title: Methods to induce primary and secondary traumatic damage in organotypic hippocampal slice cultures.
abstract: Organotypic brain slice cultures have been used in a variety of studies on neurodegenerative processes [K.M. Abdel-Hamid, M. Tymianski, Mechanisms and effects of intracellular calcium buffering on neuronal survival in organotypic hippocampal cultures exposed to anoxia/aglycemia or to excitotoxins, J. Neurosci. 17, 1997, pp. 3538-3553; D.W. Newell, A. Barth, V. Papermaster, A.T. Malouf, Glutamate and non-glutamate receptor mediated toxicity caused by oxygen and glucose deprivation in organotypic hippocampal cultures, J. Neurosci. 15, 1995, pp. 7702-7711; J.L. Perez Velazquez, M.V. Frantseva, P.L. Carlen, In vitro ischemia promotes glutamate mediated free radical generation and intracellular calcium accumulation in pyramidal neurons of cultured hippocampal slices, J. Neurosci. 23, 1997, pp. 9085-9094; L. Stoppini, L.A. Buchs, D. Muller, A simple method for organotypic cultures of nervous tissue, J. Neurosci. Methods 37, 1991, pp. 173-182; R.C. Tasker, J.T. Coyle, J.J. Vornov, The regional vulnerability to hypoglycemia induced neurotoxicity in organotypic hippocampal culture: protection by early tetrodotoxin or delayed MK 801, J. Neurosci. 12, 1992, pp. 4298-4308.]. We describe two methods to induce traumatic cell damage in hippocampal organotypic cultures. Primary trauma injury was achieved by rolling a stainless steel cylinder (0.9 g) on the organotypic slices. Secondary injury was followed after dropping a weight (0.137 g) on a localised area of the organotypic slice, from a height of 2 mm. The time course and extent of cell death were determined by measuring the fluorescence of the viability indicator propidium iodide (PI) at several time points after the injury. The initial localised impact damage spread 24 and 67 h after injury, cell death being 25% and 54%, respectively, when slices were kept at 37 degrees C. To validate these methods as models to assess neuroprotective strategies, similar insults were applied to slices at relatively low temperatures (30 degrees C), which is known to be neuroprotective [F.C. Barone, G.Z. Feuerstein, R.F. White, Brain cooling during transient focal ischaemia provides complete neuroprotection, Neurosci. Biobehav. Rev. 1, 1997, pp. 31-44; V.M. Bruno, M.P. Goldberg, L.L. Dugan, R.G. Giffard, D.W. Choi, Neuroprotective effect of hypothermia in cortical cultures exposed to oxygen glucose deprivation or excitatory aminoacids, J. Neurochem. 4, 1994, pp. 387-392; G.C. Newman, H. Qi, F.E. Hospod, K. Grundhmann, Preservation of hippocampal brain slices with in vivo or in vitro hypothermia, Brain Res. 1, 1992, pp. 159-163; J.Y. Yager, J. Asseline, Effect of mild hypothermia on cerebral energy metabolism during the evolution of hypoxic ischaemic brain damage in the immature rat, Stroke, 5, 1996, pp. 919-925.]. Low temperature incubation significantly reduced cell death, now being 9% at 24 h and 14% at 67 h. Our results show that these models of moderate mechanical trauma using organotypic slice cultures can be used to study neurodegeneration and neuroprotective strategies.
authors.affiliation: Playfair Neuroscience Unit, McL 12-413, Toronto Western Hospital, Department of Medicine (Neurology), University of Toronto, 399 Bathurst Street, Toronto, Canada.
authors.forename: Y
authors.initials: Y
authors.lastname: Adamchik
authors.forename: M V
authors.initials: MV
authors.lastname: Frantseva
authors.forename: M
authors.initials: M
authors.lastname: Weisspapir
authors.forename: P L
authors.initials: PL
authors.lastname: Carlen
authors.forename: J L
authors.initials: JL
authors.lastname: Perez Velazquez
mesh_terms: D000818:Animals; D001930:Brain Injuries / Q000209:etiology* / Q000473:pathology / Q000503:physiopathology; D016923:Cell Death; D006624:Hippocampus / Q000293:injuries* / Q000473:pathology / Q000503:physiopathology; D008297:Male; D009462:Neurology / Q000379:methods*; D009474:Neurons / Q000473:pathology / Q000502:physiology; D009924:Organ Culture Techniques; D051381:Rats; D017208:Rats, Wistar; D013696:Temperature; D013997:Time Factors; D014949:Wounds, Nonpenetrating / Q000209:etiology* / Q000473:pathology / Q000503:physiopathology