Voluntary Exercise Promotes Glymphatic Clearance of Amyloid Beta and Reduces the Activation of Astrocytes and Microglia in Aged Mice 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?
Materials and Methods
This study was approved by the Animal Research Committee of the First Affiliated Hospital of Sun Yat-sen University (Guangzhou, China; committee's reference number: [2013]97). All efforts were made to minimize the number and suffering of animals used in this study. Twenty-four male C57BL/6J mice obtained from the Animal Center of Sun Yat-sen University and 12 Thy1-GFP transgenic mice obtained from the Model Animal Research Center of Nanjing University (stock number: 003782) were used. All the animals were used at 14-16 months of age, and were housed under a 12:12 h light-dark cycle (light from 07:00 to 19:00), with controlled temperature and humidity, and given food and water ad libitum.
Voluntary Wheel Training
The animals were randomly divided into two groups, the control group and the physical training (running) group, and each group included 12 C57BL/6J and six Thy1 - GFP male mice. In the control group, the mice were housed in polypropylene cages (36 cm L × 20 cm W × 14 cm H). The mice in the training group were housed in polypropylene cages of the same size, with a 16 cm diameter running wheel, which rotated when a mouse climbed onto the wheel voluntarily. The mice were housed under these conditions for 6 weeks (Littlefield et al., ).
Morris Water Maze
The 12 C57BL/6J mice in each group performed the water-maze task, according to the protocol of van Praag et al. ( ) and Akers et al. ( ). The maze consisted of a circular tub (120 cm in diameter, 50 cm in height) with a white circular platform (10 cm). The tub was surrounded by a curtain located about 1 m from the tub wall, which was painted with distinct geometric cues. The water (24 ± 1°C) was made opaque with white tempera paint to conceal the platform. Over five consecutive days, the platform was submerged 1 cm beneath the surface of the water in the center of one quadrant of the pool. Twenty-four mice underwent four trials (up to 60 s each) per day, starting from each of four different locations in the pool. The animals that failed to locate the platform within the allotted 60 s were gently guided to the platform. All the mice remained on the platform for 10 s at the en...
In Vivo Two-Photon Imaging of Glymphatic Pathway Clearance
For two-photon imaging, the mice were anesthetized with chloral hydrate (4.2%, 0.01 ml/g), and placed securely in a stereotaxic device, with the skull level between the bregma and lambda. The stereotaxic coordinates of the right parietal cortex were 2 mm lateral to the midline and 1.7 mm ahead of the lambdoid (Harvey et al., ). A thin cranial window (3 mm in diameter; Golshani and Portera-Cailliau, ) was constructed for in vivo two-photon imaging.
In Vivo Two-Photon Imaging of Glymphatic Pathway Clearance
To evaluate the PVS-ISF exchange, fluorescein isothiocyanate (FITC; 70 kDa; Sigma-Aldrich, Darmstadt, Germany) was dissolved in artificial CSF (ACSF) at a concentration of 1%. A microsyringe (BASi, West Lafayette, IN, USA) was inserted into the cisterna magna, and 10 µl of FITC-dextran was injected within a period of 10 min. To visualize the vasculature, 0.2 ml of rhodamine B (RD; 1% in saline; Sigma) was injected intravenously immediately before imaging.
In Vivo Two-Photon Imaging of Glymphatic Pathway Clearance
Two-photon imaging was performed with a custom-built two-photon laser scanning microscope (Leica) at a wavelength of 800 nm and a laser scanning system (Coherent, Santa Clara, CA, USA) equipped with a water immersion objective (25 × ). To monitor the clearance of ovalbumin injected into the brain parenchyma, lateral images of the xyz stacks (512 × 512 pixels, 2 µm resolution) were taken up to 300 µm below the cortical surface. The stacks were collected at 5, 15, 30, 45 and 60 min after the injection of the dye.
Analysis of BBB Permeability
As described previously, RD (1% in saline) was injected intravenously to visualize the brain vasculature while evaluating the PVS-ISF exchange. Fluorescent intravascular dyes were used to detect leakage from the vasculature (Burgess et al.,; He et al., ). Therefore, we selected the red fluorescence channel for detection and analyzed the total fluorescence intensity in the extravascular compartment (Nhan et al., ). Images were collected at 0, 5, 15, 30 and 60 min after the injection of fluorescent dextran, and were shown as both three-dimensional (3D) stacks and xyz stacks.
Histology
For the histological analysis, the dendrites and dendritic spines of 12 Thy1-GFP mice ( n = 6 per group) were detected, and 12 C57BL/6J mice ( n = 6 per group) were used for other histological analyses ( n = 6 per group), including enzyme-linked immunosorbent assays (ELISAs) that detected Aβ1-40 and Aβ1-42. The mice were perfused with 50 ml of ice-cold saline, followed by 50 ml of 4% (w/v) paraformaldehyde in phosphate-buffered saline (PBS; pH 7.4). Their brains were removed and incubated overnight in 4% paraformaldehyde, and then dehydrated in 20%-30% sucrose in PBS. Coronal brain slices (40 µm or 10 µm thick) from the right parietal cortex were sectioned with a frozen microtome (Leica) at intervals of 100 µm to produce consecutive frozen sections. For immunofluorescent staining, the sections were boiled in citric acid buffer (pH 6.0)...
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)
This study was approved by the Animal Research Committee of the First Affiliated Hospital of Sun Yat-sen University (Guangzhou, China; committee's reference number: [2013]97). All efforts were made to minimize the number and suffering of animals used in this study. Twenty-four male C57BL/6J mice obtained from the Animal Center of Sun Yat-sen University and 12 Thy1-GFP transgenic mice obtained from the Model Animal Research Center of Nanjing University (stock number: 003782) were used. All the animals were used at 14-16 months of age, and were housed under a 12:12 h light-dark cycle (light from 07:00 to 19:00), with controlled temperature and humidity, and given food and water ad libitum.
The animals were randomly divided into two groups, the control group and the physical training (running) group, and each group included 12 C57BL/6J and six Thy1 - GFP male mice. In the control group, the mice were housed in polypropylene cages (36 cm L × 20 cm W × 14 cm H). The mice in the training group were housed in polypropylene cages of the same size, with a 16 cm diameter running wheel, which rotated when a mouse climbed onto the wheel voluntarily. The mice were housed under these conditions for 6 weeks (Littlefield et al., ).
The 12 C57BL/6J mice in each group performed the water-maze task, according to the protocol of van Praag et al. ( ) and Akers et al. ( ). The maze consisted of a circular tub (120 cm in diameter, 50 cm in height) with a white circular platform (10 cm). The tub was surrounded by a curtain located about 1 m from the tub wall, which was painted with distinct geometric cues. The water (24 ± 1°C) was made opaque with white tempera paint to conceal the platform. Over five consecutive days, the platform was submerged 1 cm beneath the surface of the water in the center of one quadrant of the pool. Twenty-four mice underwent four trials (up to 60 s each) per day, starting from each of four different locations in the pool. The animals that failed to locate the platform within the allotted 60 s were gently guided to the platform. All the mice remained on the platform for 10 s at the end of each trial. On day 6, the platform was removed and a single 60 s probe trial was conducted. The swim paths were recorded with an overhead video camera and tracked with automated software (San Diego Instruments, San Diego, CA, USA). The swim speed (mm/s), time to reach the platform during water-...
For two-photon imaging, the mice were anesthetized with chloral hydrate (4.2%, 0.01 ml/g), and placed securely in a stereotaxic device, with the skull level between the bregma and lambda. The stereotaxic coordinates of the right parietal cortex were 2 mm lateral to the midline and 1.7 mm ahead of the lambdoid (Harvey et al., ). A thin cranial window (3 mm in diameter; Golshani and Portera-Cailliau, ) was constructed for in vivo two-photon imaging.
To evaluate the PVS-ISF exchange, fluorescein isothiocyanate (FITC; 70 kDa; Sigma-Aldrich, Darmstadt, Germany) was dissolved in artificial CSF (ACSF) at a concentration of 1%. A microsyringe (BASi, West Lafayette, IN, USA) was inserted into the cisterna magna, and 10 µl of FITC-dextran was injected within a period of 10 min. To visualize the vasculature, 0.2 ml of rhodamine B (RD; 1% in saline; Sigma) was injected intravenously immediately before imaging.
Two-photon imaging was performed with a custom-built two-photon laser scanning microscope (Leica) at a wavelength of 800 nm and a laser scanning system (Coherent, Santa Clara, CA, USA) equipped with a water immersion objective (25 × ). To monitor the clearance of ovalbumin injected into the brain parenchyma, lateral images of the xyz stacks (512 × 512 pixels, 2 µm resolution) were taken up to 300 µm below the cortical surface. The stacks were collected at 5, 15, 30, 45 and 60 min after the injection of the dye.
As described previously, RD (1% in saline) was injected intravenously to visualize the brain vasculature while evaluating the PVS-ISF exchange. Fluorescent intravascular dyes were used to detect leakage from the vasculature (Burgess et al.,; He et al., ). Therefore, we selected the red fluorescence channel for detection and analyzed the total fluorescence intensity in the extravascular compartment (Nhan et al., ). Images were collected at 0, 5, 15, 30 and 60 min after the injection of fluorescent dextran, and were shown as both three-dimensional (3D) stacks and xyz stacks.
For the histological analysis, the dendrites and dendritic spines of 12 Thy1-GFP mice ( n = 6 per group) were detected, and 12 C57BL/6J mice ( n = 6 per group) were used for other histological analyses ( n = 6 per group), including enzyme-linked immunosorbent assays (ELISAs) that detected Aβ1-40 and Aβ1-42. The mice were perfused with 50 ml of ice-cold saline, followed by 50 ml of 4% (w/v) paraformaldehyde in phosphate-buffered saline (PBS; pH 7.4). Their brains were removed and incubated overnight in 4% paraformaldehyde, and then dehydrated in 20%-30% sucrose in PBS. Coronal brain slices (40 µm or 10 µm thick) from the right parietal cortex were sectioned with a frozen microtome (Leica) at intervals of 100 µm to produce consecutive frozen sections. For immunofluorescent staining, the sections were boiled in citric acid buffer (pH 6.0) for 5 min in a microwave oven. After the sections were cooled, they were treated with 0.3% Triton X-100 and 10% goat serum for 1 h at room temperature. The sections were then incubated overnight at 4°C with a primary antibody (1:400 anti-ionized calcium-binding adapter molecule 1 (IBA1) antibod...
Machine-readable layer
[
{
"@context": "https://schema.org",
"@type": "HowTo",
"name": "Voluntary Exercise Promotes Glymphatic Clearance of Amyloid Beta and Reduces the Activation of Astrocytes and Microglia in Aged Mice methods",
"description": "Protocol for Voluntary Exercise Promotes Glymphatic Clearance of Amyloid Beta and Reduces the Activation of Astrocytes and Microglia in Aged Mice methods",
"step": [
{
"@type": "HowToStep",
"position": 1,
"name": "Materials and Methods",
"text": "This study was approved by the Animal Research Committee of the First Affiliated Hospital of Sun Yat-sen University (Guangzhou, China; committee's reference number: [2013]97). All efforts were made to minimize the number and suffering of animals used in this study. Twenty-four male C57BL/6J mice obtained from the Animal Center of Sun Yat-sen University and 12 Thy1-GFP transgenic mice obtained from the Model Animal Research Center of Nanjing University (stock number: 003782) were used. All the animals were used at 14-16 months of age, and were housed under a 12:12 h light-dark cycle (light from 07:00 to 19:00), with controlled temperature and humidity, and given food and water ad libitum."
},
{
"@type": "HowToStep",
"position": 2,
"name": "Voluntary Wheel Training",
"text": "The animals were randomly divided into two groups, the control group and the physical training (running) group, and each group included 12 C57BL/6J and six Thy1 - GFP male mice. In the control group, the mice were housed in polypropylene cages (36 cm L × 20 cm W × 14 cm H). The mice in the training group were housed in polypropylene cages of the same size, with a 16 cm diameter running wheel, which rotated when a mouse climbed onto the wheel voluntarily. The mice were housed under these conditions for 6 weeks (Littlefield et al., )."
},
{
"@type": "HowToStep",
"position": 3,
"name": "Morris Water Maze",
"text": "The 12 C57BL/6J mice in each group performed the water-maze task, according to the protocol of van Praag et al. ( ) and Akers et al. ( ). The maze consisted of a circular tub (120 cm in diameter, 50 cm in height) with a white circular platform (10 cm). The tub was surrounded by a curtain located about 1 m from the tub wall, which was painted with distinct geometric cues. The water (24 ± 1°C) was made opaque with white tempera paint to conceal the platform. Over five consecutive days, the platform was submerged 1 cm beneath the surface of the water in the center of one quadrant of the pool. Twenty-four mice underwent four trials (up to 60 s each) per day, starting from each of four different locations in the pool. The animals that failed to locate the platform within the allotted 60 s were gently guided to the platform. All the mice remained on the platform for 10 s at the en..."
},
{
"@type": "HowToStep",
"position": 4,
"name": "In Vivo Two-Photon Imaging of Glymphatic Pathway Clearance",
"text": "For two-photon imaging, the mice were anesthetized with chloral hydrate (4.2%, 0.01 ml/g), and placed securely in a stereotaxic device, with the skull level between the bregma and lambda. The stereotaxic coordinates of the right parietal cortex were 2 mm lateral to the midline and 1.7 mm ahead of the lambdoid (Harvey et al., ). A thin cranial window (3 mm in diameter; Golshani and Portera-Cailliau, ) was constructed for in vivo two-photon imaging."
},
{
"@type": "HowToStep",
"position": 5,
"name": "In Vivo Two-Photon Imaging of Glymphatic Pathway Clearance",
"text": "To evaluate the PVS-ISF exchange, fluorescein isothiocyanate (FITC; 70 kDa; Sigma-Aldrich, Darmstadt, Germany) was dissolved in artificial CSF (ACSF) at a concentration of 1%. A microsyringe (BASi, West Lafayette, IN, USA) was inserted into the cisterna magna, and 10 µl of FITC-dextran was injected within a period of 10 min. To visualize the vasculature, 0.2 ml of rhodamine B (RD; 1% in saline; Sigma) was injected intravenously immediately before imaging."
},
{
"@type": "HowToStep",
"position": 6,
"name": "In Vivo Two-Photon Imaging of Glymphatic Pathway Clearance",
"text": "Two-photon imaging was performed with a custom-built two-photon laser scanning microscope (Leica) at a wavelength of 800 nm and a laser scanning system (Coherent, Santa Clara, CA, USA) equipped with a water immersion objective (25 × ). To monitor the clearance of ovalbumin injected into the brain parenchyma, lateral images of the xyz stacks (512 × 512 pixels, 2 µm resolution) were taken up to 300 µm below the cortical surface. The stacks were collected at 5, 15, 30, 45 and 60 min after the injection of the dye."
},
{
"@type": "HowToStep",
"position": 7,
"name": "Analysis of BBB Permeability",
"text": "As described previously, RD (1% in saline) was injected intravenously to visualize the brain vasculature while evaluating the PVS-ISF exchange. Fluorescent intravascular dyes were used to detect leakage from the vasculature (Burgess et al.,; He et al., ). Therefore, we selected the red fluorescence channel for detection and analyzed the total fluorescence intensity in the extravascular compartment (Nhan et al., ). Images were collected at 0, 5, 15, 30 and 60 min after the injection of fluorescent dextran, and were shown as both three-dimensional (3D) stacks and xyz stacks."
},
{
"@type": "HowToStep",
"position": 8,
"name": "Histology",
"text": "For the histological analysis, the dendrites and dendritic spines of 12 Thy1-GFP mice ( n = 6 per group) were detected, and 12 C57BL/6J mice ( n = 6 per group) were used for other histological analyses ( n = 6 per group), including enzyme-linked immunosorbent assays (ELISAs) that detected Aβ1-40 and Aβ1-42. The mice were perfused with 50 ml of ice-cold saline, followed by 50 ml of 4% (w/v) paraformaldehyde in phosphate-buffered saline (PBS; pH 7.4). Their brains were removed and incubated overnight in 4% paraformaldehyde, and then dehydrated in 20%-30% sucrose in PBS. Coronal brain slices (40 µm or 10 µm thick) from the right parietal cortex were sectioned with a frozen microtome (Leica) at intervals of 100 µm to produce consecutive frozen sections. For immunofluorescent staining, the sections were boiled in citric acid buffer (pH 6.0)..."
}
],
"tool": [],
"supply": [],
"isBasedOn": {
"@type": "ScholarlyArticle",
"headline": "Voluntary Exercise Promotes Glymphatic Clearance of Amyloid Beta and Reduces the Activation of Astrocytes and Microglia in Aged Mice",
"datePublished": "2017",
"author": [
{
"@type": "Person",
"name": "Xiao-fei He"
},
{
"@type": "Person",
"name": "Dong-xu Liu"
},
{
"@type": "Person",
"name": "Qun Zhang"
},
{
"@type": "Person",
"name": "Feng-ying Liang"
},
{
"@type": "Person",
"name": "Guang-yan Dai"
},
{
"@type": "Person",
"name": "Jin-sheng Zeng"
},
{
"@type": "Person",
"name": "Zhong Pei"
},
{
"@type": "Person",
"name": "Guang-qing Xu"
},
{
"@type": "Person",
"name": "Yue Lan"
}
],
"identifier": "10.3389/fnmol.2017.00144"
}
},
{
"@context": "https://schema.org",
"@type": "BreadcrumbList",
"itemListElement": [
{
"@type": "ListItem",
"position": 1,
"name": "Experiments",
"item": "https://replicatescience.com/experiments"
},
{
"@type": "ListItem",
"position": 2,
"name": "Voluntary Exercise Promotes Glymphatic Clearance of Amyloid Beta and Reduces the Activation of Astrocytes and Microglia in Aged Mice methods",
"item": "https://replicatescience.com/experiments/voluntary-exercise-promotes-glymphatic-clearance-of-amyloid-beta-and-reduces-the-activation-of-astrocytes-and-microglia-in-aged-mice-methods-xiao-fei-he-pmc5437122/voluntary-exercise-promotes-glymphatic-clearance-of-amyloid-beta-and-reduces-the-activation-of-astro-mlph8ayn"
}
]
}
]