Abstracts

Rationale: Like other neurodegenerative disorders, epilepsy is characterized by neuronal loss. Temporal lobe epilepsy is the most common form of difficult to treat epilepsy and is frequently associated with hippocampal sclerosis, a lesion characterized by reactive gliosis and loss of pyramidal- and interneurons in specific hippocampal cell layers. Replacement of lost cells seems to be a vital step for functional repair of the brain. A promising technique for replacement of lost cells is neurotransplantation. This technique tries to repair damaged neuronal networks or to deliver anti-epileptic substances by means of cell transplantation. In this study we evaluate if predifferentiated pyramidal neurons, derived from mouse embryonic stem cells, are able to survive in a kainic acid (KA) cortical lesion and in the sclerotic hippocampus of the intrahippocampal KA status epilepticus mouse model. Methods: In vitro generated tau-Green Fluorescent Protein (GFP) mouse embryonic stem cells were differentiated to precursors of pyramidal neurons and transplanted in a cortical and hippocampal lesion induced by focal injection of KA three days before grafting. In a first experiment, a cortical lesion was induced in mice by focal injection of 200 ng KA in 50 nl saline. Three different cell numbers were transplanted in the lesion [1,000 (n=4); 5,000 (n=6) and 25,000 cells (n=6) in 0.5 l medium]. In a second experiment an intrahippocampal lesion was induced by focal injection of 100 (n=5) or 200 ng KA (n=4), dissolved in 50 nl, saline and 700 cells in 0.5 l medium were grafted in the sclerotic hippocampus. Four weeks after transplantation mice were transcardially perfused to evaluate the presence of GFP-positive neuronal projections from grafted cells. Results: In the first experiment, a dense network of projections were found in KA lesioned cortex, in 5 out of 6 mice grafted with 25,000 cells, in 4 out of 6 mice grafted with 5,000 cells and in 1 out of 4 mice grafted with 1,000 cells. A high number of axonal projections, running down along the lower layers of the cortex and along the external capsule, were seen. These projections were strikingly similar to the projections of the endogeneous cortical neurons. In the second experiment, a dense network of GFP positive neuronal projections were found in the sclerotic hippocampus in 3 out of 4 mice, injected with 200 ng KA, and in 2 out of 5 mice, injected with 100 ng KA. The projection pattern of grafted neurons was very similar in the successfully grafted mice. GFP positive neuronal fibers were confined to the ipsilateral and contralateral hippocampus and found in all layers of the hippocampal formation. Conclusions: These promising results show that mouse embryonic stem cells, in vitro predifferentiated to pyramidal neurons, are able to survive in a KA cortical lesion and in a sclerotic hippocampus. Further research will be done to determine the functional integration of the grafted neuronal precursors.