Electroacupuncture promotes BDNF-dependent neurogenesis via microglial reprogramming in a chronic stress model methods
Show snapshot details
On this page
This experiment, in seven questions
Jump straight to the part of the recipe you need. Data and provenance labels stay close to the action they support.
Shopping and prep list
What do I need before I start?
Biological model pending
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
Before you run
What should be confirmed before execution?
First confirmation
Species or subject information is missing.
Confirm before execution
Protocol does not list structured equipment.
Confirm before execution
This page shows passages quoted from the paper. Equipment, materials, times, and temperatures are hidden until the page is rebuilt from the full paper and checked by a reviewer. Check the paper before using this method.
Confirm before execution
Open the source paper before finalizing run-specific details.
Procurement checkpoint
Use source-stated vendors where present. Treat mapped products as sourcing options unless the page marks an exact source match.
Open quote workflowStep-by-step procedure
What do I do, in order?
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?
Microscopy-based tissue or cell-state imaging
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
Analysis plan
How should the outputs become interpretable results?
Acquisition
Collect raw experimental outputs with enough metadata to preserve sample identity, condition, and timing.
inferred from protocolPreprocessing / cleaning
Review raw outputs for quality, remove unusable captures, and organize the data into a comparison-ready table or image set.
inferred from protocolScoring or quantification
Quantify the primary readouts for this experiment: Microscopy-based tissue or cell-state imaging.
from paperStatistical comparison
Statistical method not yet structured for this page.
inferred from protocolReporting output
Report representative outputs alongside summary comparisons for Microscopy-based tissue or cell-state imaging.
inferred from protocolSource 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
[
{
"@context": "https://schema.org",
"@type": "HowTo",
"name": "Electroacupuncture promotes BDNF-dependent neurogenesis via microglial reprogramming in a chronic stress model methods",
"description": "Protocol for Electroacupuncture promotes BDNF-dependent neurogenesis via microglial reprogramming in a chronic stress model methods",
"step": [
{
"@type": "HowToStep",
"position": 1,
"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",
"position": 2,
"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..."
},
{
"@type": "HowToStep",
"position": 3,
"name": "Experiment 1",
"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 [ ]."
},
{
"@type": "HowToStep",
"position": 4,
"name": "Behavioral testing timeline and order",
"text": "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 [ ]."
},
{
"@type": "HowToStep",
"position": 5,
"name": "Tail suspension test (TST)",
"text": "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."
},
{
"@type": "HowToStep",
"position": 6,
"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."
},
{
"@type": "HowToStep",
"position": 7,
"name": "Cell culture and drug treatment",
"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 [ ]."
},
{
"@type": "HowToStep",
"position": 8,
"name": "Golgi staining",
"text": "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..."
}
],
"tool": [],
"supply": [],
"isBasedOn": {
"@type": "ScholarlyArticle",
"headline": "Electroacupuncture promotes BDNF-dependent neurogenesis via microglial reprogramming in a chronic stress model",
"datePublished": "2026",
"author": [
{
"@type": "Person",
"name": "Lijuan Zhang"
},
{
"@type": "Person",
"name": "Ting Wei"
},
{
"@type": "Person",
"name": "Xuan Liu"
},
{
"@type": "Person",
"name": "Lifan Zhang"
},
{
"@type": "Person",
"name": "Dan Wang"
},
{
"@type": "Person",
"name": "Yucai Luo"
},
{
"@type": "Person",
"name": "Yanyan He"
},
{
"@type": "Person",
"name": "Zhaoxuan He"
},
{
"@type": "Person",
"name": "Fang Zeng"
}
],
"identifier": "10.1186/s13020-026-01334-y"
}
},
{
"@context": "https://schema.org",
"@type": "BreadcrumbList",
"itemListElement": [
{
"@type": "ListItem",
"position": 1,
"name": "Experiments",
"item": "https://replicatescience.com/experiments"
},
{
"@type": "ListItem",
"position": 2,
"name": "Electroacupuncture promotes BDNF-dependent neurogenesis via microglial reprogramming in a chronic stress model methods",
"item": "https://replicatescience.com/experiments/electroacupuncture-promotes-bdnf-dependent-neurogenesis-via-microglial-reprogramming-in-a-chronic-stress-model-methods-lijuan-zhang-pmc12866076/electroacupuncture-promotes-bdnf-dependent-neurogenesis-via-microglial-reprogram"
}
]
}
]