| PART I MULTI-TIME POINT BEHAVIORAL STUDIES IN MICE WITH LPS-INDUCED DEPRESSIONObjective: This experiment aims to validate that the rodent model’s peripheral administration of lipopolysaccharide(LPS)leads to dynamic changes in inflammatory depressive symptoms.Previous studies have shown that glial cells are activated six h after a single intraperitoneal administration of low doses of LPS in mice and that activation persists for at least 3 to 7 days just before the depression-like and anxiety-like behaviors of the mice behavior emerge concomitantly.But few studies have explained the relationship between the two.In this study,transient sepsis-induced time-dependent depressive symptoms by using peripheral administration of LPS to observe the dynamic changes of depressive-like behaviors and to detect the expression of proinflammatory cytokines and inflammasomes,including interleukin-1 β(IL-1 β),NLRP3(NLRP3-Caspase-1-ACS).Methods: C57BL/6J mice(10-12 weeks old,18-23 g)were obtained from Chongqing Medical University(Chongqing,China).Mice(2-4mice/cage)are reared with free access to water and food in a facility with a12 h regular light/dark cycle(8:00 a.m.-8:00 p.m.off).After a two-week adaptation period,mice are randomly divided into a vector control group(n=25)and an LPS(E.coli,serotype 055:B5,Sigma-Aldrich Chemical,St.Louis,USA)model group(n = 70).The mice in the model group were subdivided into the LPS-24 h group and the LPS-72 h mouse group.LPS model mice are injected intraperitoneally(IP.)with LPS(1 mg/kg in PBS),while the vehicle control group is injected with an equivalent volume of PBS(10 m L/kg).At 24 h after injection,control mice and mice in the LPS-24 h group were subjected to behavioral testing,including a tail suspension test(TST),open field test(OFT),and sucrose preference test(SPT).Each 8-10 mice group is then euthanized to collect brain tissue samples for inflammatory factors and NLRP3 q PCR analysis at the hippocampus site.At 72 h after injection,mice in the LPS-72 h group were subjected to the behavioral testing described above.Results(1)LPS exposure induces depressive-like behavior in mice Consistent with prior reports,C57BL/6J mice injected with LPS(1mg/kg)developed depressive and anxiety-like behaviors at 24 h post-injection relative to control animals.We observed LPS injection-induced depressive behaviors at 24 h,including immobility time increase and struggling time decrease in the TST(P=0.0128),reduced mobility;decreased total travel distance and rearing behavior in the OFT(P<0.0001);decreased sucrose consumption in the SPT(P<0.0001).However,these effects were reversed at 72 h post-LPS injection,with immobility time in the TST significantly decreased compared to that at the24 h time point and equivalent to controls(P=0.523)as well as a significant increase in sucrose consumption compared to that at 24 h time and no significant difference from control(P=0.736).OFT analyses conducted in mice at 72 h post-injection revealed increased total distance traveled and more stand behaviors than those observed in LPS-injected mice at the 24 h time point(P<0.0001).However,compared to the control group,the total travel distance of LPS-72 mice was still reduced(P=0.028).(2)LPS injection induces hippocampal IL-1β and NLRP3 inflammasome upregulation To evaluate the effects of LPS injection more comprehensively on the hippocampus in this murine model system,we assessed the expression of inflammation-related genes 24 h post-LPS injection.At the m RNA level,hippocampal IL-1β expression was significantly increased at 24 h post-LPS injection relative to the levels observed in control mice(P=0.0022).The NLRP3 inflammasome and downstream Caspase-1 cleavage are essential for IL-1β maturation,leading us further to evaluate the expression of this signaling pathway in mice.Hippocampal m RNA levels of the NLRP3 inflammasome components NLRP3(P=0.0001),Caspase-1(P=0.0028),ASC(P=0.0022)were markedly elevated at 24 h post-LPS injection relative to those in control mice,consistent with a role for the NLRP3 inflammasome as a regulator of LPS-induced depressive-like behavior.Conclusion:(1)LPS-induced acute depressive behavior appears 24 hours after LPS injection,and most of the depressive behaviors are relieved at 72 hours and return to normal levels.A quantitative dependence between depressive behavior and acute peripheral infection shows a dynamic change trend.(2)24 hours after LPS injection,depressive behavior appears.A reasonable explanation is that LPS intraperitoneal injection leads to a systemic inflammatory response and interacts with the ventral hippocampus,causing inflammatory factors.Thus,provocative bodies activate IL-1β-NLRP3-Caspase 1-ASC pathway activation produces neuroinflammation leading to depressive behavior.PART II MULTI-TIME POINT WHOLE BRAIN PET-CT SCAN COMBINED WITH IN VITRO HIPPOCAMPAL IMMUNOFLUORESCENCE LOCALIZATION DETECTION FOR MULTIMODAL OBSERVATION OF ACUTE INFLAMMATORY DEPRESSION MODELObjective: Activation of microglia is a common finding in patients with depression and is associated with neuronal apoptosis and inflammatory cytokine production.Positron Emission Tomography(PET)[18F] DPA-714 can be used to directly assess microglial activation in vivo using a second-generation translocation protein(TSPO)ligand,providing a powerful and effective method for monitoring neuroinflammatory activity within the CNS in a sensitive manner in the context of neurological diseases.[18F] DPA-714 exhibits longer isotopic half-lives,lower nonspecific binding,higher affinity,and superior bioavailability in the brain than other radiotracers.However,efforts to apply PET imaging in studying MDD are relatively limited.Only biased reports have been made of in vivo imaging to evaluate LPS-induced rodent depression models.This study aims to establish a multimodal longitudinal method to monitor the mechanisms that control MDD-related behavior in LPS-induced mouse model systems.The dynamics of neuroinflammation in these mice were observed over time by in vivo TSPO PET-CT scans and in vitro hippocampal microglia markers Iba-1,TSPO expression,and localization.Depressive behaviors and processes associated with microglia were assessed to elucidate the correlation between these two sets of variables.Methods: The brain tissue samples were first collected for immunofluorescence staining after the first group of 4-6 mice in LPS-72 h and LPS-72 h were euthanized.TSPO PET-CT scans were performed on nine ramdon male mice,which were scanned 24 h before LPS injection(baseline),24 h after LPS injection(LPS-24 h),and 72 h after LPS injection(LPS-72 h).We scanned each mouse twice at a time.Pet data were reconstructed and analyzed by PMOD3.8.Result:(1)[18F] DPA-714 PET-CT imaging analyses We employed a [18F] DPA-714Nano-PET imaging approach to monitor levels of TSPO in LPS-injected mice over time(baseline,24 h post-injection,72 h post-injection)as a tool to assess microglial activation.SUV values(g/m L)were used to monitor [18F] DPA-714 uptake in the whole brain and in specific regions of interest including the cortex,hippocampus,and amygdala.Quantitative analyses revealed significant increases in [18F] DPA-714 signal in the whole brain,the cortex and hippocampus at 24 h post-LPS injection and then had returned to baseline at72 h post-injection,consistent with the substantiality but transiency of microglial activation.Baseline and 24 h post-injection SUVs were as follows: cortex(0.11 ± 0.010 vs.0.16 ± 0.011 g/ml,P=0.0029),hippocampus(0.020 ± 0.003 vs.0.032 ± 0.003 g/ml,P=0.008),whole brain(0.42 ± 0.028 vs.0.62 ± 0.032 g/ml,P=0.0002),and amygdala(0.0135 ±0.003 vs.0.0173 ±0.002 g/ml,P=0.015).SUVs at 24 h and 72 h post-LPS injection were as follows: cortex(0.16 ± 0.011 vs.0.13 ± 0.007 g/ml,P=0.025),hippocampus(0.032 ± 0.003 vs.0.020 ± 0.002 g/ml,P=0.007),whole brain(0.62 ± 0.032 vs.0.46 ± 0.024 g/ml,P=0.0027),and amygdala(0.0173 ± 0.002 vs.0.0152 ±0.002 g/ml,P =0.384).There were no substantial differences between LPS-72 h and baseline SUVs,consistent with microglial activation having returned to baseline: cortex(0.13 ± 0.007 vs.0.11 ± 0.010 g/ml,P = 0.64),hippocampus(0.020 ± 0.002 vs.0.020 ±0.003 g/ml,P=0.99),whole brain(0.46 ± 0.027 vs.0.42 ± 0.028 g/ml,P =0.53),and amygdala(0.0152 ±0.002 vs 0.0135 ± 0.003 g/ml,P=0.232).(2)LPS exposure increases the expression of microglia markers and alters microglial morphology Next,immunofluorescent staining of brain tissue samples from control and LPS-injected mice(24h and 72h)was performed to evaluate correlations between [18F] DPA714-PET signals,activated microglia,and TSPO levels.A dual-staining approach was used to detect the expression of TSPO and Iba-1 or GFAP within the hippocampal region to determine whether TSPO was primarily expressed by microglia(Iba-1+)or reactive glial cells(GFAP+).LPS injection-induced significant microglial activation,with TSPO being primarily expressed on Iba-1+ microglia rather than on GFAP+ astrocytes.These data confirmed a strong correlation between [18F]DPA-714 signal and microglial activation.Visual analysis of immunofluorescent TSPO,Iba-1,and DAPI-stained tissue sections from mice in these three different treatment groups revealed that specimens collected at 24 h post-LPS injection exhibited the highest levels of microglial activation.Those from the LPS-24 h group displayed more activated microglia and significantly elevated TSPO levels than the control group.However,no evident differences in TSPO levels or microglial activation were found between the control and LPS-72 h samples,consistent with the return-to-baseline phenotypes.Quantitative analyses revealed thatrelative to control brain sections,samples from mice collected at 24 h post-LPS injection exhibited increased Iba-1+ activated microglia(P<0.01),a higher number of TSPO+ Iba-1+ cells(P<0.01),and a rise in the total number of cells in the hippocampus(P<0.05).Relative to LPS-24 h mice,LPS-72 h mice exhibited a reduction in activated microglia in the hippocampus(P<0.05)with fewer cells exhibiting dual TSPO and Iba-1positivity(P<0.05)and a reduction in total cell number(P<0.05).When comparing samples from control and LPS-72 h mice,no differences in these variables were observed.The morphology of microglia can be classified descriptively,or it can be quantified based upon continuous parameter variables,including cell branching,complexity,and shape.The Analyze Skeleton(2D/3D)plugins collect collected morphological data from fluorescent and brightfield images converted into representative binary and skeletonized images in Image J.The outputs of these plugins summarized cell morphology in terms of process endpoints as cell shape and size descriptors.The resting microglia in the control group were branched with smaller cell bodies and longer processes.After LPS stimulation,microglia were activated,exhibiting an amoeboid shape with an enlarged cell body and shortened cycles.The hippocampal tissue sections from different groups delineated have delineated the region of interest(ROI)for morphological analyses of these microglia via one-way ANOVAs;Branch endpoint analysis results showed a significant reduction in these endpoints in the LPS-24 h and LPS-72 h groups compared with the control group(P < 0.0001).As for cell area,the average size of cell soma in the control group was 640 voxels,which was significantly smaller than that observed in the LPS-24 h group(1448 voxels)and the LPS-72 h group(1809 voxels)(P < 0.05).These single-cell description studies and multicellular quantitative analyses of the hippocampal region revealed that microglia were activated after LPS injection.Conclusion:(1)The TSPO PET study showed that [18F] DPA-714 PET imaging performed a comprehensive longitudinal analysis of neuroinflammation in a mouse model of LPS-induced depression.Neuroinflammation associated with microglia activation was observed using [18F] DPA-714 PET.TSPO levels in the whole brain,hippocampus,and cortex were observed 24 hours after LPS injection.We found that this inflammation eventually returned to baseline 72 hours after injection.(2)PET imaging data and ex vivo Iba-1 and TSPO staining of brain tissue sections allowed us to conclude that activated microglia had a significant correlation with enhanced [18F] DPA-714 uptake in this model system.[18F] DPA-714 signaling can be very stable in the localization and quantification of microglia activation in the response center.(3)It was once again verified that peripheral inflammation has an activating effect on central microglia.Microglia morphological analysis,at least 72 hours after LPS injection,microglia in the brain hippocampus still have an activated state.PART III A STUDY FOR THE IMPROVEMENT OF DEPRESSIVE SYMPTOMS BY THE INFLAMMATION DISSIPATING SUBSTANCE MARESIN-1 BASED ON A MULTIMODAL OBSERVATIONAL SYSTEMObjective: Peripheral infection can initiate an inflammatory program with the production of IL-1 β through the immune response with the CNS,which directly causes damage to neurons and activates microglial M1 type so that it secretes pro-inflammatory factors and free radicals,causing further damage to neurons.Damaged neurons can activate more NLRP3 bodies and further activate more microglia,aggravating inflammation.Above inflammatory vicious circle and abnormality of the inflammatory dissipating process are related.We speculated that the inflammatory dissipating substance Ma R1 could alleviate depressive symptoms in the LPS induced murine model by reducing the activation of inflammatory factors and inflammasomes while regulating the activation of microglia to ameliorate central inflammation.Methods: Eight-week-old male C57BL/6J mice were group-housed and maintained under standard housing conditions(lights on between 7 am and 7 pm;room temperature 22 ± 2 ° C).Food and water were provided ad libitum.After two weeks of acclimation to the novel environment,mice were randomly divided into a healthy versus the control group(n = 33),an LPS + NS group(n = 35),and an LPS + Ma R1 group(n = 35).Control mice were given intraperitoneal(i.p)saline at 10 kg ml/kg body weight.Mice in the LPS group were intraperitoneally(i.p.)injected with LPS,a bacterial antigen,at a 1 mg/kg body weight.After injection of LPS(1mg/kg body weight).In parallel,mice in the Ma R1 + LPS group wereinjected intraperitoneally with Ma R1 5ug / kg.Both Ma R1 and LPS were prepared in normal saline at a 10 kg ml/kg bodyweight volume.The behavioral analysis was performed 24 hours after injection for the three groups,and the brains of sacrificed mice after behavioral analysis were removed for Western blot and QRT-PCR detection of inflammatory factors,inflammasomes,microglial phenotype markers,and pyroptosis;Formaldehyde perfusion brains were harvested,and after fixing sections,quantitative localization analysis was performed for Iba-1,a marker of microglial activation,GFAP,a marker of stellate cell activation,as well as immunofluorescence staining for TSPO.Four PET-CT scans per group were taken for three groups of mice simultaneously,and TSPO-pet scan studies of the whole brain and sensitive brain regions were carried out for analysis.Results:(1)Ma R1 intervention partially reverses LPS induced depression-like behaviors.Consistent with previous reports,we used an LPS induced protocol to explore the relationship between neuroinflammation and depression.In this protocol,mice were generated to develop depressive behavior 24 h after intraperitoneal injection of LPS(1 mg/kg).Depressive behavior was determined by TST(tail suspension test)and,SPT(sugar water preference test),OFT(open field test).As expected,mice exhibited depression-like behavioral changes 24 h after LPS intraperitoneal injection.Mice in the LPS group exhibited depression-like and anxiety-like behaviors by increasing immobility time in the TST(P < 0.0001)and decreasing total distance in the OFT(P < 0.0001)compared with normal controls.In SPT experiments,mice in the LPS model also significantly reduced the sucrose preference percentage of mice(P < 0.0001).These results indicated that 24 h after intraperitoneal injection of LPS(1 mg/kg)successfully induced depression-like behavioral changes in mice.However,compared with the LPS model group,maresin-1 treatment significantly improved immobility time in the TST(P < 0.01)and total distance in the OFT(P < 0.01),but the sucrose preference percentage was not alleviated,suggesting that the improvement of neuroinflammation may be associated with the alleviation of depression.(2)Ma R1 intervention reduced [18F] DPA-714 uptake in TSPO PET-CT in the whole brain and some depression-sensitive brain regions.The experimental radiopharmaceutical specificity of [18F] DPA-714 as a TSPO ligand allowed us to quantify microglial activation using PET imaging.To examine the effects of intraperitoneal injection of LPS on inflammatory signals,PET scans were performed on mice before LPS injection and 24 h after LPS intraperitoneal injection,and changes in DPA-714 uptake were compared.The accumulation of [18F] DPA-714 was greater in the whole brain(P < 0.0001)and in some brain regions such as the hippocampus(P < 0.01),amygdala(P < 0.01),hypothalamus(P <0.0001),striatum(P < 0.0001),and hippocampus(P < 0.0001)of mice 24 hours after i.p.Meanwhile,SUV values in the hippocampus of mice treated with maresin1 were significantly lower than those in the LPS group(P <0.01),and there was also a decline in the amygdala(P < 0.05).The maresin-1 peripheral injection can significantly reduce the activation situation of microglia in mice.(2)Ma R1 intervention reduced [18F] DPA-714 uptake.It’s the whole brain and parts of the brain region.The experimental radiopharmaceutical specificity of [18F] DPA-714 is a TSPO ligand that allowed us to quantify microglial activation using PET imaging.To examine the effects of intraperitoneal injection of LPS on inflammatory signals,PET scans were performed on mice before LPS injection,and 24 h after LPS intraperitoneal injection,and changes in [18F] DPA-714 uptake were compared.The accumulation of [18F] DPA-714 was higher in the whole brain(P < 0.0001)and in some brain regions such as the hippocampus(P < 0.01),amygdala(P < 0.01),hypothalamus(P < 0.0001),striatum(P < 0.0001)of mice 24 hours after i.p.Meanwhile,SUV values in the hippocampus of mice treated with Ma R1 were significantly lower than those in the LPS group(P < 0.01),and there was also a decline in the amygdala(P < 0.05).The maresin-1peripheral injection can significantly reduce the activation situation of microglia in mice.(3)Ma R1 intervention reduced the number and fluorescence intensity of Iba-1 + expression,a microglial marker.We then examined the number of activated microglial Iba-1 + expressing cells and the level of TSPO fluorescence by using immunohistochemical techniques.Showed that after LPS induction,the Iba-1 mean fluorescence intensity increased significantly compared with the normal group(P < 0.0001)and that the intervention of Ma R1 reduced LPS induced expression of the above fluorescence intensities(P < 0.05).Double staining for TSPO and Iba-1 in the hippocampus was performed to determine whether TSPO was mainly expressed by microglia(Iba-1 +).Pearson’s correlation coefficient yielded that TSPO + and Iba-1 + were was associated with about 60%.In contrast,the correlation between TSPO + and GFAP + was in the range(of 0-37%).Intraperitoneal injection of LPS in Iba-1 + microglia significantly induced microglial activation and TSPO expression.These results further confirmed the close correlation between [18F] DPA-714 signaling and microglial activation.Furthermore,a more nuanced immunofluorescence quantification of hippocampal structure in the hippocampal region of mouse brains revealed that Iba-1 + cells were expressed in CA1 + CA2 of hippocampal substructure areas in the LPS model group and Ma R1 intervention group compared with normal controls.The total number of Iba-1 + cells in the LPS group was increased compared with the other two groups(P < 0.05,P < 0.001).There was no significant difference between the standard control and maresin1 groups(P > 0.05).(4)Mar1 intervention reduces microglial activation and IL-1 β-Activation of the NLRP3 inflammasome pathway.Image j was used to analyze the morphology of the Microglia in the hippocampus of mice.The results showed that there was no significant difference in the Microglia soma area and branching volume between the MAR1 intervention group and the Normal Control Group(P > 0.05);compared with the LPS model,the cell area of microglia decreased significantly(P < 0.001),and its branches became thinner(P < 0.001)In the analysis of inflammatory factors and inflammatory bodies in the hippocampus,we found that the m RNA expression levels of IL-1 β and NLRP3 in the Ma R1 intervention group were significantly lower than those in the LPS model group(P <0.0001).Compared with the normal control group,IL-1 β and NLRP3 inflammatory factors were still increased(P < 0.001,P < 0.01).In the subsequent protein level analysis,the levels of IL-1β and NLRP3,ASC in the hippocampus of the Ma R1 intervention group were lower than those of the LPS group,but there was no difference.The expression level of Caspase-1 protein in the LPS group was higher than that in the normal group and Ma R1 group(P<0.05).Still,there was no difference between the Ma R1 and the normal groups.Pyrolethal protein GSDMD increased after intraperitoneal injection of LPS,although there was no difference between the normal and Ma R1 intervention groups(P > 0.05).However,after the intervention of Ma R1,the level of GSDMD in the LPS model group was significantly lower than that in the LPS model group,indicating that Ma R1 may reduce pyroptosis in hippocampal cellsConclusions(1)Intraperitoneal injection of Ma R1 partially ameliorated LPS induced acute depressive behaviors.(2)Ma R1 may act by reducing IL-1 β and activating NLRP3 bodies,while inhibition of inflammation and proptosis in the hippocampus region plays an antidepressant role.(3)Regardless of microglial activation number from hippocampal slices or microglia single morphological analysis,Ma R1 was able to inhibit microglial activation maintained from the homeostasis of microglia in the brain acting as an antidepressant.(4)PET-CT studies confirmed that Ma R1 decreased the uptake values of TSPO in both the whole brain and parts of brain regions critical for depression,such as the hippocampus,amygdala,etc.,implying the activation of microglia in brain regions with less brain inflammation.The anti-inflammatory antidepressant effect of Ma R1 was confirmed by in vivo experiments. |
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