Oenanthe Javanica Extract Protects Mouse Skin from UVB Radiation via Attenuating Collagen Disruption and Inflammation.

In recent years, the use of botanical agents to prevent skin damage from solar ultraviolet (UV) irradiation has received considerable attention. Oenanthe javanica is known to exert anti-inflammatory and antioxidant activities. This study investigated photoprotective properties of an Oenanthe javanica extract (OJE) against UVB-induced skin damage in ICR mice. The extent of skin damage was evaluated in three groups: control mice with no UVB, UVB-exposed mice treated with vehicle (saline), and UVB-exposed mice treated with 1% extract. Photoprotective properties were assessed in the dorsal skin using hematoxylin and eosin staining, Masson trichrome staining, immunohistochemical staining, quantitative real-time polymerase chain reaction, and western blotting to analyze the epidermal thickness, collagen expression, and mRNA and protein levels of type I collagen, type III collagen, and interstitial collagenases, including matrix metalloproteinase (MMP)-1 and MMP-3. In addition, tumor necrosis factor (TNF)-α and cyclooxygenase (COX)-2 protein levels were also assessed. In the UVB-exposed mice treated with extract, UV-induced epidermal damage was significantly ameliorated. In this group, productions of collagen types I and III were increased, and expressions of MMP-1 and MMP-3 were decreased. In addition, TNF-α and COX-2 expressions were reduced. Based on these findings, we conclude that OJE displays photoprotective effects against UVB-induced collagen disruption and inflammation and suggest that Oenanthe javanica can be used as a natural product for the treatment of photodamaged skin.

Effects of Scopolamine and Melatonin Cotreatment on Cognition, Neuronal Damage, and Neurogenesis in the Mouse Dentate Gyrus.

It has been demonstrated that melatonin plays important roles in memory improvement and promotes neurogenesis in experimental animals. We examined effects of melatonin on cognitive deficits, neuronal damage, cell proliferation, neuroblast differentiation and neuronal maturation in the mouse dentate gyrus after cotreatment of scopolamine (anticholinergic agent) and melatonin. Scopolamine (1 mg/kg) and melatonin (10 mg/kg) were intraperitoneally injected for 2 and/or 4 weeks to 8-week-old mice. Scopolamine treatment induced significant cognitive deficits 2 and 4 weeks after scopolamine treatment, however, cotreatment of scopolamine and melatonin significantly improved spatial learning and short-term memory impairments. Two and 4 weeks after scopolamine treatment, neurons were not damaged/dead in the dentate gyrus, in addition, no neuronal damage/death was shown after cotreatment of scopolamine and melatonin. Ki67 (a marker for cell proliferation)- and doublecortin (a marker for neuroblast differentiation)-positive cells were significantly decreased in the dentate gyrus 2 and 4 weeks after scopolamine treatment, however, cotreatment of scopolamine and melatonin significantly increased Ki67- and doublecortin-positive cells compared with scopolamine-treated group. However, double immunofluorescence for NeuN/BrdU, which indicates newly-generated mature neurons, did not show double-labeled cells (adult neurogenesis) in the dentate gyrus 2 and 4 weeks after cotreatment of scopolamine and melatonin. Our results suggest that melatonin treatment recovers scopolamine-induced spatial learning and short-term memory impairments and restores or increases scopolamine-induced decrease of cell proliferation and neuroblast differentiation, but does not lead to adult neurogenesis (maturation of neurons) in the mouse dentate gyrus following scopolamine treatment.

Neuronal loss and gliosis in the rat striatum subjected to 15 and 30 minutes of middle cerebral artery occlusion.

Selective neuronal death or loss in certain brain regions has been well characterized in animal models of transient global cerebral ischemia. However, selective neuronal death in transient focal cerebral ischemia needs more investigation. Therefore, in this study, we studied selective neuronal death in the striatum (caudate putamen) of rats subjected to 15 or 30 min middle cerebral artery occlusion (MCAO). Neuronal death occurred in the dorsolateral field, not in the medial field in 30 min, not 15 min, MCAO-operated rats 5 days after MCAO using neuronal nuclear antigen immunohistochemistry and Fluoro-Jade B histofluorescence staining. In this group, immunoreactivity of glial fibrillary acidic protein in astrocytes was hardly shown in the dorsolateral field, although the immunoreactivity increased in the medial field. In addition, immunoreactivity of ionized calcium binding adapter molecule 1 in microglia was dramatically increased in the dorsolateral, not in the medial, field only in 30 min MCAO-operated rats. Briefly, these results show that at least 30 min of MCAO can evoke selective neuronal death, astrocytic dysfunction and microglial activation in the dorsolateral field of the rat striatum and suggest that a rat model of 30 min MCAO can be used to investigate mechanisms of neuronal death and gliosis following brief transient focal cerebral ischemic events for acute transient ischemic attack.

Brain ischemic preconditioning protects against moderate, not severe, transient global cerebral ischemic injury.

Ischemic preconditioning (IPC) in the brain increases ischemic tolerance to subsequent ischemic insults. In this study, we examined whether IPC protects neurons and attenuates microgliosis or not in the hippocampus following severe transient global cerebral ischemia (TCI) in gerbils. Gerbils were assigned to 8 groups; 5- and 15-min sham operated groups, 5-min and 15-min TCI operated groups, IPC plus 5- and 15-min sham operated groups, and IPC plus 5- and 15-min TCI operated groups. IPC was induced by subjecting animals to 2-min transient ischemia 1 day before 5-min TCI for a typical transient ischemia and 15-min TCI for severe transient ischemia. Neuronal damage was examined by cresyl violet staining and Fluoro-Jade B histofluorescence staining. In addition, microglial activation was examined using immunohistochemistry for Iba-1 (a marker for microglia). Delayed neuronal death and microgliosis was found in the CA1 alone in the 5-min TCI operated group at 5 days post-ischemia, and, in the 15-min TCI operated group, neuronal death and microgliosis was shown in all CA areas (CA1-3) and the dentate gyrus. IPC displayed neuroprotection and attenuated microglial activation in the 5-min TCI operated group. However, in the 15-min TCI operated group, IPC did not show neuroprotection and not attenuate microglial activation. Our present findings indicate that IPC hardly protect against severe transient cerebral ischemic injury.

Transient Cerebral Ischemia Alters GSK-3ß and p-GSK-3ß Immunoreactivity in Pyramidal Neurons and Induces p-GSK-3ß Expression in Astrocytes in the Gerbil Hippocampal CA1 Area.

Glycogen synthase kinase 3ß (GSK-3ß) is a key downstream protein in the PI3K/Akt pathway. Phosphorylation of serine 9 of GSK-3ß (GSK-3ß activity inhibition) promotes cell survival. In this study, we examined changes in expressions of GSK-3ß and phosphorylation of GSK-3ß (p-GSK-3ß) in the gerbil hippocampal CA1 area after 5 min of transient cerebral ischemia. GSK-3ß immunoreactivity in the CA1 area was increased in pyramidal cells at 6 h after ischemia-reperfusion. It was decreased in CA1 pyramidal cells from 12 h after ischemia-reperfusion, and hardly detected in the CA1 pyramidal cells at 5 days after ischemia-reperfusion. p-GSK-3ß immunoreactivity was slightly decreased in CA1 pyramidal cells at 6 and 12 h after ischemia-reperfusion. It was significantly increased in these cells at 1 and 2 days after ischemia-reperfusion. Five days after ischemia-reperfusion, p-GSK-3ß immunoreactivity was hardly found in CA1 pyramidal cells. However, p-GSK-3ß immunoreactivity was strongly expressed in astrocytes primarily distributed in strata oriens and radiatum. In conclusion, GSK-3ß and p-GSK-3ß were significantly changed in pyramidal cells and/or astrocytes in the gerbil hippocampal CA1 area following 5 min of transient cerebral ischemia. This finding indicates that GSK-3ß and p-GSK-3ß are closely related to delayed neuronal death.

Ischemia-Induced Changes of PRAS40 and p-PRAS40 Immunoreactivities in the Gerbil Hippocampal CA1 Region After Transient Cerebral Ischemia.

Proline-rich Akt substrate of 40-kDa (PRAS40) is one of the important interactive linkers between Akt and mTOR signaling pathways. The increase of PRAS40 is related with the reduction of brain damage induced by cerebral ischemia. In the present study, we investigated time-dependent changes in PRAS40 and phospho-PRAS40 (p-PRAS40) immunoreactivities in the hippocampal CA1 region of the gerbil after 5 min of transient cerebral ischemia. PRAS40 immunoreactivity in the CA1 region was decreased in pyramidal neurons from 12 h after ischemic insult in a time-dependent manner, and, at 5 days post-ischemia, PRAS40 immunoreactivity was newly expressed in astrocytes. p-PRAS40 immunoreactivity in the CA1 pyramidal neurons was hardly found 12 h and apparently detected again 1 and 2 days after ischemic insult. At 5 days post-ischemia, p-PRAS40 immunoreactivity in the CA1 pyramidal neurons was not found. These results indicate that ischemia-induced changes in PRAS40 and p-PRAS40 immunoreactivities in CA1 pyramidal neurons and astrocytes may be closely associated with delayed neuronal death in the hippocampal CA1 region following transient cerebral ischemia.

Tanshinone I Enhances Neurogenesis in the Mouse Hippocampal Dentate Gyrus via Increasing Wnt-3, Phosphorylated Glycogen Synthase Kinase-3ß and ß-Catenin Immunoreactivities.

Tanshinone I (TsI), a lipophilic diterpene extracted from Danshan (Radix Salvia miltiorrhizae), exerts neuroprotection in cerebrovascular diseases including transient ischemic attack. In this study, we examined effects of TsI on cell proliferation and neuronal differentiation in the subgranular zone (SGZ) of the mouse dentate gyrus (DG) using Ki-67, BrdU and doublecortin (DCX) immunohistochemistry. Mice were treated with 1 and 2 mg/kg TsI for 28 days. In the 1 mg/kg TsI-treated-group, distribution patterns of BrdU, Ki-67 and DCX positive ((+)) cells in the SGZ were similar to those in the vehicle-treated-group. However, in the 2 mg/kg TsI-treated-group, double labeled BrdU(+)/NeuN(+) cells, which are mature neurons, as well as Ki-67(+), DCX(+) and BrdU(+) cells were significantly increased compared with those in the vehicle-treated-group. On the other hand, immunoreactivities and protein levels of Wnt-3, ß-catenin and serine-9-glycogen synthase kinase-3ß (p-GSK-3ß), which are related with morphogenesis, were significantly increased in the granule cell layer of the DG only in the 2 mg/kg TsI-treated-group. Therefore, these findings indicate that TsI can promote neurogenesis in the mouse DG and that the neurogenesis is related with increases of Wnt-3, p-GSK-3ß and ß-catenin immunoreactivities.

Increases of Catalase and Glutathione Peroxidase Expressions by Lacosamide Pretreatment Contributes to Neuroprotection Against Experimentally Induced Transient Cerebral Ischemia.

Lacosamide is a new antiepileptic drug which is widely used to treat partial-onset seizures. In this study, we examined the neuroprotective effect of lacosamide against transient ischemic damage and expressions of antioxidant enzymes such as Zn-superoxide dismutase (SOD1), Mn-superoxide dismutase (SOD2), catalase (CAT) and glutathione peroxidase (GPX) in the hippocampal cornu ammonis 1 (CA1) region following 5 min of transient global cerebral ischemia in gerbils. We found that pre-treatment with 25 mg/kg lacosamide protected CA1 pyramidal neurons from transient global cerebral ischemic insult using hematoxylin-eosin staining and neuronal nuclear antigen immunohistochemistry. Transient ischemia dramatically changed expressions of SOD1, SOD2 and GPX, not CAT, in the CA1 pyramidal neurons. Lacosamide pre-treatment increased expressions of CAT and GPX, not SOD1 and 2, in the CA1 pyramidal neurons compared with controls, and their expressions induced by lacosamide pre-treatment were maintained after transient cerebral ischemia. In brief, pre-treatment with lacosamide protected hippocampal CA1 pyramidal neurons from ischemic damage induced by transient global cerebral ischemia, and the lacosamide-mediated neuroprotection may be closely related to increases of CAT and GPX expressions by lacosamide pre-treatment.

Comparison of immunoreactivities of calbindin-D28k, calretinin and parvalbumin in the striatum between young, adult and aged mice, rats and gerbils.

Calcium binding proteins play important roles in all aspects of neural functioning in the central nervous system. In the present study, we examined age-related changes of three different calcium binding proteins calbindin-D28k (CB), calretinin (CR) and parvalbumin (PV) immunoreactivities in the striatum of young (1 month), adult (6 months) and aged (24 months) ages in three species of rodents (mouse, rat and gerbil) using immunohistochemistry and Western blotting. Our results show that the number of CB-immunoreactive neurons was highest in the adult mouse and rat; however, in the gerbil, the number of CB-immunoreactive neurons was not significantly different from each group although the CB immunoreactivity was significantly decreased in the aged group compared with the adult group. The number of CR-immunoreactive neurons in the striatum was significantly highest in all the adult groups, and, especially, the number of CR-immunoreactive neurons and CR immunoreactivity in the aged gerbil were significantly decreased in the aged group compared with the other groups. Finally, we did not found any significant difference in the number of PV-immunoreactive neurons in the striatum with age among the three rodents. On the other hand, we found that protein levels of three calcium binding proteins in all the mouse groups were similar to the immunohistochemical data. These results indicate that the distribution pattern of calcium binding proteins is different according to age; the adult might show an apparent tendency of high expression in the striatum.

Neuroprotection of Ischemic Preconditioning is Mediated by Anti-inflammatory, Not Pro-inflammatory, Cytokines in the Gerbil Hippocampus Induced by a Subsequent Lethal Transient Cerebral Ischemia.

Ischemic preconditioning (IPC) induced by sublethal transient cerebral ischemia could reduce neuronal damage/death following a subsequent lethal transient cerebral ischemia. We, in this study, compared expressions of interleukin (IL)-2 and tumor necrosis factor (TNF)-α as pro-inflammatory cytokines, and IL-4 and IL-13 as anti-inflammatory cytokines in the gerbil hippocampal CA1 region between animals with lethal ischemia and ones with IPC followed by lethal ischemia. In the animals with lethal ischemia, pyramidal neurons in the stratum pyramidale (SP) of the hippocampal CA1 region were dead at 5 days post-ischemia; however, IPC protected the CA1 pyramidal neurons from lethal ischemic injury. Expressions of all cytokines were significantly decreased in the SP after lethal ischemia and hardly detected in the SP at 5 days post-ischemia because the CA1 pyramidal neurons were dead. IPC increased expressions of anti-inflammatory cytokines (IL-4 and IL-13) in the stratum pyramidale of the CA1 region following no lethal ischemia (sham-operation), and the increased expressions of IL-4 and IL-13 by IPC were continuously maintained is the SP of the CA1 region after lethal ischemia. However, pro-inflammatory cytokines (IL-2 and TNF-α) in the SP of the CA1 region were similar those in the sham-operated animals with IPC, and the IL-4 and IL-13 expressions in the SP were maintained after lethal ischemia. In conclusion, this study shows that anti-inflammatory cytokines significantly increased and longer maintained by IPC and this might be closely associated with neuroprotection after lethal transient cerebral ischemia.

Adenylyl-Sulfate Kinase (Met14)-Dependent Cysteine and Methionine Biosynthesis Pathways Contribute Distinctively to Pathobiological Processes in Cryptococcus neoformans.

Blocking of nutrient uptake and amino acid biosynthesis are considered potential targets for next-generation antifungal drugs against pathogenic fungi, including Cryptococcus neoformans. In this regard, the sulfate assimilation pathway is particularly attractive, as it is only present in eukaryotes such as plants and fungi, yet not in mammals. Here, we demonstrated that the adenylyl sulfate kinase (Met14) in the sulfate assimilation pathway is not essential yet is required for the viability of C. neoformans due to its involvement in biosynthesis of two sulfur-containing amino acids, cysteine and methionine. Met14-dependent cysteine and methionine biosynthesis was found to significantly contribute to a diverse range of pathobiological processes in C. neoformans. Met14-dependent cysteine rather than methionine biosynthesis was also found to play pivotal roles in cell growth and tolerance to environmental stresses and antifungal drugs. In contrast, the Met14-dependent methionine biosynthesis was found to be more important than cysteine biosynthesis for the production of major cryptococcal virulence factors of melanin pigments and polysaccharide capsules. Finally, we also found that despite its attenuated virulence in an insect model, Galleria mellonella, the met14Δ mutant yielded no difference in virulence in a murine model of systemic cryptococcosis. Hence, clinical inhibition of Met14-dependent amino acid biosynthetic pathways may not be advantageous for the treatment of systemic cryptococcosis. IMPORTANCE Current antifungal drugs have several limitations, such as drug resistance, severe side effects, and a narrow spectrum. Therefore, novel antifungal targets are urgently needed. To this end, fungal sulfur amino acid biosynthetic pathways are considered potential targets for development of new antifungal agents. Here, we demonstrated that Met14 in the sulfate assimilation pathway promotes growth, stress response, and virulence factor production in C. neoformans via synthesis of sulfur-containing amino acids methionine and cysteine. Met14-dependent cysteine rather than methionine synthesis was found to be critical for growth and stress responses, whereas Met14-dependent methionine synthesis was more important for the production of antiphagocytic capsules and antioxidant melanin in C. neoformans. Surprisingly, deletion of the MET14 gene was found to attenuate cryptococcal virulence in an insect model, yet not in a murine model. Collectively, our results showed that Met14-dependent cysteine and methionine biosynthesis play roles that are distinct from each other in C. neoformans. Moreover, Met14 is unlikely to be a suitable anticryptococcal drug target.

Effects of Cu,Zn-superoxide dismutase on cell proliferation and neuroblast differentiation in the mouse dentate gyrus.

Oxidative stress is one of the most important factors in reducing adult hippocampal neurogenesis in the adult brain. In this study, we observed the effects of Cu,Zn-superoxide dismutase (SOD1) on lipid peroxidation, cell proliferation, and neuroblast differentiation in the mouse dentate gyrus using malondialdehyde (MDA), Ki67, and doublecortin (DCX), respectively. We constructed an expression vector, PEP-1, fused PEP-1 with SOD1, and generated PEP-1-SOD1 fusion protein. We administered PEP-1 and 100 or 500 µg PEP-1-SOD1 intraperitoneally once a day for 3 weeks and sacrificed at 30 min after the last administrations. PEP-1 administration did not change the MDA levels compared to those in the vehicle-treated group, while PEP-1-SOD1 treatment significantly reduced MDA levels compared to the vehicle-treated group. In the PEP-1-treated group, the number of Ki67-positive nuclei was similar to that in the vehicle-treated group. In the 100 µg PEP-1-SOD1-treated group, the number of Ki67-positive nuclei was slightly decreased; however, in the 500 µg PEP-1-SOD1-treated group, Ki67-positive nuclei were decreased to 78.5% of the vehicle-treated group. The number of DCX-positive neuroblasts in the PEP-1-treated group was similar to that in the vehicle-treated group. However, the arborization of DCX-positive neuroblasts was significantly decreased in both the 100 and 500 µg PEP-1-SOD1-treated groups compared to that in the vehicle-treated group. The number of DCX-positive neuroblasts with tertiary dendrites was markedly decreased in the 500 µg PEP-1-SOD1-treated group. These results suggest that a SOD1 supplement to healthy mice may not be necessary to modulate cell proliferation and neuroblast differentiation in the dentate gyrus.

Effects of sensitive to apoptosis gene protein on cell proliferation, neuroblast differentiation, and oxidative stress in the mouse dentate gyrus.

Sensitive to apoptosis gene (SAG) protein is a redox-inducible protein that protects cells against apoptosis induced by redox agents. In this study, we observed effects of SAG on cell proliferation and neuroblast differentiation in the mouse hippocampal dentate gyrus (DG) using Ki67 and doublecortin (DCX), respectively. For easy penetration into neurons, Tat-SAG expression vector was constructed by ligation with SAG and expression vector, Tat, in-frame with six histidine open-reading frames to generate the expression vector, and cloned into E. coli DH5α cells. One or 5 mg/kg Tat-SAG fusion protein (Tat-SAG) was intraperitoneally administered to mice once a day for 3 weeks. The administration of Tat-SAG significantly increased the number of 5-bromodeoxyuridine positive cells, Ki67 positive cells and DCX immunoreactive neuroblast in the mouse DG: Especially, in the 5 mg/kg Tat-SAG-treated mice, DCX positive neuroblasts showed a well-developed arborization of tertiary dendrites in the DG. On the other hand, we examined that the administration of Tat-SAG significantly reduced the DNA damage and lipid peroxidation judging from 8-hydroxy-2'-deoxyguanosine and 4-hydroxynonenal immunohistochemistry: The decrease was much more distinct in the 5 mg/kg Tat-SAG-treated mice than 1 mg/kg Tat-SAG-treated mice. This result suggests that SAG significantly increases cell proliferation, neuroblast differentiation and oxidative stress in normal states.

Neuronal damage using fluoro-jade B histofluorescence and gliosis in the striatum after various durations of transient cerebral ischemia in gerbils.

Ischemic damage occurs well in vulnerable regions of the brain, including the hippocampus and striatum. In the present study, we examined neuronal damage/death and glial changes in the striatum 4 days after 5, 10, 15 and 20 min of transient cerebral ischemia using the gerbil. Spontaneous motor activity was increased with the duration time of ischemia-reperfusion (I-R). To examine neuronal damage, we used Fluoro-Jade B (F-J B, a marker for neuronal degeneration) histofluorescence staining. F-J B positive cells were detected only in the 20 min ischemia-group, not in the other groups. In addition, we examined gliosis of astrocytes and microglia using anti-glial fibrillary acidic protein (GFAP) and anti- ionized calcium-binding adapter molecule 1 (Iba-1), respectively. In the 5 min ischemia-group, GFAP-immunoreactive astrocytes were distinctively increased in number, and the immunoreactivity was stronger than that in the sham-group. In the 10, 15 and 20 min ischemia-groups, GFAP-immunoreactivity was more increased with the duration of I-R. On the other hand, the immunoreactivity and the number of Iba-1-immunoreactive microglia were distinctively increased in the 5 and 10 min ischemia-groups. In the 15 min ischemia-group, cell bodies of microglia were largest, and the immunoreactivity was highest; however, in the 20 min ischemia-group, the immunoreactivity was low compared to the 15 min ischemia-group. The results of western blotting for GFAP and Iba-1 were similar to the immunohistochemical data. In brief, these findings showed that neuronal death could be detected only in the 20 min ischemia-group 4 days after I-R, and the change pattern of astrocytes and microglia were apparently different according to the duration time of I-R.

Chronological changes in inflammatory cytokines immunoreactivities in the mouse hippocampus after systemic administration of high dosage of tetanus toxin.

Tetanus toxin (TeT) is an exotoxin and has a capacity for neuronal binding and internalization. In the present study, we compared changes in the immunoreactivities and protein levels of interleukin (IL-) 2 as a pro-inflammatory cytokine and IL-4 as an anti-inflammatory cytokine in the hippocampus proper (HP) and dentate gyrus (DG) after systemic treatment of 10 or 100 ng/kg TeT into mice. In this study, we could not find any neuronal damage or loss in any subregions of the hippocampus after TeT treatment. In the control groups, strong IL-2 immunoreactivity was shown in the stratum pyramidal (SP) of the HP and in the granule cell layer (GCL) of the DG. At 6 h post-treatment, IL-2 immunoreactivity was hardly detected in the SP and GCL; however, strong IL-2 immunoreactivity was shown in the stratum oriens of the HP in both the groups. Thereafter, intermediate IL-2 immunoreactivity was shown in the SP and GCL. On the other hand, intermediate IL-4 immunoreactivity was detected in the SP and GCL of the control groups. At 6 h post-treatment, IL-4 immunoreactivity in the SP and GCL was apparently increased. Thereafter, IL-4 immunoreactivity was lower than that at 6 h post-treatment. In brief, IL-2 and 4 immunoreactivities were easily detected in SP and GCL in the controls and dramatically decreased and increased at 6 h post-treatment, respectively.

Extract from Terminalia chebula seeds protect against experimental ischemic neuronal damage via maintaining SODs and BDNF levels.

The fruit of Terminalia chebula Retz has been used as a traditional medicine in Asia and contains tannic acid, chebulagic acid, chebulinic acid and corilagin. Extract from T. chebula seeds (TCE) has various biological functions. We observed the neuroprotective effects of TCE against ischemic damage in the hippocampal C1 region (CA1) of the gerbil that had received oral administrations of TCE (100 mg/kg) once a day for 7 days before the induction of transient cerebral ischemia. In the TCE-treated ischemia group, neuronal neuclei (a marker for neurons)-positive neurons were distinctively abundant (62% of the sham group) in the CA1 4 days after ischemia-reperfusion (I-R) compared to those (12.2% of the sham group) in the vehicle-treated ischemia group. Four days after I-R TCE treatment markedly decreased the activation of astrocytes and microglia in the ischemic CA1 compared with the vehicle-treated ischemia group. In addition, immunoreactivities of Cu, Zn-superoxide dismutase (SOD1), Mn-superoxide dismutase (SOD2) and brain-derived neurotrophic factor (BDNF) in the CA1 of the TCE-treated ischemia group were much higher than those in the vehicle-ischemia group 4 days after I-R. Protein levels of SOD1, SOD2 and BDNF in the TCE-treated ischemia group were also much higher than those in the vehicle-ischemia group 4 days after I-R. These results indicate that the repeated supplement of TCE protected neurons from ischemic damage induced by transient cerebral ischemia by maintaining SODs and BDNF levels as well as decreasing glial activation.

Comparison of glucocorticoid receptor and ionized calcium-binding adapter molecule 1 immunoreactivity in the adult and aged gerbil hippocampus following repeated restraint stress.

Stress leads to changes in homeostasis and internal balance of the body and is known to be one of important factors in the development of several diseases. In the present study, we investigated changes in glucocorticoid receptor (GR) and ionized calcium-binding adapter molecule 1 (Iba-1) immunoreactivity in the adult and aged gerbil hippocampus after chronic restraint stress. Serum corticosterone level was much higher in both the stress-groups than the control groups. No neuronal death was found in all hippocampal subregions of the adult and aged gerbil after chronic restraint stress. GR immunoreactivity was decreased in both the adult and aged groups after repeated restraint stress; however, GR immunoreactivity in the adult-stress-group was decreased much more than that in the aged-stress-group. Iba-1 immunoreactive microglia were hypertrophied and activated in the adult group after repeated restraint stress; in the aged-stress-group, there was no any significant change in Iba-1 immunoreactive microglia. In brief, level of GR, not Iba-1, in the hippocampus was much decreased in the adult gerbil compared to the aged gerbil following chronic restraint stress.

PEP-1-frataxin significantly increases cell proliferation and neuroblast differentiation by reducing lipid peroxidation in the mouse dentate gyrus.

Frataxin plays important roles in the mitochondrial respiratory chain and in the differentiation of neurons during early development. In this study, we observed the effects of frataxin on cell proliferation and neuroblast differentiation in the mouse hippocampal dentate gyrus. For this, we constructed an expression vector, PEP-1, that was fused with frataxin to create a PEP-1-frataxin fusion protein that easily penetrated frataxin into the blood-brain barrier. Three mg/kg PEP-1-frataxin was intraperitoneally administered to mice once a day for 2 weeks. The administration of PEP-1 alone did not result in any significant changes in the number of Ki67-positive cells and doublecortin (DCX)-immunoreactive neuroblasts in the mouse dentate gyrus. However, the administration of PEP-1-frataxin significantly increased the number of Ki67-positive cells and DCX-immunoreactive neuroblasts in the mouse dentate gyrus. In addition, PEP-1-frataxin significantly reduced 4-hydroxynonenal protein levels and malondialdehyde formation, while Cu, Zn-superoxide dismutase protein levels were maintained. These results suggest that frataxin effectively increased cell proliferation and neuroblast differentiation by decreasing lipid peroxidation in the dentate gyrus.

Down-regulation of cyclin-dependent kinase 5 attenuates p53-dependent apoptosis of hippocampal CA1 pyramidal neurons following transient cerebral ischemia.

Abnormal activation of cyclin-dependent kinase 5 (Cdk5) is associated with pathophysiological conditions. Ischemic preconditioning (IPC) can provide neuroprotective effects against subsequent lethal ischemic insult. The objective of this study was to determine how Cdk5 and related molecules could affect neuroprotection in the hippocampus of gerbils after with IPC [a 2-min transient cerebral ischemia (TCI)] followed by 5-min subsequent TCI. Hippocampal CA1 pyramidal neurons were dead at 5 days post-TCI. However, treatment with roscovitine (a potent inhibitor of Cdk5) and IPC protected CA1 pyramidal neurons from TCI. Expression levels of Cdk5, p25, phospho (p)-Rb and p-p53 were increased in nuclei of CA1 pyramidal neurons at 1 and 2 days after TCI. However, these expressions were attenuated by roscovitine treatment and IPC. In particular, Cdk5, p-Rb and p-p53 immunoreactivities in their nuclei were decreased. Furthermore, TUNEL-positive CA1 pyramidal neurons were found at 5 days after TCI with increased expression levels of Bax, PUMA, and activated caspase-3. These TUNEL-positive cells and increased molecules were decreased by roscovitine treatment and IPC. Thus, roscovitine treatment and IPC could protect CA1 pyramidal neurons from TCI through down-regulating Cdk5, p25, and p-p53 in their nuclei. These findings indicate that down-regulating Cdk5 might be a key strategy to attenuate p53-dependent apoptosis of CA1 pyramidal neurons following TCI.

Pre- and Post-Treatment with Novel Antiepileptic Drug Oxcarbazepine Exerts Neuroprotective Effect in the Hippocampus in a Gerbil Model of Transient Global Cerebral Ischemia.

Oxcarbazepine, an antiepileptic drug, has been reported to modulate voltage-dependent sodium channels, and it is commonly used in epilepsy treatment. In this study, we investigated the neuroprotective effect of oxcarbazepine in the hippocampus after transient ischemia in gerbils. Gerbils randomly received oxcarbazepine 100 or 200 mg/kg before and after transient ischemia. We examined its neuroprotective effect in the cornu ammonis 1 subfield of the gerbil hippocampus at 5 days after transient ischemia by using cresyl violet staining, neuronal nuclei immunohistochemistry and Fluoro-Jade B histofluorescence staining for neuroprotection, and by using glial fibrillary protein and ionized calcium-binding adapter molecule 1 immunohistochemistry for reaction of astrocytes and microglia, respectively. Pre- and post-treatment with 200 mg/kg of oxcarbazepine, but not 100 mg/kg of oxcarbazepine, protected pyramidal neurons of the cornu ammonis 1 subfield from transient ischemic damage. In addition, pre- and post-treatment with oxcarbazepine (200 mg/kg) significantly ameliorated astrocytes and microglia activation in the ischemic cornu ammonis 1 subfield. In brief, our current results indicate that post-treatment as well as pre-treatment with 200 mg/kg of oxcarbazepine can protect neurons from ischemic insults via attenuation of the glia reaction.