| Background and Objection:In mammalian brain, there is a proliferating population of neural progenitor/stem cells which produce new neurons and incorporate into the existing neuronal circuitry in appropriate circumstances. The existence of the active process, termed neurogenesis, raises the possibility that manipulating endogenous neural progenitors may lead to successful cell replacement therapies for various degenerative neurological diseases. It is therefore important to decipher the "appropriate circumstances" regulating neurogenesis, which may allow us to translate this endogenous neuronal replacement system into therapeutic interventions.Growth factors are important components of neural stem cell niches. Multiple extracellular growth factors have been proved to promote the proliferation of progenitor cells and neurogenesis in vivo and in culture systems. Neurotrophic factors (NTFs), a family of growth factors that include nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin 4/5 (NT-4/5) are involved in the survival and proliferation of neural progenitors, contributing to differentiation and migration of newly born neurons for self-repairmen of neural system. Among them, brain-derived neurotrophic factor (BDNF) attracts the most attention because it is the most abundant and widely distributed neurotrophin in the central nervous system. BDNF regulates almost all aspects of neuronal development and function including precursor proliferation and commitment. BDNF exerts its actions through binding and activation of specific, membrane-bound tropomyosin-related kinase (Trk) receptors, or a single pan-neurotrophin receptor, the so-called p75NTR. BDNF-TrkB signaling has been hypothesized to play an important role in neurogenesi. Functional changes in BDNF or TrkB through either genetical or chemical methods positively relate to neurogenesis in the different regions of the brain. In vitro, BDNF promotes proliferation of neural progenitor cells and increases neuronal production in neurosphere cultures. Nonetheless, controversy still exists about the outcomes of BDNF therapy for promoting neurogenesis and how BDNF influences proliferation and differentiation in neural precursor cells. More research is needed to understand the determinants of BDNF in endogenous neurogenesis before it can be considered promising for therapeutic purposes.Unlike previous reports, we investigated the effects of BDNF on neurogenesis by using organotypic hippocampal slice cultures (OHSCs). In comparison with neurosphere cultures which traditionally used in in vitro neurogenesis studies, the OHSCs seem to be more suitable for studying ex vivo postnatal and adult neurogenesis because the OHSCs contain various neural and nonneural elements. Moreover, the OHSCs retain much of their anatomical circuitry and intrinsic functional activity for a number of weeks, and allow precise experimental manipulation of the concentration of BDNF that is not possible in vivo. The OHSCs preserve endogenous neuronal progenitor cells which spontaneously generates new neurons in vitro. Thus, the OHSCs are suitable for studying neurogenesis. So far, only a few growth factors, such as basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF), have been demonstrated their complex effects on neurogenesis under the OHSC system. BDNF and its associated receptors TrkB have been identified as strong candidates for mediating neurogenesis processes. We therefore applied the OHSCs to study the effects of BDNF on neurogenesis. The neurogenic abilities in hippocampal slice cultures were identified using the 5-bromodeoxyuridine (BrdU) labelling method. The neuronal maturation of the BrdU labeled cells was assessed using two neuronal markers, doublecortin (DCX) and the neuron-specific DNA-binding protein Neuronal Nuclei (NeuN). By counting the cells labeled by these markers, we tried to reevaluate the effects of BDNF on neurogenesis in the OHSCs system, and explore whether a right amount and timing of BDNF-TrkB signal activation could be one of key factors to create an optimal circumstance for neurogenesis.In the neurogenic niche of adult mammalian brain, self renewing neural stem cells (NSCs) reside at subventricular zone (SVZ) and subgranular zone (SGZ) within the hippocampus. Recent research indicates that BDNF-TrkB signal exert a neuroprotective effect against cerebral ischemia and significantly promote ischemia-increased neurogenesis, especially in the SGZ of hippocampus. Cerebral ischemia leads neuronal death of the hippocampus. On the other side, increases in cell proliferation and neurogenesis in response to cerebral ischemia was reported and considered as a compensatory mechanism of neuronal loss. More evidence is still necessary for understanding the ischemia-induced neurogenesis. The OHSCs system may serve as a convenient and reliable tool for studying the ischemia-induced neurogenesis. Injury induced by ischemia was mimicked by oxygen-glucose deprivation (OGD) in the OHSCs. We here first designed a series experiments to confirm the OGD-induced neurogenesis. It is still uncertain that whether intensity of ischemia-induced injury is associated with neurogenesis. We used different durations of OGD treatment to induce mild, normal and severe injury in the cultured slices, and tried to explore the association between injury intensity and ischemia-induced neurogenesis. Because of the importance of BDNF-TrkB signal in controlling of ischemia-induced neurogenesis, we further monitored the expression of BDNF and TrkB in the cultured hippocampal slices after OGD-induced injury, to understand what changes in the BDNF-TrkB signaling after neuronal injury and their possible association with neurogenesis.Methods and materialsAnimalC57BL mice obtained from the Laboratory Animal Center of Guangdong Province were used in this study. Procedures involving animals and their care were conducted in conformity with the Ethics Committee for the Use of Experimental Animals.Organotypic hippocampal slice cultureHippocampal organotypic slice cultures were prepared as previously described with slight modifications. The pups (postnatal 6 days) were decapitated and the hippocampi were rapidly dissected out in ice-cold artificial cerebrospinal fluid (aCSF) consisting of (in mM):NaCl 118, KCl2.5, MgSO43, NaH2PO4 1.1, NaHCO3 26, CaCl21 and glucose 11 (all reagents from Sigma), bubbled with 95% O2/5% CO2. Subsequently,400 μm thick coronal slices were cut with a vibrotome (MA752, Campden Instruments). One or two slices with dorsal hippocampal at approximal coronal section in each pup were collected for culture. The hippocampal slices were carefully dissected in cold, oxygenated Hank’s balanced salt solution (Gibco, Invitrogen) under stereo microscope. Those slices without visible blemish were transferred onto sterile porous membrane confetti (Millicell, Millipore), and cultured with serum free medium consisting of 50% MEM,18% HBSS,4 mM L-glutamine, 12 mM glucose,4.5 mM NaHCO3,20 mM sucrose,100 U/mL penicillin and 100 mg/mL streptomycin (all reagents from Gibco or Sigma). To correct the possible confounding effects of the site of origin of the culture on the number of proliferating cells, the selected slices were carefully matched for position. The position-matched slices from each hippocampus were then randomly allocated into different culture condition groups. The culture condition groups were based on the above medium with the supplementation of different concentrations of recombinant human BDNF (Immunological Sciences) and the BDNF antagonist K252a (50 nM, Sigma) or TrkB-IgG (2 μg/mL, R&D Systems), a recombinant tyrosine kinase receptor B (TrkB) engineered as an immunoadhesin to sequester BDNF.Bromodeoxyuridine administrationTo label the immediately post-mitotic cells, the DNA synthesis marker BrdU was administered as previously described with small modifications. To determine a suitable BrdU administration, we used two protocols. First, BrdU dissolved in 0.9% NaCl was injected intraperitoneally at a dose of 50 mg/kg into the pups 30 min before slice preparation, and the slices were not subjected to a second administration of BrdU. The other protocol was that BrdU was administered twice. Besides first injection of BrdU into mice,0.5 μM BrdU was administered into culture medium right after culture and maintained for the first 72 hrs.Oxygen-glucose deprivation (OGD)OGD was used as an in vitro model of cerebral ischemia. For OGD, the inserts with OHC were placed into 1 mL of culture medium containing mannitol (10 mM; Sigma) instead of glucose in sterile 6-well culture plates. Before use, OGD medium was saturated by flushing a gas mixture of 95% N2/5% CO2 for 10 min. The cultures were then placed into an airtight chamber and exposed to 10 min (mild group,5 mins; severe group,20 mins) of 95% N2/5% CO2 gas flow. After that, the chamber was sealed and placed for 40 min (mild group,20 mins; severe group,80 mins) in an incubator where the temperature was maintained at 37oC. After OGD, OHC were returned to their original culture conditions.ImmunohistochemistryAt 16 days in vitro (DIV), the cultures were fixed in 4% PFA for 1 hour at room temperature, and then transferred to a 30% sucrose solution for 24 hours. Subsequently, slices were exposed to 2 M HCl at 37℃ for 30 minutes and were subsequently washed in boric acid (0.1 M, pH 8.5) for 10 minutes. Slices were then washed thoroughly in PBS, and blocked with 5% Goat serum and 5% BSA for 30 min. For immunostaining, slices were incubated with rat anti-BrdU (1:50, Sigma) and mouse anti-NeuN (1:50, Chemicon) or mouse anti-GFAP (1:200, Sigma), or mouse anti-DCX (1:100, Sigma) in 0.1% Triton PBS overnight. After thoroughly wash in PBS 3 times for 10 min, second antibodies goat anti-rat Trite (1:50, Chemicon) and goat anti-mouse Alexa 488 (1:50, Chemicon) in PBS. Appropriate negative controls showed no nonspecific secondary antibody staining. Following multiple washes in PBS, the slices were cover-slipping with DPX (Sigma).Western blotThe cultured slice (8 slices per experimental condition) were collected and homogenized in ice-cold RIPA buffer. Protein concentration was determined using the BCA Protein Assay Kit (Thermo Scientific). Samples of 15 μg of protein from whole homogenates were subjected to SDS-PAGE (4-20% gels, Bio-Rad) and transferred to PVDF Transfer Membranes (Millipore). Immunoblots were performed with rabbit anti-TrkB (Sigma) and anti-BDNF (Sigma). Detection of antibodies was conducted with goat anti rabbit HRP conjugate (Bio-Rad). A control for protein loading was performed by reprobing membranes with an antibody against beta-actin (Sigma). Immunopositive bands were visualized using the Enhanced Chemiluminescence (ECL) plus Western blotting system. Bands were scanned at 300 dpi and the optical density of the bands was quantified using Image J.Cell counting and StatisticsSlices were examined under a DMRA2 microscope (Leica) with a DFC300FX video camera (Leica). The counting area covered whole slices. To ensure a similar total cell number in the slices, we only chose the slices in approximate size as well as in approximate average density of the nuclear stain DAPI. For all quantification, slices were coded and cell counting was carried out with the examiner blind to the treatments of each slice. Statistical analysis and graph composition was performed using the SPSS software (USA). All values are presented as means±SD. A one-way ANOVA and post hoc Bonferroni test were used for multiple group comparisons. Statistical significance was established at P<0.05.Results:1. Neurogenesis in hippocampal slice culturesTo find an optimal labeling method for neurogenesis in the OHSCs, we compared two protocols for the BrdU administration. The protocol 1 applied single injection in mice prior to the slice culture, while in the protocol 2 the BrdU was administered twice with an additional BrdU administration to the slices at DIV 0-3. At DIV 16, slices were processed for BrdU immunoreactivity. With the protocol 1 of BrdU administration, less BrdU-positive cells were observed within the slices. The vast majority (over 70%) were distributed in the granule cell layer of dentate gyrus, while a small proportion scattered in the other regions of the hippocampus such as hilus, CA1 and CA3. These BrdU-positive cells were colabeled with DCX, whereas more than 60% of the DCX-positive cells were negative to BrdU. It suggested that some newly born neurons might lose BrdU incorporation if applying the protocol 1 of BrdU administration. In comparison, there were larger amounts of BrdU-positive cells in the slices that had been treated with BrdU twice according to the protocol 2. An average of 213±29 BrdU-positive cells per slice were detected in the DG region. Almost all of the intense DCX-positive cells were positive to BrdU. We also found a large proportion of the BrdU positive cells (>85%) was immunoreactive for the astroglial marker GFAP, rather than colocalized with the neuronal nuclei marked with NeuN. Thus, a large proportion of BrdU-positive cells, if applying the BrdU labeling method according to the protocol 2, would rather represent proliferation of astrocytes in the case of in vitro culture system. A few mature newly born neurons (8±2.3) that double positive to BrdU and NeuN were consistently observed in the slices at DIV 16. We also found that the immunostain of BrdU in the GFAP+/BrdU+ cells appeared condense and equitable coloring, while the BrdU signals in the DCX+/BrdU+ or NeuN/BrdU+cells, representing the newly born neurons, dispersed in the nuclei and were relatively slight.2. BDNF-TrkB signaling impacts on neurogenesisTo date, the actions of BDNF on neurogenesis in the OHSC system have not yet been determined. In order to clarify the role of BDNF in proliferation and differentiation, the cultures were continuously administered with a series of defined doses (1,10,20,40,80,160,320 ng/mL) of exogenous recombinant BDNF. Combined with immunohistochemistry against BrdU, the DCX, NeuN, and GFAP were used to determine early and late neurogenesis, and gliogenesis respectively at DIV 16. The number of double labeled cells was counted in the whole slices from overlapping pictures.The supplementation of BDNF increased the number of BrdU+/DCX+ and BrdU+/NeuN+ cells in a concentration dependent manner. The concentration-response curves look alike. Compared to the control, the increases reached statistical significances at the high concentrations (40-320 ng/mL) of supplementation of BDNF (P<0.05, n=6), with peak responses at around 100 ng/mL. The number of BrdU+/DCX+ cells are 3-4 times as much as that of BrdU+/NeuN+ cells. When we pooled the number of the BrdU+/DCX+ and BrdU+/NeuN+ cells as indicates of all newly born neurons in different maturation, it showed that at least 20 ng/mL of BDNF could lead to a significant increase in the number of newly born neurons. When the supplementation of BDNF increased up to 160 or higher, it seemed that the number of newly born neuron tended to decrease. When analyzing the number of newly born astrocytes labeled by BrdU+/GFAP+, there were not significant changes in response to BDNF supplementation in the culture medium (P>0.05, n=6).We further used the blocking of BDNF-TrkB signal by the TrkB antagonist K252a and the BDNF scavenger TrkB-IgG to confirm the effects of BDNF. It showed that both blockings of BDNF-TrkB signal attenuated the increases in the number of newly born neurons, which was the pooled data of BrdU+/DCX+and BrdU+/NeuN+ cells (P<0.05, n=6, vs.80 ng/mL BDNF), although they could not completely abolished the effects of supplementation of BDNF (P<0.05, n=6, vs. control). To understand the importance of BDNF signaling in neurogenesis, we treated the slices with K252a and TrkB-IgG without supplementation of BDNF during culture. It showed that the BDNF antagonist K252a led to significant decrease in the number of newly born neuron when compared to the control group (P<0.05, n=6, vs. control;). The TrkB-IgG that designed to eliminate soluble BDNF had no significant effect on the neurogenesis (P>0.05, n=6, vs. control).3. Timing of BDNF administrationGiven that the enhancement of BDNF-TrkB signal could increase the neurogenesis, we further investigated the dynamic changes of BDNF and TrkB expression during culture. Using the same culture condition without BDNF supplementation just as the above control, we examined the protein levels of BDNF and TrkB in the cultures. The expression of BDNF firstly reduced to 35% at DIV2, and then rebounded to an even higher level by about 60% as compared to the acute slices (P<0.05, n=4). Different from the BDNF, the protein levels of TrkB were gradually upregulated along with maturation of the cultures, and reached significant difference from the DIV2 (P<0.05,n=4).The time-dependent changes in the expression of BDNF and TrkB during the OHSC suggested that the signal strength of BDNF-TrkB was dynamically regulated during neuronal maturation. We then tried to explore whether the pro-neurogenesis effects by up-regulation of BDNF signal were associated with neuronal maturation. By dividing the culture period into two halves, the supplementation of BDNF on slices was maintained for either the early half or the late half. The number of BrdU+/DCX+was counted at DIV 16. It showed that the late supplementation of BDNF could not significantly promote neurogenesis, while the early administration of BDNF resulted in a higher amount of the BrdU+/DCX+cells when compared to the control (P<0.05, n=5). The average amount of newly born neurons in the group of early BDNF supplementation was similar to that in the group treated with BDNF all through the 16-day culture period. There was significant difference in the amount of neurogenesis between the early and late supplementation of BDNF (P<0.05, n=5).4. OGD-induced neurogenesis in OHSCEven though it had been reported that the slices treated with OGD led to increase in the number of newly born neurons, it remained uncertain whether the newly born neurons were directly resulted by the OGD. As there are a number of newly born neurons during the OHSC, it is necessary to discriminate the part of newly born neurons which were directly induced by OGD. To make a more convincible evidence, we designed 4 experimental groups:1) Group of Control 1, BrdU was administered only in the first 3 days in vitro; 2) Group of Control 2, BrdU was administered twice with additional administration from DIV 8 to DIV 11; 3) Group of OGD 1, slices at DIV 8 received OGD on the base of Control 1; 4) Group of OGD 2, slices at DIV 8 received OGD on the base of Control 2. At DIV 16, number of newly born neurons which were double labeled by DCX+and BrdU+ was counted. In the groups that BrdU was only administered before OGD, we did not find any significant differences between the groups of OGD 1 and Control 1, indicating that OGD treatment did not impact on survival and differentiation of newly born neurons. In the groups that BrdU was designed to label the proliferating cells right after OGD, there was significant differences between the groups of OGD 2 and Control 2 (P<0.05, n=5, ANOVA and post-hoc Student-Newman-Keuls test). OGD could increase the number of newly born neurons. On the other side, the number of DCX+/BrdU+cells in the OGD 2 group was much higher that that in the OGD 1. The differences between the groups indicate OGD could induce neurogenesis mainly through promoting prolifetion of neural progenitor cells.5. Intensity of OGD-induced neuronal injury and neurongenesisThe OGD-induced injury in the OHSCs could induce neurogenesis, possibly by promoting the proliferation of progenitor cell. There might be some correlation between proliferation rate and intensity of OGD-induced neuronal injury. To prove this, we designed three groups of OGD treatments:1) Normal OGD group, in which the OGD persisted for 50 mins as previously indicated; 2) Mild OGD group, in which the duration of OGD treatment was shorten to half; 3) Severe OGD group, in which the OGD persisted as long as 100 mins. At DIV 9, the day after OGD treatment, the cultured slices were examined for their viability using PI staining. It showed that severity of OGD-induced neuronal injury was positive associated with the duration of OGD treatment. Right after OGD treatment, the BrdU was exposed to the slices for 3 days to label the OGD-induced cell proliferation and neurogenesis. It showed that the groups of mild OGD and normal OGD could significantly increase the number of the DCX+/BrdU+cells when compared with the control (P<0.05, n=5, ANOVA and post-hoc LSD test). The number of newly born neurons induced by normal OGD was also significantly higher than that in the mild OGD group. However, the group of severe OGD led to a much smaller number of the DCX+/BrdU+ cells, even less than the control (P<0.05, n=5, ANOVA and post-hoc LSD test). The reversed effect in the severe OGD group could attribute to less survival of progenitor cells or newly born neurons during the attack of OGD, finally resulting in the decrease of newly born neurons at late period after OGD.6. Dynamical changes in the expression of BDNF and TrkB after OGD in the OHSCsAs stated above, expression of BDNF and TrkB is dynamically regulated along with the culture time in the OHSCs. During the OHSCs, neurons undergo different processes, including injury, restoration, and maturation. These processes might correlate with the strength of BDNF-TrkB signaling. In the OHSCs, the ability of OGD treatment to induce neurogenesis might be somewhat correlated with the BDNF-TrkB signaling. We then used Western blot to examine the expression of BDNF and TrkB after OGD which was conducted at DIV 8. It showed that the changes in the expression of BDNF were similar to TrkB after OGD. They decreased during the early period after OGD, and rebounded to a higher level during the late period after OGD. We firstly defined the protein levels of the slices at DIV 8 which did not receive OGD as the controls. The expression levels of BDNF were significantly lower than the control at 24 hrs and 48 hrs after OGD treatment, and significantly higher than the control at 6 days and 8 days after OGD (P<0.05). The significant decrease in the expression level of TrkB was found at 48 hrs after OGD, and the significant increase in the expression level of TrkB was found at 8 days after OGD. When we compared the expression between OGD and normal control at DIV 12 and DIV 16, there was no significant difference at DIV 12 which was 4 days after OGD, while significant increases in either BDNF or TrkB were found at DIV 16 (P<0.05). The results indicated that the expression of BDNF and TrkB temporarily was downregulated during early period of OGD-induced neuronal injury, and upregulated during the restoration period (6 to 8 days after OGD). OGD treatment could result in elevation in the expression of BDNF and TrkB, which might probably be related with its beneficial effects on neurogenesis.Conclusion1. The OHSC is a convenient and reliable tool to study neurogenesis.2. Supplementation of exogenous BDNF in the OHSCs promotes neurogenesis in a concentration-dependent manner.3. Block of exogenous or endogenous BDNF inhibit neurogenesis, indicating the importance of BDNF-TrkB signaling in neurogenesis.4. The expressions of BDNF and TrkB are dynamically regulated along with neuronal maturation in the OHSCs. In the early culture period, the expression of BDNF is relatively insufficient.5. Timing of supplementation of exogenous BDNF to promote neurogenesis is crucial. Earlier supplementation of BDNF is more efficient possibly due to the insufficiency of endogenous BDNF in the early culture period of the OHSC.6. OGD treatment induces not only cell injury but also neurogenesis in the OHSC.7. Duration of OGD treatment is positively correlated with intensity of cell injury. Milder OGD results in the less number of newly born neurons. Severe cell injury induced by prolonged OGD shows however a reverse effect on the number of newly born neurons. Neurogenesis in the OHSC is correlated with the intensity of cell injury induced by OGD.8. OGD treatment leads to dynamical changes in the expression of BDNF and TrkB in the OHSC. The expression of BDNF and TrkB is temporarily downregulated during the acute phase of OGD-induced injury and rebound to higher levels during restoration. The coincidence of neurogenesis and BDNF-TrkB expression indicates the importance of BDNF-TrkB signal strength in neurogenesis. |
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