| BackgroundCerebral ischemic (CI) and neurodegenerative diseases can induce hippocampalneuron loss of varying severity which are pivotally implicated in the deficiencies of spatiallearning and memory. Although persistent progenitor cell proliferation and neurogenesiscommonly follow ischemic periods in the forebrain subventricular zone (SGZ) andhippocampal dentate gyrus (DG), a phenomenon that has been well documented in rodentmodels, the definite role of neurogenesis following CI remains controversial.Neurogenesis in response to ischemic neuron loss may be a result of neural stem cells(NSCs) participation in the endogenous repair processes of the adult mammalian brain.New neurons have been detected in the injured area of previously ischemic tissues, where these new cells were functionally integrated into existing cerebral circuits. Conversely,neurogenesis has been suppressed using X-ray irradiation prior to bilateral commoncarotid arteries occlusion (BCCAO) in gerbil models, resulting in severe spatial learningand memory deficits following ischemia. These findings underscore the importance ofneurogenesis in functional recovery. Enhanced hippocampal neurogenesis also facilitateslong-term potentiation and improves learning and memory. Therefore, facilitation ofendogenous neurogenesis may be a promising regenerative strategy for ischemic stroketreatment.Both direct and indirect approaches have been developed to stimulate proliferationand neurogenesis, attenuate infarct volume, and improve neurological deficits in theischemic stroke model. Thioredoxin (Trx), a12-kD oxidoreductase enzyme containing adithiol-disulfide active site, is a redox-sensitive molecule with pleiotropic cellular effectsinvolved in regulation of proliferation, redox states, and anti-apoptosis. Two isoforms ofTrx exist in mammalian cells. Cytosolic Trx-1, which can be translocated into the nucleusunder certain circumstances, is the dominant form, and mitochondrial Trx-2occursrelatively less frequently. Endogenous Trx-1production is induced by ischemia of braintissues, where it is responsible for alleviation of oxidative damage. Moreover,exogenously administered recombinant human Trx-1(rhTrx-1) can penetrate the bloodbrain barrier and exert a neuroprotective effect, as demonstrated in the middle cerebralartery occlusion (MCAO) model. In addition to its role as an antioxidant, rhTrx-1has alsobeen reported to promote proliferation of human adipose tissue-derived mesenchymalstem cells and angiogenesis.While the neuroprotective role of rhTrx-1on injured neurons is well-established,rhTrx-1may also promote neurogenesis and facilitate long-term recovery following CI. Inorder to investigate this hypothesis, the current study applied the BCCAO mouse model toinvestigate the role of rhTrx-1in neurogenesis and its potential effect on cognitive deficitsin learning and memory following global CI. In addition, the mechanisms associated withrhTrx-1were tentatively explored. Experiment1rhTrx-1ameliorated spatial learning and memory deficits induced byischemiaObjective: To investigate the potential role of rhTrx-1on ameliorating spatial learningand memory deficits induced by ischemiaMethods: Mice were anesthetized with2%isoflurane using a facemask, and CI wasinduced by transient BCCAO for20min.1. Male C57BL/6J mice weighing18-22gpurchased from the Laboratory Animal Center of the Fourth Military Medical University(Shaanxi, China) were used in the current in vivo studies. Mice were randomized into3groups (n=5): the sham-operated group, the cerebral ischemia plus vehicle (CI+vehicle)group, and the cerebral ischemia plus rhTrx-1(CI+rhTrx-1) group. Mice of theCI+rhTrx-1group were subjected to transient BCCAO for20min and intraperitoneal (i.p.)administration of rhTrx-1containing3mg/kg of the cytosolic form of Trx (Sigma-Aldrich,St. Louise, MO)10min before reperfusion. The CI+vehicle group was simultaneouslytreated with similar BCCAO and i.p. administration of an equal volume of a0.1Mphosphate buffered saline (PBS) solution as an inert vehicle (pH7.4). After reperfusionfor72h, mice were sacrificed and underwent Nissl Staining. The numbers of healthyneurons were counted in the hippocampus CA1among the groups.2. Male C57BL/6Jmice weighing18-22g were randomized into3groups (n=8): sham-operated group, CI+Vehicle group and CI+rhTrx-1group. Experimental C57BL/6J mice were subjectedbehavioral tests30days after ischemia. To minimize potential stress-related effects fromprevious behavioral performance testing, mice were first tested in the open field and thenin the Morris water maze.3. To exclude the occurrence of hypoxemia or hypercapniaduring the anesthetics, three groups of experimental animals were used alone (n=5):sham-operated group, CI+Vehicle group and CI+rhTrx-1group. Each mouse wascollected approximately0.2ml of blood from the left carotid artery for arterial blood gasmeasurement on set of BCCAO and on set of reperfusion. Results:1. Blood gas analysis: there was no significant difference among these groups,whenever the on set of BCCAO or the on set of reperfusion.2. Nissl staining: the resultsshowed that the percentage of healthy neurons was significantly declined in CI+vehiclegroup compared to sham-operated group (P <0.01). However, the number of healthyneurons was increased after treatment with rhTrx-1(P <0.01).3. In the Morris WaterMaze test, long latent period were initially required to find the platform for all mousesubjects, though this time decreased with progressive trial number. Sham-operated andCI+rhTrx-1groups showed no significant differences in task-learning abilities and similarlatency reduction rates (P>0.05). Mice in the CI+vehicle group, however, requiredincreased times to locate the submerged platform compared with times in the rhTrx-1andsham groups for each training day in MWM testing. Consistent with these findings,two-way ANOVA indicated that group and day exerted significant effects on escapelatency. At test day1, CI+vehicle mice required longer times to find the platform relativeto the CI+rhTrx-1group; however, no statistical difference was observed (P>0.05). Bydays2-4, the time required to find the platform in the CI+vehicle group was notablylonger than those in both the sham-operated and CI+rhTrx-1groups (Fig.2A, P <0.01atday1and3; P <0.05at day4) groups. Swimming speed during the test was alsoevaluated and analyzed by two-way ANOVA, demonstrating no group or day effects onswimming speed (P>0.05). In the probe trial test, both sham-operated and CI+rhTrx-1group mice appeared to have learned the task, demonstrating superior ability to rememberthe platform location compares to vehicle-treated mice. This effect was evidenced bysignificantly longer periods of time spent in the target quadrant in the sham-operated andCI+rhTrx-1groups compared to the CI+vehicle group (P <0.05).4. In the open field test,no significant difference in horizontal distance of travel in the chamber was observedamong the three groups (P>0.05). In addition, the number of entries to the central zoneof the open field was recorded in order to assess mouse anxiety level. No significantdifference was observed in center zonetime for any group (P>0.05).Conclusions: rhTrx-1ameliorated spatial learning and memory deficits induced byischemia. Experiment2rhTrx-1promoted ischemia-induced neurogenesis in DGObjective: To investigate the effect of rhTrx-1on proliferation of the neural stem cells inDG following forebrain ischemia.Methods: Male C57BL/6J mice weighing18-22g were randomized into3groups (n=12):sham-operated group, CI+Vehicle group, and CI+rhTrx-1group. Mice were subjected todeep anesthesia with300mg/kg i.p. chloral hydrate and treated with saline followed by acold4%paraformaldehyde during transcardiac perfusion. Brain tissues were thenextracted and post-fixed in4%paraformaldehyde overnight at4°C before undergoingsucrose infiltration (20%,30%sucrose). Coronal sections with12μm thicknesses werecollected and stored at-20°C until use. For BrdU immunohistochemistry, brain tissuesections were incubated in HCl (1N) for40min at45°C to break open the DNA structureof the labeled cells, as previously described. Immediately after the acid wash, boratebuffer (0.1M) was added to the cells. All sections were then incubated in a blockingsolution for1h prior incubation with1:100rat-anti-BrdU (Abcam, USA) diluted in PBSat4°C for48h. Following incubation with the primary antibody, these sections weretreated with1:100biotinylated goat-anti-rat (Vector, USA) at room temperature for2hfollowed by treatment with1:1000rhodamine-avidin D anti-mouse IgG (Vector, USA) atroom temperature for2h. For Ki67and DCX staining, sections were first treated with1:1000anti-rabbit Ki67(Abcam, USA) and1:2000anti-rabbit DCX (Abcam, USA)antibodies at4°C overnight. After washing in PBS, sections were then incubated in1:500Alexa Fluor488-conjugated donkey anti-rabbit (Invitrogen, USA) and1:500Alexa Fluor594-conjugated goat anti-rabbit (Invitrogen, USA) secondary antibodies at roomtemperature for4h. For double immunostaining, sections were incubated with ratanti-BrdU first, as described previously. After treatment with a biotinylated goat-anti-ratand Rhodamine-avidin D, these sections were washed in PBS and treated with1:100 mouse anti-NeuN (Chemicon, USA) at4°C overnight followed by treatment with1:500Alexa Fluor488-conjugated anti-rat IgG (Invitrogen, USA) at room temperature for4h.Finally, sections were incubated with Hoechst33342for10min at room temperature tostain cell nuclei.Results: To investigate the effect of rhTrx-1on proliferation of the neural stem cellsfollowing forebrain ischemia, the cells incorporated with BrdU and those stained withKi67in the DG were counted. At post-ischemic day14, the number of BrdU+cellsincreased approximately1.5-fold in the CI+vehicle group compared with thesham-operated group (P <0.05). In addition, administration of rhTrx-1significantlyincreased the number of BrdU+cells compared with the CI+vehicle group (P <0.01).Similarly, the CI+vehicle group showed increased numbers of Ki67+cells (P <0.05) andthis number reached approximately2-fold in CI+rhTrx-1group (P <0.01). Doublecortin(DCX) expression was used to estimate cycling precursors of neuronal lineage as well asmaturing young granule cells. The CI+rhTrx-1group demonstrated a significant increasein the number of DCX+cells in the DG (P <0.01). To estimate the effect of rhTrx-1onthe differentiation of NSCs, double labeling of BrdU and NeuN was performed30daysafter ischemia. The number of BrdU+cells in the CI+rhTrx-1group was much larger thanthat observed in the CI+vehicle group (P <0.01), indicating the survival of the majority ofrhTrx-1-induced new neural cells. Although many BrdU+cells were still found in the DG,immunofluorescence staining revealed that the proportion of BrdU+/NeuN+double-labeledcells to total BrdU+cells in both the CI+rhTrx-1and CI+vehicle groups were similar (P>0.05).Conclusion: rhTrx-1promoted the proliferation of the neural stem cells in DG followingforebrain ischemia.Experiment3rhTrx-1enhanced proliferation and differentiation of cultured NSCs Objective: To investigate the direct effects of rhTrx-1on NSCs and to study theunderlying mechanismMethods:1. Embryonic brains between E14.5and E16.5were dissected under astereomicroscope, and single cell suspensions from the hippocampus were obtained bymechanical dissociation.2. To verify the identity of NSCs, neurospheres were collectedand post fixed in4%paraformaldehyde for2h at4°C before being moved into20%sucrose in0.1M phosphate buffer (PB) overnight at4°C for cry protection. Frozensections with10μm thicknesses were cut with a cryostat and mounted onto gelatinizedslides. Single cells were then plated onto poly-L-lysine coated coverslips. After a24hattachment period, cells on the slides were fixed in4%paraformaldehyde for45min andprocessed for nestin staining.3. Proliferation assay was performed after NSCs wereincubated with rhTrx-1for2d. Cells on cover slips were incubated with the followingprimary antibodies in PBS containing1%BSA and0.3%Triton-X100at4°C overnight:mouse anti-BrdU antibody (1:500, Sigma-Aldrich, USA) or rabbit anti-Ki67antibody(1:1000, Abcam, USA). BrdU-labeled and Ki67-positive cells were counted andnormalized to the total cell count.4. NSCs differentiation was examined after cells weretreated with rhTrx-1for7d and then immunocytochemical staining was conducted. Cellswere incubated at4°C overnight with the primary antibodies composed of1:500mouseanti-Tuj1(Sigma-Aldrich, USA) and1:2000rabbit anti-GFAP (Millipore, USA) diluted inblocking reagent.5. The ERK signaling pathway was inhibited by adding4μM of U0126(Sigma-Aldrich, USA) to the medium0.5h prior to rhTrx-1administration.Results:1. To investigate the direct effects of rhTrx-1on NSCs and to study theunderlying mechanism, in vitro study was also conducted. The result showed thatneurospheres and single cells expressed nestin, a marker of NSCs.2. After treatment withdifferent concentrations of rhTrx-1for48h, the percentage of BrdU labeled cells washigher in10,50, and100μg/ml rhTrx-1treatments compared to the CI+vehicle group (P<0.01). The results of Ki67staining also revealed that rhTrx-1promoted proliferation ofNSCs at concentrations of1,10, and50μg/ml.3. After treatment with rhTrx-1for7days,expression of Tuj1and GFAP were examined. Immunocytochemical results revealed that the ratio of differentiated neurons was higher in groups treated with both1μg/ml (13.51%)and10μg/ml (18.08%) rhTrx-1compared with the vehicle group (7.21%)(P <0.05at1μg/ml; P <0.01at10μg/ml). Western blot analysis showed that Tuj1expression of NSCsin the10μg/ml treatment group was increased approximately1.5-fold compared to thevehicle group (P <0.05). The ratio of GFAP positive cells and the expression of GFAP inNSCs were decreased when cells were incubated in10μg/ml of rhTrx-1.(P <0.05).4.Both MEK/ERK and PI3K/Akt signaling pathways have been implicated in themechanism of proliferation and neurogenesis of NSCs. The activation of these pathwaysduring rhTrx-1treatment and subsequent induction of proliferation and neurogenesis wereexplored using a10μg/ml rhTrx-1treatment to further study this mechanism. Expressionof phosphorylated ERK began to increase at2h, maintain a high level to48h afterrhTrx-1administration (P <0.05at2h, P <0.01at24h and48h). The levels ofphosphorylated Akt did not significantly change after NSCs incubation with rhTrx-1(P>0.05). Western blot analysis of the protein sample, extracted from NSCs cultured for48hin proliferation medium revealed that pretreatment with the inhibitor of ERK U0126inhibited the elevation of the phosphorylated ERK induced by rhTrx-1(P <0.01).Furthermore, U0126suppressed increases in the ratio of BrdU labeled cells in theCI+rhTrx-1group (P <0.05). Also, co-administration with U0126decreased thepercentage of neuron differentiation (P <0.05) compared to levels observed in theCI+rhTrx-1group (P <0.01).Conclusions: rhTrx-1promoted NSCs proliferation and differentiating into neurons,which could be inhibited by U0126---inhibitor of MEK/ERK signaling pathway.SummaryIn our study, we found that exogenous administration of rhTrx-1promote theneurogenesis in the DG in mice, which could improve spatial learning and memorydeficits and play a neuroprotective role after global ischemia. The potential role of rhTrx-1on proliferation and neuronal differentiation may become the underlying mechanisms. Moreover, the MEK/ERK signaling pathway participated in the role of rhTrx-1, which haddemonstrated the relationship between neurogenesis of rhTrx-1and MEK/ERK. Theseresults suggest that rhTrx-1may be a potential agent for the clinical treatment of ischemicinjury in brain tissues. |
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