Embryonic organotypic cultures represent a useful model to investigate neuronal maturation and physiology of the spinal cord. In fact, organotypic slices maintain the basic cytoarchitecture of the spinal cord, including the dorsal-ventral orientation of the spinal segments, and the fundamental properties of network dynamics. Furthermore, they allow long term studies of their developmental processes and their ability to adjust to changes in the local environment (Sibilla and Ballerini, Progress in Neurobiology 2009, in press). Thus, these cultures may offer the opportunity to investigate certain mechanisms underlying the response of spinal neurons to injury in terms of alterations in molecular, cellular and circuit characteristics following experimental conditions that mimic a spinal cord lesion.

The main purpose of the present study was to evaluate the consequences of the glutamate analogue kainate on survival of embryonic organotypic spinal cord cultures and the potential use of drugs to limit any toxic effects. Indeed, it is widely believed that hyperactivation of glutamate receptors during the early phase of spinal cord injury represents a major contributor to subsequent damage even if the precise mechanisms of this effect remain poorly understood.

Previous studies by our group, using the in vitro spinal cord of the neonatal rat, show that kainate (1 mM) applied for 1 h largely destroyed neurons around the central and ventral gray matter with only modest white matter damage (Taccola et al., Neuroscience 2008, 155:538-555). Nevertheless, the in vitro spinal cord preparation is unsuitable for investigating long term consequences of the initial excitotoxicity, a phenomenon which is better explored with organotypic cultures.

Embryonic (E13) rat organotypic spinal slices at 22 days in vitro (DIV) were sensitive to kainate treatment (1 h) that significantly increased the percentage of pyknotic nuclei (observed with DAPI nuclear staining). Moreover, the damage appeared to be an ongoing process because 4 and 24 h later (despite extensive washout of kainate), histological examination demonstrated further intensification of cell loss. To characterize the topographical distribution of pyknotic nuclei, slices were divided into three regions, namely dorsal, central, and ventral. The largest number of pyknotic nuclei was predominantly found in dorsal areas. Immunohistochemistry with the neuron specific marker NeuN confirmed strong disappearance of neurons. These findings suggest that kainate-mediated injury to organotypic cultures is a reliable model to study molecular and cellular process regarding spinal injury and may be represent an advantageous test system for exploring neuroprotective drugs.