Cocultures of Rat Sensorimotor Cortex and Spinal Cord Slices to Investigate Corticospinal Tract Sprouting

Abstract

Experimental study of corticospinal axonal sprouting in an organotypic slice culture model. To develop an in vitro model that simplifies the study of various factors regulating neuronal regeneration. Spinal cord injury leads to permanent neurologic damage, mainly due to the inability of the adult central nervous system to regenerate. Much attention has been focused on promoting axonal regeneration and sprouting, either by exogenous administration of various neurotrophic factors or by the antagonization of factors inhibiting regeneration. An in vitro system that allows coculture of slices from rat sensorimotor cortex and spinal cord (p4) was established. Two groups of cultures were investigated: In the first group, intact spinal cord slices were cultured adjacent to sensorimotor cortex slices, while in the second group the spinal cord slices were sagittally cut into halves, with the sectioned interface placed directly adjacent to the sensorimotor cortex, to prevent the spinal white matter from interference. Each group was further divided into 2 subgroups: The neurotrophin-3 (NT-3) group, where the culture medium contained 50 ng/mL NT-3 and the control group treated with normal culture medium. Sensorimotor cortex pyramidal neurons were anterogradely labeled with Mini-Ruby, a 10 kD biotinylated dextran amine. Cocultures of cortical and spinal cord tissue were propagated in vitro, and axonal sprouting occurred. The group of cocultures treated with NT-3 showed an improved cortical cytoarchitecture, and sprouting axons were more frequently observed. In NT-3-treated cocultures 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 cord pia mater and white matter were 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 neurotrophic factors promote axonal sprouting. Cocultures of sensorimotor cortex and spinal cord slices combined with anterograde axonal labeling could provide a valuable in vitro model for the simplified screening of factors influencing corticospinal tract regeneration.