| CHAPTER ONE:TO EXPLORE THE CHANGES OF NLRP1 ACTIVATION AND IMMUNE INFLAMMATION IN HIPPOCAMPUS OF DEPRESSION-LIKE BEHAVIOR MICE INDUCED BY LPS BASED ON PETBackground:Neuroinflammation is considered to be one of the pathogeneses of depression.The hippocampus is closely associated with the disease,and as the major immune cells in the brain,microglial cells play an important role in this process.The application of in vivo molecular imaging technology combined with stereological analysis of brain tissue,PCR and Western blotting is of great significance to reveal the pathogenesis of depression and develop effective biological markers in the diagnosis and treatment process.Methods:The 45 8-week-old healthy male mice were randomly divided into 3 groups（Control group,lipo-polysaccharide standard group（LPS）and LPS/fluoxetine（LPS/FLX）group）.Mice in the three group were given an intraperitoneal injection of NS/FLX 2-weeks in advance and then received intraperitoneal injection of LPS with mice in the LPS group and LPS/FLX group.Before LPS injection,after 24h and 72h of injection,mice in all groups received[18F]DPA-714 positron emission computed tomography（PET-CT）imaging and behavioral tests（sucrose preference test（SPT）,open field test（OFT）,and tail suspension test（TST））,and the activation of hippocampal microglial cells in adolescent mice with acute inflammation-induced depression-like behaviors was observed in vivo（tracer uptake values）.After behavioral tests,five mice in each group were randomly selected,and microglial cells in the hippocampal CA1,CA2-3,and DG regions were evaluated by stereology.In addition,the expression conditions of hippocampal nucleotide-binding oligomerization domain（NOD）-like receptor protein 1（NLRP1）and NLRP3inflammasomes（NLRP1,apoptosis-associated speck-like protein containing a caspase recruitment domain（ASC）,caspase-1）and the inflammatory cytokine（IL-1β、IL-18、IL-4及IL-10）in all groups were detected.Results:After LPS 24h,compared to controls,LPS-stressed mice showed a lower preference rate in SPT,had shorter central area activity distances in OFT,and had a longer immobility time in TST.The results in the LPS/FLX groups were between the results in the control group and the LPS group.The trend of PET-CT results was consistent with behavioral tests.Stereological results showed that the total amount of microglial cells during LPS stress in the CA1 region were greater than that in the CA2-3region,which was greater than that in the DG region,and the total amount of microglial cells in all hippocampal subregions in the LPS/FLX group was lower than that in the LPS and higher than that in the normal group.LPS-stressed mice had stronger expression of IL-1βand NLRP1 and NLRP3 inflammasomes in hippocampus than control group.After LPS 72h,the trend of behavioral tests and PET-CT in the three groups were close to that before LPS injection.Conclusion:（1）In mice with adolescent depression-like behaviors,the hippocampus was susceptible to acute inflammatory challenges especially in the CA1 region.（2）PET molecular imaging technology combined with NLRP1inflammasome,pro-inflammatory and anti-inflammatory cytokines can suggest immune inflammatory changes in the hippocampus during depression-like behavior to a certain extent,providing potential value for evaluating the occurrence and development of depression.（3）Fluoxetine may play a role in reducing the inflammatory response during depression-like behavior induced by acute inflammation.CHAPTER TWO: TO EXPLORE THE CHANGES OF NLRP1 ACTIVATION AND IMMUNE INFLAMMATION IN HIPPOCAMPUS OF CHRONIC STRESS MICE BASED ON PETObjective: Immune response showed different pathological characteristics under different stress states.In the course of depression,there is not only acute inflammation,but also chronic stress.In chapter one,we investigated microglial activation,inflammatory bodies,and inflammatory factors during depression-like behavior induced by acute inflammation.In order to simulate different stress states,we will discuss the process in this chapter.Methods: Male C57BL/6J mice aged 4-6 weeks were selected for baseline test of sugar water preference after 1 week of adaptive feeding.The remaining mice were randomly divided into normal Control group（Control group,n = 20）and CUS model group（CUS group,n = 126）.During the experiment,the Control group was normally fed with 5 mice per cage;CUS group mice were raised in a single cage（solitary）.CUS group mice were given CUS intervention for 4 weeks,and 40 model mice were screened out according to behavioral experiment results,which were randomly divided into CUS model control group（CUS+N group,n = 20）and CUS+ fluoxetine group（CUS+FLX group,n = 20）.Mice inCUS+FLX group received intraperitoneal injection of fluoxetine for 4weeks: fluoxetine injection with a concentration of 1mg/ m L of normal saline was prepared,and the dose was 10 mg/kg/d,and continued for 28 days.CUS+N group received the same volume intraperitoneal injection of normal saline for 4 weeks.Weight measurement and sugar water preference test were performed on all mice before modeling（week0）.The body weight of mice was monitored weekly.Mice in each group were given live [18F] DPA-714 positron emission computed tomography（PET-CT）imaging and behavioral test（SPT,OFT and TST）at week2（week2）and week4（week4）after fluoxetine intervention.After behavioral test,5 mice in each group were randomly selected to sacrifice brain tissue for stereological evaluation of microglia in hippocampal CA1,CA2-3 and DG regions.Meanwhile,the expression of NLRP1 inflammasome（NLRP1,ASC,Caspase-1）,inflammatory factors（IL-1β,IL-18,IL-4,IL-10）and GSDMD in each group were detected.To evaluate the effect of fluoxetine on antidepressant.Results:（1）The results of body weight of mice showed that there was statistical difference in body weight of mice in the control group and CUS intervention group during the first four weeks of modeling（p =0.009< 0.01）,among which,the weight decrease trend was most significant in the second week of modeling;Four weeks after the intervention,the difference between the Control group and CUS/N group（p = 0.044<0.05）,Control group and CUS/FLX group（p = 0.018<0.05）there was statistical difference.The results of sugar water preference experiment showed that,after modeling,the preference percentage of sugar water in CUS group was significantly lower than that in normal group（p =0.000< 0.001）;Before fluoxetine intervention,the difference between control group and CUS+N group（p = 0.012<0.05）,Control group and CUS+F group（p = 0.02< 0.05）;Two weeks after fluoxetine intervention,CUS+N group and CUS+FLX group were significantly lower than normal group,control group and CUS+N group（p = 0.000<0.001）,control group and CUS+F group（p = 0.000< 0.001）;At 4 weeks after intervention,there was statistically significant difference in sugar water preference percentage among the three groups,CUS+N group was significantly lower than normal group,while CUS+FLX group was in between.The results of open field experiment showed that the movement distance in the central area of the open field was significantly different between Control group and CUS group（p = 0.000< 0.001）,and the residence time in the central area was significantly different（p = 0.000< 0.001）.Four weeks after fluoxetine intervention（Week8）,there were no statistically significant differences in central region travel distance and central region stay time between Control group,CUS+N group and CUS+FLX group.The results of suspended tail test showed that at the 4th week of modeling,the immobility time of mice in Control group and CUS group was significantly different（p = 0.000<0.001）;After 4 weeks of fluoxetine intervention,the immobility duration of mice in the three groups was statistically different,and the duration of incomprehension of mice in the Control group was significantly shorter than that in the CUS group（p = 0.000< 0.001）and CUS+FLX group（p=0.000<0.001）,CUS+FLX group was shorter than CUS group（p=0.011<0.05）.（2）Before modeling,there was no statistical difference in trace capture values between the Control group and CUS group.During modeling period（week0-Week4）and fluoxetine intervention period（week6-week8）,there were statistically significant differences in the whole brain tracking values between the Control group and CUS+N group（p= 0.017<0.05）,that is,the whole brain trace uptake value of CUS+N group was higher than that of Control group;CUS+FLX group was higher than Control group（p = 0.013< 0.05）.Before modeling（week0）,there was no statistical difference in the uptake of PET trace in hippocampus between Control group and CUS group.At the second week of modeling（week2）,the PET trace uptake value of CUS group was higher than that of Control group（p= 0.002< 0.05）;At the fourth week of modeling（week4）,the PET trace uptake value of CUS group was also higher than that of Control group（p= 0.003< 0.05）but the intake decreased compared with week2.There was no statistically significant difference in PET trace uptake values in hippocampus of Control group,CUS+N group and CUS+FLX group at week 2 of fluoxetine intervention（Week6）.At week 4（Week8）,there was statistical difference in the uptake values among the three groups,that is,CUS+N group was higher than Control group（p= 0.021<0.05）,CUS+FLX group was significantly lower than CUS+N group（p= 0.000<0.001）and lower than Control group（p= 0.036< 0.05）.（3）Comparison of accurate quantitative stereological results at 4weeks of modeling and 4 weeks of intervention showed that the total number of microglia（Iba1+）in the hippocampus of mice in Control group and CUS group was statistically significant（p = 0.002<0.01）,that is,the total number of microglia-positive cells in CUS group was more than that in Control group.The number of positive cells in CUS group was the highest at week 2 of modeling,and decreased significantly at week4 of modeling.The number of positive cells continued to increase from week 6to week 8 after the maintenance of modeling stress and injection of normal saline control since week4.Four weeks before modeling,accurate quantitative stereological results showed that there was a statistically significant difference in the total number of microglia（Iba1+）in the hippocampus between the Control group and CUS group,and the total number of microglia positive cells in the CUS group was more than that in the Control group（week2,p = 0.000< 0.001;Week4,p= 0.027<0.05）At week 2 of modeling,stereological accurate quantitative results showed that:There were statistically significant differences in the number of microglia （Iba1+）in CA1,CA2-3 and DG subregions of hippocampi between Control group and CUS group,and the number of microglia positive cells in CUS group was more than that in Control group（CA1,p = 0.000< 0.001;CA2-3,p = 0.006<0.01;DG,p = 0.004 <0.01）At the fourth week of modeling,accurate and quantitative stereological results showed that:There were statistically significant differences in the number of microglia（Iba1+）in each CA1 and CA2-3 subregion of hippocampus between Control group and CUS group,that is,the number of microglia positive cells in CUS group was significantly higher than that in Control group（CA1,p= 0.000<0.001）;CUS group had more positive microglia cells than Control group（p = 0.04<0.05）.In the second week（Week6）after intervention,the exact quantitative results of stereology showed that: There was statistically significant difference in the total number of microglia（Iba1+）in the hippocampus of mice in the Control group,CUS+N group and CUS+FLX group,and the total number of microglia positive cells in the CUS+N group and CUS+FLX group was more than that in the Control group（CUS+N group,p= 0.000<0.001;CUS+FLX group,p = 0.008< 0.01）.At week 4 after intervention,the total number of Iba1+ microglia in the hippocampus was significantly different among the three groups.CUS+N group was significantly higher than Control group（p= 0.000< 0.001）,CUS+FLX group was between the two groups（p = 0.006<0.01;p = 0.03<0.05）.At the second week of intervention（Week6）,the exact quantitative results of stereology showed that there were statistically significant differences in the number of microglia（Iba1+）in each CA1 and CA2-3 subregion of the hippocampus of mice in CUS+N group,CUS+FLX group and Control group.In the CA1 subregion: The number of microglia-positive cells in CUS+N group was significantly higher than that in Control group（p = 0.000<0.001）,CUS+FLX group was between the two groups（p= 0.005< 0.01）;In ca2-3 subregion,CUS+N group had more cells than Control group（p= 0.031<0.05）,CUS+FLX group was less than Control group（p = 0.006<0.01）,significantly lower than CUS+N group（p= 0.000<0.001）.At the 4th week of intervention（Week8）,accurate quantitative stereological results showed that the number of microglia（Iba1+）in hippocampal CA1 subregion of mice in CUS+N group,CUS+FLX group and Control group was statistically significant,and the CUS+N group（p= 0.000< 0.001）and CUS+FLX group（p = 0.006< 0.01）more than Control group;CUS+FLX group was significantly lower than CUS+N group（p = 0.000< 0.001）.CUS+FLX group was less than Control group in ca2-3 subregion.CUS+FLX group was less than CUS+N group.CUS+N group（p = 0.033<0.05）and CUS+FLX group（p = 0.042<0.05）were higher than those in the Control group.（4）Western blotting results showed that the relative protein level of pro-inflammatory cytokine IL-1β in CUS+N group was significantly higher than that in Control group at week6 after fluoxetine intervention（p=0.000<0.001）and higher than CUS+FLX group（p = 0.007<0.01）;The level of IL-18 in CUS+N and CUS+FLX groups was higher than that in Control group.CUS+N group had statistical difference in il-4 protein level compared with Control group.CUS+N group（p = 0.002< 0.01）and CUS+FLX group（p = 0.000<0.001）were higher than the Control group.（5）PCR results（FIG.2.14）: During the second week of fluoxetine intervention（Week6）,there were statistically significant differences in NLRP1,Caspase-1 and ASC in mouse hippocampal tissues between the Control group,CUS+N group and CUS+FLX group（p<0.05）.CUS + N group > CUS + FLX group > CUS+N group,caspase-1,one of the complex components of inflammatory bodies,was significantly higher than the Control group（p = 0.000<0.001）,CUS+N group（p = 0.04<0.05）and CUS+FLX group（p = 0.03< 0.05）.The level of GSDMD in hippocampus of mice was higher than that of Control group.（6）Immunohistochemical fluorescence staining results showed that the number of costandard cells in CUS+N group in CA1 area was higher than that in Control group at the second week of fluoxetine intervention（P= 0.00< 0.001）;There was no significant difference in the number of costandard cells between groups in CA2-3 region.The number of coreference cells in CUS/FLX group in DG region was higher than that in Control group（p= 0.041< 0.05）;The number of costandard cells in CUS+FLX group was between Control group and CUS+N group,but the ifference was not statistically significant.At week 4 of intervention（Week8）,the number of costandard cells in CUS/N group in CA1 region was higher than that in Control group（p = 0.001<0.01）and CUS+FLX group（p = 0.014<0.05）;Similarly,the number of costandard cells in CUS/N group in DG region was higher than that in Control group（p =0.022<0.05）and CUS+FLX group（p = 0.041<0.05）.Conclusion:（1）CUS induced depression model produced immune inflammatory activation,and the activation of stress was obvious in the early stage,but weakened in the later stage;（2）In CUS,the CA1 region in hippocampus was more vulnerable to chronic stress.（3）Fluoxetine has certain anti-inflammatory effect in CUS model,which may produce antidepressant effect by promoting NLRP1 inflammosome activation,microglia activation,and promoting anti-inflammatory cytokine IL-10. |
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