The potential of human milk oligosaccharides in ameliorating traumatic brain injury-induced cognitive impairment in mice methods
Aim. Evidence-backed execution summary for The potential of human milk oligosaccharides in ameliorating traumatic brain injury-induced cognitive impairment in mice methods from The potential of human milk oligosaccharides in ameliorating traumatic brain injury-induced cognitive impairment in mice.
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mouse
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
HMOs treatment can reduce colonic inflammatory response and intestinal barrier injury in TBI mice
reagent used in the protocol.
- Use
- The gut-brain axis is composed of bidirectional pathways through which neuroinflammation and neurodegeneration induced by TBI can affect intestinal function [ ]. Moreover, dynamic changes in the gut-brain-gut microbiota axis can also influence the severity of TBI [, ]. Given that HMOs primarily exert their effects...
HMOs treatment can reduce colonic inflammatory response and intestinal barrier injury in TBI mice
reagent used in the protocol.
- Use
- HMOs treatment can reduce colonic inflammatory response and intestinal barrier injury in TBI mice. (A) Measurement of serum D-Lactate among mice in different groups. (B) Representative immunofluorescence staining was performed to show the expression of occludin in the colons on the 14th day post-TBI (C) Immunoblot a...
Western blot
reagent used in the protocol.
- Use
- After removing the brain tissue, the hippocampus was carefully dissected on ice. Total lysates were prepared using the RIPA buffer (Beyotime Biotechnology) containing protease inhibitors (Beyotime Biotechnology). After sonication, the protein concentration in each sample was determined by using the BCA protein assay...
Measurement of hippocampal ROS activity and serum D-lactic acid
reagent used in the protocol.
- Use
- The ROS activity in the mouse hippocampus was detected using the Beyotime ROS Detection Kit (S0038) according to the manufacturer's instructions.
Measurement of hippocampal ROS activity and serum D-lactic acid
reagent used in the protocol.
- Use
- The serum D-lactic acid was detected using the Abcam D-Lactate Assay Kit (ab83429) according to the manufacturer's instructions.
Immunofluorescence staining
reagent used in the protocol.
- Use
- Mice were anesthetized and transcardially perfused with 0.01 M ice-cold phosphate-buffered saline (PBS), followed by ice-cold 4% paraformaldehyde in PBS. The brain and intestinal tissues were removed, incubated in 4% paraformaldehyde for 24 hours, and then subjected to gradient sucrose dehydration. After...
HMOs may ameliorate cognitive dysfunction in TBI mice by indirectly supporting hippocampal synaptic function
Synapses are the foundation for ensuring the fine functions of the nervous system. Synaptophysin is an integral protein located on the presynaptic vesicle membrane, and its deficiency leads to an imbalance in the release and uptake of neurotransmitters [ ]. Neurotrophic factors, especially brain-derived neurotrophic...
- Use
- Synapses are the foundation for ensuring the fine functions of the nervous system. Synaptophysin is an integral protein located on the presynaptic vesicle membrane, and its deficiency leads to an imbalance in the release and uptake of neurotransmitters [ ]. Neurotrophic factors, especially brain-derived neurotrophic...
Animal handling
Controlled cortical impact (CCI) was employed to establish a mild traumatic brain injury (TBI) mouse model. The specific methodology referred to a previously established experimental protocol [ ]. Briefly, the mice were anesthetized with isoflurane and then placed in a stereotaxic system. An incision was made at the...
- Use
- Controlled cortical impact (CCI) was employed to establish a mild traumatic brain injury (TBI) mouse model. The specific methodology referred to a previously established experimental protocol [ ]. Briefly, the mice were anesthetized with isoflurane and then placed in a stereotaxic system. An incision was made at the...
Western blot
After removing the brain tissue, the hippocampus was carefully dissected on ice. Total lysates were prepared using the RIPA buffer (Beyotime Biotechnology) containing protease inhibitors (Beyotime Biotechnology). After sonication, the protein concentration in each sample was determined by using the BCA protein assay...
- Use
- After removing the brain tissue, the hippocampus was carefully dissected on ice. Total lysates were prepared using the RIPA buffer (Beyotime Biotechnology) containing protease inhibitors (Beyotime Biotechnology). After sonication, the protein concentration in each sample was determined by using the BCA protein assay...
Immunofluorescence staining
Mice were anesthetized and transcardially perfused with 0.01 M ice-cold phosphate-buffered saline (PBS), followed by ice-cold 4% paraformaldehyde in PBS. The brain and intestinal tissues were removed, incubated in 4% paraformaldehyde for 24 hours, and then subjected to gradient sucrose dehydration. After...
- Use
- Mice were anesthetized and transcardially perfused with 0.01 M ice-cold phosphate-buffered saline (PBS), followed by ice-cold 4% paraformaldehyde in PBS. The brain and intestinal tissues were removed, incubated in 4% paraformaldehyde for 24 hours, and then subjected to gradient sucrose dehydration. After...
Morris water maze (MWM) test
The MWM test consists of two consecutive phases: the training phase (17th to 21th days post-TBI) and the spatial memory test phase (22th days post-TBI). The apparatus is composed of a black circular pool with a diameter of 120 cm, and there are four distinct markers on the walls of the four quadrants of the po...
- Use
- The MWM test consists of two consecutive phases: the training phase (17th to 21th days post-TBI) and the spatial memory test phase (22th days post-TBI). The apparatus is composed of a black circular pool with a diameter of 120 cm, and there are four distinct markers on the walls of the four quadrants of the po...
Morris water maze (MWM) test
In the training phase, mice are placed into the pool from any one of the four quadrants every day, with 4 training sessions per day. When a mouse reaches the platform and stays there for 5 seconds, or is manually placed on the platform to stay for 5 seconds after the 90-second time limit expires, it is t...
- Use
- In the training phase, mice are placed into the pool from any one of the four quadrants every day, with 4 training sessions per day. When a mouse reaches the platform and stays there for 5 seconds, or is manually placed on the platform to stay for 5 seconds after the 90-second time limit expires, it is t...
Morris water maze (MWM) test
In the test phase, the platform is removed, and the time taken for the mice to reach the original position of the platform after entering the water (escape latency) and the number of times they cross the platform (crossing times) are recorded.
- Use
- In the test phase, the platform is removed, and the time taken for the mice to reach the original position of the platform after entering the water (escape latency) and the number of times they cross the platform (crossing times) are recorded.
Y-maze (YM) test
The YM test was conducted on the 16th day post-TBI. The apparatus comprises three identical arms, each measuring 35 cm in length, 5 cm in width, and 10 cm in height, with an angle of 120° between each pair of arms. One arm was randomly designated as the "start arm", and the mouse w...
- Use
- The YM test was conducted on the 16th day post-TBI. The apparatus comprises three identical arms, each measuring 35 cm in length, 5 cm in width, and 10 cm in height, with an angle of 120° between each pair of arms. One arm was randomly designated as the "start arm", and the mouse w...
Statistical analyses
Software used for acquisition, scoring, statistics, or reporting.
- Use
- GraphPad Prism 9.0 was used for statistical analysis. Data obtained from three independent experiments were presented as mean ± standard deviation (SD). Variations across multiple groups were assessed using one-way analysis of variance (ANOVA) followed by Tukey's multiple comparisons test. For...
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Animal handling
Controlled cortical impact (CCI) was employed to establish a mild traumatic brain injury (TBI) mouse model. The specific methodology referred to a previously established experimental protocol [ ]. Briefly, the mice were anesthetized with isoflurane and then placed in a stereotaxic system. An incision was made at the midline of the scalp to expose the skull. A 5.0 mm craniotomy was performed between the anterior fontanel and the sagittal suture, with the dura mater kept intact. CCI modeling was conducted using a precision impact device. The parameters of this TBI model were set as follows: low pressure of 100 kPa, high pressure of 200 kPa, and impact depth of 1.0 mm. For the sham-operated group, the mice were only anesthetized, and the same part of the skull was removed, followed by scalp suturing after the operation. After surgery, the mice were placed on a hea...
Animal handling
For mice in the TBI+HMOs group, HMOs were administered by gavage at a dose of 1.6 mg/g daily, starting on the same day of TBI induction and continuing until the end of the experiment, while the other groups were gavaged with normal saline. The HMOs dose was set approximately equivalent to the daily intake from breast milk in human infants, calculated based on body surface area conversion [, ].
Western blot
After removing the brain tissue, the hippocampus was carefully dissected on ice. Total lysates were prepared using the RIPA buffer (Beyotime Biotechnology) containing protease inhibitors (Beyotime Biotechnology). After sonication, the protein concentration in each sample was determined by using the BCA protein assay kit (Beyotime Biotechnology) and boiled at 95 °C for 10 min. Equivalent amounts of protein samples were separated by SDS-PAGE and electroblotted onto a polyvinylidene fluoride membrane (Roche) using a Mini Trans-Blot Cell (Bio-Rad). The membranes were blocked at room temperature for 2 hr in PBS containing 3% bovine serum albumin (BSA), followed by incubation with the indicated primary antibodies overnight at 4 °C. After washing three times with TBS-Tween (50 mM Tris-HCl, 150 mM NaCl, and 0.1% [v/v] Tween 20, pH 7.4), the membra...
Immunofluorescence staining
Mice were anesthetized and transcardially perfused with 0.01 M ice-cold phosphate-buffered saline (PBS), followed by ice-cold 4% paraformaldehyde in PBS. The brain and intestinal tissues were removed, incubated in 4% paraformaldehyde for 24 hours, and then subjected to gradient sucrose dehydration. After the tissues were embedded in OCT embedding medium (4583, Sakura), 5 µm-thick coronal sections (brain) or cross-sections (colon) were prepared using a cryostat. The sections were fixed in 4% paraformaldehyde for 30 minutes and washed in phosphate-buffered saline plus Tween-20 (PBST) for 10 minutes. After blocking with bovine serum albumin for 2 hours, the sections were placed in BSA solution containing primary antibodies and incubated overnight at 4 °C. Following three washes with PBST, the sections were incubated with Alexa Fluor 488...
Morris water maze (MWM) test
The MWM test consists of two consecutive phases: the training phase (17th to 21th days post-TBI) and the spatial memory test phase (22th days post-TBI). The apparatus is composed of a black circular pool with a diameter of 120 cm, and there are four distinct markers on the walls of the four quadrants of the pool. A transparent circular plexiglass platform (6 cm in diameter) is set 15 cm away from the southwest wall and 25 cm away from the bottom of the pool. During the experiment, the water temperature is maintained at around 25 °C, and the water level is adjusted to 3 cm above the plexiglass platform.
Measurement outputs
What raw and processed outputs should exist?
Forty-eight C57BL/6 mice were randomly divided into the sham-operated group, TBI group, and TBI+HMOs group. The TBI model was established via controlled cortical impact (CCI). M...
- 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
Subsequently, we investigated the effect of HMOs on TBI-induced inflammatory responses in the hippocampus. Results from immunofluorescence ( ) and Western blot ( ) demonstrated...
- 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
The gut-brain axis is composed of bidirectional pathways through which neuroinflammation and neurodegeneration induced by TBI can affect intestinal function [ ]. Moreover, dynam...
- 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
HMOs treatment can reduce colonic inflammatory response and intestinal barrier injury in TBI mice. (A) Measurement of serum D-Lactate among mice in different groups. (B) Represe...
- 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
Analysis plan
How should the outputs become interpretable results?
Acquisition
Capture matched images from the relevant tissue region using the same acquisition settings across samples.
inferred from protocolPreprocessing / cleaning
GraphPad Prism 9.0 was used for statistical analysis.
from paperScoring or quantification
Quantify the primary readouts for this experiment: Forty-eight C57BL/6 mice were randomly divided into the sham-operated group, TBI group, and TBI+HMOs group. The TBI model was established via controlled cortical impact (CCI). M...; Subsequently, we investigated the effect of HMOs on TBI-induced inflammatory responses in the hippocampus. Results from immunofluorescence ( ) and Western blot ( ) demonstrated...; The gut-brain axis is composed of bidirectional pathways through which neuroinflammation and neurodegeneration induced by TBI can affect intestinal function [ ]. Moreover, dynam...; HMOs treatment can reduce colonic inflammatory response and intestinal barrier injury in TBI mice. (A) Measurement of serum D-Lactate among mice in different groups. (B) Represe....
from paperNormalization
Normalize expression or signal values against the stated control or loading reference before comparing groups.
inferred from protocolStatistical comparison
GraphPad Prism 9.0 was used for statistical analysis. Data obtained from three independent experiments were presented as mean ± standard deviation (SD). Variati...
from paperReporting output
Report representative outputs alongside summary comparisons for Forty-eight C57BL/6 mice were randomly divided into the sham-operated group, TBI group, and TBI+HMOs group. The TBI model was established via controlled cortical impact (CCI). M..., Subsequently, we investigated the effect of HMOs on TBI-induced inflammatory responses in the hippocampus. Results from immunofluorescence ( ) and Western blot ( ) demonstrated..., The gut-brain axis is composed of bidirectional pathways through which neuroinflammation and neurodegeneration induced by TBI can affect intestinal function [ ]. Moreover, dynam..., HMOs treatment can reduce colonic inflammatory response and intestinal barrier injury in TBI mice. (A) Measurement of serum D-Lactate among mice in different groups. (B) Represe....
inferred from protocolStructured statistical methods
GraphPad Prism 9.0 was used for statistical analysis. Data obtained from three independent experiments were presented as mean ± standard deviation (SD). Variati...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (5)
Controlled cortical impact (CCI) was employed to establish a mild traumatic brain injury (TBI) mouse model. The specific methodology referred to a previously established experimental protocol [ ]. Briefly, the mice were anesthetized with isoflurane and then placed in a stereotaxic system. An incision was made at the midline of the scalp to expose the skull. A 5.0 mm craniotomy was performed between the anterior fontanel and the sagittal suture, with the dura mater kept intact. CCI modeling was conducted using a precision impact device. The parameters of this TBI model were set as follows: low pressure of 100 kPa, high pressure of 200 kPa, and impact depth of 1.0 mm. For the sham-operated group, the mice were only anesthetized, and the same part of the skull was removed, followed by scalp suturing after the operation. After surgery, the mice were placed on a heating pad to maintain body temperature until they could move independently, and then they were returned to their cages.
For mice in the TBI+HMOs group, HMOs were administered by gavage at a dose of 1.6 mg/g daily, starting on the same day of TBI induction and continuing until the end of the experiment, while the other groups were gavaged with normal saline. The HMOs dose was set approximately equivalent to the daily intake from breast milk in human infants, calculated based on body surface area conversion [, ].
After removing the brain tissue, the hippocampus was carefully dissected on ice. Total lysates were prepared using the RIPA buffer (Beyotime Biotechnology) containing protease inhibitors (Beyotime Biotechnology). After sonication, the protein concentration in each sample was determined by using the BCA protein assay kit (Beyotime Biotechnology) and boiled at 95 °C for 10 min. Equivalent amounts of protein samples were separated by SDS-PAGE and electroblotted onto a polyvinylidene fluoride membrane (Roche) using a Mini Trans-Blot Cell (Bio-Rad). The membranes were blocked at room temperature for 2 hr in PBS containing 3% bovine serum albumin (BSA), followed by incubation with the indicated primary antibodies overnight at 4 °C. After washing three times with TBS-Tween (50 mM Tris-HCl, 150 mM NaCl, and 0.1% [v/v] Tween 20, pH 7.4), the membranes were incubated with secondary antibodies at RT for 45 min. Finally, the membranes were visualized with a chemiluminescence system (Tanon) after three times of wash.
Mice were anesthetized and transcardially perfused with 0.01 M ice-cold phosphate-buffered saline (PBS), followed by ice-cold 4% paraformaldehyde in PBS. The brain and intestinal tissues were removed, incubated in 4% paraformaldehyde for 24 hours, and then subjected to gradient sucrose dehydration. After the tissues were embedded in OCT embedding medium (4583, Sakura), 5 µm-thick coronal sections (brain) or cross-sections (colon) were prepared using a cryostat. The sections were fixed in 4% paraformaldehyde for 30 minutes and washed in phosphate-buffered saline plus Tween-20 (PBST) for 10 minutes. After blocking with bovine serum albumin for 2 hours, the sections were placed in BSA solution containing primary antibodies and incubated overnight at 4 °C. Following three washes with PBST, the sections were incubated with Alexa Fluor 488-conjugated secondary antibodies at room temperature for 2 hours. Afterwards, the sections were stained with DAPI (C1006, Beyotime) for 15 seconds and then washed three times with PBST. Tissue section images were observed using a digital slide scanner (3DHISTECH, P250 FLASH III).
The MWM test consists of two consecutive phases: the training phase (17th to 21th days post-TBI) and the spatial memory test phase (22th days post-TBI). The apparatus is composed of a black circular pool with a diameter of 120 cm, and there are four distinct markers on the walls of the four quadrants of the pool. A transparent circular plexiglass platform (6 cm in diameter) is set 15 cm away from the southwest wall and 25 cm away from the bottom of the pool. During the experiment, the water temperature is maintained at around 25 °C, and the water level is adjusted to 3 cm above the plexiglass platform.
Machine-readable layer
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"datePublished": "2026",
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