Abstracts

Tuberous Sclerosis Complex (TSC) is an autosomal dominant human genetic disease caused by mutations of either TSC2 (tuberin) or TSC1 (hamartin) genes. In the CNS, TSC is characterized by cortical tubers, subependymal nodules, and abnormal giant cells. Clinical manifestations invariably include seizures and mental retardation. Eker rats carry a spontaneous germline mutation of [italic]TSC2[/italic] (TSC2 ^+/-). Although renal cell carinomas, subependymal and subcortical hamartomas have been found in these animals, little evidence for cortical tubers or abnormal giant cells exists. In the present study, we attempted to induce cortical tubers or giant cells using a prenatal [quot]second hit[quot] approach. Hydroquinone, a nongenotoxic carcinogen, was chosen for these studies because of its ability to increase renal cell carcinomas in the Eker rat (Yoon et al. 2001). Detailed histological and immunohistochemical studies were performed on brain sections from [quot]second hit[quot] litters as well as aged Eker rats.
Pregnant TSC2 ^+/- females were injected once a day with hydroquinone (HQ) according to the following protocols: (i) intraperitoneal (i.p.) injections of 50, 100, 150 mg/kg of HQ between embyonic days 15 and 20 ([italic]n[/italic] = 5) or (ii) i.p. injections of 50 or 100 mg/kg HQ between E8 and E19 ([italic]n [/italic]= 9). Offspring were sacrificed at p14 or p28 and tissue sections throughout the CNS were prepared and stained for cresyl violet and tuberin. In separate studies, brains of older Eker rats (~18 months old) that died of natural causes were sectioned for anatomical analysis. Sagital and coronal sections every 40 [mu]m were stained for NeuN (neuron-specific antibody), GFAP (glial-specific antibody), vimentin, nestin, tuberin, Hematoxylin and Eosin (H[amp]E), and cresyl violet.
Tissue sections stained with cresyl violet did not reveal any difference between HQ treated Eker (TSC2^+/-) rats and siblings (TSC2^+/+) at p14 or p28. None of the hallmark traits of human TSC including cortical tubers, hamartomas, or giant cell astrogliomas were found. However, abnormal giant cells in deep cortical layers were observed in brain sections from un-treated aged Eker rats (~18 mos.) . Abnormal giant cells observed with cresyl violet or H[amp]E, were GFAP- and tuberin-positive, but did not express NeuN. In addition, large tumors connected to the brain stem and cerebellum were observed in three of four aged Eker rats. Within tumors, large ectopic GFAP-positive cells with abnormal morphology could also be found, along with increased angiogenesis.
Our data suggest that cortical tuber formation in Eker rats is a rare event (Mizuguchi et al. 2000) and that prenatal exposure to a non-genotoxic carcinogen such as hydroquinone is not sufficient to induce tuber formation. However, with adavanced age, there may be an increased likelihood of tumor formation, and the emergence of giant cells in the Eker rat brain - each of these abnormalities could contribute to neurological problems associated with TSC. As such, further analysis of this rodent model may be warranted.
[Supported by: Tuberous Sclerosis Alliance.]