Gu et al. Journal of Neuroinflammation(2024) 21:147
https://doi.org/10.1186/s12974-024-03116-5

Journal of Neuroinflammation

Open Access

RESEARCH

Prevotella copri transplantation promotes
neurorehabilitation in a mouse modelof traumatic brain injury
Nina Gu1, Jin Yan1, Wei Tang1, Zhaosi Zhang1, Lin Wang1,2, Zhao Li1,3, Yingwen Wang1, Yajun Zhu1, Shuang Tang1,4,
Jianjun Zhong1, Chongjie Cheng1*, Xiaochuan Sun1* and Zhijian Huang1*
Abstract
Background The gut microbiota plays a critical role in regulating brain function through the microbiome-gut-brain
axis (MGBA). Dysbiosis of the gut microbiota is associated with neurological impairment in Traumatic brain injury(TBI) patients. Our previous study found that TBI results in a decrease in the abundance of Prevotella copri (P. copri). P.
copri has been shown to have antioxidant effects in various diseases. Meanwhile, guanosine (GUO) is a metabolite ofintestinal microbiota that can alleviate oxidative stress after TBI by activating the PI3K/Akt pathway. In this study, weinvestigated the effect of P. copri transplantation on TBI and its relationship with GUO-PI3K/Akt pathway.
Methods In this study, a controlled cortical impact (CCI) model was used to induce TBI in adult male C57BL/6J mice.
Subsequently, P. copri was transplanted by intragastric gavage for 7 consecutive days. To investigate the effect of
the GUO-PI3K/Akt pathway in P. copri transplantation therapy, guanosine (GUO) was administered 2 h after TBI for 7
consecutive days, and PI3K inhibitor (LY294002) was administered 30 min before TBI. Various techniques were used toassess the effects of these interventions, including quantitative PCR, neurological behavior tests, metabolite analysis,
ELISA, Western blot analysis, immunofluorescence, Evans blue assays, transmission electron microscopy, FITC-dextran
permeability assay, gastrointestinal transit assessment, and 16 S rDNA sequencing.
Results P. copri abundance was significantly reduced after TBI. P. copri transplantation alleviated motor and cognitive
deficits tested by the NSS, Morris’s water maze and open field test. P. copri transplantation attenuated oxidative
stress and blood-brain barrier damage and reduced neuronal apoptosis after TBI. In addition, P. copri transplantation
resulted in the reshaping of the intestinal flora, improved gastrointestinal motility and intestinal permeability.
Metabolomics and ELISA analysis revealed a significant increase in GUO levels in feces, serum and injured brain
after P. copri transplantation. Furthermore, the expression of p-PI3K and p-Akt was found to be increased after P.
copri transplantation and GUO treatment. Notably, PI3K inhibitor LY294002 treatment attenuated the observed
improvements.

*Correspondence:
Chongjie Cheng
358187887@qq.com
Xiaochuan Sun
sunxiaochuan@cqmu.edu.cn
Zhijian Huangzhijian@cqmu.edu.cn
Full list of author information is available at the end of the article© The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use,sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) andthe source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in thisarticle are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not includedin the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you willneed to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The
Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available
in this article, unless otherwise stated in a credit line to the data.

Gu et al. Journal of Neuroinflammation(2024) 21:147

Page 2 of 25

Conclusions We demonstrate for the first time that P. copri transplantation can improve GI functions and alter gut
microbiota dysbiosis after TBI. Additionally, P. copri transplantation can ameliorate neurological deficits, possibly via the
GUO-PI3K/Akt signaling pathway after TBI.
Keywords Traumatic brain injury, Prevotella copri, Neurorehabilitation, Gut microbiota, Guanosine, GUO-PI3K/Akt
pathway

Background
Traumatic brain injury (TBI) represents an enormous
challenge to public health. More than 50 million people
worldwide suffer TBI each year, and it is estimated thatapproximately half of the world’s population will experience one or more TBI during their lifetime [1]. It is wellestablished that secondary injury is the major cause of
TBI-associated brain damage. In the past few decades,much progress has been made in studying the pathological mechanisms of TBI and in developing therapeuticstrategies [2, 3]. Targeting the unfavorable lesion microenvironment such as free radicals and neuroinflammation after TBI holds great promise for restoring neuralfunction [4]. Excessive reactive oxidative stress (ROS)
production is associated with neuroinflammation andchronic neurodegeneration following TBI [5]. Inhibition of oxidative stress after TBI markedly reduces proinflammatory activation of macrophages/microglia witha concomitant increase in anti-inflammatory responses[6].Therefore, anti-oxidative stress treatment helps alleviate neuroinflammation after TBI. Although anti-oxidative stress target and anti-neuroinflammation target
are fairly distinct, they are interwoven, interactive andinterdependent.
At present, many drugs showed good therapeutic
effects in experimental animals, but most of their clinical
trials failed, it still remains a challenging task to develop
an effective strategy for the treatment of TBI [7]. On theother hand, it has been found that some central nervous
system (CNS) diseases, including acute ischemic stroke[8, 9], Alzheimer’s disease [10] and Parkinson’s disease[11], can be treated by regulating the gut microbiome.
Likewise, it has been found that TBI can cause changes in
the intestinal microbiota, and the intestinal flora changes
significantly in number and categories, mainly involving the pseudorod Bacteria, Purpuromonas, Firmicutes,
Proteobacteria among others [12, 13]. In our previous
research, we found that fecal microbiota transplantation
can restore gut microbiota dysbiosis and alleviate neurological deficits after TBI [14]. Randall et al. found that thechange in Prevotella was the most significant microbiota
after TBI, and the abundance of P. copri in Prevotella
was significantly reduced by 4.5-fold [15]. However, indepth studies on how to affect the disease course after
TBI by regulating a specific strain of gut microbiota are
still lacking. Prevotella are Gram-negative anaerobes thathave previously been identified as potential key factors inshaping gut function and host health [16]. Prevotella canferment dietary fiber to produce the short chain fatty acid
(SCFA) acetate. They can also metabolize dietary fiberand fat to produce succinate [17, 18]. P. copri is an abundant member of the human gastrointestinal microbiota.
The effect of P. copri on human health is currently inconclusive. Its relative abundance may play a positive or
negative role in disease [19]. Whether P. copri transplantation can have a protective effect on TBI’s neurological
function is still in need of further confirmation.
Gut bacteria regulate the central nervous systems
through dynamic bidirectional communication along the‘gut–brain axis’ in a microbial metabolites-dependent
manner [20]. Guanosine (GUO) is an intermediate product of nucleotide metabolism, which is produced by thedecomposition of GMP by the 5 ‘-nucleotidase (5’ -NT)of intestinal bacteria, and is absorbed into the bloodby intestinal epithelial cells. Then GUO is transported
through the Nucleotide transporter on the surface of
cerebral microvascular endothelial cells and enters thebrain tissue rapidly through the blood brain barrier (BBB)[21, 22]. Guanosine plays an important neuroprotectiverole in CNS neural injury. Guanosine is neuroprotective
against focal cerebral ischemia by inhibiting microglia/
macrophage activation and mediating anti-inflammatory
response that ameliorates neural injury [23]. Guanosine
inhibits LPS-induced pro-inflammatory response andoxidative stress in hippocampal astrocytes [24]. Previous
studies have confirmed that GUO can alleviate oxidative
stress after TBI by activating PI3K/Akt pathway in astrocytes and hippocampus cultured in vitro [25]. GUO also
reduces mitochondrial oxidative stress damage caused byinhibition of mitochondrial complex by activating PI3K/
Akt pathway in neuroblastocytes [26].
In this study, we hypothesized that P. copri transplantation could relieve the neurological deficits in a TBI
mouse model in a GUO-dependent manner. GUO might
be upregulated by P. copri treatment and thereafter activating the antioxidant PI3K/Akt pathway.

Materials and methods
Animals

Adult C57BL/6J mice (18–22 g, 8 weeks old) were purchased from the Laboratory Animal Center of Chongqing Medical University (Chongqing, China). The groups
were housed separately. Mice were kept under standard
conditions (temperature, 22 ± 2 °C; humidity, 55 ± 10%)

Gu et al. Journal of Neuroinflammation(2024) 21:147

with a 12:12 light/dark cycle. Food and water were available ad libitum. All procedures were performed in a
specific pathogen-free (SPF) environment, and all tools
and materials were sterilized with 75% alcohol. The
type of bedding was poplar wood shavings (Chongqing Tengxin Biotechnology Co., LTD), the food wasstandard chow(Beijing Huafukang Biotechnology Co.,LTD), the water was autoclaved purified water.All procedures on animals were approved by the Institutional
Animal Care and Use Committee of Chongqing Medical University(approval No. 2,019,335) and carried out inaccordance with ARRIVE guidelines [27].
CCI model

A standard protocol and a controlled cortical impact(CCI) device (TBI-0310, Precision Systems and Instrumentation, Fair fax, VA, USA) were utilized to induce
brain injuries as described previously [28]. Mice wereanesthetized with isoflurane (3% induction, 2% maintenance) when they underwent any surgeries. Briefly, acircular craniotomy was performed in the right sensorimotor cortex and above the hippocampus (center of the
impact: A/P, − 2.00 mm; M/L, 2.50 mm from bregma).
Following craniotomy, a CCI model was established witha TBI-0310 TBI model system, and the impact parameters were set as follows: 5 m/s velocity, 100 ms dwellingtime, 2 mm depth and 3 mm diameter impactor. A pneumatic impactor was used to provide the power for impact(Jun-Air Model 3–4). Sham control group mice underwent only craniotomy without CCI. Following the injury,
the hole was sealed with bone wax, and the skin incision
was sutured. Finally, the mice were kept on an electricblanket (69,001, RWD Life Science, China) to maintaintheir body temperature at 37.5 ± 0.5 °C until completely
awakening (about 3–4 h).
Pharmacological intervention

In accordance with a previous study, antibiotics intervention was used to remove intestinal flora and minimize
the difference in intestinal flora among the experimental groups of mice before the CCI operation or P. copri
treatment [29]. Before the CCI operation, we administered antibiotics to adult mice (6 weeks of age) in allgroups through drinking water containing 0.2 g/L ampicillin (Macklin, China), neomycin (Macklin, China),
metronidazole (Macklin, China) and 0.1 g/L vancomycin (Macklin, China) daily for 2 weeks. At 2 h after TBI,
the TBI + Vehicle group received 0.1 ml of 10% glycerol
in PBS solution, while the TBI + P. copri group received
a dose of 0.1 ml containing 5 × 106 CFU/mL P. copri in
10% glycerol in PBS via intragastric gavage [30]. Then,the same perfusion method was performed for 7 consecutive days (every morning at 9–10 a.m.). As previously
described, GUO (Macklin, China) solution dissolved inPage 3 of 25

0.9% saline was maintained at 37 °C before injections toprevent any drug precipitation [31]. GUO (7.5 mg/kg)
was intraperitoneally administered (1 mL/kg) to mice atevery day for 7 days (every morning at 9–10 a.m.) andthe first administration was performed 2 h after TBI.
LY294002 (Macklin, China) solution dissolved in 5%
dimethyl sulfoxide and 25 mg/kg LY294002 were administered via intracerebroventricular injection 30 min
before TBI [32].
Experimental protocols

All experimental protocols and setups are shown in
Fig. 1.
Experiment 1

To determine the time course of P. copri abundance inthe feces of mice after TBI. Feces sample from a group of
three mice feces were collected at different time periods:
before TBI, 1 d, 3 d, 7 d, 14 d, 21 d, and 28 d after TBI. A
quantitative real-time PCR (QPCR) assay was performed
to evaluate the changes in P. copri abundance.
Experiment 2

To evaluate the effects of P. copri on neurological function in mice after TBI, 32 mice were randomly divided
into four groups: sham group, TBI group, TBI + Vehicle
group, TBI + P. copri group (n = 8 per group). All mice
were subjected to Neurological Severity Scores (NSS) andwire grip tests to evaluate short-term neurological function. Subsequently, all mice were subjected to the Morris water maze (MWM) and the open field test (OFT) toevaluate learning and memory function. The OFT and
MWM were performed up to 2 weeks after TBI.
Experiment 3

To evaluate the effects of P. copri transplantation on bodyweight, food intake, intestinal permeability and microbiome analysis in TBI, mice were randomly divided into
three groups: sham group, TBI group, TBI + P. copri
group (n = 18 per group). We randomly recorded bodyweight and food consumption of 8 mice in each group.
Four mice were subjected to immunofluorescence staining, 8 mice were used for FITC-dextran permeability
assay, and 6 mice were used for microbiome analysis. To
evaluate the effects of P. copri transplantation on gastrointestinal motility, mice were randomly divided into fourgroups: sham group, sham + P. copri group, TBI group,
TBI + P. copri group (n = 4 per group), and 4 mice in eachgroup were used for gastrointestinal transit assessment.
Experiment 4

To estimate the effects of P. copri on serum metabolites
in TBI mice, 15 mice were randomly divided into threegroups: the sham group, the TBI group, and the TBI + P.

Gu et al. Journal of Neuroinflammation(2024) 21:147

Page 4 of 25

Fig. 1 Schematic diagram of the different experimental protocols and setups used in this study. P. copri, Prevotella copri; CCI, controlled cortical impact;
TBI, traumatic brain injury; WB, western blot; IF, immunofluorescence; GUO, guanosine; BBB, blood-brain barrier; TME, transmission electron microscopyGu et al. Journal of Neuroinflammation(2024) 21:147

copri group (n = 5 per group). After 7 days of P. copri
treatment, serum from each mouse was collected formetabolite analysis.
Experiment 5

To evaluate the time course of GUO presence in the feces,serum and brain tissue of mice at the injury site after P.
copri treatment, 48 mice were used in this experiment:[1] 24 mice were continuously treated with P. copri for
7 days after TBI and randomly divided into four groups:
sham, TBI + P. copri 1d, TBI + P. copri 3d, and TBI + P.
copri 7d (n = 6 per group); [2] as a control, 24 mice were
not transplanted with P. copri after TBI and were randomly divided into four groups: sham, TBI 1d, TBI 3d,
and TBI 7d (n = 6 per group). ELISA was performed toevaluate the changes in GUO presence.
Experiment 6

To investigate the effect of P. copri transplantation onoxidative stress, blood-brain barrier (BBB) permeability
and neuronal apoptosis in TBI mice, mice were randomly
divided into four groups: sham group, TBI group, TBI + P.
copri group, and TBI + GUO group. For oxidative stressstudy, every 6 mice in each group were assigned to Catalase (CAT) measurements, Superoxide dismutase (SOD)measurements, Reactive oxygen species (ROS) measurements, Oxidized glutathione (GSSG) and Reduced
glutathione (GSH)/GSSG ratio measurements. For BBB
permeability study, every 6 mice were assigned to Western blot analysis; 4 mice were assigned to immunofluorescence staining of Occludin; 6 mice were assigned toEvans blue (EB) assay, 4 mice were assigned to EB fluorescence observation; and 4 mice were assigned to transmission electron microscopy examination. For neuronal
apoptosis study, 4 mice were assigned to immunofluorescence staining.
Experiment 7

To determine whether the GUO-PI3K/Akt pathway
plays a role in the neuroprotective effects of P. copri
transplantation, mice were randomly divided into six
groups: sham group, TBI group, TBI + P. copri group,
TBI + GUO group, TBI + P. copri + LY294002 group, and
TBI + GUO + LY294002 group. Western blot analysis was
performed to detect key proteins (p-PI3K, p-Akt) in the
GUO-PI3K/Akt pathway using 6 mice per group. For
behavioral tests, 8 mice were assigned to each group as
carried out in Experiment 2.
Experiment 8

To determine whether the GUO-PI3K/Akt pathway is
involved in the effects of P. copri on oxidative stress,
blood-brain barrier (BBB), and neuronal apoptosis in
TBI mice, mice were randomly divided into five groups:

Page 5 of 25

sham group, TBI group, TBI + P. copri group, TBI + P.
copri + LY294002 group and TBI + GUO + LY294002
group. For oxidative stress study, every 6 mice in eachgroup were assigned to SOD measurements and ROS
measurement. For BBB permeability study, every 6 mice
were assigned to Western blot analysis of ZO-1 andOccludin; 4 mice were assigned to immunofluorescencestaining of Occludin. For neuronal apoptosis study, 6
mice were assigned to Western blot analysis of Bcl-2 andBax.
Real-time quantitative PCR (qPCR)

Total DNA from mouse feces was extracted using TRIzol(Invitrogen, Carlsbad, CA, USA) based on the manufacturer’s protocol. Confirmation of colonization wasachieved with pcopri2F_pcopri2R (F: ​A​C​C​A​C​T​T​G​G​G​G​
A​T​A​A​C​C​T​T​G, R: ​T​A​C​A​T​G​C​A​A​A​A​A​G​C​C​T​C​A​C​G​A​G​
G​C). Detailed procedures are provided in Supplemental
File.
Neurobehavioral assessment

The investigators were blinded to the experimentalgroups for all tests. Tests were conducted in a quiet roomat 22 ± 2 °C and 55 ± 10% relative humidity.
NSS scores and wire grip test

NSS analyses and wire grip tests were performed inaccordance with previous research [33]. The Neurological Severity Scores (NSS) measured general behavior,alertness, balance and motor ability using ten different
tasks. Two researchers who were blinded to each groupused NSS to assess the neurologic deficits on 1 day preinjury and on days 1, 3 and 7 after TBI. One point was
obtained for each failed task. Zero points represented theminimum deficit, and ten points represented the maximum deficit. Table 1 in supplemental file shows details of
the NSS score.
Wire grip tests were used to evaluate vestibular motor
function. A 45-cm-long, 3-mm-diameter metal wire
was suspended 45 cm above the ground with two vertical wooden sticks. Mice were placed on the bar midway
between the supports and were observed for 60 s, andthen the latency to fall was recorded and assessed.
Open field test (OFT)As previously reported [34], On day 14 after TBI, we used
the open-field experiment to assess anxiety behavior inmice. The equipment used for the open-field experimentconsisted of an organic 50 cm×50 cm×50 cm polyvinyl
chloride box. The bottom was uniformly divided into 25
small compartments, and its interior and bottom werecovered with a black opaque and non-reflective coating.
A digital camera was installed 2 m above the plate. The
entire interior of the field was visible, with the 9 centerGu et al. Journal of Neuroinflammation(2024) 21:147

Page 6 of 25

Table 1 Neurological severity score (NSS) for head-injured mice
Task
Presence of mono- or hemiparesis
Inability to walk on a 3-cm-wide beam 1
Inability to walk on a 2-cm-wide beam
Inability to walk on a 1-cm-wide beam
Inability to balance on a 1-cm-wide beam
Inability to balance on a round stick (0.5 cm wide)
Failure to exit a 30-cm-diameter circle (for 2 min)Inability to walk straight
Loss of startle behavior
Loss of seeking behavior
Maximum total

NSS
1
1
1
1
1
1
1
1
1
1
10

One point is awarded for failure to perform a task

grids in the central area and the remaining grids in the
peripheral area. A video tracking system (ANY-mazeStoelting USA) was used to monitor the behavior of themice, and the entire measurement process lasted for
5 min. The overall time in the center area and the total
distance during the recording were recorded and analyzed. The experiment time is between 10am and 3pm ata quiet environment.
Morris water maze test (MWM)

Cognitive function was assessed using the Morris water
maze test for 6 consecutive days. Briefly, the latency tofind the platform was measured from day 15 to 19 postCCI using the navigation test. The time spent in the correct quadrant was measured on day 20 post-CCI using
the probe trial test [33]. The Morris water maze test was
performed using a swimming pool (1.2 m diameter,40 cm
height). A platform (8 cm diameter,20 cm height) was
placed in one of four quadrants of the pool. The pool was
filled with water (22 ± 1 °C) to a depth of 5 cm above theplatform. Visual cues (i.e., black landmark) were placedon the wall of the water maze. The mice behavior wasrecorded using an camera that was placed above the pool,and ANYmaze 7.0 software system was used for recording and analysis. The data were exported to Microsoft
Excel. The order of the animals was randomized withintrials. The test consisted of 5 days of training (four trials,
60 s each). Memory was tested at day 6. For the test, theplatform was removed, and the animals were allowed toswim in the pool for 60 s. The time to locate the platform
was measured during all training days. Swimming speed
was also recorded to assess motor performance. Data
from the spatial learning test and the memory test wererecorded and analyzed using a video-based tracking system (ANY-maze, Stoelting, USA).
FITC-dextran intestinal permeability assay

Measurement of serum concentration of 4-kDa fluorescein isothiocyanate-dextran (FITC-dextran) represents

for intestinal permeability. Seven days after TBI, mice
were fasted for 14 h and gavage with 4-kDa FITC-dextran (Sigma-Aldrich, Madrid, Spain) at a dose of 60 mg
per 100 g body weight in a volume of 0.2 ml. Four hours
later, blood was collected by cardiac puncture and clottedfor 30 min, followed by centrifugation at 6000 g for 90 s.
Serum was diluted equally with PBS, and 100 µL dilutions were measured in a 96-well plate at 481 nm excitation and 524 nm emission. FITC-dextran was quantified
by reference to standard curve measurements in thesame plate [29].
Gastrointestinal transit assessment

GI motility was assessed by radiographic methods 7
days after TBI. Specifically, animals were intragastricallygavaged with barium (0.2 ml, 2 g/ml). All mice were anesthetized with isoflurane (3% induction, 2% maintenance)
and immobilized in the supine position temporally by fixing the limbs on the exam stage with adhesive tapes. Plain
radiographs of the gastrointestinal tract were obtainedusing a digital radiographer (Siemens, Germany). To further reduce stress, mice were released immediately after
each imaging session (immobilization lasted 1–2 min).
Radiographs were taken at different times (1, 3, and 8 hafter barium administration). Analysis of the radiographs
was performed by a trained investigator blinded to thedifferent groups [35]. Changes in GI motility were determined semiquantitatively from the images by assigning acomposite value to the indicated regions of the GI tract,taking into account the following parameters: percentage of the GI region filled with contrast (0–4); intensityof the contrast (0–4); homogeneity of the contrast (0–2);
and sharpness of the profile of the GI region (0–2). Eachof these parameters was scored and a total score (0–12
points) was calculated. X-ray images were also analyzed
morphometrically using ImageJ software (ImageJ 1.4,
NIH, Bethesda, MD, USA).

Gu et al. Journal of Neuroinflammation(2024) 21:147

Page 7 of 25

Serum from each mouse was collected in eppendorf
tubes on day 7 after TBI and stored at -80 °C. Samples
were sent to Shanghai Biotree Biomedical Technology
Co., Ltd. for analysis. Targeted metabolomics analysis
was performed by the ultra-performance liquid chromatography coupled with triple-quadrupole tandem mass
spectrometry (UPLC-QqQ-MS/MS) and was provided
by BIOTREE (Shanghai, China) [37].

nonfat milk for 1 h at room temperature and then incubated overnight at 4 °C with primary antibodies, including rabbit monoclonal anti-β-actin (1:5000, ab213262,
Abcam), rabbit polyclonal anti-ZO-1 (1:1000, 21773-1AP, Proteintech), rabbit polyclonal anti-Occludin (1:1000,
27260-1-AP, Proteintech), rabbit monoclonal anti-Bcl-2(1:1000, ab182858, Abcam), rabbit monoclonal anti-Bax(1:1000, ab182733, Abcam), rabbit polyclonal anti-p-Akt(1:1000, 28731-1-AP, Proteintech), rabbit polyclonal antiAkt (1:1000, 10176-2-AP, Proteintech), rabbit polyclonalanti-p-PI3K (1:1000, #4228, Cell Signaling), and rabbit
polyclonal anti-PI3K (1:1000, 60225-1-lg, Proteintech).
After being washed with Tris-buffered saline/Tween-20,
membranes were incubated for 1 h at room temperature
with horseradish peroxidase-conjugated AffiniPure goatanti-rabbit IgG (1:5000; SA00001-2, RRID: AB_2722564,
Proteintech). Enhanced chemiluminescence was used todetect proteins in the membranes(ECL Plus, Millipore),and proteins were quantified using the Fusion system(Fusion-FX7 Spectra, Vilber, France). All raw Western
blot bands are shown in Supplemental Figures.

Enzyme-linked immunosorbent assay (ELISA)

Immunofluorescence staining

The brain tissue of ipsilateral injured hemisphere andfeces were homogenized with an appropriate amountof normal saline (saline: fecal/brain tissue = 9 ml:1 g),
and the supernatant was removed by centrifugation
at 3000 rpm for 10 min at 4℃. The amount of GUO in
the supernatant and serum was measured by a mouse
cyclic guanosine monophosphate (guanosine) ELISA kit(MEIKE Jiangsu Sumeike Biological Technology Co., Ltd)according to the manufacturer’s instructions [38]. The
amount of GUO was normalized to protein content.

As previously reported [42], the mice were euthanizedunder deep anesthesia with phenobarbital sodium(40 mg/kg, i.p.) and then perfused with phosphate buffer
saline (PBS) and 4% paraformaldehyde (PFA) for fixation.
The collected brains were postfixed overnight at 4 °C in
4% PFA and then cryoprotected in graded sucrose (20%and 30%). Next, the brains were embedded in optimal
cutting temperature compound and cut into 20-µm coronal sections. These tissues were stained using a slidemounted method. After washing with PBS and PBS + 0.4%
Triton X-100, the brain sections were blocked with 10%
goat serum for 1 h at 37 °C, incubated with primary antibodies overnight at 4 °C and washed three times with
PBS. Then, the sections were incubated with secondary
antibodies (1:400, Beyotime Institute of Biotechnology,
Shanghai, China) conjugated to Alexa Fluor 488/594 for
1 h at room temperature. Cell nuclei were stained with
4′,6-diamidino-2-phenylindole (DAPI, 1:1, Solarbio).
The primary antibodies included rabbit polyclonal antiZO-1 (1:1000, 21773-1-AP, Proteintech), rabbit polyclonal anti-Occludin (1:1000, 27260-1-AP, Proteintech),mouse monoclonal anti-CD31 (1:50, GTX20218, Genetex), and mouse monoclonal anti-NeuN (1:100, 668361-Ig, Proteintech). For each sample, 3–4 corresponding
sections at intervals of 300 μm were collected. For eachgroup, sections from 4 mice were used for analysis. Eachmeasurement was expressed as the average of all section
measurements per mouse. All sections were collected
where the lesions were located. The regions of interest(ROIs) where the images were captured are shown inthe black box of Fig. 8D. All images were captured using

Microbiome analysis

Feces from each mouse were collected in autoclaved sterile vials on day 7 after TBI and stored at − 80 °C. Samples
were shipped to Shanghai Majorbio Bio-Pharm Technology Co., Ltd. for analysis. Briefly, bacterial genomic
DNA was extracted, and the bacterial V3-V4 region was
amplified using barcode-indexed primers (338 F, ​A​C​T​C​
C​T​A​C​G​G​G​A​G​G​C​A​G​C​A​G; 806 R, ​A​C​T​C​C​T​A​C​G​G​G​A​
G​G​C​A​G​C​A​G). PCR products were sequenced using theIllumina MiSeq platform and analyzed on the Majorbio
Cloud platform [36].
Metabolite analysis

CAT, SOD, ROS and GSSG measurements

Brain samples from the ipsilateral injured hemisphere
were collected. The activities of CAT, SOD, ROS, GSSG
levels and GSH/GSSG ratio were measured using commercial kits (Nanjing Jiancheng Bioengineering Institute,
Nanjing, China) according to the manufacturer’s instructions [39, 40]. Protein concentration was measured by the
BCA kit method [41].
Western blot analysis

Western blot analysis was performed as previously
described [42]. Briefly, brain tissue including the injury
site (approximately 5 mm length ×5 mm width×3 mm
height), was collected for total protein extraction using
RIPA lysis buffer and protease and phosphatase inhibitors. The sample proteins (20 µg/lane) were separated
by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS‒PAGE) (Invitrogen) and transferred onto
polyvinylidene fluoride (PVDF) membranes (Millipore,
Boston, MA, USA). Membranes were blocked with 5%

Gu et al. Journal of Neuroinflammation(2024) 21:147

a Leica DM4 B fluorescence microscope (Leica, DM4 B,
Wetzlar, Hesse, Germany) with a 10× eyepiece and a 20×
objective and processed using ImageJ. We set the same
exposure time when quantitative analysis was involvedin each experiment. The relative immunofluorescenceintensity of Occludin was calculated by the percentage
of immunofluorescence intensity of Occludin relative toimmunofluorescence intensity of CD-31 with ImageJ.
Additionally, the relative immunofluorescence intensityof Occludin/ZO-1 in the intestinal mucosa was also calculated by the percentage of immunofluorescence intensity of Occludin/ZO-1 relative to immunofluorescenceintensity of DAPI with ImageJ software. All counts wereobtained in a blinded fashion.
TUNEL assays were performed using a one-step
TUNEL kit (C1090, Beyotime Institute of Biotechnology, Shanghai, China) according to the manufacturer’s
instructions. These tissues were stained using a slidemounted method just as above. The immune-positive cellnumbers were calculated with ImageJ, and presented as
the mean number of cells per high power field (HPF) for
single staining or presented as the percentage of the number of cells for the main marker to the number of cells forthe secondary marker per HPF for double-staining.
BBB permeability assays

To measure BBB permeability, 2% Evans blue (EB, 4 mL/kg) in sterile saline was injected through the tail vein 1 h
before the animals were sacrificed. The mice were transcardially perfused with saline, and their brains weredissected and weighed. The samples were then homogenized in PBS, sonicated for 2 min, and centrifuged at
15,000 rpm for 5 min at 4 °C, and the supernatant wasthen collected in aliquots. Next, 500 µL of 50% trichloroacetic acid was added to each 500 µL of supernatant andincubated overnight at 4 °C. Finally, these samples were
centrifuged at 15,000 rpm for 30 min at 4 °C, the supernatant was collected and detected with a spectrometer at
610 nm and quantified using a standard curve that was
normalized to tissue weight (µg/g). Then, to assess thefluorescence intensity, the brains were removed in preparation for coronal brain sectioning. Brains for Evans blue
fluorescence were removed and fixed in 4% paraformaldehyde at 4 °C for 24 h and prepared for coronal brain
sectioning (20 μm). Red auto-fluorescence of Evans blue
was observed on the Leica DM4 B fluorescence microscope with a 10× eyepiece and a 20× objective and processed using ImageJ [43]. The mean red autofluorescenceof EB was evaluated by 2 observers blinded to the groups.
Transmission electron microscopy

After anesthetization, the mice were transcardiallyperfused with ice-cold saline followed by 4% PFA and2.5% glutaraldehyde with 0.1 mol/L PBS buffer. The

Page 8 of 25

pericontusional cortex tissues were microdissected into
1 mm3 specimens. Then, these samples were immersion-fixed in 2% glutaraldehyde for 24 h. After washingwith PBS, they were fixed in 1% osmium tetroxide in
0.1 M PBS for 45 min. The specimens were dehydrated
in increasing concentrations of acetone and embedded
in Aralditeresin. Ultrathin 60-nm-thick sections were cut
using a diamond knife on a Leica UCT ultramicrotome(Diatome, Wetzlar, Germany). The prepared ultrathin
sections were mounted on a copper grid, stained withuranium acetate and lead citrate, and observed with a
JEM-1400Plus TEM (H-7500, Hitachition) as previously
described [44].
Statistical analysis

All results are presented as the means ± standard deviations (SDs). Before analysis, the Shapiro–Wilk test wasused to test the normality of the variables. Two-way
repeated-measures ANOVA with Tukey’s post hoc multiple-comparisons test was used to analyze continuously
measured data. One-way analysis of variance (ANOVA)
was used to compare the means of different groups followed by a Tukey post hoc multiple-comparisons test.
All statistical analyses were performed using GraphPad
Prism software (version 9.5.1, CA, USA). In the 16 S
rRNA gene sequencing analysis, alpha diversity indices(Ace, Chao and sobs even indices) were calculated in
QIIME (version 1.7.0) and examined using the Kruskal‒
Wallis H-test in SPSS 21.0. Principal coordinate analysis(PCoA) based on Bray‒Curtis similarities. The Kruskal‒
Wallis H-test with false discovery rate (FDR), multiple
comparisons correction, and Tukey’s post hoc comparisons test were calculated at the phylum, family, and genus
levels among each group, and corrected p < 0.05 was considered statistically significant [14]. For metabolomics
analysis, orthogonal partial least-squares discriminant
analysis (OPLS-DA), a multivariate statistical analysis
model, was conducted with SIMCA16.1 software (Umetrics, Umeå, Sweden). OPLS-DA models were validated
based on the multiple correlation coefficient (R2) andcross-validated Q2 in cross-validation and permutation
tests by applying 200 iterations. A Q2 value of more than
0.3 showed that the model was stable and reliable. The
permutation tests are deemed valid when all Q2 and R2
values show a gradual decline. The variable importance inthe projection (VIP) value of each variable in the OPLSDA model was calculated to indicate its contribution to
the classification. The model validity was evaluated by
7-fold cross-validation, and a permutation test (n = 200)
VIP > 1 and P < 0.05 were used as screening thresholds fordifferentially abundant metabolites [37]. Statistical significance was defined as p < 0.05.

Gu et al. Journal of Neuroinflammation(2024) 21:147

Results
Time course of P. Copri abundance after TBI

qPCR was used to assess the abundance of P. copri in thefeces of mice at the following timepoint: before TBI, 1
d, 3 d, 7 d, 14 d, 21 d, and 28 d post-TBI. After TBI, we
found that the abundance of P. copri decreased gradually over time; its abundance was lowest at 7 d and thenincreased gradually at 14 d, 21 d, and 28 d. However, its
abundance at 28d post-TBI still did not reach the level
before TBI (Fig. 2A).
P. Copri treatment improved neurological function in TBI
mice

The NSS score and wire grip scores were applied toevaluate the effect of P. copri transplantation on neurological deficits. The injury groups, which included the
TBI group, TBI + Vehicle group and TBI + P. copri group,
had significantly higher NSS scores than the sham groupon day 1, day 3, and day 7 after TBI. The TBI + P. copri
group had lower NSS scores than the TBI group and
TBI + Vehicle group on day 3 and 7 after TBI, while there
were no significant differences between the NSS scoresof the TBI group and TBI + Vehicle group (Fig. 2B). The
injury groups, which included the TBI, TBI + Vehicle, and
TBI + P. copri groups, had significantly lower wire grip
scores than the sham group on day 1, day 3, and day 7
after TBI. The TBI + P. copri group had higher wire grip
scores than the TBI group and TBI + Vehicle group on
day 3 and day 7 after TBI, while there were no significant
differences between TBI group and TBI + Vehicle groups(Fig. 2C).
Next, the open field test (OFT) was used to assessanxiety related behavior 14 days after TBI. Our results
showed that TBI resulted in less time spent in the center, more time spent in the periphery and immobile in the
OFT than the sham group, indicating that mice exhibited anxiety related behavior after TBI. However, P. copri
transplantation reversed this effect compared to the TBI
group and TBI + Vehicle group. In addition, there wasno significant difference between the TBI group and the
TBI + Vehicle group (Fig. 2D, E, F, H). There was no significant difference among the four groups in the total distance traveled and mean speed in the OFT (Fig. 2G, I).
In addition, the Morris water maze was performed
to evaluate spatial learning and memory on days 15
to 20 after TBI. In the learning stage, mice in the TBI,
TBI + Vehicle and TBI + P. copri groups took more time
to find the correct target than those in the sham group.
There was no significant difference between the TBI and
the TBI + Vehicle groups. However, P. copri treatment significantly decreased the latency in the learning test compared with the TBI group and the TBI + Vehicle group(Fig. 2J and K). In the probe trial, mice in the TBI and
TBI + Vehicle group took more time to enter the platform

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for the first time than those in the sham group, but P.
copri treatment decreased the latency (Fig. 2M). Meanwhile, TBI resulted in less time spent in the correct target quadrant than that in the sham group, but P. copri
treatment increased the time spent in the target quadrant when compared to the TBI group and TBI + Vehicle
group (Fig. 2L). There was no significant difference inswimming speed among the groups of mice (Fig. 2N).
P. Copri treatment improved intestinal permeability andmotility in TBI mice

P. copri transplantation has been reported to be safe inmice [30]. In our study, body weights and food intake
were monitored and compared at the indicated time
points. The body weights of the mice were greatly
reduced in the TBI group and increased gradually after
P. copri treatment (Fig. 3A). The food intakes of mice
were also greatly reduced in the TBI group and increased
gradually after P. copri treatment (Fig. 3B).
Altered intestinal barrier integrity and subsequent gastrointestinal dysfunction have been postulated as pathophysiological events in TBI [45, 46]. GI transit, as an
overall measure of GI motility, was assessed in mice byusing barium gavage followed by X-ray imaging. Representative images from different groups at different time
points are shown in Fig. 3C (1, 3, and 8 h after gavage).
Overall, TBI mice had a higher level of barium filling in
the GI tract, which indicates a slower GI transit. P. copri
treatment accelerated GI transit in mice after TBI. With
respect to gut tract filling, GI motility was significantly
delayed after TBI at 8 h after barium gavage, which was
improved in TBI + P. copri mice. Meanwhile, there was no
significant difference between the sham group and thesham + P. copri group (Fig. 3D-E). Taken together, P. copri
treatment may have contributed to the faster GI transit
following TBI.
To test whether P. copri treatment had any effect on
intestinal barrier permeability after TBI, the mice weregavaged with FITC-labeled dextran (4 kDa) on day 7 andblood FITC levels were measured. Intestinal permeability
was more severely disrupted in the TBI group than in the
TBI + P. copri group (Fig. 3F). Intestinal tight junctions(TJs) have been shown to be associated with intestinal
barrier integrity [47]. Therefore, in our study, we examined the expression and distribution of TJ proteins in thecolon. In the TBI group, the relative immunofluorescenceintensity of Occludin and ZO-1 in the intestinal mucosa
were reduced, and P. copri treatment increased the relative immunofluorescence intensity of Occludin and ZO-1
(Fig. 3G-I). The results demonstrated that P. copri treatment led to an upregulation of tight junction proteins
expression and facilitated the maintenance of intestinal
barrier integrity after TBI.

Gu et al. Journal of Neuroinflammation(2024) 21:147

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Fig. 2 P. copri treatment improved neurological function in TBI mice. A. Time course of P. copri abundance after TBI by qPCR. B-C. Quantitative analysis ofshort-term neurological function by NSS scores (B) and wire grip scores (C). *, p < 0.05 vs. sham; #, p < 0.05 vs. TBI. n = 8 per group. D. Representative imagesof the movement path of mice in the open field test 14 days after TBI. E-I. Quantitative analysis of central time (E), periphery time (F), total distance(G), time
immobile (H) and mean speed (I) travelled by mice in the open field test. *, p < 0.05 vs. Sham; #, p < 0.05 vs. TBI, n = 8 per group. J. Representative imagesof the swim path of mice in the Morris water maze. K-N. Quantitative analysis of the latency in the learning test (K), the time in the target quadrant (L), thelatency (M) and the average swimming speed (N) in the probe trial. *, p < 0.05 vs. Sham; #, p < 0.05 vs. TBI, n = 8 per group. Two-way repeated-measures
ANOVA with Tukey’s post hoc multiple-comparisons test was used to analyze continuously measured data. One-way analysis of variance (ANOVA) wasused to compare means of different groups followed by a Tukey post hoc multiple-comparisons test

Gu et al. Journal of Neuroinflammation(2024) 21:147

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Fig. 3 P. copri treatment improved body weight, food intake, intestinal permeability and intestinal motility of TBI mice. A-B. Changes of body weight(A) and food intake (B) over time. *, p < 0.05 vs. sham; #, p < 0.05 vs. TBI. n = 8 per group. C-E. Effect of P. copri on gastrointestinal motility. Representative
images after administration of barium (C), the filling of the intestinal tract was measured by radiological methods (D), the size of the intestinal tract wasdetermined using ImageJ (E). *, p < 0.05 vs. Sham; #, p < 0.05 vs. TBI. n = 4 per group. F. Intestinal permeability by measuring FITC intensity in serum after
oral gavage of FITC-dextran. *, p < 0.05 vs. sham; #, p < 0.05 vs. TBI. n = 8 per group. G-I. Effect of P. copri treatment on tight junction proteins expression.
Representative images of immunofluorescence staining for Occludin (green) and ZO-1 (red), nuclei were stained with DAPI (blue) (G). Scale bar = 100 μm.
Quantitative analysis of relative fluorescence intensity of Occludin (H) and ZO-1 (I) in different groups. *, p < 0.05 vs. Sham; #, p < 0.05 vs. TBI, n = 4 pergroup. Two-way repeated-measures ANOVA with Tukey’s post hoc multiple-comparisons test was used to analyze continuously measured data. One-way
ANOVA was used to compare means of different groups followed by a Tukey post hoc multiple-comparisons test

Gu et al. Journal of Neuroinflammation(2024) 21:147

P. Copri treatment reshaped the gut microbiota of TBI mice

To test whether P. copri treatment modulated the gut
microbiota, we performed 16 S rDNA (V3 + V4 regions)gene sequencing to analyze the bacterial taxonomic
composition following microbial therapy in TBI mice.
To measure the degree of similarity between microbialcommunities, β diversity was further evaluated by using
Bray‒Curtis principal coordinate analysis (PCoA), whichshowed apparent clustering in different groups (Suppl
Fig. 1A-C). The α-diversity was measured by the Ace,
Chao and Sobs even indices. Results from the analysis
showed that the α-diversity index was significantly different among the three groups (Suppl Fig. 1D-F). Community bar plot analysis, at the phylum level, based on
taxonomic analysis, revealed 4 different phyla whencompared with the sham and TBI + P. copri groups, suchas Proteobacteria, Firmicutes, Verrucomicrobiota, andBacteroidetes (Fig. 4A). Further analysis revealed that therelative abundances of the phyla Firmicutes and Verrucomicrobia were lower and the relative abundance of Proteobacteria was increased in the TBI group compared tothe sham or TBI + P. copri groups (Fig. 4D). At the familylevel, the TBI group had 11 different families comparedwith the sham and TBI + P. copri groups (Fig. 4B), andspecies difference analysis showed that Enterobacteriaceae, Sutterellaceae, Morganellaceae and Staphylococcaceae were higher in the TBI group than in the sham and
TBI + P.copri groups, and P. copri treatment increased therelative abundance of Enterococcus, Akkemansia, Lachnospiraceae, Lactobacillus, Erysipelotrichaceae,
Eryssipelatoclostridiaceae and Peptostreptococcaceae(Fig. 4E). At the genus level, the TBI group had 15 different bacteria when compared with the sham and
TBI + P. copri groups (Fig. 4C). Species difference analysis showed that in the TBI group induced alterations of 3
genera with high relative abundance, namely, Enterobacteriaceae, Parasulterella and Proteus, and in the TBI + P.
copri group, the relative abundances of Enterococcus,
Akkemansia, Lactobacillus, Turicibacter, EscherichiaShigella, Eryssipelatoclostridium, Morganella, Lachnospiraceae, Lachnospiraceae-NK4A136 and Romboutsia wereincreased compared to those in the TBI group (Fig. 4F).
P. Copri treatment caused changes in serum metabolomics
in TBI mice

The results of behavioral testing showed that vehicle
treatment does not impact neurological behaviors after
TBI, therefore we choose the sham group, TBI group and
TBI + P. copri groups for in-depth metabolomics analysis.
To profile differential metabolism, OPLS-DA was performed using these three groups, which were comparedin pairs and possessed satisfactory fit. The results showeda clear separation in the two groups (Suppl Fig. 2A-C).
Permutation tests with 200 iterations were performed,

Page 12 of 25

which also validated these models. In the serum, 38
metabolites were found to be differentially expressed
between the sham group and the TBI group, 37 metabolites were differentially expressed between the TBI
group and the TBI + P. copri group, 64 metabolites weredifferentially expressed between the sham group andthe TBI + P. copri group (Fig. 5A and B). Combining thematchstick analysis of the serum metabolites suggested
that guanosine (GUO) was elevated simultaneously inserum of the TBI + P. copri group when compared tothe sham group and the TBI group (VIP > 1 and p < 0.05,
Fig. 5C and D).
P. Copri treatment increased the level of GUO in the feces,serum and brain tissue of TBI mice

In metabolomics analysis, GUO was one of the mostsignificantly upregulated metabolites after P. copri treatment, thereafter ELISA analysis of GUO was subsequently performed. The level of fecal GUO after TBI
at the 1st day and 3rd day were significantly decreased,
and there is no significant difference at day 7 comparedwith sham group (Fig. 6A). The level of serum GUO after
TBI at the 1st day was significantly decreased, and there
is no significant difference at day 3 and day 7 comparedwith sham group (Fig. 6B). In the brain tissue, the change
between each group did not reach statistical significanceafter TBI (Fig. 6C). After P. copri treatment, compared tothe sham group, the level of fecal GUO was significantly
decreased on the 1st day and 3rd day. Compared withthe 1st day, the level of GUO increased gradually at 7thday (Fig. 6D). The level of serum GUO was significantly
decreased on the 1st day and 3rd day but increased at 7th
day compared to the sham group. Compared with the 1stday, the level of serum GUO increased gradually at 3rd
and 7th day (Fig. 6E). The level of brain GUO after.
P. copri treatment at 3rd day and 7th day was significantly higher than that in the sham group as well as the
1st day group (Fig. 6F).
P. copri treatment. *, p < 0.05 vs. sham; #, p < 0.05 vs. day
1. n = 6 per group. One-way ANOVA was used to compare the means of different groups followed by a Tukey
post hoc multiple-comparisons test.
P. copri treatment attenuated oxidative stress after TBI

The TBI group showed a significant decrease in CAT
and SOD activities compared to the sham group. The
TBI group showed higher levels of ROS and GSSG, alongwith a lower GSH/GSSG ratio than the sham group. After
treatment with P. copri transplantation or GUO intake,
both the TBI + P. copri group and the TBI + GUO group
showed significant increases in CAT and SOD activities compared to the TBI group (Fig. 7A and B). In addition, the TBI + P. copri group and the TBI + GUO group
had significantly reduced ROS and GSSG levels and an

Gu et al. Journal of Neuroinflammation(2024) 21:147

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Fig. 4 P. copri treatment reshaped the gut microbiota of TBI mice. A-C. Bar plot analysis of gut microbiota relative abundance of bacterial phylum (A), family (B) and genus (C) in the sham, TBI, TBI + P. copri groups. Different colours represent different phyla, family and genus. D-F. Significant differences in theabundance of phyla (D) in each group, and significant differences in the families (E) and genus (F) with high abundance (top 15) (n = 6 per group). Data
are presented as mean ± SD. *, p < 0:05. **, p < 0:01. ***, p < 0:001. The Kruskal‒Wallis H-test with false discovery rate (FDR), multiple comparisons correction
and Tukey’s post hoc comparisons test were calculated at the phylum, family, and genus levels among each groupGu et al. Journal of Neuroinflammation(2024) 21:147

Page 14 of 25

Fig. 5 P. copri treatment caused the changes in serum metabolomics in TBI mice. A. Hierarchical clustering analysis of serum metabolite biomarkers. The
abscissa is the clustering of samples and the ordinate is the clustering of differential metabolites. The coloured blocks in different positions represent thecontent of the metabolites in the corresponding parts. Red means that the substance is highly expressed in the group where it is located, while bluemeans that the sense is poorly represented in the group where it is located. B. Venn diagram of differential metabolites in serum. C-D. Matchstick analysisof serum metabolite biomarkers. TBI + P. copri group vs. sham group (C), TBI + P. copri group vs. TBI group (D). Red nodes represent significantly upregulated
metabolites (top 25), blue nodes represent significantly downregulated metabolites (top 25) (VIP > 1 and P < 0.05)

Gu et al. Journal of Neuroinflammation(2024) 21:147

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Fig. 6 P. copri treatment increased the presence of GUO in the feces, serum and brain tissue of TBI mice. A-C. The changes of GUO level in feces (A), serum(B) and brain (C) after TBI were detected by ELISA. *, p < 0.05 vs. sham; #, p < 0.05 vs. day 1. n = 6 per group. D-F. Changes in GUO level in feces (D), serum(E) and brain (F) after

increased GSH/GSSG ratio compared to the TBI group(Fig. 7C-E). All of these results indicated that P. copri or
GUO treatment can effectively reduce oxidative stressafter TBI.
P. Copri treatment attenuated BBB disruption after TBI

Evans blue assays indicated that TBI caused more Evansblue leakage than the sham group, and P. copri and GUO
visually mitigated EB dye leakage after Evans blue injection (Fig. 8A-C). Meanwhile, double-immunofluorescence staining showed that Occludin was decreased
significantly in the TBI group compared to the shamgroup, and treatment with P. copri and GUO attenuated this damage compared with the TBI group (Fig. 8E
and F). Similarly, Western blot assays revealed that theexpression of BBB-associated proteins (ZO-1, Occludin)
was decreased significantly after TBI compared to shamgroup. By contrast, both P. copri treatment and GUO
treatment showed an increase level compared to that in
the TBI group (Fig. 8G-I).

Furthermore, we investigated the ultrastructure of
the tight junctions by transmission electron microscopy analysis. In the sham group, we observed smoothand continuous TJ structures between endothelial cells,
and the basement membrane was intact. After TBI, themicrovilli of endothelial cells protruded, the continuity was interrupted, the density of intercellular TJ structures was reduced, and the opening is increased to form
cracks. After treatment with P. copri or GUO, the above
manifestations were improved: the opening of intercellular TJ structures was reduced, and the basement membrane was relatively intact (Fig. 8J). A scoring system wasused to quantitatively determine the number of intact
tight junctions, which were rated as follows: perfect tightjunctions worth 3 points; patches of blurriness worth 2
points; totally blurred worth 1 point. In the TBI group,
the number of intact tight junctions was significantly
lower than that in the sham group, while P. copri or GUO
treatment increased their expression compared to that in
the TBI group (Fig. 8K). Overall, P. copri transplantation
help to attenuated BBB disruption in mice after TBI.

Gu et al. Journal of Neuroinflammation(2024) 21:147

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Fig. 7 P. copri treatment attenuated oxidative stress after TBI. A-E. The changes of oxidative stress markers in the brain lesion area after TBI were detectedby spectrophotometer. A. SOD activity. B. CAT activity. C. ROS level. D. GSSG level. E. GSH/GSSG ratio. *, p < 0.05 vs. sham; #, p < 0.05 vs. TBI. n = 6 per group.
One-way ANOVA was used to compare the means of different groups followed by a Tukey post hoc multiple-comparisons test

P. Copri treatment attenuated neuronal apoptosis after TBI

The TUNEL-positive neurons surrounding the traumasite was significantly increased after TBI. However, they
were reduced in the TBI + P. copri group and TBI + GUO
groups compared to the TBI group as shown by double
immunofluorescence staining of TUNEL and NeuN
(Fig. 9A and B). Furthermore, Western blotting was used
to measure the expression of the apoptotic markers Bcl-2
and Bax after TBI. The results showed that the expression of Bcl-2 was significantly increased and the expression of Bax was significantly decreased with P. copri or
GUO treatment when compared with the TBI group(Fig. 9C-E).

The GUO-PI3K/Akt pathway was involved in theneuroprotective effects of P. Copri treatment

Previous research has verified that GUO can reduce oxidative stress after TBI by activating the PI3K/Akt pathway [25]. Therefore, we use Western blot analysis to
examine the expression of PI3K/Akt pathway. The results
showed that after P. copri or GUO treatment, the expression of p-PI3K and p-Akt significantly increased whencompared with both the sham group and TBI group, andLY294002 inhibited the increase in p-PI3K and p-Akt
expression caused by P. copri or GUO (Fig. 10A-C).
Furthermore, the administration of LY294002
resulted in the inhibition of the decrease in NSS

Gu et al. Journal of Neuroinflammation(2024) 21:147

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Fig. 8 P. copri treatment attenuated BBB disruption after TBI. A. Representative horizontal and coronal images of brain slices after EB injection. B. Quantitative analysis of EB leakage intensity. *p, < 0.05 vs. sham; #, p < 0.05 vs. TBI, n = 6 per group. C. Red fluorescence of EB was observed by fluorescencemicroscopy in different groups. Cell nuclei were stained with DAPI (blue). Scale bar = 100 μm. n = 4 per group. D. Brain sample with a schematic illustration
showing the area (indicated by the black box) that was used for the immunofluorescence analysis. E. Representative images of double immunofluorescence staining for Occludin and CD31, nuclei were stained with DAPI (blue). Scale bar = 100 μm. F. Quantitative analysis of relative Occludin fluorescenceintensity in different groups. *, p < 0.05 vs. sham; #, p < 0.05 vs. TBI, n = 4 per group. G. Representative Western blot bands of ZO-1, Occludin and β-actin at
the lesion sites after TBI. H-I. Quantitative analysis of relative ZO-1 (H), Occludin (I) density. *, p < 0.05 vs. sham; #, p < 0.05 vs. TBI, n = 6 per group. J. Transmission electron microscopy showed the ultrastructure of tight junctions. Scale bar = 500 nm. EC: Endothelial cells. TJ: Tight junction. K. Quantitative data
show a decreased number of organized tight junctions. *, p < 0.05 vs. sham; #, p < 0.05 vs. TBI, n = 4 per group. One-way ANOVA was used to compare themeans of different groups followed by a Tukey post hoc multiple-comparisons test

Gu et al. Journal of Neuroinflammation(2024) 21:147

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Fig. 9 P. copri treatment attenuated neuronal apoptotic death and expression of apoptosis-related proteins after TBI. A. Representative images of deadcells (TUNEL, red) and neurons (NeuN, green) surrounding the lesion sites. Scale bar = 100 μm. B. Quantitative analysis of TUNEL-positive neurons surrounding lesion sites after TBI. *, p < 0.05 vs. sham; #, p < 0.05 vs. TBI, n = 4 per group. C. Representative Western blot bands of Bcl-2, Bax and β-actin at thelesion sites after TBI. D-E. Quantitative analysis of Bcl-2 (D) and Bax (E) density at the lesion sites after TBI. *, p < 0.05 vs. sham; #, p < 0.05 vs. TBI, n = 6 pergroup. One-way ANOVA was used to compare the means of different groups followed by a Tukey post hoc multiple-comparisons testscores and enhancement in wire grip scores causedby P. copri or GUO (Fig. 11A-B). The OFT test showed
the mice in the TBI + P. copri + LY294002 group and
TBI + GUO + LY294002 groups exhibited more anxiety
related behavior as opposed to those in the TBI + P. copri
group and TBI + GUO group (Fig. 11C-H). Learning andmemory function in mice was weakened in the TBI + P.
copri + LY294002 group and TBI + GUO + LY294002
groups in comparison to the TBI + P. copri group and
TBI + GUO groups, as evidenced by the results of Morris
water maze test (Fig. 11I-M).

The GUO-PI3K/Akt axis regulated the effects of P. Copri
on oxidative stress, the blood‒brain barrier and neuronal
apoptosis in TBI mice

Compared with the sham group, all the experiment groups (including the TBI group, TBI + P.
copri + LY294002 group and TBI + GUO + LY294002
group) displayed a significant decrease in SOD activity, with no statistical significance among them. Notably,
the TBI + P. copri group showed a significant increase
in SOD activity compared to the TBI group (Fig. 12A).
At the same time, compared to the sham group, higher
levels of ROS were measured in the TBI group, TBI + P.
copri + LY294002 group and TBI + GUO + LY294002

Gu et al. Journal of Neuroinflammation(2024) 21:147

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Fig. 10 The GUO-PI3K/Akt axis was involved in the neuroprotective effects of P. copri. A. Representative Western blot bands of total PI3K, p-PI3K, total Akt,
p-Akt and β-Actin at the lesion sites after TBI. B-C. Quantitative analysis of p-PI3K (B) and p-Akt (C) density at the lesion sites after TBI. *, p < 0.05 vs. sham;#, p < 0.05 vs. TBI + P. copri or TBI + GUO, n = 6 per group. One-way ANOVA was used to compare the means of different groups followed by a Tukey post
hoc multiple-comparisons test

group, while the TBI + P. copri group showed significantly
reduced ROS compared to the TBI group (Fig. 12B).
In addition, LY294002 prevented the improvement of
P. copri on loss of Occludin after TBI (Fig. 12C and D).
Western blot assays revealed that LY294002 inhibited theincrease in ZO-1 and Occludin expression (Fig. 12E-G).
We examined Bcl-2 and Bax expression by western blot
to determine the effect of LY294002 on the apoptosis.
The results showed that LY294002 inhibited the increasein Bcl-2 expression and the decrease in Bax expression
caused by P. copri (Fig. 12E, H and I).

Discussion
Numerous studies have confirmed changes in the gut
microbiota after TBI that can produce a series of effects
on the central nervous system through the brain-gut axis
[13, 48–50]. It has been reported that Prevotella changes
significantly after TBI, in which P. copri bacteria are significantly reduced [15, 51], but it has not been reported
whether.
P. copri has an effect on the pathophysiology of TBI.
In our study, we found that P. copri experienced a continuous decline for 7 days after TBI, which was consistent with reports in the literature. We found that P. copri
treatment improved neurological dysfunction after TBI,
including learning memory function and anxiety related

behaviors in TBI mice. P. copri transplantation helps
recover dysbiosis of the gut microbiota after TBI andincreases the level of guanosine. Furthermore, we provedthat P. copri transplantation relieved neurological deficits
possibly through the GUO-PI3K/Akt signaling pathway.
The safety of P. copri transplantation in normal micehas been proved by Phebe Verbrugghe et al [30]. They
found that histological analysis of liver, pancreas, colon,
heart, lung, spleen, kidney, testis, brain and epididymal
fat showed no microscopic abnormalities after a long
period (for 29 consecutive days) P. copri oral administration. Repeated-dose oral administration of P. copri
did not result in any significant changes in the blood cellcount or serum biochemistry parameters. On the basisof this valuable study, we discovered more roles for P.
copri. We found that P. copri could improve intestinal
permeability and intestinal motility and reshape the gut
microbiota of TBI mice. Gastrointestinal motility contributes to the maintenance of a healthy intestinal barrier as it clears luminal debris and, importantly prevents
the proliferation of microbiota. Impaired motility canpotentially contribute to bacterial dysbiosis in TBI, which
may have deleterious effects on gut barrier integrity [45].
Besides, the FITC-dextran assay was used in this study
as a measure of intestinal permeability. The gut-transit
time affects the speed of movement of the FITC-dextranGu et al. Journal of Neuroinflammation(2024) 21:147

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Fig. 11 LY294002 inhibited the neuroprotective effects of P. copri. A-B. Quantitative analysis of short-term neurological function using NSS scores (A) andwire grip scores (B). *, p < 0.05 vs. sham; #, p < 0.05 vs. TBI + P. copri or TBI + GUO. n = 8 per group. C. Representative images of the movement path of micein the open field test 14 days after TBI. D-H. Quantitative analysis of central time (D), periphery time (E), total distance (F), time immobile (G) and mean
speed (H) travelled by mice in the open field test. *, p < 0.05 vs. sham; #, p < 0.05 vs. TBI + P. copri or TBI + GUO, n = 8 per group. I. Representative imagesof the swim path of mice in the Morris water maze. J-M. Quantitative analysis of latency in the learning test (J), the time in the target quadrant (K), thelatency (L) and the average swimming speed (M) in the probe trial. *, p < 0.05 vs. sham; #, p < 0.05 vs. TBI + P. copri or TBI + GUO, n = 8 per group. Two-way
repeated-measures ANOVA with Tukey’s post hoc multiple-comparisons test was used to analyze continuously measured data. One-way ANOVA wasused to compare means of different groups followed by a Tukey post hoc multiple-comparisons test

Gu et al. Journal of Neuroinflammation(2024) 21:147

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Fig. 12 The GUO-PI3K/Akt axis was involved in the effects of P. copri on oxidative stress, blood-brain barrier and neuronal apoptosis in TBI mice. A-B.
Oxidative stress after TBI. SOD activity (A) and ROS level (B). *, p < 0.05 vs. Sham; #, p < 0.05 vs. TBI + P. copri, n = 6 per group. C. Representative images ofdouble immunofluorescence staining for Occludin and CD31, nuclei were stained with DAPI (blue). Scale bar = 100 μm. D. Quantitative analysis of relative
Occludin fluorescence intensity in different groups. *, p < 0.05 vs. sham; #, p < 0.05 vs. TBI + P. copri, n = 4 per group. E. Representative Western blot bandsof ZO-1, Occludin, Bcl-2, Bax and β-Actin at the lesion sites after TBI. F-I. Quantitative analysis of the relative density of ZO-1 (F), Occludin (G), Bcl-2 (H)and Bax (I). *, p < 0.05 vs. sham; #, p < 0.05 vs. TBI + P. copri, n = 6 per group. One-way ANOVA was used to compare means of different groups followed bya Tukey post hoc multiple-comparisons test

Gu et al. Journal of Neuroinflammation(2024) 21:147

along the digestive tract [52]. FITC-dextran reached its
maximal concentration in the plasma of adult wildtype
C57BL/6 mice 4 h after its oral administration. With aphysiologic gut-transit time, FITC-dextran can reach thecolon within 4 h [53]. However, a recent study in adultwildtype C57BL/6 mice reported that FITC-dextran
reached its maximal concentration in plasma within thefirst 2 h after oral administration [54]. Therefore, measuring the plasma concentration of FITC-dextran at only
one time point cannot accurately assess the site of intestinal barrier dysfunction. Thus, we used the concentration of FITC-dextran in plasma only as a measure for theintestinal permeability assay. Together with the resultsof tight junction proteins in the intestinal wall, it is wellconvinced that P. copri treatment can improve intestinal
permeability and motility in TBI mice.
Through 16 S rDNA sequencing, we found that P. copri
treatment increased Akkemansia, Lactobacillus, Lachnospiraceae and Lachnospiraceae-NK4A136. As an important genus of intestinal bacteria, Akkermansia degradesmucin and enhances gut barrier function [55]. Lactobacillus, Lachnospiraceae and Lachnospiraceae-NK4A136
bacteria in the gastrointestinal tract produce butyrate
and other short-chain fatty acids (SCFAs) by hydrolyzing starch and other sugars, which directly interact withthe host’s immune system and regulate the surrounding
microbial environment [56]. SCFAs are not only the mainenergy source of the colon but also responsible for intestinal epithelial protection and the regulation of inflammatory intestinal responses, favoring mucus synthesis
and upregulating tight junction proteins [57]. Previous
study mentioned that Prevotella can ferment dietary fiber
to produce the SCFA acetate [17] and metabolize dietaryfiber and fat to produce succinate [18]. However, we did
not find a direct relationship between P. copri and SCFAs
alterations in the serum metabolomics (Fig. 5). Therefore,
the relationships between SCFAs and P. copri transplantation in TBI may require further detection over time.
In a study encompassing both humans and mice, P.
copri was shown to enhance the ability to utilize complex
polysaccharides, potentially by increasing glycogen storage [58]. In our study, we interestingly found that guanosine (GUO) expression was significantly increased after P.
copri treatment. It has been reported that the gut microbiota is one of the contributors to guanosine nucleotidebiosynthesis, such as Akkermansia [59] and Lachnospiraceae [60]. The increase in GUO might be related to thechange in intestinal flora. That is to say, P. copri treatment
may not only cause the remodeling of bacterial flora but
also directly lead to an increase in GUO. A large number of studies have confirmed that GUO could reduce
oxidative stress, inflammation and neuronal apoptosis after TBI and improve the destruction of the blood
brain barrier after TBI [61]. GUO is an endogenous

Page 22 of 25

neuroprotective nucleoside that provides the long-term
benefits in controlling brain neurodegeneration, mainly
due to its capacity to activate the antioxidant defense system and maintain the redox system [61]. Intraperitoneal
administration of GUO increases GUO of cerebrospinal
fluid levels approximately two to three times in 30 min[62]. GUO is effective in reducing ROS generation [63]and preventing the collapse of mitochondrial membranepotential in hippocampal slices subjected to oxygen
glucose deprivation [64]. In our study, treatment with
P. copri or GUO ameliorated oxidative stress, repaired
blood-brain barrier disruption, and reduced neuronal
apoptosis in TBI mice. Therefore, the neuroprotective
effect of P. copri treatment on TBI mice is firmly related
to the increase in GUO.
Previous studies have confirmed that GUO can reduce
oxidative stress after TBI by activating the PI3K/Akt
pathway. GUO can reduce the excessive production of
ROS by activating the PI3K/Akt pathway in astrocytes
and hippocampal tissue in vitro [25]. GUO also alleviates
mitochondrial oxidative stress injury caused by inhibitionof the mitochondrial complex by activating the PI3K/Akt
pathway in neuroblast cells [26]. In our experiment, we
detected the key proteins in the PI3K/Akt pathway andfound that the two proteins were significantly increased
after P. copri treatment. Meanwhile, a PI3K inhibitor(LY294002) significantly inhibited the neuroprotective
effect of P. copri and suppressed the effects of P. copri on
oxidative stress, the blood-brain barrier and neuronal
apoptosis in TBI mice. It can be suggested that P. copri
transplantation causes GUO elevation, which activates
the PI3K/Akt pathway and ultimately ameliorates neurological dysfunction in TBI mice.
TBI is a severe injury, and there is no effective therapeutic method that can be used in the current clinicalscenario. In recent years, certain progress has been madein research on the brain-gut axis [65, 66]. The neuroprotective effect of fecal transplantation on TBI has also
been confirmed by the literature [14, 67]. In our study, weused the common intestinal bacteria in the genus Prevotella (P. copri) for gavage treatment to further investigate the role of a specific bacterial species in TBI. There is
still great controversy about the role of P. copri in humanhealth. Even in the same disease, two opposite results
have appeared [31]. There may be many reasons for this,such as the different diets of subjects in different experiments and the large differences between P. copri strains.
All these factors affect the pathophysiological role of P.
copri in the development of diseases. We believe that itis through the synergistic effect of multiple targets that P.
copri can exert a beneficial action on TBI mice.
In this study, we have demonstrated for the first timethat P. copri can promote the recovery of neurological
function after TBI, however, there are several limitations

Gu et al. Journal of Neuroinflammation(2024) 21:147

in this study. First, antibiotics pretreatment was used toremove intestinal flora and minimize the gut microbiome difference between the experimental groups in alarge number of studies. However, recently publishedstudy find that microbiome depletion prior to brain
injury may exacerbate secondary inflammatory cascades[68]. Therefore, to further elucidate the impact of microbial alterations on the repair of brain injury, we suggestthat an integrated approach to simultaneously avoid thesecondary injury should be explored in future studies.
Meanwhile, TBI patients will not likely have a depleted
microbiome or antibiotics pretreatment upon injury inreality. In the future study, P. copri transplantation study
without prior microbiome manipulation should be considered. Another limitation is that the research is carriedout in male mice. However, sex plays a significant role in
the outcome of TBI [69]. Female mice have higher ROS
activities in the brain in the acute phase [70]. Glial cells
in female mice are also less activated in the subacutephase of brain injury [71].Meanwhile, recent studies also
found that post-CCI neurological complications may be
influenced by the differential gut microbiota perturbation in a gender-dependent manner [72]. Thus, in orderto minimize the confounding factors caused by gender,
only single-sex mice were used in this study. In the future
preclinical studies, mice of both sexes must be included
for further in-depth studies. On the other hand, we used
CCI model to mimic TBI in vivo, and craniotomy is an
essential step in the CCI procedure. There is evidence
that the craniotomy is not atraumatic and the effects ofsurgery should be controlled [73]. However, recently invivo study of CCI model showed that no pathology waspresent in sham operated mice (craniotomy only) by MRI
scan [74]. To mitigate these concerns to some extent, we
included the craniotomy surgical sham groups as control
in our study.

Conclusions
In conclusion, the current study showed that P. copri
transplantation can improve GI functions and remodel
the gut microbiota after TBI. P. copri transplantation
relieved the blood-brain barrier damage and reduced
neuronal apoptosis through elevating GUO and activating the PI3K/Akt pathway. As a result, P. copri transplantation could relieve the neurological deficits, exerts anantioxidant effect.
Abbreviations
ANOVA	Analysis of variance
APES	3-Aminopropyl-triethoxysilane
BBB	Blood-Brain Barrier
Bax	BCL2-Associated X protein
Bcl-2	B cell lymphoma/leukemia-2 gene
CNS	Central nervous systemCAT	Catalase
CCI	Controlled Cortical Impact

Page 23 of 25

DAPI	4’,6-diamidino-2-phenylindole
DMB	3,3-dimethyl-1-butanol
EB	Evans blueECs	Endothelial cells
ECL	Electro-chemi-luminescence
GSSG	Oxidized glutathione
GSH	Reduced glutathione
GM	Gut MicrobiomeIF	Immunofluorescence
MGBA	Microbiome-Gut-Brain Axis
MWM	Morris water maze test
NSS	Neurological Severity Score
OFT	Open field test
PVDF	Polyvinylidene Fluoride
PFA	Paraformaldehyde
P. copri	Prevotella copri
PBS	Phosphate buffer saline
PAGE	Polyacrylamide gel eletrophoresis
ROS	Reactive oxygen species
SDS	Sodium dodecyl sulfate
SOD	Superoxide dismutase
TBI	Traumatic brain injury
TJ	Tight junction
TME	Transmission electron microscope
WB	Western blot
ZO-1	Zona Occludens 1

Supplementary Information

The online version contains supplementary material available at https://doi.
org/10.1186/s12974-024-03116-5.
Supplementary Material 1
Acknowledgements
We would like to thank the service provided by the Chongqing Key Laboratoryof Neurology (Chongqing, China).
Author contributions
NNG, ZJH, XCS and CJC conceived and designed the study. NNG, JY, WT, ZSZ,
LW and ZL performed the experiments and analyzed the results. NNG wrote
the manuscript. ZJH, XCS and CJC revised the manuscript. YWW, YJZ and ST
participated in the data acquisition, analysis, and interpretation. ZJH, XCS
and CJC provided funding support. All authors read and approved the final
manuscript.read and approved the final manuscript.
Funding
This work was supported by the National Natural Science Foundation of China(82172193, 82102316 and 82071397).
Data availability
All data generated or analyzed during this study are included in thispublishedarticle and its supplementary files.

Declarations
Ethics approval and consent to participate
Animal protocols have been approved by the Animal Care and Use
Committee of Chongqing Medical University. Surgical interventions andpostoperative animal care were performed in accordance with the guidelines
and policies for rodent survival surgery provided by the Experimental Animal
Committee of Chongqing Medical University.
Consent for publication
All the authors consented to the publication of the study once it is accepted.
Competing interests
The authors declare that they have no competing interests.

Gu et al. Journal of Neuroinflammation(2024) 21:147

Author details
1
Department of Neurosurgery, The First Affiliated Hospital of Chongqing
Medical University, Chongqing 400016, China
2
Department of Neurosurgery, The Second Clinical Medical College ofNorth Sichuan Medical College, Nanchong Central Hospital, Nanchong,
China
3
Emergency Department, Chengdu First People’s Hospital, Chengdu,
China4
Department of Neurosurgery, Suining Central Hospital, Suining, China

Page 24 of 25

22.
23.
24.

Received: 30 January 2024 / Accepted: 30 April 2024
25.

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