Skip to main content
PMC home page
Journal of Cellular and Molecular Medicine logoLink to Journal of Cellular and Molecular Medicine
. 2007 May 1;8(2):241–248. doi: 10.1111/j.1582-4934.2004.tb00279.x

Morphological and functional changes in rat hippocampal slice cultures after short‐term oxygen‐glucose deprivation

I V Lushnikova 1,✉, K Y Voronin 1, P Y Malyarevskyy 1, G G Skibo 1
PMCID: PMC6740250  PMID: 15256072

Abstract

To study effects of short‐term cerebral ischemia, hippocampal slice cultures were subjected to oxygen and glucose deprivation (OGD) followed by a period of normoxic reoxygenation. Propidium iodide staining, and MTT/formazanassay were used to evaluate cell viability and metabolic activity. CA1 pyramidal cells were analyzed at the light‐and electron microscopic levels. Cell damage was found to be insignificant during the first hour after 10 min OGD but profound following 4 h, showing delayed neuronal cell damage caused by short‐term OGD. Our model can be used to characterize the mechanisms of cell damage caused by mild cerebral ischemia. These data might apply to further development of neuroprotective tools for the treatment of brain diseases.

Keywords: rat hippocampal slice cultures, short‐term oxygen‐glucose deprivation, cell viability, morphology

References

  • 1. Lipton, P. , Ischemic cell death in brain neurons, Physiol. Rev., 79: 1431–1568, 1999. [DOI] [PubMed] [Google Scholar]
  • 2. Zhong C., Qin Z., Zhong C.J., Wang Y., Shen X.Y., Neuroprotective effects of bone marrow stromal cells on rat organotypic hippocampal slice culture model of cerebral ischemia, Neurosci. Lett., 342: 93–96, 2003. [DOI] [PubMed] [Google Scholar]
  • 3. Jourdain P., Nikonenko I., Alberti S., Muller D., Remodelling of hippocampal synaptic network by a brief anoxia‐hypoglycemia, J. Neurosci., 22: 3108–3116, 2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4. Afsar N., Kaya D., Aktan S., Aykut‐Bingol C., Stroke and status epilepticus: stroke type, type of status epilepticus and prognosis, Seizure, 12: 23–27, 2003. [DOI] [PubMed] [Google Scholar]
  • 5. Egashira N., Iwasaki K., Ishibashi M., Hatip‐Al‐Khatib I., Wolozin B., Mishima K., Irie K., Fujiwara M., Hypoxia enhances β‐amyloid‐induced apoptosis in rat cultured hippocampal neurons, Jpn. J. Pharmacol., 90: 321–327, 2002. [DOI] [PubMed] [Google Scholar]
  • 6. Brana C., Benham C., Sundstrom L., A method for characterising cell death in vitro by combining propidium iodide staining with immunohistochemistry, Brain Res. Prot., 10: 109–114, 2002. [DOI] [PubMed] [Google Scholar]
  • 7. Buchner M., Huber R., Riepe M.W., Trans‐synaptic increase of hypoxic tolerance in hippocampus upon physical challenge with two‐photon microscopy, Hippocampus, 765–773, 2002. [DOI] [PubMed]
  • 8. Laake J.H., Haug F.‐M., Wieloch T., Ottersen O. P., A simple in vitro model of ischemia based on hippocampal slice cultures and propidium iodide fluorescence, Brain Res. Brain Res. Protoc., 4: 173–184, 1999. [DOI] [PubMed] [Google Scholar]
  • 9. Stoppini P.A., Buchs P., Muller D., A simple method for organotypic cultures of nervous tissue, J. Neurosci. Methods, 37: 173–182, 1991. [DOI] [PubMed] [Google Scholar]
  • 10. Singer C.A., Figueroa‐Masot X.A., Batchelor R.H., Dorsa D.M., The mitogen‐activated protein kinase pathway mediates estrogen neuroprotection after glutamate toxicity in primary cortical neurons, J. Neurosci., 19: 2455–63, 1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11. Connelly C., Chen L., Colquhoun S., Metabolic activity of cultured rat brainstem, hippocampal and spinal cord slices, J. Neurosci. Methods, 99: 1–7, 2000. [DOI] [PubMed] [Google Scholar]
  • 12. Mathews K.S., McLaughlin D.P., Ziabari L.H., Toner C.C., Street P.C., Hisgrove E., Bezzina E.L., Stamford J.A., Rapid quantification of ischemic injury and cerebroprotection in brain slices using densitometric assessment of 2,3,5‐triphenyltetrazolium chloride staining, J. Neurosci. Methods, 102: 43–51, 2000. [DOI] [PubMed] [Google Scholar]
  • 13. Buddle M., Eberhardt E., Ciminello L.H., Levin T., Wing R., DiPasquale K., Raley‐Susman, K.M. , Microtubule‐associated protein 2 (MAP2) associates with the NMDA receptor and is spatially redistributed within rat hippocampal neurons after oxygen‐glucose deprivation, Brain Res., 978: 38–50, 2003. [DOI] [PubMed] [Google Scholar]
  • 14. Strasser U, Fischer G., Quantitative measurement of neuronal degeneration in organotypic hippocampal cultures after combined oxygen/glucose deprivation, J. Neurosci. Methods, 57: 177–186, 1995. [DOI] [PubMed] [Google Scholar]
  • 15. Jung Y.J., Park S.J., Park J.S., Lee K.E., Glucose/oxygen deprivation induces the alteration of synapsin I and phosphosynapsin, Brain Res., 996: 47–54 2004. [DOI] [PubMed] [Google Scholar]
  • 16. Brana C., Benham C.D., Sundstrom L.E., Calpain activation and inhibition in organotypic rat hippocampal slice cultures deprived of oxygen and glucose, Eur. J. Neurosci., 11: 2375–2384, 1999. [DOI] [PubMed] [Google Scholar]
  • 17. Cimarosti H., Rodnight R., Tavares A., Paiva R., Valentim L., Rocha E., Salbego C., An investigation of the neuroprotective effect of lithium in organotypic slice cultures of rat hippocampus exposed to oxygen and glucose deprivation. Neurosci. Lett., 315: 3336, 2001. [DOI] [PubMed] [Google Scholar]
  • 18. Graulich J., Hoffmann U., Maier R.F., Ruscher K., Pomper J.K., Ko H.‐K., Gabriel S., Obladen M., Heinemann U., Acute neuronal injury after hypoxia is influenced by the reoxygenation mode in juvenile hippocampal slice cultures, Develop. Brain Res., 137: 35–42, 2002. [DOI] [PubMed] [Google Scholar]
  • 19. Peeters C., Hoelen D., Groenendaal F., van Bel F., Bar D., Deferoxamine, allopurinol and oxypurinol are not neuroprotective after oxygen/glucose deprivation in an organotypic hippocampal model, lacking functional endothelial cells, Brain Res., 963: 72–80, 2003. [DOI] [PubMed] [Google Scholar]
  • 20. Pringle A.K., Schmidt W., Deans J.K., Wulfert E., Reymann K.G., Sundstrom L.E., 7‐Hydroxylated epiandrosterone (7‐OH‐EPIA) reduces ischaemia‐induced neuronal damage both in vivo and in vitro, Eur. J. Neurosci., 18: 117–124, 2003. [DOI] [PubMed] [Google Scholar]
  • 21. Crepel V., Hammond C., Krnjevic K., Chinestra P., Ben‐Ari Y., Anoxia‐induced LTP of isolated NMDA receptormediated synaptic responses, J. Neurophysiol., 69: 1774–1778, 1993. [DOI] [PubMed] [Google Scholar]
  • 22. Melani R., Rebaudo R., Balestrino M., Cupello A., Haglid K., Hyden H., Involvement of S‐100 protein in anoxic long‐term potentiation, Brain Res. 840: 171–174, 1999. [DOI] [PubMed] [Google Scholar]
  • 23. Crepel V., Epsztein J., Ben‐Ari Y., Ischemia induces shortand long‐term remodeling of synaptic activity in the hippocampus, J. Cell. Mol. Med., 7: 401–407, 2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24. Xu G.P., Dave K.R., Vivero R., Schmidt‐Kastner R., Sick T.J., Perez‐Pinzon M.A., Improvement in neuronal survival after ischemic preconditioning in hippocampal slice cultures, Brain Res., 952: 153–158, 2002. [DOI] [PubMed] [Google Scholar]
  • 25. Valentim L.M., Rodnight R., Geyer A.B., Horn A.P., Tavares A., Cimarosti H., Netto C.A., Salbego C.G., Changes in heat shock protein 27 phosphorylation and immunocontent in response to preconditioning to oxygen and glucose deprivation in organotypic hippocampal cultures, Neurosci., 118: 379–386, 2003. [DOI] [PubMed] [Google Scholar]
  • 26. Horakova L., Stolc S., Chromikova Z., Pekarova A., Derkova L., Mechanisms of hippocampal reoxygenation injury, Mol. Chem. Neuropathol., 33: 223–236, 1998. [DOI] [PubMed] [Google Scholar]
  • 27. White B.C., Sullivan J.M., DeGracia D.J., O'Neil B.J., Neumar R.W., Grossman L.I., Rafols J.A., Krause G.S., Brain ischemia and reperfusion: molecular mechanisms of neuronal injury, J. Neurol. Sci., 179: 1–33, 2000. [DOI] [PubMed] [Google Scholar]
  • 28. Kreisman, N.R. , Soliman, S. , and Gozal, D. , Regional differences in hypoxic depolarization and swelling in hippocampal slices, J. Neurophysiol., 83: 1031–1038, 2000. [DOI] [PubMed] [Google Scholar]
  • 29. Nabetani, M. , and Okada, Y. , Developmental and regional differences in the vulnerability of rat hippocampal slices to brief and prolonged periods of hypoxia, Dev. Neurosci., 16: 301–306, 1994. [DOI] [PubMed] [Google Scholar]
  • 30. Sullivan B.L., Leu D., Taylor D.M., Fahlman C.S., Bickler P.E., Isoflurane prevents delayed cell death in an organotypic slice culture model of cerebral ischemia, Anesthesiology, 96: 189–195, 2002. [DOI] [PubMed] [Google Scholar]
  • 31. Abraham H., Losonczy A., Czeh G., Lasar G., Rapid activation of microglial cells by hypoxia, kainic acid, and potassium ions in slice preparations of the rat hippocampus, Brain Res., 906: 115–126, 2001. [DOI] [PubMed] [Google Scholar]
  • 32. Kato H., Takahashi A., Itoyama Y., Cell cycle protein expression in proliferating microglia and astrocytes following transient global cerebral ischemia in the rat, Brain Res. Bull., 60: 215–221, 2003. [DOI] [PubMed] [Google Scholar]
  • 33. Davis P.K., Johnson G.V.W., Energy metabolism and phosphorylation during apoptosis, Biochem. J., 340: 51–58, 1999. [PMC free article] [PubMed] [Google Scholar]
  • 34. Majno G., Joris I., Apoptosis, oncosis, and necrosis. an overview of cell death, Am. J. Pathol., 146: 3–15, 1995. [PMC free article] [PubMed] [Google Scholar]
  • 35. Syntichaki P., Tavernarakis N., The biochemistry of neuronal necrosis: rogue biology, Nat. Rev. Neurosci., 4: 672–684, 2003. [DOI] [PubMed] [Google Scholar]
  • 36. Choi D.W., Ischemia‐induced neuronal apoptosis, Curr. Opin. Neurobiol., 6: 667–672, 1996. [DOI] [PubMed] [Google Scholar]
  • 37. Kanduc D., Mittelman A., Serpico R., Sinigaglia E., Sinha A.A., Natale C., Santacroce R., Di Corcia M.G., Lucchese A., Dini L., Pani P., Santacroce S., Simone S., Bucci R., Farber E., Cell death: apoptosis versus necrosis (review), Int. J. Oncol., 21: 165–170, 2002. [PubMed] [Google Scholar]
  • 38. van Cruchten S., van Den Broeck W., Morphological and biochemical aspects of apoptosis, oncosis and necrosis, Anat. Histol. Embryol., 31: 214–223, 2002. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Cellular and Molecular Medicine are provided here courtesy of Blackwell Publishing

RESOURCES