Design
An in vitro cell experiment.
Time and setting
The experiment was performed at the Department of Anatomy, Li Ka Shing Faculty of Medicine, the University of Hong Kong between January and August 2012.
Materials
Ten newborn female Sprague-Dawley rats aged 7 days old and two adult female Sprague-Dawley rats aged 90 days old were used in this study. The rats were provided by Laboratory Animal Unit, the University of Hong Kong, China.
Methods
Neural stem cell harvest and primary culture
After anesthesia by intramuscular injection of 20% Dorminal (200 mg/mL, 0.5 mL/kg), newborn and adult Sprague-Dawley rats were sacrificed and their brains were placed into cold, sterile Hank’s Buffered Salt Solution (HBSS; Life TechnologiesTM Inc., Hong Kong, China) immediately. The hippocampi, identified as a seahorse-shaped structure in medial temporal lobes, were washed with HBSS once and dissociated into single cell suspensions by 2.5% trypsin (Life TechnologiesTM Inc.) for 15 minutes. The dissociated cells were re-suspended in HBSS and filtered through a sterile cell strainer (70 μm pore size) to obtain single-cell suspensions. An aliquot of dissociated cells was diluted with HBSS and counted using a hemocytometer to estimate the number of cells obtained for both rat groups (newborn and adult). Single-cell suspensions were plated onto a 25cm2 cell culture flask at a density of 1 × 105/mL in proliferation medium for neural stem cells [dulbecco's modified eagle's medium (DMEM)/F12 (1:1), B27 supplement, N2 supplement, 20 ng/mL basic fibroblast growth factor and 20 ng/mL epidermal growth factor; Life TechnologiesTM Inc.]. The cells were incubated in an incubator at 37°C with 5% CO2 for several days.
Formation of free-floating neurospheres and secondary culture of neural stem cells (passage 1)
In order to obtain sufficient number of neural stem cells, passage 1 neural stem cells were further cultured for one more generation. After primary culture, free-floating neurospheres formed. Neurospheres were collected into 15 mL centrifuge tubes and centrifuged at 800 r/min for 5 minutes. The cell pellets at the bottom of the centrifuge tube were washed once with HBSS and 0.5% trypsin (Life TechnologiesTM Inc.) was added to dissociate the cell pellets at 37°C for 3 minutes. DMEM/F12 with 10% fetal bovine serum (Life TechnologiesTM Inc.) was added to stop the enzymatic digestion. Digested neurospheres were centrifuged at 800 r/min for 5 minutes. The dissociated cells were re-suspended in DMEM/F12, and an aliquot of dissociated cells was further diluted with HBSS and the cell number was counted using a hemocytometer. Dissociated cells at the density of 1 × 105/mL were plated onto a 25 cm2 cell culture flask containing proliferation medium for neural stem cells [DMEM/F12 (1:1), B27 supplement, N2 supplement, 20 ng/mL basic fibroblast growth factor and 20 ng/mL epidermal growth factor]. The cells were incubated at 37°C with 5% CO2 for several days.
Dissociation of neurospheres
After 5–8 days, a plenty of passage 2 free-floating neurospheres formed. The neurospheres were collected and centrifuged at 800 r/min for 5 minutes. The cell pellets were washed once with HBBS and trypanized by 0.5% trypsin at 37°C. DMEM/F12 with 10% fetal bovine serum was added to stop the enzymatic digestion. Digested neurospheres were centrifuged at 800 r/min for 5 minutes. The dissociated cells were re-suspended in DMEM/F12, and an aliquot of dissociated cells was diluted with HBSS and counted using a hemocytometer to estimate the number of cells.
Characterization of neurospheres
Single cell suspensions obtained from neurospheres were plated onto the poly-L-lysine-coated coverslips at a density of 2.5 × 104 cells/cm2 per well in 500 μL proliferation medium [DMEM/F12 (1:1), B27 supplement without vitamin A, N2 supplement, 20 ng/mL basic fibroblast growth factor and 20 ng/mL epidermal growth factor]. The cells were incubated at 37°C with 5% CO2 for 12 hours to allow the cells to attach to the coverslips.
After 12 hours, the cells were fixed with 4% paraformaldehyde for 15 minutes at room temperature and permeabilized by 0.3% triton X-100 solution in PBS. Thereafter, the cells were incubated with 5% bovine serum albumin (Sigma-Aldrich China Inc., Hong Kong, China) in PBS to avoid non-specific binding of antibody. After three rinses with PBS, cells were incubated with mouse anti-nestin (a neural stem marker; Life TechnologiesTM Inc.) at 1:200 dilution in 1% bovine serum albumin/PBS for 24 hours at 4°C, washed three times with PBS and then incubated with goat anti-mouse IgG secondary antibody (Alexa Fluor®647; Life TechnologiesTM Inc.) at 1:500 dilution in 1% bovine serum albumin/PBS for 2 hours at room temperature. The stained cells were washed three times with PBS and counterstained with 4’,6-diamidino-2-phenylindole (1:1 000 dilution in 1% bovine serum albumin/PBS; Life TechnologiesTM Inc.) for 20 minutes. After being washed with PBS once, coverslips were mounted onto slides with mounting medium and allowed to dry overnight. The stained cells were viewed under light and fluorescence microscopes (Zeiss, Oberkochen, Germany) (100 ×).
Preparation of culture media with lead
In order to study the effect of Pb2+ on the survival of neural stem cells, lead acetate powder [Pb (CH3COO)2• 3H2O; Sigma-Aldrich China Inc.] was used. Lead acetate was first dissolved in warm water (37°C) and filtered through a 0.22 μm membrane filter to remove bacteria. Lead acetate solution was added into the proliferation or differentiation medium to prepare media with different concentrations of Pb2+ (0, 1, 10, 50, 100 and 200 μM).
Cell viability assay
Passage 2 cells obtained from neurospheres were seeded onto the poly-L-lysine-coated wells of a 96-well plate at a density of 2.5 × 104 cells/cm2 per well in 100 μL culture medium without Pb2+. The cells were incubated at 37°C with 5% CO2 for 24 hours. Different concentrations (0, 1, 10, 50, 100 and 200 μM) of lead acetate were prepared with proliferation medium of neural stem cells. After 24-hour incubation, the wells were washed with growth medium to remove non-adherent cells. 100 μL of culture medium with different concentrations of lead acetate was added into each well of the 96-well plate, followed by further incubation at 37°C with 5% CO2 for 48 hours. After the incubation, 20 μL 5 mg/mL 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide (MTT; Sigma-Aldrich China Inc.) solution was added into each well and the cells were further incubated for 4 hours. Then, the culture media with MTT solution were discarded and replaced with 200 μL dimethyl sulfoxide (DMSO, 1.1g/mL; Sigma-Aldrich China Inc.), followed by incubation at room temperature with shaking for 5 minutes. The absorbance of each well was measured at 570nm by enzyme-linked immunosorbent assay (ELISA) reader (Biotmed LLC, Northborogh, MA, USA).
Cell proliferation assay
Dissociated passage 2 cells were added onto the poly-L-lysine-coated coverslips in 24-well plate at a density of 2.5 × 104cells/ cm2 per well in 500 μL culture medium without lead acetate and incubated at 37°C with 5% CO2 for 24 hours. The coverslips were washed with growth medium to remove non-adherent cells. The wells were filled with the culture media with different concentrations of lead acetate (0, 1, 10, 50, 100 and 200 μM) and the cells were further incubated at 37°C with 5% CO2 for 48 hours. Then, bromodeoxyuridine solution was added into each well to a final concentration of 10 μM, followed by incubation at 37°C with 5% CO2 for additional 24 hours to allow the incorporation of bromodeoxyuridine into the DNA of the cells.
After that, the cells were fixed with 4% paraformaldehyde for 15 minutes at room temperature and permeabilized by 0.3% triton X-100 solution in PBS for 5 minutes. The DNA of the cells was denatured by 2 M hydrochloric acid at room temperature for 1 hour, followed by neutralization by 0.1 M borate buffer. The cells were then incubated with 5% bovine serum albumin/PBS for 30 minutes to prevent non-specific binding of antibodies and then rinsed by PBS three times. Afterwards, the coverslips were incubated with mouse anti-bromodeoxyuridine (1:200; Life TechnologiesTM Inc., Hong Kong, China) in 1% bovine serum albumin/PBS for 24 hours at 4°C, rinsed three times with PBS/Tween20, and stained with goat anti-mouse IgG secondary antibody (Alexa Fluor®647; Life TechnologiesTM Inc.) at 1:500 dilution in 1% bovine serum albumin/PBS for 2 hours at room temperature. The cells stained by the secondary antibody were washed with PBS three times, counterstained with 4’,6-diamidino-2-phenylindole (Life TechnologiesTM Inc.) (1:1 000 dilution in 1% bovine serum albumin/PBS) for 20 minutes and then washed once with PBS. Finally, the coverslips were mounted onto slides with mounting medium and allowed to dry overnight. The stained cells were visualized under a fluorescent microscope (100 ×). Ten random fields of view were taken for each coverslip, and the number of nuclei and bromodeoxyuridine-incorporated cells were recorded.
Immunochemistry for differentiated neural cells from neural stem cells
Different concentrations (0, 1, 10, 50, 100 and 200 μM) of lead acetate were prepared in differentiation medium of neural stem cells (neuronal differentiation: neurobasal® medium, B27supplement, 1% fetal bovine serum and GlutaMAXTM-I; astrocyte differentiation: neurobasal® medium, N2 supplement, 1% fetal bovine serum and GlutaMAXTM-I; oligodendrocyte differentiation: neurobasal® medium, B27supplement, and GlutaMAXTM-I; Life TechnologiesTM Inc.). Single cell suspensions were first seeded onto the poly-L-lysine-coated coverslips in 24-well plate at a density of 2.5 × 104cells/cm2 per well in 500 μL differentiation medium free of Pb2+, and incubated at 37°C with 5% CO2 for 24 hours to allow the cells to attach to the coverslips. After 24 hours, coverslips were rinsed with neurobasal® medium to get rid of non-adherent cells, and the wells were re-filled with lead acetate-added differentiation medium for subsequent 6-day culture. The medium was renewed every 3 days.
After 6-day culture, 4% paraformaldehyde was added to fix the cells for 15 minutes at room temperature, followed by permeabilization of the cells by 0.3% Triton X-100 in PBS for 5 minutes. The blocking agent 5% bovine serum albumin/PBS was added to block the cells thereafter. Next, the cells were incubated with primary antibodies [rabbit anti-microtubule-associated protein 2 (Life TechnologiesTM Inc.) (1:200 dilution in 1% bovine serum albumin/PBS); rabbit anti-glial fibrillary acidic protein (Life TechnologiesTM Inc.) (1:200 dilution in 1% bovine serum albumin/PBS); mouse anti-RIP (Life TechnologiesTM Inc.) (1:50 dilution in 1% bovine serum albumin/PBS)] at 4°C for 24 hours.
At the end of the incubation, the cells were rinsed three times with PBS and incubated with secondary antibodies [goat anti-rabbit Alexa Fluor®488 for rabbit anti-microtubule-associated protein 2 (1:500 dilution in 1% bovine serum albumin/PBS), goat anti-rabbit Alexa Fluor®647 for rabbit anti-glial fibrillary acidic protein (1:500 dilution in 1% bovine serum albumin/PBS) and goat anti-mouse Alexa Fluor®488 for mouse anti-RIP (1:500 dilution in 1% bovine serum albumin/PBS); Life TechnologiesTM Inc.) at room temperature for 2 hours. Following three rinses in PBS after the incubation, the cells were counterstained with 4',6-diamidino-2- phenylindole at 1:1 000 dilution in 1% bovine serum albumin/PBS for 20 minutes at room temperature. Finally, the coverslips were washed with PBS once, mounted onto the slides with mounting medium, and allowed to air-dry overnight. The stained nuclei and differentiated cells were counted in 10 random fields of view per group under a fluorescent microscope (100 ×).
Statistical analysis
Five independent samples for each concentration of lead acetate were analyzed. Data were collected in a group identity-blinded way and differences (P < 0.05 and P < 0.01) were set for intergroup comparisons. The data were expressed as mean ± SEM and analyzed by SPSS 17.0 software (SPSS, Chicago, IL, USA). One-way analysis of variance was used to analyze differences among multiple groups and P < 0.05 and P < 0.01 were regarded as statistically significant differences.
Funding: This study was supported by a grant from the University of Hong Kong, China.
Author contributions: Yan Ho Chan designed and performed the experiments, collected and analyzed the data, and prepared the manuscript. Mingyao Gao designed the experiments and provided technical support. Wutian Wu designed and supervised the experiments, data analysis and manuscript writing. All authors approved the final version of the paper.
Conflicts of interest: None declared.
Ethical approval: The use of all experimental animals in this study was carried out in accordance with the regulations of the Committee on the Use of Live Animals for Teaching and Research of the University of Hong Kong, China.
Author statements: The manuscript is original, has not been submitted to or is not under consideration by another publication, has not been previously published in any language or any form, including electronic, and contains no disclosure of confidential information or authorship/patent application/funding source disputations.
