Methodology: Eighteen rats had their gastrocnemius muscle denervated by transection of the tibial nerve. Control denervation, immediate reinnervation or sensory neurotization (sensory protection) was performed. Total RNA was extracted from the gastrocnemius muscle of the operated and control contralateral limb from 3 animals within the three experimental groups. Fluorescently labelled cDNA copies of the total RNA pool were prepared and were hybridized to the 15K mouse cDNA microarrays (UHN, Toronto, Ontario). Scanning of the microarrays was performed with Biopackard ScanArray. Quantitation of the intensities of the scanned images was performed with QuantArray Version 3 image analysis using the Adaptive method. RNA from each animal was run at least in duplicate with fluorophor reversal experiments. Ratios of the fluorescence intensity of the experimental cDNA/reference cDNA were expressed to the log base 2 for data analysis. Statistical analysis of the data was performed using Significance Analysis of Microarrays (SAM) software to look for those genes whose expression changed significantly in the muscle of the operated limb relative to the reference within each experimental group, and between experimental groups. Hierarchical clustering of genes identified as being significantly differentially expressed by SAM analysis was performed using GeneTraffic software.

Results: SAM analysis identified 367 genes as significantly differentially expressed in the gastrocnemius muscle of the Denervated group, 69 genes in the Immediate Repair group and 275 in the Sensory Protection group at the 1 month time point. Hierarchical clustering of these genes revealed the pattern of gene expression to be virtually identical between the Denervated and Sensory Protection groups, and both different from the Immediate Repair group. This visual impression was confirmed by the SAM analysis across groups. 71 genes were significantly differentially regulated in the Immediate Repair group compared to the Denervated group, but there was no statistically significant difference found between the Denervated and Sensory Protection groups. Of the significant genes 50% were known, and 50% had defined EST segments or are novel genes. Genes not previously identified as associated with muscle innervation were also identified and the subject further investigation.

Conclusion: Gene regulatory changes following denervation and reinnervation can be identified by microarray assessment and categorized. This permits identification of novel genes for evaluation.