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ABSTRACT. To elucidate the mechanisms of ischemia-mediated myopathy using in vitro model, changes of purine nucleotides, membrane lipid peroxidation(TBARS), intracellular calcium ([Ca 2+ ]i)levels, generation of free radicals, and deoxyribonucleic acid (DNA) fragmentation were examined in mouse-derived C 2 C 12 myotubes under the condition with an inhibition of glycolytic and oxidative metabolism as the ischemic condition. In purine nucleotides, intracellular adenosine triphosphate (ATP) and guanosine triphosphate (GTP) concentrations rapidly and significantly decreased after the treatment with ischemia. No remarkable differences were observed in other purine nucleotides, with the exception of inosine monophosphate (IMP) and extracellular hypoxanthine levels, both of which increased significantly during the ischemia. The lactate dehydrogenase activity in culture supernatant of C 2 C 12 myotubes increased significantly from 2 to 4 hr after the ischemia. On the generation of free radicals, no spectrum was detected in supernatants throughout the observation period, whereas supernatant TBARS concentration increased rapidly and significantly after the ischemia. The relative intensity of [Ca 2+ ]i significantly increased after the ischemia. On the fragmented deoxyribonucleic acid(DNA), no TUNEL positive cells was detected in C 2 C 12 myotubes after 1 hr of the ischemia, however the positive cell percentage subsequently increased. From these results, it was suggested that the ischemic condition induced changes of membrane permeability and increase of [Ca 2+ ]i, both of which lead to cell membrane damage, although a free radical generation was not detected. The ischemic condition also induced the release of substrate hypoxanthine for free radical generation and might initiate the apoptotic pathway in C 2 C 12 myotubes. KEY WORDS: apoptotic pathway, C 2 C 12 myotube, chemical ischemia, free radical generation, purine nucleotide.

The cerebellar vermis defect (cvd) rat is a mutant characterized by a cerebellar vermis defect and fused cerebellar hemisphere. This mutant rat also exhibits heterotopic dysplastic cerebellum, especially in the cerebello-pontine junctional zone. We focused on the auditory system of the cvd rat. The homozygous cvd rats exhibited bilaterally indistinct ABR (auditory brainstem response) waves or prolongation of latency in each ABR component. The number of the spiral ganglion neurons in the Rosenthal's canal was apparently decreased in the homozygous rats. Present study indicates the homozygous cvd rats had auditoly disorders concerned with cerebellar malformation.The cerebellar vermis defect (cvd) rat is a mutant characterized by a cerebellar vermis defect and fused cerebellar hemisphere (8). This mutant rat also exhibits heterotopic dysplastic cerebellum, especially in the cerebello-pontine junctional zone (8). These characters are inherited by a simple autosomal recessive mode (9). Therefore, the cvd mutant may provide a useful animal model for studying the pathogenesis of the cerebellar vermis defect and cerebellar cortical dysplasia.The histogenesis of cerebellum in the cvd rat has been investigated; many cerebellum-constituting cells penetrated into the pons, the external granule cells (EGCs) aggregated perivascularly, and the aggregated EGCs migrated radially around the vessels, resulting in the heterotopic and dysplastic cerebelluin (10). However, the details of the morphological and functional abnormalities have not been reported in the cvd rat except for the cerebellum. Auditoly disorders were reported in some mutant rats, such as (5) and spontaneously epileptic (SER) rat (7). In the present study, we focused on the auditory system of the cvd rat. MATERIALS AND METHODSThe cvd rats originated from spontaneously ataxic rats found in the LEW strain. The mutant colony has been kept as a hybrid strain with both LEW and Donryu genetic backgrounds (8). We mated heterozygous (+/cvd) females and heterozygous (+/cvd) or homozygous (cvd/cvd) males, and obtained the homozygous (cvri/cvd), heterozygous (+/cvd) and wild type (+/+) rats used in this study. The used rats were applied to mate with proven-l1et-erozygous rats to check the genotype. Four homozygous, 3 heterozygous, and 3 wild type rats aged at 16 weeks were examined.Auditory brainstem response (ABR) was recorded on the animals treated with a sedative xylazine hydrochloride (0.1 mg/kg, I.M.), which was known to have no effect on AER pattern (2). Click stimuli of alternating polarity generated by 4 kHz single sine waves were used at a rate of 20 Hz by an acoustic stimulator (Nihon Kohden, SMP-3100). Clicks were delivered through an inner type earphone (SONY, MDR-E464), which was attached directly to the

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