Effects Of Manganese Exposure On Neurogenesis In Mouse Brain And Intracerebral NSCs Transplantation On Mouse Manganismus Model

To explore the new mechanism of manganese neurotoxicology,this study took emphasis on the role of neurogenesis in the process of manganismus pathogenesy,found the action of manganese on NSCs in brain,treated manganismus by NSCs transplantation firstly,and gained ideal therapeutic effect, which provided a experimental base for clinical service.Our study contained two parts:Part one Changes of neurogenesis in mouse manganismus model and the relation to neurobehavioral degeneration.Objective:To observe the effects of manganismus on neurobehavior and neurogenesis,then analyze whether these effects have dose-dependence and correlation.Materials and methods:28 two-weeks-old mice were divided into 4 groups:control group(CG),low-dose group(LDG),middling-dose group(MDG) and high-dose group(HDG).They respectively received intraperitoneal injection of 0,5,20,50mg·kg^-1.d^-1manganese chloride dissolved in physiological saline. The ability of learning and memory was tested by Morris Water Maze(MWM). All mice's psychbehavior was observed in open fields tests,and the proliferation of NSCs in mouse SGZ and SVZ was detected by immunohistochemistry. Besides,horizontal cells in hippocampus and nurofilament(NF)-positive neurons were also observed.Statistical correlation analysis was performed among the average frequence((?)±S_x)in MWM test,the BrdU-labeled cells number((?)±s) and the dosage of manganese.Results:(1)Successful mouse manganismus models were built.Compared with CG,the mean latency of HDG was prolonged significantly from the third day(F_1,11=12.404,P＜0.01).Till the fifth day,mice's learning ability of all Mn-exposure group degraded greatly(F_3,21=3.105,P＜0.05)and this change of HDG was most notable(F_1,11=3.832,P＜0.01).In the retention test,both the mean frequences of MDG and HDG decreased remarkably(P＜0.01),compared with that of CG.(2)Both the mean numbers of BrdU-labeled cells in SGZ and SVZ of mouse brain in all manganismus groups were much lower than that of CG(P＜0.01),and this effect had evident dose-dependence of manganese chloride.(3)The difference of nestin-immunopositive cells' numbers of SGZ between MDG and CG mouse was statistically significant(P＜0.001),which is same to the difference of SVZ(P＜0.001).(4)GFAP-immunopositive cells of HDG mouse decreased,compared with CG(P＜0.05),while NF-positive neurons had no statistical difference among all groups.(5)The ability of memory was negatively associated with the dose of manganese chloride(r_s=-0.598,P＜0.01). Meanwhile,the decrease degree of BrdU-positive cells was positively associated with the retarded degree of learning and memory(r_s=0.734,P＜0.01).Conclusion:Manganese exposure could inhibit neurogenesis in brain, decrease the number of NSCs and prevent their proliferation,affect the ability of learning and memory in the same time,with positive dose-dependence effect of manganese.The correlation analysis suggested that inhibition on neirogenesis was an important factor to cause neurobehavioral disorder of manganismus,and this was a new mechanism.Part two Therapeutic action ofintracerebral NSCs transplantation on mouse manganismus modelObjective:To explore the feasibility and mechanism of NSCs transplantation into lateral cerebral ventricle and hippocampus for treating manganismus symptom,and observe their surviving,migration and differentiation in the host's brain.Materials and methods:Using serum free culture medium containing b27, bFGF,EGF,NSCs in new-born mouse hippocampus were isolated,exacted and cultured,of unicell clone was used to confirm the character of self-renewing. After passaged for a week,unicell suspension was made for transplantation by PBS after labeled by BrdU.48 two-weeks-old mice were divided into 6 groups including normal group(NG),manganismus group(MG),Intra-lateral-ventricular NSCs transplantation group(LNG),Intra-lateral-ventricular pseudotransplantation group(LPG),Intrahippocampus NSCs transplantation group(HNG) and Intrahippocampus pseudo-transplantation group(HPG).All manganismus mouse was induced by 2-weeks intraperitoneal administration of 20mg·kg^-1·d^-1 manganese chloride solution.Then mice in LTG and HTG were transplanted into lateral cerebral ventricle and hippocampus with unicell suspension of BrdU-labeled NSCs from new-born mouse hippocampus,mice in LPG and HPG were only injected with phosphate buffered saline from the same position,while mice in NG and MG received no treatment.After a week resting,all experimental mice's neurobehavioral scores were evaluated by MWM tests.The surviving,migration,differentiation of exogenous NSCs were detected by single-labeled and double-labeled methods of immunohistochemistry.Results:(1)Typical neurospheres formed and floated in the medum,before or after being passaged.By immunocytochemistry,most cells were found to show nestin-positive,and could be labeled by BrdU.(2)Both of the mice's latency and frequence in MG were decreased significantly,compared with that in NG(P＜0.05).(3)NSCs transplatation into lateral cerebral ventricular for treating manganismus:ⅰ)Compared with LPG,mean latency in LTG decreased significantly from the fifth day of the MWM tests(P＜0.05).In the retention tests, mean frequences of mice in LTG were more than that in both LPG and MG(P＜0.05),and it was not different from that in NG statistically,ⅱ)A great number of BrdU-positive NSCs survived in the pin hole of injection.Many of NSCs migrated into SVZ,hippocampus,olfactory bulb and survived there, which was most notable in angulus lateralis of the lateral cerebral ventricle. Some transplanted NSCs in SVZ and hippocampus still showed nestin-positive in the same time,while many others showed GFAP-positive characteristic.This phenomenon was observed notably in SGZ.(4)NSCs transplatation into hippocampus for treating manganismus:ⅰ)Compared with HPG,mean platency in HTG became short significantly constanly from the second day of the MWM tests(P＜0.05).And this enhancement facilitation also appeared when compared with MG from the fourth day(P＜0.05).In the retention test,mean frequences of mice in HTG were more than that in both HPG and MG(P＜0.05),either.There is no significant difference in frequence between HTG and NG.ⅱ)Numerous BrdU-labeled NSCs also survived in the pin hole of injection,and could move around along the SGL,more than 0.4mm far away from the termination of the channel.Many of NSCs also migrated into olfactory bulb and survived there. Some surviving NSCs in SGZ showed nestin-immunopositive and many others showed GFAP-positive or NF-positive characteristic.Conclusion:The ability of learning and memory in manganismus mouse was improved notably by NSCs transplantation,and the restore action was more notable in intra-hippocampus transplantation experiment.This effect was caused by transplanted NSCs' surviving,migrating selectively and differentiate into founctional cells for modulating the structure of some functional areas in host's brain,which suggested that stimulating the faculty of neurogenesis could improved brain functional outcome.Summary:The capacity of neurogenesis is inhibited by Mn-exposure, resulting in neural functional disturbance.There is a positive correlation between the capacity of neurogenesis and recognition,both of which have dose-dependence effect with Mn-exposure,but NSCs stereotaxistransplantation altered the symptom.It suggests that damage to neurogenesis may take an important role in neural pathopoiesis process of mangannismus,and NSCs tranplantation can be used to restore neurodegeration symptom of manganismus patient in clinical service.