3R-Project 69-99

A human neural cell line as in vitro model system for the excitotoxic cascade during stroke

Gerd Bicker
University of Veterinary Medicine, Div. Cell Biology, 30173 Hannover Germany
gerd.bicker@tiho-hannover.de

Keywords: human; brain; pharmacology; ischemia; cell cultures: organ-specific; reduction; replacement; pharmacological testing

Duration: 2 years Project Completion: 2002

Background and Aim
Stroke is the third leading cause of death and an important cause of adult disability in industrialized countries. Most strokes are caused by an acute interruption of the brain’s arterial blood supply, which leads to tissue ischaemia. Ischaemic neurons deprived of oxygen and glucose rapidly lose ATP and become depolarized. The resultant complex ischaemic cascade triggers an excessive release of the excitotoxic transmitter glutamate; this substance causes changes in intracellular calcium homeostasis and results in the production of free radicals. Apart from immediate damage, the ischaemic cascade results in a wave of delayed cell death. The latter process might be prevented by administering neuroprotective substances that are active at the various stages of the ischaemic cascade.
Currently, investigations on the cellular processes occurring in ischaemic nervous tissue and screening tests for neuroprotective compounds are carried out mainly in rodents. In order to minimize the use of experimental animals, the aim of this project is to establish a human nerve cell culture system as an alternative model.

Method and Results
NT2 cells are a clonal human teratocarcinoma cell line that yield terminally differentiated neurons after treatment with retinoic acid (Pleasure et al., 1992). We investigated a cell culture system in which the NT2 neurons are stimulated to differentiate in the presence of co-cultured glial cells, obtained from the same cell line. In addition, we have established a method for the rapid differentiation of NT2 neurons using an additional cell aggregation step. Using a variety of antibodies against neural markers, terminal differentiation to the functional neuronal phenotype can be shown. After several weeks in culture, terminally diffrentiated NT-2 neurons are subjected to anoxic and/or hypoglycemic conditions and the resultant changes in neuronal viability monitored. The influence of additional parameters on neuronal survival, such as type of culture medium, pH, glutamate concentration, and specific neuroactive drugs are presently being evaluated.
When the neurons have end-differentiated, ischaemic conditions will be simulated and the resultant changes in neuronal viability monitored (see also 3R-INFO-BULLETIN Nr. 23). We will analyze, how pharmacological agents that rescue the ischaemic neurons from cell death, regulate cytosolic calcium levels and mitochondrial potential.

Conclusions and Relevance for 3R
This human cell culture system will allow ischaemic processes and the complex cellular mechanisms involved in ischaemic cell death to be investigated in vitro. Furthermore, it will be possible to use this model to screen potential neuroprotective substances for their ability to facilitate neuronal regeneration and repair. This model will help to reduce the number of experimental animals needed in pre-clinical drug testing and the development of neuroprotective treatment strategies.

References
1) Hartley, R. S., Margulis, M., Fishman, P. S., Lee, V. M.-Y., Tang, C.-M. (1999) Functional synapses are formed between human NTera 2 (NT2N, hNT) neurons grown on astrocytes J. Comp. Neurol. 407: 1-10.

2) Pleasure, S.J., Page, C. und Lee, V. M.-Y. (1992) Pure, postmitotic, polarized human neurons derived from Ntera 2 cells provide a system for expressing exogenous proteins in terminally differentiated neurons. J. Neurosience 12(5): 1802-1815.

3) Paquet-Durand F, Tan S. and Bicker G. (2003) Turning teratoma cells into neurons: Rapid differentiation of NT-2 cells in floating spheres. Dev. Brain Res. 142 : 161-167.

4) Paquet-Durand F, Tan S. and Bicker G. (2004) Hypoxic/ischemic cell damage in cultured human NT-2 neurons. Brain research 1011, 33-47.

5) Paquet-Durand F, Gierse A., and Bicker G. (2006) Diltiazem protects human NT-2 neurons against excitoxic damage in a model of simulated ischemia. Brain Research 1124, 45-54.

6) Paquet-Durand F and Bicker G. (2007) Human model neurons in studies of brain cell damage and neural repair, Current Molecular Medicine, Vol 7 in press.