Microarray and in Situ Hybridization Analyses of Natural Spinal cord Regeneration

Salamander is a promising model for spinal cord injury (SCI) because it is the only vertebrate capable of complex organ regeneration in the adult phase. Unfortunately, little is known about mechanisms that allow salamander to naturally regenerate complex organs such as spinal cord, and how these mechanisms relate to mammalian models of SCI. Here, we propose the first genome scale analysis of gene expression during natural spinal cord regeneration in a model salamander, Mexican axolotl (Ambystoma mexicanum). The specific aims of the proposal are to: (1) Profile gene expression during the ependymal response of natural spinal cord regeneration, and (2) Optimize and implement...automated in situ hybridization on a robotic workstation to localize gene expression in the regenerating salamander spinal cord. Gene expression will be measured during the ependymal response by micro-array analysis, using custom DNA oligonucleotide chips. Transcripts that are identified as significantly up or down regulated will be examined further by in situ hybridization, which will be optimized on an existing robotic workstation to localize expression patterns within cross-sections of spinal cord collected during the first 12 days of regeneration. Analysis of gene expression data and in situ images will reveal candidate genes and gene pathways from salamander that may enhance regeneration, prevent secondary neuronal injury, and restore function following spinal cord injury in human.