DZF-Project: Richard T. Robertson

Tissue slice culture model for studies of neural degeneration and regeneration

Richard T. Robertson, Ph.D.

University of California, Irvine, California, 29697-1280, USA


Keywords: cerebral cortex, cholinergic, in vitro, organotypic, slice culture

Begin and End of the Project: 1994 - 1996

Background and Aim

Background: The study of brain circuitry has attracted the attention of a large number of neuroscientists in recent years, and we now have a good understanding of the basic morphological and physiological organization of the nervous system.  Research is moving toward understanding how the nervous system is affected by damage. Unfortunately, these studies of the recovery of nerve cells most commonly involve the intentional surgical injury to the brains of these animals, and these injuries are too often accompanied by discomfort and suffering by the animals being studied. For the scientific and ethical reasons stated above, the tissue slice culture approach is preferable. With the tissue slice culture approach, tissue from one animal can be used to produce approximately 20 slices to be used in 20 different experiments. Thus, although tissue slice culture studies will still involve the use of some animals, this approach will markedly reduce the total number of animals used in these experiments.

Aim: The plan was to study issues of neuronal injury, degeneration and regeneration using the organotypic tissue slice method originally developed by Gähwiler.

Methods and Results

Slices of basal forebrain and of cerebral cortex are taken from newborn rats and maintained in culture for periods up to one month. The newborn rats are deeply anesthetized and sacrificed without pain. Tissue from each animal produce about 20 cultures, thus providing much more experimental data than if whole animals were used in the experiments. These tissues develop quite well in culture, and express many of the features that are characteristic of these neural systems in the whole brain. After these tissue slice are established in culture, small foci of damage can be produced in parts of the tissue using electric current or specific toxins. The effects of such controlled lesions are then studied in damaged neurons by using morphological techniques.

Scientific papers resulting from this funding include:

Baratta, J., J.W.A. Marienhagen, D. Ha, J. Yu and R.T. Robertson. Cholinergic innervation of cerebral cortex in organotypic slice cultures: sustained basal forebrain and transient striatal cholinergic projections.  Neuroscience, 1996, 72, 1117-1132.

Baratta, J., D.H. Ha, J. Yu and R.T. Robertson, Evidence for target preferences by cholinergic axons originating from different subdivisions of the basal forebrain.  Developmental Brain Research, 2001, 132, 15-21.

Lee, Y-S., J. Baratta, J. Yu, V.W. Lin, and R.T. Robertson,  aFGF promotes axonal growth in rat spinal cord organotypic slice cultures.  Journal of Neurotrauma, 2002, 19, 357-367.

Eliason, D.A., S.A. Cohen, J. Baratta, J. Yu, and R.T. Robertson,  Local proliferation of microglial cells in response to neocortical injury in vitro.  Developmental Brain Research, 2002, 137, 75-79.

Oishi, Y., J. Baratta, R.T. Robertson and O. Steward,  Assessment of factors regulating axon growth between the cortex and spinal cord in organotypic co-cultures: effects of age and neurotrophic factors. Journal of Neurotrauma, 2004, 21, 339-356.

Conclusions and Relevance for 3R

The use of the tissue slice culture approach allows much greater control over the biochemical environment of the tissue under study, and will allow precise assessments of a variety of putative therapeutic agents in the recovery of these damaged neurons.