The effect of ketogenic diet on dentate gyrus and CA3 KCC2 expression in male rats with electrical amygdala kinding-induced seizures

Leticia Granados-Rojas ^1* Leonardo Hernández-López ² Emmanuel Leonardo Bahena-Alvarez ¹ Tarsila Elizabeth Juárez-Zepeda ¹ Verónica Custodio ² Joyce Graciela Martìnez-Galindo ³ Karina Jerónimo-Cruz ⁴ Miguel Tapia-Rodríguez ⁵ America Vanoye-Carlo ⁶ Pilar Duran ⁷ Carmen Rubio ^2*

• ¹ Laboratorio de Biomoléculas y Salud Infantil, México City, Mexico
• ² Departamento de Neurofisiología, Instituto Nacional de Neurología y Neurocirugía, México City, Mexico
• ³ Laboratorio de Demencias, Instituto Nacional de Neurología y Neurocirugía, México city, Mexico
• ⁴ Laboratorio de Biomoléculas, Instituto Nacional de Pediatría, México City, Mexico
• ⁵ Unidad de Microscopía, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, México city, Mexico
• ⁶ Laboratorio de Oncología Experimental, México City, Mexico
• ⁷ Laboratorio de Biología Animal Experimental, Facultad de Ciencias, Universidad Nacional Autónoma de México, México City, Mexico

Introduction: Ketogenic diet (KD), a high-fat, low-carbohydrate, and adequate-protein diet, is a non-pharmacological treatment for refractory epilepsy. However, their mechanism of action is not fully understood. The cation-chloride cotransporter, KCC2, transports chloride out of neurons, thus, contributing to the intraneuronal concentration of this ion. Modifications in the KCC2 expression by KD could explain the beneficial effect of this diet on epilepsy. This work aimed to determine the impact of the KD on the KCC2 expression in dentate gyrus layers and CA3 strata of rats with seizures induced by amygdaloid kindling.Material and methods: Male Sprague-Dawley rats were fed normal diet (ND) or KD from postnatal day 24 until the end of the experiment. At 6 weeks after the start of the diets, rats were subjected to the amygdala kindling epilepsy model, sham or remained intact. Glucose and β-hydroxybutyrate concentration were quantified. The after-discharge duration (ADD), latency and duration of stages of kindling were evaluated. In addition, KCC2 expression was evaluated using optical density. A Pearson bivariate correlation was used to determine the relationship between KCC2 expression and ADD.Results: At the end of the experiment, KD-fed groups showed a reduction in glucose and an increase in β-hydroxybutyrate. KD reduced ADD and increased latency and duration of generalized seizures. In ND-fed animals, kindling reduced KCC2 expression in all three layers of the dentate gyrus; however, in KD-fed animals no changes were observed. KD increased KCC2 expression in the kindling group. In CA3, the pyramidal and lucidum strata presented an increase of KCC2 in KD-fed groups. Besides, the kindling groups presented lower levels of KCC2 than sham or intact groups. In all the layers of dentate gyrus, and the pyramidal and lucidum CA3 strata, the correlation indicated that the higher the KCC2 expression, the shorter the ADD during generalized seizures. Conclusion: KD reduces ADD in generalized seizures. In addition, KD has a putative neuroprotective effect by preventing the kindling-induced reduction of KCC2 expression in the molecular, granule and hilar dentate gyrus layers, and the pyramidal and lucidum CA3 strata. Increased KCC2 expression levels are related to a shorter duration of generalized seizures.