Skip to main content
NCBI home page
Future Science OA logoLink to Future Science OA
. 2026 Feb 1;12(1):2622241. doi: 10.1080/20565623.2026.2622241

The potential of human milk oligosaccharides in ameliorating traumatic brain injury-induced cognitive impairment in mice

Dongliang He a,✉, Renli He a, Wei Duan b, Guilan Li a, Qin Kang c,✉
PMCID: PMC12867443  PMID: 41622772

Abstract

Objective

To investigate the ameliorative effect and underlying mechanisms of human milk oligosaccharides (HMOs) on cognitive impairment induced by traumatic brain injury (TBI) in mice.

Materials and Methods

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). Mice in the TBI+HMOs group received daily HMOs administration by gavage, while other groups were given normal saline. Relevant indicators were detected using behavioral tests, pathological staining, Western blot, and other methods.

Results

HMOs significantly improved cognitive function in TBI mice, inhibited hippocampal oxidative stress and the expression of proinflammatory cytokines (IL-1β, IL-6, TNF-α), alleviated intestinal barrier injury, and regulated the expression of synaptophysin, BDNF, and pro-BDNF.

Conclusion

HMOs exert neuroprotective effects by targeting central inflammation, oxidative stress, synaptic function, and intestinal barrier integrity, providing a novel natural therapeutic candidate for TBI treatment.

Keywords: Traumatic brain injury, human milk oligosaccharides, cognitive impairment, neuroprotection, inflammation

ARTICLE HIGHLIGHTS

HMOs significantly ameliorate traumatic brain injury (TBI)-induced cognitive impairment in mice.

HMOs exert neuroprotective effects through a multi-target mechanism: inhibiting oxidative stress and proinflammatory cytokines in the hippocampus to alleviate neuronal damage, while mitigating intestinal barrier injury by restoring occludin expression and reducing colonic IL-6.

HMOs support hippocampal synaptic function by reversing TBI-induced dysregulation of key synaptic proteins, offering a promising natural therapeutic strategy for TBI.

1. Introduction

Traumatic Brain Injury (TBI) is a common neurological trauma, which refers to brain tissue damage caused by external force impact on the head (such as impact, blow, blast wave, etc.). Among all trauma-related injuries globally, it is the leading cause of death and disability [1–3]. Studies have confirmed that TBI may increase the risk of neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease [4–6]. Moreover, TBI also has a significant impact on patients’ cognitive function and emotional state [7], and is a high-risk factor for inducing cerebral hemorrhage, cerebral infarction, cerebral vasospasm, and epilepsy [8,9]. Despite advances in healthcare and research, gaps remain in gaining a comprehensive understanding of the disease’s burden, particularly when it comes to enhancing treatments to achieve better outcomes.

Given the remarkable research progress achieved in the field of the gut-brain axis, here we aim to explore whether gavage administration of human milk oligosaccharides (HMOs) can alleviate TBI-induced cognitive impairment in mice. HMOs are a large class of indigestible oligosaccharides derived from breast milk and rank as the third most abundant solid component in breast milk [10–12]. Studies have demonstrated that HMOs contribute to the establishment of gut microbiota, thereby supporting the maturation of the intestinal and immune systems [10,13]. Additionally, HMOs play an important role in maintaining neurodevelopment, and studies have shown that they can alleviate cognitive impairment in mice with Alzheimer’s disease [14–17]. These findings suggest that HMOs may have the potential to mitigate cognitive impairment caused by factors such as disease, trauma, and genetics via the gut-brain axis.

Here, we evaluated the effects of HMOs on TBI mice and found they can alleviate TBI-induced cognitive dysfunction. Further analyses revealed that HMOs not only attenuate hippocampal damage by inhibiting oxidative stress and inflammatory responses in the hippocampus but also reduce colonic IL-6 levels and ameliorate intestinal barrier damage in TBI mice. Finally, via Western blotting, we found that HMOs can inhibit TBI-induced downregulation of BDNF expression in the cerebral cortex, indicating that HMOs may alleviate TBI-induced cognitive impairment by maintaining BDNF secretion. In conclusion, this study highlights the neuroprotective effects of HMOs in the context of TBI and provides a potential novel therapeutic strategy for neural repair after brain injury.

2. Results

2.1. HMOs treatment ameliorates cognitive dysfunction in TBI mice

First, we evaluated the effects of HMOs on neurological function after TBI through a series of behavioral experiments. As shown in Figure 1(A,B), results from the Morris Water Maze (MWM) test indicated that HMO administration alleviated TBI-induced prolongation of escape latency and reduction in the number of platform crossings. In the Novel Object Recognition (NOR) test, TBI mice showed no significant preference for the novel object, whereas HMO administration significantly increased the proportion of exploration time spent on the novel object in TBI mice (Figure 1©). Similar results were observed in the Y-maze (YM) test: without affecting the total number of arm entries, HMO administration significantly improved the TBI-induced decrease in spontaneous alternation rate in mice. Taken together, these results demonstrate that HMOs can improve cognitive dysfunction in TBI mice.

Figure 1.

Figure 1.

Open in a new tab

HMOs treatment ameliorates cognitive dysfunction in TBI mice. (A) Statistical analysis of latency in the MWM test among mice in different groups. (B) Statistical analysis of crossing numbers in the MWM test among mice in different groups. (C) Statistical analysis of recognition index in the NOR test among mice in different groups. RI = time to explore new object/(time to explore new object + time to explore familiar object). (D) Statistical analysis of spontaneous alteration in the YM test among mice in different groups. The spontaneous alteration was calculated as follows: (total alternations × 100)/(total arm entries − 2) × 100%] in different groups of mice. ****, p < 0.0001; **, p < 0.01; *, p < 0.05 and ns, no significance.

2.2. HMOs treatment alleviates hippocampal injury in TBI mice

Given the association between the hippocampus and TBI-induced cognitive dysfunction [18], we subsequently examined whether HMOs affect TBI-induced hippocampal damage. As shown in Figure 2(A), pathological examination results revealed that neurons in the hippocampal CA3 region of mice in the TBI group were loosely arranged with shrunken cell nuclei. In contrast, these phenomena were significantly alleviated in mice of the TBI+HMOs group, indicating that HMOs alleviated TBI-induced hippocampal damage to a certain extent.

Figure 2.

Figure 2.

Open in a new tab

HMOs treatment alleviates hippocampal injury in TBI mice. (A) Representative hematoxylin and eosin staining was performed to show the pathological features of hippocampus on the 14th days post-TBI. Scale bar, 20 μm. (B to D) Representative immunofluorescence staining was performed to show the inflammatory responses in the hippocampus on the 14th day post-TBI. Scale bar, 20 μm. (E) Immunoblot analyses of IL-1β, IL-6, and TNF-α in hippocampus. (F) Measurement of hippocampal ROS activity among mice in different groups. **, p < 0.01 and *, p < 0.05.

Subsequently, we investigated the effect of HMOs on TBI-induced inflammatory responses in the hippocampus. Results from immunofluorescence (Figure 2(B–D)) and Western blot (Figure 2(E)) demonstrated that HMO administration significantly attenuated TBI-induced upregulation of the proinflammatory cytokines IL-1β, IL-6, and TNF-α in the mouse hippocampus.

Increased levels of reactive oxygen species (ROS) in the brain are a key pathological feature of various neurodegenerative diseases, brain injuries, and psychiatric disorders [19]. Therefore, we further isolated mouse hippocampi and measured ROS levels. The results showed that HMOs also inhibited TBI-induced elevation of ROS levels in the mouse hippocampus (Figure 2(F)).

These findings indicate that HMOs alleviate TBI-induced hippocampal damage by inhibiting oxidative stress and inflammatory responses.

2.3. HMOs treatment can reduce colonic inflammatory response and intestinal barrier injury in TBI mice

The gut-brain axis is composed of bidirectional pathways through which neuroinflammation and neurodegeneration induced by TBI can affect intestinal function [20]. Moreover, dynamic changes in the gut-brain-gut microbiota axis can also influence the severity of TBI [21,22]. Given that HMOs primarily exert their effects in the digestive tract, we subsequently investigated whether HMOs can ameliorate TBI-induced impairment of intestinal mucosal barrier function. As shown in Figure 3(A), serum D-lactose levels were significantly increased in TBI mice, while HMOs treatment significantly reduced serum D-lactose levels. This indicates that 14 days after TBI, the intestinal mucosal barrier function of mice was impaired with increased permeability, and HMOs administration could alleviate this symptom. To further confirm this conclusion, we then detected the expression of the occludin, which plays a crucial role in the formation and regulation of the tight junction, in the colon using immunofluorescence and Western blot assays. As shown in Figure 3(B,C), the expression of occludin in colonic epithelial cells was significantly decreased in TBI mice, while HMO administration alleviated this phenomenon. Meanwhile, it was observed that HMOs administration could effectively inhibit the expression of IL-6 in the colon, suggesting that HMOs may maintain the function of the intestinal mucosal barrier by inhibiting TBI-induced intestinal ­inflammatory responses.

Figure 3.

Figure 3.

Open in a new tab

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 analyses of occluding and IL-6 in colons. ****, p < 0.0001 and *, p < 0.05.

2.4. 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 [23]. Neurotrophic factors, especially brain-derived neurotrophic factor (BDNF), have been confirmed to play an important role in the cellular events involved in the repair process after traumatic brain injury. It can antagonize with their precursor pro-BDNF to jointly regulate neural plasticity [24].

To further explore the potential mechanism by which HMOs improve cognitive dysfunction in TBI mice, we detected the expression of synaptophysin, BDNF, and Pro-BDNF in the hippocampus via Western blot, which are important for synaptic function. The results showed that the expression of synaptophysin and BDNF in the hippocampus of TBI mice was significantly downregulated, while the expression of pro-BDNF was significantly upregulated, and HMO administration could alleviate this phenomenon (Figure 4). These results indicates that HMOs may improve cognitive dysfunction in TBI mice by indirectly supporting hippocampal neural synaptic function.

Figure 4.

Figure 4.

Open in a new tab

HMOs may ameliorate cognitive dysfunction in TBI mice by indirectly supporting hippocampal synaptic function. Immunoblot analyses of synaptophysin, pro-BNDF and BNDF in hippocampus among mice in different groups. ****, p < 0.0001; ***, p < 0.001; **, p < 0.01 and *, p < 0.05.

3. Discussion

HMOs possess multiple physiological functions, including aiding infants in establishing a balanced gut microbiota, enhancing gastrointestinal barrier function, preventing pathogenic microbial infections, and potentially supporting the immune system, brain, and cognitive development [25–27]. In this study, we found that HMO administration can alleviate TBI-induced cognitive dysfunction.

The hippocampus is crucial for cognitive ability, and an increasing number of researchers have focused on its key role in TBI. TBI alters the fate of neural stem cells in the dentate gyrus of the mouse hippocampus, promoting neurogenesis at the expense of astrocyte generation [28]. Inhibiting the function of PD-1 in excitatory neurons of the mouse hippocampus can alleviate TBI-induced impairments in learning and memory, while upregulating the expression of Atox1 in the hippocampus reduces neuronal apoptosis and improves TBI-induced memory-related behavioral deficits [29]. These findings indicate that cognitive dysfunction after TBI is closely associated with hippocampal function. Hippocampal inflammatory response is a key factor contributing to TBI-induced cognitive impairment, and inhibition of hippocampal inflammation can effectively alleviate TBI-induced neurological abnormalities. For instance, the traditional Chinese medicine XFZYD exerts neuroprotective effects by downregulating the expression of proinflammatory cytokines in the hippocampus after TBI and inhibiting the activation of astrocytes and microglia [30], and it has been reported that ethanol can alleviate TBI-induced hippocampal inflammation through the SATA6 signaling pathway and prevents neurological dysfunction [31]. Here we found that HMOs can effectively inhibit oxidative stress and neuroinflammatory responses in the hippocampus of TBI mice, which provides a novel strategy for TBI treatment.

Neuroinflammation and neurodegeneration induced by TBI can affect intestinal function; secondary intestinal inflammatory stimuli prolong systemic inflammation, exacerbating TBI-induced neuropathological changes and neurobehavioral deficits [20]. This not only underscores the role of systemic care in TBI treatment but also suggests the possibility of alleviating TBI-related neurological symptoms via the gut. In fact, studies have demonstrated that oral supplementation with nutraceuticals and dietary supplements holds promising prospects in the treatment of TBI. It not only helps improve cognitive impairment and neuroinflammation caused by TBI but is also closely associated with better recovery outcomes in patients [32]. HMOs are naturally occurring endogenous substances in the human body. Compared with chemically synthesized or recombinant protein-based drugs, HMOs have a higher safety threshold and an extremely low risk of side effects with long-term use. Numerous studies have reported the preventive effects of HMOs on nervous system-related diseases. For instance, HMOs can directly interact with rat neurons, thereby inhibiting calcium ion influx, inflammatory responses, and cell apoptosis, which contributes to neural repair in the brain [33]. HMOs can also prevent neuroinflammation in the context of necrotizing enterocolitis via the gut-brain axis [34]. These studies provide indirect evidence that HMOs may have therapeutic potential for neurological damage caused by TBI, while also suggesting the possibility of incorporating HMOs into TBI treatment regimens as a nutraceutical.

Finally, we found that HMOs may improve cognitive dysfunction in TBI mice by indirectly supporting hippocampal neural synaptic function, as evidenced by the upregulated expression of hippocampal synaptophysin and BDNF, and the inhibited expression of pro-BDNF. A study using a rat model of arterial occlusion stroke showed that treatment with the human milk oligosaccharide 2′-FL significantly reduced cerebral infarct size, alleviated microglial activation, improved motor activity, and upregulated BDNF expression [33]. Moreover, the BDNF/TrkB pathway has been confirmed to promote hippocampal synaptic remodeling and improve cognitive function in TBI mice, thereby exerting neuroprotective effects [30]. These findings provide indirect support for the notion that HMOs may alleviate TBI-induced neural damage by improving synaptic function.

This study still has the following limitations. Firstly, we failed to thoroughly clarify the molecular mechanism by which HMOs alleviate TBI. If subsequent studies can correlate HMOs gavage intervention with changes in gut microbiota composition and microbial metabolites using microbiomics and metabolomics techniques, it is expected to provide new research clues for the treatment of TBI. Secondly, although this study indirectly confirmed that HMOs can ameliorate intestinal injury induced by TBI, it has not been clarified whether the neuroprotective effect of HMOs is entirely exerted through the intestinal pathway; whether HMOs can produce therapeutic effects through systemic absorption remains to be further explored. Finally, existing studies have clearly demonstrated the key role of genetic factors in the occurrence and development of TBI [35]. Therefore, clarifying whether HMOs have a regulatory effect on previously reported TBI-related genes (such as Bcl-2, APOE3, and IGF-1) can not only improve the molecular mechanism of HMOs in the treatment of TBI, but also provide an important scientific basis for the development of subsequent targeted therapy regimens.

In summary, this study demonstrates that HMOs can inhibit TBI-induced inflammatory responses in the hippocampus and colon, while alleviating associated damage. This not only broadens the potential clinical utility of HMOs but also holds promise for providing new strategies in the treatment of TBI.

4. Materials and methods

4.1. Human milk oligosaccharides (HMOs)

The preparation of HMOs was referenced to a previous study [36]. Briefly, the prepared HMOs are dissolved in saline, with a final concentration of 5 g/dL. Each 5 g of HMOs contains 2.608 g of 2′-FL (2′-Fucosyllactose, HY-N9965), 0.652 g of 3-FL (3-Fucosyllactose, HY-N10528), 1.304 g of LNT (Lacto-N-tetraose, HY-N9448), 0.174 g of 3′-SL (3′-Sialyllactose, HY-108065), and 0.260 g of 6′-SL (6′-Sialyllactose, HY-N10521). All HMOs were purchased from MCE, China.

4.2. Animal handling

A total of 48 SPF-grade C57BL/6 mice (6–8 weeks old, weighing 20–25 g) were purchased from Vital River Laboratory Animal Technology Co., Ltd. Mice were housed under a 12/12-hour light/dark cycle, with a maximum of five mice per cage, and had ad libitum access to food and water. The mice were randomly divided into a sham-operated group, a TBI group, and a TBI+HMOs group.

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 [37]. 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 [38,39].

The overall experimental design was carried out in accordance with Table 1. All animal studies were approved by Laboratory Animal Management and Use Committee, Hubei Provincial Center for Disease Control and Prevention (Assurance number: 202510038).

Table 1.

Summary table of experimental time points and detection methods.

Time point (post-TBI) Experimental/detection item
Day 0 Model Establishment
Day 0 to endpoint HMOs Administration (TBI+HMOs group only)
Day 13 Novel Object Recognition (NOR)—Habituation
Day 14 1. Novel Object Recognition (NOR)—Training Phase;
2. Tissue/Serum Sample Detection
Day 15 Novel Object Recognition (NOR)—Test Phase
Day 16 Y-Maze (YM) Test
Days 17–21 Morris Water Maze (MWM)—Training Phase
Day 22 Morris Water Maze (MWM)—Test Phase
Open in a new tab

4.3. 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 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.

The antibodies used in this study are as follows: Rabbit polyclonal anti-IL-1β (AF5103) was purchased from Affinity Biosciences. Rabbit monoclonal anti-IL-6 (#12912) was purchased from Cell Signaling TECHNOLOGY. Rabbit monoclonal anti-TNF-α (ab215188) and anti-Occludin (ab216327) were purchased from Abcam. Mouse monoclonal anti-Actin (66009-1-Ig) was purchased from Proteintech. Horseradish peroxidase-labeled goat anti-mouse (SA00001-1) or anti-rabbit (SA00001-2) secondary antibodies were obtained from Proteintech.

4.4. HE staining

For histological staining, brains were fixed in 4% paraformaldehyde overnight at 4 °C and embedded in paraffin. At least three brains from different mice were sectioned and stained with hematoxylin and eosin.

4.5. Measurement of hippocampal ROS activity and serum D-lactic acid

The ROS activity in the mouse hippocampus was detected using the Beyotime ROS Detection Kit (S0038) according to the manufacturer’s instructions.

The serum D-lactic acid was detected using the Abcam D-Lactate Assay Kit (ab83429) according to the manufacturer’s instructions.

4.6. 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-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).

4.7. 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.

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 taken out of the pool and dried. The swimming trajectory of each mouse is recorded by a camera.

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.

4.8. 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 was placed therein, with free exploration of the maze permitted for 5 minutes. The behavioral activity of the mice was analyzed using Ethovision XT software, and entries into each arm (Arms A–C) were recorded. An arm entry was counted when all four paws of the mouse entered the arm; an alternation was defined as consecutive entries into three distinct arms. The spontaneous alteration was calculated as follows: (total alternations × 100)/(total arm entries − 2). A mouse was considered to have intact spatial working memory if its spontaneous alteration exceeded 50% (the chance level for choosing the unfamiliar arm).

4.9. New object recognition (nor) test

The NOR experiment was performed on 14th to 15th days post-TBI. The test was conducted in an apparatus made of 0.75 cm thick white plastic board, with dimensions of 24 × 24 × 24 cm³. One day before the test, each mouse was individually placed into the behavioral apparatus for 1 hour to habituate to the environment.

In the habituation phase of the test, each test mouse was placed in the center of the apparatus and allowed to move freely to fully explore the entire apparatus for 10 minutes. The training phase was carried out one day after the habituation phase. Each test mouse was individually placed in the center of the apparatus and allowed to move freely for 10 minutes to fully explore, with two identical blue cylinders (object 1) placed at the two top corners of the apparatus.

The recognition phase was performed one day after the training phase. Each test mouse was placed in the center of the apparatus and allowed to move freely to fully explore for 10 minutes, with one blue cylinder (object 1) and one green cone (object 2) placed at the top corners of the apparatus.

During the test, video recordings were made using a Dahua high-definition camera; the time spent by the mice interacting with each object (object 1) or the new object (object 2) (with the distance from the nose tip to the object ≤ 3 cm) was analyzed using Ethovision XT software. Recognition index = Time to recognize new objects/(Time to recognize new objects + Time to recognize familiar objects).

4.10. Statistical analyses

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 all statistical significance indications in this manuscript, ****, p < 0.0001; ***, p < 0.001; **, p < 0.01 and *, p < 0.05.

Acknowledgments

We confirm that no generative AI tools were used in the process of writing and revising this manuscript.

Funding Statement

This manuscript was funded by: The Health Research Foundation of Hunan Provincial Health Commission (No.W20243153). The Basic & Application Research Foundation of Hengyang Science & Technology Bureau (No.202330046344). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Author contributions

Dongliang He: Conceptualization, Methodology, Supervision, Writing—original draft. Renli He: Software, Investigation. Wei Duan: Software, Investigation. Guilan Li: Software, Investigation. Qin Kang: Writing—review & editing, Supervision.

Ethical disclosure statement

All animal studies were approved by Laboratory Animal Management and Use Committee, Hubei Provincial Center for Disease Control and Prevention (Assurance number: 202510038).

Disclosure statement

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Data availability statement

The authors confirm that the data supporting the findings of this study are available within the article and its supplementary materials.

References

  • 1.Dewan MC, Rattani A, Gupta S, et al. Estimating the global incidence of traumatic brain injury. J Neurosurg. 2019;130(4):1080–1097. doi: 10.3171/2017.10.JNS17352 [DOI] [PubMed] [Google Scholar]
  • 2.Rubiano AM, Carney N, Chesnut R, et al. Global neurotrauma research challenges and opportunities. Nature. 2015;527(7578):S193–S197. doi: 10.1038/nature16035 [DOI] [PubMed] [Google Scholar]
  • 3.Yang DD, Wan XD, Chen AD, et al. Characteristics of traumatic brain injury models: from macroscopic blood flow changes to microscopic mitochondrial changes. Neural Regen Res. 2023;18(10):2268–2277. doi: 10.4103/1673-5374.369125 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Jellinger KA. Traumatic brain injury as a risk factor for Alzheimer’s disease. J Neurol Neurosurg Psychiatry. 2004;75(3):511–512. doi: 10.1186/1471-2377-1-3 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Bagnato S, Boccagni C.. Moderate/severe traumatic brain injury as a trigger of chronic neurodegeneration in humans. Neural Regen Res. 2020;15(7):1247–1248. doi: 10.4103/1673-5374.272574 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Bramlett HM, Dietrich WD.. Long-term consequences of traumatic brain injury: current status of potential mechanisms of injury and neurological outcomes. J Neurotrauma. 2015;32(23):1834–1848. doi: 10.1089/neu.2014.3352 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Muresanu IA, Grad DA, Muresanu DF, et al. The effect of cerebrolysin on anxiety, depression, and cognition in moderate and severe traumatic brain injury patients: a CAPTAIN II retrospective trial analysis. Medicina (Kaunas). 2022;58(5):648. doi: 10.3390/medicina58050648 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Al-Mufti F, Amuluru K, Changa A, et al. Traumatic brain injury and intracranial hemorrhage-induced cerebral vasospasm: a systematic review. Neurosurg Focus. 2017;43(5):E14. doi: 10.3171/2017.8.FOCUS17431 [DOI] [PubMed] [Google Scholar]
  • 9.Karlander M, Ljungqvist J, Sörbo A, et al. Risk and cause of death in post-traumatic epilepsy: a register-based retrospective cohort study. J Neurol. 2022;269(11):6014–6020. doi: 10.1007/s00415-022-11279-5 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Rijks V, Zuurveld M, Garssen J, et al. The potential immunomodulatory role of human milk oligosaccharides in prevention of viral infections and development of asthma in early life. Front Immunol. 2025;16:1572787. doi: 10.3389/fimmu.2025.1572787 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Ayechu-Muruzabal V, van Stigt AH, Mank M, et al. Diversity of human milk oligosaccharides and effects on early life immune development. Front Pediatr. 2018;6:239. doi: 10.3389/fped.2018.00239 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Wang J, Chen MS, Wang RS, et al. Current advances in structure-function relationships and dose-dependent effects of human milk oligosaccharides. J Agric Food Chem. 2022;70(21):6328–6353. doi: 10.1021/acs.jafc.2c01365 [DOI] [PubMed] [Google Scholar]
  • 13.Zuurveld M, van Witzenburg NP, Garssen J, et al. Immunomodulation by human milk oligosaccharides: the potential role in prevention of allergic diseases. Front Immunol. 2020;11:801. doi: 10.3389/fimmu.2020.00801 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Sato K, Nakamura Y, Fujiyama K, et al. Absolute quantification of eight human milk oligosaccharides in breast milk to evaluate their concentration profiles and associations with infants’ neurodevelopmental outcomes. J Food Sci. 2024;89(12):10152–10170. doi: 10.1111/1750-3841.17597 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Falsaperla R, Sortino V, Gambilonghi F, et al. Human milk oligosaccharides and their pivotal role in gut-brain axis modulation and neurologic development: a narrative review to decipher the multifaceted interplay. Nutrients. 2024;16(17):3009. doi: 10.3390/nu16173009 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Gao H, Fang B, Sun Z, et al. Effect of human milk oligosaccharides on learning and memory in mice with Alzheimer’s disease. J Agric Food Chem. 2024;72(2):1067–1081. doi: 10.1021/acs.jafc.3c05949 [DOI] [PubMed] [Google Scholar]
  • 17.Jia M, Wang X, Ning F, et al. Human milk oligosaccharide 2’-fucosyllactose alleviates cognitive impairment via the vagal afferent pathway in Alzheimer’s disease mice. Food Funct. 2025;16(13):5345–5362. doi: 10.1039/d4fo06272h [DOI] [PubMed] [Google Scholar]
  • 18.Brezova V, Moen KG, Skandsen T, et al. Prospective longitudinal MRI study of brain volumes and diffusion changes during the first year after moderate to severe traumatic brain injury. Neuroimage Clin. 2014;5:128–140. doi: 10.1016/j.nicl.2014.03.012 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Kim S, Jung UJ, Kim SR.. Role of oxidative stress in blood-brain barrier disruption and neurodegenerative diseases. Antioxidants (Basel). 2024;13(12):1462. doi: 10.3390/antiox13121462 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Hanscom M, Loane DJ, Shea-Donohue T.. Brain-gut axis dysfunction in the pathogenesis of traumatic brain injury. J Clin Invest. 2021;131(12):e143777. doi: 10.1172/JCI143777 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Taraskina A, Ignatyeva O, Lisovaya D, et al. Effects of traumatic brain injury on the gut microbiota composition and serum amino acid profile in rats. Cells. 2022;11(9):1409. doi: 10.3390/cells11091409 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Ma EL, Smith AD, Desai N, et al. Bidirectional brain-gut interactions and chronic pathological changes after traumatic brain injury in mice. Brain Behav Immun. 2017;66:56–69. doi: 10.1016/j.bbi.2017.06.018 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Wang C, Jiang W, Leitz J, et al. Structure and topography of the synaptic V-ATPase-synaptophysin complex. Nature. 2024;631(8022):899–904. doi: 10.1038/s41586-024-07610-x [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Gustafsson D, Klang A, Thams S, et al. The role of BDNF in experimental and clinical traumatic brain injury. Int J Mol Sci. 2021;22(7):3582. doi: 10.3390/ijms22073582 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Dinleyici M, Barbieur J, Dinleyici EC, et al. Functional effects of human milk oligosaccharides (HMOs). Gut Microbes. 2023;15(1):2186115. doi: 10.1080/19490976.2023.2186115 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Sprenger N, Tytgat HLP, Binia A, et al. Biology of human milk oligosaccharides: from basic science to clinical evidence. J Hum Nutr Diet. 2022;35(2):280–299. doi: 10.1111/jhn.12990 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Hill DR, Chow JM, Buck RH.. Multifunctional benefits of prevalent HMOs: implications for infant health. Nutrients. 2021;13(10):3364. doi: 10.3390/nu13103364 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Bielefeld P, Martirosyan A, Martín-Suárez S, et al. Traumatic brain injury promotes neurogenesis at the cost of astrogliogenesis in the adult hippocampus of male mice. Nat Commun. 2024;15(1):5222. doi: 10.1038/s41467-024-49299-6 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Zhao J, Bang S, Furutani K, et al. PD-L1/PD-1 checkpoint pathway regulates hippocampal neuronal excitability and learning and memory behavior. Neuron. 2023;111(17):2709–2726.e9. doi: 10.1016/j.neuron.2023.05.022 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Pei Z, Guo X, Zheng F, et al. Xuefu Zhuyu decoction promotes synaptic plasticity by targeting miR-191a-5p/BDNF-TrkB axis in severe traumatic brain injury. Phytomedicine. 2024;129:155566. doi: 10.1016/j.phymed.2024.155566 [DOI] [PubMed] [Google Scholar]
  • 31.Olde Heuvel F, Holl S, Chandrasekar A, et al. STAT6 mediates the effect of ethanol on neuroinflammatory response in TBI. Brain Behav Immun. 2019;81:228–246. doi: 10.1016/j.bbi.2019.06.019 [DOI] [PubMed] [Google Scholar]
  • 32.Lucke-Wold B, Zasler ND, Ruchika F, et al. Supplement and nutraceutical therapy in traumatic brain injury. Nutr Neurosci. 2025;28(6):709–743. doi: 10.1080/1028415X.2024.2404782 [DOI] [PubMed] [Google Scholar]
  • 33.Wu KJ, Chen YH, Bae EK, et al. Human milk oligosaccharide 2’-fucosyllactose reduces neurodegeneration in stroke brain. Transl Stroke Res. 2020;11(5):1001–1011. doi: 10.1007/s12975-019-00774-z [DOI] [PubMed] [Google Scholar]
  • 34.Sodhi CP, Ahmad R, Fulton WB, et al. Human milk oligosaccharides reduce necrotizing enterocolitis-induced neuroinflammation and cognitive impairment in mice. Am J Physiol Gastrointest Liver Physiol. 2023;325(1):G23–G41. doi: 10.1152/ajpgi.00233.2022 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Lakshmipathy D, Rangarajan S, Barreau A, et al. Genetic contributions to recovery following brain trauma: a narrative review. Front Biosci (Landmark Ed). 2024;29(3):103. doi: 10.31083/j.fbl2903103 [DOI] [PubMed] [Google Scholar]
  • 36.Sodhi CP, Scheese DJ, Tragesser C, et al. Necrotizing enterocolitis: specific human milk oligosaccharides prevent enteric glia loss and hypomotility. Pediatr Res. 2025;98(4):1500–1510. doi: 10.1038/s41390-025-04077-y [DOI] [PubMed] [Google Scholar]
  • 37.Huang X, An Y, Liu J, et al. The neuroprotective effect of 10-hydroxy-2-decenoic acid in traumatic brain injury by inhibiting copper-mediated neuronal pyroptosis. Phytomedicine. 2025;142:156816. doi: 10.1016/j.phymed.2025.156816 [DOI] [PubMed] [Google Scholar]
  • 38.Wu X, Cao T, Ye J, et al. Supplementation of 2’-fucosyllactose during the growth period improves neurodevelopmental disorders in offspring mice induced by maternal immune activation. J Agric Food Chem. 2025;73(20):12292–12307. doi: 10.1021/acs.jafc.5c01184 [DOI] [PubMed] [Google Scholar]
  • 39.Brosseau C, Rousseaux A, Le Romancer M, et al. “Microbial and immune modulation by 2’-fucosyllactose supplementation during gestation: a strategy to prevent food allergies. Gut Microbes. 2025;17(1):2523813. doi: 10.1080/19490976.2025.2523813 [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The authors confirm that the data supporting the findings of this study are available within the article and its supplementary materials.

Articles from Future Science OA are provided here courtesy of Taylor & Francis

RESOURCES