Increased nitric oxide caused by the ketogenic diet reduces the onset time of kainic acid-induced seizures in ICR mice

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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s

Research Report

Increased nitric oxide caused by the ketogenic diet reduces the onset time of kainic acid-induced seizures in ICR mice Hae Sook Noh a , Dong Wook Kim b , Gyeong Jae Cho a , Wan Sung Choi a , Sang Soo Kang a,⁎ a

Department of Anatomy and Neurobiology, Institute of Health Science, College of Medicine, Gyeongsang National University, 92 Chilam-dong, Jinju, Kyungnam 660-751, South Korea b Department of Pediatrics, Inje University Ilsan Paik Hospital, Goyang, Gyeonggi 411-706, South Korea

A R T I C LE I N FO

AB S T R A C T

Article history:

Although the antiepileptic effects of the ketogenic diet (KD) are well documented, the

Accepted 6 December 2005

mechanisms underlying this action remain obscure. Nitric oxide (NO) has long been thought

Available online 3 February 2006

to play a role in regulating seizures. However, the effects of the KD on endogenous NO production have not been characterized. Therefore, the present study was designed to

Keywords:

examine the effect of the KD on endogenous NO production, as well as the precise role

Kainic acid

of NO in kainic acid (KA)-induced seizures, in male ICR mice. We first found that

Ketogenic diet

preadministration of the KD for 4 weeks increased endogenous NO generation in the

Nitric oxide

hippocampus. We also demonstrated that the increase in NO induced by the KD resulted

N-ω-nitro-L-arginine methyl ester

from increased neuronal NO synthase (nNOS) activity and exerted an antiepileptic effect on

7-nitroindazole

KA-induced seizures, based on the results of experiments using NOS-knockout mice and

NOS-knockout mice

two NOS inhibitors, N-ω-nitro-L-arginine methyl ester (L-NAME) and 7-nitroindazole (7-NI). These data suggest that the antiepileptic effects of the KD might be mediated, at least in part, by increased NO levels in the hippocampus. © 2005 Elsevier B.V. All rights reserved.

1.

Introduction

The ketogenic diet (KD) was originally formulated to mimic the effects of fasting, which has been known since biblical times to have a beneficial effect on epilepsy (Swink et al., 1997; Wilder, 1921). The KD produces ketosis (that is, elevated blood levels of four-carbon ketone bodies). Ketosis alters cerebral glucose metabolism, resulting in an elevation of the seizure threshold (De Vivo et al., 1978; Schwartzkroin, 1999). However, the mechanism by which this increase in energy reserves reduces the propensity towards seizure is unclear. Recently, animal models have been developed in many laboratories in an attempt to elucidate the relationship between the KD and the seizure threshold (Freeman et al., 1998; Stafstrom, 1999).

Recently, we reported that the KD delays the kainic acid (KA)-induced seizure-onset time in ICR mice and changes many gene-expression profiles in the hippocampus (Noh et al., 2003, 2004, 2005). Nitric oxide (NO) is known to act as a neuronal messenger in the central nervous system and has been implicated in several neurological disorders, including epilepsy and cerebral ischemia (Bredt and Snyder, 1992; Nathan, 1992; Snyder and Bredt, 1991). A growing number of reports have described the relationship between NO and experimentally induced seizures, but their results have been conflicting. Several previous studies have suggested an anticonvulsant effect of NO, while others have pointed to a proconvulsant effect (De Sarro et al., 1993; Kashihara et al., 1998; Kirkby et al., 1996a,b;

⁎ Corresponding author. Fax: +82 55 759 0779. E-mail address: [email protected] (S.S. Kang). 0006-8993/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2005.12.017

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Mollace et al., 1991; Osonoe et al., 1994; Penix et al., 1994; Przegalinski et al., 1994; Starr and Starr, 1993). Although the exact reason for this discrepancy is currently unknown, it is clear that NO is, at least in part, responsible for the epileptogenesis. Previous studies have suggested that NO precursors, such as L-arginine (L-ARG), exert an anticonvulsant effect on KA-induced seizures, while NO synthase (NOS) inhibitors, such as N-ω-nitro-L-ARG (L-NAME) and 7-nitroindazole (7-NI), increase the severity of KA-induced seizures in rodents (Kashihara et al., 1998; Penix et al., 1994; Przegalinski et al., 1994). We therefore hypothesized that the antiepileptic effects of the KD might be closely related to the synthesis of NO. The present study was designed to examine the role of NO in the antiepileptic action of KD in male ICR mice. We evaluated the synthesis of NO and the activity of NOS in the hippocampus of the mouse brain after KD treatment. In addition, to determine the precise role of NO in the KAinduced seizure model, we investigated the effects of inhibitors of NOS (L-NAME and 7-NI) prior to KA administration. We also used NOS-knockout mice to further clarify the role of NO in this model.

2.

Results

2.1. Effects of the KD on hippocampal NOx content and the KA-induced seizure-onset time In the KD-fed mice, the blood β-OHB levels increased progressively to a significantly greater extent than that observed in the ND-fed mice at all time points during the treatment (Fig. 1A; P b 0.005 for days 14 and 21; P b 0.0005 for day 28). When the difference in the β-OHB blood levels was at its highest (day 28), the hippocampal NOx content in the KD-fed mice was significantly greater than that of the ND-fed mice (Fig. 2H; P b 0.05). The hippocampal NOx concentrations in the ND- and KD-fed mice were 88.7 ± 8.38 and 128.99 ± 14.99 pmol/ mg−1 wet weight, respectively. All of the mice developed typical seizure behaviors when they were injected i.p. with an epileptogenic dose of KA (25 mg/kg) after 4 weeks on the respective diets. Within 18 min, the ND-fed mice began to display staring and hind-limbpawing behaviors; however, it took 37 min before the KD-fed mice started to show these activities (Fig. 1B). Despite the fact that the KD markedly retarded the latency to the seizures elicited by KA (P b 0.0005), it did not significantly attenuate the severity of the seizures (data not shown). During the 3 h of observation, all of the mice in both groups displayed wobbling, jumping or status epilepticus, corresponding to grade IV.

2.2. Effects of KD on NOS immunoreactivity in the hippocampus In the KD-fed mice, the number of nNOS positive neurons was increased in the stratum pyramidal layer (sp) and the stratum radiatum (sr) of the CA1/2 (Figs. 2B and G) and CA3 (Figs. 2D and G) regions, and the hilus (h) of the dentate

Fig. 1 – (A) Serum β-OHB levels over the 4-week treatment period. The respective diets were started on postnatal day 21. β-OHB levels were significantly higher in the KD-fed mice than in the ND-fed mice at all time points. There was a significant difference between the two diet groups (*P b 0.05 for days 14 and 21; **P b 0.0005 for day 28) according to the Student's t test. (B) Effect of the KD on KA-induced seizure-onset time in male ICR mice at day 28 of the respective diet treatments. In the KD-fed mice, the latency to seizure elicited by KA was delayed compared with the ND-fed mice. There was a significant difference between the two diet groups (**P b 0.0005) according to the Student's t test. Each data point represents the mean ± SEM of 15 mice.

gyrus (Figs. 2F and G) compared with the ND-fed mice (Figs. 2A, C, E and G). Regarding the other NOS isoforms, eNOS activity was found in the endothelial cells of the blood vessels of the hippocampus, and there was no apparent difference between the ND- and KD-fed mice (data not shown). iNOS immunoreactivity was not detected in any experimental group (data not shown).

2.3.

Effects of NO on seizure latency elicited by KA

In order to determine whether NO had a pro- or anticonvulsant effect in the ND-fed mice, we administered inhibitors of NOS (L-NAME, a general NOS inhibitor (32) or 7-NI, a specific antagonist of nNOS (15)) prior to the KA administration. L-NAME at doses of 5 and 25 mg/kg (i.p.) shortened the onset time of KA-induced seizures dosedependently (Fig. 3A) and elevated the seizure score (Fig. 3B) and mortality (Table 1) in the vehicle-pretreated NDfed mice. Consistent with the L-NAME results, when 25 and 50 mg/kg of 7-NI were injected before the administration of KA, the latency to seizure was dose-dependently shortened compared with that of vehicle-pretreated ND-

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Fig. 2 – Effects of KD on the immunoreactivity of nNOS (A–G) and levels of NOx (H) in the hippocampus of ND- and KD-fed mice. (A–F) Photomicrographs of immunoreactive nNOS in the hippocampus of ND- (A, C and E) and KD-fed mice (B, D and F). nNOS positive cells were found in the stratum oriens (so), stratum pyramidal (sp) and stratum radiatum layer (sr) of the CA1 (A and B), in the CA3 (C and D), and in the granule cell layer (G) and hilus (h) of dentate gyrus (E and F) in the ND- and KD-fed mice; M, molecular cell layer of dentate gyrus. Arrows indicate nNOS positive cells that had strong immunoreactivity within soma and arrowhead indicates nNOS positive cells that had strong immunoreactivity within dendrites. A significant increase in nNOS immunoreactivity in KD-fed mice was observed in the sp and sr in the CA1/3, and in the h of the dentate gyrus compared with the ND-fed mice. Scale bar = 100 μm. (G) Quantitative analysis of changes in nNOS positivity in the hippocampus of ND- and KDfed mice. Data represent the means ± SEM of 30 sections/10 animals/group. *P b 0.005 and **P b 0.0005 (statistically significant differences compared with ND-fed mice). (H) Levels of NOx in hippocampus at day 28 of the respective diet treatments. The content of NOx in KD-fed mice was higher than that of the ND-fed mice. There was a significant difference between the two diet groups (*P b 0.05) according to the Student's t test. Each data point represents the mean ± SEM of 15 mice.

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Fig. 3 – Effects of NOS inhibitors on seizure latencies (A and C) and seizure score (B and D) elicited by KA. L-NAME (A and B) and 7-NI (C and D) were injected (i.p.) 30 min prior to the administration of KA (25 mg/kg). The resultant seizures were categorized into six different grades and scored according to described methods in the Experimental procedures section. *, Statistically significant differences compared with ND-fed mice pretreated with the same drug, *P b 0.05 and **P b 0.005. ˙, Significantly different from the value of vehicle-treated ND-fed mice, ˙P b 0.05. †, Significantly different from the value of vehicle-treated KD-fed mice †P b 0.05 and ‡P b 0.005. Data represent the means ± SEM (each n = 10 except 7-NI pretreated group (each n = 15)). All statistical analyses were performed using one-way ANOVA followed by the Tukey's multiple comparison tests.

fed mice (Fig. 3C) and elevated the seizure score (Fig. 3D) mortality (Table 1). In the KD-fed mice, pretreatment with 5 or 25 mg/kg of L-NAME before the KA injection shortened the latency to seizure compared with that of vehicle-pretreated KD-fed mice (Fig. 3A). However, L-NAME did not have a dosedependent effect on the seizure latency elicited by KA in the KD-fed mice compared with that of L-NAME pretreated NDfed mice (Fig. 3A). Similar latency to seizure resulted from high (25 mg/kg) and low (5 mg/kg) dose L-NAME pretreatment in the KD-fed mice. Pretreatment with L-NAME in the ND-fed mice resulted in 100% mortality after 3 h of the KA administration, while KD-fed mice showed a lower mortality rate within 3 h of KA administration (Table 1). In the case of 5 mg/kg L-NAME, KD-fed mice significantly decreased seizure score compared with that of L-NAME pretreated ND-fed mice (Fig. 3B, P b 0.05). When 25 and 50 mg/kg of 7-NI were administered before KA treatment, the seizure latency in the KD-fed mice was shortened dose-dependently compared with that of the vehicle-pretreated KD-fed mice (Fig. 3C). But KD did not decrease seizure score (Fig. 3D) and mortality elicited by KA when 7-NI was pretreated (Table 1).

2.4.

Effects of NOS gene-knockout on KA-induced seizure

We used NOS gene-knockout mice to further evaluate the effects of NO on KA-induced seizures (Fig. 4). Wild-type ND-fed

Table 1 – Effects of NOS inhibitors and NOS gene-knockout on the mortality elicited by KA Experimental group (ICR mice)

Vehicle (ICR)

Mortality (died/total) ND

KD

0/10

0/10

L-NAME

(5 mg/kg)

10/10

6/10

L-NAME

(25 mg/kg)

10/10

8/10

13/15 15/15

12/15 15/15

7-NI (25 mg/kg) 7-NI (50 mg/kg) L-NAME

Experimental group

Wild type (C57BL/6) nNOS-knockout mice iNOS-knockout mice

Mortality (died/total) ND

KD

0/20

0/20

3/20

4/20

7/20

5/20

and 7-NI were injected (i.p.) 30 min prior to the administration of KA (25 mg/kg). The mortality was measured as the number of dead mice counted within 3 h of the administration of KA.

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Fig. 4 – Effects of NOS gene-knockout on seizure latencies (A) and seizure score (B) elicited by KA. All NOS gene-knockout mice showed reduced latencies to seizure (A) and increased seizure score (B) elicited by KA compared with wild type. But in the KD-fed group, NOS gene-knockout mice showed delayed latencies to seizures compared with that of the ND-fed mice. *, Statistically significant differences between ND- and KD-fed mice, *P b 0.05. ˙, Significant difference compared with wild-type ND-fed mice, ˙P b 0.005. †, Significant difference compared with wild-type KD-fed mice, † P b 0.005. Data represent the means ± SEM (each n = 20). All statistical analyses were performed using one-way ANOVA followed by the Tukey's multiple comparison tests.

C57BL/6 mice began to display hind-limb pawing within 13 min of KA administration. And KD-fed C57BL/6 mice showed that within 16 min of KA administration (Fig. 4A). The most severe behavioral responses during the 3-h observation period were seen in wild-type ND- or KD-fed C57BL/6 mice scoring seizure score 2 (Fig. 4B). The latency to seizure elicited by KA was markedly shortened in the ND-fed nNOS-knockout mice (P b 0.005) and iNOS-knockout mice (P b 0.0005) compared with the wild-type mice (Fig. 4A). During the 3-h observation period, almost nNOS- and iNOS-knockout mice showed severe behavioral responses corresponding to grade V. And seizure score (Fig. 4B) and mortality (Table 1) of the NOS-knockout mice were increased compared with wild-type C57BL/6 mice. KDfed iNOS-knockout mice showed significantly delayed latency to seizure compared with ND-fed iNOS-knockout mice (Fig. 4A, P b 0.05). However, latency to seizure in the KD-fed nNOSknockout mice did not differ from that of the ND-fed nNOSknockout mice. And KD-fed nNOS-knockout mice showed a significantly shortened latency to seizure onset compared with KD-fed iNOS-knockout mice (P b 0.05).

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3.

Discussion

3.1.

The KD delays the KA-induced seizure-onset time

Numerous clinical and experimental reports have addressed the antiepileptic effects of the KD (Freeman et al., 1998; Noh et al., 2003; Rho et al., 1999; Vining et al., 1998). Although several animal models and experimental hypotheses have been developed in order to evaluate the mechanisms underlying the antiepileptic efficacy of KD, these approaches require further testing; the reported effects of the KD on seizure resistance vary between researchers who applied different high-fat diets, induced seizures by divergent means and used animals of different genders and ages (Bough et al., 1999; Su et al., 2000; Thavendiranathan et al., 2000). In the present study, we used young ICR mice and a KA-induced-seizure model to clarify the efficacy of KD in suppressing seizure. The effect of the KD became apparent when mice were injected i.p. with a convulsant dose of KA. We showed recently that the KD delays the KA-induced seizure-onset time in male ICR mice, reduces KA-induced hippocampal cell death and decreases the expression of caspase-3, which is thought to be a necessary component of KA-induced seizures (Noh et al., 2003). The present study confirmed our previous finding that the KD delays the KA-induced seizure-onset time in male ICR mice and C57BL/6 mice.

3.2. The KD increases the NOx content in the hippocampus through increased nNOS activity NO is thought to be involved in various pathophysiological processes, including epilepsy, cerebral ischemia and certain neurodegenerative diseases (Bredt and Snyder, 1992; Nathan, 1992; Snyder and Bredt, 1991). One possible explanation for the differential susceptibility of KD-fed mice to KA is that the NO system might act as a potent intracellular mediator of KA excitotoxicity. In order to test this idea, we measured hippocampal NO generation and three types of NOS immunoreactivity after the administration of KD. The results demonstrated that endogenous NO generation and nNOS immunoreactivity were both elevated in the hippocampus of the KD-fed mice. This result is consistent with a previous clinical report that showed that eNOS expression does not change, and iNOS activity is not detected, in the epileptic brain (Gonzalez-Hernandez et al., 2000). In the present study, nNOS positive immunoreactivities were found within the soma of hippocampal neurons (especially in the stratum pyramidal layer (sp) and the stratum radiatum (sr) of the CA1/2 and CA3 regions, and hilus (h) of the dentate gyrus) and dendrites of the granule cell layer (G) of the dentate gyrus. The expression patterns of nNOS in the hippocampus were similar to those reported previously (Jinno et al., 1999; Jinno and Kosaka, 2004). Previous studies reported that nNOS positive neurons in the mouse hippocampus represent a subpopulation of gamma-aminobutyric acid (GABA)ergic neurons using immunofluorescent colocalization of nNOS and glutamic acid decarboxylase 67 (GAD67) or somatostatin (Jinno et al., 1999; Jinno and Kosaka, 2004). And they suggest that

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nNOS positive neurons may be related to modulation GABAergic neurotransmission in hippocampal layer. In the present study, we showed that KD increased the number of nNOS positive neurons and immuoreactivities of their dendrites. From these results, we can assume that increased nNOS immunoreactivity by the KD may, at least in parts, modulate GABAergic local circuit in the hippocampus and results retarded the latency to the seizure elicited by KA.

3.3. KA

Increased NO delays the seizure-onset time induced by

The role of NO in convulsive phenomena has been controversial (Kirkby et al., 1996a; Kirkby et al., 1996b; Smith et al., 1996). In order to understand this activity, most experiments have used inhibitors of NOS, namely, L-NAME and 7-NI (Kashihara et al., 1998; Penix et al., 1994; Przegalinski et al., 1994; Rundfeldt et al., 1995). L-NAME is generally used nonselectively as an inhibitor of both eNOS and nNOS (Takei et al., 2001), while 7-NI is a relatively selective nNOS inhibitor (Moore et al., 1993). In the present study, we injected KA with or without pretreatment by NOS inhibitors, in order to clarify the relationship between seizures and NO in our experimental animal model. We found that pretreatment of the ND-fed mice with L-NAME and 7-NI delayed the seizure-onset time induced by KA compared with the vehicle-pretreated ND-fed mice. Moreover, the mortalities of the NOS inhibitor-pretreated mice were increased compared to the vehiclepretreated ND-fed mice. These results are consistent with previous reports showing that L-NAME and 7-NI reduce the latency to convulsions following the systemic administration of KA (Osonoe et al., 1994; Penix et al., 1994). Recently, Kashihara et al. (1998) reported that L-NAME inhibited KAinduced NO generation while enhancing seizure activities (Kashihara et al., 1998), suggesting that the NO generated after KA injection might be involved mainly in seizure termination. However, other studies have implied that NO acts as a proconvulsant substance (Bagetta et al., 1992; De Sarro et al., 1993; Mollace et al., 1991). The discrepancies among these results may be explained by variability in the types and doses of NOS inhibitor, the route of administration and the model of seizures used (Kirkby et al., 1996a; Penix et al., 1994; Rho et al., 1999; Rundfeldt et al., 1995). For example, L-NAME potentiated KA-induced seizures (Kirkby et al., 1996a), but attenuated pentetrazole (PTZ)-induced seizures in rats (Osonoe et al., 1994). Here, we used NOS-knockout mice (nNOS and iNOS) to further understand the role of NO in the regulation of seizures. As a result, we can find that the nNOS and iNOS-knockout mice fed a normal diet have more severe seizures and higher mortality than wild-type mice. And we also found that KD-fed iNOS-knockout mice showed significantly delayed latency to seizure compared with that of ND-fed iNOS-knockout mice or KD-fed nNOS-knockout mice. KD-fed iNOS-knockout mice may have increased NO production to a certain degree through endogenous nNOS and this may result in delayed latency to seizure, though latencies to seizure are still shorter than wild type. From these iNOS-knockout data, it is capable of suggesting the ketogenic diet worked by a mechanism independent of NO. Because nNOS expression should be intact in the iNOS

knockout, the iNOS-knockout and wild-type mice should have the same seizure latency. However, NOS-knockout data can be interpreted that the seizure severity and mortality are very high in ND-fed NOS-knockout mice compared with wildtype mice; therefore, even if the ketogenic diet increased NO through nNOS, its effect might not be observed because of the severity of the seizure model. Our findings may indicate that endogenous NO exerts an inhibitory effect on KAinduced seizures in ICR and C57BL/6 mice. In L-NAME-treated KD-fed mice, the seizure-onset times were delayed compared with the L-NAME-pretreated ND-fed mice. However, L-NAME did not show dose-dependent latency of the KA-induced seizure-onset time compared with the LNAME-pretreated ND-fed mice. These results suggest that the increase in NO caused by the KD might be mediated mainly by nNOS, because the seizure latency responded more sensitively to the nNOS-specific blocker, 7-NI, than to the general NOSinhibitor, L-NAME. In conclusion, we investigated the mechanism of the antiepileptic activity of KD and demonstrated for the first time that the KD increases endogenous NO, which is mediated by increased nNOS expression. Our results are important from a clinical perspective, as it is widely believed that the KD is partly antiepileptic. The present findings highlight the need for a further exploration of this hypothesis.

4.

Experimental procedures

4.1.

Animals and treatments

Male ICR and C57BL/6 mice were purchased from Samtako (Gyeonggi, Korea). nNOS- and iNOS-knockout mice were inbred at our animal center from stock obtained from the Korea Research Institute of Bioscience and Biotechnology (KRIBB, Taejon, Korea), which were originally purchased from the Jackson Laboratory (Jackson Laboratories, MI, USA). These knockout mice were an inbred mutant strain of C57BL/6 mice. The animals were housed five per cage and maintained at 22 ± 0.5 °C with an alternating 12h light/dark cycle. All procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health. The mice were fed ad libitum for 4 weeks on either the Harlan Teklad TD 96355 diet for the KD groups or standard rodent chow for the normal diet (ND) groups. The respective diets were begun at postnatal day 21. The KD-fed mice were fasted overnight prior to the initiation of the diet in order to facilitate the development of ketonemia (Todorova et al., 2000). The composition of the KD used in our experiments was as reported previously (Rho et al., 1999). Seizures were induced by KA (Tocris Cookson Ltd., UK), which was freshly dissolved in 0.9% NaCl prior to each experiment and injected intraperitoneally (i.p.) at a volume of 2.5 ml/kg body weight. Seizure was always induced between 10:00 and 16:00 h in order to minimize any possible complications caused by circadian rhythms (Woolley and Timiras, 1962). After KA administration (25 mg/kg), the mice were monitored for 3 h and the number of dead mice was counted during this period. The resultant seizures were categorized into six different grades and scored according to a previously defined scale (Hu et al., 1998) and some modified: grade 0 (seizure score 0), no response; grade I (seizure score 1), staring, a rigid posture, tail extension, front pawing or hind-limb pawing (scratching) and/or staring; grade II (seizure score 2), grade I + head nodding, rearing, repetitive movement; grade III (seizure score 3), grade II + jumping, wobbling and/or falling; grade IV (seizure score 4), non-intermittent seizure activity persisting for

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N30 min; grade V (seizure score 5), death. When mice displayed a behavioral response of grade I, we determined that time as “latency to seizure onset”. To examine the effects of NO on KA-induced seizures, we treated the animals with two NOS inhibitors, L-NAME and 7-NI, which were purchased from Sigma (St Louis, MO). L-NAME was dissolved in 0.9% NaCl, while 7-NI was dissolved in peanut oil (Sigma). All of the drugs were freshly dissolved prior to each experiment and were injected i.p. at a volume of 2.5 ml/kg body weight 30 min prior to KA treatment. This experiment was repeated three times and showed reproducible results. 4.2.

Measurement of blood β-hydroxybutyrate (β-OHB) level

One of the clinical hallmarks of success for the KD is a notable accumulation of ketone bodies in the blood plasma, particularly βOHB (Rho et al., 1999). In order to ensure ketosis, we measured the plasma levels of β-OHB using a Keto-Site reflectance meter and test cards (GDS Diagnostics, Elkhart, IN) as described previously (Bough et al., 1999). Blood samples were obtained via the tail vein at the same time of day (between 11:00 and 16:00 h) upon the initiation of the respective diets (for basal values) and again after 2, 3 and 4 weeks. 4.3.

Measurement of NOx metabolites

After 4 weeks on their respective diets, the animals were decapitated and the hippocampi were separated. Brain samples were weighed, homogenized in 400 μl deionized water and centrifuged at 20,000 × g for 10 min at 4 °C. Then, 80 μl of each supernatant was mixed with 10 μl of 5 mM NADPH and 10 μl of nitrate reductase (10 U) in a 96-well plate. Samples were incubated for 1 h at 37 °C, following which 100 μl of Griess reagent (a 1:1 mixture of 1% sulfanilamide in 5% H3PO4 and 0.1% N-(1-naphthyl) ethylenediamine dihydrochloride) was added and the mixture was incubated for an additional 10 min at room temperature. Absorbances were measured at 550 nm on a plate reader and converted into the NOx content using a nitrate standard curve. This experiment was repeated twice and showed reproducible results. 4.4.

NOS immunohistochemistry

After 4 weeks on their respective diets, the animals (10 mice per group) were deeply anesthetized and intracardially perfused with 0.9% saline followed by 4% paraformaldehyde in 0.1 M phosphatebuffered saline (PBS). The brains were removed, postfixed for 6 h in the same fixative at 4 °C, cryoprotected for 24 h in PBS containing 20% sucrose and sectioned (10 μm thickness) in the coronal plane using a cryostat. Serial sections were stained for neuronal NOS (nNOS; 30 sections/10 animals/group), endothelial NOS (eNOS; 15 sections/5 animals/group) or inducible NOS (iNOS; 15 sections/5 animals/group) immunohistochemistry. For NOS immunohistochemistry, we used the avidin–biotin–peroxidase method (ABC kit; Vectastain, Vector laboratories, Burlingame, CA). After blocking endogenous peroxidase, the sections were incubated with 2% normal goat serum and exposed overnight at 4 °C with the appropriate primary antibodies. The nNOS, eNOS and iNOS polyclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) were used at a 1:1000 dilution in PBS. Sections were washed, incubated for 1 h with biotinylated anti-rabbit immunoglobulin G (IgG) antibody and then incubated in the ABC reagent for 1 h at room temperature. The peroxidase reaction was visualized with 0.05% diaminobenzidine (Sigma) and 0.01% hydrogen peroxide (Fluka, Switzerland). 4.5.

Data analysis

All data are presented as the mean ± the standard error of the mean (SEM) from three or more independent experiments. The

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nNOS positive cells were counted by observers blinded to the treatment conditions using light microscopy at a 40–200× magnification. Middle region of the hippocampi was typically compared and statistically analyzed. All statistical analyses were performed using one-way analysis of variance (ANOVA) followed by the Tukey's multiple comparison test. Differences with a probability (P) value b 0.05 were considered to be statistically significant.

Acknowledgments This work was supported by Korea Science and Engineering Foundation (R13-2005-012-01001) and partially supported by a grant of the Brain Korea 21 project of the Ministry of Education of Republic of Korea.

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