The aim of this experimental study was to provide an in vitro model suitable for the investigation of the complex interactions of neurons with non-neuronal cells that take place throughout the degenerative and regenerative processes induced by spinal cord injury.

Organotypic spinal cord slice cultures (OSCSC) were prepared from postnatal Wistar rats (p0–12), were sustained in vitro up to 12 days and characterized by immunohistochemistry by well-established markers such as NeuN, Calbindin, GFAP, IB4 and Nestin.

Calbindin+ neurons, distributed across the entire gray matter, were visible also after longer culture periods. NeuN+ neurons were best preserved in the dorsal horn, whereas large NeuN+ and ChAT+ motoneurons in the ventral horn vanished after 3 days in vitro. GFAP+ astro-cytes, initially restricted to the white matter, invaded the gray matter of OSCSC early during the culture period. Microglial cells, stained by Griffonia simplicifolia isolectin B4, were rapidly activated in the dorsal tract and in the gray matter, but declined in number with time. Nestin-immunoreactivity was found in animals of all age groups, either in cells interspersed in the ependymal lining around the central canal, or in cells resembling protoplasmic astrocytes. OSCSC derived from p0 or p3 animals showed a better preservation of the cytoarchitecture than cultures derived from older animals.

In summary, OSCSC contain defined neuronal populations, the cytoarchitecture is partially preserved, and the glial reaction is self-limited. Our model of OSCSC could prove useful in future experiments on the patho-physiology of spinal cord injury

Aim of this experimental study was to develop an in vitro model that simplifies the study of various factors regulating neuronal regeneration.

An in vitro-system that allows co-culture of slices from rat motorcortex and spinal cord (p4) was established. Two groups of cultures were investigated: In the first group, intact spinal cord slices were cultured adjacent to motorcortex slices, while in the second group the spinal cord slices were sagitally cut into halves, with the sectioned interface placed directly adjacent to the motorcortex, in order to prevent the spinal white matter from interference. Each group was further divided into two subgroups: The NT-3 group, where the culture medium contained 50 ng/ml NT-3 and the control group treated with normal culture medium. Motorcortex pyramidal neurons were anterogradely labelled with MiniRuby, a 10 kD biotinylated dextran amine.

After 4 days the co-cultures were propagated, and axonal sprouting occurred. The group of co-cultures treated with NT-3 showed an improved cortical cytoarchitecture, and sprouting axons were more frequently observed. In NT-3-treated co-cultures where spinal cord gray matter was directly opposed to cortical slices sprouting axons entered the adjacent spinal cord tissue. This phenomenon was not observed if spinal white matter was opposed to the cortical slices, or if NT-3 was absent.

Our data suggest that the absence of repellent factors such as white matter and the presence of neuro-trophic factors promote axonal sprouting. Co-cultures of motorcortex and spinal cord slices combined with anterograde axonal labelling could provide a valuable in vitro model for the simplified screening of factors influencing corticospinal tract regeneration