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. 2007 May 1;6(4):555–569. doi: 10.1111/j.1582-4934.2002.tb00454.x

Dantrolene protects neurons against kainic acid induced apoptosis in vitro and in vivo

B O Popescu 1,2,✉, M Oprica 3, Maria Sajin 4, Cristina L Stanciu 2, O Bajenaru 1, Andreea Predescu 1, Cristina Vidulescu 2,5, L M Popescu 2,5
PMCID: PMC6741407  PMID: 12611640

Abstract

Apoptotic cell death induced by kainic acid (KA) in cultures of rat cerebellar granule cells (CGC) and in different brain regions of Wistar rat pups on postnatal day 21 (P21) was studied. In vitro, KA (100–500 μM) induced a concentration‐dependent loss of cell viability in MTT assay and cell death had apoptotic morphology as studied by chromatin staining with propidium iodide (PI). In vivo, twenty‐four hours after induction of status epilepticus (SE) by an intraperitoneal KA injection (5 mg/kg) we quantified apoptotic cells in hippocampus (CA1 and CA3), parietal cortex and cerebellum using PI staining and terminal deoxynucleotidyl transferase‐mediated dUTP nick end labeling (TUNEL) technique. We report that dantrolene, a specific ryanodine receptor antagonist, was able to significantly reduce the apoptotic cell death in CGC cultures and in hyppocampal CA1 and parietal cortex regions. Our finding can be valuable for neuroprotective therapy strategies in patients with repeated generalized seizures or status epilepticus.

Keywords: apoptosis, epilepsy, kainic acid, dantrolene, ryanodine receptors, cerebellar granule cells

References

  • 1. Lipton S.A., Rosenberg P.A., Excitatory amino acids as a final common pathway for neurologic disorders, N. Engl. J. Med., 330: 613–622, 1994. [DOI] [PubMed] [Google Scholar]
  • 2. Kemp J.A., McKernan R.M., NMDA receptor pathways as drug targets, Nat. Neurosci. S: 1039–1042, 2002. [DOI] [PubMed] [Google Scholar]
  • 3. Simonian N.A., Getz, R.L. , Leveque J.C., Konradi C., Coyle J.T., Kainic acid induces apoptosis in neurons, Neuroscience, 75: 1047–1055, 1996. [DOI] [PubMed] [Google Scholar]
  • 4. Sheardown M., Nielsen E., Hansen A., Jacobsen P., Honore T., Dihydro‐6‐nitro‐sulfamoyl‐benzo(F)quinoxline: a neuroprotectant for cerebral ischemia, Science, 247: 571–573, 1990. [DOI] [PubMed] [Google Scholar]
  • 5. Weiss J., Yin H., Choi D., Basal forebrain cholinergic neurons are selectively vulnerable to AMPA/kainate receptor‐mediated toxicity, Neuroscience, 60: 659–664, 1994. [DOI] [PubMed] [Google Scholar]
  • 6. White H.S., Animal models of epileptogenesis, Neurology, 59: S7–S14, 2002. [DOI] [PubMed] [Google Scholar]
  • 7. Holmes, G.L. , Seizure‐induced neuronal injury, Neurology, 59: S3–S6, 2002. [DOI] [PubMed] [Google Scholar]
  • 8. Marks J.D., Friedman J.E., Haddad G.G., Vulnerability of CA1 neurons to glutamate is developmentally regulated, Dev. Brain Res., 97: 194–206, 1996. [DOI] [PubMed] [Google Scholar]
  • 9. Liu Z., Stafstrom C.E., Sarkisian M., Tandon P., Yang Y., Hori A., Holmes G.L., Age‐dependent effects of glutamate toxicity in the hippocampus, Dev. Brain Res., 97: 178–184, 1996. [DOI] [PubMed] [Google Scholar]
  • 10. Sankar R., Shin D.H., Liu H., Mazarati A., Pereira de Vasconcelos A., Wasterlain C.G., Patterns of status epilepticus‐induced neuronal injury during development and long‐term consequences, J. Neurosci., 18: 8382–8393, 1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11. Humphrey W.M., Dong H.X., Csernansky C.A., Csernansky J.G., Immediate and delayed hippocampal neuronal loss induced by kainic acid during early postnatal development in the rat, Dev. Brain Res., 137: 1–12, 2002. [DOI] [PubMed] [Google Scholar]
  • 12. Henshall D.C., Clark R.S., Adelson P.D., Chen M., Watkins S.C., Simon R.P., Alterations in bcl‐2 and caspase gene family protein expression in human temporal lobe epilepsy, Neurology, 55: 250–257, 2000. [DOI] [PubMed] [Google Scholar]
  • 13. Rabinowicz A.L., Correale B., Boutros R.B., Couldwell W.T., Henderson C.W., DeGirogio C.M., Neuron‐specific enolase is increased after single seizures during inpatient video/EEG monitoring, Epilepsia, 37: 122–125, 1996. [DOI] [PubMed] [Google Scholar]
  • 14. Dodrill C.B., Progressive cognitive decline in adolescents and adults with epilepsy, Prog. Brain Res., 135: 397–408, 2002. [DOI] [PubMed] [Google Scholar]
  • 15. Kalviainen R., Salmenpera T., Partanen K., Vainio P., Riekkinen P., Pitakanen A., Recurrent seizures may cause hippocampal damage in temporal lobe epilepsy, Neurology, 50: 1377–1382, 1998. [DOI] [PubMed] [Google Scholar]
  • 16. Tasch E., Cendes F., Li L.M., Dubeau F., Andermann F., Arnold D.L., Neuroimaging evidence of progressive neuronal loss and dysfunction in temporal lobe epilepsy, Ann. Neurol., 45: 568–576, 1999. [DOI] [PubMed] [Google Scholar]
  • 17. Sloviter R.S., Dean E., Sollas A.L., Goodman J.H., Apoptosis and necrosis induced in different hippocampal neuronal populations by repetitive perforant path stimulation in the rat, J. Comp. Neurol., 366: 516–533, 1996. [DOI] [PubMed] [Google Scholar]
  • 18. Bengzon J., Kokaia Z., Elmer E., Nanobashvili A., Kokaia M., Lindvall O., Apoptosis and proliferation of dentate gyrus neurons after single and intermittent limbic seizures, Proc. Natl. Acad. Sci. USA, 94: 10432–10437, 1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19. Henshall D.C., Chen I., Simon R.P., Involvement of caspase‐3 like protease in the mechanism of cell death following focally evoked limbic seizures, J. Neurochem, 74: 1215–1223, 2000. [DOI] [PubMed] [Google Scholar]
  • 20. Henshall D.C., Bonislawski D.P., Skradski S.L., Araki T., Lan J.Q., Schindler C.K., Meller R., Simon R.P., Formation of Apaf‐1/cytochrome c complex precedes activation of caspase‐9 during seizure‐induced neuronal death, Cell Death Differ, 8: 1169–1181, 2001. [DOI] [PubMed] [Google Scholar]
  • 21. Henshall D.C., Araki T., Schindler C.K., Lan J.Q., Tiekoter K.L., Taki W., Simon R.P., Activation of Bcl‐2‐associated death protein and counter‐response of Akt within cell populations during seizure‐induced neuronal death, J. Neurosci., 22: 8458–8465, 2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22. Leski M.L., Valentine S.L., Coyle T., L‐type voltage‐gated calcium channels modulate kainic acid neurotoxicity in cerebellar granule cells, Brain Res., 828: 27–40, 1999. [DOI] [PubMed] [Google Scholar]
  • 23. Brines M.L., Ling Z., Broadus A.E., Parathyroid hormone‐related protein protects against kainic acid excitotoxicity in rat cerebellar granule cells by regulating L‐type channel calcium flux, Neurosci. Lett., 274: 13–16, 1999. [DOI] [PubMed] [Google Scholar]
  • 24. Blackshaw S., Sawa A., Sharp A.H., Ross C.A., Snyder S.H., Khan A.A., Type 3 inositol 1,4,5‐triphosphate receptor modulates cell death, FASEB J., 14: 1375–1379, 2000. [DOI] [PubMed] [Google Scholar]
  • 25. Zhao F., Li P., Chen S.R.W., Louis C.F., Fruen B.R., Dantrolene inhibition of ryanodine receptor Ca2+ release channels, J. Biol. Chem., 276: 13810–13816, 2001. [DOI] [PubMed] [Google Scholar]
  • 26. Popescu A.T., Vidulescu C., Stanciu C.L., Popescu B.O., Popescu L.M., Selective protection by phosphatidic acid against staurosporine‐induced apoptosis, J. Cell. Mol. Med., 6: 433–438, 2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27. Cedazo‐Minguez A., Popescu B.O., Ankarcrona M., Nishimura T., Cowburn R.F., The presenilin 1 ΔE9 mutation gives enhanced basal phospholipase C activity and a resultant increase in intracellular calcium concentrations, J. Biol. Chem., 277: 36646–36655, 2002. [DOI] [PubMed] [Google Scholar]
  • 28. Mattson M.P., Zhu H., Yu J., Kindy M.S., Presenilin‐1 mutation increases neuronal vulnerability to focal ischemia and to hypoxia and glucose deprivation in cell culture: involvement of perturbed calcium homeostasis, J. Neurosci., 20: 1358–1364, 2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29. Mosmann T., Rapid colorimetric assay for cellular growth and survival: application to proliferation and citotoxicity assays, J. Immunol. Methods, 65: 55–63, 1983. [DOI] [PubMed] [Google Scholar]
  • 30. Popescu B.O., Popescu L.M., Neuronal apoptosis triggered by anti‐Fas (CD95, APO‐1) antibody is enhanced by dexamethasone, J. Med. Biochem., 4: 1–14, 2000. [Google Scholar]
  • 31. Schneider I., Reverse D., Dewachter I., Ris L., Caluwaerts N., Kuiperi C., Gilis M., Geerts H., Kretzschmar H., Godaux E., Moechars D., Van Leuven F., Herms J., Mutant presenilins disturb neuronal calcium homeostasis in the brain of transgenic mice, decreasing the threshold for excitotoxicity and facilitating long‐term potentiation, J. Biol. Chem., 276: 11539–11544, 2001. [DOI] [PubMed] [Google Scholar]
  • 32. Zagrean L., Moldovan M., Munteanu A.M., Spulber S., Voiculescu B.A., Popescu B.O., Early electrocortical changes consistent with ischemic preconditioning in rat, J. Cell. Mol. Med., 4: 215–223, 2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33. Nat R., Voiculescu B., Stanciu C., Vidulescu C., Cergan R., Badiu C., Popescu L.M., Apoptosis in human embryo development: 2. Cerebellum, J. Cell. Mol. Med., 5: 179–187, 2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34. Beal M.F., Mechanisms of excitotoxicity in neurological diseases, FASEB J., 6: 3338–3344, 1992. [PubMed] [Google Scholar]
  • 35. Ankarcrona M., Dypbukt J.M., Bonfoco E., Zhivotovsky B., Orrenius S., Lipton S.A., Nicotera P., Glutamate‐induced neuronal death: a succession of necrosis or apoptosis depending on mitochondrial function, Neuron, 15: 961–973, 1995. [DOI] [PubMed] [Google Scholar]
  • 36. Malva J.O., Carvalho A.P., Carvalho C.M., Kainate receptors in hippocampal CA3 subregion: evidence for a role in regulating neurotransmitter release, Neurochem. Int., 32: 1–6, 1998. [DOI] [PubMed] [Google Scholar]
  • 37. Zagulska‐Szymczak S., Filipkowski R.K., Kaczmarek L., Kainate‐induced genes in the hippocampus: lessons from expression patterns, Neurochem. Int., 38: 485–501, 2001. [DOI] [PubMed] [Google Scholar]
  • 38. Nicotera P., Bellomo G., Orrenius S., The role of Ca2+ in cell killing, Chem. Res. Toxicol., 3: 484–494, 1990. [DOI] [PubMed] [Google Scholar]
  • 39. Friberg H., Wieloch T., Mitochondrial permeability transition in acute neurodegeneration, Biochimie, 84: 241–250, 2002. [DOI] [PubMed] [Google Scholar]
  • 40. Verdaguer E., Garcia‐Jorda E., Jimenez A., Stranges A., Sureda F.X., Canudas A.M., Escubedo E., Camarasa J., Pallas M., Camins A., Kainic acid‐induced neuronal cell death in cerebellar granule cells is not prevented by caspase inhibitors, Br. J. Pharmacol., 135: 1297–1307, 2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41. Hirata H., Cadet J.L., Kainate‐induced hippocampal DNA damage is attenuated in superoxide dismutase transgenic mice, Mol. Brain Res., 48: 145–148, 1997. [DOI] [PubMed] [Google Scholar]
  • 42. Nicotera P., Leist M., Fava E., Berliocchi L., Volbracht C., Energy requirement for caspase activation and neuronal cell death, Brain Pathol., 10: 276–282, 2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43. Nicotera P., Apoptosis and age‐related disorders: role of caspase‐dependent and caspase‐independent pathways, Toxicol. Lett., 127: 189–195, 2002. [DOI] [PubMed] [Google Scholar]
  • 44. Popescu B.O., Cedazo‐Minguez A., Popescu L.M., Winblad B., Cowburn R.F., Ankarcrona M., Caspase cleavage of exon 9 deleted presenilin‐1 is an early event in apoptosis induced by calcium ionophore A 23187 in SH‐S5Y neuroblastoma cells, J. Neurosci. Res., 66: 122–134, 2001. [DOI] [PubMed] [Google Scholar]
  • 45. Schwob J.E., Fuller T., Price J.L., Olney J.W., Widespread patterns of neuronal damage following systemic or intracerebral injections of kainic acid: a histological study, Neuroscience, 5: 991–1014, 1980. [DOI] [PubMed] [Google Scholar]
  • 46. Berg M., Bruhn T., Fransen A., Schousboue A., Diemer N.H., Kainic acid‐induced seizures and brain damage in the rat: role of calcium homeostasis, J. Neurosci. Res., 40: 641–646, 1995. [DOI] [PubMed] [Google Scholar]
  • 47. Clausen T., Bullock R., Medical treatment and neuroprotection in traumatic brain injury, Curr. Pharm. Des., 7: 1517–1532, 2001. [DOI] [PubMed] [Google Scholar]
  • 48. Rees K., Beranek‐Stanley M., Burke M., Ebrahim S., Hypothermia to reduce neurological damage following coronary artery bypass surgery, Cochrane Database Syst. Rev. 1: CD002138, 2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49. Gunn A.J., Cerebral hypothermia for prevention of brain injury following perinatal asphyxia, Curr. Opin. Pediatr., 12: 111–115, 2000. [DOI] [PubMed] [Google Scholar]
  • 50. Thoresen M., Cooling the asphyxiated brain — ready for clinical trials?, Eur. J. Pediatr., 158: S5–S8, 1999. [DOI] [PubMed] [Google Scholar]
  • 51. Liu Z., Gatt A., Mikati M., Holmes G. L., Effect of temperature on kainic acid‐induced seizures, Brain Res., 631: 51–58, 1993. [DOI] [PubMed] [Google Scholar]
  • 52. Maeda T., Hashizume K., Tanaka T., Effect of hypothermia on KA‐induced limbic seizures: an electroencephalographic and 14C‐deoxyglucose autoradiographic study, Brain Res., 818: 228–235, 1999. [DOI] [PubMed] [Google Scholar]
  • 53. Turski L., Kleinrok Z., Effects of kainic acid on body temperature of rats: role of catecholaminergic and serotoninergic systems, Psychopharmacol., 71: 35–39, 1980. [DOI] [PubMed] [Google Scholar]
  • 54. Oprica M., Forslin Aronsson Å., Post C., Eriksson C., Ahlenius S., Popescu L. M., Schultzberg M., Effects of α‐MSH on kainic acid induced changes in core temperature in rats, Peptides, 23: 143–149, 2002. [DOI] [PubMed] [Google Scholar]
  • 55. Ahlenius S., Oprica M., Eriksson C., Winblad B., Schultzberg M., Early hypothermic effect of kainic acid: unmasking by dizocilpine, Neuropharmacol., 43: 28–35, 2002. [DOI] [PubMed] [Google Scholar]
  • 56. Wu C.C., Yen M.H., Beneficial effects of dantrolene on lipopolysaccharide‐induced haemodynamic alterations in rats and mortality in mice, Eur. J. Pharmacol., 327: 17–24, 1997. [DOI] [PubMed] [Google Scholar]
  • 57. Nisijima K., Yoshino T., Yui K., Katoh S., Potent serotonin (5‐HT)(2A) receptor antagonists completely prevent the development of hyperthermia in an animal model of the 5‐HT syndrome, Brain Res., 890: 23–31, 2001. [DOI] [PubMed] [Google Scholar]

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