Abstracts

RATIONALE: Epilepsy, a frequent sequala of penetrating head injury, becomes manifest after a latent period. In the rat undercut model of posttraumatic epileptogenesis, epileptiform potentials can be evoked in neocortical slices in vitro after a latency of ~ 2 weeks. However, focal treatment with tetrodotoxin (TTX) in vivo during a critical period of the first three days following injury, prevents epileptogenesis in this model. We examined mRNA changes occurring during this critical period with and without treatment, to better understand epileptogenesis and help identify potential new treatment targets.
METHODS: Partial neocortical isolations were performed in 4-week-old male Sprague/Dawley rats, and Elvax resin with or without TTX was placed subdurally over the lesions. Control animals underwent anesthesia, but no surgery. Treated animals were selected that demonstrated some, but not severe, neurological deficits, to ensure effective TTX-treatment. Three days post injury, cortical isolations and control cortices were excised under a surgical microscope. RNA was isolated, and labeled cRNA synthesized and fragmented using standard protocols, for hybridization to probes on Rat Genome U34A gene microarrays (Affymetrix). RNA from at least two different animals was combined per array, and three separate arrays (different animals) were used per each test condition: untreated undercut neocortex, TTX-treated undercuts, and na[iuml]ve controls. To find significant changes in the amounts of transcripts present, nine individual comparisons of the three arrays of a test condition to each of the three arrays of other test conditions were performed using Microsuite Array 5.0 (Affymetrix) and analyzed with Data Mining Tool 3.0 (Affymetrix).
RESULTS: Comparisons of individual undercut arrays to each array of naive control cortex revealed a significant increase of signal intensity in 11.3 [plusminus] 0.3 % of transcripts, and a significant decrease in 10.5 [plusminus] 0.4%. Comparisons of epileptogenic (untreated) undercut cortex versus nonepileptogenic (TTX-treated) cortex revealed significant increases in 4.6 [plusminus] 0.6 % of transcripts and significant decreases in 9.0 [plusminus] 1.2 %. ([plusminus] values are standard error of the mean). Using more stringent criteria requiring changes to be present in all possible (nine) comparisons between individual arrays of different groups, comparison of TTX-treated undercut to untreated undercut arrays revealed significant increases only in 12 transcripts, and significant decreases in 87 transcripts. Similar stringent comparisons between arrays of undercut and na[iuml]ve neocortex revealed significant increases in 561 transcripts, and decreases of 372 transcripts.
CONCLUSIONS: Although there are numerous changes in RNA expression following injury, not all changes appear to be critical to epileptogenesis as there are fewer differences between epileptogenic and nonepileptogenic-lesioned cortex, than between epileptogenic and na[iuml]ve cortex.
[Supported by: NIH Grants NS 02167, NS12151 , and the Phil N. Allen Trust.]