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Effects of the ketogenic diet on neurogenesis after kainic acid-induced seizures in mice
Epilepsy Research (2008) 78, 186—194
journal homepage: www.elsevier.com/locate/epilepsyres
Effects of the ketogenic diet on neurogenesis after kainic acid-induced seizures in mice Young Se Kwon a,1, Sang-Wuk Jeong b,1, Dong Wook Kim c,∗, Eun Sil Choi c, Byong Kwan Son a a
Department of Pediatrics, College of Medicine, Inha University, Incheon, Republic of Korea Department of Neurology, Dongguk University Hospital, Goyang, Republic of Korea c Department of Pediatrics, Clinical Research Center, FIRST Mitochondrial Research Group, Ilsan Paik Hospital, Inje University College of Medicine, Goyang, Republic of Korea b
Received 5 February 2007; received in revised form 23 November 2007; accepted 29 November 2007
KEYWORDS Ketogenic diet; Neurogenesis; Epileptogenesis; Seizure; Kainic acid; Mice
Summary The ketogenic diet (KD) remains a therapy in search of explanation although it is an established treatment of intractable epilepsy. Recent studies suggest that the KD may be both anticonvulsant and antiepileptogenic. Epileptic seizures have been shown to stimulate the proliferation rate of neuronal progenitor cells in adult animals, which may be related to epileptogenesis. It is known that calorie restriction (CR) increases neurogenesis. The KD was originally formulated to reproduce the biochemical changes seen upon fasting (extreme CR). Thus, we investigated the effects of the KD on neurogenesis after kainic acid (KA)-induced seizures in mice. In the present study, quantitative analysis of BrdU labeling revealed a significant increase in the proliferation rate of neuronal progenitor cells after KA-induced seizures in the KD-fed mice. This finding may provide a clue to explain how the KD exerts antiepileptogenic effects although further studies are mandatory to elucidate the relationship between seizureinduced neurogenesis augmented by the KD and its antiepileptogenic properties. In conclusion, our results suggest that the KD enhances neurogenesis, which may be related to its beneficial effects on epilepsy. © 2007 Elsevier B.V. All rights reserved.
1. Introduction ∗
Corresponding author. Tel.: +82 31 910 7106; fax: +82 31 910 7108. E-mail address: [email protected] (D.W. Kim). 1 They contributed equally and should be considered as first authors.
The ketogenic diet (KD) is a high-fat, low-carbohydrate, adequate-protein diet that has been used for more than 80 years for the treatment of medically intractable epilepsy. The clinical efficacy of the KD has now been well doc-
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Effects of the ketogenic diet on neurogenesis umented (Hemingway et al., 2001; Kim et al., 2004). However, little is known about its underlying mechanisms. Numerous animal studies also have demonstrated the anticonvulsant effects of the KD (Bough et al., 2002; Noh et al., 2003). Furthermore, the KD may possess not only anticonvulsant effects but also antiepileptogenic properties, i.e., effects that may ameliorate or even cure the disease process itself, because clinical data suggest that a period of time on the KD may permanently decrease seizure activity or cause its cessation entirely (Freeman et al., 1998; Vining et al., 1998; Hemingway et al., 2001; Rho, 2004). Results obtained from the animal studies also have suggested the antiepileptogenic properties of the KD (Muller-Schwarze et al., 1999; Bough et al., 2003). Epileptogenesis refers to the events by which the normal brain becomes capable of producing epileptic seizures, i.e., the process by which neural circuits are converted from normal excitability to chronic hyperexcitability (Stafstrom, 2004). This chronic hyperexcitability may result from the combined effects of several structural alterations such as neuronal death, gliosis or mesial temporal sclerosis, and the growth of new aberrant axonal connections. Furthermore, epileptic seizures can induce neurogenesis, i.e., the birth of new neurons, in adult animals. It is especially prominent in the hippocampal dentate gyrus (Parent et al., 1998; Scott et al., 1998). Again, it can give rise to increased or decreased excitability depending on the connectivity and type of newborn neurons. Recently, many investigators have made efforts to shed light on the role of neurogenesis in epileptogenesis (Parent, 2002; Scharfman, 2004; Parent et al., 2006). Evidence accumulated over the past four decades or more has dispelled the long-held dogma that the adult mammalian brain cannot generate new neurons (Parent, 2003). Among the principal neuronal populations within the hippocampus, the dentate granule cells (DGCs) have the unusual property of prolonged postnatal neurogenesis (Eckenhoff and Rakic, 1988) that persists into adulthood in the rodent (Kuhn et al., 1996). DGC neurogenesis during postnatal life appears to be influenced, at least in part, by factors such as aging, stress, exercise, environmental enrichment, and genetic background (Parent, 2003). Furthermore, recent research demonstrated that calorie restriction (CR) enhanced neurogenesis in the brain (Lee et al., 2000, 2002). The KD was originally formulated to mimic the effects of fasting (an extreme form of CR), as it had been known since biblical times that fasting had a beneficial effect on epilepsy (Swink et al., 1997; Wheless, 2004). It has been assumed ever since that the fasting and the KD share a common mechanism in alleviating seizures, although this assumption has not been tested rigorously (Stafstrom, 1999). The KD mimics fasting that induces ketosis and then relieves seizures (Swink et al., 1997; Schwartzkroin, 1999). Thus, we can hypothesize that the KD may enhance neurogenesis similar to the effects of CR. In the present study, we investigated the effects of the KD on neurogenesis after kainic acid (KA)-induced seizures in mice, which may be related to its antiepileptogenic properties.
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2. Methods 2.1. Animals and diet regime Normal male Institute for Cancer Research (ICR) mice (Folas International, Korea) were used for all the experiments. Animals were housed, 4 per cage, in a room maintained at 22 ± 3 ◦ C with an alternating 12-h light/dark cycle. Mice were divided into four groups: (1) seizure-free normal diet (ND) group (n = 11), (2) seizure-free KD group (n = 11), (3) KA-seizure ND group (n = 14), and (4) KA-seizure KD group (n = 14). For 4 weeks, beginning on postnatal day 21, mice of the KD groups were fed the Harlan Teklad TD 96355 (USA), in which most of fat source is polyunsaturated fatty acids, while ND groups were fed a standard rodent chow (Samtako, Korea). The KD composition, which we used for this study, was reported previously (Rho et al., 1999). All mice were fed ad libitum and were weighed at least twice weekly. All procedures were performed in accordance with the Guide for Care and Use of Laboratory Animals published by the National Institutes of Health.
2.2. Blood BHB level After the 4 weeks of dietary treatment, blood -hydroxybutyrate (BHB) levels were measured before seizure induction from blood obtained via tail vein using Keto-SiteTM test kit (GDS Diagnostics, USA).
2.3. Seizure induction After the 4 weeks of dietary treatment, seizures were chemically induced by a single intraperitoneal (i.p.) injection of KA (Sigma, dissolved in physiological saline) with a dose of 30 mg/kg in the KAseizure groups. Animals in the seizure-free groups were injected with equal volume of physiological saline. Mice were monitored for seizure behavior for 2 h after KA injection, and seizures were categorized into five different grades according to a previously defined scale (Hu et al., 1998)—–Grade 0: no response; Grade I: front or hind limb pawing or staring; Grade II: rearing, nodding or bilateral pawing; Grade III: jumping, wobbling or falling; Grade IV: status epilepticus or death. All mice were graded according to the maximal behavioral response observed within 2 h after KA administration.
2.4. BrdU labeling The thymidine analog 5-bromodeoxyuridine (BrdU, Sigma, was dissolved in saline) was administered i.p. with a dose of 50 mg/kg per injection for BrdU labeling. Animals received BrdU injection once a day for 6 consecutive days, starting at 24 h after KA or saline administration. Twenty-three mice were sacrificed 24 h after the last BrdU injection, which allowed us to measure the number of cells that had incorporated BrdU, i.e., the proliferation rate of neuronal progenitor cells. For the double fluorescent immunohistochemistry experiment, 24 mice were allowed to survive for 28 days after the last injection of BrdU. This allowed us to investigate the phenotype, survival, and migration pattern of newborn cells. All the mice in these experiment were allowed to be fed either ND or KD until sacrifice.
2.5. Tissue fixation, immunohistochemistry and quantification Animals were anesthetized with 1% ketamine (30 mg/kg, Ketara; Yuhan Yanghang, Seoul, Korea) and xylazine hydrochloride (4 mg/kg, Rompun; Bayer Korea, Seoul, Korea) at the corresponding time after the BrdU injection, and then perfused through the heart with 50 ml cold saline and 50 ml of 4% paraformaldehyde (PFA) in 0.1 mol/l
188 phosphate-buffered saline (PBS). After 24 h of fixation in 4% PFA, the brains were cryoprotected with PBS containing 30% sucrose for 24 h. Coronal sections through the entire hippocampus were cut at 30 m on a freezing microtome and sections were stored in PBS (pH 7.4). Tissue sections were incubated in 2M HCl for 30 min in order to denature the DNA into single-stranded DNA at room temperature (the BrdU antibody recognizes only single-stranded DNA). Tissue sections were then rinsed three times for 10 min each in PBS, and treated with 3% H2 O2 (in 30% methanol) to block endogenous peroxidase. After they were washed for 30 min each in PBS, free-floating sections were incubated in PBS containing 10% normal goat serum and 0.5% BSA/0.3% Triton X-100 for 1 h. Section were then incubated with a mouse monoclonal anti-BrdU antibody (1:300, Pharmigen, USA) at 4 ◦ C for 24 h. The antibody solutions also contained goat serum and BSA/Triton X-100. On the next day, after washing all tissue sections three times in PBS, the sections were incubated in biotinylated secondary antibody (EnvisionTM /HRP, rabbit anti-mouse, DAKO, USA) for 2 h at room temperature. After three 10-min washes, sections were reacted for peroxidase enzyme activity by using 3,3 -diaminobenzidine-tetrahydrochloride (DAB, DAKO, USA). The reaction was terminated by transferring the sections to PBS. Sections were mounted on gelatinized slides, dehydrated, and coverslipped. The number of BrdU-positive cells in the combined hippocampal dentate gyrus and hilar regions was counted in every seventh section in a series of 30 m coronal sections (210 m apart) covering the complete right and left hippocampi. Cell counting was performed by an experimenter who was not informed of group assignment. Cells were counted with 40× lens on microscope (Nikon, Japan) equipped with a magnifier digital camera and a computer-assisted image analysis system (Image J, NIH, USA).
2.6. Double fluorescent immunohistochemistry and confocal microscopy In order to identify the BrdU-labeled cells as either neurons or GFAPpositive glia, BrdU fluorescent immunohistochemistry was combined with NeuN (neuron) and GFAP (glia) fluorescent immunohistochemistry in conjunction with confocal microscopy. Coronal free-floating sections (30 m) from the whole brain were used. DNA denaturation was performed as described above. Tissue sections then were rinsed three times for 10 min each in PBS. Free-floating sections were incubated in PBS containing 10% normal rabbit serum and 0.3% Triton X-100 for 1 h. Sections were then incubated with sheep polyclonal anti-BrdU (1:300, Biodesign, USA) antibody at 4 ◦ C for 24 h. On the next day, after washing all tissue sections three times in PBS, the sections were incubated with FITC-conjugated anti-sheep IgG (1:100, Biodesign, USA) secondary antibody for 2 h at room temperature. After three 10-min washes, sections were incubated in PBS containing 10% normal goat serum and 0.5% BSA for 1 h. Sections were then incubated with mouse antiNeuN (1:200, Chemicon, USA) or mouse anti-GFAP (1:1000, Sigma, USA) at 4 ◦ C for 24 h. On the next day, after washing all tissue sections three times in PBS, the sections were incubated with Cy3-conjugated anti-mouse IgG (1:300, Biodesign, USA) secondary antibody for 2 h at room temperature. After washes in PBS, sections were mounted on gelatinized slides in anti-fade medium and coverslipped. Immunofluorescent images were obtained under a confocal laser scanning microscope (Carl Zeiss LSM510) and processed using Adobe Photoshop v7.0 (Adobe Systems, Mountain View, CA, USA). BrdUpositive cells from combined hippocampal dentate gyrus and hilar regions, chosen at random from each hippocampus, were examined for coexpression of BrdU and NeuN (a neuronal phenotype), BrdU and GFAP (a GFAP-positive glial phenotype). We counted 100 BrdU-positive cells per animal to obtain the percentages of coexpression.
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Fig. 1 Influence of the ketogenic diet (KD) on body weight in male ICR mice. During the dietary treatment, mean body weights of the normal diet (ND)-fed and KD-fed mice (each n = 25) showed no significant differences. Bars represent mean ± S.D.
2.7. Statistical analysis All data in this study are presented as mean ± S.D. We used the SPSS 11.0 program. Data were analyzed by unpaired Student’s t-test if they were normally distributed (Kolmogorov-Smirov test, P > 0.05). Numbers of BrdU-positive cells were examined by two-way ANOVA with interaction. Percentages of BrdU-positive cells that were colabeled with Neu-N were examined by ANOVA. A value of P < 0.05 was considered significant.
3. Results 3.1. The KD was well tolerated, increased blood BHB level and seizure threshold To perform a satisfactory study using a KD, the diet should be well tolerated, induce ketosis, and show anticonvulsant
Fig. 2 Blood -hydroxybutyrate (BHB) levels in male ICR mice treated with the ketogenic diet (KD). Bars represent mean ± S.D. The asterisk (*) indicates a significant difference (P < 0.001, Student’s t-test) between the normal diet (ND)-fed mice and the KD-fed mice (each n = 25).
Effects of the ketogenic diet on neurogenesis
189 old. All mice were graded according to the maximal seizure behavior observed for 2 h after KA administration. Every seizure behavior was grade II or more. However, the severity represented as seizure behavior grade showed no significant differences between the KD-fed and ND-fed animals (data not shown) as reported previously (Noh et al., 2003, 2005a,b, 2006a). The mortality of mice following the administration of KA was 10.7%.
3.2. The KD increased neurogenesis after KA-induced seizures
Fig. 3 Influence of the ketogenic diet (KD) on the kainic acid (KA)-induced seizure onset time in male ICR mice. In the KDfed mice (n = 14), the latency to a seizure onset was delayed compared with the normal diet (ND)-fed mice (n = 14). Bars represent mean ± S.D. The asterisk (*) indicates a significant difference (P < 0.01, Student’s t-test) between the ND-fed and the KD-fed mice.
effects. In this study, the KD-fed mice tolerated their diet well for 4 weeks without oily appearance of the fur. The KDfed mice showed no difference in their behavior and health compared with the ND-fed mice. Mice in both groups had a similar weight gain within the time allotted (Fig. 1). We confirmed ketosis induced by the KD, because the blood BHB levels in the KD-fed mice were significantly higher than those of the ND-fed ones (2.52 ± 1.16 mM vs. 0.33 ± 0.13 mM, P < 0.001, Fig. 2). After the systemic administration of KA (30 mg/kg), all mice developed typical seizure behaviors. We found that the KD delays the KA-induced seizure onset time in male ICR mice (Fig. 3). These results were consistent with the previous reports (Noh et al., 2003, 2005a,b, 2006a,b), and indicate that the KD increased KA-induced seizure thresh-
After BrdU labeling, KA-induced seizures caused a marked increase in the number of BrdU-positive cells in the hippocampal dentate gyrus in contrast to the seizure-free animals (Fig. 4). Moreover, BrdU labeling revealed a significant increase in mitotic activity of neuronal progenitor cells in the KA-seizure KD group in comparison with the KA-seizure ND group (Fig. 4C and D). In the ND-fed animals, BrdU-positive cells increased significantly after KA administration compared with saline administration (77.28 ± 19.55 vs. 43.28 ± 12.64, P < 0.05 by ANOVA with interaction). In the KD-fed mice, BrdU-positive cells also increased significantly after KA administration compared with saline administration (111.41 ± 32.12 vs. 42.90 ± 23.94, P < 0.001 by ANOVA with interaction). These findings indicate that KA-induced seizures in mice increased the rate of neurogenesis within the hippocampal dentate gyrus. Measurement of BrdU-positive cells showed no significant differences between the seizure-free ND and KD groups. However, BrdU-positive cells after KA-induced seizures significantly increased in the KD-fed mice compared with the ND-fed ones (111.41 ± 32.12 vs. 77.28 ± 19.55, P < 0.05 by ANOVA with interaction). In summary, quantitative analysis of BrdU labeling after KA-induced seizures revealed a significant increase in the proliferation rate of neuronal progenitor cells in the KD-fed mice compared with the ND-fed ones (Fig. 5). It indicates that the KD increased neurogenesis after KA-induced seizures.
Fig. 4 BrdU-positive cells in the hippocampal dentate gyrus of male ICR mice. Mice were divided into four groups: (1) seizure-free normal diet (ND), (2) seizure-free ketogenic diet (KD), (3) KA-seizure ND, and (4) KA-seizure KD groups. (A) Baseline mitotic activity in the hippocampal dentate gyrus of group (1). (B) No remarkable difference in BrdU-positive cells of group (2) compared with (1). (C) Increased BrdU-positive cells of group (3) compared with (1) and (2). (D) Remarkable increase in BrdU-positive cells of group (4) compared with (1), (2), and (3). Scale bars: 200 m.
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Fig. 5 The number of BrdU-positive cells in the male ICR mice. Mice were divided into four groups: (1) seizure-free normal diet (ND) group (n = 5), (2) seizure-free ketogenic diet (KD) group (n = 5), (3) KA-seizure ND group (n = 6), and (4) KA-seizure KD group (n = 7). Bars represent mean. Although measurement of BrdU-positive cells showed no significant differences between the seizure-free ND and KD groups, BrdU-positive cells after KA-induced seizures significantly increased in the KD-fed mice compared with the ND-fed ones (P < 0.05, ANOVA with interaction).
3.3. The KD showed no difference in the long-term fate of seizure-induced neurogenesis We next examined the long-term fate of BrdU-positive cells in the dentate gyrus after KA-induced seizures in the KD-fed mice. This was done by injecting BrdU once a day for 6 consecutive days after KA or saline administration (i.e., within the period of significantly increased mitosis) and then sacrificing the animals 28 days later. We used markers of neuronal and glial phenotypes to examine the identity of the proliferating cells located in the hippocampal dentate gyrus. Cells that co-localized both BrdU and NeuN were classified as neurons. BrdU-positive cells that co-localized with GFAP were classified as GFAP-positive glia. We found that most of BrdUlabeled cells expressed neuronal marker NeuN (Fig. 6E and G), but the astrocytic marker GFAP was rarely co-localized with BrdU-immunostained nuclei (Fig. 6F and H) in the KAseizure KD group. Similar results have also been observed in other experimental animal groups. The majority of all BrdU-positive cells are of a neuronal phenotype. There were no significant differences among the experimental groups in the cellular phenotype (percentages) of BrdU-positive cells (F = 1.35, P = 0.28 by ANOVA, Fig. 7).
4. Discussion Our major findings in the present study were that (1) the KD-induced ketosis; (2) KA-induced seizures increased neurogenesis; (3) the KD does not alter neurogenesis in seizure-free mice; and (4) the KD increases the enhancement of neurogenesis induced by KA-seizure. Although the efficacy of the KD for the treatment of epilepsy is already documented through many clinical and experimental studies, its underlying mechanisms are largely
Y.S. Kwon et al. unknown. However, it is well acknowledged that ketosis, i.e., the systemic elevation of the ketone bodies (BHB, acetoacetate, and acetone), is necessary for seizure control (Swink et al., 1997; Schwartzkroin, 1999). Thus, in the first place, we confirmed that the KD regime used in the present study was truly ketogenic and had anticonvulsant effects as it increased blood BHB level and KA-induced seizure threshold in the KD-fed mice. This diet regime was used in numerous previous studies (Rho et al., 1999; Noh et al., 2003, 2005a,b, 2006a,b). Our colleagues previously demonstrated that the KD-induced ketosis, delayed the KAinduced seizure onset time, and prevented the hippocampal neuronal death in the male ICR mice (Noh et al., 2003). In the present study, to investigate whether the KD influences seizure-induced neurogenesis, we used the same experimental model. As a matter of course, we reproduced the result that the KD-induced ketosis and delayed the KA-induced seizure onset time in the present study. In animal studies, KA-induced seizure model has been widely used because of several advantages. Systemic administration of KA readily produces various motor signs including convulsive seizures (Sperk, 1994). KA, an agonist for kainate and AMPA receptors, is an excitotoxin in the hippocampus. It induces ongoing seizures, degeneration of cornu ammonis (CA) neurons, and hyperexcitability of surviving CA neurons in the hippocampus. Furthermore, recent studies have demonstrated that KA administration in adult rodents increased the rate of neurogenesis within the hippocampal dentate gyrus (Parent and Lowenstein, 1997; Gray and Sundstrom, 1998). In the present study, we also observed that KA-induced seizures resulted in a significant increase in the proliferation rate of neuronal progenitor cells in the hippocampal dentate gyrus. Although the birth of new neurons, neurogenesis, in mammalian CNS is confined largely to the embryonic period, accumulating evidence indicates that certain germinative zones persist throughout life and continue to generate neurons and glia in specific brain regions, including the hippocampal dentate gyrus (Parent, 2002). The study of this phenomenon offers the opportunity to identify and characterize circumstances that activate neuronal progenitor cells to form new neurons and the conditions required for these cells to mature and establish functional connections. Neurogenesis in the hippocampal dentate gyrus during postnatal life can be modulated by hormones, pharmacological actions, stress, levels of physical activity, and the complexity of the environment (Parent, 2003). Evidence from previous studies suggests that specific growth or neurotrophic factors influence neuronal progenitor cell proliferation in the brain (Benraiss et al., 2001; Pencea et al., 2001). In addition, CR has been shown to enhance hippocampal neurogenesis through recent studies (Lee et al., 2000, 2002). On the other hand, it has been well known that the KD was originally formulated to mimic the beneficial effects of fasting (an extreme form of CR) on epilepsy (Swink et al., 1997; Wheless, 2004). Recent research demonstrates that CR alone can reduce seizure susceptibility in rodents (Greene et al., 2001; Eagles et al., 2003). Thus, we can assume that the KD, as well as CR, may also enhance neurogenesis. In the present study, we tried to demonstrate whether the KD indeed increases neurogenesis in the hippocampal den-
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Fig. 6 The neuronal phenotype of proliferating cells in the hippocampal dentate gyrus of a ketogenic diet (KD)-fed mouse after kainic acid (KA)-induced seizure. (A) and (B) BrdU-positive cells (green) in the hippocampal dentate gyrus. (C) Cells immunostained by neuronal marker NeuN (red). (D) Cells immunostained with astrocytic marker GFAP (red). (E) Colocaliztion of BrdU with NeuN shown by yellow nuclei. (F) BrdU rarely colocalized with GFAP. (G) High-power photograph of (E). (H) High-power photograph of (F). Scale bars: (A—F) 200 m; (G) 25 m; (H) 20 m.
tate gyrus. Then, we failed to show that the KD significantly enhanced neurogenesis in the case of seizure-free (salinetreated) groups. However, neurogenesis after KA-induced seizures increased significantly in the KD-fed mice in comparison with the ND-fed ones. These results suggest that the KD does not increase neurogenesis in the seizure-free mice but it augments seizure-induced neurogenesis significantly. From our results, it remains a question to be answered why there is difference of the effect between the basal and the seizure-induced neurogenesis. Since our assumption was that the KD and CR operate with a similar mechanism and both would show increased neurogenesis, our results also contradict that assumption. Perhaps the mechanisms are different. Another remaining question is how the KD augments seizure-induced neurogenesis. Further researches into these questions are clearly needed. In recent years, there appeared interesting reports suggesting that the KD may have antiepileptogenic effects as well as anticonvulsant properties (Hemingway et al., 2001; Bough et al., 2003; Rho, 2004). The most persuasive clin-
ical data suggesting a potential antiepileptogenic effect were presented in 2001 by Hemingway et al. (2001). They investigated the long-term outcome of 150 children with intractable epilepsy who were enrolled prospectively in a KD trial. One year after starting the diet, 7% had become seizure free, and 20% had a greater than 90% reduction in seizure frequency; interestingly, 3—6 years after beginning the KD, a greater percentage of patients (13%) became seizure free. Intriguing as such observations may be, it is difficult to determine whether patients who remain seizure free after cessation of the KD (following full seizure control for at least 2 years) have had a spontaneous remission of their epilepsy, have had amelioration due to lack of damaging repetitive seizure activity, or have experienced a true direct antiepileptogenic effect of the KD (Rho, 2004). Despite the limitations of clinical speculation, results obtained from the animal studies have raised the possibility that the KD may possess antiepileptogenic properties (Rho, 2004). The first report that the KD may impede epileptogenesis in an animal model was provided by Muller-Schwarze et
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Fig. 7 Alterations in the percentages of BrdU-positive cells that were neurons (NeuN) or ‘‘other’’ cells in the male ICR mice. Mice were divided into four groups: (1) seizure-free normal diet (ND), (2) seizure-free ketogenic diet (KD), (3) KA-seizure ND, and (4) KA-seizure KD groups (each n = 6). Cells that were BrdU-positive, but lacked a co-localization of NeuN were classified as ‘‘other’’. There were no significant differences among respective groups in the cellular phenotype (percentages) of BrdU-positive cells (F = 1.35, P = 0.28, ANOVA). Bars represent mean ± S.D.
al. (1999). They found that intervention with a KD reduced the expected frequency of spontaneous recurrent seizures and the degree of supragranular dentate mossy fiber sprouting in the KA-induced seizure model in adult rats. If they had further placed the animals on a regular chow diet after the KD and continued to see fewer spontaneous recurrent seizures, then a stronger argument for an antiepileptogenic effect could be made. Unfortunately, they did not do that in this experimental design. However, a lowering of spontaneous recurrent seizure frequency could represent a retardation of the epileptogenic process. Recently, Bough et al. (2003) reported that the rate of increase in electrographic seizure duration after repeated stimuli was markedly reduced in the KD-fed animals with maximal dentate activation protocol. These results suggest that the KD may be not only anticonvulsant but also antiepileptogenic. However, it is not yet known how the KD exerts antiepileptogenic action. In the present study, we found that neurogenesis after KA-induced seizures was significantly augmented in the KD-fed mice. This finding may provide a clue to explain how the KD exerts its antiepileptogenic action. Further studies, of course, are expected to reveal the relationship between seizure-induced neurogenesis augmented by the KD and its antiepileptogenic properties. Meanwhile, it remains still a mystery in neuroscience how the brain becomes epileptogenic. Sometimes an etiology or structural cause can be determined, but often no explanation is found (Stafstrom, 2004). Temporal lobe epilepsy can be a consequence of structural alterations to the hippocampus, one of the most epilepsy-prone areas of the brain. Hippocampal injury, such as caused by status epilepticus, may produce persistent hyperexcitability long after the end of prolonged seizure. This chronic hyperexcitability may be a result of the combined effects of several structural alterations: neuronal death, gliosis or mesial temporal sclerosis, and supragranular dentate mossy fiber sprouting. To make matters even more complicated, seizures or seizure-induced injury can stimulate DGC neurogenesis in the adult hippocampus (Parent et al., 1998; Scott et al.,
1998). Then, increased neurogenesis can make the brain become epileptogenic or antiepileptogenic by leading to increased or decreased excitability depending on the connectivity and type of newborn neurons. On the contrary, McCabe et al. (2001) reported a reduction in newly born granule cells after recurrent seizures in the neonatal rat between postnatal day 0 and 4. Despite recent vigorous efforts to investigate the role of neurogenesis in epileptogenesis, it has been still poorly understood (Parent, 2002; Scharfman, 2004; Parent et al., 2006). Most studies which examined alterations of neurogenesis in animal seizure models suggest that increased neurogenesis seems to be a general response to seizures and moreover may contribute to the development of epilepsy (i.e., epileptogenesis). Therefore, intervention to block neurogenesis could be a prudent decision to prevent subsequent seizures (Scharfman, 2004). Recently, our colleagues (Jung et al., 2004) reported that blocking neurogenesis following pilocarpine-induced status epilepticus by cytosine-b-D-arabinofuranoside attenuated spontaneous recurrent seizures. On the contrary, Parent et al. (1999) reported that inhibition of DGC neurogenesis following pilocarpine-induced status epilepticus with brain irradiation did not block seizures. Thus, these may reflect that there are many potential foci after pilocarpine-induced status epilepticus, and that neurogenesis does not explain seizures and epilepsy completely (Scharfman, 2004). Although it may be possible that increased neurogenesis could act as a protective mechanism following seizures, it is only speculation at present. Up to now, we could not find any research evidence suggesting that increased neurogenesis may have the potential for compensatory effects after seizure-induced neuronal injury and thus may be beneficial. Existing evidence, however, suggests that neurogenesis stimulated by brain insults may be beneficial in some contexts or maladaptive in other situations (Parent, 2003). Likewise, increased neurogenesis may have beneficial (antiepileptogenic) as well as harmful (pro-epileptogenic) effects on the subsequent seizure generation. In the present study,
Effects of the ketogenic diet on neurogenesis we found that neurogenesis increased following KA-induced seizures and this increased neurogenesis was significantly augmented by the KD. Because the efficacy of the KD for the treatment of epilepsy is well-documented now, our findings suggest that at least the augmented portion of increased neurogenesis affected by the KD may be beneficial although the major portion of increased neurogenesis affected only by seizures may be harmful. Significant augmentation of seizure-induced neurogenesis in the KD-fed animals found in the present study may contribute to reestablishment of normal neuronal networks, thus may exert antiepileptogenic effects. Although this assumption was not examined in the present study, there is no doubt that it should be verified by elucidating whether the type and connectivity of these increased newborn neurons. Increased neurogenesis following seizures might be triggered by seizure-induced hippocampal cell death. Then, it may also be speculated that the KD increases neurogenesis following seizures via increasing the susceptibility to seizure-induced cell death. This possibility, however, is thought be low because recent studies showed that the KD reduced seizure-induced hippocampal cell death in mice (Noh et al., 2003, 2005b). There are some reports showing a phenotypic shift towards the production of new neurons following seizures (Scott et al., 1998, 2000). For example, Scott et al. (2000) examined the effects of eletroconvulsive shock (ECS) seizures on neurogenesis in the dentate gyrus of the adult rat using BrdU immunohistochemistry to identify newly generated cells. Cells were also labeled for NeuN to identify neurons. One month following eight ECS seizures, ECStreated rats had approximately twice as many BrdU-positive cells as sham-treated controls. Eighty-eight percent of newly generated cells co-labeled with NeuN in ECS-treated subjects, compared to 83% in sham-treated controls. These data suggest that there is a net increase in neurogenesis within the hippocampal dentate gyrus following ECS treatment. However, our study showed no significant phenotypic differences of newly generated cells following seizures. In summary, we found for the first time that the KD increased neurogenesis after KA-induced seizures in the male ICR mice. This finding supports our suggestion that the KD enhances seizure-induced neurogenesis. This increased seizure-induced neurogenesis by the KD may be a structural substrate for its antiepileptogenic properties. This assumption, of course, should be verified through further studies. Although there remain still unanswered questions about the relationship between the increased neurogenesis and the antiepileptogenic properties of the KD, we suggest that the KD may exert its beneficial effects on epilepsy, at least partially, by enhancing neurogenesis after seizures.
Acknowledgement This work was supported by Korea Research Foundation Grant (KRF-2004-002-E00088).
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194 D28k in the hippocampi of male ICR mice with kainic acid seizures. Epilepsy Res. 65, 153—159. Noh, H.S., Kim, D.W., Cho, G.J., Choi, W.S., Kang, S.S., 2006a. Increased nitric oxide caused by the ketogenic diet reduces the onset time of kainic acid-induced seizures in ICR mice. Brain Res. 1075, 193—200. Noh, H.S., Kim, Y.S., Kim, Y.H., Han, J.Y., Park, C.H., Kang, A.K., Shin, H.S., Kang, S.S., Cho, G.J., Choi, W.S., 2006b. Ketogenic diet protects the hippocampus from kainic acid toxicity by inhibiting the dissociation of bad from 14-3-3. J. Neurosci. Res. 84, 1829—1836. Parent, J.M., Lowenstein, D.H., 1997. Mossy fiber reorganization in the epileptic hippocampus. Curr. Opin. Neurol. 10, 103—109. Parent, J.M., Janumpalli, S., McNamara, J.O., Lowenstein, D.H., 1998. Increased dentate granule cell neurogenesis following amygdala kindling in the adult rat. Neurosci. Lett. 247, 9—12. Parent, J.M., Tada, E., Fike, J.R., Lowenstein, D.H., 1999. Inhibition of dentate granule cell neurogenesis with brain irradiation does not prevent seizure-induced mossy fiber synaptic reorganization in the rat. J. Neurosci. 19, 4508—4519. Parent, J.M., 2002. The role of seizure-induced neurogenesis in epileptogenesis and brain repair. Epilepsy Res. 50, 179—189. Parent, J.M., 2003. Injury-induced neurogenesis in the adult mammalian brain. Neuroscientist 9, 261—272. Parent, J.M., Elliott, R.C., Pleasure, S.J., Barbaro, N.M., Lowenstein, D.H., 2006. Aberrant seizure-induced neurogenesis in experimental temporal lobe epilepsy. Ann. Neurol. 59, 81—91. Pencea, V., Bingaman, K.D., Wiegand, S.J., Luskin, M.B., 2001. Infusion of brain-derived neurotrophic factor into the lateral ventricle of the adult rat leads to new neurons in the parenchyma of the striatum, septum, thalamus, and hypothalamus. J. Neurosci. 21, 6706—6717. Rho, J.M., Kim, D.W., Robbins, C.A., Anderson, G.D., Schwartzkroin, P.A., 1999. Age-dependent differences in flurothyl seizure sen-
Y.S. Kwon et al. sitivity in mice treated with a ketogenic diet. Epilepsy Res. 37, 233—240. Rho, J.M., 2004. The neuroprotective and antiepileptogenic effects of the ketogenic diet. In: Stafstrom, C.E., Rho, J.M. (Eds.), Epilepsy and the Ketogenic Diet. Humana Press Inc., Totowa, NJ, pp. 289—301. Scharfman, H.E., 2004. Functional implications of seizure-induced neurogenesis. Adv. Exp. Med. Biol. 548, 192—212. Schwartzkroin, P.A., 1999. Mechanisms underlying the anti-epileptic efficacy of the ketogenic diet. Epilepsy Res. 37, 171—180. Scott, B.W., Wang, S., Burnham, W.M., De Boni, U., Wojtowicz, J.M., 1998. Kindling-induced neurogenesis in the dentate gyrus of the rat. Neurosci. Lett. 248, 73—76. Scott, B.W., Wojtowicz, J.M., Burnham, W.M., 2000. Neurogenesis in the dentate gyrus of the rat following electroconvulsive shock seizures. Exp. Neurol. 165, 231—236. Sperk, G., 1994. Kainic acid seizures in the rat. Prog. Neurobiol. 42, 1—32. Stafstrom, C.E., 1999. Animal models of the ketogenic diet: what have we learned, what can we learn? Epilepsy Res. 37, 241— 259. Stafstrom, C.E., 2004. An introduction to seizures and epilepsy: cellular mechanisms underlying classification and treatment. In: Stafstrom, C.E., Rho, J.M. (Eds.), Epilepsy and the Ketogenic Diet. Humana Press Inc., Totowa, NJ, pp. 3—29. Swink, T.D., Vining, E.P., Freeman, J.M., 1997. The ketogenic diet: 1997. Adv. Pediatr. 44, 297—329. Vining, E.P., Freeman, J.M., Ballaban-Gil, K., Camfield, C.S., Camfield, P.R., Holmes, G.L., Shinnar, S., Shuman, R., Trevathan, E., Wheless, J.W., 1998. A multicenter study of the efficacy of the ketogenic diet. Arch. Neurol. 55, 1433—1437. Wheless, J.W., 2004. History and origin of the ketogenic diet. In: Stafstrom, C.E., Rho, J.M. (Eds.), Epilepsy and the Ketogenic Diet. Humana Press Inc., Totowa, NJ, pp. 31—50.
journal homepage: www.elsevier.com/locate/epilepsyres
Effects of the ketogenic diet on neurogenesis after kainic acid-induced seizures in mice Young Se Kwon a,1, Sang-Wuk Jeong b,1, Dong Wook Kim c,∗, Eun Sil Choi c, Byong Kwan Son a a
Department of Pediatrics, College of Medicine, Inha University, Incheon, Republic of Korea Department of Neurology, Dongguk University Hospital, Goyang, Republic of Korea c Department of Pediatrics, Clinical Research Center, FIRST Mitochondrial Research Group, Ilsan Paik Hospital, Inje University College of Medicine, Goyang, Republic of Korea b
Received 5 February 2007; received in revised form 23 November 2007; accepted 29 November 2007
KEYWORDS Ketogenic diet; Neurogenesis; Epileptogenesis; Seizure; Kainic acid; Mice
Summary The ketogenic diet (KD) remains a therapy in search of explanation although it is an established treatment of intractable epilepsy. Recent studies suggest that the KD may be both anticonvulsant and antiepileptogenic. Epileptic seizures have been shown to stimulate the proliferation rate of neuronal progenitor cells in adult animals, which may be related to epileptogenesis. It is known that calorie restriction (CR) increases neurogenesis. The KD was originally formulated to reproduce the biochemical changes seen upon fasting (extreme CR). Thus, we investigated the effects of the KD on neurogenesis after kainic acid (KA)-induced seizures in mice. In the present study, quantitative analysis of BrdU labeling revealed a significant increase in the proliferation rate of neuronal progenitor cells after KA-induced seizures in the KD-fed mice. This finding may provide a clue to explain how the KD exerts antiepileptogenic effects although further studies are mandatory to elucidate the relationship between seizureinduced neurogenesis augmented by the KD and its antiepileptogenic properties. In conclusion, our results suggest that the KD enhances neurogenesis, which may be related to its beneficial effects on epilepsy. © 2007 Elsevier B.V. All rights reserved.
1. Introduction ∗
Corresponding author. Tel.: +82 31 910 7106; fax: +82 31 910 7108. E-mail address: [email protected] (D.W. Kim). 1 They contributed equally and should be considered as first authors.
The ketogenic diet (KD) is a high-fat, low-carbohydrate, adequate-protein diet that has been used for more than 80 years for the treatment of medically intractable epilepsy. The clinical efficacy of the KD has now been well doc-
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Effects of the ketogenic diet on neurogenesis umented (Hemingway et al., 2001; Kim et al., 2004). However, little is known about its underlying mechanisms. Numerous animal studies also have demonstrated the anticonvulsant effects of the KD (Bough et al., 2002; Noh et al., 2003). Furthermore, the KD may possess not only anticonvulsant effects but also antiepileptogenic properties, i.e., effects that may ameliorate or even cure the disease process itself, because clinical data suggest that a period of time on the KD may permanently decrease seizure activity or cause its cessation entirely (Freeman et al., 1998; Vining et al., 1998; Hemingway et al., 2001; Rho, 2004). Results obtained from the animal studies also have suggested the antiepileptogenic properties of the KD (Muller-Schwarze et al., 1999; Bough et al., 2003). Epileptogenesis refers to the events by which the normal brain becomes capable of producing epileptic seizures, i.e., the process by which neural circuits are converted from normal excitability to chronic hyperexcitability (Stafstrom, 2004). This chronic hyperexcitability may result from the combined effects of several structural alterations such as neuronal death, gliosis or mesial temporal sclerosis, and the growth of new aberrant axonal connections. Furthermore, epileptic seizures can induce neurogenesis, i.e., the birth of new neurons, in adult animals. It is especially prominent in the hippocampal dentate gyrus (Parent et al., 1998; Scott et al., 1998). Again, it can give rise to increased or decreased excitability depending on the connectivity and type of newborn neurons. Recently, many investigators have made efforts to shed light on the role of neurogenesis in epileptogenesis (Parent, 2002; Scharfman, 2004; Parent et al., 2006). Evidence accumulated over the past four decades or more has dispelled the long-held dogma that the adult mammalian brain cannot generate new neurons (Parent, 2003). Among the principal neuronal populations within the hippocampus, the dentate granule cells (DGCs) have the unusual property of prolonged postnatal neurogenesis (Eckenhoff and Rakic, 1988) that persists into adulthood in the rodent (Kuhn et al., 1996). DGC neurogenesis during postnatal life appears to be influenced, at least in part, by factors such as aging, stress, exercise, environmental enrichment, and genetic background (Parent, 2003). Furthermore, recent research demonstrated that calorie restriction (CR) enhanced neurogenesis in the brain (Lee et al., 2000, 2002). The KD was originally formulated to mimic the effects of fasting (an extreme form of CR), as it had been known since biblical times that fasting had a beneficial effect on epilepsy (Swink et al., 1997; Wheless, 2004). It has been assumed ever since that the fasting and the KD share a common mechanism in alleviating seizures, although this assumption has not been tested rigorously (Stafstrom, 1999). The KD mimics fasting that induces ketosis and then relieves seizures (Swink et al., 1997; Schwartzkroin, 1999). Thus, we can hypothesize that the KD may enhance neurogenesis similar to the effects of CR. In the present study, we investigated the effects of the KD on neurogenesis after kainic acid (KA)-induced seizures in mice, which may be related to its antiepileptogenic properties.
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2. Methods 2.1. Animals and diet regime Normal male Institute for Cancer Research (ICR) mice (Folas International, Korea) were used for all the experiments. Animals were housed, 4 per cage, in a room maintained at 22 ± 3 ◦ C with an alternating 12-h light/dark cycle. Mice were divided into four groups: (1) seizure-free normal diet (ND) group (n = 11), (2) seizure-free KD group (n = 11), (3) KA-seizure ND group (n = 14), and (4) KA-seizure KD group (n = 14). For 4 weeks, beginning on postnatal day 21, mice of the KD groups were fed the Harlan Teklad TD 96355 (USA), in which most of fat source is polyunsaturated fatty acids, while ND groups were fed a standard rodent chow (Samtako, Korea). The KD composition, which we used for this study, was reported previously (Rho et al., 1999). All mice were fed ad libitum and were weighed at least twice weekly. All procedures were performed in accordance with the Guide for Care and Use of Laboratory Animals published by the National Institutes of Health.
2.2. Blood BHB level After the 4 weeks of dietary treatment, blood -hydroxybutyrate (BHB) levels were measured before seizure induction from blood obtained via tail vein using Keto-SiteTM test kit (GDS Diagnostics, USA).
2.3. Seizure induction After the 4 weeks of dietary treatment, seizures were chemically induced by a single intraperitoneal (i.p.) injection of KA (Sigma, dissolved in physiological saline) with a dose of 30 mg/kg in the KAseizure groups. Animals in the seizure-free groups were injected with equal volume of physiological saline. Mice were monitored for seizure behavior for 2 h after KA injection, and seizures were categorized into five different grades according to a previously defined scale (Hu et al., 1998)—–Grade 0: no response; Grade I: front or hind limb pawing or staring; Grade II: rearing, nodding or bilateral pawing; Grade III: jumping, wobbling or falling; Grade IV: status epilepticus or death. All mice were graded according to the maximal behavioral response observed within 2 h after KA administration.
2.4. BrdU labeling The thymidine analog 5-bromodeoxyuridine (BrdU, Sigma, was dissolved in saline) was administered i.p. with a dose of 50 mg/kg per injection for BrdU labeling. Animals received BrdU injection once a day for 6 consecutive days, starting at 24 h after KA or saline administration. Twenty-three mice were sacrificed 24 h after the last BrdU injection, which allowed us to measure the number of cells that had incorporated BrdU, i.e., the proliferation rate of neuronal progenitor cells. For the double fluorescent immunohistochemistry experiment, 24 mice were allowed to survive for 28 days after the last injection of BrdU. This allowed us to investigate the phenotype, survival, and migration pattern of newborn cells. All the mice in these experiment were allowed to be fed either ND or KD until sacrifice.
2.5. Tissue fixation, immunohistochemistry and quantification Animals were anesthetized with 1% ketamine (30 mg/kg, Ketara; Yuhan Yanghang, Seoul, Korea) and xylazine hydrochloride (4 mg/kg, Rompun; Bayer Korea, Seoul, Korea) at the corresponding time after the BrdU injection, and then perfused through the heart with 50 ml cold saline and 50 ml of 4% paraformaldehyde (PFA) in 0.1 mol/l
188 phosphate-buffered saline (PBS). After 24 h of fixation in 4% PFA, the brains were cryoprotected with PBS containing 30% sucrose for 24 h. Coronal sections through the entire hippocampus were cut at 30 m on a freezing microtome and sections were stored in PBS (pH 7.4). Tissue sections were incubated in 2M HCl for 30 min in order to denature the DNA into single-stranded DNA at room temperature (the BrdU antibody recognizes only single-stranded DNA). Tissue sections were then rinsed three times for 10 min each in PBS, and treated with 3% H2 O2 (in 30% methanol) to block endogenous peroxidase. After they were washed for 30 min each in PBS, free-floating sections were incubated in PBS containing 10% normal goat serum and 0.5% BSA/0.3% Triton X-100 for 1 h. Section were then incubated with a mouse monoclonal anti-BrdU antibody (1:300, Pharmigen, USA) at 4 ◦ C for 24 h. The antibody solutions also contained goat serum and BSA/Triton X-100. On the next day, after washing all tissue sections three times in PBS, the sections were incubated in biotinylated secondary antibody (EnvisionTM /HRP, rabbit anti-mouse, DAKO, USA) for 2 h at room temperature. After three 10-min washes, sections were reacted for peroxidase enzyme activity by using 3,3 -diaminobenzidine-tetrahydrochloride (DAB, DAKO, USA). The reaction was terminated by transferring the sections to PBS. Sections were mounted on gelatinized slides, dehydrated, and coverslipped. The number of BrdU-positive cells in the combined hippocampal dentate gyrus and hilar regions was counted in every seventh section in a series of 30 m coronal sections (210 m apart) covering the complete right and left hippocampi. Cell counting was performed by an experimenter who was not informed of group assignment. Cells were counted with 40× lens on microscope (Nikon, Japan) equipped with a magnifier digital camera and a computer-assisted image analysis system (Image J, NIH, USA).
2.6. Double fluorescent immunohistochemistry and confocal microscopy In order to identify the BrdU-labeled cells as either neurons or GFAPpositive glia, BrdU fluorescent immunohistochemistry was combined with NeuN (neuron) and GFAP (glia) fluorescent immunohistochemistry in conjunction with confocal microscopy. Coronal free-floating sections (30 m) from the whole brain were used. DNA denaturation was performed as described above. Tissue sections then were rinsed three times for 10 min each in PBS. Free-floating sections were incubated in PBS containing 10% normal rabbit serum and 0.3% Triton X-100 for 1 h. Sections were then incubated with sheep polyclonal anti-BrdU (1:300, Biodesign, USA) antibody at 4 ◦ C for 24 h. On the next day, after washing all tissue sections three times in PBS, the sections were incubated with FITC-conjugated anti-sheep IgG (1:100, Biodesign, USA) secondary antibody for 2 h at room temperature. After three 10-min washes, sections were incubated in PBS containing 10% normal goat serum and 0.5% BSA for 1 h. Sections were then incubated with mouse antiNeuN (1:200, Chemicon, USA) or mouse anti-GFAP (1:1000, Sigma, USA) at 4 ◦ C for 24 h. On the next day, after washing all tissue sections three times in PBS, the sections were incubated with Cy3-conjugated anti-mouse IgG (1:300, Biodesign, USA) secondary antibody for 2 h at room temperature. After washes in PBS, sections were mounted on gelatinized slides in anti-fade medium and coverslipped. Immunofluorescent images were obtained under a confocal laser scanning microscope (Carl Zeiss LSM510) and processed using Adobe Photoshop v7.0 (Adobe Systems, Mountain View, CA, USA). BrdUpositive cells from combined hippocampal dentate gyrus and hilar regions, chosen at random from each hippocampus, were examined for coexpression of BrdU and NeuN (a neuronal phenotype), BrdU and GFAP (a GFAP-positive glial phenotype). We counted 100 BrdU-positive cells per animal to obtain the percentages of coexpression.
Y.S. Kwon et al.
Fig. 1 Influence of the ketogenic diet (KD) on body weight in male ICR mice. During the dietary treatment, mean body weights of the normal diet (ND)-fed and KD-fed mice (each n = 25) showed no significant differences. Bars represent mean ± S.D.
2.7. Statistical analysis All data in this study are presented as mean ± S.D. We used the SPSS 11.0 program. Data were analyzed by unpaired Student’s t-test if they were normally distributed (Kolmogorov-Smirov test, P > 0.05). Numbers of BrdU-positive cells were examined by two-way ANOVA with interaction. Percentages of BrdU-positive cells that were colabeled with Neu-N were examined by ANOVA. A value of P < 0.05 was considered significant.
3. Results 3.1. The KD was well tolerated, increased blood BHB level and seizure threshold To perform a satisfactory study using a KD, the diet should be well tolerated, induce ketosis, and show anticonvulsant
Fig. 2 Blood -hydroxybutyrate (BHB) levels in male ICR mice treated with the ketogenic diet (KD). Bars represent mean ± S.D. The asterisk (*) indicates a significant difference (P < 0.001, Student’s t-test) between the normal diet (ND)-fed mice and the KD-fed mice (each n = 25).
Effects of the ketogenic diet on neurogenesis
189 old. All mice were graded according to the maximal seizure behavior observed for 2 h after KA administration. Every seizure behavior was grade II or more. However, the severity represented as seizure behavior grade showed no significant differences between the KD-fed and ND-fed animals (data not shown) as reported previously (Noh et al., 2003, 2005a,b, 2006a). The mortality of mice following the administration of KA was 10.7%.
3.2. The KD increased neurogenesis after KA-induced seizures
Fig. 3 Influence of the ketogenic diet (KD) on the kainic acid (KA)-induced seizure onset time in male ICR mice. In the KDfed mice (n = 14), the latency to a seizure onset was delayed compared with the normal diet (ND)-fed mice (n = 14). Bars represent mean ± S.D. The asterisk (*) indicates a significant difference (P < 0.01, Student’s t-test) between the ND-fed and the KD-fed mice.
effects. In this study, the KD-fed mice tolerated their diet well for 4 weeks without oily appearance of the fur. The KDfed mice showed no difference in their behavior and health compared with the ND-fed mice. Mice in both groups had a similar weight gain within the time allotted (Fig. 1). We confirmed ketosis induced by the KD, because the blood BHB levels in the KD-fed mice were significantly higher than those of the ND-fed ones (2.52 ± 1.16 mM vs. 0.33 ± 0.13 mM, P < 0.001, Fig. 2). After the systemic administration of KA (30 mg/kg), all mice developed typical seizure behaviors. We found that the KD delays the KA-induced seizure onset time in male ICR mice (Fig. 3). These results were consistent with the previous reports (Noh et al., 2003, 2005a,b, 2006a,b), and indicate that the KD increased KA-induced seizure thresh-
After BrdU labeling, KA-induced seizures caused a marked increase in the number of BrdU-positive cells in the hippocampal dentate gyrus in contrast to the seizure-free animals (Fig. 4). Moreover, BrdU labeling revealed a significant increase in mitotic activity of neuronal progenitor cells in the KA-seizure KD group in comparison with the KA-seizure ND group (Fig. 4C and D). In the ND-fed animals, BrdU-positive cells increased significantly after KA administration compared with saline administration (77.28 ± 19.55 vs. 43.28 ± 12.64, P < 0.05 by ANOVA with interaction). In the KD-fed mice, BrdU-positive cells also increased significantly after KA administration compared with saline administration (111.41 ± 32.12 vs. 42.90 ± 23.94, P < 0.001 by ANOVA with interaction). These findings indicate that KA-induced seizures in mice increased the rate of neurogenesis within the hippocampal dentate gyrus. Measurement of BrdU-positive cells showed no significant differences between the seizure-free ND and KD groups. However, BrdU-positive cells after KA-induced seizures significantly increased in the KD-fed mice compared with the ND-fed ones (111.41 ± 32.12 vs. 77.28 ± 19.55, P < 0.05 by ANOVA with interaction). In summary, quantitative analysis of BrdU labeling after KA-induced seizures revealed a significant increase in the proliferation rate of neuronal progenitor cells in the KD-fed mice compared with the ND-fed ones (Fig. 5). It indicates that the KD increased neurogenesis after KA-induced seizures.
Fig. 4 BrdU-positive cells in the hippocampal dentate gyrus of male ICR mice. Mice were divided into four groups: (1) seizure-free normal diet (ND), (2) seizure-free ketogenic diet (KD), (3) KA-seizure ND, and (4) KA-seizure KD groups. (A) Baseline mitotic activity in the hippocampal dentate gyrus of group (1). (B) No remarkable difference in BrdU-positive cells of group (2) compared with (1). (C) Increased BrdU-positive cells of group (3) compared with (1) and (2). (D) Remarkable increase in BrdU-positive cells of group (4) compared with (1), (2), and (3). Scale bars: 200 m.
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Fig. 5 The number of BrdU-positive cells in the male ICR mice. Mice were divided into four groups: (1) seizure-free normal diet (ND) group (n = 5), (2) seizure-free ketogenic diet (KD) group (n = 5), (3) KA-seizure ND group (n = 6), and (4) KA-seizure KD group (n = 7). Bars represent mean. Although measurement of BrdU-positive cells showed no significant differences between the seizure-free ND and KD groups, BrdU-positive cells after KA-induced seizures significantly increased in the KD-fed mice compared with the ND-fed ones (P < 0.05, ANOVA with interaction).
3.3. The KD showed no difference in the long-term fate of seizure-induced neurogenesis We next examined the long-term fate of BrdU-positive cells in the dentate gyrus after KA-induced seizures in the KD-fed mice. This was done by injecting BrdU once a day for 6 consecutive days after KA or saline administration (i.e., within the period of significantly increased mitosis) and then sacrificing the animals 28 days later. We used markers of neuronal and glial phenotypes to examine the identity of the proliferating cells located in the hippocampal dentate gyrus. Cells that co-localized both BrdU and NeuN were classified as neurons. BrdU-positive cells that co-localized with GFAP were classified as GFAP-positive glia. We found that most of BrdUlabeled cells expressed neuronal marker NeuN (Fig. 6E and G), but the astrocytic marker GFAP was rarely co-localized with BrdU-immunostained nuclei (Fig. 6F and H) in the KAseizure KD group. Similar results have also been observed in other experimental animal groups. The majority of all BrdU-positive cells are of a neuronal phenotype. There were no significant differences among the experimental groups in the cellular phenotype (percentages) of BrdU-positive cells (F = 1.35, P = 0.28 by ANOVA, Fig. 7).
4. Discussion Our major findings in the present study were that (1) the KD-induced ketosis; (2) KA-induced seizures increased neurogenesis; (3) the KD does not alter neurogenesis in seizure-free mice; and (4) the KD increases the enhancement of neurogenesis induced by KA-seizure. Although the efficacy of the KD for the treatment of epilepsy is already documented through many clinical and experimental studies, its underlying mechanisms are largely
Y.S. Kwon et al. unknown. However, it is well acknowledged that ketosis, i.e., the systemic elevation of the ketone bodies (BHB, acetoacetate, and acetone), is necessary for seizure control (Swink et al., 1997; Schwartzkroin, 1999). Thus, in the first place, we confirmed that the KD regime used in the present study was truly ketogenic and had anticonvulsant effects as it increased blood BHB level and KA-induced seizure threshold in the KD-fed mice. This diet regime was used in numerous previous studies (Rho et al., 1999; Noh et al., 2003, 2005a,b, 2006a,b). Our colleagues previously demonstrated that the KD-induced ketosis, delayed the KAinduced seizure onset time, and prevented the hippocampal neuronal death in the male ICR mice (Noh et al., 2003). In the present study, to investigate whether the KD influences seizure-induced neurogenesis, we used the same experimental model. As a matter of course, we reproduced the result that the KD-induced ketosis and delayed the KA-induced seizure onset time in the present study. In animal studies, KA-induced seizure model has been widely used because of several advantages. Systemic administration of KA readily produces various motor signs including convulsive seizures (Sperk, 1994). KA, an agonist for kainate and AMPA receptors, is an excitotoxin in the hippocampus. It induces ongoing seizures, degeneration of cornu ammonis (CA) neurons, and hyperexcitability of surviving CA neurons in the hippocampus. Furthermore, recent studies have demonstrated that KA administration in adult rodents increased the rate of neurogenesis within the hippocampal dentate gyrus (Parent and Lowenstein, 1997; Gray and Sundstrom, 1998). In the present study, we also observed that KA-induced seizures resulted in a significant increase in the proliferation rate of neuronal progenitor cells in the hippocampal dentate gyrus. Although the birth of new neurons, neurogenesis, in mammalian CNS is confined largely to the embryonic period, accumulating evidence indicates that certain germinative zones persist throughout life and continue to generate neurons and glia in specific brain regions, including the hippocampal dentate gyrus (Parent, 2002). The study of this phenomenon offers the opportunity to identify and characterize circumstances that activate neuronal progenitor cells to form new neurons and the conditions required for these cells to mature and establish functional connections. Neurogenesis in the hippocampal dentate gyrus during postnatal life can be modulated by hormones, pharmacological actions, stress, levels of physical activity, and the complexity of the environment (Parent, 2003). Evidence from previous studies suggests that specific growth or neurotrophic factors influence neuronal progenitor cell proliferation in the brain (Benraiss et al., 2001; Pencea et al., 2001). In addition, CR has been shown to enhance hippocampal neurogenesis through recent studies (Lee et al., 2000, 2002). On the other hand, it has been well known that the KD was originally formulated to mimic the beneficial effects of fasting (an extreme form of CR) on epilepsy (Swink et al., 1997; Wheless, 2004). Recent research demonstrates that CR alone can reduce seizure susceptibility in rodents (Greene et al., 2001; Eagles et al., 2003). Thus, we can assume that the KD, as well as CR, may also enhance neurogenesis. In the present study, we tried to demonstrate whether the KD indeed increases neurogenesis in the hippocampal den-
Effects of the ketogenic diet on neurogenesis
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Fig. 6 The neuronal phenotype of proliferating cells in the hippocampal dentate gyrus of a ketogenic diet (KD)-fed mouse after kainic acid (KA)-induced seizure. (A) and (B) BrdU-positive cells (green) in the hippocampal dentate gyrus. (C) Cells immunostained by neuronal marker NeuN (red). (D) Cells immunostained with astrocytic marker GFAP (red). (E) Colocaliztion of BrdU with NeuN shown by yellow nuclei. (F) BrdU rarely colocalized with GFAP. (G) High-power photograph of (E). (H) High-power photograph of (F). Scale bars: (A—F) 200 m; (G) 25 m; (H) 20 m.
tate gyrus. Then, we failed to show that the KD significantly enhanced neurogenesis in the case of seizure-free (salinetreated) groups. However, neurogenesis after KA-induced seizures increased significantly in the KD-fed mice in comparison with the ND-fed ones. These results suggest that the KD does not increase neurogenesis in the seizure-free mice but it augments seizure-induced neurogenesis significantly. From our results, it remains a question to be answered why there is difference of the effect between the basal and the seizure-induced neurogenesis. Since our assumption was that the KD and CR operate with a similar mechanism and both would show increased neurogenesis, our results also contradict that assumption. Perhaps the mechanisms are different. Another remaining question is how the KD augments seizure-induced neurogenesis. Further researches into these questions are clearly needed. In recent years, there appeared interesting reports suggesting that the KD may have antiepileptogenic effects as well as anticonvulsant properties (Hemingway et al., 2001; Bough et al., 2003; Rho, 2004). The most persuasive clin-
ical data suggesting a potential antiepileptogenic effect were presented in 2001 by Hemingway et al. (2001). They investigated the long-term outcome of 150 children with intractable epilepsy who were enrolled prospectively in a KD trial. One year after starting the diet, 7% had become seizure free, and 20% had a greater than 90% reduction in seizure frequency; interestingly, 3—6 years after beginning the KD, a greater percentage of patients (13%) became seizure free. Intriguing as such observations may be, it is difficult to determine whether patients who remain seizure free after cessation of the KD (following full seizure control for at least 2 years) have had a spontaneous remission of their epilepsy, have had amelioration due to lack of damaging repetitive seizure activity, or have experienced a true direct antiepileptogenic effect of the KD (Rho, 2004). Despite the limitations of clinical speculation, results obtained from the animal studies have raised the possibility that the KD may possess antiepileptogenic properties (Rho, 2004). The first report that the KD may impede epileptogenesis in an animal model was provided by Muller-Schwarze et
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Fig. 7 Alterations in the percentages of BrdU-positive cells that were neurons (NeuN) or ‘‘other’’ cells in the male ICR mice. Mice were divided into four groups: (1) seizure-free normal diet (ND), (2) seizure-free ketogenic diet (KD), (3) KA-seizure ND, and (4) KA-seizure KD groups (each n = 6). Cells that were BrdU-positive, but lacked a co-localization of NeuN were classified as ‘‘other’’. There were no significant differences among respective groups in the cellular phenotype (percentages) of BrdU-positive cells (F = 1.35, P = 0.28, ANOVA). Bars represent mean ± S.D.
al. (1999). They found that intervention with a KD reduced the expected frequency of spontaneous recurrent seizures and the degree of supragranular dentate mossy fiber sprouting in the KA-induced seizure model in adult rats. If they had further placed the animals on a regular chow diet after the KD and continued to see fewer spontaneous recurrent seizures, then a stronger argument for an antiepileptogenic effect could be made. Unfortunately, they did not do that in this experimental design. However, a lowering of spontaneous recurrent seizure frequency could represent a retardation of the epileptogenic process. Recently, Bough et al. (2003) reported that the rate of increase in electrographic seizure duration after repeated stimuli was markedly reduced in the KD-fed animals with maximal dentate activation protocol. These results suggest that the KD may be not only anticonvulsant but also antiepileptogenic. However, it is not yet known how the KD exerts antiepileptogenic action. In the present study, we found that neurogenesis after KA-induced seizures was significantly augmented in the KD-fed mice. This finding may provide a clue to explain how the KD exerts its antiepileptogenic action. Further studies, of course, are expected to reveal the relationship between seizure-induced neurogenesis augmented by the KD and its antiepileptogenic properties. Meanwhile, it remains still a mystery in neuroscience how the brain becomes epileptogenic. Sometimes an etiology or structural cause can be determined, but often no explanation is found (Stafstrom, 2004). Temporal lobe epilepsy can be a consequence of structural alterations to the hippocampus, one of the most epilepsy-prone areas of the brain. Hippocampal injury, such as caused by status epilepticus, may produce persistent hyperexcitability long after the end of prolonged seizure. This chronic hyperexcitability may be a result of the combined effects of several structural alterations: neuronal death, gliosis or mesial temporal sclerosis, and supragranular dentate mossy fiber sprouting. To make matters even more complicated, seizures or seizure-induced injury can stimulate DGC neurogenesis in the adult hippocampus (Parent et al., 1998; Scott et al.,
1998). Then, increased neurogenesis can make the brain become epileptogenic or antiepileptogenic by leading to increased or decreased excitability depending on the connectivity and type of newborn neurons. On the contrary, McCabe et al. (2001) reported a reduction in newly born granule cells after recurrent seizures in the neonatal rat between postnatal day 0 and 4. Despite recent vigorous efforts to investigate the role of neurogenesis in epileptogenesis, it has been still poorly understood (Parent, 2002; Scharfman, 2004; Parent et al., 2006). Most studies which examined alterations of neurogenesis in animal seizure models suggest that increased neurogenesis seems to be a general response to seizures and moreover may contribute to the development of epilepsy (i.e., epileptogenesis). Therefore, intervention to block neurogenesis could be a prudent decision to prevent subsequent seizures (Scharfman, 2004). Recently, our colleagues (Jung et al., 2004) reported that blocking neurogenesis following pilocarpine-induced status epilepticus by cytosine-b-D-arabinofuranoside attenuated spontaneous recurrent seizures. On the contrary, Parent et al. (1999) reported that inhibition of DGC neurogenesis following pilocarpine-induced status epilepticus with brain irradiation did not block seizures. Thus, these may reflect that there are many potential foci after pilocarpine-induced status epilepticus, and that neurogenesis does not explain seizures and epilepsy completely (Scharfman, 2004). Although it may be possible that increased neurogenesis could act as a protective mechanism following seizures, it is only speculation at present. Up to now, we could not find any research evidence suggesting that increased neurogenesis may have the potential for compensatory effects after seizure-induced neuronal injury and thus may be beneficial. Existing evidence, however, suggests that neurogenesis stimulated by brain insults may be beneficial in some contexts or maladaptive in other situations (Parent, 2003). Likewise, increased neurogenesis may have beneficial (antiepileptogenic) as well as harmful (pro-epileptogenic) effects on the subsequent seizure generation. In the present study,
Effects of the ketogenic diet on neurogenesis we found that neurogenesis increased following KA-induced seizures and this increased neurogenesis was significantly augmented by the KD. Because the efficacy of the KD for the treatment of epilepsy is well-documented now, our findings suggest that at least the augmented portion of increased neurogenesis affected by the KD may be beneficial although the major portion of increased neurogenesis affected only by seizures may be harmful. Significant augmentation of seizure-induced neurogenesis in the KD-fed animals found in the present study may contribute to reestablishment of normal neuronal networks, thus may exert antiepileptogenic effects. Although this assumption was not examined in the present study, there is no doubt that it should be verified by elucidating whether the type and connectivity of these increased newborn neurons. Increased neurogenesis following seizures might be triggered by seizure-induced hippocampal cell death. Then, it may also be speculated that the KD increases neurogenesis following seizures via increasing the susceptibility to seizure-induced cell death. This possibility, however, is thought be low because recent studies showed that the KD reduced seizure-induced hippocampal cell death in mice (Noh et al., 2003, 2005b). There are some reports showing a phenotypic shift towards the production of new neurons following seizures (Scott et al., 1998, 2000). For example, Scott et al. (2000) examined the effects of eletroconvulsive shock (ECS) seizures on neurogenesis in the dentate gyrus of the adult rat using BrdU immunohistochemistry to identify newly generated cells. Cells were also labeled for NeuN to identify neurons. One month following eight ECS seizures, ECStreated rats had approximately twice as many BrdU-positive cells as sham-treated controls. Eighty-eight percent of newly generated cells co-labeled with NeuN in ECS-treated subjects, compared to 83% in sham-treated controls. These data suggest that there is a net increase in neurogenesis within the hippocampal dentate gyrus following ECS treatment. However, our study showed no significant phenotypic differences of newly generated cells following seizures. In summary, we found for the first time that the KD increased neurogenesis after KA-induced seizures in the male ICR mice. This finding supports our suggestion that the KD enhances seizure-induced neurogenesis. This increased seizure-induced neurogenesis by the KD may be a structural substrate for its antiepileptogenic properties. This assumption, of course, should be verified through further studies. Although there remain still unanswered questions about the relationship between the increased neurogenesis and the antiepileptogenic properties of the KD, we suggest that the KD may exert its beneficial effects on epilepsy, at least partially, by enhancing neurogenesis after seizures.
Acknowledgement This work was supported by Korea Research Foundation Grant (KRF-2004-002-E00088).
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