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Cathepsins are families of proteases that have been reported to play the key roles in neuroexcitotoxicity. The present study was sought to determine the effect of CBI, a cathepsin B inhibitor, in the prevention of neurobehavioral deficits after inhalant flurothyl-induced recurrent neonatal seizures in rats. We examined the expression pattern of autophagy-related genes at acute phase after the last seizures using western blot method, and evaluated behavioral deficits during postnatal day 28 (P28) to P35. The results showed improved neurological scores and learning ability in CBI-treated rats compared with the nontreated control. Flurothyl-induced increases in the ratio of LC3-II/LC3-I, Beclin-1 and Cathepsin-B were blocked by pre-treatment with CBI at 1.5, 3, 6 and 24 h after the last seizures in hippocampus and cerebral cortex by western blot analysis. Meanwhile, CBI also reversed flurothyl-induced down-regulation of Bcl-2 protein levels. Furthermore, in the long-term time point of 35 days (P35), PRG-1 mRNA and protein level in hippocampus and cerebral cortex of recurrent seizure group were up-regulated when compared to the control rats; meanwhile, the up-regulated expression of PRG-1 were robustly inhibited by CBI. These date demonstrated, for the first time, that lysosomal enzymes participate in neonatal seizure-induced brain damage and that modulation of cathepsin B may offer a new strategy for the development of therapeutic interventions for treatment of developmental seizure-induced brain damage. [ABSTRACT FROM AUTHOR]

Abstract: Zinc transporters (ZnTs) and plasticity-related genes (PRGs) both play the key roles in the formation of hippocampal mossy fiber sprouting, which is associated with cognitive deficits following developmental seizures. Here, for the first time, we report the timing of expression pattern of ZnT-1, ZnT-3 and PRG-1 in hippocampus and cerebral cortex following developmental seizures. A seizure was induced by inhalant flurothyl daily in neonatal Sprague–Dawley rats from postnatal day 6 (P6). Rats were assigned into the recurrent-seizure group (RS, seizures induced in 6 consecutive days) and the control group. At 1.5h, 3h, 6h, 12h, 24h, 48h, 7d and 14d after the last seizures, the mRNA level was detected using RT-PCR method; PRG-1 protein level was examined by Western blotting analysis. At an early period of 12h and 48h after the last seizures, both ZnT-1 and ZnT-3 showed significantly down-regulated mRNA level in the cerebral cortex of RS group than those at the corresponding time point in control group. In the long-term time point of 14d after the last seizure, ZnT-3 mRNA and PRG-1 protein level in hippocampus were up-regulated while the mRNA level of ZnT-1 down-regulated; in addition, there were up-regulated level of both the mRNA and protein level of PRG-1 and down-regulated mRNA level of ZnT-3 in the cerebral cortex of RS group when compared to the control. Taken together, these dates are consistent with an important role for ZnT-1, ZnT-3 and PRG-1 in the pathophysiology of the long-term adverse effects of recurrent neonatal seizure-induced hippocampal mossy fiber sprouting and cognitive deficit. [Copyright &y& Elsevier]

Abstract: Epilepsy in children is associated with a broad spectrum of cognitive deficits, which is associated with hippocampal mossy fiber sprouting. The underlying molecular mechanisms involved in mossy fiber sprouting in hippocampus following developmental seizures are not completely known. We studied the timing of cognitive dysfunction following neonatal seizures and the relation of this cognitive impairment to zinc transporter 1 (ZnT-1), 3 (ZnT-3), calcium/calmodulin-dependent protein kinase II (CaMK II), plasticity-related gene 1 (PRG-1) expression in hippocampus. A seizure was induced by inhalant flurothyl daily in neonatal Sprague-Dawley rats from postnatal day 6 (P6). Rats were assigned into the single-seizure group (SS), the recurrent-seizure group (RS, seizures induced in six consecutive days), and the control group. During P41–P46 and P85–P90, the rats were tested for spatial learning and memory abilities with automatic Morris water maze task. At P90, mossy fiber sprouting and gene expression in hippocampus were determined subsequently by Timm staining and RT-PCR methods. The escape latencies from the water maze were significantly longer in rats of RS group than those of the control and SS groups at d4 of the first maze test and at d3, d4 of the second maze test. As far as Spatial Probe Test was concerned, the frequency of passing through the platform quadrant was significantly decreased in RS group than that in control and SS groups in the entire two probe tests. In rats with recurrent seizures (RS group), there was an increased distribution of Timm granules in both the supragranular region of the dentate gyrus and the stratum pyramidale of CA3 subfield in RS group, while remaining barely visible in control and SS groups; the Timm scores in CA3 and dentate gyrus in the RS animals were significantly higher than that in the control and SS groups. RT-PCR densitometry analysis showed that the ratios of hippocampal ZnT-1 to β-actin of SS and RS group were decreased significantly compared with that of control group. Meanwhile, CaMK II to β-actin of RS group was markedly lower compared with those of SS and control groups. Our results suggest that the long-term adverse effects of recurrent neonatal seizures on cognition and mossy fiber sprouting may be associated with the down-regulated expression of ZnT-1 and CaMK II in hippocampus. [Copyright &y& Elsevier]