Axonal regeneration in hippocampal and spinal cord organotypic slice cultures

Under normal conditions, axonal regeneration after lesions is not possible in mature CNS but can occur in embryonic and early postnatal nervous systems. In recent years, a number of possible strategies to enhance axonal regeneration and eventually treat spinal cord and brain injuries have been identified, some of which have been used successfully in animal experiments, but till now there is still no successful treatment available for patients. This problem is partly due to the complexity of the animal experiments which makes it difficult to compare different treatment strategies. In this project, we have used organotypic slice culture models to test the effectiveness of pharmacological compounds that interfere with various signal transduction mechanisms, to promote axonal regeneration. We used the entorhino hippocampal slice cultures to assess regeneration of entorhinal fibers projecting to the dentate gyrus after mechanical lesions and treatment. It was previously shown (Prang et. al., 2001) that there is a marked decrease in regenerating fibers when a lesion is made at 6 7 days in vitro or later in slices derived from postnatal day 5 6 mice. We took this as a control model where there is little spontaneous axonal regeneration, added treatments on the day of lesion and later traced for entorhinal axons with biotinylated dextran amine (BDA). In this study it was shown that compounds acting on the cAMP, PKC and G proteins can promote regeneration. Furthermore, we have identified the inhibition of the PI3 kinase pathway and the IP 3 receptor as potential drug targets that promote axonal regeneration.
In order to study axonal growth in a spinal cord environment we have developed a spinal cord longitudinal organotypic slice culture model which allowed us to follow axons along the rostro caudal extension of the spinal cord. Slices of cervical spinal cord were cut in the sagittal plane from early postnatal mice and were maintained in culture for various time periods up to 4 weeks. Histological and immunohistochemical stainings of the cultures have shown that these slice cultures maintain the ventro dorsal polarity of the spinal cord and that an intrinsic fibre projection develops which runs along the rostro caudal extension of the spinal cord slice culture. After mechanical lesion, these fibres have the ability to regenerate spontaneously demonstrating the intrinsic ability of the spinal cord for repair, but this ability is decreased with increasing time in culture. During the culture period the axons became myelinated and expressed synaptic markers. These cultures could thus serve also as a model for myelin formation and synaptogenesis.
We have analyzed the potential of axons from longitudinal spinal cord cultures to grow into an adjacent slice of cerebellar tissue. We could show that spinal cord axons do enter the cerebellar slice in particular when early postnatal spinal cord is combined with postnatal cerebellum. Pharmacological treatments were used to enhance axonal growth. Similarly to our findings in the entorhino hippocampal model, cAMP activators and PKC inhibitors promoted axonal growth from the spinal cord to the cerebellum. In cocultures of longitudinal spinal cord slices with cortical slices we have shown that fibers from the cortical slices grew extensively into the spinal cord slice and extended caudally for substantial distances. Our results demonstrate that organotypic slice cultures can be a useful tool to study axonal growth and regeneration. Intrinsic spinal cord axons have a considerable potential for spontaneous regeneration in the early postnatal period and are able to grow both through a mechanical lesion and into another tissue. Moreover, compounds interfering with signal transduction mechanisms, particularly cAMP, PKC, PI3 Kinase, G proteins and IP3 receptors, were able to promote axonal growth and regeneration in diverse slice culture models making them interesting drug candidates for the promotion of axonal regeneration.