Roles Of CXCL12/CXCR4 In The Systemic Inflammation-induced Anxiety

BACKGROUNDAnxiety, also known as anxiety disorder, is a mental disorder which characterized as persistent anxiety, fear, tension and obstacle of plant nerve activity, as well as brain dysfunction. Anxiety does harm to the health of body and mind severely, and the incidence rate of anxiety is about 4%, life-time prevalence is about 13.6%-28.8%, and the recurrence rate is high as 23.5%. The pathogenic factors are complex in anxiety development, and it is always the hotspot in this field to explore the mechanisms involved in anxiety development as well as prevention. Current strategy for anxiety clinic treatment mainly used is the combination of psychological therapy with medication. While, many Anti-anxiety medications anxiety drugs is the most common serious anxiety. The long-term use of anti-anxiety medications cannot effectively control the clinical symptoms because of the side effects including addiction and the lowering therapeutic effects etc., therefore, it is very important to explore a new potential therapeutic target for anxiety disorder.AIMS1. To clarify the roles of inflammatory response system CXCL12/CXCR4 in systemic inflammation induced-anxiety, and to reveal the relationship between anxiety and CXCL12/CXCR4.2. To explore the potential treatment targets for anxiety disorder based on CXCL12/CXCR4.METHODS1. To establish the systemic inflammation model in mice by LPS injection, anxiety like behaviors of mice were observed by behavioral experiments including the elevated plus maze and open field test; to check whether glial cells are activated under systemic inflammation in some brain regions closely related to anxiety development including hippocampus and amygdala by immune fluorescence method; the expression levels of CXCL12 in plasma, hippocampus and amygdala were detected by ELISA and Western blot in the mouse model; to further determine the expression levels of CXCL12 after administration of AMD3100, an antagonist for CXCL12 receptor CXCR4;To check the changes of mouse behaviors after BLA stereotactic microinjection of recombinant CXCL12 protein with or without pretreatment with CXCR4 inhibitor AMD3100; and to further observe animal behaviors induced by LPS after AMD3100 pretreatment.3. To exclude the potential nonspecific effects of antagonists AMD3100, and further confirm the anxiety behaviors mediated CXCL12/CXCR4 in LPS induced-systemic inflammation mouse model, lentivirus vectors for CXCR4 knockdown were designed and synthesized; the infection efficiency and effect of down-regulation of CXCR4 by were measured by immunofluorescence and Western blot both in vitro and in vivo; to observe the effects of CXCL12 on the anxiety behaviors in mice by BLA amygdala stereotactic microinjection of LV-sh RNACXCR4 after 72 hours;4. Whole-cell patch clamp technique was used to study the effects of CXCL12 on the excitability of pyramidal neurons, after hyperpolarization(AHP) and synaptic plasticity in hypothalamic pathway; and the effects of AMD3100 were observed by pre-perfusion of AMD3100 in artificial cerebrospinal fluid before CXCL12 usage.RESULTS1. LPS induced mice anxiety like behavior after intraperitoneal injection for 6 h, as showed increased time spent in open arms and entries into the open arms 6 h after i.p injection compared to saline-injected controls using EPM behavioral test, and decreased time spent in central area using OFT; immunofluorescence staining showed that after LPS injection, the glial cells were activated; CXCL12 expression increased significantly in the plasma and brain regions including the amygdala and hippocampus by ELISA and Western blot; while, pretreatment with CXCR4 antagonist AMD3100, had no effects on the expression level of CXCL12 induced by LPS.Mice anxiety like behaviors were induced by stereotactic microinjection of CXCL12 in amygdala BLA of mice, while, pretreatment with antagonist AMD3100 abolished CXCL12-induced mouse anxiety like behaviors; further behaviors tests showed that antagonist AMD3100 could relieve anxiety behavior of mice induced by LPS, suggesting that CXCL12/CXCR4 mediated animal anxiety behavior in systemic inflammation induced by LPS.3. Lentiviral vectors specific for CXCR4 knockdown(LV-sh CXCR4) were designed and synthesized, LV-sh RNACXCR4 infected nerve cells efficiently both in vitro and in vivo using immunofluorescence observation, and LV-sh RNACXCR4 effectively down-regulated CXCR4 expression level by Western blot determination after BLA local injection of LV-sh RNA CXCR4 for 72 hours, and ameliorated mice anxiety like behaviors induced by CXCL12.4. Whole-cell patch clamp results indicated that CXCL12 regulated neuronal excitation by increasing s EPSC firing frequency, for the distribution of amplitude little effect; at the same time, perfusion AMD3100 reversed the increased s EPSC firing frequency, also had no effects on after hyperpolarization(AHP) and synaptic plasticity in hypothalamic pathway. alter the probability of presynaptic transmitter releaseCONCLUSIONSThe study showed that systemic inflammation mediated by LPS induces animal anxiety-like behaviors; CXCL12/CXCR4 pathway of amygdala mediates the anxiety like behaviors under systemic inflammation of mice; electrophysiological data indicate that CXCL12 regulates neuronal excitation through its receptor CXCR4. The results indicate that CXCL12/CXCR4 play a key role in anxiety development.