Journal of Neuroinflammation

Zhao et al. Journal of Neuroinflammation(2024) 21:138
https://doi.org/10.1186/s12974-024-03135-2

Open Access

RESEARCH

NHH promotes Sepsis-associatedEncephalopathy with the expression of AQP4
in astrocytes through the gut-brain Axis
Lina Zhao1, Zhen Zhang1, Pei Wang1, Nannan Zhang1, Hao shen1, Hening Wu1, Zhiyong Wei1, Fei Yang2,
Yunying Wang2, Zhijie Yu2, Haibo Li3, Zhanfei Hu3, Hongyan Zhai4, Zhiwei Wang1, Fuhong Su5, Keliang Xie1,6* andYun Li6*
Abstract
Sepsis-associated encephalopathy (SAE) is a significant cause of mortality in patients with sepsis. Despite extensive
research, its exact cause remains unclear. Our previous research indicated a relationship between non-hepatic
hyperammonemia (NHH) and SAE. This study aimed to investigate the relationship between NHH and SAE and thepotential mechanisms causing cognitive impairment. In the in vivo experimental results, there were no significant
abnormalities in the livers of mice with moderate cecal ligation and perforation (CLP); however, ammonia levels
were elevated in the hippocampal tissue and serum. The ELISA study suggest that fecal microbiota transplantationin CLP mice can reduce ammonia levels. Reduction in ammonia levels improved cognitive dysfunction andneurological impairment in CLP mice through behavioral, neuroimaging, and molecular biology studies. Further
studies have shown that ammonia enters the brain to regulate the expression of aquaporins-4 (AQP4) in astrocytes,
which may be the mechanism underlying brain dysfunction in CLP mice. The results of the in vitro experiments
showed that ammonia up-regulated AQP4 expression in astrocytes, resulting in astrocyte damage. The results ofthis study suggest that ammonia up-regulates astrocyte AQP4 expression through the gut-brain axis, which may be
a potential mechanism for the occurrence of SAE.
SummaryThis study elucidates the intricate relationship between gut microbiota, ammonia production, and brain
dysfunction in sepsis associated encephalopathy.
Keywords Sepsis-associated encephalopathy, AQP4, GFAP, Gut-brain axis, Astrocyte*Corresponding author: Keliang Xie and Yun Li.
*Correspondence:
Keliang Xie
xiekeliang2009@hotmail.com
Yun Licfsyy_liyun@126.com
1
Department of Critical Care Medicine, Tianjin Medical University General
Hospital, Tianjin 300052, China
2
Department of Critical Care Medicine, Chifeng Municipal Hospital,
Chifeng Clinical Medical College of Inner Mongolia Medical University,
Chifeng 024000, China

3

Department of Anesthesiology, Chifeng Municipal Hospital, Chifeng
Clinical Medical College of Inner Mongolia Medical University,
Chifeng 024000, China
4
Department of Ultrasound, Tianjin Medical University General Hospital,
Tianjin 300052, China
5
Experimental Laboratory of the Department of Intensive Care, Erasme
Hospital, Université Libre de Bruxelles, Brussels 1070, Belgium
6Department of Anesthesiology, Tianjin Institute of Anesthesiology,
Tianjin Medical University General Hospital, Tianjin 300052, China© 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.

Zhao et al. Journal of Neuroinflammation(2024) 21:138

Introduction
Sepsis-associated encephalopathy (SAE) is a major causeof both short- and long-term mortality in patients withsepsis. Its incidence and mortality rates are increasing
globally, posing a serious threat to public health thaturgently needs to be solved [1–3]. SAE manifests as
cognitive dysfunction and changes in the levels of consciousness and behavior [3]. Although inflammation,
mitochondrial dysfunction, and microglial cell activation
have been reported as potential mechanisms of SAE, theexact pathogenesis remains unclear, and effective prevention and intervention measures are still lacking.
Ammonia serves as a crucial neurotoxin, playing asignificant role in hepatic encephalopathy [4]. Hyperammonemia, stemming from non-hepatic conditions,is increasingly documented among critically ill patients,
notably those afflicted by sepsis [5–7]. Our previous
investigations have revealed a correlation between nonhepatic hyperammonemia (NHH) and sepsis-associatedencephalopathy (SAE). The etiology of NHH in septic patients may stem from factors such as heightenedammonia generation within the intestine and amplified
breakdown of amino acids [8, 9]. The gastrointestinal
tract, a vital component of the immune system, is susceptible to injury following sepsis, particularly in instancesof septic shock. Subsequent to intestinal damage, dysbiosis in the gut microbiota is prone to occur [10]. Ammonia
is generated from amino acids in response to intestinalbacteria; Therefore, the disruption of the gut microbiota
and the upgrading of amino acid metabolism after sepsis
may contribute to NHH production.
Astrocyte edema stands as a pivotal mechanism contributing to the onset of sepsis-associated encephalopathy (SAE) alongside neuronal damage [11].
Additionally, astrocytes play a crucial role in the development of hepatic encephalopathy induced by ammonia[12]. Aquaporin 4 (AQP4) is a specialized membranebound channel highly expressed in astrocytes, governing water movement in and out of these cells. Moreover,
AQP4 activation elevates astrocyte activity throughincreased AQP4 expression [11, 13]. Given its significance in astrocytic function, AQP4 emerges as a key
player in astrocytic injury.Given its importance in astrocyte function, the study have found AQP4 to be a key
player in ammonia-induced astrocyte injury in hepatic
encephalopathy [12]. However, it is unclear whether they
are involved in the occurrence of SAE.
This study aimed to explore the origins of ammonia ina mouse model of cecal ligation and puncture (CLP) by
examining the potential increase in ammonia-producing
bacteria in the intestine, utilizing 16 S rDNA sequencing and metabolomics technology. Pathophysiologicalalterations and the underlying mechanisms of ammonia-induced brain injury in a CLP mouse model were

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investigated at both animal and cellular levels. This wasachieved through fecal microbiota transplantation anda comprehensive array of analyses including morphological, functional, molecular biology, and neuroimaging
techniques (refer to Fig. 1).

Results
Elevated levels of NHH in serum and hippocampal tissuesof CLP mice

Our previous studies found that patients with sepsis andnormal liver function had elevated ammonia levels andthat the serum ammonia level correlated with impaired
consciousness [14]. We speculate that non-hepatic
hyperammonemia (NHH) may be a potential mechanism
underlying SAE [9]. To verify the change in ammonia in
SAE, we used CLP surgery to evaluate the mice’s response
to SAE to avoid liver damage, in this study, a moderate
CLP model was used [15]. In this study, the mortality rateof moderate CLP model mice was 48%. We also evaluated HE staining and measured alanine aminotransferase(p < 0.01), glutamate aminotransferase (p < 0.01), and total
bilirubin (p > 0.05) levels in the livers of the three groups(n = 6) at 24 h, 48 h, and 72 h after CLP (Fig. 2a and b). In
three groups, CLP resulted in mildly abnormal liver function; however, the hepatocytes of the rats were arranged
neatly, the hepatic lobule structure was intact, and there
were no significant abnormalities in the liver function
indices of the three groups (Fig. 2b). However, serum(p < 0.01) and hippocampal (p < 0.001) ammonia levels in
the CLP group were significantly higher than those in thecontrol and sham groups (Fig. 2c).
Fecal microbial transplantation in CLP mice can reduceammonia levels and may be associated with bacilli
Increased abundance of bacilli in CLP mice

After the CLP model was established, the fecal microorganisms of the mice in the control group were transplanted into the CLP mice for 3 consecutive days. HE
staining was used to observe the morphological changes
in the ileum and intestinal pH test in the three groups ofmice (n = 6). After CLP, the ileum villus length and cryptdepth were decreased, and pH was increased comparedto the control and sham groups (p < 0.0001 or p < 0.01)(Fig. 3a and b). CLP disrupts the intestinal structureof mice and pH. After fecal microbe transplantation(FMT), the feces of mice in each group (n = 6) were collected, and 16 S rDNA sequencing technology was used
to observe the changes in microbial species and abundance in each group. After CLP, at the class level, themost abundant microbes of Bacteroidia decreased,
while Bacilli increased compared to the control groupand sham group. After FMT, the abundance of Bacteroidia microbes increased, and Bacilli decreased in thecommunity plot (p < 0.05) (Fig. 3c). The diversity of theZhao et al. Journal of Neuroinflammation(2024) 21:138

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Fig. 1 Experimental flow chart. The mice were allowed to acclimatize to the experimental environment for 1 week before the start of the experiment.
After CLP or sham surgery in C57BL6/J mice, CLP mice were transplanted with fecal bacteria from the control group mice for 3 consecutive days. (a) On
the 3 days, the species and abundance of intestinal microbiota were detected (16 S rDNA), neuroimaging (b), mettabomics (c), including T-2 weighting, DTI, ASL, 1 H-MRS, and molecular biology tests including ELISA (d), Western Blot, QT-PCR, immunofluorescence (e), HE staining (f), Nissl staining andEvans blue staining (g). CLP: cecal ligation and perforation; ASL: Arterial spin labeling; DTI: Diffusion Tensor Imaging; 1 H-MRS: proton magnetic resonancespectroscopy; T2WI: T2-weighted imaging; WB: Western Blot. * p < 0.05, **p < 0.01, *** p < 0.001, *** p < 0.0001

microbiota was significantly lower in the CLP groupthan in the control and sham groups, as determined by
the Chao1 index (p < 0.05); however, there were no significant differences in the Shannon and Simpson indices (Fig. 3d). Additionally, the analysis of microbiome
beta diversity using Unweighted Unifrac and principal

coordinate analysis (PCoA) indicated significant differences in community components between the CLPgroup and the other three groups, indicating that CLP
affected gut microbiota composition (p < 0.01) (Fig. 3e
and f ). The LDA effect size (LEfSe) analysis showed thatthe key bacteria in the sham group were Bacteroidales,Zhao et al. Journal of Neuroinflammation(2024) 21:138

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Fig. 2 Assessing liver function after mild CLP model in three groups of mice. (a) : HE staining of the liver at 24 h, 48 h, 72 h after CLP in the three groups.
In the three groups, the hepatocytes of the rats were arranged neatly, and the hepatic lobule structure was intact. (b) : CLP caused a slight increase in alanine aminotransferase and aspartate aminotransferase, but the total bilirubin level did not change significantly at 24 h, 48 h, 72 h after CLP. (c) : Ammonia
levels in serum and hippocampal tissues were significantly higher in the CLP group than in the control and sham groups. *p < 0.05, **p < 0.01, ***p < 0.001,****
p < 0.0001

Muribaculaceae, Erysipelotrichaceae, Allobaculum, Prevotellaceae, and Alloprevotella. The key bacteria in CLP
mice were Bacilli, Clostridium, Firmicutes, Lactobacillales, Enterobacteriales, Streptococcus, and Enterococcus
were predominant (Fig. 3g and Supplementary Materials 1). Fecal transplantation resulted in decreased relative abundance of Bacilli and Clostridium and increased
relative abundance of Rhodospirillales, Moraxellaceae,
Clostridia_vadinBB60_group, Gastranaerophilales, andClostridia_UCG_014 in CLP group (Fig. 3h and Supplementary Materials 2). These results showed that CLP significantly altered the type and abundance of the intestinal
flora.

Functional annotation and differential analyses wereconducted to investigate the effects of modified gut
microbiota. The Kyoto Encyclopedia of Genes andGenomes (KEGG) and Gene Ontology (GO) functionalannotation results showed that the relative abundancevalues of amino acid metabolism in the CLP group werehigher than those in the other groups(Fig. 3i). Functional difference analysis also showed that “Carbohydrate
metabolism, “Amino acid metabolism,” “ Metabolism ofother amino acids”, and “Infectious disease: bacterial”
were stimulated in the CLP group (Fig. 3j) (p < 0.05) after
FMT, they were suppressed (Fig. 3k) (p < 0.05). Principal Component Analysis (PCA) analysis of microbiota

Zhao et al. Journal of Neuroinflammation

Fig. 3 (See legend on next page.)(2024) 21:138

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Zhao et al. Journal of Neuroinflammation(2024) 21:138

Page 6 of 17(See figure on previous page.)
Fig. 3 Fecal microbial transplantation in CLP mice can reduce ammonia levels and may be associated with Bacilli. (a) : HE staining was used to observe
the morphological changes of the ileum. (b) : Intestinal pH test in the three groups of mice. (c) : Observe the changes in microbial species and abundance in each group at the class level through 16 S rDNA technology. (d) : Comparison of microbial α diversity among groups. (e) and (f): Comparison of
microbial β diversity among groups. (g) : LDA effect size (LEfSe) analysis CLP group compared with sham group. (h) : LEfSe analysis CLP group comparedwith (CLP + FMT) group. (i) : The functional annotation and differential analyses by KEGG and Go. (j) and (k) : Functional difference was analyzed for eachgroup. Figure 3l: PCA analysis of microbiota function. KEGG: The Kyoto Encyclopedia of Genes and Genomes; GO: the Gene Ontology. *p < 0.05, **p < 0.01,***
p < 0.001, ****p < 0.0001

function showed that there was a significant difference inmicrobiota function between the CLP and sham groups,
and microbiota function was similar after FMT (Fig. 3l).
Therefore, it is speculated that amino acid levels may berelated to changes in the intestinal flora.
FMT reduced amino acid metabolism and ammonia levelsin CLP mice

Metabolomic analysis by LC-MS/MS showed thatamino acids accounted for 55% of all identified metabolites (Fig. 4a). The amino acid levels in the CLP groupwere significantly higher than those in the sham group,with Log2FC values ranging from 1.15 to 60.87 in volcano plots (p < 0.05). The identified amino acids included
5-Aminolevulinic acid, 5-Hydroxylysine, Alloisoleucine,
Alpha-aminobutyric acid, Arginine, Asparagine, Glutamine, Histidine, Homoserine, Lysine, Methionine,
N-Acetylalanine, Phenylalanine, Proline, Serine, Threonine, Tryptophan, Tyrosine, and Valine (Fig. 4b). After
FMT, the amino acid substance of the CLP group was significantly reduced, with Log2FC values ranging from 0.10
to 0.49 (p < 0.05), the reduced of amino acids included
Arginine, Asparagine, Creatine, Glutamine, Homoserine,
N-acetylalanine, Serine, and Threonine (Fig. 4c). Additionally, the chord diagrams the four groups further confirmed that there was a significant difference in aminoacid levels (p < 0.05), with a correlation between aminoacids (r > 0.8) (Fig. 4d, Supplementary material 3). KEGG
pathway analysis of metabolites showed that amino acidmetabolism increased after CLP, and the difference inabundance was higher than that of the sham group,
including phenylalanine, tyrosine, and tryptophan biosynthesis; glycine, serine, and threonine metabolism after
Fecal microbial transplantation, arginine, and proline
metabolism; and glycine, serine, and threonine metabolism were reduced (Fig. 4e). Additionally, after FMT in
CLP mice, ammonia levels in the serum and hippocampal tissues decreased (Fig. 5d). These results showed that
FMT reduced amino acid metabolism and ammonia levels in the CLP group. Amino acid metabolism is the primary source of ammonia.
Bacilli has a significant correlation with amino acidmetabolism and ammonia

In this study, the association between intestinal microbes
and metabolic products was analyzed in each group. The
Bacilli of CLP group and Sham group differentiating

bacteria is positively correlated with the metabolismof Arginine, Asparagine, Cysteine, Glutamine, Glycylleucine, Histidine, Homoserine, Isoleucine, Lysine,
N − Acetylalanine, N − acetyltryptophan, Norleucine, Phenylalanine, Proline, Serine, Tyrosine, and Threonine. The
Bacilli of CLP and Sham group differentiating bacteria
is positively correlated with the metabolism of Arginine,
Asparagine, Creatine, Creatinine, Glutamine, Homoserine, N − Acetylalanine, Serine, Threonine (Fig. 4f ). Therefore, we speculate that the increase in Bacilli after CLP
may be related to the occurrence of NHH.
Decreased ammonia levels improved cognitive
dysfunction and neuronal damage in CLP mice
Neuroimaging

T2-weighted Arterial spin labeling (ASL), Diffusion
Tensor Imaging (DTI), and Proton magnetic resonancespectroscopy (1 H-MRS) were performed 72 h after CLP
surgery (n = 6). T2-weighted test results showed thatthe signal intensity of the CLP group was significantly
higher than that of the sham and control groups, whichshowed an increase in water content in mouse brain tissue after CLP. Furthermore, FMT reduced ammonia
levels, resulting in decreased water content of brain tissue in CLP mice (p < 0.001 or p < 0.05). ASL examination
results showed a significant decrease in blood flow inthe hippocampal region of CLP mice. After FMT in CLP
mice, cerebral blood flow improved (p < 0.01 or p < 0.05
or p < 0.0001). DTI test results showed that the fractional
anisotropy (FA) and Axial Diffusion (AD) of hippocampal
tissues in CLP mice were significantly higher than thosein the sham and control groups, while the Radical Diffusion (RD) and Mean Diffusivity (MD) of hippocampal
tissues in CLP mice were significantly lower than thosein the sham and control groups (p < 0.01, p < 0.05, or
p < 0.0001), suggesting that CLP could lead to impaired
nerve fiber integrity in mice, with improved nerve fiber
integrity observed after reducing ammonia level (Fig. 5a).
Imaging studies have shown that CLP can lead to cerebral edema, decreased cerebral blood flow, and destruction of the nerve fiber bundle integrity. However, after
FMT and reduction of ammonia levels in mice, cerebral
edema, decreased cerebral blood flow, and nerve fiber
incompleteness improved.

Zhao et al. Journal of Neuroinflammation(2024) 21:138

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Fig. 4 Fecal microbial transplantation reduced amino acid metabolism and ammonia levels in CLP mice. Bacilli has a significant correlation with aminoacid metabolism and ammonia. (a) : Metabolomic analysis by LC-MS/MS showed that amino acid counts accounted for 55% of all identified metabolite
numbers. (b) : The amino acid levels in the CLP group were significantly higher than those in the sham group in Volcano plots. (c) : after fecal microbial
transplantation, the amino acid substance of the CLP group was significantly reduced in volcano plots and heat maps. (d) : The metabolite comparisonof the chord diagrams of the four groups. (e) : KEGG pathway analysis of metabolites in CLP group, sham group, CLP + FMT group. (f) : The association
between intestinal microbes and metabolic products in the CLP group, sham group, CLP + FMT group. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Zhao et al. Journal of Neuroinflammation(2024) 21:138

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Fig. 5 The decrease in ammonia levels improved cognitive dysfunction and neuronal damage in CLP mice. (a) : T2WI, ASL, and DTI were perfected for 72 h
after CLP surgery in each group. The red box shows the comparison of T2WI signal density and hippocampal rCBF. (b) : The Nissl staining of hippocampal
tissues(DG, CA1, CA3) was perfected in each group. (c) : Evans blue dye extravasation was used to assess changes in vascular permeability in each group.
(d) : Ammonia levels in the hippocampus and serum were compared between the CLP group and the CLP + FMT group. (e) : The novel object recognition
was completed in each group of mice. (f) : The Morris Water Maze was completed in each group of mice.T2WI: T2-weighted imaging *p < 0.05, **p < 0.01,***
p < 0.001, ****p < 0.0001

Nissl staining of hippocampal tissues

Nissl staining of hippocampal tissues was performed 72 h
after CLP surgery (n = 6). The results showed that CLP
can lead to a decrease in hippocampal neuronal cells.
Moreover, after reducing ammonia levels, the damage

caused by CLP to neuronal cells was mitigated (p < 0.01,
p < 0.05, and p < 0.0001, respectively) (Fig. 5b).
Blood-brain barrier permeability

Evans blue dye extravasation was used to evaluate alterations in vascular permeability 72 h after CLP (n = 6).

Zhao et al. Journal of Neuroinflammation(2024) 21:138

Evans blue extravasation into the entire brain was measured in the CLP group. The large increase in Evans blue
extravasation in the CLP mice was significantly reducedin the CLP + FMT mice (Fig. 5c). Pathological studies
have shown that CLP not only damages neuronal cells
but also destroys the blood-brain barrier and reduces
brain damage caused by CLP after reducing ammonia
levels (p < 0.01, p < 0.05, and p < 0.0001).
Behavioral

On days 7 and 8 after CLP [35], new-object recognition was evaluated for cognitive memory in each mousegroup (n = 8). The results showed a reduction in the discrimination index of mice after CLP, after FMT in the
CLP group, the discrimination index improved (p < 0.001,
p < 0.05, and p < 0.0001, respectively) (Fig. 5e). From days
9 to 13 after CLP, the Morris Water Maze was employed
to evaluate the spatial learning and memory function ofeach group of mice. The number of platform crossingsand the time spent in the target quadrant in the CLPgroup were significantly reduced compared to the shamgroup and the control group, while the escape latency
was prolonged (p < 0.001, p < 0.05, and p < 0.0001, respectively) (Fig. 5f ). The behavioral study results showed
impaired cognitive function of mice after CLP, and the
reduction of ammonia levels could improve the cognitive function of CLP mice, thereby further confirming the
success of the SAE model.
Ammonia up-regulates astrocytes AQP4 may be themechanism of SAE

The western blot (WB) analysis (n = 6) and Quantitative
Real-time PCR (QT-PCR) (n = 6), immunofluorescencestaining (n = 6) results showed that 72 h after CLP, theexpression of AQP4 and Glial fibrillary acidic protein(GFAP) proteins and genes, as well as the number of
GFAP-positive cells, and the positive rate of AQP4 cellnumber as a proportion of GFAP cell number in hippocampal tissues were significantly increased (p < 0.0001
or p < 0.001 or p < 0.01 or p < 0.05) (Fig. 6a, b and c).
Additionally, 1 H- MRS study found that after CLP,
the levels of glutamate(Glu)/creatine(Cr) and N-acetyl
aspartate(NAA)/Cr in the hippocampus of mice weredecreased, indicating neuronal damage, while the levelof Glx (glutamate + glutamine)/Cr was increased. After
FMT, the serum ammonia level was decreased, neuronal damage was improved, and the level of Glx/Cr was
decreased (p < 0.0001 or p < 0.001 or p < 0.01 or p < 0.05)(Fig. 6d). Simultaneously, the use of the Elisa method todetect glutamine levels in serum and hippocampal tissues of mice in each group was consistent with the resultsof 1 H-MRS (Fig. 6e). A previous study had found thatammonia enters the brain and binds to GLU to produce
glutamine, further activating astrocytic AQP4 expression

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and leading to brain damage [16]. To further verify themechanism of action of ammonia in the brains of CLPtreated mice, cellular experiments were performed.
Ammonia up-regulates astrocytes’ AQP4 expression by
activating astrocytes, resulting in astrocytes damage

Astrocytes were treated with NH4Cl for 72 h (n = 3) totest the effect of ammonia on them. Immunofluorescence
results showed that astrocytes were activated, and thenumber of GFAP-positive cells increased significantly
compared to the control group (Fig. 7a). Subsequently,
the number of AQP4 and GFAP co-expressed cells also
increased (Fig. 7a) (p < 0.0001 or p < 0.001 or p < 0.01).
Additionally, compared with the control group, astrocyte
viability decreased after NH4Cl treatment (Fig. 7d). WB
and QT-PCR results showed that the expression levels of
AQP4 protein and gene increased after NH4Cl stimulation (p < 0.0001, p < 0.001, p < 0.01, and p < 0.05, respectively) (Fig. 7b and c). To further demonstrate the effectof AQP4 on astrocytes, we treated astrocytes with the
AQP4 inhibitor TGN20, which showed decreased astrocyte activation levels, decreased GFAP-positive cell numbers, decreased levels of co-expression with AQP4, andincreased astrocyte viability (Fig. 7a and d). Therefore,
cell experiments confirmed that ammonia can induce anincrease in AQP4 expression and activate astrocytes.

Discussion
The incidence of NHH is as high as 76% in critically illpatients [17]. Particularly, it has a high incidence among
patients with sepsis in critical care settings [6, 8, 18].
In this study, NHH also occurred in the acute phase of
CLP in mice, and the results were consistent with thoseobserved in patients with sepsis. Ammonia is primarily
produced by the urease generated by intestinal bacteria,
serving as the main source of serum ammonia.
After sepsis, the intestinal barrier becomes susceptible to damage, and such damage can contribute to theprogression of sepsis [19, 20]. Consistent with thesefindings, in this study, the ileum villus length and cryptdepth were decreased, and pH was increased after CLP,
indicating disruption of the intestinal structure. Moreover, CLP led to intestinal dysbiosis, as evidenced bysignificant changes in the species and abundance ofintestinal microbiota. Specifically, the Chao1 index andbeta diversity in the CLP group were significantly different from those in the Sham group and the control
group. The main manifestations included an increase in
Bacteroidia and a decrease in Bacilli. The gut microbiota is closely related to changes in ammonia levels [21].
In this study, both the serum ammonia and brain tissueammonia levels of the CLP group decreased after Fecal
microbial transplantation, aimed at restoring the normal intestinal microbial state of the CLP group mice.

Zhao et al. Journal of Neuroinflammation(2024) 21:138

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Fig. 6 Ammonia up-regulates astrocytes AQP4 may be the mechanism of SAE. (a) : The expression of AQP4 and GFAP protein in hippocampal tissue was
detected by western blot. (b) : The expression of AQP4 and GFAP gene in hippocampal tissue was detected by QT-PCR. (c) : Hippocampal tissue AQP4
co-expression with GFAP was detected by immunofluorescence. (d) : The changes in the level of metabolites in the hippocampus of mice in each groupwere detected by 1 H-MRS. (e) : Comparison of glutamine levels in serum and hippocampal tissues of mice in each group. *p < 0.05, **p < 0.01, ***p < 0.001,****
p < 0.0001

Metabolomic analysis by liquid chromatography-tandem
mass spectrometry showed that the amino acid levels in
the CLP group were significantly higher than those in thesham group. Moreover, the level of amino acid metabolism in the CLP group decreased after fecal transplantation. Enrichment and correlation analyses showed that

Bacilli regulate amino acid metabolism and are closely
related. Bacilli, known as a representative urease-producing strain [22], promotes an increase in ammonia levels. Therefore, we hypothesize that the increase in NHHlevels in CLP model mice is associated with the mostsignificant change in the abundance of Bacilli. Based on

Zhao et al. Journal of Neuroinflammation(2024) 21:138

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Fig. 7 Ammonia up-regulates astrocytes’ AQP4 expression by activating astrocytes, resulting in astrocytes damage. (a) : Immunofluorescence co-expression of AQP4 and GFAP in astrocytes in each group. (b): AQP4 protein expression in astrocytes was detected by western blot. (c) : AQP4 gene expressionin astrocytes was detected by QT-PCR. (d): Astrocyte viability was measured. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

the changes in amino acid levels observed in CLP micemodel and after Fecal microbial transplantation, as wellas the correlation analysis results between gut microbiota
and metabolomics, we deduced that Bacilli regulate theincreased metabolism of Arginine, Asparagine, Glutamine, Homoserine, N-Acetylalanine, Serine, and Threonine. These pathways represent potential mechanisms
underlying the occurrence of NHH in CLP mice model.
Ammonia is an important neurotoxic substance,
which is also an important mechanism for the occurrence of hepatic encephalopathy [4]. In recent years, therise in ammonia levels unrelated to liver dysfunction hasreceived increased attention due to its effect on the nervous system. Ammonia produced by the gut microbiome
can help buffer stress in the host, thereby providing a
gut-brain signaling basis for emotional behavior [21]. In
this study, we observed increased ammonia levels in CLPmodel mice despite their normal liver function. However,after Fecal microbial transplantation, ammonia levels
were reduced in CLP model mice. This reduction was
associated with improved cognitive function (MWM andNOR), cerebral edema (T2-weighted imaging, T2WI),
cerebral ischemia (ASL), improvement of nerve fiber
structural integrity (DTI), blood-brain barrier permeability (Evans blue), and increased neuronal cells in the
hippocampus (Nissl staining). Therefore, NHH also plays
an important role in brain injury in CLP mice based on
multimodal brain evaluation in CLP mouse models. Several studies have shown that the gut-brain axis plays animportant role in the pathogenesis of SAE, demonstrating that the gut microbiota facilitates SAE susceptibility[23, 24]. However, in the present study, we found that gut
microbes increased ammonia levels by regulating aminoacid metabolism, resulting in nervous system damage in
SAE mice.

Zhao et al. Journal of Neuroinflammation(2024) 21:138

After ammonia enters the brain tissue, it is rapidly
taken up by astrocytes and synthesized into glutamineby glutamine synthetase. The inhibition of astrocyte
proliferation by ammonia is mediated by l-methionine
sulfoximine, oxidative stress, and p38(MAPK) -MAPKdependent activation of p53 in hepatic encephalopathy[25]. Additionally, the research has shown that ammonia
causes neuronal disinhibition and seizures by disrupting
astrocyte potassium buffering [26]. However, the mechanism of ammonia in SAE is still unclear. In this study,
cerebral edema occurred in CLP mice in T2-weighted
imaging images of CLP mouse models, and cerebral
edema was reduced after reducing ammonia level expression. Astrocytic edema is an important mechanism of
cerebral edema. AQP4 in astrocytes is the main protein
controlling the inflow and outflow of water [27]. In thisstudy, the expression of GFAP and AQP4 proteins andgenes in CLP mouse models increased, indicating thatastrocytes and aquaporin cells were activated in CLP
mice. Additionally, the AQP4-GFAP/GFAP ratio in CLP
mice was significantly increased, and GFAP and AQP4
were co-expressed. 1 H- MRI studies showed that after
CLP, the levels of Glu/Cr and NAA/C in the brain of mice
were decreased, indicating neuronal damage. Meanwhile, the levels of glutamine were increased. However,after reducing ammonia levels, the expression of GFAP
and AQP4 proteins, genes, and cell counts of CLP micedecreased, and neuronal damage improved. Additionally,
although there was no significant difference in glutaminelevels, it was reduced in the serum and hippocampus.
Based on these findings, we speculate that NHH induces

Page 12 of 17

the upregulation of AQP4 expression in astrocytes, leading to astrocyte swelling, reduced cerebral blood flow,
and neuronal damage, potentially contributing to themechanism of brain injury in CLP mice (Fig. 8).
To further clarify the mechanism of NHH-induced
brain injury in the mouse model of CLP, cell experiments
were performed. The results showed that the numberof GFAP and AQP4 co-expressing cells increased significantly after the NH4Cl culture of astrocytes. Furthermore, GFAP expression was reduced when the AQP4
inhibitor TGN020 was added. These findings suggestthat ammonia entry into the brain leads to the upregulation of AQP4 expression in astrocytes, which represents an important mechanism underlying brain injury.
Ammonia is reportedly involved in the pathogenesis ofhepatic encephalopathy by regulating astrocytic AQP4
expression in the brain [28]. Zhu DD and colleagues
found that AQP4 aggravates cognitive impairment in
SEA by inhibiting Nav1.6-mediated astrocyte autophagy[11]. In this study, we found that ammonia entering thebrain increased the expression of AQP4 in astrocytes ina mouse model of CLP. However, the potential mechanism by which AQP4 causes brain injury in this model
remains unclear and requires further exploration in
future research.
Limitations of our study including (1) healthy rodent
animals do not represent the patients; (2) the CLP model
was used in the SAE model of mice, which showed
abdominal infection, and the infection sites of SAE
patients in clinical practice often included the brain,lungs, abdomen, bloodstream infection and other sites,

Fig. 8 A diagram of the mechanism of NHH leads to SAE. After sepsis, ammonia-producing Bacilli increase in the intestine, promoting the breakdownof amino acids, ammonia increases, ammonia enters the brain and aggregates in astrocytes, up-regulating the expression of astrocyte hydration protein
AQP4, resulting in astrocyte activation, edema, promoting cerebral edema, cerebral ischemia, and nerve damage, leads to the occurrence of SAE

Zhao et al. Journal of Neuroinflammation(2024) 21:138

therefore, the results of this study may be more relevantto those of patients with SAE with abdominal infection in
the clinic. (3) clinical relevance including the fluid resucistaiton, antibiotics, vasopressor and source control inthis model were not reflected, therefore, the results ofthis study may be biased against patients with SAE inclinical practice. In this study, we wanted to observe theeffect of CLP on intestinal microbial species, but, antibiotics would a significant effect intestinal microbial species [33], therefore in this study did not use antibiotic
refer to other literature related to SAE [11, 34]. (4) thechanges in ammonia levels and imaging in CLP were notdynamically monitored and observed in the long term.
In summary, the disturbance of the intestinal flora after
sepsis, characterized by an increase in urease-producing
bacilli, leads to increased amino acid metabolism andammonia production. These changes result in the upregulation of AQP4 levels in astrocytes, facilitating the entryof ammonia into the brain. Consequently, CLP mice
exhibit cerebral edema, impaired structural integrity ofnerve fibers, neuronal cell damage, and cognitive dysfunction, potentially representing an underlying mechanism for the occurrence of SAE. This study provides new
insights into the pathological mechanisms and potential
new drug targets for the treatment of SAE.

Materials and methods
AnimalThe experimental procedures and animal care protocols were approved by the Committee on Ethical Use of
Animals of Tianjin Medical University General Hospital. The experimental procedures followed the National
Institutes of Health Guidelines for the Care and Use ofLaboratory Animals. Male C57BL/6J mice, aged 8–10
weeks and weighing 20–25 g, were obtained from theSpecific Pathogen-Free (SPF) Model Animal Center ofthe Military Medical Science Academy in Beijing, China.
The mice were categorized into control, sham, CLP, and
CLP + FMT groups.
CLP model

The experiment utilized the classical CLP model [29].
Briefly, after administering 2% isoflurane anesthesia anddisinfecting the skin, a 1-cm incision was made along
the abdominal midline to reveal the cecum. The cecum
ligation was about 50% ligated between the distal poleand base, 21G needle size was used to passed throughthe cecum, to induce moderate sepsis. The incision wasclosed using a sterile 6 − 0 silk suture. Mice subjected tothe sham operation had their cecum exposed in the samemanner as those subjected to CLP, but the cecum wasneither ligated nor punctured. The mice in the control
group did not undergo surgery. On the day of surgery,
fresh FMT was prepared by collecting the cecal contents

Page 13 of 17

from healthy mice. On postoperative days one, two, andthree, the selected CLP group of mice was administered
2 mL of FMT via rectal enema using a blunt vein irrigation cannula. The FMT protocol was based on the studyof Kim et al. [20].
Genomic DNA extraction

Genomic DNA was extracted from the samples using
an OMEGA Soil DNA Kit (D5625-01) (Omega Bio-Tek,
Norcross, GA, USA). The selected V3-V4 variable region
was amplified by PCR using specific primers with barcodes and high-fidelity DNA polymerase. The PCR products were detected by 2% agarose gel electrophoresis,
and the fragments of interest were recollected using the
Quant-IT Pico Green dsDNA Assay Kit. The PCR-amplified recovered products were assayed and quantified
using a microplate reader (BioTek, FLx800) and a fluorescence quantitative system. Library-building reagentswere obtained using Illumina TruSeq Nano DNA LT
Library Prep Kit Box. Quality assessment of the built
library was performed using a Bioanalyzer 2100 and Promega Quant Fluor.
16 S rDNA processing and analysis

The 16 S analysis uses the Silva (version138) database for
species comparison and annotation. Qiime2 software wasused to calculate the α β diversity indices. The analysis
included the Wilcoxon rank sum test for assessing differences in α diversity (Shannon, Simpson, Chao 1) and β
diversity (based on Weighted Unifrac and Unweighted U
distances). Differences in community composition wereanalyzed using principal coordinate analysis (PCoA) or
PCA.
LEfSe (LDA Effect Siz) analysis was conducted to identify species significantly impacting the differentiation ofsamples between groups. STAMP difference analysis wasused to compare the abundance of species and the differential species functions between two sets of samples(Wilcox test analysis) or multiple sets of samples (Kruskal–Wallis test analysis). Piecrust software was used toanalyze functional differences between different groups
by comparing species composition information obtained
from 16 S sequencing data, including KEGG and COG.
LC/MS untargeted metabolomics analysis

Following sample pretreatment, metabolite analysis was
conducted using a UHPLC system (1290 Infinity LC,
Agilent Technologies) combined with a QTRAP MS
instrument (6500, Scitex) at Shanghai Applied Protein
Technology Co., Ltd. The analytes were separated using
HILIC and C18 columns in UPLC with specific column
specifications (HILIC: Waters UPLC BEH Amide column, 2.1 mm × 100 mm, 1.7 μm; C18: Waters UPLC
BEH C18-2.1 × 100 mm, 1.7 μm). Multiquanta was used

Zhao et al. Journal of Neuroinflammation(2024) 21:138

Page 14 of 17

for quantitative data processing. The processed data were
uploaded to SIMCA-P (version 14.1, Umetrics, Umea,
Sweden) for multivariate data analysis, which included
Pareto-scaled principal component analysis (PCA) andother analyses.

a dehydrated transparent mount. Intestinal villus lengthand crypt depth and liver tissue were measured fromphotomicrographs captured with an OLYMPUS eclipse
inverted Microscope (OLYMPUS CKX43-LP) using
image software.

Magnetic resonance imaging (MRI)

Nissl staining

MRI of each group of mice was conducted using a 9.4T
magnetic resonance small animal scanner (Bruker Bio
spin, Germany) at Tianjin Medical University General
Hospital. In this experiment, MRI techniques, including
T2-weighted imaging, ASL, TDI, and 1 H-MRS, wereused to observe changes in cerebral edema, cerebral
blood flow, nerve fiber integrity, and metabolites in eachgroup of mice.

After routine treatment in paraffin, Sect. (8 μm) wereobtained using a microtome (Leica Histoscore MULTICUT). Paraffin sections were then dewaxed to water.
Subsequently, they were stained with methylene blue
staining solution for 10 min, differentiated in Nicholas
differentiation solution, and processed in ammonium
molybdate solution for 1 min. Following this, sections
were dehydrated and transparently sealed. Observationand photography were conducted using a panoramicscanner (Panoramic Scan).

T2-weighted imaging

The Parameter settings for T2-weighted imaging(T2WI) were as follows: repetition time (TR)/echo time(TE) = 4808/33 ms, field of view (FOV) = 20 × 20 mm2,
slice thickness = 0.5 mm, number of averages (NA) = 2,
and slices = 50.
1 H-MRSThe parameter settings for 1 H-MRS were as follows:
RT/TE = 1500/16.5 ms, averages = 256, target voxel ofinterest, 4 × 1 × 2 mm3. Data analyses were performed
using the Bruker Para Vision 360 and Mestre Nova software (V12.0.0, Mestre Lab Research, Spain). The process of analyzing the metabolites used Cr as a standard
reference.
ASL

The parameter settings for 1 H-MRS were as follows: RT/TE = 4808/33 ms, FOV = 20 × 20 mm2, image
size = 128 × 128, and slice = 1. The ASL perfusion image
was obtained using Bruker Para Vision 360 software.
DTI

The DTI parameter settings include TR of 3,000 ms, TEof 19.6 ms, 85 gradient directions, diffusion gradient ontime of 4 ms, FOV = 20 × 20 mm², 0.5 mm slice thickness,
and a 256 × 256 matrix. DTI images were analyzed using
ParaVision 360 V3.0 software (Bruker Biospin, Germany)
and DSI Studio.
HE staining

Samples from the ileum region were taken for histopathology, while the liver tissue was placed in neutral
buffered formalin for fixation, with a slice thicknessof 4 μm. Paraffin sections were then deparaffinized inwater stained with Harris hematoxylin for 5–10 min for
nuclear staining, followed by staining with eosin staining solution for 1–3 min for cytoplasmic staining using

BBB permeability

The four groups of mice were injected with 4% Evans blue
(EB; 2 mL/kg, Aladdin) through their tail veins. After
3 h, the mouse brain was removed, and the tissue wascut and homogenized in an ice water bath at 1000 g. The
homogenate was then centrifuged for 15 min, and 750 µLof the supernatant was collected. Acetone was added tothe supernatant in a ratio of supernatant to acetone = 3:7,
followed by incubation at room temperature for 24 h.
Thereafter, the mixture was centrifuged at 2000 g for
15 min, and the supernatant was collected. The absorbance value of both the standard and the sample was readat 620 nm using a microplate reader, and the EB concentration was calculated accordingly.
ELISA

Blood and hippocampal tissue samples were collectedfrom each mouse centrifuged at 3000 rpm for 10 min.
Serum and hippocampal tissue samples were collectedaccording to weight (mg) to volume (mL) ratio of 1:9,
with the corresponding amount of normal saline added.
Grinding beads were introduced, and the samples were
thoroughly ground in a high-speed and low-temperature
tissue grinder. Subsequently, centrifugation at 3000 rpm
for 10 min was performed, and the supernatant was
immediately tested following the instructions provided
for the ammonia (A086-1-1, 202,305) and glutamine(F30107-A, 202,307) assays.
Biochemical testing

After administering anesthesia, about 0.8 ml of bloodfrom blood vessels is extracted from the heart, and thesupernatant was obtained by centrifugation at 3000 rpm
for 15 min at room temperature. Serum was used to measure liver function levels in mice. Alanine aminotransferase, aspartate aminotransferase, and total bilirubin levels

Zhao et al. Journal of Neuroinflammation(2024) 21:138

were measured using an automatic biochemical analyzer(XN-530, Sysmex).Quality control testing is performed
prior to the application of automatic biochemical analyze. After the application of automatic biochemical analyze quality control test is passed, the prepared samples
are sampled for test, in this process, the machine takes
about 25µL sample. After interrupting sampler mode,
perform sample analysis.When analysis of the sample iscompleted, close the sampler cover and press the modeswitch.
Morris water maze (MWM)

The cognitive dysfunction test was conducted using theMWM [30, 31]. The apparatus had a diameter of 120 cm
and a height of 45 cm, with the water temperature maintained at 20 ± 2 °C. The pool was divided into four quadrants of equal area, and the platform was placed at thecenter of the target quadrant. Each mouse in all groups
was trained for four consecutive days (9–12 days after
CLP) to find the platform and reach a plateau. The time
taken by the mice to reach the platform was separately
recorded after they entered the water in each quadrant,
serving as the latency period. If the mouse failed to findthe platform within 60 s, the experimenter guided it
to the platform and allowed it to remain there for 10 s,with an escape latency of 60 s. In the probe test (13 days
after CLP), mice were placed in water opposite the target
quadrant, and the number of times they passed throughthe original plateau area within 60 s was recorded, alongwith the time spent in the target quadrant.

(Novel object recognition) NOR
The NOR was used to evaluate the cognitive functionof exploring new object abilities [32]. Before the experiment, the mouse was gently placed in a square box(50 × 50 × 40 cm3) for 3 min to acclimate to the environment. The box was equipped with a camera directly
above it, connected to the Super Maze Animal Behavior
Video Analysis System (Xinruan Information Technology
Ltd, Shanghai, China).
Subsequently, the mice were randomly placed in thecenter of the arena, where two identical objects were
explored for 5 min each (day 7 after CLP). On day 8 after
CLP, the two familiar objects were replaced with a novel
object, and the mice were allowed to explore for 5 min.
The novel object and discrimination index [(time spent
exploring the novel object/time spent exploring the two
objects) × 100%] were recorded and analyzed.
QT-PCR

Hippocampal samples were collected from the brain
tissue, and cell experiments were performed using
C8-D1A (mouse cerebellar astrocytes)/CL-0506. TRIzol
lysate was added, and total RNA was extracted. RNA

Page 15 of 17

was synthesized using a ReverTra Ace qPCR RT Kit
to synthesize cDNA. A denaturation buffer was added
to the cell sample to induce cell lysis, followed by RNA
extraction, precipitation, washing, solubilization, andassessment of RNA integrity. Real-time fluorescencequantification PCR was performed using specific primers. For the gene of interest, the upstream primers of
GFAP were 5’- ​A​A​C​A​A​C​C​T​G​G​C​T​G​C​G​T​A​T​A​G​A​C − 3,’and the downstream primers of GFAP: 5’- ​A​T​C​T​C​C​T​C​C​T​C​C​A​G​C​G​A​T​T​C​A​A − 3.’ Similarly, for AQP4, upstream
primers were 5’- ​G​C​A​T​C​G​C​T​A​A​G​T​C​C​G​T​C​T​T​C​T − 3,’and the downstream primers were 5’- ​G​A​G​G​T​G​T​G​A​C​C​A​G​G​T​A​G​A​G​G​A​T − 3.’ For B-actin, the upstream primers
were 5’- ​G​T​A​C​T​C​T​G​T​G​T​G​G​A​T​C​G​G​T​G​G − 3,’ and thedownstream primers were 5’- ​G​C​A​G​C​T​C​A​G​T​A​A​C​A​G​T​
C​C​G − 3.’ The PCR protocol included pre-denaturation at
95 °C for 10 min, followed by denaturation at 95 °C for
15 s, annealing at 62 °C with elongation for 1 min, and a
total of 40 cycles.
Immunofluorescence

Briefly, the brain samples were fixed overnight in 4%
paraformaldehyde and subjected to immunofluorescencestaining. The astrocytes were pre-treated with NH4Cl(Chemuza, 12125-02-9) or TGN020 (Selleck, S0158).
Primary astrocytes were then removed from the cellincubator and fixed in 4% paraformaldehyde after washing, followed by permeabilization in 0.3% TX-100 andblocking with 2% bovine serum albumin (BSA). The cells
were treated with primary antibodies, including mouseanti-GFAP (1:300, Wuhan Mitaka, 60190-1-Ig), rabbit
anti‐AQP4 (1:300, Wuhan Mitaka, 16473-1-AP) overnight at 4 °C. Subsequently, the cells were then incubatedwith secondary antibodies: goat anti-rabbit IgG/TRITC(1:100; Nakasugi Golden Bridge, ZF-0316) and goatanti-mouse IgG/FITC (1:100; Nakasugi Golden Bridge,
ZF-0312). All images were acquired using a panoramicscanner (3DHISTECH Panoramic Scan). Positive fluorescence staining was calculated using image software.
Western blot analysis

Brain tissue and astrocyte samples were homogenized in
RIPA buffer to extract the proteins. Different groups ofbrain tissues and astrocytes were treated with different
drugs. The primary antibodies used included mouse antiGFAP (1:10000, Wuhan Mitaka, 60190-1-Ig), rabbit anti‐
AQP4 (1:15000, Wuhan Mitaka, 16473-1-AP), Mouse
Anti-β-tubulin (1:2000, Nakasugi Kanabashi, TA-10). The
sample‐loaded membranes were incubated overnight at
4 °C and then, post 8–12 h of incubation, treated withsecondary antibody, Goat Anti-Rabbit IgG(1:5000, Nakasugi Golden Bridge, ZB-2301), and Goat Anti-MOUSE
IgG(1:5000, Nakasugi Golden Bridge, ZB-2301). A gelZhao et al. Journal of Neuroinflammation(2024) 21:138

imaging analysis system (Beijing Saizhi, Champchemi
610 plus) was used to detect band signals.
Astrocytes CCK8 assay

Astrocyte viability was determined using a CCK8 kit(BS350A, Biosharp). The cell suspension was seeded ina 96-well plate with 100 µL per well, containing 5,000
cells per well. Subsequently, 10 µL of CCK8 solution wasadded to each well, and the plate was further incubated in
the cell culture incubator for 1–4 h.

Astrocyte culture
The brain tissues of C57BL/6J mice were extracted anddigested with 0.125% trypsin in Dulbecco’s modified
Eagle’s medium (DMEM) in a cell culture incubator.
DMEM containing 10% fetal bovine serum (FBS) wasused to terminate the digestion. Single cells were seeded
at a density of 1 × 10 cells/well in a 24-well culture plate
pre-coated with polylysine, with a density of approximately 4 cm-2. After 72 h, the medium was replaced witha fresh, complete medium, and the cells were cultured forapproximately 7 days to achieve 90% cell growth coverage. The plate was then spun at 260 rpm (24 h, 37 °C) tocollect purified astrocytes [11].
Statistical analysis

All data were expressed as the mean ± standard deviation (SD). GraphPad Prism 10.0.2 software (San Diego,
CA, USA) was used for data analysis. Statistical significance was assessed using Student’s t-test, Mann–Whitney U test, ANOVA for multiple comparisons, two-way
ANOVA with Tukey’s post hoc test, and one-way
ANOVA with Tukey’s post hoc test. p < 0.05 was considered statistically significant.
Abbreviations
SAE	Sepsis-associated encephalopathy
NHH	Non-hepatic hyperammonemia
CLP	Cecal ligation and perforation
ASL	Arterial spin labeling
DTI	Diffusion Tensor Imaging
1H-MRS	Proton magnetic resonance spectroscopy
AQP4	Aquaporin4channel
GFAP	Glial fibrillary acidic protein
KEGG	Kyoto encyclopedia of genes and genomes
GO	Gene ontology
PCoA	Principal coordinate analysis
LEfSe	LDA effect sizeFA	Fractional anisotraphy
AD	Axial diffusion
RD	Radical diffusion
MD	Mean Diffusivity
Glu	Glutamate
Cr	Creatine
NAA	N-acetylaspartate
Glx	Glutamate + glutamine, T2WI: T2 weighted image

Page 16 of 17

Supplementary Information

The online version contains supplementary material available at https://doi.
org/10.1186/s12974-024-03135-2.
Supplementary Material 1
Supplementary Material 2
Acknowledgements
We would like to thank to “Zhongke New Life Biotechnology Co., Ltd.” forproviding intestinal microbiota and metabolomics analysis and departmentof imaging of General Hospital of Tianjin Medical University for providing micefMRI and thank Editage (www.editage.cn) for English language editing.
Author contributions
L.N.Z, and K.L.X, Y.L. contributed to the conception and design of the project,they played a major role in performing experiments and analyzing the data, as
well as writing the initial draft of the manuscript. L.N.Z., Z.Z, H.N.W., J.Y.Z., P.W.,
N.N.Z., F.Y., Z.W.W., Y.Y.W., H.Y.Z., H.B.L., Z.F.H., H.S., Z.Y.W., and F.H. S contributed
to conducting the experiments. K.L.X., and Y.L modified the language. All
authors read and approved the final version of the manuscript.
Funding
This work was supported by the grant from the Natural Science Foundationof Inner Mongolia Autonomous Region (serial number 2022MS08002); Tianjin
Municipal Health Commission (serial number TJWJ2023QN007); National
Natural Science Foundation of China (82302413).
Data availability
No datasets were generated or analysed during the current study.

Declarations
Ethical approval
All animal experiments were carried out according to the Institutional Animal
Care and Use Committee guidelines of the NIH, USA, and approved by theInstitutional Ethics Committee of Tianjin Medical University General Hospital.
Competing interests
The authors declare no competing interests.
Received: 26 March 2024 / Accepted: 20 May 2024

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