| Objective:Nicotine from smoking and environment or maternal nicotine replacement during preganacy and lactation period affects hippocampal neurogenesis and long-term behavior,learning and memory in offsprings.To explore the effect and the underlying mechanism of nicotine on hippocampal neurogenesis,neural plasicity,and CX3CL1/CX3CR1 signaling in hippocampus,we performed in vitro and in vivo experiment using C57BL/6 mouse and primary hippocampal neural progenitor cells.Methods:1.Seven-week-old male(n=4)and female(n=12)C57BL/6 mice were acclimated to the environment for two weeks.Female mice(n=6)were administrated with nicotine(200 μg/ml in 1%saccharin)by drinking water starting from 2 weeks pre-mating until the offspring were weaned,while control female mice(n=6)were received 1%saccharin.Female and male mice were paired with 3:1.Offspring mice were separated from mother on postnatal day 20.Some of the offspring were used for open field tests and the other ones were used for BrdU injection in P35.2.All offspring received an intraperitoneal injection of 5-Bromo-2’-Deoxyuridine Injection on P35.Male and female offspring were given two injections of BrdU(100 mg/kg)at intervals of 6 hours for proliferation assay.Animals were sacrificed within 24h after BrdU injection.The mice were sacrificed and the brains were perfused,fixed and were kept in 30%sucrose solution before IHC staining.While the other male and female offspring were injected with BrdU(50 mg/kg,i.p.)once a day for five consecutive days for survival assay.Mice were sacrificed on day 7(D7),day 21(D21)after the last-injection of BrdU.The mouse brain was cut into coronal sections with 40μm thickness on the frozen slicer.Every 12th section was selected and processed to make a series of slices for immunohistochemistry staining.BrdU positive cells were counted for hippocampal neural progenitor proliferation at 24 hours,and determing newborn cell survival at day 7 and 21.3.BrdU positive newborn neural cells.BrdU+/DCX+immunofluorescence double staining newborn immature neuron.BrdU+/Ibal+microglia and BrdU+/GFAP+astrocytes were observed under confocal microscope,and analyzed for their morphological and quantitative changes of newborn neural cells,newborn neurons and microglia cells in hippocampal of adolescent mice in the nicotine group and control group were observed by immunofluorescence assay.4.The protein expression levels of CX3CL1,CX3CR1 and downstream molecules PKA and p-ErK in the hippocampus were analyzed quantitatively by Western blot in adolescent nicotine offspring(P35)and the vehicle group.5.The percentage of survival newborn neural cells was determined in one and three weeks after BrdU labeled in nicotine group and control group.6.Primary microglia and hippocampal neurons were isolated,and were treated with nicotine for detecting the nicotine affection on CX3CL1/CX3CR1 signaling.Microglia were treated with 10 μM nicotine for 12 hours and 24 hours,respectively.The supemaatant was used for conditioned medium supplemented into hippocampal neurons culture for 48 hours.Total proteins of microglia and hippocampal neurons with or without nicotine were collected at each time point.7.The protein levels of CX3CL1 and CX3CR1 in microglia and hippocampal neurons with or without nicotine were detected by Western blot.Result:1.The righting reflex test showed that the time of righting in nicotine group was significantly longer than that in control group(P<0.05).2.The open-field test showed that the total distance moved in nicotine female offspring was less than that in the vehicle group(P<0.05),the center time was less than that in the vehicle group(P<0.01),but the immobility time was longer than that in the control group(P<0.05).The trend of male offspring was consistent with that of female offspring,but there was no statistical significance..3.The immunofluorescence staining in the hippocampal sections of offspring mice showed that the proliferation of newborn neurons increased in both male and female adolescent offspring with nicotine treatment relative to the vehicle group(P<0.05).However,the survival rate at one week was lower than that in the vehicle group,and significant differences was found in the female offspring(P<0.05),and a large number of newborn neurons died after three weeks from BrdU injection.4.The BrdU/DCX immunofluorescence double labeling showed that the number of neonatal neurons in both male and female offspring with nicotine treatment was higher than that in the vehicle group in adolescent offspring.However,female offspring,not male offspring,have significant difference in BrdU+/DCX+ double staining number(P<0.05).The incidence of disarray of newborn DCX+ immature neuron increased in both male and female nicotine offspring.5.The BrdU/Ibal immunofluorescence double labeling showed that the hippocampal microglia of male mice in the nicotine group was higher than that in the control group(P<0.01),and that the number of hippocampal microglia of female mice in the nicotine group was significantly higher than that in the vehicle group(P<0.001).6.Western blot results showed that the expression of microglia marker Ibal in hippocampus of adolescent male mice(P35)in the nicotine group was higher than that in the vehicle group(P<0.05),and Ibal expression in female mice was higher than that in the Vehicle group(P<0.01).7.Westem blot results showed that the expressions of CX3CL1,CX3CR1and downstream molecules PKA and p-ErK in the nicotine group were increased compared with the Vehicle group.The expressions of CX3CL1(P<0.01),CX3CR1(P<0.01)and downstream molecules PKA and p-Erk(P<0.001)in nicotine group were significantly higher than those in the Vehicle group.8.In vitro microglia experiments showed the expression of CX3CL1 in microglia was up-regulated after 12 hours of nicotine treatment(P<0.05),and was significantly higher than that in the control group after 24 hours of nicotine treatment(P<0.01).The expression of CX3CR1 in microglia was up-regulated 12 hours after nicotine treatment(P<0.01)and decreased 24 hours after nicotine treatment when compared with the blank.9.In vitro hippocampal neuron experiments,showed that,compared with the control group,the expression of CX3CL1 in primary neurons gradually decreased after 12 hours and 24 hours of nicotine treatment.The expression of CX3CR1 in primary neurons was significantly decreased after 12 hours of nicotine treatment(P<0.05),whereas CX3CR1 expression slightly increased after 24 hours of nicotine treatment.Conclusion:In this study,it was found that maternal chronic nicotine exposure to offspring in the early life exhibited neurodevelopmental retardation.After nicotine withdrawl,axiety-like behavioral was shown in nicotine-exposed adolescent offspring.The hippocampal neurogenesis increases and survival decreases in one and three weeks.This suggests that maternal exposure to nicotine during pregnancy and lactation may have adverse effects on their adolescent offspring,s neurogenesis,and CX3CL1/CX3CR1 signaling pathway was involved.Figure[17]table[0]reference[41]... |
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