| Parkinson’s disease(PD)is the second most common neurodegenerative disease,with motor symptoms such as,as well as non-motor symptoms such as olfactory dysfunction and rapid eye movement sleep behavior disorder.The major pathological features are degeneration of dopaminergic neurons and iron deposition in the pars compacta of substantia nigra(SNc).Olfactory dysfunction is one of the important biomarkers of early onset of PD,and 75-90% of PD patients suffer olfactory dysfunction to certain extent.Olfactory sensory neurons in the olfactory epithelium are susceptible to inflammation in the connected olfactory bulb due to exposure to environmental toxins.Intranasal administration may evade the blood-brain barrier and enter the central nervous system directly.However,how ferric ammonium citrate(FAC)enters the brain after intranasal administration and whether it can reach the SN and cause iron deposition and dopaminergic neurodegenetation as well as whether microglia are involved in this process need further investigation.In this study,we established the intranasal FAC administration model in mice.FAC was delivered with bilateral nostril administration(200 mg/kg),and the control group was given equal amount of saline,every other day for 0.5,1,2,3 or 6 weeks.The olfactory function of mice was tested by the olfactory discrimination test,and the motor function of mice was tested by the Cat Walk XT system,pole test and rotarod test;the changes of tyrosine hydroxylase(TH),light chain ferritin and alpha-synuclein(α-syn)protein expression were detected by western blotting;Immunofluorescence was applied to detect the number of TH-positive neurons and IBA-1(microglia marker)positive cells in the SN;Perl’s-DAB was used to detect the number of iron deposition in the olfactory bulb(OB),SN,globus pallidum(GP),dorsal striatum and other brain regions;and iron assay kit was used to analyze iron content in cerebrospinal fluid(CSF).1.The motor function and olfactory function of C57BL/6 mice were evaluated at 1,2,3 and 6 weeks by intranasal FAC administration.The climbing time of pole test increased significantly in the FAC group at 2 weeks and more dramatically at 3 weeks.Cat Walk showed that in mice with intranasal FAC administration for 3 weeks,the Swinging Speed of right frontpaw and left hindpaw,The Max Contact Area of frontpaws,Print Area and Mean Intensity all decreased.Print Length of left frontpaw reduced,The Base of support of frontpaws were increased.The Step Cycle and Initial Dual Stance of left paws were prolonged.The support mode changed.There is a decrease in the accumulated time spent in familiar compartments in mice with FAC administration for 6 weeks.The results indicated that motor incoordination was observed in mice with intranasal FAC administration for 2 weeks,before the occurrence of olfactory dysfunction at 6 weeks.2.The expression of light chain ferritin in olfactory bulb increased by 60% in the OB of mice with intranasal FAC administration for half a week,and TH expression decreased by 20.8%.Light chain ferritin expression increased by 105%,151% and 245% at 1,3,6 weeks respectively.TH expression decreased by 25%,63% and 86% accordingly.With a 6-week interval time at the end of of 3-week FAC administration,light chain ferritin expression increased by 135% in the OB and then 39.6% after another more 3 weeks.TH expression decreased by 48% and then 38% accordingly.However,α-syn expression increased by 72% at 6 weeks and then 432% at 9 weeks.The results suggested that iron deposition and loss of dopaminergic neuron occurred in the OB of mice with FAC administration for half a week and then were aggravated with longer exposure.These two events in the OB were restored after 6 or 9 weeks of interval with the cease of FAC administration,however,α-syn expression continued to be upregulated.3.There was no difference in the number of iron-positive cells in external globus pallidus(GPe)and ventral globus pallidum(VP)in mice with or without FAC administration for 1 week.The number of iron-positive cells in GPe and VP increased by 29% and 28% at 2 weeks,and 90.2% and 40.6% at 3 weeks respectively.Pearson correlation analysis showed that there was a significant positive correlation between average number of iron-positive cells in GPe and the climbing time of pole test(P=0.0008).The number of iron-positive cells in the dorsal striatum did not change significantly at 1,2,3 and 6 weeks.The results indicated that iron deposition occured in the GPe of mice with FAC administration for 2 weeks and even more at 3 weeks.4.CSF iron levels were elevated in mice with FAC administration for 3 weeks.The number of iron-positive cells and TH positive cells in the SN did not change significantly compared with the control group.With a 9-week interval time at the end of of 3-week FAC administration,the number of iron-positive cells increased by 67.5% and the number of TH positive cells decreased by 26.7%.The number of iron-positive cells in the SN increased by 77.6% in mice with FAC administration for 6 weeks,and the number of TH positive cells in dorsolateral side of SNc decreased by 40.2%,as well as the number of IBA-1 positive cells increased.The results of Pearson correlation analysis showed that there was a significant negative correlation between the number of iron-positive cells in the SN and the time of staying familiar compartments(P=0.0032).The results suggest that intranasal FAC administration for 6 weeks was able to induce iron deposition,dopaminergic neuron loss and microglia activation in the SN of mice.5.PLX5622(Csf1r inhibitor,working concentration of 1200 ppm)was applied to clear microglia in the brain 7 days prior to FAC administration and co-administration for 6 weeks.The removal of microglia was able to restore olfactory dysfunction and motor incoordination induced by intranasal administration of FAC,the removal of microglia exerted no effects on iron deposition and the loss of dopaminergic neurons in the OB induced by intranasal administration of FAC.FAC and PLX5622 co-administration effectively blocked the increase in the number of iron-positive cells in the GPe and VP,as well as the increase in the number of iron-positive cells and the decrease in the number of TH-positive cells in the SN iinduced by FAC.The results suggest that microglia may be involved in the process of iron deposition in the GP and SN.We reported intranasal FAC administration caused iron deposition and loss of dopaminergic neurons in the OB at half a week,followed by the occurrence of motor incoordination and iron deposition in the Gpe and VP at 2 weeks,and then olfactory dysfunction and significant iron deposition and loss of dopaminergic neurons in the SN appeared at 6 weeks.The application of PLX5622 was able to restore the motor incoordination.and olfactory dysfunction,and was able to block the iron deposition in the GP and SN triggered by intranasal FAC administration.The results suggest that intranasal FAC administration causes loss of dopaminergic neurons in the SN and microglia may be involved in iron deposition in the GP and SN.In this study,we established mice model using intranasal administration of FAC to investigate the effects and mechanisms of dopaminergic neuron loss,and the results provide new evidence for specific iron deposition and neurodenegeration in the SN of PD. |
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