Introduction
Gene expression changes with potential effects on cortical plasticity have not been studied in great detail. We postulated that genes that are implied in plasticity or recovery are characterized by 1.) their persitent regulation over long time intervals after ischemia, or their induction in a delayed mode after ischemia, and/or 2.) their regulation in the periinfarct area, or in the contralateral homotopic cortex. In order to study these events we chose the photothrombotic model of cortical ischemia, as the infarct is small and defined, does not interfere with survival of the animals, and animals are known to recuperate from the lesion, implying active recovery mechanisms. We applied a sensitive fragment display technique, restriction-mediated differential display (RMDD) to systematically study gene expression changes following the lesion at 6 h, 2 days, and 3 weeks to observe long-term changes.
Material and Methods
Photothrombotic ischemia was induced using an i.v. infusion of the photosensitive dye rose bengal, and illumination of the sensory-motor cortex with a light source. Using coronal sections, periinfarct areas were dissected by punch biopsies, and processed for RNA extraction. The RMDD protocol was performed as described 1 . Quantitative PCR was done using the Lightcycler system (Roche Diagnostics). Immunohistochemistry was performed on 2 μm paraffin sections.
Results
We were able to identify over 50 genes that were regulated at each time point examined, a surprisingly high number of which were regulated in the contralateral homotopic cortex. Quantitative PCR was used to confirm the regulation of selected genes, and immunohistochemistry was used to demonstrate the pattern and cellular origin. Several candidates for involvement in cortical plasticity processes could be identified that follow the requirements outlined above. Further work is needed to indeed prove the contribution of these genes to plasticity.
Conclusion
The extent of differential gene regulation even at late intervals after cortical ischemia was unexpected and indicates strong plastic reactions 3 weeks after ischemia in the ipsilateral infarct-adjacent cortex, and in the homotopic contralateral context. Two groups of genes that appear particularly interesting are those related to neurogenic events (e.g. nestin and stathmin), and synaptic events (e.g. synaptotagmin 4). Our data have potential implications for strategies to enhance post-stroke rehabilitation.