Abstracts

RATIONALE: Tuberous sclerosis (TSC) is a multiorgan autosomal dominant disorder caused by mutations of either TSC1 or TSC2 genes. In the brain, manifestations typical of TSC include cortical tubers (i.e., clusters of heterotopic neurons) and subependymal nodules or giant cell astrocytomas. These structural malformations are thought to cause neurological dysfunctions, including epilepsy. The Eker rat, which carries a spontaneous germline mutation of the TSC2 gene, has been studied as an animal model of TSC. Although pathological analysis of Eker rat brain reveals subependymal and subcortical hamartomas, none of these animals develops cortical tubers with [dsquote]giant[dsquote] or [dsquote]balloon[dsquote] cells typical of TSC tubers. In the present study, we used postnatal irradiation as a [dsquote]second hit,[dsquote] in an attempt to induce [dsquote]loss of heterozygosity[dsquote] in Eker TSC2 +/- cells, and thus exacerbate cortical abnormalities that might be linked to seizures.
METHODS: Wildtype and Eker (TSC2 +/-) rats were subjected to full body irradiation within 3 days of birth. Twenty-one rats (5 wildtype (WT) non-irradiated, 8 WT irradiated, 3 Ekers non-irradiated, and 5 Ekers irradiated) were examined at postnatal age 3 months. All rats were tested for seizure susceptibility by measuring latencies to flurothyl-induced seizures. Rats were subsequently prepared for histological/ immunocytochemical processing, using markers for neuronal and glial characterization.
RESULTS: Seizure threshold testing revealed that for both WT and Eker rats, irradiation led to significantly shorter seizure latencies (p=.004 and p[lt].001, respectively). While there was no latency difference between WT and Eker non-irradiated rats, irradiated Ekers showed slightly shorter latencies than irradiated WT rats (p=.056). Except for a reduction in the size of the dentate gyrus (seen in both groups), irradation generated no gross brain abnormalities, and no aberrations in development. However, all of the irradiated Eker brains exhibited at least two abnormal histological features in their morphology: 1) Subcortical/subependymal hamartoma-like aggregates of large, irregularly-shaped cells, which stain lightly with hematoxylin and eosin; cell bodies and their large processes show immunoreactivity for GFAP and GABA. 2) Abnormal large cell bodies (cytomegalic neurons) with long extended processes in normal-appearing neocortical and subcortical regions; although usually seen as isolated cells, they sometimes appeared in clusters of 2 to 6 cells. None of these findings were seen in non-radiated Ekers, or in irradiated or non-irradiated WT controls.
CONCLUSIONS: Irradiation produces malformations in TSC2 +/- (Eker) rats which resemble some features of TSC in humans, and which may contribute to greater seizure susceptibility. These results suggest that a [dsquote]second hit[dsquote] approach may provide insight into the generation of TSC-related brain abnormalities that are correlated with seizure activity.
[Supported by: NIH NS 18895.]