Journal of Neuroinflammation

Xu et al. Journal of Neuroinflammation(2024) 21:195
https://doi.org/10.1186/s12974-024-03187-4

Open Access

RESEARCH

Bruton’s tyrosine kinase inhibition ameliorated
neuroinflammation during chronic whitematter ischemia
Lu-Lu Xu1,2,3,4†, Sheng Yang1,2,3,4†, Luo-Qi Zhou1,2,3,4, Yun-Hui Chu1,2,3,4, Xiao-Wei Pang1,2,3,4, Yun-Fan You1,2,3,4,
Hang Zhang1,2,3,4, Lu-Yang Zhang1,2,3,4, Li-Fang Zhu1,2,3,4, Lian Chen1,2,3,4, Ke Shang1,2,3,4, Jun Xiao1,2,3,4, Wei Wang1,2,3,4,
Dai-Shi Tian1,2,3,4* and Chuan Qin1,2,3,4*Abstract
Chronic cerebral hypoperfusion (CCH), a disease afflicting numerous individuals worldwide, is a primary causeof cognitive deficits, the pathogenesis of which remains poorly understood. Bruton’s tyrosine kinase inhibition(BTKi) is considered a promising strategy to regulate inflammatory responses within the brain, a crucial processthat is assumed to drive ischemic demyelination progression. However, the potential role of BTKi in CCH has notbeen investigated so far. In the present study, we elucidated potential therapeutic roles of BTK in both in vitro
hypoxia and in vivo ischemic demyelination model. We found that cerebral hypoperfusion induced white matterinjury, cognitive impairments, microglial BTK activation, along with a series of microglia responses associatedwith inflammation, oxidative stress, mitochondrial dysfunction, and ferroptosis. Tolebrutinib treatment suppressedboth the activation of microglia and microglial BTK expression. Meanwhile, microglia-related inflammation andferroptosis processes were attenuated evidently, contributing to lower levels of disease severity. Taken together,
BTKi ameliorated white matter injury and cognitive impairments induced by CCH, possibly via skewing microglia
polarization towards anti-inflammatory and homeostatic phenotypes, as well as decreasing microglial oxidative
stress damage and ferroptosis, which exhibits promising therapeutic potential in chronic cerebral hypoperfusioninduced demyelination.
Keywords Chronic cerebral hypoperfusion, Tolebrutinib, Microglia, Inflammation, Ferroptosis†

Lu-Lu Xu and Sheng Yang contributed equally to this work.

*Correspondence:
Dai-Shi Tian
tiands@tjh.tjmu.edu.cn
Chuan Qinchuanqin@tjh.tjmu.edu.cn
1
Department of Neurology, Tongji Hospital, Tongji Medical College,
Huazhong University of Science and Technology, Wuhan 430030, China

2

Hubei Key Laboratory of Neural Injury and Functional Reconstruction,
Huazhong University of Science and Technology, Wuhan 430030, China
3
Key Laboratory of Vascular Aging, Tongji Hospital of Tongji Medical
College, Ministry of Education, Huazhong University of Science andTechnology, Wuhan 430030, China
4
Key Laboratory of Neurological Diseases of the Chinese Ministry of
Education, School of Basic Medicine, Tongji Medical College, Huazhong
University of Science and Technology, Wuhan 430030, China© The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0
International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you
give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified thelicensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it.The images orother third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to thematerial. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation
or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.To view a copy of this licence, visit http://
creativecommons.org/licenses/by-nc-nd/4.0/.

Xu et al. Journal of Neuroinflammation(2024) 21:195

Introduction
The cerebral white matter is prone to ischemic injury
when cerebral blood flow sustains inadequate perfusion,
particularly due to lower blood supply from distal partsof long deep white matter arteries [1, 2]. Accumulating
evidence suggests that chronic cerebral hypoperfusion(CCH) serves as a primary driver of vascular pathology
and clinical manifestations of vascular cognitive impairment (VCI) [3, 4]. As the second most common typeof dementia worldwide, VCI delivers a great burden tosociety for its high mortality and disability rate [5, 6].
Accordingly, attempts to investigate the pathologicalmechanisms and seek new therapeutic targets of CCH
are of critical importance for both preventing the occurrence and mitigating the development of VCI.
A cascade of molecular and cellular events such asionic imbalance, oxidative stress, mitochondrial dysfunction, and inflammation, are involved in the pathogenesis of CCH [7–9]. These cascades subsequently
trigger blood-brain barrier (BBB) breakdown, endothelial dysfunction, and glial activation, creating an ongoinginflammatory microenvironment [10–12]. In general, as
the major resident immune cells in the central nervous
system (CNS), microglia are documented to play anessential role in both the acute and chronic phase of neuroinflammation [13]. Activated microglia are responsible
for combating harmful external stimuli, which contributeto homeostasis in the CNS microenvironment. However,dysfunctional and sustained microglial activation often
leads to chronic inflammation in the CNS, accompanied
by release of a large amount of pro-inflammatory cytokines and chemokines, leading to cell death and myelin
injury, which eventually exacerbates ischemic demyelination [14, 15]. Thus, interventions that suppress chronic
inflammation mediated by microglia dysfunctions may
be appropriate strategies against CCH.
Bruton’s tyrosine kinase (BTK), a member of the cytoplasmic non-receptor tyrosine kinase Btk/Tec family,is involved in the activation and function of B cells andmyeloid cells, and is currently regarded as a drug target tolighten both dysregulated innate and adaptive immunity[16]. BTK inhibitors (BTKi), which could penetrate theblood-brain barrier (BBB), may act on brain-infiltrated
B cells or brain resident microglia to affect inflammationand neurodegeneration within the CNS, respectively.
However, little is known about the effects of BTKi on
microglia status and functions in CCH, and the related
mechanisms remain to be further elucidated.
Tolebrutinib is an irreversible, selective, effective, andorally active small-molecule inhibitor of BTK, which
can breach the BBB [17]. In this study, utilizing a modelof bilateral carotid artery stenosis (BCAS), we exploredthe potential regulative role of tolebrutinib in microglia
inflammation, oxidative stress, and ferroptosis. We found

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that BTK was remarkably activated, which was responsible for microglia activation and a series of subsequent
inflammatory reactions following hypoxic-ischemic
injury. The treatment of tolebrutinib restored myelination and ameliorated cognitive impairment in chronic
cerebral ischemia, indicating a new therapeutic strategy.

Experimental animals and subject details
Animals and study designs

Wild-type (WT) C57BL/6J mice (20–25 g, 8–12 weeks)
were purchased from Vital River Laboratory Animal
Technology Co. Ltd., Beijing, China. Male and female
animals were assigned into different groups randomly
and housed in a specific pathogen-free (SPF) environment, at generally 25 °C and 55–60% relative humidity
with a 12-hour cycle of light alternating with darkness,
fed with free access to food and water. Emphatically, allexperiments involving animals were consented by the
Animal Care Committee of Tongji Medical College,
Huazhong University of Science. Technology, China.
A total of 57 mice were consumed in our study. Animals were randomized using random numbers generated
by GraphPad Prism. In principle, the sample size for eachexperimental group was used according to existed studies[18]. Meanwhile, the sample size estimation for comparison of means of multiple samples in completely randomized designs with a statistical power of 0.8 and statisticalsignificance of 0.05 was performed via PASS software,
which the mean and standard deviation were referred toprevious literature on BCAS models [2]. Consequently,
for most animal experiments, n = 6 per group was sufficient. For several experiments such as the eight-arm
maze experiment and the new object recognition experiment, n = 9 mice per group was otherwise used to maximally meet statistical requirements. All analyses were
executed by experienced and blinded researchers who
abide by the guidelines of ARRIVE completely.
Bilateral carotid artery stenosis (BCAS) surgery

The BCAS model was established comply with previous
methods [19]. Briefly, mice were anesthetized with 5%
isoflurane and kept with 2% isoflurane in oxygen supplied
from RWD inhalation anesthesia machine. Then, themiddle cervical line was made to separate bilateral common carotid arteries (CCAs) carefully. One side CCA wastwined around a delicate miniature spring with an internal diameter of 0.18 mm (Sawane Spring Co, Shizuoka,
Japan) gently, and the same procedure was repeated on
the other side. The sham-operated group received a
middle cervical line incision only without twisting coils
around common CCAs. All animals were housed in an
SPF environment and provided with abundant food andwater after operations.

Xu et al. Journal of Neuroinflammation(2024) 21:195

Brain simple preparation

At 3, 30, and 90 days after BCAS surgery, mice wereanesthetized with 5% isoflurane, then phosphate buffer(PBS) (0.1%, 30mL, 4 °C) were transcardially perfused,followed by PFA (4%, 30mL, 4 °C). Dissecting brain tissues, soaking in cold 4% PFA all night and dehydrating in
30% sucrose for 3 days. The dehydrated brain tissues wereembedded in Tissue-Tek O.C.T compound (Sakura Finetek, USA) to freeze, sliced into 20 μm-thick serial sections and stored at -80 °C subsequently.
Luxol fast blue staining

White matter Luxol fast blue staining (LFB) staining was
carried out as previously described to evaluate the degreeof myelin damage [20, 21]. The 20 μm frozen slices wereincubated with 0.1% LFB dye (G1030, Servicebio) at 60 °C
for 8 h, then differentiated in 0.05% lithium carbonate
solution and 70% ethanol by turn, dehydrated with gradient ethanol (70%, 95%, 100%) eventually. The staining
position of brain sections were same in each group andthe site was 1.0 mm lateral, 0.8 mm anterior, and 2.2 mm
deep to the bregma [22]. The demyelinated corpus callosum images were captured by a microscope (BX51,
Olympus, Japan) and sorted into 0–3 grades: normal(grade 0), disarranged myelin fiber (grade 1), marked vacuoles (grade 2), and disappearance of nerve fibers (grade
3) [23, 24].
Flow cytometry

Mice were anesthetized with 5% isoflurane and transcardially perfused with pr-cold PBS, then taken out brain
samples. The brain dissociation kit (Miltenyi Biotec,
Germany) was utilized to make single cell suspensions.
Briefly, the cerebral hemisphere was cut into pieces andincubated with enzyme A and enzyme P and digested
for 30 min at 37 °C. The 10 ml PBS was added to theabove mixed suspension. The enzyme mixture was filtered with a 70 mm cell strainer into a 15 ml centrifuge
tube and centrifuged at 300xg, 4 C for 5 min. Subsequently, myelin debris was removed by debris removal
solution. Afterward, cell pellets were re-suspended by
100uL PBS containing 2% FBS (FACS), then added 1ul
Fvs-700 (1:100000, 546996, BD Biosciences) and incubated for 10 min at 4 °C. The suspensions were further
washed by PBS and re-suspended with 100ul FACS again.
2ul Fc block (1:50, 553142, BD Biosciences) was added
to cell pellets, and incubated for 10 min at 4 °C. Then,
1ul each of CD45-PERCP-Cy5.5 (1:100, 103131, Biolegend), CD11b-FITC (1:100,101206, Biolegend), CD4-APC(1:100, 553051, BD Biosciences), Ly6G-PE (1:100, 551461,
BD Biosciences), CD3-PE-Cy7(1:100, 552774, BD Biosciences), CD19-BV650(1:100, 563148, BD Biosciences) andCD8-BV510 (1:100, 563068, BD Biosciences) were added
to the suspension and incubated for 30 min at 4 °C. The

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mixture was washed with PBS twice. Finally, the live cells
were performed through a flow cytometer (CytoFLEX,
Beckman) and analyzed by Flowjo.
Tolebrutinib administration

Tolebrutinb was orally administered to wild-type micewith a gastric needle at the dosage of 5 mg/kg per day for
30 consecutive days after BCAS surgery two hours. The
vehicle group was treated with 200ul excipient containing
10% DMSO per day for 30 consecutive days after BCAS
surgery two hours, which was a therapeutic strategy
for ischemic white matter injury. The sham group also
received oral vehicle. The method and dosage of administration followed the manufacturer’s instructions for tolebrutinb (SAR442168, MCE).
Immunofluorescence staining

For immunofluorescent staining, the brain slices were
washed with 0.1% PBS thrice. Immediately, these sections were permeabilized with 0.25% Triton-X100 for
10 min at room temperature, followed by blocking with
QuickBlock for 15 min. After that, brain sections wereincubated with primary antibodies at 4 °C overnightand subsequently incubated with secondary antibodies at room temperature for 1 h under darkness. At last,
the slices were stained with a 4,6-diamidino-2-phenylindole (DAPI) medium. Primary antibodies including MBP (1:200, ab7349, abcam), Iba1(1:300,17198,
CST), MAP2(1:200,ab254143, Abcam), GFAP (1:500,
ab4648, Abcam), OLIG2(1:200, 13999-1-AP, Proteintech), 4HNE (1:100,bs-6313R, Bioss), 8-OHDG (1:100,bs1278R, Bioss), BTK (1:200, 8647, CST), pBTK (1:200,
NBP178295, NOVUS), CD16/32(1:50, 553142, BD Biosciences), CD68 (1:200, MCA1957, Bio-rad), Clec7a(1:50, mabg-mdect-2, InvivoGen). Secondary antibodies
including Secondary antibodies include AlexaFluor488
Donkey anti Goat-IgG (Yeasen), AlexaFluor488 Donkey
anti Rat-IgG (Yeasen) and Cy3 Donkey anti Rabbit IgG(Jackson).
Images were captured by confocal microscope(FV1200, OLYMPUS,) and uploaded to Image J for analysis. The microglial morphology analysis and the skeletonized images were performed as previous study [25].
Behavioral tests

The working and reference memory in mice wereaccessed with the eight-arm maze experiment as previous described [18]. Mice to be trained were deprived offood and water for eight hours. For spatial working memory test, mice were placed in the central starting platform
and allowed to consume food pellets that placed in the
distal end of eight arms for 15 min or until food in eacharm were disappeared. The working memory error wasrecorded when a mouse reentered an arm. All animals

Xu et al. Journal of Neuroinflammation(2024) 21:195

Page 4 of 15

completed a trial per day, lasting for 7 days. In reference
memory task, only four of the eight arms had food pellets
and the trial ceased immediately after pellets in four foodwells were consumed. All animals completed three trials
per day, lasting for 3 days.
The new object recognition experiment (NOR) was
performed to evaluate novelty preference. Briefly, mice
were habituated in a square box and familiar with twosquare objects the day before the test. After 24 h, theexploring functions were evaluated by replacing one of
the familiar square objects with a novel rounded object.
All exploring time were 5 min [26, 27].

RT Master Mix (RR036A, Takara) following manufacturer’s protocol. Subsequently, the mixed reaction system for quantitative RT-PCR was performed using Hieff
qPCR SYBR Green Master Mix (11201ES03, Yeasen).
The RT-PCR related primers were listed in Table 1. The
expression levels of the relevant genes were referred to𝛽-actin and calculated utilizing 2ΔΔCT method.
Table 1Related primers of target genes in quantitative
RT PCR. All base sequences can be acquired from on
Genbank: (http://www.ncbi.nlm.nih.gov/GenBank/index.
html) and PrimerBank (https://pga.mgh.harvard.edu/
primerbank/).

Primary microglia culture

Western blot

As previous methods, primary brain microglia were separated from 0 to 3 days neonate WT mice [18]. Basically,
the P1-2 postnatal mouse pup skull was stripped and thebrain was removed. Then, the brain was cut into pieces
and digested with 0.125% trypsin for 15 min at 37 °C. The
mixed glial cells were cultured with DMEM/F12 medium
containing 10%FBS for 3 days at 37 °C with 5% CO2 and95%O2. For remaining 9 days, the cell mixture was cultured in high glucose DMEM containing 10% FBS andthe medium was renewed every three days. Finally, themicroglia were separated from by shuddering at 200 rpm
at 37 ◦C for 1 h [28, 29].

Samples from harvested primary microglia were digested
with RIPA lysis buffer (P0013K, Beyotime) supplemented
with phosphatase inhibitors (HYK0031, MCE). Briefly,
the concentration of extracted protein was quantified
using BCA Protein Assay Kit (Promoter), then transferred to Nitrocellulose (NC, Satrorius) membranes andblocked with 5% BSA for 1 h, followed by incubated withprimary antibodies including BTK (1:1000, 8647, CST)
and pBTK (1:1000, NBP178295, NOVUS) overnight at
4 °C. After incubation with anti-rabbit (H + L) secondary
antibody (GB23303, Servicebio), the special protein signals were captured by GelView 6000 Pro (BLT PHOTONTECHNOLOGY).

Cell treatment

For tolebrutinib treatment group, the dense microglia
adhered to the 6-well plates for 24 h. Then, the primary
microglia that previous cell culture medium was replacedwith low glucose DMEM containing 1 μm Tolebrutinb
was placed in an incubator for 4 h at 37 °C with 5% CO2
and 1% O2 concentration for oxygen-glucose deprivation(OGD) without reoxygenation. As for vehicle group, theprimary microglia were performed with OGD with low
glucose DMEM containing 0.1% DMSO.
Cell staining

For cell staining, the treated microglia were added fluorochromes including Bodipy C11(1:2000,D3861, Invitrogen), Mitosox (1:10000, 40742ES50, Yeasen), Mitotracker(1:2000,40742ES50, Yeasen), JC-1(1:500,40705ES03,
Yeasen), FerroOrange (1:1000, F374, Dojindo) and ROS(1:2000,HY-D0940) severally and incubated at 37 °C
for 30 min before the study time point. The washed cellslice was observed under a confocal microscope. Moreover, the flow cytometer was used to analyze the stained
microglia suspensions.
RNA extraction and real-time PCR

Total RNA from primary microglia cultures wereextracted by Trizol (9109, Takara). The cDNA wasreversely transcribed from isolated RNA via PrimeScript™

Cell viability

The cell viability was conducted using Cell Counting
Kit-8 (HY-K0301, MCE) and Cytotoxicity LDH Assay Kit(CK12, Dojindo) according to manufacturer’s instructions. Briefly, for CCK8, the suspended microglia wereplanted in 96-well plates with a density of 10,000/welland added 10ul CCK8 solutions to incubate for 1–4 h at
37 °C. The absorbance of the mixed solution at 450 nm
was measured by an enzyme-labeled instrument (Thermo
Scientific). For LDH release test, the 96-well plates wereincorporated in 100ul working solution and incubatedunder darkness at room temperature for 30 min. Finally,
the above mixtures were added 50ul stop solution anddetected at 490 nm immediately.
Statistics analysis

All data were analyzed by GraphPad Prism software version 9. Comparisons were analyzed using Unpaired twosided t’s t-test, One-way ANOVA followed by Tukey post
hoc test and Two-way ANOVA followed by Tukey post
hoc test. The results were statistically significant whenp < 0.05.

Xu et al. Journal of Neuroinflammation(2024) 21:195

Page 5 of 15

Table 1 Related primers of target genes in quantitative RT PCR
Geneβ-actin
Nos2
Tnf-α
Il6
Il1b
Cd86
Nlrp3
Tgfb
Il10
Arg1
Spp1
Il4
Cd68
Lgals3
Clec7a
Acsl4
Ncoa4
Ptgs2
Fth1
Alox12
Gpx4
Fsp1
Hspb1

Forward-Primer​G​G​C ​T​G​T​A​T ​T​C​C​C​C ​T​C​C​A​T​C​G​G​T ​T​C ​T​C​A​G​C​C​C​A​A​C​A​A​T​A​C​A​A​G​A​C​C​C ​T​C​A​C​A​C ​T​C​A​G​A​T​C​A​T​C ​T ​T​C ​T​T​A​G​T​C​C ​T ​T​C​C ​T​A​C​C​C​C​A​A​T ​T ​T​C​C​G​C​A​A​C ​T​G​T ​T​C​C ​T​G​A​A​C ​T​C​A​A​C ​T​T​G​T ​T ​T​C​C​G​T​G​G​A​G​A​C​G​C​A​A​G​A​T ​T​A​C​C​C​G​C​C​C​G​A​G​A​A​A​G​G​C ​T​C​C​C​G​T​G​G​C ​T ​T​C ​T​A​G​T​G​C​G​C ​T​C ​T ​T​A​C ​T​G​A​C ​T​G​G​C​A​T​G​A​G​C ​T​C​C​A​A​G​C​C​A​A​A​G​T​C​C ​T ​T​A​G​A​G​A​G​C​A​A​G​A​A​A​C ​T​C ​T ​T​C​C​A​A​G​C​A​A​G​G​T​C ​T​C​A​A​C​C​C​C​C​A​G​C ​T​A​G​T​T​G​T​C ​T​G​A​T​C ​T ​T​G​C ​T​A​G​G​A​C​C​G​A​G​A​C​A​G​C ​T ​T ​T ​T​C​G​C ​T ​T​A​A​C​G​A​G​A​C ​T ​T​C​A​G​C​A​C ​T​C​A​A​G​A​C​A​T​C​C​C ​T​C​A​C​C​A​T ​T​A​T​A​T ​T​G​C ​T​G​C​C ​T​G​T​G​A​A​C​C​A​T​C​A​G​G​A​C​A​C​A​T​G​G​A​A​A​T ​T​C​A​A​C​A​C​A​C ​T​C ​T​A​T​C​A​C ​T​G​G​C​C​A​A​G​T​G​C​G​C​C​A​G​A​A​C ​T​A​C​C​A​T​C​C​C ​T​C​A​A​C​C ​T​A​G​T​G​C​G​T ​T ​T​G
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Results
Chronic hypoperfusion induced white matter injury,
microglia, and BTK activation

The corpus callosum (CC) containing myelinated axons
is susceptible to demyelination after hypoperfusion [30].
Accordingly, we analyzed the time-course changes in
myelin damage in the corpus callosum (CC) at 3, 30, and90 days after BCAS surgery. After 1 m (1 month) post
BCAS, luxol fast blue (LFB) staining revealed a significant
myelin loss compared with sham, 3and 90 days groups, inwhich the white matter damage score ascended to nearly
2.5 points (Fig. 1A-B). Additionally, the relative intensityof myelin basic protein (MBP) decreased since BCAS
3d and came to the lowest level at 1 m (Fig. 1A-B). The
results of LFB and MBP staining both confirmed whitematter damage reached its peak at 1 m after BCAS.
To figure out the alterations in the proportions ofvarious immune cells in the brain, we investigated theimmune cell type composition using flow cytometry in
BCAS 1 m brains. CD3+T, CD4+T, B, Nature killer cells(NK), NKT, and neutrophils were rarely found in thewhite matter of BCAS brains, while microglia take uphigh proportion in the live cells (Fig. 1C). Apparently,
microglia occupied a dominant position in the immune
cells of the BCAS brains.
We then performed various approaches based on
immunofluorescence to characterize microglia activities at different time points after BCAS (3d, 1 m,3 m).

Reverse Primer​C​C​A​G​T ​T​G​G​T​A​A​C​A​A​T​G​C​C​A​T​G​T​G​T​G​G​A​C​G​G​G​T​C​G​A​T​G​T​C​A​C​G​C ​T​A​C​G​A​C​G​T​G​G​G​C ​T​A​C​A​G​T ​T​G​G​T​C​C ​T ​T​A​G​C​C​A​C ​T​C​C ​T ​T​C
​A​T​C ​T ​T ​T ​T​G​G​G​G​T​C​C​G​T​C​A​A​C ​T​T ​T​G​A​G​C​C ​T ​T ​T​G​T​A​A​A​T​G​G​G​C​A​T​C​G​C​A​G​C​A​A​A​G​A​T​C​C​A​C​A​C​A​G​G​C​C ​T ​T​A​G​T ​T ​T​G​G​A​C​A​G​G​A​T​C ​T​G
​C​G​C​A​G​C ​T​C ​T​A​G​G​A​G​C​A​T​G​T​G​A​G​G​A​G​C ​T​G​T​C​A​T ​T​A​G​G​G​A​C​A​T​C​G​T​G​A​G​A​T ​T​C​G​T​C​A​G​A​T ​T​C​A​T​C​C​G​G​C​C​G​A​T​G​A​T​C ​T​C ​T​C ​T​C​A​A​G​T​G​A​T​G​A​G​A​G​T​A​A​C​G​G​C​C ​T ​T ​T ​T ​T​G​T​G​A​G​G​G​T​A​G​G​C​A​C ​T​A​G​G​A​G​G​A​G​C​T ​T​G​T​G​T​C​G​C​C​A​A​A​A​T​G​C ​T​A​G​G​T​C​T​C​T​T​T​G​C​C​A​T​A​G​C​G​T​T​T​T​T​C​T​A​G​G​A​G​C​C​A​T​A​G​C​C ​T ​T​G​G​G​T​A​G​A​A​G​C​G​T ​T ​T​G​C​G​G​T​A​C ​T​C​A​T​G​C​C​A​C​A​T​C​A​T​C ​T​C​G​G​T​C​A​A​A​A​G​T ​T​G​C​A​G​C ​T​C​C​A​G​T ​T ​T​C​G​C​C​C​C ​T​G​T​A​C ​T ​T​A​T​C​C​A​G​G​C​A​G​A​G​C​A​G​C ​T​C​C​C ​T​G​G​T​C​A​G​T​A​G​G​G​A​A​T​G​G​T​G​A​T​C ​T​C​C​G​C ​T​G​A​C

AmpliconSize(bp)
154
127
61
76
89
70
141
133
105
185
134
102
75
210
164
114
265
271
122
64
185
81
75

The number of microglia gradually rise from 3d to 1 m
post BCAS (Fig. 1D-E). Consistently, although no significant changes in microglia branches, branch length, andend-point voxels were found (Figure S1A), soma area,
roundness, and solidity of microglia were also increased,suggesting microglia activation during hypoperfusion(Fig. 1D-E). We then probed the expression of BTK andphosphorylated BTK (pBTK) post BCAS, which wasreported to be involved in microglia activation [16, 17].
The ratio of BTK+Iba1+ and pBTK+Iba1+ cells increased
gradually at 3 days, peaked at 30 days, and remained
relatively high up to 90 days after BCAS (Fig. 1D-E).
Furthermore, we found BTK or pBTK were rarely presented in neurons, oligodendrocytes or astrocytes (Figure
S1B). These results illustrated that BTK is activated significantly in microglia, along with relatively high level ofmicroglial activation at BCAS 1 m.
Tolebrutinib suppressed the activation of BTK
and microglia and reduced oxidative stress andneuroinflammation

Given its promising prospect as a drug target, realizing
the potential role of BTKi in microglia functions holds
great interest [31]. Then, we asked about the impacts of
tolebrutinib treatment on microglia changes in ischemic
demyelination. The wild BCAS mice were intragastrically administrated 5 mg/kg tolebrutinib per day for 1 m(Fig. 2A). We found the percentage of BTK+Iba1+ and
pBTK+Iba1+ cells were decreased obviously followingXu et al. Journal of Neuroinflammation(2024) 21:195

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Fig. 1 Ischemic demyelination and activation of BTK in microglia induced by BCAS. Ischemic demyelination and activation of BTK in microglia induced by
BCAS. (A) The degree of white matter damage and the relative intensity of MBP in the CC at different time points after BCAS. Scale bar = 200 μm. (B) Summarized data display the severity of CC myelin damage. One-way ANOVA and Tukey post hoc test, n = 6 mice/ group (biological replicates). (C) Proportionsof various immune cells in BCAS 1 m brains. (D) Representative images of microglia immunofluorescence, skeletonized microglia, and the expression of
BTK and p-BTK in microglia at several time points post BCAS. Scale bar = 10 μm. (E) Quantification analysis of the number, soma area, solidity, round of
microglia and the percentage of BTK+microglia and p-BTK+microglia. One-way ANOVA and Tukey post hoc test, n = 6 mice/ group (biological replicates)

Xu et al. Journal of Neuroinflammation

Fig. 2 (See legend on next page.)(2024) 21:195

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Xu et al. Journal of Neuroinflammation(2024) 21:195

Page 8 of 15(See figure on previous page.)
Fig. 2 Tolebrutinib therapy attenuated the myelin damage via reducing neuroinflammation. Tolebrutinib therapy attenuated the myelin damage andcognitive deficits via reducing neuroinflammation. (A) Experimental flow chart. (B) The representative images of microglia immunofluorescence, skeletonized microglia, and the expression of BTK and p-BTK in microglia of Sham, BCAS + Vehicle and BCAS + Tolebrutinb mice. Scale bar = 10 μm. (C) Quantifications of microglial density, soma area, solidity, round, and the percentage of BTK+microglia and p-BTK+microglia. One-way ANOVA and Tukey post
hoc test, n = 6 mice/ group (biological replicates). (D-E) The representative immunofluorescent images and quantitative analysis of microglia and proinflammatory marker or phagocytosis related markers co-localization in the CC. One-way ANOVA and Tukey post hoc test, n = 6 mice/ group (biologicalreplicates). Scale bar = 10 μm. (F-G-H) Representative LFB and MBP staining and quantifications of Sham, Vehicle and Tolebrutinb group. One-way ANOVA
and Tukey post hoc test, n = 6 mice/ group (biological replicates). Scale bar = 200 μm

tolebrutinb treatment, proving that tolebrutinb canpenetrate the BBB to exert effects (Fig. 2B-C). Besides,
microglia activation was inhibited in tolebrutinb treatment group, characterized by reduced microglia celldensity, smaller soma area, and decreased solidity androundness (Fig. 2B-C).
The oxidative stress damage occurred in BCAS withincreased intensity of 4HNE and 8-OHDG in Iba1+
cells. After tolebrutinb treatment, the oxidative stressinjury was significantly mitigated (Figure S1C). Furthermore, we examined the microglia-mediated inflammatory response by coimmunostaining pro-inflammatory
marker Fc RII/III receptor (CD16/32), phagocytosis
related makers CD68 antigen (CD68) and C-type lectin domain family 7 (Clec7a) with Iba-1 (biomarkers of
microglia). Compared with BCAS vehicle group, theproportion of CD16/32 was markedly decreased in tolebrutinb treatment group (Fig. 2D-E). Additionally, CD68
and Clec7a positive microglia also exhibit significantly
lower proportions after tolebrutinb treatment (Fig. 2DE). Taken together, the interference of tolebrutinib effectively inhibited the activation of BTK in microglia andmitigated neuroinflammation mediated by microglia
during cerebral hypoperfusion.
Myelin injury and cognitive deficits were attenuated after
tolebrutinib treatment

Neuroinflammation induced by CCH is also a crucial
mechanism of myelin injury and cognitive impairments.
To further validate whether tolebrutinib was associatedwith white matter integrity and cognitive function post
BCAS, LFB and MBP staining were firstly used to evaluate degree of myelin injury. We observed that tolebrutinb
taken group exhibited improved white matter integrity compared with vehicle treatment group (Fig. 2F-H).
Then, eight-arm maze experiment and new object recognition experiment were carried out to assess cognitive
functions of mice. For revisiting errors, the total numberof times was calculated for the mice entered different
arms until food pellets were consumed in eight arms. The
different arms that mice entered in the first eight entries
were also counted. Furthermore, for reference memory
errors, the total number of times was calculated for themice entered different arms until food pellets were consumed in four of eight arms. Mice treated with tolebrutinib showed decreased revisiting errors and more different

arm choice in the first eight entries, which indicating an
improved working memory (Fig. 3A). Moreover, the mice
exhibited increased interest of exploring new objects
after tolebrutinib treatment (Fig. 3B-C). Conclusively,
tolebrutinib can ameliorate ischemic myelin damage andcognitive impairments distinctly after BCAS.
Inhibition of BTK activation alleviated microglial
inflammation in vitro

To fully elucidate the role of tolebrutinib in microglia,
we utilized oxygen-glucose deprivation (OGD, 4 h) to
induce hypoxia in vitro (Fig. 4A). The viability of microglia remains unaffected until the concentration of tolebrutinib exceeds 25 μm (Fig. 4B). To address the effects of
tolebrutinib on microglia in vitro, we conducted western
blotting and demonstrated the BTK phosphorylation on
Tyr223 in microglia is inhibited following treatment with
1 μm of tolebrutinb (Fig. 4C-D, S2).
Afterward, we employed quantitative RT-PCR toevaluate the expression levels of various classic proinflammatory and anti-inflammatory genes in microglia.
Tolebrutinib-OGD treatment decreased the mRNA levels of pro-inflammatory markers, including Nos2, Tnf-a,
IL-1b, Cd86, and Nlrp3 in vitro (Fig. 4E). Conversely, themRNA levels of anti-inflammatory genes, such as Tgfb,
Il-10, Arg1, Spp1, and Il-4, were increased significantly(Fig. 4E). Meanwhile, the expression levels of markers related to microglial phagocytosis (Cd68, Lgal3 andClec7a) were pronouncedly downregulated in tolebrutinib treatment group (Fig. 4E). Consistent with the resultsin vivo, these observations verified that tolebrutinb skews
microglia towards an anti-inflammatory and homeostatic
phenotype in in vitro hypoxia.
Tolebrutinib suppressed microglial oxidative stress,
mitochondrial dysfunction and ferroptosis

Recently, emerging studies pointed out that ferroptosis,
characterized by an imbalance of redox function was
powerfully associated with the pathologic physiology of
CCH [32, 33]. To further understand the neuroprotective mechanisms of tolebrutinib in CCH mice, oxidative stress, mitochondrial function, ferroptosis-related
markers, and cell viability by qPCR, immunofluorescence staining, flow cytometry, and lactic dehydrogenase(LDH) release test in vitro. The expression of pro-ferroptosis markers Acsl4, Noca4, Ptgs2 and Fth1 were notably

Xu et al. Journal of Neuroinflammation(2024) 21:195

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Fig. 3 Tolebrutinib treatment improved cognitive deficits resulted from cerebral hypoperfusion. (A) The cognitive function was evaluated using theeight-arm maze test. Two-way ANOVA followed by Tukey post hoc test, n = 9 per group. (B) The representative images and quantification of the interestof exploring new object. (C) The quantitative analysis of frequency of mice exploring new objects. One-way ANOVA and Tukey post hoc test, n = 6 mice/group

upregulated and the mRNA level of anti-ferroptosis
indicators Alox12, Gpx4, Fsp1 and Hspb1 were downregulated in vehicle + OGD group microglia compared tothe control group (Fig. 4E). However, these effects weresignificantly reversed with the addition of tolebrutinib,suggesting that tolebrutinib suppressed ferroptosis in
microglia under hypoxic circumstances.
To further confirm the effects of suppressing ferroptosis by microglia, we detected the intensity of Mitosox,
C11 Bodipy, ROS, FerroOrange, and JC1. Comparedwith the control group, the intensity of Mitosox, ROS,
and FerroOrange were significantly increased in OGDVehicle group, while tolebrutinb treatment decreased theintensity of Mitosox, ROS and FerroOrange (Fig. 5A-B).
In addition, the proportion of oxidation state BODIPY
and JC1 monomer presented a lower level after tolebrutinib treated in vitro (Fig. 5A-B).
Correspondingly, by flowcytometry, we found that theMitosox+ cells account for nearly 87% in Vehicle-OGD
group, yet the ratio of the Mitosox+ cells was reduced toabout 65% after tolebrutinb treatment (Fig. 5C-D). Similarly, the ratio of JC-1 monomer+ cells decreased fromapproximately 60% to about 40% (Fig. 5E). Concurrently,
the mean fluorescence intensity of oxidized BODIPY,ROS and FerroOrange had a decrease in Tolebrutinib-OGD group compared with Vehicle-OGD group(Fig. 5C-D). As expected, LDH release was released in the

Tolebrutinib-OGD group (Fig. 5F), implying that tolebrutinib may be an important inhibition of cell death in
the duration of ischemic injury. Collectively, these findings delineated that tolebrutinb may rescue white matter microglia damage by restraining microglial oxidative
stress, mitochondrial dysfunction and ferroptosis during
hypoperfusion.

Discussion
At present, the importance of BTKi therapy in the fieldof CNS diseases has not been fully elucidated [17]. Here,
we extended insights into chronic white matter ischemia
and identified a role of BTKi in regulating neuroinflammation. Along with sustained myelin damage, microglia
and its expressions of BTK were remarkably activatedduring CCH (Figs. 1 and 2). Consistently, the tolebrutinib therapy led to alleviated microglia-related oxidative
stress and inflammation, ameliorated myelin injury andimproved cognitive impairments during ischemic demyelination (Figs. 2 and 3 and Figure S1). Furthermore, it
has also been confirmed that tolebrutinib could alleviate
microglial pro-inflammatory responses in vitro (Fig. 4).
Impressively, oxidative stress, mitochondrial dysfunction, and ferroptosis occurred in microglia in in vitro
hypoxia, which was rescued by tolebrutinib effectively(Figs. 4 and 5). Our results exhibit promising prospective

Xu et al. Journal of Neuroinflammation(2024) 21:195

Page 10 of 15

Fig. 4 Tolebrutinib treatment alleviates inflammatory response and ferroptosis in microglia. Tolebrutinib treatment alleviates inflammatory response
and ferroptosis in microglia. (A) Experimental flow chart. (B) Primary microglia were treated with tolebrutiinb at different concentrations for 4 h, andCCK8 were performed. One-way ANOVA and Tukey post hoc test. n = 6 per group. (C-D) Representative image and quantification of pBTK protein levels
in primary microglia. One-way ANOVA and Tukey post hoc test, n = 6 per group. (E) Normalized mRNA levels of genes related to inflammatory response,
phagocytosis and ferroptosis were measured. One-way ANOVA and Tukey post hoc test. n = 6 per groupXu et al. Journal of Neuroinflammation(2024) 21:195

Page 11 of 15

Fig. 5 Tolebrutinib therapy attenuated oxidative stress, mitochondrial dysfunction and ferroptosis in microglia. Tolebrutinib therapy attenuated oxidative
stress, mitochondrial dysfunction and ferroptosis in microglia. (A-B) Immunofluorescence staining and quantitative analysis of oxidative stress, mitochondrial dysfunction and ferroptosis related markers. One-way ANOVA and Tukey post hoc test, n = 6 per group. Scale bar = 10 μm. (C-D-E) Representative
images of flow cytometry and quantitative analysis of oxidative stress, mitochondrial dysfunction and ferroptosis related markers. One-way ANOVA andTukey post hoc test, n = 4 per group. (D) The cell viability was detected by Cytotoxicity LDH Assay. One-way ANOVA and Tukey post hoc test, n = 6 pergroup

Xu et al. Journal of Neuroinflammation(2024) 21:195

of tolebrutinib in the treatment of CCH, possibly via regulation of microglia activities and functions in the CNS
in CCH.
Microglia are the first responders to active demyelinated lesions even lesions get inactive or lightened, which
are responsible for immune surveillance, host defense,and brain tissue repair [34, 35]. Previous studies fromwhite matter ischemic lesions have already shown thereactive microglia exist in the core and surrounding
areas of myelin injury, along with up-regulated biomarkers of oxidative stress and inflammation [36–38]. On
the other hand, these reactive microglia is also involvedin the pathophysiology of CCH through several mechanisms, including recruiting infiltrating immune cells [39,40] and releasing numerous proinflammatory cytokinesand chemokines that cause BBB dysfunctions and hinder
remyelination [41–45]. Additionally, our previous studies
have revealed that inhibition of microglial inflammatory
response alleviated ischemic demyelination injury andcognitive dysfunction effectively [18, 46, 47]. In line withthese previous results, we first showed that microglia
were activated in chronic white matter ischemia, as has
been shown by significant alterations in microglia density
and morphology, along with activation of BTK.
We then tried to further elucidate therapeutic potentials of targeting BTK in CCH. It has been reported that
BTKi possesses the ability to penetrate the BBB and accumulate in the CNS against possible pathophysiologicalprocesses in CNS diseases by directly affecting resident
or infiltrated immune cells, such as microglia and B cells[17, 48, 49]. The potential role of BTKi in microglia has
been explored in several neurodegenerative and inflammatory demyelinating diseases. Keaney et al. stated thatmicroglial activation, phagocytosis, and uptake of synapsis can be relieved by inhibiting microglial BTK, contributing to improved cognitive performance in Alzheimer’s
disease (AD) [31]. Meanwhile, Martin et al. proved that
BTKi had promising therapeutic potential in myelin
repair in ex vivo and in vivo lysophosphatidylcholine(LPC)-induced demyelination models [50]. However,
the effect of BTKi on microglial function has not been
investigated during chronic white matter ischemia. By
utilizing tolebrutinib, a second-generation BTKi with astronger capacity to penetrate the BBB, lower off-target
effects and fewer adverse events, including headaches,
nasopharyngitis, and elevations in liver aminotransferase levels than the first generation BTKi [17, 51], wethen demonstrated that the tolebrutinib therapy caninhibit BTK activation in microglia, which subsequently
led to ameliorated microglia inflammatory response andmitigated myelin damage in mouse BCAS models, suggesting its therapeutic potentials in CCH. In our results,
we observed that BCAS induced microglia activation,
along with enhanced levels of phagocytosis, which wasPage 12 of 15

alleviated with tolebrutinib. We hypothesize that this isbecause microglial phagocytosis also serves as indicatorsof microglia-related neuroinflammation (e.g. alterations
in microglial morphology, biomarkers including CD68,
CD16/32 and others), which induces dysfunctions in the
CNS microenvironment after BCAS. Taken together,phagocytosis of microglia may potentially serve as a double-edged sword in BCAS-induced white matter injury.
Combining these previous findings and our results, we
proposed that tolebrutinib exerts its neuroprotectiveroles during CCH via suppressing BTK activation andsubsequently the release of proinflammatory cytokines
from microglia, which thus contribute to ameliorated
neuroinflammation and demyelination in CCH.
Previous studies emphasized solely on BTKi restricting microglia inflammation, however, the role of BTKi
in microglia ferroptosis has not been elucidated until
now. Of note, we first identified that the tolebrutinib
treatment significantly ameliorated microglia ferroptosis induced by in vitro hypoxia. Recent studies have shedlight on ferroptosis in CCH pathogenesis, for it is recognized that oxidative stress and ferroptosis were inducedin CCH brains and brains of subcortical ischemic vascular dementia patients exhibited iron deposits [33, 52, 53].
Ferroptosis serves as a form of cell death, with characteristics including accumulated Fe2+, peroxidized lipids andincreased ROS, which are also closely associated withenhanced levels of oxidative stress. Our results suggestthat BCAS-induced white matter injury contribute toenhanced levels of microglial ferroptosis-related oxidative stress, which was, on the other hand, alleviated via
tolebrutinib. From this aspect, the white matter injury
induced by BCAS more possibly contribute to a proferroptosis state in microglia, which is also related withexacerbated levels of neuroinflammation, mitochondrial
dysfunction and oxidative stress.
In our present study, we found microglial ferroptosis
was significantly enhanced, which is characterized byperoxidized lipids, increased ROS, mitochondrial superoxide, disrupted mitochondrial membrane potential,
accumulated Fe2+, and obvious cell death in hypoxia.
Ferroptosis has been demonstrated to be potential therapeutic target against cognitive impairments induced byhypoperfusion, as emerging evidence suggests that, indegenerative microglia of aging population, abundant
Fe2+ and myelin debris were enriched, the suppressionof may reduce the effects of white matter damage andmyelin loss on the progression of cognitive deficits [54].
Therefore, we then further explored the effects of tolebrutinib on microglia ferroptosis under hypoxic circumstances. We identified that tolebrutinib treatment skewed
microglia towards anti-inflammatory and homeostatic phenotype, along with relieving hypoxia-induced
unbalanced redox function, mitochondria damage andXu et al. Journal of Neuroinflammation(2024) 21:195

ferroptosis effectively. Combining our results and theprevious studies, it is convincing that the alleviation of
myelin injury and cognitive dysfunction by suppressing
microglial oxidative stress, mitochondrial dysfunction
and ferroptosis is one of the mechanisms of neuroprotective effect of tolebrutinib.
There’ve already been several studies showing themechanisms of BTK on demyelination injuries. Elkjaer et
al. have found that enhanced BTK expression was associated with elevated levels of both complement and Fc
gamma receptors, as well as higher number of microglia, B lymphocytes and other immune cells [55]. On
the other hand, Steinmauer et al. have also shown that
BTK + cells were significantly increased in both active
and inactive demyelination lesions, along with iron+ andCD68+ microglia-like cells, showing that expression andactivities of BTK are potentially associated with microglial iron accumulation in demyelination lesions [56]. In
line with these previous studies, our results also showedthat activation of BTK in microglia was accompanied
by significant alterations in both microglia density andmorphology in ischemic myelin injury, while inhibitionof BTK not only alleviated microglial phagocytosis andpro-inflammatory phenotypic transitions, but also significantly inhibited ferroptosis and oxidative stress in
microglia in demyelination injury.
In summary, we demonstrated that tolebrutinib ameliorates white matter lesions and cognitive dysfunction via
relieving microglia inflammation, oxidative stress, mitochondrial dysfunction and ferroptosis during chronic
white matter ischemia. Our study provides novel insights
into the role of tolebrutinib in improving ischemic whitematter injury, by weakening microglia inflammationand ferroptosis and facilitating its conversion from proinflammatory microglia towards anti-inflammatory andhomeostatic phenotypes, which suggest the promisingtherapeutic potential of tolebrutinib in the treatment of
CCH.
Limitations

The study still has some limitations. The staining pattern may depend on the degree of pathologic damage in
different models. Therefore, the immunofluorescenceof several indicators may not be pretty evident in BCAS
model, in which pathologic damage was much slighter
than NMO or MCAO model. This shortcoming is what
we endeavor to overcome in future studies.

Supplementary Information

The online version contains supplementary material available at https://doi.
org/10.1186/s12974-024-03187-4.
Supplementary Material 1
Supplementary Material 2

Page 13 of 15

Acknowledgements
We acknowledge the support of Experimental Medicine Center, Tongji
Hospital, Tongji Medical College, Huazhong University of Science andTechnology.
Author contributions
QC and TD designed the project conception. LQ, CY, PX, YY, ZH, ZL, ZL andCL performed the literature search. XL conducted the experiment. XL andYS drafted the paper and analyzed the data. YS, SK, XJ, QC, TD and WW
revised the manuscript. All authors read and approved the final version of themanuscript.
Funding
This study was funded by National Natural Science Foundation ofChina (Grants: 82271341, 82371404, 82071380, 81873743), Knowledge
Innovation Program of Wuhan Shuguang Project (2022020801020454),
Tongji Hospital (HUST) Foundation for Excellent Young Scientist (Grant No.
2020YQ06), Ministry of Science and Technology China Brain Initiative Grant(2022ZD0204704), National Natural Science Foundation of China 82301510,
and Tongji Hospital (HUST) Foundation for Excellent Young Scientist
24-2KYC13057-12.
Data availability
Data is provided within the manuscript or supplementary information files.

Declarations
Ethics approval and consent to participate
All experiments involving animals were consented by the Animal Care
Committee of Tongji Medical College, Huazhong University of Science.
Technology, China.
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Received: 8 February 2024 / Accepted: 25 July 2024

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