Differences in the response to periarterial nerve stimulation or exogenous noradrenaline infusion in the mesenteric vascular bed with the intestinal tract harvested from commonly used rat models of hypertension.

Many hypertensive animal models have been developed and used to elucidate the pathophysiology of hypertension and to develop antihypertensive drugs. Among them, the spontaneous hypertensive rat (SHR), deoxycorticosterone acetate (DOCA)-treated and high salt intake rat (DOCA-salt), and high sodium-fed Dahl salt-sensitive rat (HS) models are commonly used. Multiple studies have been conducted, however, elevation in blood pressure in these models due to the reactivity of adrenergic vasoconstriction has not been well characterized in a centralized experiment. In this study, the pressor responses to periarterial nerve stimulation (PNS) or exogenous noradrenaline (NA) infusion were measured in the isolated mesenteric vascular bed with the intestinal tract to investigate the reactivity of mesenteric adrenergic vasoconstriction. The systemic arterial blood pressure of the hypertensive rat models was uniformly elevated compared with their respective controls. However, the changes in perfusion pressure in the mesenteric vascular bed in response to PNS and exogenous NA infusion were quite different depending on the model. The pressor responses to PNS in SHRs and Dahl S HS rats were significantly higher, and those in DOCA-salt rats were significantly lower than those in the controls. The pressor responses to exogenous NA infusion in SHRs were significantly higher, and those in Dahl S HS rats were significantly lower than those in their respective controls. No difference was observed in the pressor responses to the exogenous NA between the DOCA-salt and sham groups. These results demonstrate that the reactivity of adrenergic vasoconstriction is different for each type of experimental hypertensive model rat.

Diabetes Mellitus Induces Hyperreactivity of 5-Hydroxytryptamine (5-HT)-Induced Constriction in Human Internal Thoracic Artery and Is Associated with Increase in the Membrane Protein Level of 5-HT2A Receptor.

Studies indicate that 5-hydroxytryptamine (5-HT) released from activated platelets in coronary artery bypass grafting (CABG) induces 5-HT2A receptor-mediated graft spasm. We previously reported that 5-HT-induced constriction of human endothelium-denuded saphenous vein (SV) was significantly augmented in patients with diabetes mellitus (DM) than in patients without DM (non-DM), without changes in the levels of the membrane-bound 5-HT2A receptor of their smooth muscle cells. Although the internal thoracic artery (ITA) is the key graft conduit for CABG, the effect of DM on the ITA graft spasm is still unclear. Therefore, in this study, we investigated the effect of DM on 5-HT-induced vasoconstriction and the level of membrane-bound 5-HT2A receptor in ITA grafts. 5-HT-induced constriction of the isolated human endothelial-denuded ITA was significantly higher in patients with DM than in patients without DM. In addition, the level of the 5-HT2A receptor in the membrane fraction of human ITA smooth muscle cells was significantly higher in patients with DM than in those without DM. These results demonstrate that DM is a risk factor for CABG in both venous and arterial conduits, and that it differentially affects the level of the membrane-bound 5-HT2A receptor in the venous and arterial smooth muscle cells.

Protease-activated receptor-1 negatively regulates proliferation of neural stem/progenitor cells derived from the hippocampal dentate gyrus of the adult mouse.

Thrombin-activated protease-activated receptor (PAR)-1 regulates the proliferation of neural cells following brain injury. To elucidate the involvement of PAR-1 in the neurogenesis that occurs in the adult hippocampus, we examined whether PAR-1 regulated the proliferation of neural stem/progenitor cells (NPCs) derived from the murine hippocampal dentate gyrus. NPC cultures expressed PAR-1 protein and mRNA encoding all subtypes of PAR. Direct exposure of the cells to thrombin dramatically attenuated the cell proliferation without causing cell damage. This thrombin-induced attenuation was almost completely abolished by the PAR antagonist RWJ 56110, as well as by dabigatran and 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), which are selective and non-selective thrombin inhibitors, respectively. Expectedly, the PAR-1 agonist peptide (AP) SFLLR-NH2 also attenuated the cell proliferation. The cell proliferation was not affected by the PAR-1 negative control peptide RLLFT-NH2, which is an inactive peptide for PAR-1. Independently, we determined the effect of in vivo treatment with AEBSF or AP on hippocampal neurogenesis in the adult mouse. The administration of AEBSF, but not that of AP, significantly increased the number of newly-generated cells in the hippocampal subgranular zone. These data suggest that PAR-1 negatively regulated adult neurogenesis in the hippocampus by inhibiting the proliferative activity of the NPCs.

Angiotensin II, as well as 5-hydroxytriptamine, is a potent vasospasm inducer of saphenous vein graft for coronary artery bypass grafting in patients with diabetes mellitus.

Diabetes mellitus (DM) is an important risk factor for adverse outcomes of coronary artery bypass grafting. The bypass grafts harvested from patients with DM tend to go into spasm after their implantation into the coronary circulation. To clarify the contribution of 5-hydroxytriptamine (5-HT) and angiotensin II (AngII) in the bypass graft spasm, we examined the contractile reactivity to 5-HT or AngII of isolated human endothelium-denuded saphenous vein (SV) harvested from DM and non-DM patients. The 5-HT-induced constriction of the SV was significantly augmented in the DM group than in the non-DM group, which is similar to our previous report. AngII-induced constriction of the SV was also significantly augmented in the DM group than the non-DM group. Especially in the non-DM group, the AngII-induced maximal vasoconstriction was markedly lower than the 5-HT-induced one. Meanwhile, the increasing rates of AngII-induced vasoconstriction in the DM group to the non-DM group were significantly greater than those of 5-HT-induced vasoconstriction. These results indicate that 5-HT is a potent inducer of SV graft spasm in both DM and non-DM patients, while AngII is a potent inducer of SV graft spasm only in patients with DM. Furthermore, the protein level of AngII AT1 receptor (AT1R), but not the protein level of 5-HT2A receptor, in the membrane fraction of the SV smooth muscle cells of DM patients was significantly increased as compared with that of the non-DM patients. These results suggest that the mechanism for hyperreactivity to AngII in the SV from DM patients is due to, at least in part, the increase in the amount of AT1R on membrane of the SV smooth muscle cells.

Beneficial effect of cilostazol-mediated neuronal repair following trimethyltin-induced neuronal loss in the dentate gyrus.

Cilostazol acts as an antiplatelet agent and has other pleiotropic effects based on phosphodiesterase-3-dependent mechanisms. We evaluated whether cilostazol would have a beneficial effect on neuronal repair following hippocampal neuronal damage by using a mouse model of trimethyltin (TMT)-induced neuronal loss/self-repair in the hippocampal dentate gyrus [Ogita et al. (2005) J Neurosci Res 82:609-621]; these mice will hereafter be referred to as impaired animals. A single treatment with cilostazol (10 mg/kg, i.p.) produced no significant change in the number of 5-bromo-2'-deoxyuridine (BrdU)-incorporating cells in the dentate granule cell layer (GCL) or subgranular zone on day 3 after TMT treatment. However, chronic treatment with cilostazol on days 3-15 posttreatment resulted in an increase in the number of BrdU-incorporating cells in the dentate GCL of the impaired animals, and these cells were positive for neuronal nuclear antigen or doublecortin. Cilostazol was effective in elevating the level of phosphorylated cyclic adrenosine monophosphate response element-binding protein (pCREB) in the dentate gyrus of impaired animals. The results of a forced swimming test revealed that the chronic treatment with cilostazol improved the depression-like behavior seen in the impaired animals. In the cultures of hippocampal neural stem/progenitor cells, exposure to cilostazol produced not only enhancement of proliferation activity but also elevation of pCREB levels. Taken together, our data suggest that cilostazol has a beneficial effect on neuronal repair following neuronal loss in the dentate gyrus through promotion of proliferation and/or neuronal differentiation of neural progenitor cells in the subgranular zone.

Possible involvement of caspases in proliferation of neocortical neural stem/progenitor cells in the developing mouse brain.

Caspases are well-known enzymes that work as initiators and effectors of apoptosis. To elucidate the role of caspases in neurodevelopment, we sought to determine if caspases are involved in the proliferation of neural stem/progenitor cells (NPCs) in the developing mouse brain. Labeling with 5-bromo-2'-deoxyuridine (BrdU) from days 14 to 18 of pregnant mice revealed that the 18-d old fetus had many BudU-positive cells in its brain. Double-labeling revealed that active caspase-3 was co-localized with these BrdU-positive cells in the neocortex, hippocampus, and subventricular zone of the fetal brain. Active caspase-3 was detected in cultures of NPCs derived from the neocortex of 15-d old fetuses during culture periods. Importantly, the pan-caspase inhibitor z-VAD-FMK was effective at completely inhibiting neurosphere formation by the NPCs. These results suggest the possibility that the caspase cascade is essential for the proliferation of neocortical NPCs in the developing mouse brain.

Disruption of ion-trafficking system in the cochlear spiral ligament prior to permanent hearing loss induced by exposure to intense noise: possible involvement of 4-hydroxy-2-nonenal as a mediator of oxidative stress.

Noise-induced hearing loss is at least in part due to disruption of endocochlear potential, which is maintained by various K(+) transport apparatuses including Na(+), K(+)-ATPase and gap junction-mediated intercellular communication in the lateral wall structures. In this study, we examined the changes in the ion-trafficking-related proteins in the spiral ligament fibrocytes (SLFs) following in vivo acoustic overstimulation or in vitro exposure of cultured SLFs to 4-hydroxy-2-nonenal, which is a mediator of oxidative stress. Connexin (Cx)26 and Cx30 were ubiquitously expressed throughout the spiral ligament, whereas Na(+), K(+)-ATPase α1 was predominantly detected in the stria vascularis and spiral prominence (type 2 SLFs). One-hour exposure of mice to 8 kHz octave band noise at a 110 dB sound pressure level produced an immediate and prolonged decrease in the Cx26 expression level and in Na+, K(+)-ATPase activity, as well as a delayed decrease in Cx30 expression in the SLFs. The noise-induced hearing loss and decrease in the Cx26 protein level and Na(+), K(+)-ATPase activity were abolished by a systemic treatment with a free radical-scavenging agent, 4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl, or with a nitric oxide synthase inhibitor, N(ω)-nitro-L-arginine methyl ester hydrochloride. In vitro exposure of SLFs in primary culture to 4-hydroxy-2-nonenal produced a decrease in the protein levels of Cx26 and Na(+), K(+)-ATPase α1, as well as Na(+), K(+)-ATPase activity, and also resulted in dysfunction of the intercellular communication between the SLFs. Taken together, our data suggest that disruption of the ion-trafficking system in the cochlear SLFs is caused by the decrease in Cxs level and Na(+), K(+)-ATPase activity, and at least in part involved in permanent hearing loss induced by intense noise. Oxidative stress-mediated products might contribute to the decrease in Cxs content and Na(+), K(+)-ATPase activity in the cochlear lateral wall structures.

Lithium promotes neuronal repair and ameliorates depression-like behavior following trimethyltin-induced neuronal loss in the dentate gyrus.

Lithium, a mood stabilizer, is known to ameliorate the stress-induced decrease in hippocampal neurogenesis seen in animal models of stress-related disorders. However, it is unclear whether lithium has beneficial effect on neuronal repair following neuronal damage in neuronal degenerative diseases. Here, we evaluated the effect of in vivo treatment with lithium on the hippocampal neuronal repair in a mouse model of trimethyltin (TMT)-induced neuronal loss/self-repair in the hippocampal dentate gyrus (such mice referred to as "impaired animals") [Ogita et al. (2005) J Neurosci Res 82: 609-621]. The impaired animals had a dramatically increased number of 5-bromo-2'-deoxyuridine (BrdU)-incorporating cells in their dentate gyrus at the initial time window (days 3 to 5 post-TMT treatment) of the self-repair stage. A single treatment with lithium produced no significant change in the number of BrdU-incorporating cells in the dentate granule cell layer and subgranular zone on day 3 post-TMT treatment. On day 5 post-TMT treatment, however, BrdU-incorporating cells were significantly increased in number by lithium treatment for 3 days. Most interestingly, chronic treatment (15 days) with lithium increased the number of BrdU-incorporating cells positive for NeuN or doublecortin in the dentate granule cell layer of the impaired animals, but not in that of naïve animals. The results of a forced swimming test revealed that the chronic treatment with lithium improved the depression-like behavior seen in the impaired animals. Taken together, our data suggest that lithium had a beneficial effect on neuronal repair following neuronal loss in the dentate gyrus through promoted proliferation and survival/neuronal differentiation of neural stem/progenitor cells in the subgranular zone.

Beneficial in vivo effect of aripiprazole on neuronal regeneration following neuronal loss in the dentate gyrus: evaluation using a mouse model of trimethyltin-induced neuronal loss/self-repair in the dentate gyrus.

Aripiprazole is used clinically as an atypical antipsychotic. We evaluated the effect of in vivo treatment with aripiprazole on the proliferation and differentiation of neural stem/progenitor cells in a mouse model, trimethyltin-induced neuronal loss/self-repair in the hippocampal dentate gyrus (referred as "impaired animals") [Ogita et al., J Neurosci Res. 82, 609 - 621 (2005)]. In the impaired animals, an increased number of 5-bromo-2'-deoxyuridine (BrdU)-positive cells was seen in the dentate gyrus at the initial time window of the self-repair stage. At the same time window, a single treatment with aripiprazole significantly increased the number of cells positive for both BrdU and nestin in the dentate gyrus of the impaired animals. Chronic treatment with aripiprazole promoted the proliferation/survival and neuronal differentiation of the cells newly-generated following the neuronal loss in the dentate gyrus of the impaired animals. The chronic treatment with aripiprazole improved depression-like behavior seen in the impaired animals. Taken together, our data suggest that aripiprazole had a beneficial effect on neuronal regeneration following neuronal loss in the dentate gyrus through indirectly promoted proliferation/survival and neuronal differentiation of neural stem/progenitor cells in the subgranular zone of the dentate gyrus.

In vivo and in vitro treatment with edaravone promotes proliferation of neural progenitor cells generated following neuronal loss in the mouse dentate gyrus.

Edaravone is clinically used in Japan for treatment of patients with acute cerebral infarction. To clarify the effect of edaravone on neurogenesis in the hippocampus following neuronal injury in the hippocampal dentate gyrus, we investigated the effect of in vitro and in vivo treatment with edaravone on the proliferation of neural stem/progenitor cells prepared from the mouse dentate gyrus damaged by trimethyltin (TMT). Histological assessment revealed the presence of large number of nestin(+) cells in the dentate gyrus on days 3 - 5 post-TMT treatment. We prepared cells from the dentate gyrus of naïve, TMT-treated mice or TMT/edaravone-treated mice. The cells obtained from the dentate gyrus of TMT-treated animals were capable of BrdU incorporation and neurosphere formation when cultured in the presence of growth factors. The TMT-treated group had a larger number of nestin(+) cells and nestin(+)GFAP(+) cells than the naïve one. Under the culture condition used, sustained exposure of the cells from the damaged dentate gyrus to edaravone at 10(-11) and 10(-8) M promoted the proliferation of nestin(+) cells. The systemic in vivo treatment with edaravone for 2 days produced a significant increase in the number of nestin(+) cells among the cells prepared from the dentate gyrus on day 4 post-TMT treatment, and as well as one in the number of neurospheres formed from these cells in the culture. Taken together, our data indicated that edaravone had the ability to promote the proliferation of neural stem/progenitor cells generated following neuronal damage in the dentate gyrus.

Opposing roles of glucocorticoid receptor and mineralocorticoid receptor in trimethyltin-induced cytotoxicity in the mouse hippocampus.

The organotin trimethyltin (TMT) is known to cause neuronal degeneration in the murine brain. Earlier studies indicate that TMT-induced neuronal degeneration is enhanced by adrenalectomy and prevented by exogenous glucocorticoid. The aim of this study was to investigate the regulation of TMT neuroxicity by corticosterone receptors including type I (mineralocorticoid receptor, MR) and type II (glucocorticoid receptor, GR) in adult mice. The systemic injection of TMT at the dose of 2.0 or 2.8 mg/kg produced a marked elevation in the level of plasma corticosterone that was both dose and time dependent. The MR agonist aldosterone had the ability to exacerbate TMT cytotoxicity in the dentate granule cell layer, whereas its antagonist spironolactone protected neurons from TMT cytotoxicity there. In contrast, the GR antagonist mifepristone exacerbated the TMT cytotoxicity. Taken together, our data suggest TMT cytotoxicity is oppositely regulated by GR and MR signals, being exacerbated by MR activation in adult mice.

[Activated microglial cells trigger neurogenesis following neuronal loss in the dentate gyrus of adult mice].

Neurological injuries are widely known to promote neurogenesis in the adult hippocampal dentate gyrus. Our previous studies demonstrated that the granule cells in the hippocampal dentate gyrus are injured and eradicated by treatment with trimethyltin (TMT), with being regenerated in the dentate granule cell layer (GCL) after neuronal loss. Recent collective reports indicate that during brain injury and in neurodegenerative disorders, neurogenesis is controlled by cytokines, chemokines, neurotransmitters, and reactive oxygen/nitrogen species, which are released by dying neurons as well as by activated macrophages, micro-glia, and astrocytes. To elucidate the role of activated microglia in the neuroregeneration following the dentate granule cell loss, in this study, we evaluated the involvement of activated microglial cells and a related factor in the generation of newly-generated cells of the hippocampal dentate gyrus following neuronal loss induced by TMT. Our results support the possibility that pro-inflammatory cytokines released from activated microglial cells may be involved in promotion of the neurogenesis mechanism through activation of the NF-kappaB signaling pathway following the dentate neuronal loss induced by TMT treatment.

Indomethacin ameliorates trimethyltin-induced neuronal damage in vivo by attenuating oxidative stress in the dentate gyrus of mice.

The organotin trimethyltin (TMT) is well known to cause neuronal degeneration in the hippocampal dentate gyrus of mice. The first purpose of the present study was to examine whether the cyclooxygenase (COX) inhibitor indomethacin could ameliorate neuronal degeneration in the dentate gyrus of mice following TMT treatment in vivo. The systemic injection into mice of TMT at 2.8 mg/kg produced activation of endogenous caspase-3 and calpain, enhanced the gene expression of COX-1 and COX-2, activated microglial cells, and caused the formation of the lipid peroxidation product 4-hydroxynonenal in the hippocampus. Given at 12-h post-TMT treatment, the systemic injection of indomethacin (5 or 10 mg/kg, subcutaneously) significantly decreased the TMT-induced damage to neurons having active caspase-3 and single-stranded DNA in the dentate granule cell layer of the hippocampus. The results of the α-Fodrin degradation test revealed that the post-treatment with indomethacin was effective in attenuating TMT-induced activation of endogenous caspases and calpain in the hippocampus. In TMT-treated animals, interestingly, the post-treatment with indomethacin produced not only activation of microglial cells in the dentate gyrus but also the formation of 4-hydroxynonenal in the dentate granule cell layer. Taken together, our data suggest that COX inhibition by indomethacin ameliorated TMT-induced neuronal degeneration in the dentate gyrus by attenuating intensive oxidative stress.

Adult neurogenesis is regulated by endogenous factors produced during neurodegeneration.

Adult neurogenesis is the process of generating new neurons that become integrated into existing circuits after fetal and early postnatal development has ceased. In most mammalian species, adult neurogenesis only appears to occur in the olfactory bulb and the hippocampus, where neural stem/progenitor cells (NPCs) exist to create new neurons. In adult neurogenesis, microenviromental change is thought to provide a specific modulation for maintaining the multi-potent state of these NPCs. Neurodegeneration is driven by the activation of resident microglia, astrocytes, and infiltrating peripheral macrophages, which release a plethora of cytokines, chemokines, neurotransmitters, and reactive oxygen species. These endogenous factors cause further bystander damage to neurons and produces both detrimental and favorable conditions for neurogenesis. Interestingly, these endogenous factors also affect the proliferation, migration, differentiation, and survival of the NPCs, as well as regulate the incorporation of newly formed neurons into the brain circuitry. The unique profile of the endogenous factors released can vary the degree of neuroregeneration after neurodegeneration. This current review summarizes recent knowledge in the emerging field that is showing that adult neurogenesis is regulated by endogenous factors produced during neurodegeneration.

Endogenous nitric oxide generation linked to ryanodine receptors activates cyclic GMP / protein kinase G pathway for cell proliferation of neural stem/progenitor cells derived from embryonic hippocampus.

Nitric oxide (NO) activates the cyclic GMP (cGMP) / protein kinase G (PKG) pathway during physiological processes in numerous types of cells. Here, we evaluated whether this NO/cGMP/PKG pathway is involved in the proliferation of neural stem/progenitor cells (NPCs) derived from the hippocampus of embryonic mice. In culture, the exposure to the NO synthase inhibitor N(ω)-nitro-L-arginine methyl ester (L-NAME) significantly decreased the number of viable cells and 5-bromo-2'-deoxyuridine (BrdU) incorporation into the cells, as well as the levels of intracellular reactive oxygen species, extracellular NO(2), and intracellular cGMP. Like L-NAME, the soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and PKG inhibitor KT5823 also decreased cell viability and BrdU incorporation. The membrane-permeable cGMP analogue 8-bromo-cGMP partially abolished the L-NAME-induced decrease in the BrdU incorporation. BrdU incorporation was decreased by Ca(2+)-channel blockers, including dantrolene, MK-801, ifenprodil, and nifedipine. Interestingly, the NO(2) level was decreased by dantrolene, but not by the other 3 blockers. L-NAME and ODQ attenuated phosphorylation of Akt, but not that of extracellular signal-regulated kinases or epidermal growth factor receptors. Our data suggest that endogenous NO generation linked to dantrolene-sensitive ryanodine receptors activates the cGMP/PKG signaling pathway for positive regulation of proliferation of hippocampal NPCs derived from embryonic mice.

Endogenous Nitric Oxide Generation Linked to Ryanodine Receptors Activates Cyclic GMP / Protein Kinase G Pathway for Cell Proliferation of Neural Stem/Progenitor Cells Derived From Embryonic Hippocampus.

Nitric oxide (NO) activates the cyclic GMP (cGMP) / protein kinase G (PKG) pathway during physiological processes in numerous types of cells. Here, we evaluated whether this NO/cGMP/PKG pathway is involved in the proliferation of neural stem/progenitor cells (NPCs) derived from the hippocampus of embryonic mice. In culture, the exposure to the NO synthase inhibitor Nω-nitro-l-arginine methyl ester (l-NAME) significantly decreased the number of viable cells and 5-bromo-2'-deoxyuridine (BrdU) incorporation into the cells, as well as the levels of intracellular reactive oxygen species, extracellular NO2, and intracellular cGMP. Like l-NAME, the soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and PKG inhibitor KT5823 also decreased cell viability and BrdU incorporation. The membrane-permeable cGMP analogue 8-bromo-cGMP partially abolished the l-NAME-induced decrease in the BrdU incorporation. BrdU incorporation was decreased by Ca2+-channel blockers, including dantrolene, MK-801, ifenprodil, and nifedipine. Interestingly, the NO2 level was decreased by dantrolene, but not by the other 3 blockers. l-NAME and ODQ attenuated phosphorylation of Akt, but not that of extracellular signal-regulated kinases or epidermal growth factor receptors. Our data suggest that endogenous NO generation linked to dantrolene-sensitive ryanodine receptors activates the cGMP/PKG signaling pathway for positive regulation of proliferation of hippocampal NPCs derived from embryonic mice.

Enhanced expression of glutathione S-transferase in the hippocampus following acute treatment with trimethyltin in vivo.

We investigated the effects of treatment with trimethyltin (TMT) on the expression of glutathione-related enzymes in mouse hippocampus. TMT promoted the expression of glutathione S-transferase (GST) Ya/Yc mRNA, and GSTA2 protein, but not that of glutamate-cysteine ligase catalytic subunit mRNA, 1 day after injection. TMT produced a slight but significant elevation of GST activity during the period from day 1 to 7 post-treatment. No significant change was seen in the activity of glutathione peroxidase at anytime post-TMT treatment. Our data suggest the prolonged elevation of GST activity in the hippocampus following TMT treatment through enhanced expression of the GST Ya/Yc.

Endogenous reactive oxygen species are essential for proliferation of neural stem/progenitor cells.

It is widely thought that accumulation of reactive oxygen species (ROS) causes injury to cells. In this study, we investigated the effect of endogenous ROS on the proliferation of neural stem/progenitor cells derived from the hippocampus of embryonic mice. The cells were treated with free radical-scavenging agents [3-methyl-1-phenyl-2-pyrazolin-5-one (edaravone) or 4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl (tempol)], an NADPH oxidase inhibitor (apocynin), catalase, a nitric oxide synthase inhibitor [N(omega)-nitro-L-arginine methyl ester hydrochloride (L-NAME)] or a peroxynitrite generator (SIN-1) during the culture period. Edaravone and tempol had the ability to decrease endogenous ROS in the cells exposed for periods from 1 to 24h, with attenuation of the proliferation activity of the cells during culture. Apocynin and L-NAME were also effective in attenuating cell proliferation but not cellular damage. Conversely, SIN-1 was capable of promoting the proliferation activity. However, catalase had no effect on the proliferation activity of the cells during culture. Furthermore, tempol significantly decreased the level of NFkappaB p65, phospho-cyclic AMP response element-binding protein, and beta-catenin within the nucleus of the cells. These data suggest that endogenous ROS and nitric oxide are essential for the proliferation of embryonic neural stem/progenitor cells.