An anticonvulsant profile of the ketogenic diet in the rat

Epilepsy Research 50 (2002) 313 /325 www.elsevier.com/locate/epilepsyres

An anticonvulsant profile of the ketogenic diet in the rat Kristopher J. Bough, Kirana Gudi, Frederick T. Han, Alyssa H. Rathod, Douglas A. Eagles * Department of Biology, Georgetown University, Box 571229, Washington, DC 20057-1229, USA Received 30 November 2001; received in revised form 23 April 2002; accepted 1 May 2002

Abstract The present study was designed to evaluate the anticonvulsant effects of a high-fat ketogenic diet (KD) in rats. Animals were maintained on one of four experimental diets: (1) calorie-restricted ketogenic (KCR); (2) calorie-restricted normal (NCR); (3) ad libitum ketogenic (KAL); or (4) ad libitum normal (NAL). The calorie-restricted diets were fed in quantities such that they were calorically equivalent. All animals began diet treatment at age P37 and each was subjected to one of five chemically-induced seizure tests: bicuculline (BIC; s.c.), picrotoxin (PIC; s.c.), kainate (KA, i.p. or s.c.) and g-butyrolactone (GBL, i.p.), strychnine (s.c.). Bipolar epidural electrodes were implanted under ketamine/ xylazine anesthesia to permit recording the spike and wave discharges (SWD) characteristic of electroencephalograms during absence seizures. Ketonemia was assayed by measuring blood levels of b-hydroxybutyrate (BHB) spectrophotometrically prior to induction of seizures in each experiment. Animals fed ketogenic diets (i.e. either calorie restricted or ad libitum) exhibited greater blood levels of BHB compared to control groups. Seizure results show that treatment with a KD: (1) reduced the incidence of bicuculline-induced convulsions; (2) diminished the number of picrotoxin-induced seizures (KCR group only); (3) increased latency to GBL-induced SWD and reduced both the number and duration of SWD; but (4) conferred no protection from strychnine-induced seizures; and (5) made KAinduced seizures more severe. Together these results indicate a spectrum of anticonvulsant action for the KD in rats that includes threshold seizures induced via GABA receptors (BIC, PIC, GBL) but not those induced at glycine (strychnine) or the KA-subclass of glutamate receptors. Uniquely, the KD is the only treatment described that protects against both convulsive and non-convulsive (absence) seizures in rats. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Ketogenic diet; Epilepsy; Bicuculline; Picrotoxin; Kainic acid; Strychnine; Rat

1. Introduction In 1921, Geyelin first reported improved seizure control associated with fasting (Geyelin, 1921). In

* Corresponding author. Tel.:
/1-202-687-5905; fax:
/1202-687-5662 E-mail address: [email protected] (D.A. Eagles).

an effort to mimic the physiology of prolonged starvation and acquire the seizure protective effects associated with abstinence from food, Wilder (1921) developed a high-fat (ketogenic) diet that was low in carbohydrates and proteins (i.e. less than 20% by weight, combined). The development of effective antiepileptic drugs in the late 1930s, however, provided an easier means of seizure control than the exacting regimen of

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ketogenic diet (KD) treatment (Merritt and Putnam, 1938). Since that time, despite a resurgence of interest into the anticonvulsant action(s) of the KD in the mid-1990s (Wheless, 1995), the KD has been used predominantly as a treatment of last resort for children with difficult-to-manage seizures. The effectiveness of the KD as a treatment for human epilepsy has been well established. Numerous clinical reports have described its usage and validated its efficacy for over 75 years (e.g. Peterman, 1925; Helmholtz and Keith, 1930; Mike, 1965; Livingston, 1972; Huttenlocher, 1976; Dodson et al., 1976; Schwartz et al., 1989a,b; Kinsman et al., 1992; Freeman et al., 1994; Swink et al., 1997; Tallian et al., 1998; Freeman et al., 1998; Vining et al., 1998). For children maintained on the diet for at least 1 year, 40 /50% of children exhibit greater than 50% reduction in seizure frequency (Freeman et al., 1998; Vining et al., 1998). Of those children who show marked improvement, 7 /10% may become seizure-free (Freeman et al., 1998; Vining et al., 1998) and success does not appear to be restricted to particular seizure types (Schwartz et al., 1989a; Vining et al., 1998; Freeman et al., 1998). Despite the clinical successes of KD treatment, the action(s) of the KD remain largely unexplored. Although several studies have described an anticonvulsant role for the KD in mice (Millichap et al., 1964; Uhlemann and Neims, 1972; Otani et al., 1984; Nakazawa et al., 1983; Rho et al., 1999) and in rats (Appleton and DeVivo, 1974; DeVivo et al., 1978; Mahoney et al., 1983; Hori et al., 1997; Muller-Schwarze et al., 1999), variations in diet, age, strain and protocol have rendered comparisons difficult. Here we extend a single KD protocol (Bough and Eagles, 1999; Bough et al., 1999) using a standard battery of tests (Krall et al., 1978; White et al., 1998) to differentiate anticonvulsant effects of the KD.

2. Methods 2.1. Animals and drugs Three hundred and seventy-two male Sprague/ Dawley rats (Harlan Sprague/Dawley, Indiana-

polis, IN) were housed 3/4 to a cage at a temperature of 219/1 8C on a 12:12 h light:dark cycle with lights on at 07:00 h. Picrotoxin (PIC) (1.92 mg/ml), strychnine (0.60 mg/ml) and gammahydroxybutyric acid lactone (GBL, 100 mg/ml) (all from Sigma, St. Louis, MO), and kainate (KA, 10 mg/ml, Tocris-Cookson, Ballwin, MO) were dissolved in 0.9% NaCl and filtered through a 0.22 mm filter (Costar) into 30 ml vials of sterile bacteriostatic 0.9% NaCl (Abbott Laboratories, Chicago, IL). Bicuculline (BIC, Sigma) was first dissolved in 1 ml of warmed 0.1 N HCl, then into 0.9% NaCl, and was subsequently filtered into sterile bacteriostatic 0.9% NaCl vials to a final concentration of 1.73 mg/ml (Bailleux et al., 1995). All solutions were made immediately prior to testing and were used within 2 h of preparation. All were injected sub-cutaneously (s.c.) except for those injected with GBL (intraperitoneally, i.p.) and half of the animals injected with kainate (i.p.) (8 in each diet group). 2.2. Establishment of the 97% convulsive dose for bicuculline, picrotoxin and strychnine Whereas anticonvulsant drug screening has typically been conducted in mice and we knew of no 97% convulsive dosages (CD97) for BIC, PIC and strychnine established for rats (Krall et al., 1978; White et al., 1998), relevant CD97 values were determined prior to initiating experimental diet treatment. Effective doses of GBL in rats are well described (Snead, 1988). Animals used for CD97 determination (n /162) were maintained on a normal, ad libitum diet with free access to water at all times. Animals were segregated into groups (n /4 /8) and were age-matched to experimental conditions (i.e. as though animals had been diet treated and were to be seizure-tested at P57 /P68). Injections of various dosages of the convulsant drugs (i.e. BIC, PIC or strychnine) were given subcutaneously in a fold of skin between the scapulae. Each group of animals received only one of the various doses of convulsant drugs (BIC, PIC or strychnine), seizures were scored behaviorally (see Section 2.4 below for scoring) and the percent response for each dosage group was recorded. Initial dosages injected into the rats

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were based upon the doses known to produce seizures in approximately 50% of mice (EC50; Bailleux et al., 1995). All animals were seizurenaı¨ve when tested and subjected to seizure testing only once. Depending upon the response of the initial group, subsequent convulsant doses were prepared to be either one-third greater or onethird less than the initial dose. This process was repeated until endpoints (i.e. 0 and 100% response) were observed. Remaining groups were used to establish at least two intermediate points. The CD97 was interpolated from the regression analysis of these data (Piredda et al., 1985; White et al., 1998).

2.3. Diet treatment Animals not used to establish the CD97 for BIC, PIC or strychnine (n/175) were divided into four groups at age P37 and, following a 6 /8 h fast, were fed one of the following diets: (1) a calorierestricted ketogenic diet (KCR); (2) a calorierestricted normal diet (NCR); (3) an ad libitum ketogenic diet (KAL); or (4) an ad libitum normal diet (NAL). Rats used in the kainate study (n /32) also began experimental diets at P37 following fasting, but were divided into only two diet groups, NCR and KCR (n /16 each). The three rats used in the GBL study also began their diets at P37 after fasting and were fed either the NAL (n /1) or the KCR (n/2) diets. A detailed description of the constituents of both ketogenic (BioServe #F3666, Frenchtown, NJ) and normal (Purina 5001) diets has been reported previously (Bough and Eagles, 1999). Rats were fed individually once each day, beginning between 15:00 and 17:00 h and were allowed to feed for 2.5 /3.5 h. Calorierestricted diets were fed in quantities calculated to make both diets calorically equivalent and were limited to less than 90% of the normal daily requirement for rats (Appleton and DeVivo, 1974; Rogers, 1979). Experimental diets were continued for at least 20 days before seizure testing began and seizures were evaluated at ages P57 / P68. Water was provided ad libitum throughout each experiment.

315

2.4. Seizure testing Animals injected with the CD97 for BIC or PIC (as determined above) were observed for 30 or 45 min, respectively, for the presence or absence of a threshold seizure. A threshold seizure (i.e. positive response) was defined as facial and/or forelimb clonus lasting for longer than 5 s. In some instances, as BIC administration produced maximal seizures (i.e. tonic /clonic), seizure behavior was semi-quantitatively scored using the following four-point scale: 0/no seizure activity, 1 /uni- or bi-lateral forelimb clonus for /5 s, 2/stage 1 plus rearing and falling, 2 /stages 1 and 2 plus development of tonic forelimb or tonic hindlimb extension. Similarly, for strychnine-induced seizures, rats were observed for the presence or absence of a maximal (i.e. tonic) seizure. Those animals that demonstrated tonic hindlimb extension were considered to exhibit a positive convulsive response (Bailleux et al., 1995; White et al., 1998). 2.5. Determination of ketonemia Blood b-hydroxybutyrate (BHB) levels were measured spectrophotometrically using a StatSite meter (GDS Technologies, Elkhart, IN) and taken as measures of ketonemia. Blood samples (0.1 ml) were collected at the same time of day (13:00 / 17:00 h) from the tail vein of the rat after 19 days of diet treatment and immediately analyzed (25 ml per assay). 2.6. Surgery and EEG Aseptic sterile conditions were used for implantation of cranial electrodes and all surgical instruments, cranial screws and electrodes were gassterilized. At age P30, animals were anesthetized with Ketamine/Xylazine (85/8 mg/kg, ip), the head was shaved, and a mid-sagittal incision was made on a line between the orbits caudally for approximately 4 cm. A cautery tool was used to minimize bleeding. Three holes were drilled with a sterile drill bit (Plastics One, Roanoke, VA) to a depth of 1 mm. Two holes for the electrodes were drilled at AP:
/2 and /2 mm; M: /1 mm; DV: /1 mm

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relative to bregma following Banerjee et al. (1993). A third hole, drilled contralaterally (AP: /2, M:
/1, DV: /1), was used to place a screw (0 /80/ 1/16 in., Plastics One) to which cranioplastic cement (Plastics One) would anchor. Baytril antibiotic (0.1 ml, s.c., Bayer, Shawnee Mission, KS) was given to prevent infection. Rats were provided rodent chow and water ad libitum for 7 days, after which experimental diets were initiated as described above. Following injection of GBL (100 mg/kg, i.p.), a shielded cable was connected to the electrode pedestal and the rat was free to move about within a large colony cage placed inside a Faraday cage. The cable was connected to an optically isolated amplifier (Isodam, World Precision Instruments, Sarasota, FL) and data were collected on computer using CODAS software (DATAQ, Akron, OH) at an acquisition rate of 100 Hz per channel for up to 90 min post-injection. Absence seizures were scored quantitatively, based upon the number of observed 4/6 Hz spike and wave discharges (SWD).

from regression analyses were calculated to be 1.80, 1.89 and 1.80 mg/kg for BIC, PIC and strychnine, respectively. 3.2. Ketonemia Animals maintained on the KD (either calorierestricted or ad libitum) exhibited markedly greater levels of ketonemia than did control animals fed a normal (rodent chow) diet (either calorie restricted or ad libitum). Animals fed the KD commonly showed a 3/4-fold increase in blood BHB compared to controls fed normal diets (Tables 1 /5). In addition, calorie restriction (CR) affected the level of ketonemia. Animals fed a NCR diet were more ketonemic than were animals fed the same diet on an ad libitum basis (Tables 2 and 3). A similar trend was noted for animals fed the KD. Although the levels of ketonemia were not always significantly different in the two groups fed the KD, the blood level of BHB exhibited by rats fed the KCR diet was consistently higher than in those maintained on the KAL diet.

2.7. Statistics 3.3. Diet and kainic acid Regression analyses of the data obtained for establishing the 97% convulsive dosages were completed using SigmaPlot/SigmaStat (SPSS). Statistical differences between diet treatment groups were reported as group means (9/S.E.M.) and were analyzed either by Students t -test or by Student Newman /Keuls (SNK) tests. Comparisons of non-parametric data (e.g. Mann /Whitney) were computed using SigmaStat whereas Chisquare analyses were performed with SAS (Tables 2 /4). Statistical significance was taken at P B/ 0.05.

3. Results 3.1. Establishment of the CD97 Age-matched animals used in this part of the study did not receive any experimental diet treatment and all animals appeared healthy prior to convulsive testing. The CD97 values interpolated

In general, as shown in Table 1, rats fed the KCR diet experienced more severe seizures and more deaths than did those fed the NCR diet. The route of injection also was a factor, as animals injected subcutaneously experienced more seizures than did those injected intraperitoneally, regardless of diet, except for those having the most severe seizures, injected intraperitoneally, and fed the NCR diet. 3.4. Mean latency to seizure onset Mean latency to BIC- and PIC-induced seizures was greater for those animals maintained on the NAL diet than it was for those fed any of the other diets (Tables 2 and 3). By comparison, mean latency to strychnine-induced seizures tended to be shorter for NAL-fed animals. For this convulsive challenge, those animals fed calorie restricted diets tended to exhibit the greatest time to seizure onset (Table 4).

Diet group

Dosage used (mg/kg)

[b-OHB] (mM)

Mean latency to stage-1 onset (min)

S stage 1 events Wet-dog shakes

S stage 2 events Scratching

S stage 3 events Unilateral forelimb clonus

S stage 4 events S stage 5 events Bilateral foreRearing & limb clonus falling

S stage 6 events Maximal clonic / tonic seizures

No. deaths

K, CR (i.p.) (s.c.)

10 10

2.22a (9/0.5)

47.59/9 31.59/2

267 412

4 7

3 14

1 24

94 186b

5 37

2/8 1/8

N, CR (i.p.) (s.c.)

10 10

0.75 (9/0.07)

43.29/5 33.69/2

444 574

26 58

1 72

5 4

27 26

12 0

0/8 0/8

a

P B/0.05. P B/0.05, Mann /Whitney test. Animals (n/32) were maintained on either a calorie-restricted ketogenic (KCR) or calorie-restricted normal (N, CR) diet for 20 /21 days prior to seizure testing. The levels of b-hydroxybutyrate (b-OHB) were measured after 19 days of diet treatment. All animals began diet treatment at age P37 and were seizure-naı¨ve when tested. Convulsions were chemically evoked by 10 mg/kg KA (s.c. [n/16] or i.p. [n/16]) and observed for 3 h. b-hydroxybutyrate levels are reported9/S.E.M. Mean latency refers to the time to stage-3 seizure onset (min)9/S.E.M. (see Section 2). b

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Table 1 Pro-convulsant action of the ketogenic diet in kainic acid-induced seizures

317

318

Table 2 Ketogenic diet (KD)-induced anticonvulsant activity against the proconvulsant drug, bicuculline (BIC) BIC CD97 (n /20) (mg/kg)

[b-OHB] (mM)

Mean latency to seizure onset No. animals exhibiting threshold (min) seizuresf

K, CR

1.80

N, CR

1.80

K, ad lib

1.80

N, ad lib

1.80

2.141a (9/0.18) 0.52b (9/0.04) 1.58c (9/0.14) 0.33d (9/0.03)

2.50 (9/0.29) 2.45 (9/0.41) 1.63 (9/0.37) 4.79e (9/0.39)

No. animals exhibiting maximal seizuresg

No. deaths

3/10a

1/10

1/10

10/10b

8/10

4/10

4/10a

4/10

4/10

7/10a,b

3/10

3/10

1 ad Different letters represent means that are significantly different from one another. (For b-OHB, P B/0.05, Student Newman /Keuls test). (For seizure response, P B/0.01, Chi-square analysis). e P/0.008 f Threshold seizures were defined as at least one incident of facial and forelimb clonus sustained for greater than 5 s (see Section 2). g Seizures were considered maximal with an exhibition of tonic (either forelimb or hindlimb) extension. Animals (n/40) were maintained on one of four diets for 20 days prior to seizure testing: a calorie-restricted ketogenic (K, CR), a calorie-restricted normal (N, CR), an ad libitum ketogenic (K, ad lib), or an ad libitum normal (N, ad lib) diet. The levels of b-hydroxybutyrate (b-OHB) were measured after 19 days of diet treatment. All animals began diet treatment at age P37 and were seizure-naı¨ve when tested. Convulsions were induced as described in Section 2. The convulsive dose reported (CD97 / dose that induced seizures in 97% of the animals tested) was established as described in Section 2. For b-OHB levels, blood concentrations are reported9/S.E.M. Mean latency refers to the time to seizure onset (min)9/S.E.M. The n reported in column 2 is representative of the total number of animals used to determine the CD97 value.

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Diet group

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Table 3 Ketogenic diet (KD)-induced anticonvulsant activity against the proconvulsant drug, picrotoxin (PIC) Diet group

PIC CD97 (n/69) (mg/ [b-OHB] kg) (mM)

K, CR

1.89

N, CR

1.89

K, ad lib

1.89

N, ad lib

1.89

4.141a (9/0.78) 0.54b (9/0.02) 1.75c (9/0.26) 0.36d (9/0.02)

Mean latency to seizure onset (min)

No. animals exhibiting threshold seizuresf

No. deaths

12.0 (9/0.39) 11.53 (9/0.47) 12.44 (9/0.58) 14.60e (9/0.78)

10/15a

0/10

14/15b

0/10

10/10b

0/10

10/10b

0/10

1 ad

Different letters represent means that are significantly different from one another. (For b-OHB, P B/0.05, Student Newman / Keuls test). (For seizure response, P B/0.05, Chi-square analysis). e P B/0.05. f Threshold seizures were defined as at least one incident of facial and forelimb clonus sustained for greater than 5 s (see Section 2). Animals (n/50) were maintained on one of four diets for 20 days prior to seizure testing: a calorie-restricted ketogenic (K, CR), a calorie-restricted normal (N, CR), an ad libitum ketogenic (K, ad lib) or an ad libitum normal (N, ad lib) diet. The levels of bhydroxybutyrate (b-OHB) were measured after 19 days of diet treatment. All animals began diet treatment at age P37 and were seizurenaı¨ve when tested. Convulsions were induced and scored as described in Section 2. The convulsive dose reported (CD97 /dose that induced seizures in 97% of the animals tested) was previously established (see Section 2). Blood concentrations of b-OHB are reported9/S.E.M. Mean latency refers to the time to seizure onset (min)9/S.E.M. The n reported in column 2 is representative of the total number of animals used to determine the CD97 value.

Table 4 Ketogenic diet-induced anticonvulsant activity against the proconvulsive agent, strychnine Diet group

Strychnine CD97 (n/73) (mg/kg)

[b-OHB] (mM)

Mean latency to seizure onset No. animals exhibiting maximal (min) seizuresf

No. deaths

K, CR

1.80

10/15

1.80

14/15

1/15e

K, ad lib

1.80

9/10

7/10

N, ad lib

1.80

3.58b,c (9/0.14) 4.21c (9/0.26) 3.40a,b (9/0.27) 2.67a (9/0.29)

12/15

N, CR

2.051a (9/0.28) 0.35b (9/0.06) 1.87a (9/0.33) Not tested

10/10

9/10

1 ad Different letters represent means that are significantly different from one another. (For b-OHB and mean latency, P B/0.05, Student Newman /Keuls test). e P B/0.005, Chi-square analysis. f Maximal seizures were defined as a tonic extension of the hindlimbs sustained for greater than 5 s (see Section 2). Animals (n/50) were maintained on one of four diets for 20 days prior to seizure testing: a calorie-restricted ketogenic (K, CR), a calorie-restricted normal (N, CR), an ad libitum ketogenic (K, ad lib) or an ad libitum normal (N, ad lib) diet. The levels of bhydroxybutyrate (b-OHB) were measured after 19 days of diet treatment. All animals began diet treatment at age P37 and were seizurenaı¨ve when tested. Convulsions were induced and scored as described in Section 2. The convulsive dose reported (CD97 /dose that induced seizures in 97% of the animals tested) was established for rats as described in Section 2. For b-OHB levels, blood concentrations are reported9/S.E.M. Mean latency refers to the time to seizure onset (min)9/S.E.M. The n reported in column 2 is representative of the total number of animals used to determine the CD97 value.

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320

Table 5 Ketogenic diet (KD)-induced anticonvulsant activity against g-hydroxybutyrolactone (GBL) Diet group

[b-OHB] (mM)

Latency to SWD onset (min)

No. SWD events

Mean duration of SWD (s)

S duration of SWD events (min)

K, CR (n/2)

1.83a (9/0.14)

44.94

11

13.67

60.73

8

12.15

33

1.24 (9/0.21) 1.14 (9/0.17) 1.71 (9/0.21)

N, ad lib (n/1)

0.25

9.15 56.28

a

P B/0.05. Seven days prior to diet onset, electrodes were surgically implanted into three rats. Animals were fed either a calorie-restricted ketogenic (K, CR; n /2) or an ad libitum normal (N, ad lib; n/1) diet. All diets were begun at P37 and maintained for 20 days prior to seizure testing. The levels of b-hydroxybutyrate (b-OHB) were measured spectrophotometrically after 19 days of diet treatment and are reported9/S.E.M. All animals began diet treatment at age P37 and were seizure-naı¨ve when tested. Convulsions were evoked chemically by injection with 100 mg/kg GBL (i.p.) as described in Section 2. Latency to onset was defined as the time until the initial spike-and-wave (SWD) event was electrographically noted (min)9/S.E.M. (see Section 2).

3.5. Diet-induced effects on seizure expression and mortality

exception that the death rate was remarkably low for rats fed an NCR diet.

3.5.1. Bicuculline-induced seizures Rats fed the KD were protected from BICinduced threshold seizures (Table 2). Those fed a KD, either KCR or KAL, exhibited fewer BICinduced threshold seizures than did control animals fed an NCR diet. Animals fed the NAL diet had an incidence of threshold seizures that was statistically indistinguishable from either the NCR or KCR groups. While calorie restriction of the KD tended to reduce the incidence of threshold seizures, maximal seizures, and number of deaths compared to animals fed the KAL diet, calorie restriction of the ND tended to increase the number of threshold seizures, maximal seizures, and number of deaths compared to NAL-fed animals.

3.5.4. Absence seizures Although the number of animals from which we were able to obtain data was too small to permit any conclusions, we thought it worth reporting that the latency to SWD onset was increased in the two rats fed the KCR diet and the number and total duration of SWD events were both reduced, suggesting a protective effect of the ketogenic diet as shown in Table 5. More remarkable was the profound lethargy shown by the ketogenic rats following injection of GBL. For more than 1 h following injection, each of the animals lay prostrate and showed labored breathing but, otherwise, did not move. The single animal fed the normal diet was not visibly affected by the GBL injection.

3.5.2. Picrotoxin-induced seizures The KCR diet, but not the KAL diet, reduced the incidence of threshold seizures following injection of PIC (Table 3). No other dietary differences in seizure activity or death were observed.

3.5.5. Mortality There were no deaths associated with diet alone in any of the experiments. All diets were well tolerated and animals appeared to be in good health (i.e. exhibited normal exploratory behavior, remained well groomed). There were, however, seizure-induced deaths. For BIC, all of the animals from the KCR, KAL, and NAL groups that developed maximal, tonic seizures subsequently died whereas only 50% (4/8)

3.5.3. Strychnine-induced seizures There were no differences among any of the diets for the number of animals experiencing maximal seizures or death, with the notable

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of the NCR animals that developed maximal, tonic seizures died (Table 2). There were no deaths in any diet group following PIC injection (Table 3), but there was very high mortality in all but the NCR group following injection of strychnine (Table 4). None of the three animals from which EEGs were obtained and into which GBL was injected died, though the ketogenic rats showed a profound catatonia lasting more than an hour (Table 5). Both recovered fully, with no apparent ill effects.

4. Discussion The purpose of these investigations was to: (i) evaluate systematically anticonvulsant effects of the KD in a variety of chemically-induced seizure models; and (ii) examine the role of calorie restriction in a standard battery of seizure tests. Published experimental studies investigating the effects of the KD have employed a wide variety of diet compositions, diet treatments (varying degrees of calorie restriction vs. ad libitum administration), species, age at onset of diet, age at seizure testing, and duration of diet treatment, making it difficult to compare results across studies and to glean a broad profile for KD efficacy. In these experiments, each of these variables remained consistent with most of our published work, including the nature of the KD, caloric restriction, duration of feeding, and ages of treatment and testing (Bough et al., 1999, 2000a,b). The principal finding of these studies is that the KD most effectively protects against clonic-type seizure activity, but not tonic seizure activities. 4.1. Ketonemia */was the KD working in each test? Treatment with a KD (either KCR or KAL) produced significant increases in blood levels of BHB compared to both NCR and NAL controls (Tables 1 /5), indicating that the KD was inducing ketonemia, the clinical hallmark of KD efficacy (Freeman et al., 1994). More importantly, consistent levels of ketonemia (i.e. 29/0.5 mM) were observed for rats fed the KCR diet (Tables 1, 2, 4 and 5) and are similar to levels observed for other

321

age-matched experiments (Bough et al., 2000a,b). The mean level of ketonemia produced in KCR animals fell within a range of 1.83 /2.25 mM BHB, with one exception. Calorie-restricted ketogenic animals shown in Table 3 demonstrated a mean BHB level of 4.149/0.78 despite beginning the diet at the same age (P37) as all other animals, a consequence of an unusually high level of BHB (10.38 mM) in one animal. Animals fed a KCR diet exhibited higher levels of ketonemia than those fed the KAL diet (Tables 2 and 3). Although such differences are not always evident for rodent chow diets, animals maintained on the NCR diet also had elevated levels of BHB compared to NAL controls (Tables 2 and 3). Collectively, these findings suggest that calorie restriction augments the level of ketone bodies in the blood. 4.2. Effectiveness of the KD against threshold vs. maximal seizures Although early studies in mice suggested that the KD decreased seizure severity (Millichap et al., 1964; Uhlemann and Neims, 1972), more recent studies in rats have shown that the KD elevates seizure threshold (Appleton and DeVivo, 1974; DeVivo et al., 1978; Hori et al., 1997; Bough and Eagles, 1999; Rho et al., 1999) and perhaps retards epileptogenesis (Muller-Schwarze et al., 1999; Su et al., 2000). The KD does not appear to reduce either convulsive severity or duration once a seizure begins (Bough et al., 2000a,b). Findings presented here support more recent studies. Ketogenic diet-fed animals showed more severe seizure activity than did controls following kainate injection (Table 1) and showed no protection against strychnine-induced maximal seizures (Table 4). Curiously, the route of kainate administration had differential effects upon seizure generation in KCR- and NCR-fed animals. As Sperk (1994) had reported that from 60 to 80% of rats injected (i.p.) with 10 mg/kg KA experienced maximal seizures, we chose to also employ subcutaneous injection to reduce seizure severity. As shown in Table 1, there were no stage 6 seizures in NCR rats injected by this route but KCR-fed rats experienced more seizures in response to subcutaneous KA. We cannot explain this observa-

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tion. There was a low incidence of both maximal and threshold seizures in KCR rats injected with BIC (Table 2) and a significant reduction in threshold seizures following challenge with PIC (Table 3). These results suggest that the KD acts to increase the resistance to multiple models of acute, drug-induced seizure activity with a profile comparable to that of several anticonvulsant drugs, particularly valproate (VPA) (Table 6), although it exacerbates maximal seizure activity induced by kainate (Table 1) and maximal electroshock (Bough et al., 2000b).

DeVivo et al. (1978) investigated the cerebral metabolism in Sprague /Dawley rats maintained on the KD for 20 days and reported significant increases in alpha-ketoglutarate (P B/0.005). They speculated this was important because GABA concentrations might be globally or regionally elevated if ‘‘increased alpha-ketoglutarate levels. . .increased flux through the GABA shunt’’ (Nordli and DeVivo, 1997). Whereas our results do not address GABA levels in the brain, our data are consistent with these aforementioned studies and the possibility that elevated levels of GABA might explain (at least in part) KD-conferred resistance to BIC- and PIC-induced seizures.

4.3. A possible link between the KD and GABA? As these results show that the KD affords protection against BIC- and PIC-induced seizures and it is well documented that both drugs antagonize the inhibitory action of the g-aminobutyric acid receptor (GABAA) (e.g. Collins and Hill, 1974; Schwartzkroin and Prince, 1980; Olsen, 1981), it seems possible that the KD acts to enhance GABAergic function. High concentrations of ketone bodies can increase GABA synaptosomal content in vitro (Erecin´ska et al., 1996). In vivo, elevated GABA content has been reported within the dorsal hippocampus of mice (Rho et al., 1999) and brain homogenates taken from KD-fed mice have been shown to exhibit elevated levels of GABA (Yudkoff et al., 2001), although Al-Mudallal et al. (1996) found no changes in GABA levels in the cerebral cortex of rats fed the KD.

5. Summary Overall, we have found that animals fed a KAL diet do not respond as robustly as do those fed the KCR diet. Although there are some differences across seizure tests, we commonly observe that seizure threshold for various treatment groups is best represented by the following relationship: KCR /KAL /NCR /NAL. In Table 6 we profile our work with the KD relative to published findings for other common AEDs in rats. From these comparisons, the KD appears to present a profile most similar to valproic acid (VPA), a broad-spectrum anticonvulsant. Indeed, it is interesting to note that recent experimental evidence indicates that VPA and the

Table 6 The anticonvulsant profile of the ketogenic diet in comparison to other antiepileptic drugs (Swinyard et al., 1986; Velı´sˇek et al., 1992, 1995; Chronopoulos et al., 1993; Macdonald and Kelly, 1994; Frey and Bartels, 1997; White, 1997)

Ketogenic diet Phenytoin Valproic acid Ethosuximide Felbamate Phenobarbital a

MES

PTZb

BIC

PIC

Strych

KA

GBL

/a
/
/ /
/
/


/
/ /
/
/
/
/
/


/ /
/
/ /
/


/// /
/
/
/
/

/
/
/
/// / /

/1 /
/ /
/
/


/? /
/
/
/
/// /a

Evidence of proconvulsant effects. Threshold PTZ administered either s.c. or via timed i.v. infusion. Abbreviations used: MES, maximal electroconvulsive shock; PTZ, pentylenetetrazole; BIC, bicuculline; PIC, picrotoxin; KA, kainic acid; GBL, g-butyrolactone. These findings represent the findings collected from both rats and mice. KEY:
/
/, strongly anticonvulsant;
/, anticonvulsant;
///, evidence of anticonvulsant effects; /, ineffective. b

K.J. Bough et al. / Epilepsy Research 50 (2002) 313 /325

KD work synergistically to increase PTZ seizure resistance (Bough et al., 2000b) and clinical reports describe severe side-effects for the combination of KD and VPA in children (Ballaban-Gil et al., 1998). The significance of the present study is the differentiation of KD effects for a single animal and diet model against a variety of standard proconvulsant drugs. To date, we know of few studies that have characterized the actions of the diet using a single KD protocol. We asked whether such a KD model would demonstrate anticonvulsant action in a variety of acute, chemicallyinduced seizure models. We observed that the KD acted to reduce the incidence of BIC- and PIC-evoked seizures (Tables 2 and 3) but did not provide protection from either kainate- or strychnine-induced seizures. Taken together, these findings suggest that KD protection is model-specific in rats, but has an anticonvulsant profile similar to that of drugs having a wide spectrum of anticonvulsant action (Table 6). As the KD has been shown to be effective in cases of medically intractable epilepsy (Freeman et al., 1994; Swink et al., 1997; Freeman et al., 1998), the elucidation of the mechanism(s) of the KD will likely lead to the development of uniquely potent AEDs.

Acknowledgements The authors wish to acknowledge gratefully the Department of Biology at Georgetown University and the ARCS Foundation (KJB) for their generous support of this work. In addition, we wish to thank O. Carter Snead for helpful advice regarding the g-hydroxybutyrate model of absence seizures, and Jong M. Rho and Philip A. Schwartzkroin for helpful comments in the preparation of this manuscript.

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