Effects of CUMS combined with CRS on hippocampal glial cells and synaptic plasticity in depressed mice

LI Xin, WANG Qiong-ying, MA Zhao-tian, SUN Hong-hao, YU Xue, REN Xiao-qiao

School of Traditional Chinese Medicine, Beijing University of Chinese Medicine Beijing, Beijing 100029, China

Keywords:

ABSTRACT

1.Introduction

Depression is a widespread class of mental disorders mainly characterized by depressed mood, reduced interest, delayed thinking, etc., and in severe cases, it can even be accompanied by self-injurious or suicidal tendencies.According to the World Health Organization (WHO) survey, depression will be the number one contributor to the world’s burden of disease in 2030 and has become a global public health problem leading to human disability and death[1].Currently, the understanding of the pathogenesis of depression has not been fully clarified.In recent years, a large number of studies[2,3] have confirmed that neuroinflammation has a significant impact on the occurrence of depression.However,when the level of neuroinflammation in the brain increases, it can exert a series of effects on glial cells, synaptic plasticity, and so on.However, at present, there are no definite reports on whether glial cell damage and reduced synaptic plasticity caused by neuroinflammation are necessarily associated with the occurrence of depression.

Chronic unpredictable mild stress (CUMS) and chronic restraint stress (CRS) are commonly used in the preparation of animal models of depression.For depression, CUMS (usually performed by cold stimulation, tail pinch, and swimming) was mostly somatic stress, while restraint for CRS was mainly psychological stress.Some scholars[4,5] have pointed out that patients with depression tend to have mixed somatic and psychological effects, and none of the animal models prepared by the application of CUMS or CRS alone can reflect the clinical reality of patients.Therefore, in this study, BALB/c mice were selected to establish a mouse model of depression by continuous stimulation through the method of CUMS combined with CRS for 7 weeks and based on this, the behavior of the model mice and the related pathological changes, so as a reference for the study of the pathogenesis of depression.

2.Materials and methods

2.1 Reagents

Rabbit monoclonal anti-Iba1 antibody (ab178846), Rabbit polyclonal anti-GFAP antibody (ab7260), Rabbit monoclonal anti-PSD-95 antibody (ab238135), Rabbit monoclonal anti-SYP antibody (ab254349) were purchased from Abcam Company in the United States; Mouse monoclonal anti-β-actin antibody(66009-1-Ig), HRP-conjugated Affinipure Goat Anti-Rabbit IgG(H+L) (SA00001-2), HRP-conjugated Affinipure Goat Anti-Mouse IgG (H+L) (SA00001-1) were purchased from Proteintech Company in the United States.Mouse TNF-a ELISA Kit (E-ELM3063) was purchased from Elabscience Company in Wuhan,China.RNA extraction kit was purchased from Magen Company in China.Reverse transcription kit (K1622) and RT-PCR detection kit (A25742) were purchased from Thermo Fisher Company in the United States.

2.2 Main instruments

Ultrasonic cell disruptor (SONICS, USA), Biofuge Stratos highspeed Refrigerated Centrifuge (HERAEUS, Germany), Bio-Rad PowerPace Basic system (Bio-Rad, USA), Paraffin Slicing Machine(Leica, Shanghai China), Panoramic section scanner (3Dhistech,Hungary), Automatic chemiluminescence analyzer (Tanon, Shanghai China), Microplate reader (Molecular Devices, USA), Thermal Cycler PCR instrument (Bio-Rad, USA), qPCR instrument (Bio-Rad, USA).

2.3 Animals

42 SPF healthy BALB/c mice (male, 20～23 g) , which were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.(Beijing, China).License No:SCXK (JING) 2016-0006.And fed in a standard animal room (4 mice/cage; room temperature:25±1 ℃; relative humidity: 65±5%; light condition: 12 h dark/light cycle).Adaptive feeding for 1 week, drinking and eating freely during the adaptation period.The experiment was approved by the animal ethics committee of Beijing University of traditional Chinese medicine and complied with the Animal Management Rules of the Chinese Ministry of Health and existing current animal welfare guidelines (NO.BUCM-4-2021091001-3058).

2.4 Modeling and drug interventions

Before the experiment, the Sucrose preference test was conducted,and the mice with abnormal Sucrose preference rate were removed.The eligible 40 mice were randomly divided into two groups by body weight: normal group (n=20), and model group (n=20).The method of CRS combined with CUMS was applied to model the model group.CRS: bound in centrifuge tubes (50 mL) daily at 9 am for 6 h; CUMS: 1 stimulus was given daily and each stimulus could not be presented continuously (food deprivation, water deprivation).CUMS: (1) food deprivation 24 h (2) water deprivation 24 h (3)continuous light 24 h (4) tail clipped 3 min (5) damp bedding 24 h (6) cage tilt 45° 24 h (7) Circadian inversion.After 7 weeks of modeling, mice models of depression were assessed by three behavioral tests (Sucrose preference test, Tail suspension test, and Open field test).

2.5 Detection index

2.5.1 Body weight

Body weight of mice were weighed at the end of each week.In this study, the growth of mice were evaluated by calculating the initial body weight and the body weight of mice at each weekend.

2.5.2 Behavioral Tests

2.5.2.1 Sucrose preference test (SPT)

Sucrose preference rate can applicant the degree of anhedonia in experimental animals[6].The SPT were performed before modeling,at the end of 3 and 7 weeks of modeling.Before the experiment,1% sucrose solution was applied to train the mice.Day 1: 2 bottles of 1% sucrose solution per cage for 24 h; Day 2: one bottle of 1%sucrose water and one bottle of distilled water were placed per cage for 24 h.Day 3: mice were transferred to a single cage, food and water deprivation for 12 h.During the SPT procedure, mice were allowed access to one bottle of 1% sucrose solution and one bottle of distilled water.The weights of the sucrose solution and distilled water were weighed after 2 h.Sucrose preference rate (%) was calculated as sucrose consumption/(sucrose consumption+distilled water consumption)×100%[6].

2.5.2.2 Tail suspension test (TST)

The immobility time of tail suspension test can reflect the state of depression despair in experimental mice[7].Tail suspension test was performed at the end of 3 and 7 weeks of modeling.In the tail suspension test, the caudal end of the mouse (1 cm from the tail tip)was fixed on top of the box with medical tape.The mice were placed with their heads down, about 20 cm from the bottom of the box.The experiment lasted 5 min.Mice were considered stationary when they did not struggle appreciably.The cumulative immobility time of mice within 4 min after the experiment were recorded.

2.5.2.3 Open field test (OFT)

Open field test can reflect the exploratory behavior and autonomous activity of experimental mice in an unfamiliar environment[8].The open field test was performed at the end of 3 and 7 days after modeling.The tested mice were acclimated within the behavioral laboratory for 1 h before testing.For formal experiments, each mouse was gently placed in the center of the open-field box (50 cm×50 cm×40 cm) and could explore the arena for 5 min freely.The video camera was placed on the top of the box and the activity of the mice was recorded for 5 min, and the main observation indexes were as follows: total traveling distance, the times of standing (mouse standing on its hind limbs), cumulative duration of central area, and velocity of motion.At the end of each experiment,the floor of the box was cleaned with 75% ethanol and the whole experiment was kept quiet.

2.6 TNF-a content of hippocampal tissue was determined by double antibody sandwich enzyme-linked immunosorbent assay (ELISA)

Five mice were selected for ELISA in each group.After behavioral test, mice were anesthetized and brains were decapitated.Hippocampal tissues were carefully separated on ice, weighed,and put into the homogenizer, and the corresponding ratio of PBS solution was added according to the instructions and homogenized on ice.After homogenization, the lysate was centrifuged at 10 000 r/min for 10 min at 4 ℃, and the supernatant was collected for further use.The detection of TNF-a content was performed by the double antibody sandwich ELISA method, and the actual operation was strictly according to the reagent use instructions.In the first step, added 100 μL of standard working solution or sample into corresponding plate wells, and incubated at 37 ℃ for 90 min.In the second step, the liquid was discarded and added 100 μL of biotinylated antibody working solution, and incubated at 37 ℃ for 60 min.In the third step, the liquid was discarded, and the plates were washed 3 times before adding 100 μL HRP enzyme conjugate working solution, and then incubated at 37 ℃ for 30 min.In the fourth step, the liquid was discarded and the plate was washed five times, and 90 μL substrate solution was added to each well, and incubated at 37 ℃ for 20 min.For the fifth step, 50 μL stop solution was added to each well and immediately read at 450 nm wavelength on a microplate reader for data analysis.

2.7 Iba-1, GFAP MOD values were determined by immunohistochemistry (IHC) in CA1, CA3, and DG regions of the hippocampus

Five mice were selected for IHC in each group.At the end of the behavioral tests, mice were anesthetized and transcardially perfused with 0.9% pre-cooled saline and 4% paraformaldehyde.Brains were decapitated after successful perfusion, fixed in 4% paraformaldehyde for 48 h, and embedded in paraffin.For immunohistochemistry,paraffin sections of brain tissues were deparaffinized to water,antigen retrieval was performed in citrate buffer, cooled down naturally to room temperature, rinsed 3 times in PBS, and blocked by dropwise addition of 3% H2O2for 10 min.PBS was rinsed again three times and blocked by dropwise addition of 5% goat serum for 1h.After it's dried, sections were incubated overnight at 4 ℃ with Rabbit anti-GFAP (1:1 000), and rabbit anti-iba-1 (1:1 000), and secondary antibodies were added for 30 min at room temperature.Secondary antibodies were removed, washed for 3 min in PBS,and repeated 5 times.Sections were then stained with DAB and hematoxylin counterstain.Finally, after dehydration and drying, the sections were mounted with neutral glue and scanned with a scanner.The mean optical density (MOD) of GFAP, Iba-1 was analyzed using image pro plus 6.0.

2.8 The protein expression of Iba-1, GFAP, PSD95, and SYP in the hippocampus were determined by Western blot (WB)

Five mice were selected for WB in each group.After behavioral tests, mice were anesthetized and brains were decapitated.Hippocampal tissues were carefully separated on ice, weighed,and placed into a homogenizer, and the corresponding proportion of Ripa lysis solution was added according to the instructions and homogenized on ice.The supernatant was collected by centrifugation at 14 000 r/min for 10 min at 4 ℃ in a refrigerated centrifuge.Total protein concentrations of tissue samples were determined by the BCA method.SDS-PAGE gels preparation, sample injection, and then proteins were transferred onto polypropylene fluoride (PVDF)membranes and blocked in 5% nonfat milk (1×TBST) for 2 h.Corresponding primary antibodies were added: rabbit monoclonal anti-iba-1 (1:1 000), rabbit polyclonal anti-GFAP (1:10 000), rabbit monoclonal anti-PSD-95 (1:1 000), rabbit monoclonal anti-SYP(1:1 000), mouse monoclonal anti β- actin monoclonal antibody(1:30 000) and incubated overnight in the refrigerator at 4 ℃.The second day, the membranes were washed 5 times with 1×TBST,5 minutes each time.The following secondary antibodies were added: Goat anti-rabbit IgG (1:10 000), Goat anti-mouse IgG (1:10 000), incubated for 1.5 h at room temperature with 1×TBST membrane washing 5 times, 5 minutes each time.ECL reagent was added dropwise to develop color, and the gray values of the bands were analyzed using Image J software to β- actin was the internal reference protein, and the relative expression of the proteins were calculated.

2.9 mRNA expression of PSD-95 and SYP in the hippocampus was determined by quantitative real-time PCR (qRT-PCR)

Five mice were selected for qRT-PCR in each group.Total RNA was extracted and reverse transcribed to cDNA.Using cDNA as a template and adding corresponding primers, a 20 μL reaction system was prepared.And amplified in a PCR machine.Amplification was performed at 60-95 ℃ with primers for the target gene and primers for the internal reference gene, and dissociation curves were analyzed.Target genes were determined by qRT-PCR.Primer sequences are shown in Table1.Relative gene expression was analyzed by the2-△△CTmethod.

Tab1 Primer sequences of PSD-95、SYP and β-actin

2.10 Statistical processing

Statistical analysis of the experimental data was performed by SPSS 20.0 Metrology data were presented as, and comparisons between two groups were performed with an independent samples t-test when the data fit a normal distribution and the variances were homogeneous; When noncompliance, nonparametric tests were applied.P＜0.05 was considered statistically significant.

3.Results

3.1 Effects of CUMS combined with CRS on body weight in depression mice

There was no difference in body weight between the two groups before the experiment (P＞0.05).After modeling, the body weight of mice in both groups showed an increasing trend.Compared with normal group, the body weight of mice in model group were increased slowly, and the body weight of mice in model group was significantly lower than that in normal group every weekend from the first week of modeling (P＜0.01).As shown in Table 2.

Tab2 Changes of body weight of mice between the two group(n=20,)

Tab2 Changes of body weight of mice between the two group(n=20,)

Note: Compared with normal group, **P＜0.01.

Group Initial 1-Week 2-Week 3-Week 4-Week 5-Week 6-Week 7-Week normal group 21.09±0.25 22.21±0.23 23.25±0.25 24.62±0.23 25.17±0.26 25.42±0.23 27.47±0.25 27.56±0.26 model group 20.91±0.29 19.86±0.24**20.50±0.23**20.60±0.35**21.61±0.29**22.91±0.28**23.63±0.23**24.26±0.27**t 0.464 7.188 8.065 9.489 9.255 6.939 11.379 8.779 P 0.645 0.000 0.000 0.000 0.000 0.000 0.000 0.000

3.2 Effects of CUMS combined with CRS on Sucrose preference test in depression mice

There was no difference in the initial Sucrose preference rate between the two groups before the experiment (P＞0.05).At the 3rd week and 7th week during the modeling, the sucrose preference rate in model group was significantly decreased compared with that in normal group (P＜0.01).As shown in Table 3.

Tab3 Changes of Sucrose preference rate between the two group(%, n=20, )

Tab3 Changes of Sucrose preference rate between the two group(%, n=20, )

Note: Compared with normal group, **P＜0.01.

Group Initial 3-week 7-week normal group 62.09±1.70 66.92±3.14 65.08±2.35 model group 63.40±1.62 43.69±2.77**44.79±2.74**t-0.558 5.546 5.623 P 0.580 0.000 0.000

3.3 Effect of CUMS combined with CRS on Tail suspension test in depression mice

At the 3rd week and 7th week during the modeling, the immobile time of mice in model group was significantly increased than that in normal group (P＜0.01).As shown in Table 4.

Tab4 Changes of immobile time between the two group(s, n=20,)

Tab4 Changes of immobile time between the two group(s, n=20,)

Note: Compared with normal group, **P＜0.01.

Group 3-week 7-week normal group 102.06±10.69 100.47±4.02 model group 181.07±16.83**155.99±8.81**t-3.963 -5.734 P 0.000 0.000

3.4 Effect of CUMS combined with CRS on Open field test in depression mice

At the 3rd week and 7th week during the modeling, the total traveling distance in model group was significantly shorter than that in normal group (P＜0.05); the times of standing in model group was significantly decreased than that in normal group (P＜0.05 or P＜0.01); the cumulative duration of central area in model group was significantly shorter than that in normal group (P＜0.05 or P＜0.01);The velocity of motion in model group was not significantly different from that in normal group at the 3rd week (P＞0.05), and the velocity of motion in model group was significantly lower than that in normal group at the 7th week (P＜0.01).As shown in Table 5.

Tab5 Changes of total traveling distance, times of standing, cumulative duration of central area and velocity of motion of mice between the two group(n=20, )

Tab5 Changes of total traveling distance, times of standing, cumulative duration of central area and velocity of motion of mice between the two group(n=20, )

Note: Compared with normal group, *P＜0.05; **P＜0.01.

Groupnormal group total traveling distance (cm) times of standing cumulative duration of central area (s) velocity of motion(cm/s)3-week 7-week 3-week 7-week 3-week 7-week 3-week 7-week model group 2 789.68±129.46 3 874.18±260.05 23.05±2.80 25.40±2.57 132.63±10.11 53.63±8.34 9.30±0.43 13.73±0.68 Group 2 283.09±199.75*3 045.03±174.14*16.65±1.19*11.05±1.26**84.58±10.45**33.72±4.93*9.79±0.32 10.90±0.45**t 2.128 2.649 2.101 5.007 3.304 2.056 -0.915 3.450 P 0.041 0.012 0.046 0.000 0.002 0.048 0.336 0.002

3.5 Effects of CUMS combined with CRS on TNF-a content in hippocampus

At the end of the 7th week, the content of TNF-a in hippocampus in model group was significantly higher than that in normal group(P＜0.05).As shown in Table 6.

Tab6 Changes of TNF-a content in hippocampal tissue of mice between the two group()

Tab6 Changes of TNF-a content in hippocampal tissue of mice between the two group()

Note: Compared with normal group, *P＜0.05.

Group n TNF-a(pg/mL)normal group 5 247.41±18.21 model group 5 394.45±45.08*t--3.024 P-0.016

2.6 Effects of CUMS combined with CRS on Mean optical density (MOD) values of Iba-1 and GFAP in hippocampus

Immunohistochemical positive staining is defined as the expression of yellow or tan particles in the section.After 7 weeks of modeling,the MOD values of Iba-1 in hippocampal CA1, CA3 and DG regions in model group were significantly higher than those in normal group(P＜0.05 or P＜0.01).The MOD values of GFAP in hippocampal CA1, CA3 and DG regions in model group were significantly lower than those in normal group (P＜0.05 or P＜0.01).As shown in Table 7.

3.7 Effect of CUMS combined with CRS on protein expression of Iba-1, GFAP, PSD-95 and SYP in hippocampus

After 7 weeks of modeling, the protein level of iba-1 in hippocampus in model group was significantly increased compared with that in normal group (P＜0.05).The protein level of GFAP,PSD-95 and SYP in hippocampus in model group were significantly decreased compared with that in normal group (P＜0.01 or P＜0.05).As shown in Table 8.

Tab7Changes of mean optical density (MOD) values of Iba-1 and GFAP in hippocampal between the two group(n=5, )

Tab7Changes of mean optical density (MOD) values of Iba-1 and GFAP in hippocampal between the two group(n=5, )

Note: Compared with normal group, *P＜0.05; **P＜0.01.

Groupnormal group Iba-1 GFAP CA1 CA3 DG CA1 CA3 DG model group 0.19±0.02 0.20±0.01 0.18±0.02 0.29±0.02 0.28±0.03 0.28±0.02 Group 0.33±0.04*0.33±0.04*0.32±0.02**0.17±0.01**0.18±0.01*0.19±0.01**t-3.481 -3.186 -4.952 5.835 3.555 3.541 P 0.014 0.027 0.001 0.000 0.022 0.008

Fig1 Immunohistochemical images of Iba-1 and GFAP in each hippocampus region (400×)

Tab8 Changes of protein expression of Iba-1, GFAP, PSD-95 and SYP in hippocampal tissue between the two group(n=5, )

Tab8 Changes of protein expression of Iba-1, GFAP, PSD-95 and SYP in hippocampal tissue between the two group(n=5, )

Note: Compared with normal group, *P＜0.05; **P＜0.01.

Group Iba-1 GFAP PSD-95 SYN1 normal group 0.98±0.08 1.03±0.09 1.00±0.12 1.32±0.10 model group 1.82±0.27*0.64±0.08*0.50±0.05**0.90±0.09*t-2.998 3.201 3.751 2.985 P 0.032 0.013 0.006 0.017

Fig2 Western blotting pictures of Iba-1, GFAP, PSD-95 and SYP in the hippocampal tissue between the two group

3.8 Effect of CUMS combined with CRS on mRNA expression of PSD-95 and SYP in hippocampus

After 7 weeks of modeling, the mRNA expression of PSD-95 and SYP in hippocampus in model group were significantly decreased compared with those in normal group (P＜0.05).

Tab9 mRNA expression changes of PSD-95 and SYP in hippocampal tissues between the two group(n=5, )

Tab9 mRNA expression changes of PSD-95 and SYP in hippocampal tissues between the two group(n=5, )

Note: Compared with normal group, *P＜0.05.

Group PSD-95 SYN1 normal group 1.16±0.06 3.12±0.65 model group 1.00*1.00*t 2.810 3.261 P 0.048 0.031

4.Discussion

Currently, there are more than 20 methods for laboratory preparation of animal models of depression, comparing common chronic unpredictable mild stress (CUMS), chronic restraint stress(CRS), social defeat stress (SDS), and so on.The preparation of animal models of depression by methods of chronic stress is well recognized, but the behavioral alterations and associated pathological changes in animal models of depression prepared for this class of methods are not fully defined.Chronic stress can cause mood changes, behavioral changes, and altered energy metabolism,which can then lead to the development of depression.But different stressors (physiology and psychological stress) can cause different physiological responses and activate different neural regions.CUMS and CRS, which belong to chronic stress, are widely used in animal modeling of depression.The CUMS model was first proposed by Katz et al [9] and developed with further refinements by Willner et al[10].The method is to inflict the occurrence of depression by administering to experimental animals various stressors, generally belonging to physiology stress.First proposed by Renaud[11],the CRS model refers to the generation of depression that can be well modeled by confining rodents to a specific space for a while,dominated by psychological stress.Because depressed patients tend to have mixed somatic and psychological effects in the clinic,none of the animal models prepared by the application of CUMS or CRS alone can truly reflect the clinical reality of patients.Therefore, based on the above reasons and the accumulation of previous experimental experience in our subject group, to establish a more suitable clinically practical mouse model of depression, we used CUMS combined with CRS to construct a mouse model of depression, and then the body weight, corresponding behavioral changes, and pathological changes were investigated.

Body weight was able to reflect the growth performance and the state of the body of the experimental animals during modeling procedure.Studies have shown that animals experience loss of body mass during the construction of chronic stress models [12-14].The experimental results showed that, since the 1st week of modeling,the body weight of the mice in model group on each weekend was significantly lower than that in normal group, and through the data, it can be observed that the mice in model group grew relatively slowly compared with the normal group, consistent with the manifestation of reduced body weight in patients with clinical depression.

Behavioral tests such as sugar water preference, hanging tail, and open field tests are currently important in evaluating depression models.Sucrose preference rate can reflect the degree of anhedonia in rodents; Immobility time of TST can reflect the level of despair of rodents; And, OFT reflects the autonomic behavior of mice in an unfamiliar environment, their exploratory behavior, and tension.In SPT, after 3 weeks after CUMS+CRS, the mice in model group showed a significantly lower sucrose preference rate than those of the normal group; After 7 weeks of modeling, the sucrose preference rate in model group was also significantly lower than that in the normal group (P＜0.01).The above results indicate that modeling for 3 and 7 weeks, show no significant changes in anhedonia.In the TST,the immobility time of mice in model group was significantly higher than that in normal group after CUMS + CRS modeling for 3 weeks;After 7 weeks of modeling, the immobility time in model mice was also significantly higher than that in normal group (P＜0.01).The above results indicated that the mice which were modeled for 3 and 7 weeks, did not show significant changes in the degree of despair.In OFT, after 3 weeks of CUMS+CRS, the total traveling distance(P＜0.05), times of standing (P＜0.05), and cumulative duration of central area (P＜0.01) in model group were significantly lower than those in normal group, whereas no significant difference was observed in velocity of motion (P＞0.05); After 7 weeks of modeling,the total traveling distance (P＜0.05), ttimes of standing (P＜0.01),cumulative duration of central area (P＜0.05), and velocity of motion(P＜0.01) in model group were significantly lower than those in normal group.The results described above indicate that mice that had been modeled for 3 and 7 weeks had significant alterations in velocity of motion and that increasing the duration of modeling led to significantly slower velocity of motion.A comprehensive analysis of the above behavioral results found that the mice in model group for 3 and 7 weeks had some differences in behavioral performance,which was mainly reflected in the velocity of motion.However, in clinical studies, the degree of depression in patients with depression was also classified as mild and severe.Illustrates the ability to cause broader and more severe effects on the behavior of laboratory animals as the duration of stress increases.Therefore, the mice were subjected to 3 weeks of modeling by CUMS combined with the CRS method, a successful mouse model of depression could be established.However, by extending the modeling period to 7 weeks,the mice were allowed to show some differences in velocity of motion, making the depression-like behavior more significantly.

Neuroinflammation, a complex immune response in the central nervous system (CNS), is caused by dysregulated synthesis and(or) release of inflammatory cytokines in the central or peripheral after body injury[15].Studies in recent years[16,17] have shown that“neuroinflammation”is one of the important mechanisms leading to the occurrence of depression, and the level of body inflammation in depression patients is positively correlated with the degree of improvement of depressive symptoms[18].The CNS contains a large number of glial cells, mainly including microglia (MG), astrocytes(AS) and others.Modern studies[19,20] have confirmed that glial cells are widely involved in depression’s physiological and pathological activities.Microglia serve as the innate immune regulatory cells of the CNS, and their activation toward a proinflammatory type is the most predominant manifestation of neuroinflammation.Because Iba-1 can specifically bind to microglia, it is often used as a marker to identify microglial activation, and when microglia are activated, their morphology changes, and the expression of Iba-1 is significantly upregulated[21].Astrocytes are the most abundant class of glial cells in the CNS.When the center is in an inflammatory state, it can lead to dysfunction of AS and result in the disturbance of the glutamate-glutamine cycle in the brain, which increases the risk of developing depression[22].Glial fibrillary acidic protein(GFAP) is a specific marker for as and reflects the amount of as.The hippocampus plays an important role in learning, memory, emotion,and visceral activity, and is also one of the important brain regions in the pathological changes of depression[23], the hippocampus is mainly divided into three subregions, the CA1 region is mainly responsible for the time-related information processing; The CA3 region is primarily responsible for space-related information processing and the formation of short-term memory; The DG region is primarily responsible for information input to the hippocampus.Numerous studies have shown that long-term chronic stress causes pathological changes in various subregions of the hippocampus.The experimental results showed that the amount of TNF-a in the hippocampus of the model mice was significantly increased compared with that in the normal group (P＜0.05); MOD values of Iba-1 in hippocampal CA1, CA3, and DG regions of the model mice were significantly increased compared with those of the normal group (P＜0.05 or P＜0.01; MOD values of GFAP were significantly lower (P＜0.05 or P＜0.01) than those of the normal group; The protein expression of Iba-1 in the hippocampus of the model mice was significantly increased compared with that in the normal group (P＜0.05); The protein expression of GFAP was significantly decreased (P＜0.05) compared with the normal group.The above results indicated that CUMS combined with CRS could lead to the appearance of inflammation in the hippocampus of mice, which was able to promote the over-activation of microglia and induce the apoptosis of astrocytes.

Glial cells play an important role in the regulation of synaptic plasticity[24].And whether synaptic development is normal or not,especially whether synaptic plasticity is normal is closely related to the occurrence and development of depression[25].There are multiple ways in which glial cells can regulate synaptic plasticity.Among them, are the mechanisms of synaptic pruning by microglia,a key regulator of synaptic plasticity during development and adulthood; Astrocytes integrate and process synaptic information,control synaptic transmission, and plasticity, and are important effectors in the regulation of synaptic plasticity[26].When microglia as well as astrocytes are damaged, it can lead to reduced synaptic plasticity[27,28].Postsynaptic density-95 (PSD-95) and synaptophysin(SYP) are important molecular markers associated with synaptic plasticity.Among them, PSD-95 is one of the most abundant proteins in the postsynaptic dense matter, and SYP is a presynaptic membrane vesicle fusion protein, both of which play important roles in the process of synaptic plasticity regulation[29,30].When synaptic plasticity is reduced, it can lead to deficits in hippocampal function and loss of normal control of mood and emotion in depressed patients[31], while abnormalities in synaptic morphology or structure that lead to disturbances in synaptic function are one of the important pathogenesis of depression.The results of this study showed that the relative expression of PSD-95 and SYP proteins in the hippocampus of the model mice decreased significantly, and the relative expression of PSD-95 and SYP mRNA decreased significantly (P＜0.05 or P＜0.01).The above results suggest that CUMS combined with CRS can lead to decreased synaptic plasticity in depressed mice.

Considering the body weight, behavioral performance, and the related changes of inflammation levels in the hippocampus,microglia, astrocytes, and synaptic plasticity-related proteins in the depression mice based on the experimental results, in this study,we conclude that the hippocampus of depressed mice prepared by CUMS combined with CRS produces neuroinflammation, which leads to microglial overactivation and astrocyte loss in number, It further affects the regulatory function of glial cells on synapses,causing reduced synaptic plasticity, which leads to the occurrence of depression.

Author’s contribution:

Conceived and designed the experiments, conducted molecular experiment, analyzed the data and writed the article: Li Xin.Carried out the animal experiment: Xin Li, Wang Qiong-ying, Ma Zhao-tian,Sun Hong-hao.Xue Yu Participated in the guidance of molecular experiment:.Guide experimental design and article modification:Ren Xiao-qiao.

All authors declare no conflict of interest.