3R-Project 121-10

A new in vitro model to study therapeutic approaches to improve spinal cord regeneration and repair after injury

Roman Chrast¹ and Josef Kapfhammer²
¹Department of Medical Genetics, University of Lausanne, 1005, Lausanne, Switzerland
²Anatomical Institute, University of Basel, 4056, Basel, Switzerland
roman.chrast@unil.ch, josef.kapfhammer@unibas.ch

Keywords: mice; spinal cord; spinal cord repair / diseases; slices; reduction; replacement

Duration: 2 years Project Completion: 2012

Background and Aim
Spinal cord injury (SCI) affects millions of people worldwide with serious clinical consequences such as paraplegia and neuropathic pain. It is a topic of intense basic and applied research. Animal models of SCI have allowed a number of findings in the fields of repair and regeneration. However, such in-vivo models are extremely distressful for animals. Moreover, the use of a wide variety of lesion models, and their variability and complexity, limit the ability to identify new therapies. Up to now, no real in-vitro alternative exists. We suggest using organotypic cultures of mouse spinal cord slices (“slice model”) that we have recently developed (Bonnici and Kapfhammer, 2008). This “slice model” allows to generate reproducible SCI-like lesions and to study axonal regeneration. In the experiments covered by this grant, we plan to characterize our “slice model” at the histological and functional levels. To confirm its relevance in SCI research, we will experiment on therapeutic strategies which have been successfully tested in vivo. By means of these experiments, we hope to demonstrate the possibility to perform SCI-related research without the need of in-vivo models.

Method and Results
in progress (present status)
We plan to start with the characterization of the “slice model” at the cytoarchitectonic and functional levels. We will use retrograde tracing and immunostaining of neurons and their axons to characterize the slice and the lesion site. These experiments will be complemented by functional characterization of the “slice model” by recordings of compound action potentials (CAPs) to test neuronal activity generation and conduction through the slice. Finally, we plan to validate our “slice model” by studying the effect of pharmacological treatments, previously shown to enhance regeneration after SCI in vivo (anti-Nogo A antibodies and Cethrin (a Rho antagonist)), on axonal regeneration in vitro.

Conclusions and Relevance for 3R
Successful characterization and implementation of the proposed “slice model” may substantially help to reduce animal experimentation in the field of SCI where any in-vivo model is by definition extremely debilitating.

References
1. Bonnici B, Kapfhammer JP (2008) Spontaneous regeneration of intrinsic spinal cord axons in a novel spinal cord slice culture model. Eur J Neurosci 27:2483-2492.

Figures

Figure 1: Spinal cord cultures mature and have the same ventro-dorsal polarity as adult tissue acute slices. Sagittal spinal cord organotypic slice culture from P4 mouse pups after 14 days in vitro. The calretinin-positive neurons (red) are distributed in the dorsal (D) part of the culture. Neurofilament H (non phosphorylated)-positive motoneurons (green, arrowheads) are present in the ventral (V) part of the culture. Scale bar: 200 µm.