Effect of ketogenic diet on hippocampus mossy fiber sprouting and GluR5 expression in kainic acid induced rat model

Ketogenic diet (KD) is a high fat, low protein, low carbohydrate diet. Its antiepileptic effect is certain but the underlying mechanism is unknown.1 Mossy fiber sprouting in the inner molecular layer of the dentate gyrus causes the synaptic reorganization in the hippocampus, which is an important cause of temporal lobe epilepsy in animals and humans.1,2 It is also essential to the genesis and development of epilepsy. As the predominant excitatory neurotransmitter in the central nervous system, glutamate plays a role in synaptic reorganization and development of epilepsy. In recent years, the role of glutamate receptor 5 (GluR5) in the genesis of seizures has attracted more and more attention of researchers and this receptor has become a candidate target of new antiepileptic drugs.3,4 In this study, we investigated the possible antiepileptic mechanism of KD in terms of synaptic reorganization and GluR5 expression, attempting to provide a theoretical basis for the development of new antiepileptic drugs. METHODS KA-induced epileptic model Male Sprague-Dawley rats obtained from the Experimental Animal Center of Shandong University were divided into four groups: kainic acid (KA) + normal diet (ND), KA + KD, saline (NS) + ND, and NS + KD. On day 28 after birth, the rats of the experimental groups received KA (10 mg/kg, i.h., Sigma, USA), and the saline control groups received an equivalent volume of saline. All rats were fasted for 24 hours, after which on day 30 they had free access to their respective diet. The experimental KD resembled the classic 4:1 diet used clinically (fat 70%, protein 20%, and carbohydrates 0%).5 The diet was given for the next 8 weeks. At the end of the experiment, the number of animals in each group was 13 (KA + ND group), 12 (KA + KD group), 4 (NS + ND group), and 4 (NS + KD group). Behavior observation During the 8-week dietary treatment, spontaneous recurrent seizure (SRS) was recorded by observing each KA-induced animal for 1 hour everyday. SRS was defined as an unprovoked seizure involving unilateral or bilateral forelimb clonus, with or without loss of posture. Material extraction and brain slice preparation At the end of the experiment, 3 ml blood was withdrawn from the heart of the animal after deep anesthesia. β-hydroxybutyrate (β-OHB) level was assessed with a commercially available kit (Randox, UK). After blood extraction, brains were removed from some rats, and bilateral hippocampus was immediately dissected, put in liquid nitrogen, then stored for later use in a -80°C freezer. The other rats were perfused transcardially with 200 ml sodium sulfide medium (2.925 g Na2S, 2.975 g NaH2PO4/H2O in 500 ml H2O) followed by 200 ml 4% phosphate buffered paraformaldehyde (PFA, 0.1 mol/L, pH 7.4). The brains were postfixed in 4% PFA overnight and then placed in a 30% sucrose phosphate buffer until they sank to the bottom of the vial. The fourth coronal section of 25 μm was used for Timm staining and the adjacent section was for Nissl staining. Timm staining and Nissl staining Sections were developed in the dark for 60 minutes in a 12:6:2 mixture of 50% gum arabic, 5.6% hydroquinone, and citric acid-sodium citrate buffer with 1.5 ml 17% AgNO3 solution. Toluidin blue was used for Nissl staining. All images were processed with Image-pro plus 5.0 software. For Timm stained sections, the mean density (A) of Timm granules in the inner molecular layer (IML) of the dentate gyrus and Timm granules in the stratum pyramidal and stratum orient of the CA3 region was calculated. For Nissl stained sections, A of neurons in the hilus, CA3 and CA1 regions was analyzed. Five brain slices of the bilateral hippocampus for each rat were examined. Protein extracts and Western blot analysis One side of the hippocampus was homogenized. The cell lysates were cleansed by a centrifugation at 15 000 r/min for 20 minutes at 4°C, and protein concentration was determined by BCA assay (Pierce, Rockford, USA). For immunoblots, 40 μl of protein extract was loaded on a 10% SDS-PAGE and transferred onto nitrocellulose membranes. Blots were detected with 1:50 primary antibody against GluR5, and specific signals were detected with 1:1000 horseradish peroxidase secondary antibodies by a chemiluminescence reaction (Santa Cruz, California, USA). RNA preparation and RT-PCR The other side of the hippocampus was used for RNA isolation by the guanidinium thiocyanatephenol-chloroform method. The RT-PCR procedure was performed according to the manufacturer's recommendation (RT-PCR Kit, Fermentas Life Science, Ontario, Canada). The primers used in this study were as follows: GluR5: 5′-GGTATAACCCCC ACCCATGCAACC-3′ (forward), 5′-GAAGGTCATCG TCGAGCCATCTCTG-3′ (Reverse); β-actin: 5′-AAGA TCCTGACCGAGCGTGG-3′ (Forward), 5′-CAGCAC TGTGTTGGCATAGAGG-3′ (Reverse). PCR procedures (50 μl) contained 30 cycles, with each cycle of 40 seconds at 94°C, 40 seconds at 56°C, and 40 seconds at 72°C. The products were GluR5 313 bp, and β-actin 326 bp. Statistical analysis Differences between means of the two groups were compared by Student's t/t' test. The comparison of GluR5 mRNA and protein used Wilcoxon's rank-sum test. Significance level was 0.05 for all comparisons. RESULTS Behavior observation Three weeks after KA injection, KA-induced rats started SRS in succession. Eleven rats in the KA + ND group experienced SRS, while eight rats in the KA + KD group had SRS. SRS frequency per rat was significantly less in the KD-fed animals (1.40±1.03) than in the controls (7.36±3.75, t' =5.024, P<0.05). No SRS was observed in saline injected animals. β-OHB determination In KD-fed (KA + KD and NS + KD groups) rats, β-OHB rose to the level [(4.51 ± 2.32) mmol/L] significantly higher than that in standard diet-fed (KA + ND and NS + ND groups) rats [(0.11 ± 0.03) mmol/L, t=6.83, P<0.05]. Histology Timm staining reflects mossy fiber sprouting into the dentate IML. The A of Timm granules in the IML of the dentate gyrus in the KA-induced groups (KA + KD and KA + ND) was respectively higher than that in the saline control groups (NS + KD and NS + ND) (P<0.01, Fig. 1A-1D, Table 1), but no significant difference was found between the KD-fed groups (KA + KD and NS + KD) and the normal diet control groups (KA + ND and NS + ND) (Table 1). There was no statistically significant difference in Timm staining of the CA3 region between the groups (t values were 0.35 and 0.08, both P>0.05, Fig. 1E). Granular cells and pyramidal cells were densely arranged in the dentate and CA3, CA1 regions, as demonstrated in toluidin blue stained sections. There was no significant difference between gross neuron damages in subfields of the CA3, CA1, and hilus (Fig. 1F).Fig. 1.: Examples of Timm staining and Nissl staining in the hippocampus. A and B: Mossy fiber sprouting in IML of the dentate gyrus of rats from the KA + ND group. C and D: Timm-stained sections reveal a normal pattern of staining in the NS + ND group. E: The distribution of Timm granules in the stratum pyramidale and stratum oriens of the CA3 region of rats from the KA + KD group. F: The distribution of neurons in the CA3 region of rats from the KA + ND group. The magnification of A, C, E, and F is 100 and that of B and D is 200.Table 1: Comparison of Timm stained granular density in IML of dentate gyrusGluR5 mRNA and its expression The results of Western blot indicated that GluR5 expression in KA+ KD rats was significantly higher than that in KA +ND rats (Fig. 2, Table 2), although there was no significant difference in GluR5 mRNA between the two groups, shown by RT-PCR (Fig. 3, Table 2).Fig. 2.: Western blot detection of GluR5.Table 2: Hippocampus GluR5 mRNA and its protein in KA-induced different diet fed groupsFig. 3.: RT-PCR detection of GluR5 mRNA. Lanes 1 and 4: GluR5; Lanes 3 and 6: β-actin.DISCUSSION In recent years, a number of new antiepileptic drugs have been used in the treatment of epilepsy, but about 25% -30% patients are intractable to conventional medications and can be considered to have refractory epilepsy. Because of its special effect on this group of patients, KD has become popular since the 1970s of the last century.6 KD action is age-dependent since several studies have shown that KD is more efficient in fetuses, infants and children than in adults.7 In this study, KA-induced on day 28, young rats were used and results showed KD-fed rats had significantly fewer SRS than did standard diet-fed rats, indicating the definite antiepileptic effect of KD on KA-induced animals. MFS refers to the synaptic reorganization of the mossy-fiber axons of dentate granule cells into the inner molecular layer of the dentate gyrus of the hippocampus.8 The formation of recurrent excitatory synapses between dentate granule cells, as is thought to occur after MFS, may transform the dentate granule cells into an epileptogenic population of neurons and could promote seizure initiation.9 Muller-Schwarze et al1 found in KA-induced adult rats, KD could decrease the frequency of SRS and prevent MFS in the dentate gyrus, suggesting that KD might play an antiepileptogenic role by suppressing MFS. Although in our study, MFS in the dentate gyrus was different between KA-induced and saline control groups, no significant difference was found in KD-fed groups and standard diet-fed groups. Both MFS in the CA3 region and neuron damage in different regions of the hippocampus were similar in different groups. These data were in accord with the results reported by Zhao,10 which were drawn from experiments of pilocarpine-induced animals on day 20. Therefore, MFS may be responsible for epileptogensis in KA-induced rats, but for young rats, impeding MFS is not the antiepileptic mechanism of KD. The difference in antiepileptic mechanism between young and adult rats may be due to the characterization of the developing brain. Thus, we propose that for young rats, KD's antiepileptic effect may be age-dependent and related to neurogenesis and synaptic plasticity. Glutamate plays a crucial role in the ontogeny of the nervous system and synaptic formation. As a type of ionotropic glutamate receptor, KA receptors can be divided into five types of KA1–2 and GluR5–7, and they modulate synaptic transmission by pre- and post-synaptic mechanism.11 It was reported that in KA induced adult rats, GluR5 mRNA and protein increased significantly at 72 hours and 180 days.12 The study on temporal lobe epilepsy patients found decreased GluR5 mRNA densities per pyramid cell and increased GluR5 hybridization densities per granule cell in patients with hippocampus sclerosis (HS). Mathern et al13 hypothesized that the former change was a consequence of seizures and the latter was in association with MFS and/or hippocampal neuronal loss. The results of our study showed increased GluR5 expression in KA induced KD-fed rats although no difference was found of GluR5 mRNA in the hippocampus. The different expression of GluR5 and its mRNA in the hippocampus may be due to their different expression phases and different factors regulating the transcription and translation processes. In addition, the different expression may also be related to the neuron protection effect of KD. According to Noh's findings,14 KD has an antiepileptic effect via a neuroprotective action involving the inhibition of caspase-3-mediated apoptosis of hippocampal neurons. Despite no significant neuron damage of the hippocampus found between different diet-fed groups may result from neurogenesis after repetitive seizures or KD's neuroprotective effect, further investigation is required to clarify whether there are differences in GluR5 expression between neurons from neurogenesis and those protected from seizure through KD. GluR5 mRNA is confined predominantly to hippocampal interneurons.15 In GluR6-deficient animals, currents mediated by kainate receptors in CA1 interneurons are not eliminated, supporting the role of GluR5 in interneuronal kainate receptors.16 Our findings indicate that KD can not inhibit MFS in KA-induced young rats. The high expression of GluR5 does not result from MFS, otherwise it might be the reason for decreased SRS in KD-fed animals since the activation of GluR5 on GABAergic interneurons can lead to an increased inhibition in the hippocampus to prevent the propagation of seizure from one hemisphere to the other.17,18 In conclusion, in young rats, KD may increase GluR5 expression in interneurons in the CA1 region as well as the inhibitory transmission in the hippocampus to play its antiepileptic action. Acknowledgement: This study was accomplished in Cardiovascular Reorganization and Function Key Lab in Shandong University of the Ministry of Education. We deeply appreciate LIU Chun-xi, JIANG Hong and FENG Jin-bo for their technical assistance.

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