NeuroReport 2009, 20: a Department of Neurology, Children s Hospital of Chongqing Medical University

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1 Regeneration and transplantation 295 In-vitro effects of brain-derived neurotrophic factor on neural progenitor/stem cells from rat hippocampus Tingsong Li a,b, Li Jiang a, Xiaoping Zhang b and Hengsheng Chen b Mounting evidences from in-vivo studies showed that brain-derived neurotrophic factor (BDNF) plays an important role in the neurogenesis, but the effects of BDNF are controversial because of neurogenesis affected by many factors in vivo. In this study, we investigated the effects of BDNF on the survival, proliferation, and differentiation of the neural progenitor/stem cells (NPCs) in vitro. The results showed that 40 ng/ml of BDNF significantly increased the number and diameter of neurospheres formed by NPCs; meanwhile, the TUNEL rates and lactate dehydrogenase release of NPCs were also inhibited. Tuj-1 + immunostaining showed that BDNF obviously induced the NPCs to differentiate into neurons and elongated the neurite. Our results implied that BDNF promotes the proliferation of NPCs and induces them to differentiate into neurons, and enhancement of the survival of NPCs probably is one of the mechanisms. NeuroReport 20: c 2009 Wolters Kluwer Health Lippincott Williams & Wilkins. NeuroReport 2009, 20: Keywords: brain-derived neurotrophic factor, cell differentiation, cell proliferation, cell survival, hippocampus, stem cell, Wistar rat a Department of Neurology, Children s Hospital of Chongqing Medical University and b Research Institute of Pediatric Medicine, Chongqing Medical University, Chongqing, PR China Correspondence to Dr Li Jiang, PhD, MD, Department of Neurology, Children s Hospital of Chongqing Medical University, 136# Zhongshan 2 Road, Chongqing , PR China Tel: ; fax: ; neurojiang@gmail.com Received 18 September 2008 accepted 29 October 2008 Introduction Adult neural progenitor/stem cells (NPCs) are multipotent cells found in two principal neurogenic regions of the adult brain, the subventricular zone lining the lateral ventricles and the subgranular zone of the dentate gyrus in the hippocampus [1]. To date, however, the microenvironment present in neurogenic regions of the adult brain still remains largely unknown, and regional environmental cues present in the adult brain play a strong role in determining the lineage potential of adult neural progenitor cells [2]. Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophin family, and is highly expressed in hippocampus. In the dentate gyrus, BDNF protein is strongly expressed in granule cells, where it seems to be anterogradely transported to the axons of the granule cells, called the mossy fibers [3,4]. Functionally, BDNF seems to have several effects in the dentate gyrus. It influences the growth and survival of granule cells in primary culture [5,6] and the morphology of adult granule cells [7]. Owing to the extensive literature showing that BDNF is important to the growth and development of the central nerve system [8 10] and the evidence that granule cells of the hippocampus undergo neurogenesis throughout life, several studies have investigated whether BDNF might influence neurogenesis. For example, Pencea et al. [11] showed that intra-cerebro-ventricular infusion of BDNF led to the increase in number of new neurons in several areas adjacent to the ventricles, such as the striatum, septum, and thalamus. In the dentate gyrus, Lee et al. [12] showed that dietary restriction increased BDNF, and that there was an increase in dentate gyrus neurogenesis as well. Consistent with the results, they found that infusion of an antibody to BDNF into the ventricles blocked the increase in proliferation. Interestingly, the results of other studies of BDNF, in the context of ischemia, have not led to similar conclusions. In these studies, BDNF after ischemia seemed to block the neurogenesis that ischemia produces [13]. The significant differences between the above results may lie in the complexity of neuroregenesis microenvironment in the brain, where some uncontrolled factors might dissimulate the actual effects of BDNF on NPCs. Shetty and Turner [14] found that BDNF can support survival of hippocampal stem cell-derived neurons and also can induce differentiation of these cells into pyramidal-like neurons by in-vitro study. In this study, however, they did not rule out whether BDNF has early effects on proliferation of hippocampal stem cells and possible mechanism. In this study, at first, we confirmed the proliferation enhancement of BDNF, and further investigated the probable mechanisms by TUNEL staining and LDH assay, then the ratio and neurite length of differentiated neurons were measured to study the effects of BDNF on the differentiation. Our results implied that BDNF promotes the proliferation of NPCs and induces them to differentiate into neurons, and enhancement of the survival of NPCs probably is one of the mechanisms. Materials and methods Cell culture Adult pregnant Wistar rats were anesthetized and sacrificed with 100% CO 2, embryos (embryonic stage c 2009 Wolters Kluwer Health Lippincott Williams & Wilkins DOI: /WNR.0b013e c8

2 296 NeuroReport 2009, Vol 20 No 3 E15 16 days) were removed from the uterus and stripped of the meninges in an ice-cold Hank s balanced salt solution, then the hippocampus were taken out under a surgical microscope, dissociated into single cells mechanically. Cells at a density of /ml were seeded into a 6-well plate without adhesion substrate, cultured with Dulbecco s modified eagle s medium (DMEM)/F12 medium containing 2% B27 (v/v; Invitrogen Corp., Carlsbad, California, USA), basic fibroblast growth factor (bfgf) (20 ng/ml; PeproTech, Inc., New Jersey, USA), and epidermal growth factor (EGF) (20 ng/ml; Peprotech) at 371C incubator with 5% CO 2. The medium was changed twice a week. Neurospheres which grew for 3 5 days were dissociated gently into the single cells, and cells at a density of /ml were seeded in a new 6-well plate. NPCs from two to three passages were used for experiments. Proliferation of NPCs Dissociated NPCs from neurospheres were seeded in 96-well (4.5 mm in diameter) plates at a density of /ml. Cells were grown in conditioned DMEM/F12 medium containing BDNF with different concentration of 10, 20, 40, 80, 160, and 200 ng/ml at 371C incubator for 3 days, then the number of newly formed neurosphere was estimated, and the diameter of a sphere was measured with Image-Pro Plus version software. (Media Cybernetics Inc., Bethesda, Maryland, USA). At least five independent experiments were carried out. In this experiment, the results showed that with the concentration of 40 ng/ml, BDNF has the greatest effect on the enhancement of proliferation at the cell density of /ml. Thus, in this study, 40 ng/ml was taken as the final experimental concentration of BDNF. Transferase-mediated biotinylated UTP nick end labeling NPCs were mechanically dissociated and plated at a density of /ml in 24-well plates in the presence of 40 ng/ml BDNF medium containing EGF and bfgf and cultured for 3 days. For the terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL) experiments, cells and neurospheres were first washed, fixed, and then permeabilized with 0.1% triton X-100 in 0.1% sodium citrate for 2 min on ice (2 81C). TUNEL reaction was carried out for 1 hr at 371C. TUNEL reaction mixture of 50 ml contained 5 ml enzyme solution and 45 ml label solution (nucleotide mixture containing fluorescein isothiocyanate-dutp) (Roche Diagnostics GmbH, Mannheim, Germany). After the TUNEL reaction, preparations were washed with phosphate buffered Saline (PBS). The cells were stained with 4 0,6-diamino-2-phenylindole (DAPI) to determine the total number of cells. Seven independent experiments were carried out. Lactate dehydrogenase release assay The injury of NPCs was quantitatively assessed by the measurement of lactate dehydrogenase leakage in the cell supernatant. Lactate dehydrogenase release in the control group and experimental group was measured using a lactate dehydrogenase diagnostic kit (Promega Corp., Madison, Wisconsin, USA) according to manufacturer s instructions. Lactate dehydrogenase activity was calculated by measuring absorbance at 492 nm. Six independent experiments were carried out. Differentiation of neural stem/progenitor cells Neurospheres were dissociated mechanically into single cells and seeded onto glass coverslips precoated by poly- L-lysine in 24-well plates (15.6 mm in diameter) at a density of /ml. Cells were cultured in two conditioned media without EGF and bfgf contained, but with 1% fetal calf serum and 40 ng/ml BDNF, respectively, at 371C incubator for 7 days, then were identified for neurons by immunocytochemistry. Five independent experiments were carried out. Quantification of neurites length Neurites were identified from images of neurons stained with anti-b III tubulin (Tuj-1). Neurites were defined as those processes with length equivalent to at least one cell body diameter, and only neurites without contacting with other cells were evaluated [15]. About 50 cells were chosen randomly from every group for measurement. Measurements were carried out by Image Plus Pro software Immunocytochemistry Cells on coverslips were washed with 0.02 mol/l of PBS and fixed with 4% paraformaldehyde, blocked with 10% normal goat serum in a 0.02 mol/l PBS, and then incubated with primary antibody in 1% normal goat serum at 41C overnight followed with fluorescein isothiocyanate-labeled or tetramethylrhodamine isothiocyanate-labeled secondary antibody (1 : 200, Santa Cruz Biotechnology, Inc., California, USA). The primary antibodies were used for the identification of NPCs (anti-nestin, 1 : 500, Chemicon, Illinois, USA), neuron (anti-tuj-1, 1 : 500, Promega), astrocyte (anti-glial fibrillary acidic protein, 1 : 500, Chemicon), after mounting in the fluorescent mounting medium (Beyotime, China), cells were visualized with fluorescent microscope (Olympus, Japan). Statistics analysis All data were presented as the mean ± SEM. Statistical analysis was performed with one-way analysis of variance test with Bonferroni s corrections and two-tailed independent-samples t-test, using SPSS 13.0 for Windows (SPSS Inc., Chicago, Illinois, USA). The level of statistical significance is defined as P value less than 0.05.

3 Effects of BDNF on neural progenitors Li et al. 297 Results Cell division, morphology, and immunoreactivity After seeding into growth medium, in response to the mitogens EGF and bfgf, aggregates of dividing cells grew in size over time for 3 to 5 days and formed into spheres. The spheres could be gently mechanically dissociated into a mixed suspension of single cells before replated into growth medium. In addition, these cells could be passaged for 4 5 times. As shown in Fig. 1, the nestin immunostaining of neurosphere was positive, whereas Tuj-1 was negative; furthermore, cultures of hippocampal NPCs could be induced to differentiate into neurons and astrocytes by adding the DMEM/F12 medium containing 2% B27 and 1% fetal calf serum, which indicated that the cultured cells in this experiment have two essential characteristics of stem cells: selfrenewal and multipotency. Proliferation of NPCs was enhanced by BDNF As shown in Fig. 2, BDNF have significant effects on the proliferation of NPCs as compared with the control group (P < 0.05). With the concentration of 40 ng/ml, BDNF has the greatest effect on the enhancement of proliferation at the cell density of /ml. Thus, in this study, 40 ng/ml was taken as the final experimental concentration of BDNF. To verify the proliferation promoting effects of BDNF, we measured the diameter of neurosphere which indirectly reflected the number of NPCs between the control group and experiment group with 40 ng/ml BDNF. The results showed that the average diameter of the control and experiment group was ± and ± 5.02 mm, respectively (P < 0.05). Thus, BDNF contributes significantly to the proliferation of NPCs. BDNF was essential for the survival of NPCs We examined the effects of BDNF on cell survival by TUNEL staining and lactate dehydrogenase release measurement in supernatant. The TUNEL staining and 4 0,6-diamino-2-phenylindole staining were shown in Fig. 3, and analysis revealed that NPCs in the presence of BDNF at the concentration of 40 ng/ml significantly decreased apoptosis with 19 ± 5.8% of cells being TUNEL positive. As in the control group, a large increase in TUNEL-positive cells with 39 ± 5.7% was observed (P < 0.05). To confirm the results from TUNEL staining, we measured the release of lactate dehydrogenase in supernatant. Lactate dehydrogenase is a high molecular weight protein (140 kda) that is abundant in cells and is released only when cellular injury occurred. Sustained exposure to BDNF at the concentration of 40 ng/ml for 3 days caused decreased cell injury. The results showed that the leakage of lactate dehydrogenase was significantly decreased in BDNF group (20.56 ± 0.86 U/l) than that in control group (26.08 ± 0.49 U/l) (P < 0. 01). Consistent with the results obtained in TUNEL staining, BDNF inhibited cell injury and promoted the cell survival in the NPCs prepared from rat hippocampus. Fig. 2 Number of neurosphere The concentration of BDNF (ng/ml) Effects of brain-derived neurotrophic factors (BDNF) on the proliferation of neural progenitor/stem cells (NPCs). Data are expressed at mean ± SEM. BDNF have significant effects on the cell proliferation as compared with the control group, and 40 ng/ml BDNF has the greatest effect on the proliferation (One-way analysis of variance, *P < 0.05) Fig. 1 (a) (b) (c) Markers and micrographs of undifferentiated and differentiated neural progenitor/stem cells (NPCs). (a) NPCs of the second passage formed neurosphere under phase contrast microscopy. (b) The NPCs of the second passage were nestin immunostaining positive. (c) NPCs were seeded onto coverslips precoated by poly-l-lysine hydrochloride in DMEM/F12 containing 1% fetal calf serum and 2% B27, and cultured 7 days, then were immunostained for neurons (Tuj-1, green), astrocyte (glial fibrillary acidic protein, red) and nuclei (6-diamino-2-phenylindole, blue).

4 298 NeuroReport 2009, Vol 20 No 3 Fig. 3 (a) (b) Control group (c) (d) BDNF group Effects of brain-derived neurotrophic factor (BDNF) on the apoptosis of neural progenitor/stem cells (NPCs). The apoptosis of the NPCs was expressed as the percentages of TUNEL + cells of 6-diamino-2-phenylindole (DAPI) + cells in the same vision. Fluorescence micrographs of TUNEL + cells (green) in the control group (a) and BDNF group (c), DAPI + cells (blue) in the control group (b) with the same vision as (a) and BDNF group (d) with the same vision as (c). Differentiation of NPCs into neurons were enhanced by BDNF We next tested whether BDNF could influence the differentiation of NPCs. We plated single NPC onto coverslips precoated with poly-l-lysine and cultured for 7 days, then visualized by immunocytochemistry. We use anti-tuj-1 to identify neurons (Fig. 4). In 40 ng/ml BDNF group, more neurons (Tuj-1 + cells) were found as compared with the control group (15 ± 0.2% vs. 10 ± 0.5%, P < 0.01). So these results illustrated that BDNF is able to induce NPCs to differentiate into neurons. Neurite processes of neurons generated from NPCs were elongated by BDNF Morphologically, as showing in Fig. 4, neurons (Tuj-1 + cells) derived from NPCs cultured in BDNF had long processes and more branches, whereas neurons in the control group had shorter processes. Furthermore, the total neurite length was measured, the results showed that neurite length in BDNF group was significantly longer than in the control group ( ± 7.53 vs ± 6.14 mm, P < 0.01). Combining with the results that BDNF was able to affect differentiation of NPCs into neurons and increase neurite outgrowth. Discussion Stem cells have two essential characteristics: self-renewal and multipotency. NPCs can generate multiple cell types including neurons, astrocytes and oligodendrocytes. Mounting evidences have shown that many growth factors including BDNF play an important role in the proliferation and differentiation of neural progenitor cells in vivo; however, the effects of BDNF on the neurogenesis are controversial [11 13]. Thus, the effects of BDNF-regulating neurogenesis are still not very clear. On the basis of the fact that the culture condition in vitro is easy to control, we used cell culture to confirm the effects of BDNF on the NPCs, which excluded the other factors in vivo that might interfere with the results. In this study, our results showed that BDNF significantly enhanced the proliferation and survival of NPCs, and also obviously induced differentiation of NPCs into neurons. Furthermore, neurite processes of neurons generated from NPCs were promoted by BDNF. In addition, the proliferation effect and the BDNF concentration were not in linear relationship, and this may be concerned with that when the BDNF concentration is at 40 ng/ml and the cell density is /ml, the binding receptor related with BDNF in the NPCs get saturation status.

5 Effects of BDNF on neural progenitors Li et al. 299 Fig. 4 (a) that BDNF is able to promote neurite extension, combining with the finding by Naidu et al. [20] that BDNF stimulate neurite extension through TrkB receptor activation in PC12 cell transfected with c-myc-trkb, we suppose that through TrkB activation, BDNF not only promotes neuron differentiation, but also promotes the neurite outgrowth of newly differentiated neurons. In conclusion, our data showed that the survival, proliferation, and neuronal differentiation of progenitor cells in hippocampus can be enhanced by BDNF, which may provide a clue and means for promoting neurogenesis in some pathological conditions to rescue the brain injury, such as stroke and brain trauma. (b) Acknowledgements This study was supported by grants from the National Natural Science Foundation of China (No ), the Project of Natural Science Foundation of Chongqing Educational Department (No ), and the Science Foundation of Chongqing Health Bureau (No ). The neurons differentiated from neural progenitor/stem cells with fetal calf serum (FCS) and brain-derived neurotrophic factor (BDNF). The neurons were immunostained by Tuj-1 (green) and all the cell nuclei were stained by 6-diamino-2-phenylindole (DAPI). The percentages of Tuj-1 + cells of DAPI + cells were measured. (a) Fluorescence overlap micrographs of Tuj-1 + cells (green), nuclei (blue) induced differentiation by (a) 1% FCS and(b)1%fcsadding40ng/mlofbdnffor7days. BDNF along with its preferred tyrosine kinase receptors (TrkB), is expressed in the cortical ventricular/subventricular zone at the onset of cortical neurogenesis [16]. Data presented that TrkB expressed both on cortical progenitor cells [17] and human embryonic stem cells [18]. Furthermore, TrkB ligands and BDNF are survival factors for human embryonic stem cells, which was consistent with the results found in cortical progenitors [17]. Here, we used TUNEL staining and lactate dehydrogenase assay to verify that BDNF has a neuroprotective effect, which may promote the proliferation of neural progenitors through this mechanism. In addition, BDNF enhances survival and synaptic actions in neuron [19] and significantly increased neuronal survival differentiated from mouse hippocampal stem cell [14], so in the BDNF group, more neurons are observed and this probably related with the survival action of BNDF on the newly differentiated neurons. Our results showed References 1 Abrous DN, Koehl M, Le Moal M. Adult neurogenesis: from precursors to network and physiology. Physiol Rev 2005; 85: Chen K, Henry RA, Hughes SM, Connor B. Creating a neurogenic environment: the role of BDNF and FGF2. Mol Cell Neurosci 2007; 36: Conner JM, Lauterborn JC, Yan Q, Gall CM, Varon S. Distribution of brain-derived neurotrophic factor (BDNF) protein and mrna in the normal adult rat CNS: evidence for anterograde axonal transport. J Neurosci 1997; 17: Yan Q, Rosenfeld RD, Matheson CR, Hawkins N, Lopez OT, Bennett L, et al. Expression of brain-derived neurotrophic factor protein in the adult rat central nervous system. Neuroscience 1997; 78: Holtzman DM, Lowenstein DH. Selective inhibition of axon outgrowth by antibodies to NGF in a model of temporal lobe epilepsy. J Neurosci 1995; 15: Patel MN, McNamara JO. Selective enhancement of axonal branching of cultured dentate gyrus neurons by neurotrophic factors. Neuroscience 1995; 69: Danzer SC, Crooks KR, Lo DC, McNamara JO. Increased expression of brain-derived neurotrophic factor induces formation of basal dendrites and axonal branching in dentate granule cells in hippocampal explant cultures. J Neurosci 2002; 22: Lu B. Acute and long-term synaptic modulation by neurotrophins. Prog Brain Res 2004; 146: Eisch AJ. Adult neurogenesis: implications for psychiatry. Prog Brain Res 2002; 138: Gould E, Beylin A, Tanapat P, Reeves A, Shors TJ. Learning enhances adult neurogenesis in the hippocampal formation. Nat Neurosci 1999; 2: Pencea V, Bingaman KD, Wiegand SJ, Luskin MB. Infusion of brain-derived neurotrophic factor into the lateral ventricle of the adult rat leads to new neurons in the parenchyma of the striatum, septum, thalamus, and hypothalamus. J Neurosci 2001; 21: Lee J, Duan W, Long JM, Ingram DK, Mattson MP. Dietary restriction increases the number of newly generated neural cells, and induces BDNF expression, in the dentate gyrus of rats. J Mol Neurosci 2000; 15: Larsson E, Mandel RJ, Klein RL, Muzyczka N, Lindvall O, Kokaia Z. Suppression of insult-induced neurogenesis in adult rat brain by brain-derived neurotrophic factor. Exp Neurol 2002; 177: Shetty AK, Turner DA. In vitro survival and differentiation of neurons derived from epidermal growth factor-responsive postnatal hippocampal stem cells: inducing effects of brain-derived neurotrophic factor. J Neurobiol 1998; 35:

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