Bull. Egypt. Soc. Physiol. Sci. 31 (1) 2011 Shehata Neurophysiological Studies on the Effect of Acetone on Pentylenetetrazole-Induced Seizure in Rats Ahmed M. Shehata Department of Physiology- National Organization for Drug Control and Research ABSTRACT Recent interest in the anticonvulsant effects of acetone has stemmed from studies related to the ketogenic diet (KD). Despite knowledge of acetone's anticonvulsant properties, the neurochemical basis for this effect is not well known. The present study aimed to explore the neurochemical basis underlying the anticonvulsant effect of acetone in pentylenetetrazol (PTZ) - induced convulsions. This was achieved through determining the neurochemical changes of acetone in pentylenetetrazol (PTZ) – treated rats. Male adult rats received either saline, acetone (15 m mol/kg i.p.), PTZ (60 mg/kg, i.p.), or acetone 3 h before PTZ injection. Result showed that the maximum concentration of acetone reached about 3 h after acetone administration. Thus, the animals were administered pentylentetrazole three hours after acetone treatment. Pentylenetetrazole treated rats exhibited epilepsy, increased brain levels of excitatory amino acids (glutamate and aspartic acids) and decreased levels of inhibitory amino acid (γ-aminobutyric acid and glycine). In addition, pentylenetetrazole treatment decreased total antioxidant activity and reduced glutathione (GSH) in brain. Acetone pretreatment remarkably decreased seizure incidence rate and increased seizure latency. Moreover, acetone significantly minimized the disturbing effect of pentylenetetrazole on the redox status and the balance between excitatory and inhibitory amino acids. The study indicated that the epilepsy might be mediated, at least partially, through the disturbance in the redox status and imbalance between excitatory and inhibitory amino acids in the brain. Moreover, the study indicated that the antiepileptic effect of acetone might be due to its antioxidant effect and sustaining the balance between excitatory and inhibitory amino acids. Moreover, the study might recommend the concurrent intake of ketogenic diets with the conventional antiepileptic drugs. In addition, synthesizing new chemical entities yielding acetone during its metabolism in the body might provide new candidates as antiepileptic drugs. INTRODUCTION awaiting safer drugs with improved antiepileptic effectiveness. Moreover, Epilepsy is considered one of the combinations of conventional most common neurological disorders antiepileptic drugs (AEDs) might fail (2) worldwide, with a prevalence of 0.5- to effectively control seizures . 1% in the general population(1). Consistently, about 30% of epileptic Epilepsy continues to be a disease patients do not respond to clinically 135 Bull. Egypt. Soc. Physiol. Sci. 31 (1) 2011 Shehata established AEDs(3). The the brain with a ketogenic diet neurobiological relationships between intake(16). The ketone bodies, b- epilepsy and affective disorders are hydroxybutylate, acetoacetate and receiving increased attention(4,5). acetone are significantly increased in Epilepsy is a neurological disorder the plasma of patients receiving a characterized by spontaneous, ketogenic diet(17). Among these three recurrent and paroxysmal cerebral ketone bodies, acetoacetate and discharge leading to persistent acetone easily pass through the blood alterations in function and brain barrier, and partly replace morphology of neurons(6). The burst glucose as fuel in the brain(18), leading firing associated with prolonged to the changes in energy metabolism epileptic discharges could lead to a in the brain(14,18). These changes might large number of changes and cascades be related to the anticonvulsive effect of events at the cellular level. These associated with a ketogenic diet in effects include activation of glutamate animal models of neurodegenerative receptors, changes in composition of diseases. Acetone is the principal glutamate and γ-aminobutyric acid ketone body elevated in the ketogenic (GABA) receptors, cytokine diet (KD), with demonstrated robust activation and oxidative stress, anticonvulsant properties across a modulation in neurogenesis and variety of seizure tests and models of neuroplasticity or activation of some epilepsy(19). In addition, the late cell death pathways(1,7,8). anticonvulsant effects of acetone have Consistently, N-methyl-D-aspartate been reported in various animal (NMDA) receptor antagonists were models of epilepsy (19, 20). shown to possess anticonvulsant The present study was conducted properties against several insults to explore the neurochemical basis including pentylenetetrazole (PTZ)- underlying the antiepileptic effect of induced seizures and to enhance the acetone in rats. This was achieved effects of AEDs (9,10). through determining the effect of Despite progress in current acetone on the brain content of the antiepileptic therapy, neither are excitatory (glutamate and aspartic) seizures adequately controlled nor and inhibitory (GABA and glycine) medications free of untoward side amino acids and the redox status (total effects(11). On the other hand, a antioxidant activity and reduced ketogenic diet has been used glutathione). successfully to treat patients with (12) intractable epilepsy . In addition, MATERILAS & METHODS many studies on the anticonvulsant effects of a ketogenic diet have been Experimental animals: male adult performed, however, the mechanism Sprague Dawley rats (150-200 g) (13,14) remains unknown . Several were kindly provided from our hypotheses have been proposed to breeding center at NODCAR and kept explain the anticonvulsant effects of a for a week for acclimatization under (15) ketogenic diet. , such as the normal conditions and constant alterations in energy metabolism in temperature (25±1C°) with ad libitum 136 Bull. Egypt. Soc. Physiol. Sci. 31 (1) 2011 Shehata water and food until starting the euthanized by decapitation after experiment. seizures assessment; brains were Chemicals: All chemicals, unless removed and dissected bilaterally. specified other-wise, were purchased One brain half was homogenized in form Sigma-Aldrich Chemical Co. 75% (v/v) aqueous methanol (HPLC (St. Louis, MO). grade, Sigma-Aldrich, MO, USA), Drug: Pentylenetetrazole was homogenates were centrifuged at purchased from Sigma-Aldrich 4000×g, 20 min, 4 °C, and Chemical Co. (St. Louis, MO). supernatants were employed for the A total number of forty rats with determination of brain amino acids an average weight of 175g, were contents. The second brain half was administered an i.p. dose of acetone homogenized in ice cold saline and (15 mmol/kg) and blood samples were was used for the estimation of brain taken at time intervals 1, 2, 3, 5 and redox status (total antioxidant activity 10 h to asses the time corresponding and reduced glutathione). to the highest concentration of Methods: acetone. The rats were decapitated and Level of acetone in plasma was trunk blood was collected in 1 ml determined by HPLC method heparinized tubes. The heparinized according to Brega et al.(22). Free samples were centrifuged at 3,000 amino acids were determined using rpm for 20 min at 4°C. Plasma was precolumn derivatization HPLC-UV aspirated from the centrifuged method(23). Total antioxidant activity specimen using a transfer pipette. was determined using the Plasma samples were used to colourimetric method of Blois(24). determine acetone. Reduced glutathione was determined In addition, a total number of by HPLC method according to thirty two rats were divided into four Jayatilleke and Shaw (25). groups, group I received saline and Statistical Analysis. served as control. Group II received Data presented as means ± SE. single intraperitoneal injection of PTZ One-way ANOVA followed by LSD in convulsive dose of 60 mg/kg(21). test were used to evaluate significant Group III was injected differences from the control group. P< intraperitoneally with acetone (ACET, 0.05 was considered to be statistically 15 mmol/kg). Group IV was injected significant. Statistical processor intraperitoneally PTZ, 3h following system support (SPSS) for Windows acetone injection. After PTZ software, release 10.0 (SPSS, Inc, injection, rats were placed singly in Chicago, IL) was used. plexiglass cages and were observed for 30 min. Incidences and latency of RESULT clonic convulsive attacks, which lasts over 3 s with an accompanying loss of As shown in Figure 1. the righting reflex were recorded. Seizure maximum concentration of acetone latency for rats showing no convulsive attained after 3h of acetone attacks within the observation period administration to give 800.0 ± 95.5 was taken as 30 min(21). Rats were 137 Bull. Egypt. Soc. Physiol. Sci. 31 (1) 2011 Shehata mg/L and gradually declined to give decreased glutamate content. Acetone 100 .8 ± 35 mg/L after 10 h. pretreatment remarkably prevented Table 1 depicts that PTZ- treated the disturbing effect of PTZ on the rats exhibited clonic convulsions with free amino acid levels 3.5-min average seizure latency. (Table 2). Data in tables 3 shows Acetone pretreatment significantly that pentylenetetrazole treatment decreased the convulsions' incidence significantly decreased both total and prolonged seizure latency. antioxidant activity and GSH content Data in table 2 shows that PTZ in rat brain. Acetone treatment significantly (P <0.05) reduced brain significantly (P <0.05) increased both GABA and glycine contents and total antioxidant activity and GSH increased glutamate and aspartate content in rat brain. Acetone