Metabolic Brain Disease

, Volume 27, Issue 4, pp 471–478 | Cite as

A ketogenic diet did not prevent effects on the ectonucleotidases pathway promoted by lithium-pilocarpine-induced status epilepticus in rat hippocampus

  • Vanessa Gass da Silveira
  • Rosane Souza da Silva
  • Giana de Paula Cognato
  • Katiucia Marques Capiotti
  • Fabrício Figueiró
  • Mauricio Reis Bogo
  • Carla Denise Bonan
  • Marcos Luis Santos Perry
  • Ana Maria Oliveira BattastiniEmail author
Original Paper


A Ketogenic Diet (KD) mimics the anticonvulsant effects of fasting, which are known to suppress seizures. The purinergic system has been investigated in the matter of epilepsy development, especially the nucleoside adenosine, which has been considered a natural brain anticonvulsant. During epileptic seizures, extracellular adenosine concentration rises rapidly to micromolar levels. Adenosine can exert its anticonvulsant functions, after its release by nucleoside bidirectional transport, or by production through the sequential catabolism of ATP by ectonucleotidases, such as E-NTPDases (ectonucleoside triphosphate diphosphohydrolases) and ecto-5′-nucleotidase. Here, we have investigated the effect of a ketogenic diet on the nucleotide hydrolysis and NTPDases expression in the lithium-pilocarpine (Li-Pilo) model of epilepsy. For the induction of Status Epileticus (SE), 21-day-old female Wistar rats received an i.p. injection of lithium chloride (127 mg/kg) and 18–19 h later an i.p. injection of pilocarpine hydrochloride (60 mg/kg). The control groups received an injection of saline. After induction of SE, the control and Li-Pilo groups received standard or ketogenic diets for 6 weeks. The lithium-pilocarpine exposure affected the ATP (a decrease of between 8 % and 16 %) and ADP (an increase of between 18 % and 22 %) hydrolysis in both groups whereas the diet did not impact the nucleotide hydrolysis. NTPDase2 and 3 mRNA expressions decreased in the Li-Pilo group (41 % and 42 %). This data highlights the participation of the purinergic system in the pathophysiology of this model of epilepsy, since nucleotide hydrolysis and NTPDase expressions were altered by Li-Pilo exposure, with no significant effects of the ketogenic diet. However, the interaction between purinergic signaling and a ketogenic diet on epilepsy still needs to be better elucidated.


Ketogenic diet Ecto-nucleotidases Status epilepticus Rat hippocampus 



This work was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). Vanessa Gass da Silveira was recipient of a CNPq fellowship.


  1. Abbracchio MP, Burnstock G, Verkhratsky A, Zimmermann H (2009) Purinergic signaling in the nervous system: an overview. Trends Neurosci 32(1):19–29PubMedCrossRefGoogle Scholar
  2. Battastini AM, Da Rocha JB, Barcellos CK (1991) Characterization of an ATP diphosphohydrolase (EC in synaptosomes from cerebral cortex of adult rats. Neurochem Res 16:1303–1310PubMedCrossRefGoogle Scholar
  3. Berman RF, Fredholm BB, Aden U, O’Connor WT (2000) Evidence for increased dorsal hippocampal adenosine release and metabolism during pharmacologically induced seizures in rats. Brain Res 872(1–2):44–53PubMedCrossRefGoogle Scholar
  4. Bianchi V, Spychala J (2003) Mammalian 5′-nucleotidases. J Biol Chem 278(47):46195–46198PubMedCrossRefGoogle Scholar
  5. Boison D (2005) Adenosine and epilepsy: from therapeutic rationale to new therapeutic strategies. Neuroscientist 11:25–36PubMedCrossRefGoogle Scholar
  6. Boison D (2008) The adenosine kinase hypothesis of epileptogenesis. Prog Neurobiol 84:249–262PubMedCrossRefGoogle Scholar
  7. Boison D, Stewart K-A (2009) Therapeutic epilepsy research: from pharmacological rationale to focal adenosine augmentation. Biochem Pharmacol 78(12):1428–1437PubMedCrossRefGoogle Scholar
  8. Bonan CD, Walz R, Pereira GS, Worm PV, Battastini AM, Cavalheiro EA, Izquierdo I, Sarkis JJ (2000) Changes in synaptosomal ectonucleotidase activities in two rat models of temporal lobe epilepsy. Epilepsy Res 39(3):229–238PubMedCrossRefGoogle Scholar
  9. Borowicz KK, Luszczki J, Czuczwar SJ (2002) 2-Chloroadenosine, a preferential agonist of adenosine A1 receptors, enhances the anticonvulsant activity of carbamazepine and clonazepam in mice. Eur Neuropsychopharmacol 12:173–179PubMedCrossRefGoogle Scholar
  10. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254PubMedCrossRefGoogle Scholar
  11. Bruno AN, Oses JP, Amaral O, Coitinho A, Bonan CD, Battastini AM, Sarkis JJ (2003) Changes in nucleotide hydrolysis in rat blood serum induced by pentylenetetrazol-kindling. Brain Res Mol Brain Res 114(2):140–145PubMedCrossRefGoogle Scholar
  12. Cavalheiro EA (1995) The pilocarpine model of epilepsy. Ital J Neurol Sci 16:33–37PubMedCrossRefGoogle Scholar
  13. Cavalheiro EA, Silva DF, Turski WA, Calderazzo-Fihlo LS, Bortolotto A, Turski L (1987) The susceptibility of rats to pilocarpine-induced seizures is age-dependent. Dev Brain Res 37:43–58CrossRefGoogle Scholar
  14. Chan K, Delfert D, Junges KD (1986) A direct colorimetric assay for Ca2+-ATPase activity. Anal Biochem 157:375–380PubMedCrossRefGoogle Scholar
  15. Da Silveira VG, Cognato GP, Müller AP, Figueiró F, Bonan DC, Perry MLS, Battastini AMO (2010) Effect of ketogenic diet on nucleotide hydrolysis and hepatic enzymes in blood serum of rats in a lithium-pilocarpine-induced status epilepticus. Metab Brain Dis 25:211–217PubMedCrossRefGoogle Scholar
  16. Dubé C, Da Silva Fernandes MJ, Nehlig A (2001a) Age-dependent consequences of seizures and the development of temporal lobe epilepsy in the rat. Dev Neurosci 23:219–223PubMedCrossRefGoogle Scholar
  17. Dubé C, Boyet S, Marescaux C, Nehlig A (2001b) Relationship between neuronal loss and interictal glucose metabolism during the chronic phase of the lithium-pilocarpine model of epilepsy in the immature and adult rat. Exp Neurol 167:227–241PubMedCrossRefGoogle Scholar
  18. Dunwiddie TV, Masino SA (2001) The role and regulation of adenosine in the central nervous system. Annu Rev Neurosci 24:31–55PubMedCrossRefGoogle Scholar
  19. Fredholm BB, Ijzerman AP, Jacobson KA, Klotz KN, Linden J (2001) International Union of Pharmacology. XXV. Nomenclature and classification of adenosine receptors. Pharmacol Rev 53(4):527–552PubMedGoogle Scholar
  20. Guzman M, Blazquez C (2001) Is there an astrocyte-neuron ketone body shuttle? Trends Endocrinol Metab 12:169–173PubMedCrossRefGoogle Scholar
  21. Heymann D, Reddington M, Kreutzberg GW (1984) Subcellular localization of 5′-nucleotidase in rat brain. J Neurochem 43:971–978PubMedCrossRefGoogle Scholar
  22. Honchar MP, Olney JW, Sherman WR (1983) Systemic cholinergic agents induce seizures and brain damage in lithium-treated rats. Science 2388:323–325CrossRefGoogle Scholar
  23. Janigro D (1999) Blood-brain barrier, ion homeostatis and epilepsy: possible implications towards the understanding of ketogenic diet mechanisms. Epilepsy Res 37(3):223–232PubMedCrossRefGoogle Scholar
  24. Jutila L, Ylinen A, Partanen K, Alafuzoff I, Mervaala E, Partanen J, Vapalahti M, Vainio P, Pitkänen A (2001) MR volumetry of the entorhinal, perirhinal, and temporopolar cortices in drug-refractory temporal lobe epilepsy. AJNR Am J Neuroradiol 22(8):1490–1501PubMedGoogle Scholar
  25. Langer D, Hammer K, Koszalka P, Schrader J, Robson S, Zimmermann H (2008) Distribution of ectonucleotidases in the rodent brain revisited. Cell Tissue Res 334:199–217PubMedCrossRefGoogle Scholar
  26. Langston JL, Myers TM (2011) Diet composition modifies the toxicity of repeated soman exposure in rats. Neurotoxicology 32(6):907–915PubMedCrossRefGoogle Scholar
  27. Masino SA, Geiger JD (2008) Are purines mediators of the anticonvulsivant/neuroprotective effects of ketogenic diet? Trends in Neuroscience 31(6):273–278Google Scholar
  28. Masino SA, Li T, Theofilas P, Sandau US, Ruskin DN, Fredholm BB, Geiger J, Aronica E, Boison D (2011) A ketogenic diet suppresses seizures in mice through adenosine A1 receptors. J Clin Invest 121(7):2679–2683PubMedCrossRefGoogle Scholar
  29. Morrisett RA, Jope RS, Snead OC 3rd (1987a) Effects of drugs on the initiation and maintenance of status epilepticus induced by administration of pilocarpine to lithium-pretreated rats. Exp Neurol 97(1):193–200PubMedCrossRefGoogle Scholar
  30. Morrisett RA, Jope RS, Snead OC 3rd (1987b) Status epilepticus is produced by administration of cholinergic agonists to lithium-treated rats: comparison with kainic acid. Exp Neurol 98(3):594–605PubMedCrossRefGoogle Scholar
  31. Nagy A, Delgado-Escueta AV (1984) Rapid preparation of synaptosomes from mammalian brain using nontoxic isoosmotic gradient material (Percoll). J Neurochem 43(4):1114–1123PubMedCrossRefGoogle Scholar
  32. Nakazawa M, Kodama S, Matsuo T (1983) Effects of ketogenic diet on electroconvulsive threshold and brain contents of adenosine nucleotides. Brain Dev 5(4):375–380PubMedCrossRefGoogle Scholar
  33. Oses JP, Viola GG, de Paula Cognato G, Júnior VH, Hansel G, Böhmer AE, Leke R, Bruno AN, Bonan CD, Bogo MR, Portela LV, Souza DO, Sarkis JJ (2007) Pentylenetetrazol kindling alters adenine and guanine nucleotide catabolism in rat hippocampal slices and cerebrospinal fluid. Epilep Res 75(2–3):104–111CrossRefGoogle Scholar
  34. Patel S, Chapman AG, Millan MH, Meldrum BS (1988) Epilepsy and excitatory amino acid antagonists. In: Lodge D (ed) Excitatory amino acids in health and disease. Wiley, London, pp 353–378Google Scholar
  35. Pedata F, Corsi C, Melani A, Bordoni F, Latini S (2001) Adenosine extracellular brain concentrations and role of A2A receptors in ischemia. Ann N Y Acad Sci 939:74–84, ReviewPubMedCrossRefGoogle Scholar
  36. Rigoulot MA, Koning E, Ferrandon A, Nehlig A (2004) Neuroprotective properties of topiramate in the lithium-pilocarpine model of epilepsy. J Pharmacol Exp Ther 308:787–795PubMedCrossRefGoogle Scholar
  37. Robson SC, Sévigny J, Zimmermann H (2006) The E-NTPDase family of ectonucleotidases: structure function relationships and pathophysiological significance. Purinergic Signal 2(2):409–430PubMedCrossRefGoogle Scholar
  38. Schwartzkroin PA (1999) Mechanisms underlying the anti-epileptic efficacy of the ketogenic diet. Epilepsy Res 37(3):171–180PubMedCrossRefGoogle Scholar
  39. Shukla V, Zimmermann H, Wang LP, Kettenmann H, Raab S, Hammer K, Sévigny J, Robson SC, Braun N (2005) Functional expression of the ecto-ATPase NTPDase2 and of nucleotide receptors by neuronal progenitor cells in the adult murine hippocampus. J Neurosci Res 80:600–610PubMedCrossRefGoogle Scholar
  40. Tagliabue A, Bertoli S, Trentani C, Borrelli P, Veggiotti P (2012) Effects of the ketogenic diet on nutritional status, resting energy expenditure, and substrate oxidation in patients with medically refractory epilepsy: A 6-month prospective observational study. Clin Nutr 31(2):246–249Google Scholar
  41. Turski L, Ikonomidou C, Turski WA, Bortolotto ZA, Cavalheiro EA (1989) Review: cholinergic mechanisms and epileptogenesis. The seizures induced by pilocarpine: a novel experimental model of intractable epilepsy. Synapse 3:154–171PubMedCrossRefGoogle Scholar
  42. Wetherington J, Serrano G, Dingledine R (2008) Astrocytes in the epileptic brain. Neuron 58:16CrossRefGoogle Scholar
  43. Wieraszko A, Seyfried TN (1989) Increased amount of extracellular ATP in stimulated hippocampal slices of seizure prone mice. Neurosci Lett 106:287–293PubMedCrossRefGoogle Scholar
  44. Wilder RM (1921) The effect of ketonemia on the course of epilepsy. Mayo Clin Bull 2:307–308Google Scholar
  45. Wilot LC, Da Silva RS, Ferreira OJ, Bonan CD, Sarkis JJF, Rocha E, Battastini AMO (2004) Chronic treatment with lithium increases the ecto-nucleotidase activities in rat hippocampal synatosomes. Neurosc Letters 368:167–170CrossRefGoogle Scholar
  46. Zhao Q, Stafstrom CE, Fu DD, Hu Y, Holmes GL (2004) Detrimental effects of the ketogenic diet on cognitive function in rats. Pediatric Res 55(3):498–506Google Scholar
  47. Zimmermann H (2001) Ecto-nucleotidases: some recent developments and a note on nomenclature. Drug Dev Res 52:44–56CrossRefGoogle Scholar
  48. Zimmermann H (2006) Ectonucleotidases in the nervous system. Novartis Found Symp 276:113–128PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Vanessa Gass da Silveira
    • 1
  • Rosane Souza da Silva
    • 2
  • Giana de Paula Cognato
    • 1
  • Katiucia Marques Capiotti
    • 2
  • Fabrício Figueiró
    • 1
  • Mauricio Reis Bogo
    • 2
  • Carla Denise Bonan
    • 2
  • Marcos Luis Santos Perry
    • 1
  • Ana Maria Oliveira Battastini
    • 1
    Email author
  1. 1.Departamento de Bioquímica, Instituto de Ciências Básicas da SaúdeUniversidade Federal do Rio Grande do SulPorto AlegreBrazil
  2. 2.Departamento de Biologia Celular e Molecular, Faculdade de BiociênciasPontifícia Universidade Católica do Rio Grande do SulPorto AlegreBrazil

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