Animals and Treatments
A total of 40 C57BL/6J male mice (8weeks, weight 20-23g, SPF) and 20 pregnant C57BL/6J mice used in this study were purchased from Hunan SJA Laboratory Animal Company of China. All the mice were housed in a temperature-controlled SPF room with free access to water and food. And all experimental procedures were approved by the Laboratory Animal Welfare and Ethics Committee of Third Military Medical University (Approval ID: SYXK (Yu) 20170002).
40 male mice were randomly assigned into four groups: Control group (n = 10) received physiological saline, ART group (n = 10) received 50mg/Kg body weight artesunate (Aladdin, China), Cd group (n = 10) received 1mg/Kg body weight cadmium chloride (Aladdin, China), and Cd + ART group (n = 10) received 50mg/Kg body weight artesunate. Cadmium chloride was dissolved by physiological saline and a 10mg/ml stock solution was made for store, until treatment, 10mg/ml stock solution was diluted into 0.5mg/ml and about 50µl was given via intraperitoneal injection. Artesunate was dissolved by DMSO and a 100mg/ml stock solution was made for store, until treatment, 100mg/ml stock solution was diluted into 12.5mg/ml and about 100µl was given via intraperitoneal injection. For Cd and Cd + ART groups, cadmium chloride was given once a day, 5 days per week consecutively for 4 weeks. After 4 weeks cadmium chloride treatment, Cd + ART and ART groups received 50mg/Kg body weight artesunate for additional 4 weeks, once a day and 5 days per week.
Behavior Tests
To examine the effect of artesunate on the learning and memory of cadmium exposed mice, Y-maze and Morris water maze was utilized after drugs treatment. 36 mice (Control group: 10, ART group: 10, Cd group: 8, Cd + ART: 8, 2 mice of Cd group and 2mice of Cd + ART group died during treatment period) were underwent Y-maze before Morris water maze.
Y-maze Test
Y-maze test was adapted to evaluate the memory of different group mice, and the test procedure was performed as previous report [20]. The apparatus makes a Y shape with three same arms (40cm long, 15cm wide, 50cm high and with a 120° angel between each arms). Each animal was tested 8 minutes and a camera was used to record the movement trajectory. 75% alcohol was used to wipe the mess when an animal test was completed, and until the equipment was dry, the next animal was test. The data of each mouse was extract from second minutes to six minutes of the video by a blind observer. And the spontaneous alteration rate was calculated as follow equation: (spontaneous alteration/ (total number of arm enties-2)).
Morris Water Maze Test
Morris water maze (MWM) test was performed to assess the spatial learning and memory of all groups. The test was carried out as previous report [21]. Briefly, the equipment consists of a black circular pool with a diameter of 1.7m and a depth of 60 cm. During the test, the pool was filled with 25℃ water supplied with TiO2. The circular pool was divided into four quarter and a platform about 8cm in diameter was set in first quarter and below the water about 1cm. The next day after the Y-maze test, all animals of each group received the spatial learning trails (navigation test). For this part, every mouse was released into water at 4 distinct quarters and was given 60 seconds to seek the platform for a consecutive 5 days. The time mouse spent to arrival the platform was recorded as the escape latency and if the mouse failed, the escape latency was recorded as 60 seconds, and the failed mouse was placed on the platform to rest 10 seconds. On the sixth day, the platform in first quarter was removed and mouse of each group was released into water at third quarter and was allowed to swim 60 seconds to seek the removed platform to examine the spatial memory of mouse (probe test). The number of crossing the platform and the time spent in first quarter were recorded.
Primary Neural Stem/progenitor Cells Culture And Treatments
20 pregnant pregnant C57BL/6J mice were used to isolate primary NSPCs to perform the in vitro study. NSPCs were isolated and cultured as our previous work [22]. As a brief, pregnant mouse was anesthetic deeply by isoflurane, E12.5 fetal mice were taken out and washed with cold sterile PBS. Cortices were isolated by sterile microscopic shear, after the mengminges were cleared by micro tweezers under microscope, then tissues were digested by 0.25% trypsin-EDTA (Gibco, USA) at 37℃ for 12 minutes. Then, tissues were washed twice in DMEM/F12 (Gibco, USA) consists of 10% FBS (Gibco, USA). After washed twice with DMEM/F12, tissues were triturated by a pipette, and suspensions were filtrated by a 100µm strainer (NEST, China).Then, the suspensions were collected and cultured in NSPCs culture medium (DMEM/F12 supplemented with 2% B27 (Gibco, USA), 20ng/ml EGF (Peprotech, USA), 20ng/ml bFGF (peprotech, USA)) at 37℃ with a humidified 5% CO2. Suspended cells were passaged when the majority of them got the diameter about 100µm. Neurospheres were collected and digested by accutase (Gibco, USA), and then suspended into single cell that were seeded and grew to first passage neurospheres. All NSPCs used in this study were passage 2 to passage 4. Besides, 10µg/ml poly-L-ornithine (Sigma, USA) was used to pre-coat culture plate before cells were seeded. The experiment groups were set as following: 1) Control, 0.5, 1, 2, 4, 8µM cadmium chloride. 2) Control (equal DMSO), ART (0.5µM artesunate), Cd (4µM cadmium chloride), Cd + ART (0.5µM artesunate and 4µM cadmium chloride).
Cck-8 Assay
CCK-8 assay was adapted to evaluate the proliferation of NSPCs in vitro. According to manufacturer’s instrument, after the drugs treatment, 110µl NSPCs culture medium which contains 10µl CCK-8 solution (Boster, China) was added into each well and incubated at 37℃ with a humidified 5% CO2 for 2 hours. Then, the absorbance was measured at 450nm by microplate reader and a reference wavelength of 620nm was measured too.
Western Blots
After drugs treatment, bilateral hippocampus of animals or NSPCs in vitro were harvested and lysed by RIPA (Beyotime, China) with addition of protease and phosphatase inhibitor (Beyotime, China). And protein concentrations were determined by Enhanced BCA Protein Assay Kit (Beyotime, China). Mouse hippocampi tissues protein (40µg/lane) and NSPCs protein (10µg/lane) were separated by 6–10% SDS-PAGE under reducing conditions and transferred to polyvinylidene difluoride membranes (Millipore, USA). The membranes were washed by PBST to remove the 5% milk after incubated at room temperature for 2 hours. Then, they were incubated separated in mouse polyclonal to Nestin (1:1000, Abcam, UK), rabbit polyclonal to DCX (1:1000, Proteintech, China), rabbit polyclonal to MAP2 (1:2000, Preoteintech, China), rabbit polyclonal to AKT (1:1000, Bioss, China), rabbit polyclonal to phosphor-AKT (1:500, Bioss, China) and mouse polyclonal to GAPDH (1:2000, Proteintech, China) as a loading control. Membranes were incubated with these antibodies at 4℃ overnight, and after washed three times by PBST, they were incubated with relative HRP-conjugated secondary antibodies. All membranes were detected by ChemiDoc™ XRS + imaging system (Biorad, USA) with addition of WesternBright ECL (Advansta, USA). And the relative density of all membranes was analyzed by Image Lab™ softmare (Biorad, USA).
Immunofluorescence Staining
After behavior test, the mice were anesthetized and perfused via cardiac with ice-cold PBS and followed with 4% paraformaldehyde. Brain tissues were collected and dehydrated using 30% sucrose solution at 4℃ until they sank into the bottom. Brains were embedded by OCT and coronal sections were prepared (20µm) by freezing microtome (Leica, Germany). NSPCs after treatment with drugs were fixed with 4% paraformaldehyde at room temperature for 15 minutes. Then, samples were incubated using 0.3% triton X-100 (Sigma, USA) to penetrate the membranes at room temperature for 30 minutes. After washed with PBS three times, samples were rinsed in 5% BSA to block the non-specific site at room temperature for 2 hours. Then, samples were incubated with following primary antibodies: mouse polyclonal to Nestin (1:100, Abcam, UK), mouse monoclonal to SOX2 (1:100, Proteintech, China), rabbit polyclonal to DCX (1:100, Proteintech, China), rabbit polyclonal to Olig2 (1:100, Proteintech, China), goat polyclonal to GFAP (1:200, Abcam, UK), rabbit polyclonal to Ki67 (1:500, Abcam, UK), rabbit polyclonal to MAP2 (1:100, Proteintech, China) at 4℃ overnight. Later, relative fluorescence-conjugated secondary antibodies (1:400, Proteintech, China) were incubated at room temperature for 2 hours in the condition of avoiding light. DAPI was incubated to counteract the nucleus. Images were captured through Olympus inverted fluorescence microscope (Olympus, Japan) for NSPCs in vitro and Zeiss fluorescence microscope (Zeiss, Germany) for brain slices.
Edu Staining
To assess the proliferation of NSPCs in vitro, 5-ethynyl-2'-deoxyuridine (EDU) label was performed. EDU (Beyotime, China) was administered into culture medium of NSPCs and incubated 2 hours at 37℃ with a humidified 5% CO2 after drugs treatment 3 days. Then, EDU staining was performed according to manufactures’ instrument, and DAPI was incubated to counteract the nucleus.
Statics
All data were analysed by Graphpad Prism 6.0 and presented as mean ± standard error mean (SEM). One-way ANOVA with Bonferroni post-hoc test was used for comparisons, as appropriate. p < 0.05 indicated statistically significant difference.