3rd O.C. A model of atypical absence seizures: EEG, pharmacology and developmental characterization.Neurology. 2001; 56: 341-349Crossref PubMed Scopus (87) Google Scholar). We also predicted that an early-stage cholesterol synthesis inhibitor that blocks synthesis of both cholesterol and its precursors (i.e., one that does not raise 7-dehydrocholesterol) would not induce AY-like seizures. BM 15.766 (BM) and U18666A (UA) were chosen as examples of late-stage cholesterol synthesis inhibitors that would mimic AY; lovastatin (LV), an HMG-CoA reductase inhibitor, was chosen as the early-stage cholesterol synthesis inhibitor. Known sites of action of these drugs in the cholesterol synthesis pathway are shown in Fig. 1. Three experiments are reported here. Experiment 1 was a comparison of the effects of our standard dose of AY to effects of BM or UA. Experiment 2 was an AY dose-response curve to determine whether brain sterols and SWD duration are equally affected at the same dose of AY. Experiment 3 was a comparison of our standard dose of AY to two different doses of LV or a combination of AY and LV. The Animal Care Committee of the Research Institute of the Hospital for Sick Children, Toronto, approved all experimental procedures. Untimed pregnant Long-Evans hooded rats were obtained from Charles River (St. Constant, Quebec, Canada) and housed in the animal facility at the Hospital for Sick Children. Long-Evans rats were used in these experiments because, unlike most other rat strains, they have a spontaneous tendency to produce SWD lasting ∼25 s/h (1Cortez M.A. McKerlie C. Snead 3rd O.C. A model of atypical absence seizures: EEG, pharmacology and developmental characterization.Neurology. 2001; 56: 341-349Crossref PubMed Scopus (87) Google Scholar). All offspring were weaned at postnatal day 21 (P21), and then three animals of the same gender were housed per cage until the recording electrodes were implanted (2Cortez M.A. Cunnane S.C. Snead 3rd O.C. Brain sterols in the AY-9944 rat model of atypical absence seizures.Epilepsia. 2002; 43: 3-8Crossref PubMed Scopus (21) Google Scholar). All animals were housed in a controlled environment with a 12 h light/dark cycle, with lights on at 06:00 h. They were given ad libitum access to food and water. Body weight of test and control animals was monitored throughout the study. AY (trans-1,4-bis [2-chloro-benzylaminomethyl] cyclohexane dihydrochloride) was a gift from Wyeth-Ayerst (Philadelphia, PA). BM (4-[2-[4-(4-chlorocinnamyl) piperazine-1-yl]ethyl]benzoic acid, sulfate) was a gift from Dr. G. S. Tint, New Jersey Medical School (Newark, NJ). Both drugs inhibit conversion of 7-dehydrocholesterol to cholesterol at the 7-dehydrocholesterol Δ7-reductase (Fig. 1). UA (3β-[2-diethylaminoethoxy]androst-5-en-17-one hydrochloride) was provided by Dr. R. J. Cenedella, Kirksville College of Osteopathic Medicine, Kirksville, MO. LV (1S-[1α(R*), 3α,7β,8β,(2S*,4S*),8a β]2-methylbutanoic acid-1,2,3,7,8a-hexahydro-3,7-dimethyl-8[2-(tetrahydro-4-hydroxy-6-oxo-2H-pyran-2-yl) ethyl]1-naphthalenyl ester) was purchased from Sigma-Aldrich (St. Louis, MO). In Experiment 1, AY (7.5 mg/kg; AY7.5), UA (10 mg/kg; UA10), or BM (20 mg/kg; BM20) was injected subcutaneously at P2, P8, P14, and P20. AY was diluted in distilled water, and UA and BM were suspended in olive oil. In Experiment 2, suckling rats were injected subcutaneously with one of six different doses of AY (2.5, 5.0, 7.5, 10.0, 15.0, or 22.5 mg/kg; AY2.5 to AY22.5, respectively), at P2, P8, P14, and P20. In Experiment 3, the standard AY protocol was compared with either 50 mg/kg (LV50) or 100 mg/kg (LV100) gavaged on P2, P8, P14, and P20 or was compared with 7.5 mg/kg AY given subcutaneously plus 50 mg/kg LV (AY+LV) gavaged on P2, P8, P14, and P20. Control rats received an equivalent volume of distilled water or olive oil subcutaneously on the same postnatal day as the drug-treated litters. Seizure severity was assessed by measuring SWD duration in seconds for three consecutive 20 min intervals over each 1 h recording period, as described previously (1Cortez M.A. McKerlie C. Snead 3rd O.C. A model of atypical absence seizures: EEG, pharmacology and developmental characterization.Neurology. 2001; 56: 341-349Crossref PubMed Scopus (87) Google Scholar). For implantation of skull seizure recording electrodes, rats were anesthetized with a single intraperitoneal injection of sodium pentobarbital (35 mg/kg) that lasted 3–4 h. Surgeries were performed in all animals at P55 and consisted of chronic implants containing two frontal and two parietal monopolar electrodes placed 1 mm deep and 2.2 mm anterior or posterior to the frontal parietal suture and 3 mm lateral to midline. Skull coordinates were measured relative to bregma at 0.0 (12Paxinos G. Watson C. The Rat Brain in Stereotaxic Coordinates. 2nd edition. Academic Press, San Diego1986Google Scholar). Electrodes were secured with dental cement and two screws attached to the parietal regions of the skull without penetrating the dura mater. All animals were then returned to the animal facility for a 4 day recovery period. ECoG recordings to assess baseline SWD duration in male and female rats from all treatment groups were performed at P59–P60. On seizure test days, the rats were placed freely moving in individual, warm Plexiglas chambers (Harvard Apparatus, Holliston, MA) and were connected to the recording equipment. There was an initial 20 min adaptation period to minimize movement artifact prior to ECoG recordings (1 h long), using a Grass Polysomnograph (Grass Instruments, Quincy, MA). All ECoG recordings were performed from 10:00 h to 14:00 h to minimize circadian variations (13Löscher W. Fiedler M. The role of technical, biological and pharmacological factors in the laboratory evaluation of anticonvulsant drugs. VI. Seasonal influences on maximal electroshock and pentylenetetrazol seizure thresholds.Epilepsy Res. 1996; 25: 3-10Crossref PubMed Scopus (72) Google Scholar). Twenty-four hours after each injection (P3, P9, P15, or P21), two to eight rats from each group were anesthetized with sodium pentobarbital (35 mg/kg) and the brains were quickly removed. Brains were homogenized in 20 volumes of 2:1 chloroform/methanol to extract brain total lipids. 5-α-Cholestane was added as an internal standard for sterol quantification (2Cortez M.A. Cunnane S.C. Snead 3rd O.C. Brain sterols in the AY-9944 rat model of atypical absence seizures.Epilepsia. 2002; 43: 3-8Crossref PubMed Scopus (21) Google Scholar). Brain sterols were obtained after saponification of the total lipid extracts in 1 N methanolic KOH. Sterols were derivatized using trimethylsilyl chloride and were analyzed by capillary gas chromatography as previously described (2Cortez M.A. Cunnane S.C. Snead 3rd O.C. Brain sterols in the AY-9944 rat model of atypical absence seizures.Epilepsia. 2002; 43: 3-8Crossref PubMed Scopus (21) Google Scholar, 14Corey E.J. Venkateswarlu A. Protection of hydroxyl groups as tert-butyldimethylsilyl derivatives (Abstract).J. Am. Chem. Soc. 1972; 94: 17-18Crossref Scopus (2377) Google Scholar). Data were expressed as the mean ± 1 SD for both the ECoG recording experiments and the brain sterol studies. Comparisons of group means were performed and analyzed by the two-tailed Student's t-test with a probability value of P < 0.05 chosen as an index of statistical significance. ANOVA for repeated was used to the in SWD duration and brain sterols as a of and All rats were prior to each injection to determine the P20 in Experiment 1, were similar in the control and groups mean ± ± 4 but were than control in the and groups ± 4 P < In Experiment 2, weight at P20 was in the and groups compared with < In Experiment 3, in the different groups did not from control throughout the The ECoG recordings in all test rats similar recurrent SWD at and of (Fig. SWD duration was not different in the or groups (Fig. the and groups but not the group SWD duration than did the < In brain cholesterol from at to at not Brain cholesterol in the and rats than in with the inhibition at was in at at and decreased to by not brain cholesterol was and 7-dehydrocholesterol was to in the and groups than in the (Fig. the of seizure (i.e., after the brain cholesterol returned to or in the control rats of the same and 7-dehydrocholesterol to control In levels of were and 1). was not after in the but was at and in the group and to in the group discharge (SWD) duration at postnatal and brain cholesterol and 7-dehydrocholesterol at postnatal day 21 in and rats injected with either 20 mg/kg BM 15.766 mg/kg or 7.5 mg/kg AY-9944 at P2, P8, P14, and P20 SWD duration was in each treatment group than in < but did not significantly Student's Brain cholesterol was significantly reduced and brain 7-dehydrocholesterol was significantly in all three treatment brain on postnatal from to ± SD ± ± < 0.05 control at ± < 0.05 control at ± < 0.05 control at ± < 0.05 control at ± < 0.05 control at ± < 0.05 control at ± not standard Brains were h after rats on postnatal P2, P8, P14, or P20 with or with one of three late-stage cholesterol AY, 7.5 mg/kg 20 mg/kg BM UA, mg/kg P < 0.05 control at in a not standard Brains were h after rats on postnatal P2, P8, P14, or P20 with or with one of three late-stage cholesterol AY, 7.5 mg/kg 20 mg/kg BM UA, mg/kg SWD duration with AY dose from s/h at AY2.5 to s/h at and (Fig. was in all rats in the AY dose-response at after AY data not with AY but at AY doses of 7.5 mg/kg and Brain cholesterol to of control as the AY dose from to 7.5 mg/kg and did not decrease at AY doses of mg/kg and (Fig. Brain 7-dehydrocholesterol the to as the AY dose from mg/kg to similar mg/kg and and then at 22.5 mg/kg (Fig. SWD duration was by treatment with LV or AY (Fig. SWD were with than with but these did not significantly from or Brain sterols did not change significantly in the cholesterol was but significantly reduced in the group (Fig. Brain 7-dehydrocholesterol in the group to brain cholesterol was reduced by of 50 mg/kg LV to the standard protocol did not significantly the brain sterol changes compared with (Fig. we reported previously (2Cortez M.A. Cunnane S.C. Snead 3rd O.C. Brain sterols in the AY-9944 rat model of atypical absence seizures.Epilepsia. 2002; 43: 3-8Crossref PubMed Scopus (21) Google the changes in brain sterols after the not was a the injection and seizure we compared the SWD duration to the brain sterol levels on which was h after the injections were In the or rats in Experiment 1, SWD duration with brain 7-dehydrocholesterol levels P In Experiment 2, SWD duration was with AY dose P and with brain cholesterol P SWD duration with AY but did not at AY doses mg/kg (Fig. There was significant brain sterols and SWD duration in Experiment that injection of either of the two late-stage cholesterol synthesis inhibitors or during the suckling period induces recurrent atypical absence seizures from those seen in AY-treated rats (1Cortez M.A. McKerlie C. Snead 3rd O.C. A model of atypical absence seizures: EEG, pharmacology and developmental characterization.Neurology. 2001; 56: 341-349Crossref PubMed Scopus (87) Google Scholar, 2Cortez M.A. Cunnane S.C. Snead 3rd O.C. Brain sterols in the AY-9944 rat model of atypical absence seizures.Epilepsia. 2002; 43: 3-8Crossref PubMed Scopus (21) Google Scholar). The conversion to cholesterol is inhibited by AY and BM at the (Fig. 1). 20 BM similar to that of in its effect on brain but SWD duration by of that induced by (Fig. BM has not previously shown to cause seizures, and UA not previously used in this seizure the doses we were on or brain effects seen in other G. Salen G. Shefer S. Ness G.C. Chen T.S. Zhao Z. Salen L. Tint G.S. Treatment of the cholesterol biosynthetic defect in Smith-Lemli-Opitz syndrome reproduced in rats by BM 15.766.Gastroenterology. 1995; 109: 1301-1307Abstract Full Text PDF PubMed Scopus (26) Google Scholar, G.G. of chronic activity in the rat by an inhibitor of cholesterol Res. PubMed Scopus Google Scholar). UA is a inhibitor of conversion to cholesterol G.G. of chronic activity in the rat by an inhibitor of cholesterol Res. PubMed Scopus Google Scholar, W. of a chronic epilepsy in the with an inhibitor of cholesterol Res. PubMed Scopus Google Scholar, during in of the PubMed Scopus Google Scholar). injections of UA at mg/kg and P20 did not brain but did raise brain 7-dehydrocholesterol (Fig. the effect of UA on brain sterol is not to the conversion of to cholesterol but inhibition of 7-dehydrocholesterol to In the SWD duration caused by UA to its inhibition of 7-dehydrocholesterol than to inhibition of conversion to In the rats with AY, UA, or a was seen brain elevated brain and SWD duration in our that the seizure and duration induced by AY in on these changes in brain sterols (2Cortez M.A. Cunnane S.C. Snead 3rd O.C. Brain sterols in the AY-9944 rat model of atypical absence seizures.Epilepsia. 2002; 43: 3-8Crossref PubMed Scopus (21) Google Scholar). the brain sterols and SWD duration with AY, and UA and a AY brain sterol and SWD duration our LV raise the of these LV blocks conversion of to in cholesterol synthesis by HMG-CoA reductase (Fig. 1). has other effects on that for the effect than inhibition of HMG-CoA In LV HMG-CoA reductase its its effect is not to inhibition of HMG-CoA In the doses of LV we tested effects on brain sterols but significantly SWD analogous to the effects of the doses of AY In a we that the in SWD duration did not whether LV mg/kg) was injected day or 4 during not Hence, the injection was injections of LV at 100 mg/kg did not SWD duration significantly than injections at 50 mg/kg and did not the effects of 7.5 mg/kg AY (Fig. Hence, LV did not have a dose-response effect on SWD elevated brain 7-dehydrocholesterol is responsible for the SWD duration caused by AY the in brain LV to have inhibited the effects of AY. reduced brain cholesterol were responsible for the SWD caused by AY, we would have predicted that LV would AY-induced seizures. LV data that effect at two our experimental sterol on the brain h after each has to brain sterol changes after AY, UA, or this h and/or sterol in the brain not of a or reduction in brain cholesterol after LV if during this the effect of cholesterol in a in the brain has A of this is that the effect of AY on brain sterols is than is to induce seizures. our dose-response data that the dose of AY we used mg/kg) seizure duration by than brain cholesterol by (Fig. changes in brain sterols h or in the brain a of the by which these seizures are effects of cholesterol synthesis inhibitors or on SWD duration in this model of cholesterol that these inhibitors induce seizures they inhibit or synthesis or action of from have previously in seizures, and seizures V. an effect of on in male Res. 2001; PubMed Scopus Google Scholar, A. and in adult female Res. PubMed Scopus Google Scholar). The effects of cholesterol synthesis inhibitors changes in of whether brain levels of cholesterol or cholesterol are This was in by the Hospital the Hospital for Sick Children, the and Research of and the of The are to and the at Animal Hospital for Sick Children, for and to for in of this C. and provided

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