3R-Project 64-97

Use of three-dimensional brain cell cultures as a model for ischemia-related research

Paul Honegger and Beatriz Pardo
Department of Physiology, University of Lausanne, 1005 Lausanne, Switzerland.
paul.honegger@unil.ch

Keywords: rat; brain; ischemia; cell cultures: co-cultures; cell cultures: organ-specific; refinement

Duration: 2 years Project Completion: 2000

Background and Aim
Ischemic brain damage in human due to stroke or cardiac arrest is one of the most serious halth problems in our society. Adequate protective and therapeutic strategies require knowledge about the mechanisms involved. Most of the relevant research is carried out in animal models. The nature of these interventions and the lesions inflicted involve a high degree of distress. Models in vitro require a three-dimensional arrangement of the cells in order to reproduce the situation in a tissue. In this respect, aggregate cell cultures represent a highly promising in vitro system. These cultures prepared from fetal rat telencephalon and grown in a chemically defined medium reach a high degree of cellular differentiation and organization, ressembling in many aspects brain tissue in vivo. The present investigations were designed to test the suitability of this in vitro system for ischemia-related investigations.

Method and Results
Aggregate cell cultures containing either mixed rat brain cells or predominantly neurons derived from freshly isolated embryonic brain tissue can be obtained routinely by established procedures (1). Using biochemical and immunocytochemical criteria, we showed ischemia could be simulated in these cultures by a transient switch from rotatory to stationary culture conditions. This increased the medium-to-tissue glucose and oxygen gradients and caused selective neural cell death. The extent of the ischemia-induced damage correlated with the observed degree of glucose depletion (2). A similar pattern of adverse effects was obtained by transient glucose restriction. Further investigations showed that these deleterious effects were attenuated by antagonists of either NMDA receptors or L-type voltage-gated calcium channels, as well as by chelation of extracellular Ca²⁺. These results are in accord with the view that the influx of extracellular Ca ²⁺ is a critical event in ischemia-induced neuronal cell death (3, 4).
Work is now in progress to examine the implications of metabolic perturbations and glial reactivity in ischemia-induced neurodegeneration, and to evaluate potential strategies for neuroprotection.

Conclusions and Relevance for 3R
The results obtained so far indicate that aggregating brain cell cultures offer a useful in vitro model to investigate the mechanisms involved in ischemia. Promotion of this in vitro approach could lead to a reduction in the use of animal models for ischemia research in the future.

References
1. Honegger P, Monnet-Tschudi F. Aggregating neural cell cultures. In: Fedoroff S, Richardson A (eds) Protocols for for Neural Cell Culture 2nd edn. Totowa NJ: Humana Press, 1997; 25-49.

2. Pardo B, Honegger P. Selective neurodegeneration induced in rotation-mediated aggregate cell cultures by a transient switch to stationary culture conditions: a potential model to study uschemia-related pathogenic mechanisms. Brain Res 1999; 818:84-95.

3. Pardo B, Honegger P. Aggregating brain cell cultures as a model to study selective neurotoxic ischemia-related mechanisms. Toxicology in Vitro 1999; 13,543-547.

4. Honegger P, Pardo B. Separate neuronal and glial Na+,K+-ATPase isoforms regulate glucose utilization in response to membrane depolarization and elevated extracellular potassium. J Cerebr Blood Flow Metab 1999;19 (9): 1051-9.

5. Pardo B., and P. Honegger. Evaluation of aggregating brain cell cultures as a model for studying ischaemia-related neurodegenerative processes. In: M. Balls, A.-M. van Zeller and M.E. Halder, eds., Progres in the Reduction, Refinement and Replacement of Animal Experimentation. Elsevier Sci., 2000, pp. 241-247.

6. Honegger et al. Alteration of amino acid metabolism in neuronal aggregate cultures exposed to hypoglycemic conditions. J. Neurochem. 2002; 81, 1141-1151.