The potential of human milk oligosaccharides in ameliorating traumatic brain injury-induced cognitive impairment in mice methods
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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.
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Protein expression by western blot
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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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