Hydrogen-Mediated Activation of the Nrf2/ HO -1 Signaling Pathway Improves Cognitive Impairment in Sleep-Deprived Mice methods
Aim. Evidence-backed execution summary for Hydrogen-Mediated Activation of the Nrf2/ HO -1 Signaling Pathway Improves Cognitive Impairment in Sleep-Deprived Mice methods from Hydrogen-Mediated Activation of the Nrf2/ HO -1 Signaling Pathway Improves Cognitive Impairment in Sleep-Deprived Mice.
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mouse
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
Methods
reagent used in the protocol.
- Use
- A chronic sleep deprivation (SD) model was established using the modified multiple platform method, with 18 h of deprivation daily for 28 consecutive days. Cognitive function was evaluated using the Morris water maze and novel object recognition (NOR) test. Histopathological and biochemical analyses, including...
Hematoxylin and Eosin (H&E) Staining
reagent used in the protocol.
- Use
- Following behavioral assessments, brain samples were collected after euthanasia. Tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and sliced into 5 µm sections. Hippocampal sections were incubated overnight at 60°C, then deparaffinized through three 20-min xylene washes. Rehydra...
Nissl Staining
reagent used in the protocol.
- Use
- After routine deparaffinization and rehydration, tissue sections were treated with Nissl staining solution for 30-60 s. Excess stain was removed by washing with distilled water, followed by differentiation in 1% glacial acetic acid. Processed sections were coverslipped with buffered glycerol mounting med...
Enzyme-Linked Immunosorbent Assay ( ELISA )
reagent used in the protocol.
- Use
- Brain tissue samples were mechanically homogenized and stored at -20°C for 24 h. After two complete freeze-thaw cycles at 2°C-8°C to disrupt cells, homogenates were centrifuged at 5000 × g for 5 min to collect supernatant. ELISA was conducted following kit instruction...
Immunohistochemistry
reagent used in the protocol.
- Use
- Following standard deparaffinization and rehydration steps, tissue sections underwent antigen retrieval by heating in 0.01 M citrate buffer (pH 6.0) followed by gradual cooling to ambient temperature. After three 3-min washes with phosphate-buffered saline (PBS, 0.01 M), endogenous pero...
Cell Culture and Treatment
reagent used in the protocol.
- Use
- PC12 cells were cultivated under standard culture conditions [ ]. Cells were grown in RPMI-1640 medium at 37°C in a humidified incubator with 5% CO 2, with medium refreshed every 2 days. An in vitro neuronal injury model was produced by exposing PC12 cells to 200 µM corticosterone (...
Cell Viability Assay
reagent used in the protocol.
- Use
- Viability was determined using a CCK-8 kit (Beyotime, China) as per manufacturer's instructions. PC12 cells were seeded in a 96-well plate and cultured for 72 h. Then, 10 µL of CCK-8 solution was introduced into each well. After 2 h incubation, absorbance at 450 nm was...
Cell Apoptosis Assay
reagent used in the protocol.
- Use
- Apoptosis was evaluated with an Annexin V-FITC/PI Apoptosis Detection Kit (Keygen, China) following the manufacturer's protocol. Cells were resuspended in binding buffer and stained with Annexin V-FITC and propidium iodide (PI) in darkness for 20 min. Apoptotic rate was then assessed by flow cytome...
Methods
A chronic sleep deprivation (SD) model was established using the modified multiple platform method, with 18 h of deprivation daily for 28 consecutive days. Cognitive function was evaluated using the Morris water maze and novel object recognition (NOR) test. Histopathological and biochemical analyses, including...
- Use
- A chronic sleep deprivation (SD) model was established using the modified multiple platform method, with 18 h of deprivation daily for 28 consecutive days. Cognitive function was evaluated using the Morris water maze and novel object recognition (NOR) test. Histopathological and biochemical analyses, including...
Morris Water Maze Test
A black cylindrical pool (150 cm diameter, 50 cm height) was used. The tank was notionally separated into four quadrants (I-IV). A concealed circular platform (10 cm diameter) was placed 1 cm under the water surface in quadrant I's center. Fixed visual cues were positioned around the po...
- Use
- A black cylindrical pool (150 cm diameter, 50 cm height) was used. The tank was notionally separated into four quadrants (I-IV). A concealed circular platform (10 cm diameter) was placed 1 cm under the water surface in quadrant I's center. Fixed visual cues were positioned around the po...
Morris Water Maze Test
Navigation phase: Mice received 4 trials daily for 5 successive days (1-h intervals between trials). Escape latency (time from entering water at varying start locations to finding the platform) was recorded. The mean daily value from four trials was analyzed.
- Use
- Navigation phase: Mice received 4 trials daily for 5 successive days (1-h intervals between trials). Escape latency (time from entering water at varying start locations to finding the platform) was recorded. The mean daily value from four trials was analyzed.
Morris Water Maze Test
Spatial probe phase: On Day 6, the platform was withdrawn. Mice were released from the quadrant opposite the original platform site. Their swimming path over 60 s was tracked, and time spent in the target quadrant and platform crossings were quantified.
- Use
- Spatial probe phase: On Day 6, the platform was withdrawn. Mice were released from the quadrant opposite the original platform site. Their swimming path over 60 s was tracked, and time spent in the target quadrant and platform crossings were quantified.
Novel Object Recognition ( NOR ) Test
Two identical objects were securely placed in an open arena with double-sided tape, positioned 6 cm from adjacent walls in two non-release corners to prevent displacement. All sessions were video-recorded and analyzed with behavior-tracking software.
- Use
- Two identical objects were securely placed in an open arena with double-sided tape, positioned 6 cm from adjacent walls in two non-release corners to prevent displacement. All sessions were video-recorded and analyzed with behavior-tracking software.
Hematoxylin and Eosin (H&E) Staining
Following behavioral assessments, brain samples were collected after euthanasia. Tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and sliced into 5 µm sections. Hippocampal sections were incubated overnight at 60°C, then deparaffinized through three 20-min xylene washes. Rehydra...
- Use
- Following behavioral assessments, brain samples were collected after euthanasia. Tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and sliced into 5 µm sections. Hippocampal sections were incubated overnight at 60°C, then deparaffinized through three 20-min xylene washes. Rehydra...
Immunohistochemistry
Following standard deparaffinization and rehydration steps, tissue sections underwent antigen retrieval by heating in 0.01 M citrate buffer (pH 6.0) followed by gradual cooling to ambient temperature. After three 3-min washes with phosphate-buffered saline (PBS, 0.01 M), endogenous pero...
- Use
- Following standard deparaffinization and rehydration steps, tissue sections underwent antigen retrieval by heating in 0.01 M citrate buffer (pH 6.0) followed by gradual cooling to ambient temperature. After three 3-min washes with phosphate-buffered saline (PBS, 0.01 M), endogenous pero...
Preparation of Hydrogen-Rich Saline
Hydrogen-rich saline was generated according to a published method [ ]. Briefly, hydrogen gas was dissolved into saline under high pressure (0.4 MPa) for 2 h using specialized equipment to attain supersaturation. Hydrogen concentration was determined via gas chromatography (Biogas Analyzer Systems&...
- Use
- Hydrogen-rich saline was generated according to a published method [ ]. Briefly, hydrogen gas was dissolved into saline under high pressure (0.4 MPa) for 2 h using specialized equipment to attain supersaturation. Hydrogen concentration was determined via gas chromatography (Biogas Analyzer Systems&...
Statistical Analysis
Software used for acquisition, scoring, statistics, or reporting.
- Use
- Statistical analyses were conducted using GraphPad Prism 10 software (GraphPad Software Inc., USA). Data are expressed as mean ± standard deviation. Comparisons between two groups were analyzed by Student's t -test, while multiple-group comparisons were evaluated by one-way analysi...
Before you run
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Open quote workflowStep-by-step procedure
What do I do, in order?
Nissl Staining
After routine deparaffinization and rehydration, tissue sections were treated with Nissl staining solution for 30-60 s. Excess stain was removed by washing with distilled water, followed by differentiation in 1% glacial acetic acid. Processed sections were coverslipped with buffered glycerol mounting medium and examined by light microscopy.
Enzyme-Linked Immunosorbent Assay ( ELISA )
Brain tissue samples were mechanically homogenized and stored at -20°C for 24 h. After two complete freeze-thaw cycles at 2°C-8°C to disrupt cells, homogenates were centrifuged at 5000 × g for 5 min to collect supernatant. ELISA was conducted following kit instructions. Commercial kits were utilized to detect cytokines: mouse tumor necrosis factor-α (TNF-α; KE10002), interleukin (IL)-1β (KE10003), and IL-6 (KE10007) from Proteintech (USA), as well as oxidative stress markers glutathione peroxidase (GSH-PX, A005-1), malondialdehyde (MDA, A003-1), and superoxide dismutase (SOD, A003-1) from Nanjing Jiancheng Bioengineering Institute (China).
Immunohistochemistry
Following standard deparaffinization and rehydration steps, tissue sections underwent antigen retrieval by heating in 0.01 M citrate buffer (pH 6.0) followed by gradual cooling to ambient temperature. After three 3-min washes with phosphate-buffered saline (PBS, 0.01 M), endogenous peroxidase activity was blocked with 1% periodic acid solution (10 min incubation) and subsequent PBS rinses (3 × 3 min). Primary antibody incubation occurred at 4°C for 12-16 h using these antibodies (all from Proteintech): postsynaptic density protein 95 (PSD95, 1:200), synaptophysin (SYN, 1:200), Nrf2 (1:200), and HO-1 (1:200). Secondary detection involved 30-min incubation at 37°C with horseradish peroxidase-conjugated anti-rabbit immunoglobulin G (IgG). Color development employed a 3,3′-...
Measurement outputs
What raw and processed outputs should exist?
Navigation phase: Mice received 4 trials daily for 5 successive days (1-h intervals between trials). Escape latency (time from entering water at varying start locations to...
- 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
Two identical objects were securely placed in an open arena with double-sided tape, positioned 6 cm from adjacent walls in two non-release corners to prevent d...
- 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
Brain tissue samples were mechanically homogenized and stored at -20°C for 24 h. After two complete freeze-thaw cycles at 2°C-8°C to disru...
- 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
Total RNA was isolated with TRIzol (Invitrogen, USA). cDNA was synthesized using the MonScript RT III Kit (Monad). Gene expression analysis was performed on a real-time PC...
- 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
Statistical analyses were conducted using GraphPad Prism 10 software (GraphPad Software Inc., USA).
from paperScoring or quantification
Quantify the primary readouts for this experiment: Navigation phase: Mice received 4 trials daily for 5 successive days (1-h intervals between trials). Escape latency (time from entering water at varying start locations to...; Two identical objects were securely placed in an open arena with double-sided tape, positioned 6 cm from adjacent walls in two non-release corners to prevent d...; Brain tissue samples were mechanically homogenized and stored at -20°C for 24 h. After two complete freeze-thaw cycles at 2°C-8°C to disru...; Total RNA was isolated with TRIzol (Invitrogen, USA). cDNA was synthesized using the MonScript RT III Kit (Monad). Gene expression analysis was performed on a real-time PC....
from paperStatistical comparison
Statistical analyses were conducted using GraphPad Prism 10 software (GraphPad Software Inc., USA). Data are expressed as mean ± standard deviation. Comparisons...
from paperReporting output
Report representative outputs alongside summary comparisons for Navigation phase: Mice received 4 trials daily for 5 successive days (1-h intervals between trials). Escape latency (time from entering water at varying start locations to..., Two identical objects were securely placed in an open arena with double-sided tape, positioned 6 cm from adjacent walls in two non-release corners to prevent d..., Brain tissue samples were mechanically homogenized and stored at -20°C for 24 h. After two complete freeze-thaw cycles at 2°C-8°C to disru..., Total RNA was isolated with TRIzol (Invitrogen, USA). cDNA was synthesized using the MonScript RT III Kit (Monad). Gene expression analysis was performed on a real-time PC....
inferred from protocolStructured statistical methods
Statistical analyses were conducted using GraphPad Prism 10 software (GraphPad Software Inc., USA). Data are expressed as mean ± standard deviation. Comparisons...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (3)
After routine deparaffinization and rehydration, tissue sections were treated with Nissl staining solution for 30-60 s. Excess stain was removed by washing with distilled water, followed by differentiation in 1% glacial acetic acid. Processed sections were coverslipped with buffered glycerol mounting medium and examined by light microscopy.
Brain tissue samples were mechanically homogenized and stored at -20°C for 24 h. After two complete freeze-thaw cycles at 2°C-8°C to disrupt cells, homogenates were centrifuged at 5000 × g for 5 min to collect supernatant. ELISA was conducted following kit instructions. Commercial kits were utilized to detect cytokines: mouse tumor necrosis factor-α (TNF-α; KE10002), interleukin (IL)-1β (KE10003), and IL-6 (KE10007) from Proteintech (USA), as well as oxidative stress markers glutathione peroxidase (GSH-PX, A005-1), malondialdehyde (MDA, A003-1), and superoxide dismutase (SOD, A003-1) from Nanjing Jiancheng Bioengineering Institute (China).
Following standard deparaffinization and rehydration steps, tissue sections underwent antigen retrieval by heating in 0.01 M citrate buffer (pH 6.0) followed by gradual cooling to ambient temperature. After three 3-min washes with phosphate-buffered saline (PBS, 0.01 M), endogenous peroxidase activity was blocked with 1% periodic acid solution (10 min incubation) and subsequent PBS rinses (3 × 3 min). Primary antibody incubation occurred at 4°C for 12-16 h using these antibodies (all from Proteintech): postsynaptic density protein 95 (PSD95, 1:200), synaptophysin (SYN, 1:200), Nrf2 (1:200), and HO-1 (1:200). Secondary detection involved 30-min incubation at 37°C with horseradish peroxidase-conjugated anti-rabbit immunoglobulin G (IgG). Color development employed a 3,3′-diaminobenzidine substrate system (ZSGB-BIO, China). Final processing included sequential dehydration in graded ethanols (60-100%, 5 min per concentration), xylene clearing (10 min), and mounting with neutral gum for light microscopic assessment.
Machine-readable layer
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