Electroacupuncture promotes BDNF-dependent neurogenesis via microglial reprogramming in a chronic stress model methods
Aim. Evidence-backed execution summary for Electroacupuncture promotes BDNF-dependent neurogenesis via microglial reprogramming in a chronic stress model methods from Electroacupuncture promotes BDNF-dependent neurogenesis via microglial reprogramming in a chronic stress model.
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mouse
Subject model for the experiment.
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- confirm full cohort details in the source paper
Experiment 3
reagent used in the protocol.
- Use
- To explore the role of BDNF signaling in the therapeutic effects of EA, mice were allocated to four groups: Control (n = 8); CUMS (n = 8); CUMS/EA (n = 8); CUMS/EA/ANA-12 (n = 8). Mice in the CUMS/EA/ANA-12 group were administered ANA-12 (MCE, 0.5 mg/kg) [ ] via i...
EA enhances AHN and ameliorates depressive-like behaviors in a microglia-dependent manner
reagent used in the protocol.
- Use
- To investigate the necessity of microglia for the beneficial effects of EA on neurogenesis and depressive-like behaviors, we depleted microglia using PLX5622 prior to EA treatment (Fig. A). Three weeks of PLX5622 treatment effectively eliminated approximately 90% of microglia (Fig. S1A-B). We first asses...
EA enhances BDNF signaling through convergent pathways in CUMS-exposed mice
reagent used in the protocol.
- Use
- To elucidate the molecular mechanisms through which EA promotes neurogenesis, we systematically examined key pathways both upstream and downstream of BDNF signaling (Fig. A). At the transcriptional level, one-way ANOVA indicated that CUMS exposure significantly decreased the mRNA expression of Bdnf (F =&...
Chronic unpredictable mild stress (CUMS) procedure
reagent used in the protocol.
- Use
- The CUMS protocol was optimized from an established rodent depression model [ ] to induce behavioral and neurobiological deficits. Stressors were administered in a randomized daily sequence and included: 24-h continuous light exposure; 12-h water/food restriction; 45° cage inclination for 12 h; 4-h confine...
Sucrose preference test (SPT)
reagent used in the protocol.
- Use
- Sucrose preference was assessed using a two-bottle free-choice paradigm according to the CUMS protocol. After 24 h of water and food deprivation, mice were provided with two separate bottles of the same size: one containing a 1% sucrose solution and the other containing tap water. The containers were weighed an...
Real-time quantitative fluorescence PCR (qRT-PCR)
reagent used in the protocol.
- Use
- Total RNA was extracted from brain lysate using FastPure tissue total RNA isolation kit (Vazyme, #RC112-01) following the protocol. First-strand cDNA synthesis was performed with 1 µg total RNA in a 20 µL reaction volume using HiScript IV All-in-One Ultra RT SuperMix for qPCR Reagent (vazyme, Cat# R43...
Immunofluorescence
reagent used in the protocol.
- Use
- The hippocampus was separated and immersed in 4% paraformaldehyde for 12 h before being embedded in paraffin and sectioned, and immersed in 30% sucrose solution until the tissue was sunken to the bottom of the tube. Each sample was cut into a 35 µm thick section (RWD, #FS800). The sections were incuba...
Western blotting
reagent used in the protocol.
- Use
- Cerebral tissue samples were mechanically lysed in ice-cold RIPA buffer (Thermo Fisher, #89,900) containing protease/phosphatase inhibitors (Thermo Fisher, #78,440). Protein quantification was performed using a bicinchoninic acid (BCA) assay kit (Thermo Fisher, #23,225), with equalized lysates resolved on 4-12...
Methods
Male C57BL/6 J mice were subjected to chronic unpredictable mild stress (CUMS) for three weeks. Following this period, the mice were divided into groups receiving either EA at the Yintang (GV29) and Baihui (GV20) acupoints, imipramine (IMI) as a positive control, or no treatment (vehicle control) for an additio...
- Use
- Male C57BL/6 J mice were subjected to chronic unpredictable mild stress (CUMS) for three weeks. Following this period, the mice were divided into groups receiving either EA at the Yintang (GV29) and Baihui (GV20) acupoints, imipramine (IMI) as a positive control, or no treatment (vehicle control) for an additio...
EA treatment diminishes anxiety-like behaviors in CUMS-exposed mice
The anxiolytic effects of EA and IMI were evaluated using the open field test (OFT) and the elevated plus maze (EPM) in mice subjected to CUMS. In the OFT, no significant differences in total distance traveled were observed among groups (Fig. A-B), indicating that locomotor activity remained unaffected....
- Use
- The anxiolytic effects of EA and IMI were evaluated using the open field test (OFT) and the elevated plus maze (EPM) in mice subjected to CUMS. In the OFT, no significant differences in total distance traveled were observed among groups (Fig. A-B), indicating that locomotor activity remained unaffected....
ANA-12 reversing EA improves anxiodepressive behavior in CUMS mice
To determine whether BDNF-TrkB signaling is essential for the sustained antidepressant effects of EA at the behavioral level, ANA-12 was administered to inhibit TrkB prior to each EA session throughout the three-week CUMS and treatment period (Fig. A). A one-way ANOVA on body weight showed a significant...
- Use
- To determine whether BDNF-TrkB signaling is essential for the sustained antidepressant effects of EA at the behavioral level, ANA-12 was administered to inhibit TrkB prior to each EA session throughout the three-week CUMS and treatment period (Fig. A). A one-way ANOVA on body weight showed a significant...
EA intervention
Following three-week of CUMS induction, mice received standardized EA therapy during the light cycle (09:00-11:00) over a three-week regimen (5 sessions/week). EA was applied at Yintang (GV29; located in the midline depression between the nasal apex and the superior labial margin) and Baihui (GV20; located at...
- Use
- Following three-week of CUMS induction, mice received standardized EA therapy during the light cycle (09:00-11:00) over a three-week regimen (5 sessions/week). EA was applied at Yintang (GV29; located in the midline depression between the nasal apex and the superior labial margin) and Baihui (GV20; located at...
Behavioral testing timeline and order
Prior to behavioral testing, all mice were handled for three consecutive days to reduce handling stress. On each test day, mice were transferred to the testing room 1 h in advance for acclimation. To minimize stress carryover effects, tests were administered in a sequence progressing from the least to the most...
- Use
- Prior to behavioral testing, all mice were handled for three consecutive days to reduce handling stress. On each test day, mice were transferred to the testing room 1 h in advance for acclimation. To minimize stress carryover effects, tests were administered in a sequence progressing from the least to the most...
Tail suspension test (TST)
The TST is a desperation behaviors test that assesses the immobility time of mice under uninvited conditions. The tail of mouse was suspended at a height of 20-25 cm by wrapping a piece of tape around the tail, 1 cm from the tip of tail. Each mouse was suspended with the end of its tail for 6 mi...
- Use
- The TST is a desperation behaviors test that assesses the immobility time of mice under uninvited conditions. The tail of mouse was suspended at a height of 20-25 cm by wrapping a piece of tape around the tail, 1 cm from the tip of tail. Each mouse was suspended with the end of its tail for 6 mi...
Force swimming test (FST)
The FST assesses the degree of desperation behaviors in an inescapable space. Each mouse was filmed in a transparent acrylic cylinder (High: 25 cm, Depth: 10 cm) filled with 800 mL water (23-25 °C). All trials were filmed by video camera (VisuTrack, shanghai, XR-VT). Immobility was def...
- Use
- The FST assesses the degree of desperation behaviors in an inescapable space. Each mouse was filmed in a transparent acrylic cylinder (High: 25 cm, Depth: 10 cm) filled with 800 mL water (23-25 °C). All trials were filmed by video camera (VisuTrack, shanghai, XR-VT). Immobility was def...
Open field test (OFT)
The OFT assesses general locomotor activity and anxiety-like behavior. Locomotor activity was recorded for 5 min in a white, rectangular open-field arena (50 × 50 × 50 cm) using video tracking software (VisuTrack, Shanghai, XR-VT). The total distance traveled, number of zo...
- Use
- The OFT assesses general locomotor activity and anxiety-like behavior. Locomotor activity was recorded for 5 min in a white, rectangular open-field arena (50 × 50 × 50 cm) using video tracking software (VisuTrack, Shanghai, XR-VT). The total distance traveled, number of zo...
Data analysis and statistics
Software used for acquisition, scoring, statistics, or reporting.
- Use
- All data analyses were performed in a blinded manner. Statistical analyses were conducted using GraphPad Prism. Data are presented as the mean ± SEM, and the number of independent replicates (n) is specified in the figure legends. The normality of data distribution was assessed using the D'Ago...
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EA enhances AHN and ameliorates depressive-like behaviors in a microglia-dependent manner
To investigate the necessity of microglia for the beneficial effects of EA on neurogenesis and depressive-like behaviors, we depleted microglia using PLX5622 prior to EA treatment (Fig. A). Three weeks of PLX5622 treatment effectively eliminated approximately 90% of microglia (Fig. S1A-B). We first assessed hippocampal neurogenesis via DCX immunofluorescence in the DG (Fig. B). A one-way ANOVA revealed a significant effect of treatment on the number of DCX⁺ cells (F = 31.18, p < 0.001). Post hoc analysis using Tukey's test confirmed that CUMS exposure significantly reduced the number of DCX⁺ cells compared to control (p < 0.001). EA treatment effectively reversed this CUMS-induced decrease (p < 0.001). Notably, this pro-neurogenic effect of EA was abolished in mice receiving PLX5622, as the CUMS/EA/PLX...
Limitations of the study
Although our study delineates a microglia-dependent mechanism through which EA enhances BDNF signaling and promotes neurogenesis, several key mechanistic questions remain. First, a potential limitation lies in our behavioral testing paradigm. While the FST and TST were conducted on separate days, using both tests in the same cohort carries a risk of mutual influence. This is because both tests measure a similar form of "passive coping" behavior. Second, although our data firmly establish that microglia are necessary for the BDNF-mediated effects of EA, we do not definitively distinguish between two plausible cellular mechanisms: (1) that reprogrammed microglia directly upregulate and release BDNF, or (2) that they create a permissive environment (e.g., via anti-inflammatory cytokines) which in turn enables neurons or astrocytes to become the primary source of BDNF. Elucida...
Experiment 1
To investigate whether EA improves depressive-like behaviors in CUMS mice, mice were randomly assigned to five groups: Control (n = 8); CUMS (n = 10); CUMS/EA (n = 9); CUMS/sham (n = 8); CUMS/IMI (n = 9). Pharmacological intervention involved daily intraperitoneal injections of imipramine (IMI, Sigma-Aldrich, # 113-52-0, 10 mg/kg), following established protocols with dose optimization [ ].
Behavioral testing timeline and order
Prior to behavioral testing, all mice were handled for three consecutive days to reduce handling stress. On each test day, mice were transferred to the testing room 1 h in advance for acclimation. To minimize stress carryover effects, tests were administered in a sequence progressing from the least to the most aversive paradigm: sucrose preference test (SPT) → open field test (OFT) → elevated plus maze (EPM) → tail suspension test (TST) → forced swim test (FST). A minimum interval of 24 h was maintained between any two tests, a duration established in the literature as sufficient for stress hormone levels to return to baseline [ ].
Tail suspension test (TST)
The TST is a desperation behaviors test that assesses the immobility time of mice under uninvited conditions. The tail of mouse was suspended at a height of 20-25 cm by wrapping a piece of tape around the tail, 1 cm from the tip of tail. Each mouse was suspended with the end of its tail for 6 min in a dark box with little background. Duration of immobility, recorded by a video camera (VisuTrack, shanghai, XR-VT), was monitored during the last 4 min of suspension. In addition, mouse was ensured to be undisturbed throughout the test.
Immunofluorescence
The hippocampus was separated and immersed in 4% paraformaldehyde for 12 h before being embedded in paraffin and sectioned, and immersed in 30% sucrose solution until the tissue was sunken to the bottom of the tube. Each sample was cut into a 35 µm thick section (RWD, #FS800). The sections were incubated in 0.01 M phosphate-buffer saline (PBS), washed with PBS Tween-20 (PBST) for three times and blocked with 5% normal donkey serum in 0.05% PBST for 2 h at room temperature, then the sections were incubated in primary antibodies overnight. The secondary antibody incubated for 2 h at room temperature. The primary antibody and second antibody are detailed in Additional file Table S10.
Cell culture and drug treatment
BV2 microglial cells, kindly provided by Professor Zili You from the University of Electronic Science and Technology of China, and cultured in DMEM medium (Gibco, USA) containing 10% fetal bovine serum (FBS, Gibco) under 37 °C and 5% CO 2. BV2 cells were treated with the PKA inhibitor H89 (5 µM; MedChemExpress, HY-15979) for 5 h. The dose for H89 was based on previous studies [ ].
Golgi staining
Golgi staining was performed using the FD Rapid Golgi Stain™ kit (FD Neuro Technologies, PK401) according to the manufacturer's instructions. In brief, freshly dissected mouse brains were immersed in an impregnation solution (a mixture of equal volumes of Solutions A and B) and stored in the dark at room temperature for 2 weeks. The brains were then transferred into Solution C and stored under the same conditions for an additional 72 h. Subsequently, the impregnated tissues were sectioned coronally at a thickness of 100 µm using a freezing microtome (Leica CM1950, Germany). The sections were mounted onto gelatin-coated slides with Solution C and subsequently stained by applying Solutions D and E according to the kit protocol. For each mouse, 13-20 dendritic segments were randomly selected for spine density quantification. Dendritic spines were cou...
Measurement outputs
What raw and processed outputs should exist?
Male C57BL/6 J mice were subjected to chronic unpredictable mild stress (CUMS) for three weeks. Following this period, the mice were divided into groups receiving either EA...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
The anxiolytic effects of EA and IMI were evaluated using the open field test (OFT) and the elevated plus maze (EPM) in mice subjected to CUMS. In the OFT, no significant differ...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Emerging evidence indicates that hippocampal neurogenesis enhances stress resilience [ ]. Thus, we investigated whether the antidepressant effect of EA is associated with the pr...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
A growing body of research suggested that depression was often accompanied by microglial overactivation [ ]. Given that microglia play a critical role in shaping the neurogenic...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Analysis plan
How should the outputs become interpretable results?
Acquisition
Collect the raw assay or blot output and retain identifiers for each sample and experimental group.
inferred from protocolPreprocessing / cleaning
The anxiolytic effects of EA and IMI were evaluated using the open field test (OFT) and the elevated plus maze (EPM) in mice subjected to CUMS.
from paperScoring or quantification
Quantify the primary readouts for this experiment: Male C57BL/6 J mice were subjected to chronic unpredictable mild stress (CUMS) for three weeks. Following this period, the mice were divided into groups receiving either EA...; The anxiolytic effects of EA and IMI were evaluated using the open field test (OFT) and the elevated plus maze (EPM) in mice subjected to CUMS. In the OFT, no significant differ...; Emerging evidence indicates that hippocampal neurogenesis enhances stress resilience [ ]. Thus, we investigated whether the antidepressant effect of EA is associated with the pr...; A growing body of research suggested that depression was often accompanied by microglial overactivation [ ]. Given that microglia play a critical role in shaping the neurogenic....
from paperNormalization
Normalize expression or signal values against the stated control or loading reference before comparing groups.
inferred from protocolStatistical comparison
The anxiolytic effects of EA and IMI were evaluated using the open field test (OFT) and the elevated plus maze (EPM) in mice subjected to CUMS. In the OFT, no significant differ...; Emerging evidence indicates that hippocampal neurogenesis enhances stress resilience [ ]. Thus, we investigated whether the antidepressant effect of EA is associated with the pr...; A growing body of research suggested that depression was often accompanied by microglial overactivation [ ]. Given that microglia play a critical role in shaping the neurogenic...; To investigate the necessity of microglia for the beneficial effects of EA on neurogenesis and depressive-like behaviors, we depleted microglia using PLX5622 prior to EA treatme...
from paperReporting output
Report representative outputs alongside summary comparisons for Male C57BL/6 J mice were subjected to chronic unpredictable mild stress (CUMS) for three weeks. Following this period, the mice were divided into groups receiving either EA..., The anxiolytic effects of EA and IMI were evaluated using the open field test (OFT) and the elevated plus maze (EPM) in mice subjected to CUMS. In the OFT, no significant differ..., Emerging evidence indicates that hippocampal neurogenesis enhances stress resilience [ ]. Thus, we investigated whether the antidepressant effect of EA is associated with the pr..., A growing body of research suggested that depression was often accompanied by microglial overactivation [ ]. Given that microglia play a critical role in shaping the neurogenic....
inferred from protocolStructured statistical methods
The anxiolytic effects of EA and IMI were evaluated using the open field test (OFT) and the elevated plus maze (EPM) in mice subjected to CUMS. In the OFT, no significant differ...; Emerging evidence indicates that hippocampal neurogenesis enhances stress resilience [ ]. Thus, we investigated whether the antidepressant effect of EA is associated with the pr...; A growing body of research suggested that depression was often accompanied by microglial overactivation [ ]. Given that microglia play a critical role in shaping the neurogenic...; To investigate the necessity of microglia for the beneficial effects of EA on neurogenesis and depressive-like behaviors, we depleted microglia using PLX5622 prior to EA treatme...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (8)
To investigate the necessity of microglia for the beneficial effects of EA on neurogenesis and depressive-like behaviors, we depleted microglia using PLX5622 prior to EA treatment (Fig. A). Three weeks of PLX5622 treatment effectively eliminated approximately 90% of microglia (Fig. S1A-B). We first assessed hippocampal neurogenesis via DCX immunofluorescence in the DG (Fig. B). A one-way ANOVA revealed a significant effect of treatment on the number of DCX⁺ cells (F = 31.18, p < 0.001). Post hoc analysis using Tukey's test confirmed that CUMS exposure significantly reduced the number of DCX⁺ cells compared to control (p < 0.001). EA treatment effectively reversed this CUMS-induced decrease (p < 0.001). Notably, this pro-neurogenic effect of EA was abolished in mice receiving PLX5622, as the CUMS/EA/PLX group exhibited a significant reduction compared to the CUMS/EA group (p < 0.001) (Fig. D). Western blot analysis corroborated these findings at the protein level. A one-way ANOVA indicated a significant effect of treatment on DCX protein expression (F = 8.814, p...
Although our study delineates a microglia-dependent mechanism through which EA enhances BDNF signaling and promotes neurogenesis, several key mechanistic questions remain. First, a potential limitation lies in our behavioral testing paradigm. While the FST and TST were conducted on separate days, using both tests in the same cohort carries a risk of mutual influence. This is because both tests measure a similar form of "passive coping" behavior. Second, although our data firmly establish that microglia are necessary for the BDNF-mediated effects of EA, we do not definitively distinguish between two plausible cellular mechanisms: (1) that reprogrammed microglia directly upregulate and release BDNF, or (2) that they create a permissive environment (e.g., via anti-inflammatory cytokines) which in turn enables neurons or astrocytes to become the primary source of BDNF. Elucidating this precise cellular crosstalk and the integrated molecular circuitry will be a critical focus of our subsequent research.
To investigate whether EA improves depressive-like behaviors in CUMS mice, mice were randomly assigned to five groups: Control (n = 8); CUMS (n = 10); CUMS/EA (n = 9); CUMS/sham (n = 8); CUMS/IMI (n = 9). Pharmacological intervention involved daily intraperitoneal injections of imipramine (IMI, Sigma-Aldrich, # 113-52-0, 10 mg/kg), following established protocols with dose optimization [ ].
Prior to behavioral testing, all mice were handled for three consecutive days to reduce handling stress. On each test day, mice were transferred to the testing room 1 h in advance for acclimation. To minimize stress carryover effects, tests were administered in a sequence progressing from the least to the most aversive paradigm: sucrose preference test (SPT) → open field test (OFT) → elevated plus maze (EPM) → tail suspension test (TST) → forced swim test (FST). A minimum interval of 24 h was maintained between any two tests, a duration established in the literature as sufficient for stress hormone levels to return to baseline [ ].
The TST is a desperation behaviors test that assesses the immobility time of mice under uninvited conditions. The tail of mouse was suspended at a height of 20-25 cm by wrapping a piece of tape around the tail, 1 cm from the tip of tail. Each mouse was suspended with the end of its tail for 6 min in a dark box with little background. Duration of immobility, recorded by a video camera (VisuTrack, shanghai, XR-VT), was monitored during the last 4 min of suspension. In addition, mouse was ensured to be undisturbed throughout the test.
The hippocampus was separated and immersed in 4% paraformaldehyde for 12 h before being embedded in paraffin and sectioned, and immersed in 30% sucrose solution until the tissue was sunken to the bottom of the tube. Each sample was cut into a 35 µm thick section (RWD, #FS800). The sections were incubated in 0.01 M phosphate-buffer saline (PBS), washed with PBS Tween-20 (PBST) for three times and blocked with 5% normal donkey serum in 0.05% PBST for 2 h at room temperature, then the sections were incubated in primary antibodies overnight. The secondary antibody incubated for 2 h at room temperature. The primary antibody and second antibody are detailed in Additional file Table S10.
BV2 microglial cells, kindly provided by Professor Zili You from the University of Electronic Science and Technology of China, and cultured in DMEM medium (Gibco, USA) containing 10% fetal bovine serum (FBS, Gibco) under 37 °C and 5% CO 2. BV2 cells were treated with the PKA inhibitor H89 (5 µM; MedChemExpress, HY-15979) for 5 h. The dose for H89 was based on previous studies [ ].
Golgi staining was performed using the FD Rapid Golgi Stain™ kit (FD Neuro Technologies, PK401) according to the manufacturer's instructions. In brief, freshly dissected mouse brains were immersed in an impregnation solution (a mixture of equal volumes of Solutions A and B) and stored in the dark at room temperature for 2 weeks. The brains were then transferred into Solution C and stored under the same conditions for an additional 72 h. Subsequently, the impregnated tissues were sectioned coronally at a thickness of 100 µm using a freezing microtome (Leica CM1950, Germany). The sections were mounted onto gelatin-coated slides with Solution C and subsequently stained by applying Solutions D and E according to the kit protocol. For each mouse, 13-20 dendritic segments were randomly selected for spine density quantification. Dendritic spines were counted and analyzed using ImageJ software.
Machine-readable layer
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"name": "Electroacupuncture promotes BDNF-dependent neurogenesis via microglial reprogramming in a chronic stress model methods",
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{
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"name": "EA enhances AHN and ameliorates depressive-like behaviors in a microglia-dependent manner",
"text": "To investigate the necessity of microglia for the beneficial effects of EA on neurogenesis and depressive-like behaviors, we depleted microglia using PLX5622 prior to EA treatment (Fig. A). Three weeks of PLX5622 treatment effectively eliminated approximately 90% of microglia (Fig. S1A-B). We first assessed hippocampal neurogenesis via DCX immunofluorescence in the DG (Fig. B). A one-way ANOVA revealed a significant effect of treatment on the number of DCX⁺ cells (F = 31.18, p < 0.001). Post hoc analysis using Tukey's test confirmed that CUMS exposure significantly reduced the number of DCX⁺ cells compared to control (p < 0.001). EA treatment effectively reversed this CUMS-induced decrease (p < 0.001). Notably, this pro-neurogenic effect of EA was abolished in mice receiving PLX5622, as the CUMS/EA/PLX..."
},
{
"@type": "HowToStep",
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"name": "Limitations of the study",
"text": "Although our study delineates a microglia-dependent mechanism through which EA enhances BDNF signaling and promotes neurogenesis, several key mechanistic questions remain. First, a potential limitation lies in our behavioral testing paradigm. While the FST and TST were conducted on separate days, using both tests in the same cohort carries a risk of mutual influence. This is because both tests measure a similar form of \"passive coping\" behavior. Second, although our data firmly establish that microglia are necessary for the BDNF-mediated effects of EA, we do not definitively distinguish between two plausible cellular mechanisms: (1) that reprogrammed microglia directly upregulate and release BDNF, or (2) that they create a permissive environment (e.g., via anti-inflammatory cytokines) which in turn enables neurons or astrocytes to become the primary source of BDNF. Elucida..."
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"text": "To investigate whether EA improves depressive-like behaviors in CUMS mice, mice were randomly assigned to five groups: Control (n = 8); CUMS (n = 10); CUMS/EA (n = 9); CUMS/sham (n = 8); CUMS/IMI (n = 9). Pharmacological intervention involved daily intraperitoneal injections of imipramine (IMI, Sigma-Aldrich, # 113-52-0, 10 mg/kg), following established protocols with dose optimization [ ]."
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"name": "Immunofluorescence",
"text": "The hippocampus was separated and immersed in 4% paraformaldehyde for 12 h before being embedded in paraffin and sectioned, and immersed in 30% sucrose solution until the tissue was sunken to the bottom of the tube. Each sample was cut into a 35 µm thick section (RWD, #FS800). The sections were incubated in 0.01 M phosphate-buffer saline (PBS), washed with PBS Tween-20 (PBST) for three times and blocked with 5% normal donkey serum in 0.05% PBST for 2 h at room temperature, then the sections were incubated in primary antibodies overnight. The secondary antibody incubated for 2 h at room temperature. The primary antibody and second antibody are detailed in Additional file Table S10."
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"text": "BV2 microglial cells, kindly provided by Professor Zili You from the University of Electronic Science and Technology of China, and cultured in DMEM medium (Gibco, USA) containing 10% fetal bovine serum (FBS, Gibco) under 37 °C and 5% CO 2. BV2 cells were treated with the PKA inhibitor H89 (5 µM; MedChemExpress, HY-15979) for 5 h. The dose for H89 was based on previous studies [ ]."
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