Neuregulin-1/PI3K signaling effects on oligodendrocyte proliferation, remyelination and behaviors deficit in a male mouse model of ischemic stroke.

In this study, we investigated the effect of neuregulin-1 (NRG1) on demyelination and neurological function in an ischemic stroke model, and further explored its neuroprotective mechanisms. Adult male ICR mice underwent photothrombotic ischemia surgery and were injected with NRG1 beginning 30 min after ischemia. Cylinder and grid walking tests were performed to evaluate the forepaw function. In addition, the effect of NRG1 on neuronal damage/death (Cresyl violet, CV), neuronal nuclei (NeuN), nestin, doublecortin (DCX), myelin basic protein (MBP), non-phosphorylated neurofilaments (SMI-32), adenomatous polyposis coli (APC), erythroblastic leukemia viral oncogene homolog (ErbB) 2, 4 and serine-threonine protein kinase (Akt) in cortex were evaluated using immunohistochemistry, immunofluorescence and western blot. The cylinder and grid walking tests exposed that treatment of NRG1 observably regained the forepaw function. NRG1 treatment reduced cerebral infarction, restored forepaw function, promoted proliferation and differentiation of neuron and increased oligodendrogliogenesis. The neuroprotective effect of NRG1 is involved in its activation of PI3K/Akt signaling pathway via ErbB2, as shown by the suppression of the effect of NRG1 by the PI3K inhibitor LY294002. Our results demonstrate that NRG1 is effective in ameliorating the both acute phase neuroprotection and long-term neurological functions via resumption of neuronal proliferation and differentiation and oligodendrogliogenesis in a male mouse model of ischemic stroke.

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.

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.

Intermittent fasting increases the expressions of SODs and catalase in granule and polymorphic cells and enhances neuroblast dendrite complexity and maturation in the adult gerbil dentate gyrus.

Intermittent fasting (ImF) is known to reduce oxidative stress and affects adult neurogenesis in the hippocampal dentate gyrus. However, it is unknown how ImF affects endogenous antioxidants expressions, cell proliferation, and neuroblast differentiation and their dendrite remodeling over 3 months in the dentate gyrus of adult gerbils. The present study subjected 6­month old male gerbils to a normal diet or alternate­day ImF for 1, 2 and 3 months. Changes in body weight were not significantly different between gerbils fed a normal diet and on ImF. The present study also investigated the effects of ImF on antioxidant enzymes [superoxide dismutase (SOD)­1, SOD2 and catalase] using immunohistochemistry, and endogenous cell proliferation, neuroblast differentiation and neuroblast dendrite complexity by using Ki67 (a cell proliferation marker) and doublecortin (neuroblast differentiation marker) immunohistochemistry in the dentate gyrus. SOD1, SOD2 and CAT immunoreactivities were shown in cells in the granule cell and polymorphic layers. SOD1, SOD2 and catalase immunoreactivity in the cells peaked at 2, 1 and 1 month, respectively, following ImF. Cell proliferation was ~250, 129 and 186% of the control, at 1, 2 and 3 months of ImF, respectively. Neuroblast differentiation was ~41, 32 and 12% of the control, at 1, 2 and 3 months of ImF, respectively, indicating that dendrites of neuroblasts were more arborized and developed at 3 months of ImF. Taken together, these results indicate that ImF for 3 months improves endogenous SOD1, SOD2 and catalase expressions and enhances cell proliferation, and neuroblast dendrites complexity and maturation in the adult gerbil dentate gyrus.

Intermittent fasting increases SOD2 and catalase immunoreactivities in the hippocampus but does not protect from neuronal death following transient ischemia in gerbils.

Intermittent fasting has been shown to have neuroprotective effects against transient focal cerebral ischemic insults. However, the effects of intermittent fasting on transient global ischemic insult has not been studied much yet. The present study examined effects of intermittent fasting on endogenous antioxidant enzyme expression levels in the hippocampus and investigated whether the fasting protects neurons 5 days after 5 min of transient global cerebral ischemia. Gerbils were randomly subjected to either ad libitum or alternate­day intermittent fasting for two months and assigned to sham surgery or transient ischemia. Changes of antioxidant enzymes were examined using immunohistochemistry for cytoplasmic superoxide dismutase 1 (SOD1), mitochondrial (SOD2), catalase (CAT), and glutathione peroxidase (GPX). The effects of intermittent fasting on ischemia­induced antioxidant changes, neuronal damage/degeneration and glial activation were examined. The weight of fasting gerbils was not different from that of control gerbils. In controls, SOD1 and GPX immunoreactivities were strong in pyramidal neurons of filed cornu ammonis 1 (CA1). Transient ischemia in controls significantly decreased expressions of SOD1 and GPX in CA1 pyramidal neurons. Intermittent fasting resulted in increased expressions of SOD2 and CAT, not of SOD1 and GPX, in CA1 pyramidal neurons. Nevertheless, CA1 pyramidal neurons were not protected in gerbils subjected to fasting after transient ischemia, and inhibition of glial­cell activation was not observed in the gerbils. In summary, intermittent fasting for two months increased SOD2 and CAT immunoreactivities in hippocampal CA1 pyramidal neurons. However, fasting did not protect the CA1 pyramidal neurons from transient cerebral ischemia. The results of the present study indicate that intermittent fasting may increase certain antioxidants, but not protect neurons from transient global ischemic insult.

Melatonin improves vascular cognitive impairment induced by ischemic stroke by remyelination via activation of ERK1/2 signaling and restoration of glutamatergic synapses in the gerbil hippocampus.

Vascular dementia affects cognition by damaging axons and myelin. Melatonin is pharmacologically associated with various neurological disorders. In this study, effects of melatonin on cognitive impairment and related mechanisms were investigated in an animal model of ischemic vascular dementia (IVD). Melatonin was intraperitoneally administered to adult gerbils after transient global cerebral ischemia (tGCI) for 25 days beginning 5 days after tGCI. Cognitive impairment was examined using a passive avoidance test and the Barnes maze test. To investigate mechanisms of restorative effects by melatonin, neuronal damage/death, myelin basic protein (MBP, a marker for myelin), Rip (a marker for oligodendrocyte), extracellular signal-regulated protein kinase1/2 (ERK1/2) and phospho-ERK1/2 (p-ERK1/2), and vesicular glutamate transporter (VGLUT)-1 (a glutamatergic synaptic marker) in the hippocampal Cornu Ammonis 1 area (CA1) were evaluated using immunohistochemistry. Melatonin treatment significantly improved tGCI-induced cognitive impairment. Death of CA1 pyramidal neurons after tGCI was not affected by melatonin treatment. However, melatonin treatment significantly increased MBP immunoreactivity and numbers of Rip-immunoreactive oligodendrocytes in the ischemic CA1. In addition, melatonin treatment significantly increased ERK1/2 and p-ERK1/2 immunoreactivities in oligodendrocytes in the ischemic CA1. Furthermore, melatonin treatment significantly increased VGLUT-1 immunoreactive structures in the ischemic CA1. These results indicate that long-term melatonin treatment after tGCI improves cognitive deficit via restoration of myelin, increase of oligodendrocytes which is closely related to the activation of ERK1/2 signaling, and increase of glutamatergic synapses in the ischemic brain area.

Melatonin Improves Cognitive Deficits via Restoration of Cholinergic Dysfunction in a Mouse Model of Scopolamine-Induced Amnesia.

Melatonin is known to improve cognitive deficits, and its functions have been studied in various disease models, including Alzheimer's disease. In this study, we investigated effects of melatonin on cognition and the cholinergic system of the septum and hippocampus in a mouse model of scopolamine-induced amnesia. Scopolamine (1 mg/kg) and melatonin (10 mg/kg) were administered intraperitoneally to mice for 2 and 4 weeks. The Morris water maze and passive avoidance tests revealed that both treatments of scopolamine significantly impaired spatial learning and memory; however, 2- and 4-week melatonin treatments significantly improved spatial learning and memory. In addition, scopolamine treatments significantly decreased protein levels and immunoreactivities of choline acetyltransferase (ChAT), high-affinity choline transporter (CHT), vesicular acetylcholine transporter (VAChT), and muscarinic acetylcholine receptor M1 (M1R) in the septum and hippocampus. However, the treatments with melatonin resulted in increased ChAT-, CHT-, VAChT-, and M1R-immunoreactivities and their protein levels in the septum and hippocampus. Our results demonstrate that melatonin treatment is effective in improving the cognitive deficits via restoration of the cholinergic system in the septum and hippocampus of a mouse model of scopolamine-induced amnesia.

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.

Neuroprotective effects of ischemic preconditioning on hippocampal CA1 pyramidal neurons through maintaining calbindin D28k immunoreactivity following subsequent transient cerebral ischemia.

Ischemic preconditioning elicited by a non-fatal brief occlusion of blood flow has been applied for an experimental therapeutic strategy against a subsequent fatal ischemic insult. In this study, we investigated the neuroprotective effects of ischemic preconditioning (2-minute transient cerebral ischemia) on calbindin D28k immunoreactivity in the gerbil hippocampal CA1 area following a subsequent fatal transient ischemic insult (5-minute transient cerebral ischemia). A large number of pyramidal neurons in the hippocampal CA1 area died 4 days after 5-minute transient cerebral ischemia. Ischemic preconditioning reduced the death of pyramidal neurons in the hippocampal CA1 area. Calbindin D28k immunoreactivity was greatly attenuated at 2 days after 5-minute transient cerebral ischemia and it was hardly detected at 5 days post-ischemia. Ischemic preconditioning maintained calbindin D28k immunoreactivity after transient cerebral ischemia. These findings suggest that ischemic preconditioning can attenuate transient cerebral ischemia-caused damage to the pyramidal neurons in the hippocampal CA1 area through maintaining calbindin D28k immunoreactivity.

Pretreated quercetin protects gerbil hippocampal CA1 pyramidal neurons from transient cerebral ischemic injury by increasing the expression of antioxidant enzymes.

Quercetin (QE; 3,5,7,3',4'-pentahydroxyflavone), a well-known flavonoid, has been shown to prevent against neurodegenerative disorders and ischemic insults. However, few studies are reported regarding the neuroprotective mechanisms of QE after ischemic insults. Therefore, in this study, we investigated the effects of QE on ischemic injury and the expression of antioxidant enzymes in the hippocampal CA1 region of gerbils subjected to 5 minutes of transient cerebral ischemia. QE was pre-treated once daily for 15 days before ischemia. Pretreatment with QE protected hippocampal CA1 pyramidal neurons from ischemic injury, which was confirmed by neuronal nuclear antigen immunohistochemistry and Fluoro-Jade B histofluorescence staining. In addition, pretreatment with QE significantly increased the expression levels of endogenous antioxidant enzymes Cu/Zn superoxide dismutase, Mn superoxide dismutase, catalase and glutathione peroxidase in the hippocampal CA1 pyramidal neurons of animals with ischemic injury. These findings demonstrate that pretreated QE displayed strong neuroprotective effects against transient cerebral ischemia by increasing the expression of antioxidant enzymes.

Effects of long­term post­ischemic treadmill exercise on gliosis in the aged gerbil hippocampus induced by transient cerebral ischemia.

Therapeutic exercise is an integral component of the rehabilitation of patients who have suffered a stroke. The objective of the present study was to use immunohistochemistry to investigate the effects of post­ischemic exercise on neuronal damage or death and gliosis in the aged gerbil hippocampus following transient cerebral ischemia. Aged gerbils (male; age, 22­24 months) underwent ischemia and were subjected to treadmill exercise for 1 or 4 weeks. Neuronal death was detected in the stratum pyramidale of the hippocampal CA1 region and in the polymorphic layer of the dentate gyrus using cresyl violet and Fluoro­Jade B histofluorescence staining. No significant difference in neuronal death was identified following 1 or 4 weeks of post­ischemic treadmill exercise. However, post­ischemic treadmill exercise affected gliosis (the activation of astrocytes and microglia). Glial fibrillary acidic protein­immunoreactive astrocytes and ionized calcium binding adaptor molecule 1­immunoreactive microglia were activated in the CA1 and polymorphic layer of the dentate gyrus of the group without treadmill exercise. Conversely, 4 weeks of treadmill exercise significantly alleviated ischemia­induced astrocyte and microglial activation; however, 1 week of treadmill exercise did not alleviate gliosis. These findings suggest that long­term post­ischemic treadmill exercise following transient cerebral ischemia does not influence neuronal protection; however, it may effectively alleviate transient cerebral ischemia­induced astrocyte and microglial activation in the aged hippocampus.

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.

Pre-treated Populus tomentiglandulosa extract inhibits neuronal loss and alleviates gliosis in the gerbil hippocampal CA1 area induced by transient global cerebral ischemia.

The genus Populus (poplar) belonging to the Salicaceae family has been used in traditional medicine, and its several species show various pharmacological properties including antioxidant and anti-inflammatory effects. No study regarding protective effects of Populus species against cerebral ischemia has been reported. Therefore, in the present study, we examined neuroprotective effects of ethanol extract from Populus tomentiglandulosa (Korea poplar) in the hippocampal cornu ammonis (CA1) area of gerbils subjected to 5 minutes of transient global cerebral ischemia. Pretreatment with 200 mg/kg of P. tomentiglandulosa extract effectively protected CA1 pyramidal neurons from transient global cerebral ischemia. In addition, glial fibrillary acidic protein immunoreactive astrocytes and ionized calcium binding adapter molecule 1 immunoreactive microglia were significantly diminished in the ischemic CA1 area by pretreatment with 200 mg/kg of P. tomentiglandulosa extract. Briefly, our results indicate that pretreatment with P. tomentiglandulosa extract protects neurons from transient cerebral ischemic injury and diminish cerebral ischemia-induced reactive gliosis in ischemic CA1 area. Based on these results, we suggest that P. tomentiglandulosa can be used as a potential candidate for prevention of ischemic injury.

Intravenously Infused F3.Olig2 Improves Memory Deficits via Restoring Myelination in the Aged Hippocampus Following Experimental Ischemic Stroke.

Oligodendrocytes play a crucial role in creating the myelin sheath that is an important component in neural transmission. In an animal model of transient cerebral ischemia, application of oligodendrocyte progenitor cells (OPCs) has not yet been reported. In this study, the effects of F3.Olig2 transplantation on memory and cognitive dysfunction were investigated in the aged gerbil in which ischemic stroke was induced. To investigate the possible mechanisms underlying repair, changes in the expression of myelin basic protein (MBP), oligodendrocyte-specific protein (OSP), and brain-derived neurotrophic factor (BDNF) were examined. Experimental ischemic stroke was induced by occlusion of bilateral common carotid arteries in aged gerbils. Gerbils (n=31 per group) were randomly divided into three groups: (1) vehicle sham group, (2) vehicle ischemia group, and (3) F3.Olig2 ischemia group. After 1, 3, and 7 days of ischemiareperfusion (I-R), saline or F3.Olig2 cells (1106 cells in 100 l) were injected into the gerbils intravenously. The gerbils were sacrificed 10 days after I-R for identification of grafted F3.Olig2 cells, and 15 and 30 days after I-R for tissue analysis after conducting passive avoidance and novel object recognition test. Injected F3.Olig2 cells and MBP, OSP, and BDNF were detected by specific antibodies using immunohistochemistry and/or Western blots. Memory and cognition were significantly increased in the F3.Olig2 ischemia group compared with the vehicle ischemia group. In the F3.Olig2 ischemia group, the neurons were not protected from ischemic damage; however, MBP, OSP, and BDNF expressions were significantly increased. Our results show that injection of F3.Olig2 cells significantly improved impaired memory and cognition, which might be related to increased MBP expression via increasing OSP and BDNF expression in the aged gerbil hippocampus following transient cerebral ischemia.

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 catalase immunoreactivity in the hippocampus between young, adult and aged mice and rats.

Catalase (CAT) is an important antioxidant enzyme and is crucial in modulating synaptic plasticity in the brain. In this study, CAT expression as well as neuronal distribution was compared in the hippocampus among young, adult and aged mice and rats. Male ICR mice and Sprague Dawley rats were used at postnatal month (PM) 1, PM 6 and PM 24 as the young, adult and aged groups, respectively (n=14/group). CAT expression was examined by immunohistochemistry and western blot analysis. In addition, neuronal distribution was examined by NeuN immunohistochemistry. In the present study, the mean number of NeuN­immunoreactive neurons was marginally decreased in mouse and rat hippocampi during aging, although this change was not identified to be significantly different. However, CAT immunoreactivity was significantly increased in pyramidal and granule neurons in the adult mouse and rat hippocampi and was significantly decreased in the aged mouse and rat hippocampi compared with that in the young animals. CAT protein levels in the hippocampus were also lowest in the aged mouse and rat hippocampus. These results indicate that CAT expression is significantly decreased in the hippocampi of aged animals and decreased CAT expression may be closely associated with aging.

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.

Long-term observation of neuronal degeneration and microgliosis in the gerbil dentate gyrus after transient cerebral ischemia.

Ischemic insults in the central nervous system evoke activation of microglia. In this study, we investigated long-term changes of neuronal damage and microglial activation in the gerbil dentate gyrus for 60 days after transient cerebral ischemia using immunohistochemistry and western blot. Neuronal damage or death was hardly found in the dentate gyrus after transient ischemia using cresyl violet staining and NeuN immunohistochemistry; however, neuronal degeneration was detected in the polymorphic layer of the dentate gyrus using Fluoro-Jade (F-J) B staining. F-J B-positive cells were significantly increased after ischemia-reperfusion (I-R) and peaked at 3 days post-ischemia, thereafter, F-J B-positive cells were decreased in a time-dependent manner and shown until 30 days post-ischemia; no F-J B-positive cells were observed 60 days after I-R. On the other hand, Iba-1-immunoreactive microglia were hypertrophied after I-R, and numbers of Iba-1-immunoreactive microglia were significantly increased along with the neuronal degeneration and highest 7 days after I-R, thereafter, numbers of Iba-1-immunoreactive microglia were decreased with time, although microglia activation lasted up to 60 days after I-R. In addition, Iba-1 protein level in the dentate gyrus after I-R was changed like immunohistochemical change. Our results, in brief, indicate that transient ischemia-induced neuronal degeneration in the dentate gyrus is maintained for about 30 days after I-R and that microglial activation lasts up to, at least, 60 days after I-R in the gerbil dentate gyrus after transient cerebral ischemia.

Neuroprotection and reduced gliosis by atomoxetine pretreatment in a gerbil model of transient cerebral ischemia.

Atomoxetine (ATX) is a non-stimulant selective norepinephrine reuptake inhibitor that is widely used for the treatment of attention-deficit/hyperactivity disorder (ADHD). In this study, we firstly examined neuroprotective effects of pre- or post-treatment with 15 and 30 mg/kg ATX against ischemic damage in the gerbil hippocampal cornus ammonis 1 (CA1) region subjected to 5 min of transient cerebral ischemia using cresyl violet staining, neuronal nuclei immunohistochemistry and Fluoro-J B histofluorescence staining. We found that only pre-treatment with 30 mg/kg ATX protected CA1 pyramidal neurons from ischemic insult. In addition, pre-treatment with 30 mg/kg ATX, which had neuroprotective effect against ischemic damage, distinctly attenuated the activation of astrocytes and microglia in the ischemic CA1 region compared with the vehicle-treated ischemia group by glial fibrillary acidic protein (for astrocytes) and ionized calcium-binding adapter molecule 1 (for microglia) immunohistochemistry. In brief, our present results indicate that ATX has neuroprotective effect against transient cerebral ischemic insult and that the neuroprotective effect of ATX may be closely associated with attenuated glial activation.

Delayed hippocampal neuronal death in young gerbil following transient global cerebral ischemia is related to higher and longer-term expression of p63 in the ischemic hippocampus.

The tumor suppressor p63 is one of p53 family members and plays a vital role as a regulator of neuronal apoptosis in the development of the nervous system. However, the role of p63 in mature neuronal death has not been addressed yet. In this study, we first compared ischemia-induced effects on p63 expression in the hippocampal regions (CA1-3) between the young and adult gerbils subjected to 5 minutes of transient global cerebral ischemia. Neuronal death in the hippocampal CA1 region of young gerbils was significantly slow compared with that in the adult gerbils after transient global cerebral ischemia. p63 immunoreactivity in the hippocampal CA1 pyramidal neurons in the sham-operated young group was significantly low compared with that in the sham-operated adult group. p63 immunoreactivity was apparently changed in ischemic hippocampal CA1 pyramidal neurons in both ischemia-operated young and adult groups. In the ischemia-operated adult groups, p63 immunoreactivity in the hippocampal CA1 pyramidal neurons was significantly decreased at 4 days post-ischemia; however, p63 immunoreactivity in the ischemia-operated young group was significantly higher than that in the ischemia-operated adult group. At 7 days post-ischemia, p63 immunoreactivity was decreased in the hippocampal CA1 pyramidal neurons in both ischemia-operated young and adult groups. Change patterns of p63 level in the hippocampal CA1 region of adult and young gerbils after ischemic damage were similar to those observed in the immunohistochemical results. These findings indicate that higher and longer-term expression of p63 in the hippocampal CA1 region of the young gerbils after ischemia/reperfusion may be related to more delayed neuronal death compared to that in the adults.