Neurons were evaluated by NeuN staining 1 week after seizure. NeuN (+) neurons disappeared in the hippocampal CA1, CA3 and hilus in vehicle treated rats at this time point. Compared with vehicle-treated rats, CQ-treated rat showed a similar number of NeuN (+) neurons in the hippocampal CA1, CA3, hilus and subiculum area, suggesting neuronal death is not prevented by CQ (Fig. 2).Results Seizure-induced Hippocampal Neuronal Death is not Prevented by CQTo test whether CQ treatment shows neuroprotective effects after 56-59-7 pilocarpine-induced seizure, rats were sacrificed 1 week after insult with or without CQ injection. Neuronal injury was evaluated by FJB staining. Widespread FJB (+) neurons wereCQ Decreased Hippocampal Vesicular Zinc Level in Normal or after Pilocarpine-induced SeizureTo test whether CQ treatment decreases vesicular zinc intensity in the mossy fiber of hippocampus, brain sections were stained by TSQ. Consistent with previous observations [5,23], the intensity of mossy fiber zinc in the rat hippocampus was 48.3 lower in CQ treatment group than in vehicle treated controls (Fig. 3).Zinc and Hippocampal Neurogenesis after SeizureFigure 7. Intracellular zinc JI-101 custom synthesis chelator, TPEN, reduced the number of newly generated cells in the dentate gyrus. (A) Light microscope images show BrdU (+) cells, Ki67 (+) cells and DCX (+) cells. One week injection of intracellular zinc chelator, TPEN, reduced the number of BrdU (+) cells, Ki67 (+) cells and DCX (+) cells with or without seizure. Scale bar = 200 mm. (B) Bar graph represents number of BrdU, Ki67 and DCXimmunoreactive cell in the subgranular zone of DG (n = 5). Data are means 6 SE. *P,0.05. doi:10.1371/journal.pone.0048543.gZinc and Hippocampal Neurogenesis after SeizureProgenitor Cell Proliferation in the Subgranular Zone of Dentate Gyrus is Reduced by CQ in Normal and Postseizure SubjectsTo test whether CQ affects progenitor cell proliferation in the adult brain, rats were sacrificed 1 week after continuous CQ treatment without seizure. Rats were injected with BrdU twice per day for 4 days in both vehicle or CQ treated group. Cell proliferation was assessed by Ki67 and BrdU immunohistochemistry. We found decreased number of Ki67 and BrdU labeled cells in rats without seizure (Fig. 4). To investigate how CQ affected seizure-induced progenitor cell proliferation and neurogenesis, rats were injected with BrdU twice per day from 4 days after pilocarpine-induced seizure until to sacrifice. Rats were injected 
with CQ from 2 hours after seizure twice per day for 1 week. Cell proliferation was assessed by Ki67 and BrdU immunohistochemistry. We observed increase in the number of cells labeled by both Ki67 and BrdU staining in rats that underwent pilocarpineinduced seizure at 1 week after seizure compared to sham operation. However, a group of 1 week CQ treated rats showed lower number of Ki67 and BrdU immunoreactive cells in the DG of hippocampus after seizure compared to vehicle treated group (Fig. 5).Neuroblast Production in the Subgranular Zone of Dentate Gyrus is Reduced by CQ in Normal and 16574785 Postseizure SubjectsTo investigate how CQ affects neuroblast migration, normal or seizure-experienced rats were continuously injected with CQ. Doublecortin (DCX) is a microtubule-associated protein expressed by immature neurons. The levels of DCX expression increase in response to seizure, which occurs in parallel with BrdU labeling in measuring neurogenesis. In normal rats, CQ or vehicle was injected into th.Neurons were evaluated by NeuN staining 1 week after seizure. NeuN (+) neurons disappeared in the hippocampal CA1, CA3 and hilus in vehicle treated rats at this time point. Compared with vehicle-treated rats, CQ-treated rat showed a similar number of NeuN (+) neurons in the hippocampal CA1, CA3, hilus and subiculum area, suggesting neuronal death is not prevented by CQ (Fig. 2).Results Seizure-induced Hippocampal Neuronal Death is not Prevented by CQTo test whether CQ treatment shows neuroprotective effects after pilocarpine-induced seizure, rats were sacrificed 1 week after insult with or without CQ injection. Neuronal injury was evaluated by FJB staining. Widespread FJB (+) neurons wereCQ Decreased Hippocampal Vesicular Zinc Level in Normal or after Pilocarpine-induced SeizureTo test whether CQ treatment decreases vesicular zinc intensity in the mossy fiber of hippocampus, brain sections were stained by TSQ. Consistent with previous observations [5,23], the intensity of mossy fiber zinc in the rat hippocampus was 48.3 lower in CQ treatment group than in vehicle treated controls (Fig. 3).Zinc and Hippocampal Neurogenesis after SeizureFigure 7. Intracellular zinc chelator, TPEN, reduced the number of newly generated cells in the dentate gyrus. (A) Light microscope images show BrdU (+) cells, Ki67 (+) cells and DCX (+) cells. One week injection of intracellular zinc chelator, TPEN, reduced the number of BrdU (+) cells, Ki67 (+) cells and DCX (+) cells with or without seizure. Scale bar = 200 mm. (B) Bar graph represents number of BrdU, Ki67 and DCXimmunoreactive cell in the subgranular zone of DG (n = 5). Data are means 6 SE. *P,0.05. doi:10.1371/journal.pone.0048543.gZinc and Hippocampal Neurogenesis after SeizureProgenitor Cell Proliferation in the Subgranular Zone of Dentate Gyrus is Reduced by CQ in Normal and Postseizure SubjectsTo test whether CQ affects progenitor cell proliferation in the adult brain, rats were sacrificed 1 week after continuous CQ treatment without seizure. Rats were injected with BrdU twice per day for 4 days in both vehicle or CQ treated group. Cell proliferation was assessed by Ki67 and BrdU immunohistochemistry. We found decreased number of Ki67 and BrdU labeled cells in rats without seizure (Fig. 4). To investigate how CQ affected seizure-induced progenitor cell proliferation and neurogenesis, rats were injected with BrdU twice per day from 4 
days after pilocarpine-induced seizure until to sacrifice. Rats were injected with CQ from 2 hours after seizure twice per day for 1 week. Cell proliferation was assessed by Ki67 and BrdU immunohistochemistry. We observed increase in the number of cells labeled by both Ki67 and BrdU staining in rats that underwent pilocarpineinduced seizure at 1 week after seizure compared to sham operation. However, a group of 1 week CQ treated rats showed lower number of Ki67 and BrdU immunoreactive cells in the DG of hippocampus after seizure compared to vehicle treated group (Fig. 5).Neuroblast Production in the Subgranular Zone of Dentate Gyrus is Reduced by CQ in Normal and 16574785 Postseizure SubjectsTo investigate how CQ affects neuroblast migration, normal or seizure-experienced rats were continuously injected with CQ. Doublecortin (DCX) is a microtubule-associated protein expressed by immature neurons. The levels of DCX expression increase in response to seizure, which occurs in parallel with BrdU labeling in measuring neurogenesis. In normal rats, CQ or vehicle was injected into th.
