Neuroinflammation induced by lipopolysaccharide causes cognitive impairment in 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
C57BL/6J male mice (11-12 weeks old) were purchased from the Guangdong Medical Laboratory Animal Center. All animal experiments were approved and carried out according to the Animal Ethics Committee of Jinan University (CBNUA-436-12-02). All mice were housed in a room with automatically controlled temperature (21-25 °C), relative humidity (45-65%), and light-dark (12-12 h) cycles. The mice in each cage were divided into the following treatment groups for each of the five experiments: (I) i.p. saline group (control), (II) i.p. LPS (500 µg/kg) group, (III) i.p. LPS (750 µg/kg) group, (IV) i.c.v. saline group, and (V) i.c.v. LPS (12 µg) group. Each group consisted of ten male mice. The i.p. LPS injections were administered at doses of 500 or 750 µg/kg in saline for seven consecutive days, and the saline (0.9%...
Morris water maze test
The Morris water maze (MWM) test, which is a commonly accepted method of testing memory, was performed as described by Morris et al.. The MWM program and equipment were purchased from ChengDu Technology & Market Co, LTD. A circular pool (height: 35 cm, diameter: 120 cm) was filled with water rendered opaque with whole milk and maintained at 23 ± 2 °C. An escape platform (height: 14 cm, diameter: 4-5 cm) was submerged 1-1.5 cm below the surface of the water in a specific position. During the training trials, the mice were placed into the water in a random quadrant and allowed to locate the hidden platform for 60 s. Then, the mice were allowed to remain on the platform or were placed on the platform for 10 s at the end of each trial. The mice were trained with three trials per day for 7 days. After the t...
Passive avoidance performance test
The passive avoidance test (PAT) is widely accepted as a simple memory testing method. The PAT was performed using a "step-through" apparatus (ChengDu Technology & Market Co, LTD) composed of six reaction boxes, allowing for six mice to be tested simultaneously. Here, the latency to enter the dark box for the first time and the latency to enter the dark box after an electric shock was applied to the feet were observed and automatically recorded to probe the learning and memory abilities of the mice. The mice were placed in the illuminated compartment facing away from the dark compartment during the first three days of the training trials. Then, LPS (i.p. 500 µg/kg or 750 µg/kg) was injected 6 h before each daily test during the training phase (7 days). All mice were placed in the illuminated compartment, and upon moving completely into the dark...
Climbing pole test
All mice were submitted to the "Pole test" to measure their motor coordination. Briefly, the mice were subjected to five days of training on a pole that was 60 cm in length and 1 cm in diameter with two layers of gauze on the outside. On the sixth day, LPS (i.p. 500 µg/kg or 750 µg/kg) was injected 6 h before the climbing pole test, and the following three times were recorded: the time it took for the mouse to climb down the upper half, the time it took for the mouse to climb down the lower half, and the time it took for the mouse to complete the total length of the pole. If the mouse completed the above three steps within 3 s, 6 s or longer than 6 s, the motor coordination score was 3 points, 2 points, or 1 point, respectively.
Immunofluorescence staining
To measure the microglia activation and neuronal cell loss in the mouse hippocampus, the mice were perfused with normal saline, followed by 4% paraformaldehyde (PFA) in 0.1 M sodium phosphate buffer (PBS), pH = 7.4. The cerebral tissues were removed, incubated overnight in fixatives, and stored in a 30% sucrose solution. After being transferred to a 30% sucrose solution, the brains were cut into 10-µm-thick sections using a cryostat microtome (Leica CM 1850; Leica Microsystems, Seoul, Korea) and incubated at 37 °C overnight. After three 5-min washes with PBS (pH = 7.4) and permeabilization with Triton X-100 (0.3% in TBST) at room temperature, the brains sections were blocked for 1 h in 1% bovine serum albumin (BSA) solution and overnight at 4 °C with a 1:100 dilution of an antibody against microtubule-associated protein 2...
Western blot analysis
The hippocampus tissues were homogenized in RIPA lysis buffer (Bioteke Co, Beijing, China) containing 1 mM phenylmethylsulfonyl fluoride and then centrifuged at 12,000 × g for 15 min at 4 °C. The cytoplasmic and nuclear p65 detection were performed according to the NE-PER ® instructions (Thermo Scientific, Rockford, IL, USA) to obtain cytoplasmic and nuclear protein. An equal amount of protein was separated via SDS-PAGE and transferred onto a nitrocellulose membrane. The membranes were blocked for 1 h with a 5% skim milk solution and incubated overnight at 4 °C with specific antibodies (1:1000 dilution, Cell Signaling Technology Inc, MA, USA). Then, the membranes were incubated in a 1:15000 dilution of a horseradish peroxidase-conjugated secondary antibody (Cell Signaling Technology Inc, MA, USA) for 1 h at room temp...
Measurement outputs
What raw and processed outputs should exist?
Protein expression by western blot
- Raw artifact
- Membrane or gel image with visible bands for target and control proteins
- Processed artifact
- Band quantification and normalized densitometry values
- Reported as
- Relative expression values or fold-change comparisons across groups
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: Protein expression by western blot; 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 Protein expression by western blot, Microscopy-based tissue or cell-state imaging.
inferred from protocolSource and audit
What supports the facts on this page?
Evidence quotes (6)
C57BL/6J male mice (11-12 weeks old) were purchased from the Guangdong Medical Laboratory Animal Center. All animal experiments were approved and carried out according to the Animal Ethics Committee of Jinan University (CBNUA-436-12-02). All mice were housed in a room with automatically controlled temperature (21-25 °C), relative humidity (45-65%), and light-dark (12-12 h) cycles. The mice in each cage were divided into the following treatment groups for each of the five experiments: (I) i.p. saline group (control), (II) i.p. LPS (500 µg/kg) group, (III) i.p. LPS (750 µg/kg) group, (IV) i.c.v. saline group, and (V) i.c.v. LPS (12 µg) group. Each group consisted of ten male mice. The i.p. LPS injections were administered at doses of 500 or 750 µg/kg in saline for seven consecutive days, and the saline (0.9% NaCl) control mice received saline injections each day of testing. The i.c.v. injections of 12 µg of LPS (in 3 µL of saline) and saline (control) injections were administered on one day using a microsyringe and stereotaxic coordinates (-2.6 mm dorsal/ventral, -1.5 m...
The Morris water maze (MWM) test, which is a commonly accepted method of testing memory, was performed as described by Morris et al.. The MWM program and equipment were purchased from ChengDu Technology & Market Co, LTD. A circular pool (height: 35 cm, diameter: 120 cm) was filled with water rendered opaque with whole milk and maintained at 23 ± 2 °C. An escape platform (height: 14 cm, diameter: 4-5 cm) was submerged 1-1.5 cm below the surface of the water in a specific position. During the training trials, the mice were placed into the water in a random quadrant and allowed to locate the hidden platform for 60 s. Then, the mice were allowed to remain on the platform or were placed on the platform for 10 s at the end of each trial. The mice were trained with three trials per day for 7 days. After the training, on the final day of testing, we administered LPS or saline 6 h before conducting the spatial probe test. The escape latency and escape distance displayed by each mouse were recorded by a computer.
The passive avoidance test (PAT) is widely accepted as a simple memory testing method. The PAT was performed using a "step-through" apparatus (ChengDu Technology & Market Co, LTD) composed of six reaction boxes, allowing for six mice to be tested simultaneously. Here, the latency to enter the dark box for the first time and the latency to enter the dark box after an electric shock was applied to the feet were observed and automatically recorded to probe the learning and memory abilities of the mice. The mice were placed in the illuminated compartment facing away from the dark compartment during the first three days of the training trials. Then, LPS (i.p. 500 µg/kg or 750 µg/kg) was injected 6 h before each daily test during the training phase (7 days). All mice were placed in the illuminated compartment, and upon moving completely into the dark compartment, they received an electric shock (39 V, 3-s duration).
All mice were submitted to the "Pole test" to measure their motor coordination. Briefly, the mice were subjected to five days of training on a pole that was 60 cm in length and 1 cm in diameter with two layers of gauze on the outside. On the sixth day, LPS (i.p. 500 µg/kg or 750 µg/kg) was injected 6 h before the climbing pole test, and the following three times were recorded: the time it took for the mouse to climb down the upper half, the time it took for the mouse to climb down the lower half, and the time it took for the mouse to complete the total length of the pole. If the mouse completed the above three steps within 3 s, 6 s or longer than 6 s, the motor coordination score was 3 points, 2 points, or 1 point, respectively.
To measure the microglia activation and neuronal cell loss in the mouse hippocampus, the mice were perfused with normal saline, followed by 4% paraformaldehyde (PFA) in 0.1 M sodium phosphate buffer (PBS), pH = 7.4. The cerebral tissues were removed, incubated overnight in fixatives, and stored in a 30% sucrose solution. After being transferred to a 30% sucrose solution, the brains were cut into 10-µm-thick sections using a cryostat microtome (Leica CM 1850; Leica Microsystems, Seoul, Korea) and incubated at 37 °C overnight. After three 5-min washes with PBS (pH = 7.4) and permeabilization with Triton X-100 (0.3% in TBST) at room temperature, the brains sections were blocked for 1 h in 1% bovine serum albumin (BSA) solution and overnight at 4 °C with a 1:100 dilution of an antibody against microtubule-associated protein 2 (MAP-2, Millipore Corp, Billerica, MA, USA), 1:200 dilution of an antibody against ionized calcium-binding adapter protein 1 (IBA-1, Millipore Corp, Billerica, MA, USA) and 1:100 dilution of an antibody against amyloid beta 1-42 (Aβ 1-42, Abcam, Cambridge, UK). On the following day...
The hippocampus tissues were homogenized in RIPA lysis buffer (Bioteke Co, Beijing, China) containing 1 mM phenylmethylsulfonyl fluoride and then centrifuged at 12,000 × g for 15 min at 4 °C. The cytoplasmic and nuclear p65 detection were performed according to the NE-PER ® instructions (Thermo Scientific, Rockford, IL, USA) to obtain cytoplasmic and nuclear protein. An equal amount of protein was separated via SDS-PAGE and transferred onto a nitrocellulose membrane. The membranes were blocked for 1 h with a 5% skim milk solution and incubated overnight at 4 °C with specific antibodies (1:1000 dilution, Cell Signaling Technology Inc, MA, USA). Then, the membranes were incubated in a 1:15000 dilution of a horseradish peroxidase-conjugated secondary antibody (Cell Signaling Technology Inc, MA, USA) for 1 h at room temperature. The signals were measured with an enhanced chemiluminescence kit (ECL, Millipore, USA) using a gel imaging system (Millipore, Billerica, MA, USA), and the results were visualized using Quantity One software.
Machine-readable layer
[
{
"@context": "https://schema.org",
"@type": "HowTo",
"name": "Neuroinflammation induced by lipopolysaccharide causes cognitive impairment in mice methods",
"description": "Protocol for Neuroinflammation induced by lipopolysaccharide causes cognitive impairment in mice methods",
"step": [
{
"@type": "HowToStep",
"position": 1,
"name": "Materials and Methods",
"text": "C57BL/6J male mice (11-12 weeks old) were purchased from the Guangdong Medical Laboratory Animal Center. All animal experiments were approved and carried out according to the Animal Ethics Committee of Jinan University (CBNUA-436-12-02). All mice were housed in a room with automatically controlled temperature (21-25 °C), relative humidity (45-65%), and light-dark (12-12 h) cycles. The mice in each cage were divided into the following treatment groups for each of the five experiments: (I) i.p. saline group (control), (II) i.p. LPS (500 µg/kg) group, (III) i.p. LPS (750 µg/kg) group, (IV) i.c.v. saline group, and (V) i.c.v. LPS (12 µg) group. Each group consisted of ten male mice. The i.p. LPS injections were administered at doses of 500 or 750 µg/kg in saline for seven consecutive days, and the saline (0.9%..."
},
{
"@type": "HowToStep",
"position": 2,
"name": "Morris water maze test",
"text": "The Morris water maze (MWM) test, which is a commonly accepted method of testing memory, was performed as described by Morris et al.. The MWM program and equipment were purchased from ChengDu Technology & Market Co, LTD. A circular pool (height: 35 cm, diameter: 120 cm) was filled with water rendered opaque with whole milk and maintained at 23 ± 2 °C. An escape platform (height: 14 cm, diameter: 4-5 cm) was submerged 1-1.5 cm below the surface of the water in a specific position. During the training trials, the mice were placed into the water in a random quadrant and allowed to locate the hidden platform for 60 s. Then, the mice were allowed to remain on the platform or were placed on the platform for 10 s at the end of each trial. The mice were trained with three trials per day for 7 days. After the t..."
},
{
"@type": "HowToStep",
"position": 3,
"name": "Passive avoidance performance test",
"text": "The passive avoidance test (PAT) is widely accepted as a simple memory testing method. The PAT was performed using a \"step-through\" apparatus (ChengDu Technology & Market Co, LTD) composed of six reaction boxes, allowing for six mice to be tested simultaneously. Here, the latency to enter the dark box for the first time and the latency to enter the dark box after an electric shock was applied to the feet were observed and automatically recorded to probe the learning and memory abilities of the mice. The mice were placed in the illuminated compartment facing away from the dark compartment during the first three days of the training trials. Then, LPS (i.p. 500 µg/kg or 750 µg/kg) was injected 6 h before each daily test during the training phase (7 days). All mice were placed in the illuminated compartment, and upon moving completely into the dark..."
},
{
"@type": "HowToStep",
"position": 4,
"name": "Climbing pole test",
"text": "All mice were submitted to the \"Pole test\" to measure their motor coordination. Briefly, the mice were subjected to five days of training on a pole that was 60 cm in length and 1 cm in diameter with two layers of gauze on the outside. On the sixth day, LPS (i.p. 500 µg/kg or 750 µg/kg) was injected 6 h before the climbing pole test, and the following three times were recorded: the time it took for the mouse to climb down the upper half, the time it took for the mouse to climb down the lower half, and the time it took for the mouse to complete the total length of the pole. If the mouse completed the above three steps within 3 s, 6 s or longer than 6 s, the motor coordination score was 3 points, 2 points, or 1 point, respectively."
},
{
"@type": "HowToStep",
"position": 5,
"name": "Immunofluorescence staining",
"text": "To measure the microglia activation and neuronal cell loss in the mouse hippocampus, the mice were perfused with normal saline, followed by 4% paraformaldehyde (PFA) in 0.1 M sodium phosphate buffer (PBS), pH = 7.4. The cerebral tissues were removed, incubated overnight in fixatives, and stored in a 30% sucrose solution. After being transferred to a 30% sucrose solution, the brains were cut into 10-µm-thick sections using a cryostat microtome (Leica CM 1850; Leica Microsystems, Seoul, Korea) and incubated at 37 °C overnight. After three 5-min washes with PBS (pH = 7.4) and permeabilization with Triton X-100 (0.3% in TBST) at room temperature, the brains sections were blocked for 1 h in 1% bovine serum albumin (BSA) solution and overnight at 4 °C with a 1:100 dilution of an antibody against microtubule-associated protein 2..."
},
{
"@type": "HowToStep",
"position": 6,
"name": "Western blot analysis",
"text": "The hippocampus tissues were homogenized in RIPA lysis buffer (Bioteke Co, Beijing, China) containing 1 mM phenylmethylsulfonyl fluoride and then centrifuged at 12,000 × g for 15 min at 4 °C. The cytoplasmic and nuclear p65 detection were performed according to the NE-PER ® instructions (Thermo Scientific, Rockford, IL, USA) to obtain cytoplasmic and nuclear protein. An equal amount of protein was separated via SDS-PAGE and transferred onto a nitrocellulose membrane. The membranes were blocked for 1 h with a 5% skim milk solution and incubated overnight at 4 °C with specific antibodies (1:1000 dilution, Cell Signaling Technology Inc, MA, USA). Then, the membranes were incubated in a 1:15000 dilution of a horseradish peroxidase-conjugated secondary antibody (Cell Signaling Technology Inc, MA, USA) for 1 h at room temp..."
}
],
"tool": [],
"supply": [],
"isBasedOn": {
"@type": "ScholarlyArticle",
"headline": "Neuroinflammation induced by lipopolysaccharide causes cognitive impairment in mice",
"datePublished": "2019",
"author": [
{
"@type": "Person",
"name": "Jiayi Zhao"
},
{
"@type": "Person",
"name": "Wei Bi"
},
{
"@type": "Person",
"name": "Shu Xiao"
},
{
"@type": "Person",
"name": "Xin Lan"
},
{
"@type": "Person",
"name": "Xiaofeng Cheng"
},
{
"@type": "Person",
"name": "Jiawei Zhang"
},
{
"@type": "Person",
"name": "Daxiang Lu"
},
{
"@type": "Person",
"name": "Wei Wei"
},
{
"@type": "Person",
"name": "Yanping Wang"
},
{
"@type": "Person",
"name": "Hongmei Li"
},
{
"@type": "Person",
"name": "Yongmei Fu"
},
{
"@type": "Person",
"name": "Lihong Zhu"
}
],
"identifier": "10.1038/s41598-019-42286-8"
}
},
{
"@context": "https://schema.org",
"@type": "BreadcrumbList",
"itemListElement": [
{
"@type": "ListItem",
"position": 1,
"name": "Experiments",
"item": "https://replicatescience.com/experiments"
},
{
"@type": "ListItem",
"position": 2,
"name": "Neuroinflammation induced by lipopolysaccharide causes cognitive impairment in mice methods",
"item": "https://replicatescience.com/experiments/neuroinflammation-induced-by-lipopolysaccharide-causes-cognitive-impairment-in-mice-methods-jiayi-zhao-pmc6453933/neuroinflammation-induced-by-lipopolysaccharide-causes-cognitive-impairment-in-mice-mlpgw9ec"
}
]
}
]