Hou et al. Journal of Neuroinflammation(2024) 21:148
https://doi.org/10.1186/s12974-024-03143-2

Journal of Neuroinflammation

Open Access

RESEARCH

CD36 deletion prevents white matter injury
by modulating microglia polarization throughthe Traf5-MAPK signal pathway
Xiaoxiang Hou1†, Xiaolin Qu2†, Wen Chen1†, Xianzheng Sang1, Yichao Ye1, Chengqing Wang1, Yangu Guo1,
Hantong Shi1, Chengzi Yang1, Kaixin Zhu3, Yelei Zhang4, Haoxiang Xu1, Liquan Lv1, Danfeng Zhang1* and Lijun Hou1*
Abstract
Background White matter injury (WMI) represents a significant etiological factor contributing to neurologicalimpairment subsequent to Traumatic Brain Injury (TBI). CD36 receptors are recognized as pivotal participants in thepathogenesis of neurological disorders, including stroke and spinal cord injury. Furthermore, dynamic fluctuationsin the phenotypic polarization of microglial cells have been intimately associated with the regenerative processes
within the injured tissue following TBI. Nevertheless, there is a paucity of research addressing the impact of CD36
receptors on WMI and microglial polarization. This investigation aims to elucidate the functional role and mechanistic
underpinnings of CD36 in modulating microglial polarization and WMI following TBI.
Methods TBI models were induced in murine subjects via controlled cortical impact (CCI). The spatiotemporal
patterns of CD36 expression were examined through quantitative polymerase chain reaction (qPCR), Western blot
analysis, and immunofluorescence staining. The extent of white matter injury was assessed via transmission electron
microscopy, Luxol Fast Blue (LFB) staining, and immunofluorescence staining. Transcriptome sequencing was
employed to dissect the molecular mechanisms underlying CD36 down-regulation and its influence on white matterdamage. Microglial polarization status was ascertained using qPCR, Western blot analysis, and immunofluorescencestaining. In vitro, a Transwell co-culture system was employed to investigate the impact of CD36-dependent
microglial polarization on oligodendrocytes subjected to oxygen-glucose deprivation (OGD).
Results Western blot and qPCR analyses revealed that CD36 expression reached its zenith at 7 days post-TBI andremained sustained at this level thereafter. Immunofluorescence staining exhibited robust CD36 expression inastrocytes and microglia following TBI. Genetic deletion of CD36 ameliorated TBI-induced white matter injury, as
evidenced by a reduced SMI-32/MBP ratio and G-ratio. Transcriptome sequencing unveiled differentially expressed
genes enriched in processes linked to microglial activation, regulation of neuroinflammation, and the TNF signaling†

Xiaoxiang Hou, Xiaolin Qu, and Wen Chen contributed equally tothis work.
*Correspondence:
Danfeng Zhang
dfzhangsmmu@163.com
Lijun Houlijunhoucz@126.com
Full list of author information is available at the end of the article© The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use,sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) andthe source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in thisarticle are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not includedin the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you willneed to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The
Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available
in this article, unless otherwise stated in a credit line to the data.

Hou et al. Journal of Neuroinflammation(2024) 21:148

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pathway. Additionally, bioinformatics analysis pinpointed the Traf5-p38 axis as a critical signaling pathway. In vivo andin vitro experiments indicated that inhibition of the CD36-Traf5-MAPK axis curtailed microglial polarization toward
the pro-inflammatory phenotype. In a Transwell co-culture system, BV2 cells treated with LPS + IFN-γ exacerbated thedamage of post-OGD oligodendrocytes, which could be rectified through CD36 knockdown in BV2 cells.
Conclusions This study illuminates that the suppression of CD36 mitigates WMI by constraining microglial
polarization towards the pro-inflammatory phenotype through the down-regulation of the Traf5-MAPK signaling
pathway. Our findings present a potential therapeutic strategy for averting neuroinflammatory responses and ensuing
WMI damage resulting from TBI.
Keywords Traumatic brain injury, White matter injury, CD36, Microglial polarization
Graphical Abstract

Introduction
Traumatic brain injury (TBI) represents a globally significant source of both mortality and morbidity [1]. It inflicts
damage not only upon gray matter but also results in theoccurrence of severe white matter injury (WMI) [2]. The
profound white matter injury leads to substantial oligodendrocyte loss, a critical cell type responsible for myelin
production, maintenance of axonal metabolism, and theregulation of neuroplasticity. This, in turn, precipitatescatastrophic neurological impairment. A substantial
body of preclinical research has concentrated primarily on the treatment of gray matter injury, with relatively
fewer investigations into white matter injury. This underrepresentation may, in part, contribute to the suboptimal
therapeutic outcomes observed.
TBI comprises two sequential phases of injury: the initial mechanical trauma to the head caused by external

forces resulting in direct brain tissue damage, and thesecondary biochemical cascades, including inflammatory responses and other pathophysiological complications [3]. WMI is exacerbated by potent and persistent
inflammatory reactions following TBI [4]. Consequently,
the management of secondary biochemical cascades,
particularly inflammation, is of paramount importancefor neurological recovery. Microglia, as pivotal players in the neuroinflammatory response following TBI[5], undergo a phenotypic shift from a resting state toeither pro-inflammatory or anti-inflammatory phenotype upon stimulation [6, 7]. Pro-inflammatory microglia
are capable of promoting the production of inflammatory factors, thereby exacerbating neurological damage[8]. In contrast, anti-inflammatory phenotype facilitates
the release of pro-neural repair neurotrophic factors [9].
Pro-inflammatory microglia, induced by LPS and IFN-γ,

Hou et al. Journal of Neuroinflammation(2024) 21:148

generate substantial inflammatory factors and oxidative
metabolites, while anti-inflammatory microglia, stimulated by IL-4, promote vascular regeneration [10]. In
vitro experiments reveal that pro-inflammatory microglia
induce apoptosis in neurons and oligodendrocyte precursor cells (OPCs), and hinder the maturation of oligodendrocytes. Conversely, anti-inflammatory microglia
promote neuronal survival, synapse formation, the generation of mature oligodendrocytes, and the regenerationof myelin sheaths, thus implicating their potential role inwhite matter repair [11]. Therefore, the manipulation of
the balance between pro- and anti-inflammatory phenotypes offers promising avenues for novel neuroprotective
strategies to mitigate secondary WMI in TBI patients.
CD36, a type B scavenger receptor, is predominantly
expressed on various cell types including monocytes/
macrophages, dendritic cells, microglia, hepatocytes, andmicrovascular endothelial cells [12]. Depending on theligand, CD36 activates a multitude of intracellular signaling pathways. While earlier research emphasized CD36’s
involvement in lipid metabolism, recent studies haveidentified its role in the neuroinflammatory processes
underlying several central nervous system (CNS) diseases such as ischemic brain injury, Alzheimer’s disease,and spinal cord injury. CD36 has been found to contribute to neuroinflammatory reactions, including oxidative
stress and endoplasmic reticulum responses, through themediation of NF-κB, MAPK, and other pathways following various CNS injuries [13–15]. Notably, the inhibitionof CD36 by SSO has been demonstrated to significantly
reduce the levels of inflammatory factors possibly by
influencing microglial polarization, thus alleviating neurological damage [16, 17].
In light of these considerations, our study was devised
to investigate the role and underlying mechanisms of
CD36 in microglial polarization and WMI subsequent to
TBI.

Materials and methods
Experimental design

To elucidate the influence and underlying mechanismsof CD36 receptors on white matter injury and neurological function in murine models post-TBI, C57BL/6J wildtype mice were randomly allocated into three cohorts: asham group, a TBI group, and a TBI group treated with
SB242235. Additionally, CD36-deficient C57BL/6J mice
were randomly distributed into a sham group and a TBI
group. Lentiviral constructs designed to overexpress orknockdown CD36, denoted as lenti-CD36OE-shRNA
and lenti-CD36KD-shRNA respectively, as well as lentiTraf5KD-shRNA, were engineered. BV2 microglial cells
were stably transfected with one or more of the aforementioned lentiviruses. These cells, alongside non-transfected and SB242235-treated counterparts, were exposed

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to LPS (100ng/ml) and IFN-γ (20ng/ml) for 24 h, thencategorized into distinct experimental groups. Concurrently, primary oligodendrocytes were divided into acontrol group and an OGD group, with the latter further
subdivided based on co-culture conditions with microglia under various treatment scenarios. Randomization ofanimal and cell groupings was executed using a lotterydrawing box method. All primary outcome measures
were conducted by researchers blinded to group allocation and experimental conditions. Animals that perishedduring or subsequent to the injury were omitted from theanalysis. The time points of the experiments corresponding to different results are shown in Sch. 1.
Animals

Male C57BL/6J wild-type and CD36 knockout mice, aged
8 weeks, were procured from MODEL ORGANISMS(Shanghai, China). In total, 148 mice were utilized in ourstudy. Of these, 107 underwent TBI procedures, with 3
succumbing to surgical complications. Consequently,
data from 145 mice were incorporated into our analysis.
All experimental procedures were rigorously conductedin accordance with the Guide for the Care and Use ofLaboratory Animals as promulgated by the US National
Institutes of Health (NIH publication No.85 − 23,revised 1996) and received approval from the Animal
Care and Use Committee of the Naval Medical University (Approval ID:82,201,519). Measures were taken tominimize both the number of animals utilized and theirsuffering.
TBI and sham injury

TBI was induced using a controlled cortical impact (CCI)
methodology as previously described [18, 19]. Animals
were randomized into experimental groups and anesthetized with 1.5% isoflurane in a 30% O2/68.5% N2O
mixture, allowing for spontaneous respiration. A craniotomy was performed, and the CCI was administered
2 mm lateral to the midline and 1.0 mm anterior to thebregma using a pneumatically driven device with a 3-mm
flat-tipped impounder (velocity, 3.5 m/s; duration, 150
ms; depth, 1.5 mm). Post-impact, the bone flap was repositioned and sealed. Rectal temperature was maintained
at 37 °C ± 0.5 °C during and up to 30 min post-procedureusing a heating pad. Sham-operated animals underwent
all surgical procedures except the CCI.
Drug administration

In this study, SB242235 was used as an inhibitor of p38
MAPK due to its characteristics as a selective inhibitor of
the p38 MAPK, with minimal influence on other MAPK
pathways [20, 21]. SB242235 (MedChemExpress, USA)was suspended in 0.5% methyl cellulose (MedChemExpress, USA) and administered via a single oral gavage atHou et al. Journal of Neuroinflammation(2024) 21:148

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Sch. 1 Schematic diagram of time points for experiments of this study. CCI: controlled cortical impact; OGD: oxygen-glucose deprivation

a dose of 100 mg/kg immediately after the mice regained
consciousness post-TBI. The in vivo administration protocols for SB242235 have been detailed previously [20]. A
dose-response study confirmed that optimal p-p38 inhibition was observed at a dose of 100 mg/kg for in vivo
experiments and at a concentration of 5µM for in vitro
experiments (Fig. S1).

sensory (visual, tactile and proprioceptive), balance,and reflex functions of mice. Neurological function was
graded from 0 to 18 (0 = normal function; 18 = maximal
deficit). One point was scored for each abnormal behavior or for the lack of a tested reflex. Therefore, higherscores implying greater neurological injury.

Intracerebral lentiviral shRNA infection

Briefly, mice were forced to run on a rotating drum (IITC
Life Science, Woodland Hills, CA, USA) with speeds
starting at 4 rpm and accelerating to 40 rpm within 300 s.
Three consecutive trials were conducted for each mousewith an interval of 15 min. The time at which a mouse
fell off the drum was recorded as the latency to fall. Data
were expressed as mean values from three trials.

For intracerebral lentiviral shRNA administration, viralparticles containing shRNA targeting Traf5 or a nontargeting sequence were stereotactic injected into theright corpus callosum via cranial drilling (0.75 mm lateraland 0.82 mm posterior to the bregma, 1.75 mm ventral
to the skull surface) 7 days prior to TBI. The infusion
was conducted at a rate of 0.1 µL/min over 50 min using
a microinfusion system connected to a microsyringe
pump (UMP2; World Precision Instruments). This procedure did not increase mortality rates among the animal
subjects.
Behavioral tests
The mNSS test

The mNSS test consists of ten different tasks that canevaluate the motor (muscle status, abnormal movement),

The rotarod test

Foot fault test

Mice were placed on hexagonal wire mesh grids of different sizes. As the mice moved along the wire mesh, their
forepaws either stepped or slipped. The number of times
these footsteps are recorded, as well as the total numberof steps the mouse takes across the grid. After recording
the data, the ratio of the number of forepaw steps missed
to the total number of forepaw steps was calculated, withlarger ratios representing greater injury.

Hou et al. Journal of Neuroinflammation(2024) 21:148

Wire hang test

The wire hang test was performed as described previously[22]. The experimental apparatus was a stainless-steel bar(50 cm long, 2-mm diameter) resting on two vertical supports and elevated 37 cm above a flat surface. Mice wereplaced on the bar midway between the supports and wereobserved for 30 s in four trials. The amount of time spent
hanging was recorded and scored according to the following criteria: 0, fell off; 1, hung onto the bar with two
forepaws; 2, hung onto the bar with an added attempt toclimb onto the bar; 3, hung onto the bar with two forepaws and one or both hind paws; 4, hung onto the bar
with all four paws and with tail wrapped around the bar;5, escaped to one of the supports.
Morris water maze test

Cognitive function was analyzed with the Morris water
maze test 10–14 d after CCI, as described previously[23]. In the learning test, a square platform (11 × 11 cm2)
was submerged 2 cm beneath the water surface in a circular pool (diameter = 109 cm) of opaque water. Mice
were placed into the pool from one of the four locations
and allowed to locate the hidden platform for 90 s. Eachmouse was trained on 4 trials (with randomly assigned
starting positions) per day to locate the platform for four
consecutive days. At the end of each trial, the mouse wasplaced on the platform or allowed to stay on the platform
for 30 s with prominent spatial cues displayed around theroom. 4 trials were performed on each day. The memory
test was performed on day 5. The platform was removed,
and a single 60-s probe trial was conducted. Time spentand traveling distance in the goal quadrant where theplatform was previously located was recorded.
BV2 cell cultures

BV2 microglial cells, procured from the National Infrastructure of Cell Line Resource, were cultured at a density of 1–2 × 10^5 cells per well in a 6-well plate format.
The culture medium consisted of Dulbecco’s Modified
Eagle Medium (DMEM, Gibco, 11965092, USA) supplemented with 0.1% penicillin-streptomycin (Sigma, P4333,
St. Louis, MO, USA) and 10% fetal bovine serum (Gibco,
10099141 C, USA). The cells were incubated at 37 °C in ahumidified atmosphere containing 5% CO2 for 48 h prior
to subsequent experimental procedures.
Primary OPC and oligodendrocyte cultures

Primary oligodendrocyte precursor cell (OPC) cultures were derived using the differential adhesion technique. Brains from neonatal C57 mice (postnatal days
1–2) were triturated post-meningeal and vascular tissue removal. The dissociated brain tissues were treatedwith 0.01% Trypsin (Sigma, T4049, USA) at 37 °C for
15 min. Following trypsinization, the cells were washed

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with ice-cold DMEM, filtered through a 70-µm cellstrainer (CORNING, USA), and seeded onto poly-Dlysine (Sigma, P6307, USA)-coated T-flasks (CORNING, USA) containing culture media (DMEM/F12(Gibco, 11320033, USA) supplemented with 10% heatinactivated FBS (Gibco, 10099141 C, USA), 2 mM
L-glutamine (Gibco, A2916801, USA), 1 mM sodium
pyruvate (Gibco, 11360070, USA), 100 µM nonessential
amino acids (Gibco,11,140,050, USA), 50 U/mL penicillin (Sigma, 76427, USA), and 50 µg/mL streptomycin(Sigma, 85886, USA) [24]. The cultures were incubated at
37 °C in a 5% CO2 atmosphere for 10–12 days. Microglia
were detached by shaking the flasks at 180 rpm for 1 h.
After a resting period of 1 h in the incubator, the flasks
were shaken overnight at 200 rpm to separate OPCs fromthe astrocyte layer. OPCs were cultured for 3–5 days inserum-free basal defined medium (BDM: DMEM, 0.1%
bovine serum albumin (Gibco, 11021037, USA), 50 µg/
mL human apo-transferrin (Sigma, 616395, USA), 50 µg/
mL insulin (Sigma, I9278, USA), 30 nM sodium selenite(Sigma, S5261,USA), 10 nM D-biotin (Invitrogen, B1595,
USA), 10 nM hydrocortisone (Sigma, 3867, USA) supplemented with 10 ng/mL platelet-derived growth factor (Gibco, 31517, USA) and 10 ng/mL basic fibroblast
growth factor (Gibco, PMG0031, USA). For differentiation into mature oligodendrocytes, OPCs were treatedwith triiodothyronine (Sigma, T2877, USA) and ciliary
neurotrophic factor (Sigma, 01-195, USA) at 10 ng/mL
each. The culture medium was refreshed every two days.
Sources of material for cell culture are listed in Table. S1.
Co-culture of BV2 cells with oligodendrocytes

Oligodendrocytes were subjected to oxygen-glucose
deprivation (OGD) for 2 h and then returned to a normoxic medium for 24 h. In a Transwell system, which
permits communication via soluble factors, pre-treated
BV2 cells were cultured in the upper chamber, while control or post-OGD oligodendrocytes were placed in thelower chamber for an additional 24 h.
Transmission electron microscopy

Following perfusion with ice-cold saline and fixatives(4% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M
phosphate-buffered saline), corpus callosum (CC) tissue proximal to the injury site was excised into 1 mm^3
blocks. These samples were post-fixed in 2% glutaraldehyde for 24 h, washed in PBS, and further fixed in 1%
osmium tetroxide in 0.1 M PBS for 45 min. The tissue wasdehydrated in a graded series of acetone and embedded
in Araldite resin. Ultrathin Sect. (60 nm) were preparedusing a Leica UCT ultramicrotome with a diamond knife(Diatome, Wetzlar, Germany), stained with uranyl acetate
and lead citrate, and examined with a JEM1400 transmission electron microscope (JEOL, Akishima, Japan). Each

Hou et al. Journal of Neuroinflammation(2024) 21:148

dissected 1 mm^3 blocks tissue block was bisected, andone section from each half was processed for electron
microscopy. Subsequently, two sections from each animal
were analyzed, capturing 3–5 images (600 μm^2 each) at
random within the CC at 50,000× magnification. At least
200 axons were analyzed per animal. Images were analyzed using ImageJ software by an investigator blinded tothe experimental groups. G-ratios were calculated as theratio of the inner axonal diameter to the total outer diameter (axonal diameter plus myelin sheath thickness).
Luxol fast blue staining

Coronal brain sections of 25 μm thickness were subjected
to Luxol Fast Blue staining as per the protocol provided
by American Mastertech to assess myelin integrity. Sections were incubated for 1 h in a preheated Luxol Fast
Blue solution at 60 °C. Subsequently, sections were rinsed
with water and differentiated in a lithium carbonate
solution for 30 s, followed by a 30-second immersion in
70% ethanol. After rinsing, differentiation was assessed
microscopically. The sections were then dehydrated withalcohol and coverslipped for microscopic evaluation.
Western blot analysis

Protein extracts were obtained from the corpus callosumof mice, BV2 cells, or primary oligodendrocytes. Protein
concentrations were quantified using the Bio-Rad Protein
Assay with bovine serum albumin as the standard. Fifty
micrograms of protein per sample were resolved by SDSPAGE and transferred onto PVDF membranes. These
membranes were incubated with primary antibodies at
4 °C overnight, followed by incubation with secondary
antibodies (1:2000, AS014, Abclonal). Primary antibodies
used included Anti-CD36 (1:1000, GB112562-100, Servicebio), Anti-iNOS (1:1000, ab178945, Abcam), AntiArg1 (1:1000, A4923, Abclonal), Anti-p38 (1:1000, 8690,
CST), Anti-phospho-p38 (1:1000, 4511, CST), AntiTraf5 (1:1000, 12868-1-AP, Proteintech), and Anti-MBP(1:1000, A11162, Abclonal). β-actin (1:100000, AC026,
Abclonal) served as the loading control. Blots were visualized using a BIO-RAD imaging system, and band intensities were quantified using ImageJ software, normalized
to β-actin.
Real-time fluorescent quantitative PCR (RT-qPCR)

Total RNA was isolated using an RNA-extraction
kit (Fastagen, China) following the manufacturer’s
Table 1 qPCR primer sequences
Gene
CD36
iNOS
Arg1
β-actin

Forward primer (5′-3′)​C​A​T ​T ​T​G​C​A​G​G​T​C ​T​A​T​C ​T​A​C​C​A​A​G​C​A​C​C ​T ​T​G​G​A​A​G​A​G​G​A​G​T​C​A​C​C ​T​G​A​G​C ​T ​T ​T​G​A​T​G​T​C​G​G​C ​T​C ​T​C​C ​T​A​T​G​T ​T​G​C ​T​C ​T​A​G

Reverse primer (5′-3′)​C​A​A​T​G​T​C ​T​A​G​C​A​C​A​C​C​A​T​A​A​G​A​A​G​G​C​C​A​A​A​C​A​C​A​G​C​A​T​A​C​C​C ​T​G​A​A​A​G​G​A​G​C​C​C ​T​G​T​C ​T ​T​G
​C​G​C ​T​C​C ​T ​T​G​C​C​A​A​T​A​C ​T​C

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instructions and reverse-transcribed into cDNA
(321,392, Promega, Beijing, China). The cDNA was combined with gene-specific primers and SYBR Premix Ex
Taq™ (Takara, Japan) for qPCR quantification using an
Applied Biosystems instrument. Primer sequences aredetailed in Table 1. The relative expression levels of target
mRNAs were calculated using the 2–ΔΔCt method, withnormalization to the housekeeping gene β-actin.
Immunofluorescence staining

Brain sections were permeabilized with 0.4% Triton X-100 for 10 min, followed by three 5-minute PBS
washes. Sections were then blocked with 2% bovine
serum albumin for 35 min and incubated overnightat 4 °C with primary antibodies including anti-MBP(1:200, Abclonal), anti-SMI-32(1:200, SMI-32P, BioLegend), anti-iNOS (1:400, ab178945, Abcam), anti-Arg1(1:200, A4923, Abclonal), Anti-CD36 (1:500, GB112562100, Servicebio), anti-GFAP (1:500, ab279289, Abcam),anti-Olig2 (1:200, ab109186, Abcam), anti-NeuN (1:100,
ab104224, Abcam), and anti-Iba-1 (1:100, ab283319,
Abcam). After washing with PBS, sections were incubated with appropriate secondary antibodies (secondary antibodies included the following: Alexa Fluor 488
secondary antibodies or Alexa Fluor 594 secondary
antibodies (1:400, Jackson ImmunoResearch) for 2 h atroom temperature and counterstained with DAPI for
6 min. Cultured cells were fixed with 4% PFA for 30 min,blocked, and incubated with primary antibodies overnight at 4 °C. Following secondary antibody incubation,
cells were counterstained with DAPI and analyzed using
ImagePro Plus 6.0.
For identifying pro-inflammatory and anti-inflammatory microglia, double-positive cells (iNOS/iba1 forpro-inflammatory microglia and ARG1/iba1 for antiinflammatory microglia) were identified as co-localized
cells and were counted using image software [25]. In Shollanalysis, 200 suitable oligodendrocytes were selected forevaluation in each independent experiment per group.
For the cell count, a consistent area size was uniformly
selected for cell counting in each group, ensuring a minimum count of 500 cells per independent experiment.
Cell viability and death assays

Cell viability was assessed using the CCK-8 assay. Oligodendrocytes in 96-well plates were incubated with
20 µl of CCK-8 reagent at 37 °C in a 5% CO2 incubatorfor 2 h. After color development, absorbance at 450 nm
was measured using a microplate reader (Elx800; BioTek
Instruments).
Cell death was evaluated using the LDH assay, whichmeasures LDH release into the culture medium indicative of membrane integrity loss. Aliquots of 100 µL fromthe culture medium were mixed with 150 µL of LDH

Hou et al. Journal of Neuroinflammation(2024) 21:148

reagent (Sigma, USA). The absorbance of the reaction
was measured spectrophotometrically by monitoring
the reduction of NAD + at 340 nm at 25 °C over 5 min.
Data were expressed as a percentage of maximum LDH
activity from control wells where all cells were lysed with
Triton.
mRNA high throughput sequencing and analysis

mRNA high throughput sequencing was performed
by NewCore Biotech. (Shanghai, China). Briefly, total
RNA was extracted using Trizol Reagent (ThermoFisher,
15,596,026). The mRNAs were purified using a NEBNext
Poly(A) mRNA Magnetic Isolation Module Kit (NEB,
E7490) according to the user manual. mRNA libraries were constructed by using Illumina TrueSeq mRNA
sample preparation kit (Cat. No. RS-122-2101) according
to the manufacturer’s instructions. Library sequencing
was performed on an Illumina NovaSeq 6000 instrumentwith 151 bp paired end reads.
Bioinformatics data was analyzed. Briefly, fastp software (v0.20.0) was used to trim adaptor and remove
low quality reads to get high quality clean reads. STAR
software (v2.7.9a) was used to align the high-quality
clean reads to the human reference genome (hg38). featureCounts software (v2.0) was used to get the raw genelevel mRNA read counts as the mRNA expression profile.
DESeq2 software (v1.30.1) was used to normalize andcalculate the fold change and Pvalue between two groups.
Ensembl GTF gene annotation database (v104) was used
to annotate the mRNA. Gene Ontology and KEGG pathway enrichment analysis were performed with clusterProfiler R package (v3.18.1) based on the differentiallyexpressed mRNAs. rMATS software (v4.1.1) was used
to predict the alternative splicing events between two
groups.
Construction of plasmids and development of shRNA
transfected BV2 cells

CD36 shRNA plasmids, CD36 plasmids Traf5 shRNA
plasmids, and negative control plasmids were constructed by Genomeditech (Shanghai, China) (Table 2).
The corresponding vector was pGMLV-hU6-MCS-CMVPuromycin. BV2 cells were stably transfected by the lentiviruses (Genomeditech, Shanghai, China) following
the manufacturer’s protocol. The positive clones werescreened by puromycin.
Table 2 Target sequences
Gene
CD36 Knock-down #1
CD36 Knock-down #2
Traf5 Knock-down #1
Traf5 Knock-down #2

TargetSeq (5′-3′)​G​C ​T​A​T ​T​G​C​G​A​C​A​T​G​A​T ​T​A​A​T​G​G​C​C ​T​G​T​G​T​A​T​A​T ​T ​T​C​G​C ​T ​T​C​C​G​C​G​G​C​A​A​G​A​A​G​A​A​C​C​A​T​A​T ​T​G
​G​C​A​C​C ​T​G​T​C​C​C ​T​G​T​A​C ​T ​T ​T​G​T

Page 7 of 20

Statistical analysis

Results were presented as mean ± standard error of themean (SEM). GraphPad Prism software (version 8.1.0,
La Jolla, CA, USA) was used for statistical analyses. The
distribution normality and variance equality of the data
were assessed using the Shapiro-Wilk and Levene’s variance equality tests, respectively. If the data adhered tonormal distribution and the P value of Levene varianceequality test was greater than 0.05, Student’s t-test wasused for two-group comparisons and one-way ANOVA
with Tukey’s post hoc test for multiple groups. Otherwise, non-parametric tests such as the Mann-Whitney U
rank sum test or the Kruskal-Wallis rank sum test were
employed. Differences were considered significant at
P < 0.05.

Results
Temporal and spatial patterns of CD36 receptor expression
post-TBI

The temporal and spatial expression patterns of CD36
receptors in the brain were examined by collecting corpus callosum tissues from control mice and those subjected to TBI at multiple time points: 1, 3, 7, 14, and 28
days post-injury. Western blot (WB) and quantitative
PCR (qPCR) analyses were employed to detect CD36
expression in the peri-injury zone of brain tissue. The
results indicated a progressive increase in CD36 receptor
expression post-TBI, reaching a peak at day 7, as demonstrated by immunoblotting (Fig. 1A, B) and corroborated
by qPCR findings (Fig. 1C). Based on these findings,
day 7 post-injury was selected as the optimal time point
for further observation. Immunofluorescence staining
on day 7 post-injury revealed that CD36 was predominantly expressed in astrocytes (GFAP+) and microglia(Iba-1+), with minimal expression in oligodendrocytes(Olig2+) and neurons (NeuN+) within the corpus callosum (Fig. 1D).
CD36 knockout mitigates white matter damage andenhances neurological recovery in TBI mice

The deletion of CD36 in mice was confirmed (Fig. 2A,
B; Fig. S2). To evaluate whether CD36 inhibition could
mitigate white matter damage post-TBI, myelin loss inthe corpus callosum (CC) was assessed using Luxol fast
blue staining, and alterations in myelin basic protein(MBP) and abnormally dephosphorylated neurofilament
protein (detected with SMI-32 antibody) were examined.
In wild-type (WT) mice subjected to controlled cortical
impact (CCI), a significant loss of MBP and an increasein abnormally dephosphorylated neurofilament protein
were observed. However, the ratio of SMI-32 to MBP was
significantly reduced in the TBI KO group compared to
the TBI WT group (Fig. 2C–F), and similar results wereobtained from immunofluorescence detection of theHou et al. Journal of Neuroinflammation(2024) 21:148

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Fig. 1 Temporal and spatial expression patterns of CD36 receptor expression after TBI. (A, B) Western blot analysis and quantification depicting the temporal expression of CD36 receptors in brain tissue post-TBI. (C) Quantitative PCR results showing CD36 expression trends following TBI. (D) Immunofluorescence images illustrating CD36 localization in neurons, microglia, astrocytes, and oligodendrocytes at day 7 post-injury within the injury and peri-injury
zones. Scale bar: 20 μm. Data represent mean ± SEM for 4 mice per group. *P < 0.05, **P < 0.01, ***P < 0.001

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Hou et al. Journal of Neuroinflammation(2024) 21:148

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Fig. 2 CD36 knockout mitigates white matter injury. (A, B) Validation of CD36 knockout efficiency via western blot and associated quantification, n = 3/
group. (C) Luxol fast blue staining demonstrating reduced myelin loss in the corpus callosum of CD36 knockout mice at day 7 post-TBI. Scale bar: 50 μm.
(D) Transmission electron microscopy images showing demyelination in the corpus callosum at day 7 post-TBI. Scale bar: 1 μm. (E) Axonal diameter
distribution. (F) G-ratio analysis reflecting myelin thickness relative to axonal diameter. (G) Dual immunofluorescence staining for dephosphorylated
neurofilament protein (SMI-32, red) and myelin basic protein (MBP, green) in the corpus callosum at day 7 post-TBI, with DAPI staining nuclei (blue). Scalebar: 50 μm. (H–J) Quantitative analysis of white matter injury, indicated by changes in MBP (H), SMI-32 (I), and their ratio (J). Data represent mean ± SEM
for 4 mice per group. *P < 0.05, **P < 0.01, ***P < 0.001

ipsilateral striatum (Fig. S3). Luxol fast blue staining also
indicated partial restoration of myelin in the CD36 KO
group after TBI (Fig. 2G). Additionally, electron microscopy of the CC post-injury revealed a substantial reduction in axon diameters in the TBI group compared toboth the TBI KO and sham groups (Fig. 2H, I). G-ratio
analysis further confirmed a significant decrease in
myelin thickness in the TBI group relative to the shamand TBI KO groups (Fig. 2J).
Neurological function was evaluated by investigators
blinded to group assignments. The modified Neurological Severity Score (mNSS) was used to gauge neurological impairment, with higher scores indicating greater
impairment. Post-TBI, mNSS scores surged and thengradually declined (Fig. 3A). Notably, the decline in
mNSS scores was more pronounced in the TBI KO groupthan in the TBI WT group. Sensorimotor functions wereassessed using the foot fault test, wire hang, and rotarod
tests (Fig. 3B–D), which revealed that CD36 knockout
mitigated sensorimotor deficits. Moreover, CD36 KOmice demonstrated cognitive improvements in the Morris water maze test (Fig. 3E), evidenced by a decrease intime spent on escape during the training phase (Fig. S4A
and B) and an increase in swimming distance and time in
the target quadrant after platform removal (Fig. 3G, H).
No differences in swimming speed were observed among
the groups during both the training and testing phases,suggesting comparable gross motor abilities (Fig. 3F, Fig.
S4C).
The neuroprotective mechanism of CD36 inhibition
involves microglia activation

To understand how CD36 inhibition exerts its neuroprotective effects after traumatic brain injury (TBI), atranscriptome analysis was performed on brain tissue
collected from the peri-injury area of mice 7 days after
controlled cortical impact (CCI). This analysis identified 1212 differentially expressed genes (DEGs), with
334 upregulated and 878 downregulated genes in the
CD36 knockout (KO) group compared to the wild-type(WT) group after CCI injury (Fig. 4A). Gene Ontology(GO) enrichment analysis showed that these DEGs wereinvolved in biological processes related to cytokine production, inflammatory response regulation, neuroinflammatory response, microglia activation, and interleukin
production (Fig. 4B). Additionally, KEGG enrichment
analysis revealed their involvement in various signaling

pathways, including cytokine-cytokine receptor interaction, TNF signaling, NF-kappa B signaling, and MAPK
signaling (Fig. 4C). This suggested that the neuroprotective effects of CD36 deletion might be linked to microglia
activation and related inflammatory signaling pathways.
To delve deeper into the mechanism of CD36 in
microglial activation, BV2 and CD36 knockdown (KD)
BV2 cells were treated with LPS (lipopolysaccharide) and
IFN-γ for 24 h, and their transcriptomes were sequenced.
A total of 1079 DEGs were identified, with 308 upregulated and 771 downregulated genes in the CD36-KD
BV2 group compared to the BV2 group (Fig. 4D). KEGG
enrichment analysis of these DEGs revealed their involvement in signaling pathways like TNF signaling, NF-kappa
B signaling, and MAPK signaling (Fig. 4E). Gene Set
Enrichment Analysis (GSEA) indicated that genes weresignificantly enriched in the TNF signaling pathway after
CD36 knockdown (Fig. 4F), and the core gene Traf5 wasdetermined (Fig. 4G). Interestingly, KEGG enrichment
analysis also showed that Traf5 and phosphorylated p38(p-p38) in the MAPK signaling pathway were notably
downregulated (Fig. S5).
CD36 inhibition modulates microglia polarization via Traf5MAPK signaling pathway

To confirm the conclusions drawn from sequencing, BV2 cells were modified with overexpression andknockdown plasmids for CD36 and Traf5. Western blot
analysis demonstrated the efficiency of overexpression
and knockdown (Fig. 5A–C). The effects of CD36 inhibition or overexpression on the Traf5/MAPK axis in
LPS-IFN-γ-treated microglia were evaluated. LPS-IFN-γ
treatment led to the upregulation of Traf5 and p-p38,
and these effects were attenuated by CD36 knockdown
and enhanced by CD36 overexpression. Knockdown ofTraf5, either alone or in combination with CD36 overexpression, reduced p-p38 levels induced by LPS-IFN-γ(Fig. 5D–F).
The role of the CD36-Traf5-MAPK axis in microglia
polarization was further confirmed through Western blot
analysis, qPCR, and immunofluorescence staining. Proinflammatory microglia were characterized by high iNOS
expression, while anti-inflammatory microglia were
defined by high Arg1 expression. LPS-IFN-γ treatment
induced pro-inflammatory polarization, as evidenced byelevated iNOS expression, but CD36 and Traf5 knockdown, as well as p38 inhibition, mitigated this effect. On

Hou et al. Journal of Neuroinflammation(2024) 21:148

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Fig. 3 Neurological function enhancement in CD36 knockout mice. (A) Modified Neurological Severity Scores (mNSS) over time post-TBI. (B–D) Sensorimotor assessments including the foot fault, wire hang, and rotarod tests. (E–H) Cognitive function evaluation in the Morris water maze, with swim paths
depicted during the probe trial (E), distance traveled (H), and time spent in the target quadrant (G), without differences in swim speed (F). Data represent
mean ± SEM for 10 mice per group. *P < 0.05, **P < 0.01, ***P < 0.001

Hou et al. Journal of Neuroinflammation(2024) 21:148

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Fig. 4 Transcriptomic analysis post-CD36 inhibition. (A) Volcano plot illustrating differentially expressed genes (DEGs) between CD36(-/-) and wild-typemice post-TBI. (B) Gene Ontology (GO) enrichment analysis highlighting biological processes among DEGs. (C) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of DEGs. (D) Volcano plot of DEGs between CD36-knockdown (KD) BV2 cells and control BV2 cells treatedwith LPS + IFN-γ. (E) KEGG pathway enrichment analysis for DEGs in CD36-KD BV2 cells. (F) Gene Set Enrichment Analysis (GSEA) for the TNF signaling
pathway. (G) Identification of core genes via GSEA.

the other hand, LPS-IFN-γ inhibited anti-inflammatorypolarization, demonstrated by a decreased Arg1/Iba-1
ratio, and this effect was reversed by CD36 knockdown,
Traf5 knockdown, and p38 inhibition (Fig. 5G). Immunofluorescence staining also supported these findings.
Moreover, in vivo experiments conducted 7 days postTBI showed increased density of pro-inflammatory(Iba1+/iNOS+ co-localized cells) and anti-inflammatory (Iba1+/Arg1+ co-localized cells) microglia in the
injury area. In line with the in vitro results, CD36, Traf5
knockdown, and p38 inhibition promoted microglia
polarization toward anti-inflammatory phenotype anddecreased polarization toward pro-inflammatory phenotype (Fig. 6A–D). Additionally, Western blot and qPCR

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Fig. 5 CD36 knockdown reduces pro-inflammatory polarization in BV2 cells via the Traf5-MAPK axis. (A–C) Validation of stable transfection in BV2 cells bywestern blot and quantification, n = 3/group. (D–F) Assessment of the Traf5-MAPK signaling pathway by western blot and quantification. (G–I) Western
blot analysis of iNOS and ARG1 expression. (J, K) qPCR validation of iNOS and ARG1 expression. (L, M) Immunofluorescence images depicting pro-inflammatory (L) and anti-inflammatory (M) polarization in BV2 cells across different experimental conditions. (N, O) Proportional analysis of polarized BV2 cells
relative to the total BV2 population. Pro-inflammatory phenotype/Total (N); Anti-inflammatory phenotype/Total (O). Data represent mean ± SEM from 4
independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001

analysis confirmed the increased expression of bothiNOS and Arg1 post-TBI, and CD36 inhibition, Traf5
knockdown, and p38 inhibition attenuated this effect(Fig. 6E–I).
CD36-mediated microglia polarization contributes to whitematter protection after TBI

The impact of CD36-mediated microglia polarization on
white matter injury was further validated. Luxol fast blue
(LFB) staining revealed significant myelin loss after TBI,
which was attenuated by CD36 knockout or SB242235
treatment (Fig. 7A). This finding was consistent with the
SMI-32/MBP ratio, which increased after TBI but was
reversed by CD36 knockdown and SB242235 (Fig. 7B–E).
To investigate the role of CD36-regulated microglia
polarization in oligodendrocyte injury, oligodendrocytes(OLs) cultured in a Transwell system were analyzed after
various treatments. Since Oligodendrocytes convert froma simple bipolar morphology to a complex branchedmorphology during differentiation [26], the morphological complexity of differentiating Oligodendrocytes processes was quantified using Sholl analysis (Fig. 8A, B),
which revealed that OGD led to a significant decrease in
the number of intersections, indicating inhibition of differentiation. Co-culture with untreated BV2 cells slightly
mitigated this effect, but co-culture with LPS-IFN-γtreated BV2 cells exacerbated the inhibition. Notably,
co-culture with LPS-IFN-γ-treated CD36-KD BV2 cells
resulted in more frequent intersections compared to BV2cells (Fig. 8A-B). This suggests that CD36 knockdown canpartially restore oligodendrocyte morphology during differentiation. Furthermore, co-culture with BV2 cells did
not increase the number of MBP + multipolar cells. However, BV2 cells pretreated with LPS-IFN-γ significantly
reduced the number of MBP + multipolar cells, which
was reversed by CD36 knockdown (Fig. 8C). The expression levels of MBP were also reduced in OGD OLs cocultured with LPS-IFN-γ-treated BV2 cells. As expected,
CD36 knockdown in BV2 cells restored MBP expression
levels in OGD OLs (Fig. 8D-E). These findings were further supported by cell viability assays and LDH releaseassays, which showed that CD36 knockdown mitigatedthe detrimental effect of LPS-IFN-γ-pretreated BV2 cells
on OGD OLs (Fig. 8F–G).

Discussion
In this study, we elucidate, for the first time, the ameliorative impact of CD36 receptor inhibition on WMI
precipitated by TBI, along with its underlying cellularand molecular mechanisms. Subsequent to TBI, resting
microglia undergo activation and polarization. Notably, a
pronounced shift towards the pro-inflammatory phenotype exacerbates WMI. Our investigations in vivo and invitro, for the first time, reveal that suppression of CD36
and its downstream Traf5-MAPK signaling pathway
diminishes the prevalence of pro-inflammatory polarized
microglia while augmenting anti-inflammatory polarization, thereby mitigating WMI post-TBI. These findings
suggest that CD36 receptor inhibition may represent a
viable therapeutic strategy to prevent the induction ofdeleterious pro-inflammatory microglia polarization following TBI.
The pathophysiology of traumatic brain injury (TBI) is
characterized by a high level of complexity. Key mechanisms involved in secondary damage post-TBI, such as
inflammatory responses, excitotoxicity, oxidative stress,and mitochondrial dysfunction, often intertwine, collectively impacting the brain [27–29]. Focusing on
inflammation, microglia and macrophages, as primarydefensive cells in cranial trauma, are rapidly recruitedand activated at the injury site. This activation leads to
the release of pro-inflammatory mediators, initiating an
inflammatory response [30]. This response is intricately
connected with other damage mechanisms. For example,
mitochondrial dysfunction and oxidative stress can exacerbate inflammation [31, 32], and in some cases, inflammation can amplify excitotoxicity [33]. Recent studies
have illuminated the remarkable plasticity of microglia/
macrophages, which, upon activation, can adopt two
principal phenotypes with distinct functional roles [34,35]. The pro-inflammatory phenotype microglia secrete
inflammatory cytokines, whereas the anti-inflammatory
phenotype exhibit enhanced phagocytic capabilities
and release a plethora of neurotrophic factors [36]. The
pro-inflammatory phenotype is associated with exacerbation of neurological damage, while the anti-inflammatory phenotype is considered neuroprotective [25,37]. Intriguingly, the dynamic balance between pro- andanti-inflammatory phenotype shifts over time following
TBI and stroke, with anti-inflammatory cells predominating in the early phase post-injury, but a subsequent
shift towards pro-inflammatory polarization becomingHou et al. Journal of Neuroinflammation(2024) 21:148

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Fig. 6 The CD36-Traf5-MAPK axis influences microglial polarization post-TBI. (A, B) Immunofluorescence images of pro-inflammatory (A) and anti-inflammatory (B) microglia polarization in the corpus callosum following TBI in different experimental groups. (C, D) Quantification of Iba1+/iNOS+ doublepositive cells (C) and Iba1+/Arg1+ double-positive cells (D). (E, F) mRNA expression levels of iNOS (E) and Arg1 (F) in the corpus callosum at 7 days post-TBI.
(G–I) Western blot analysis and quantification of iNOS and ARG1 expression. Data represent mean ± SEM for 4 mice per group. *P < 0.05, **P < 0.01,***P < 0.001

Hou et al. Journal of Neuroinflammation(2024) 21:148

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Fig. 7 CD36-MAPK pathway inhibition reduces white matter injury associated with TBI. (A) Luxol Fast Blue (LFB) staining in the corpus callosum at day 7
post-TBI across different groups. Scale bar: 50 μm. (B) Dual immunofluorescence staining for SMI-32 (red) and MBP (green) in the corpus callosum at day 7
post-TBI, with DAPI-stained nuclei (blue). Scale bar: 50 μm. (C–E) Quantitative assessment of white matter injury, indicated by changes in MBP (C), SMI-32(D), and their ratio (E). Data represent mean ± SEM for 4 mice per group. *P < 0.05, **P < 0.01, ***P < 0.001

evident from day 7 onwards [38, 39]. This temporal pattern suggests an initial state of neural repair that gradually transitions to a state of sustained neuroinflammation.
Moreover, our findings indicate that CD36 expression peaks on day 7 post-injury, which, given the temporal pattern of microglia polarization [38, 39], suggests arobust association between CD36 and the activation of
pro-inflammatory microglia. Interestingly, microglia arerecognized as a specialized subset of macrophages [40].
CD36 receptors in macrophages were found to mediate inflammatory responses via mitochondrial oxidative
stress or activation of the AMPK pathway [41, 42], andit was also revealed that LPS induces pro-inflammatorypolarization of macrophages via CD36 in acute lunginjury [43]. Collectively, these findings further support
the link between CD36 and microglia polarization into
pro-inflammatory phenotype. CD36 is implicated in a

myriad of neuropathological processes, including cerebral ischemia [44, 45], neurovascular dysfunction [46],and atherosclerosis [47]. The CD36 antagonist SSO has
been shown to mitigate stroke-induced neuroinflammation and confer neuroprotection in murine models
of ischemic stroke [16]. Corroborating these findings,
our study demonstrates that CD36 deletion attenuates
myelin damage and oligodendrocyte loss following TBI.
To assess the impact on neurological function post-WMI,
we conducted a series of neurobehavioral tests to evaluate cognitive and motor functions in mice. Mice deficientin CD36 exhibited superior neurological recovery postTBI compared to their wild-type counterparts. This is
in alignment with Scott et al.‘s findings, which indicatedenhanced neurological recovery in CD36 knockout micefollowing spinal cord injury [48]. CD36 is predominantly
expressed in microglia and astrocytes within the brainHou et al. Journal of Neuroinflammation(2024) 21:148

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Fig. 8 Modulation of oligodendrocyte damage by CD36-MAPK axis inhibition in a BV2 cell co-culture system under oxygen-glucose deprivation (OGD).
(A) Immunofluorescence staining of primary oligodendrocytes in various experimental conditions. Scale bar: 50 μm. (B) Sholl analysis of MBP + oligodendrocyte process branching. (C) Count of MBP + multipolar oligodendrocytes. (D, E) Western blot validation of MBP expression levels. (F, G) Oligodendrocyte viability and cell death assessed by CCK-8 assay (F) and lactate dehydrogenase (LDH) release (G), respectively. Data represent mean ± SEM from 4
independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001

Hou et al. Journal of Neuroinflammation(2024) 21:148[49], as confirmed by our experiments, and is known tomodulate inflammatory responses and glial scarring [44,45, 50, 51]. Furthermore, CD36 expression in vascularendothelial cells has been implicated in fatty acid transport [52] and the regulation of angiogenesis [13]. Notably, CD36-expressing macrophages can also infiltrate into
the injured area and perform functions after spinal cord
injury [48]. To elucidate the mechanisms by which CD36
inhibition ameliorates TBI-induced WMI, we performed
transcriptome sequencing comparing CD36(-/-) and wildtype mice post-TBI. The data suggest that modulationof microglial activation and neuroinflammation is a key
mechanism by which CD36 inhibition exerts protective effects on white matter integrity. This substantiates
the link between CD36 receptor activity and microglial
activation. Through immunofluorescence, immunoblotting, and other experimental modalities, we have further
confirmed that CD36 inhibition curtails the polarizationof microglia towards the pro-inflammatory phenotype,thereby reducing myelin damage.
Subsequent transcriptome sequencing analysis of
BV2 cells and molecular assays have corroborated themodulatory influence of the Traf5-MAPK (p38) axis on
microglial polarization subsequent to TBI. The attenuation of the Traf5-MAPK axis was observed to inhibit
the emergence of pro-inflammatory polarized microglia, thereby mitigating the WMI induced by TBI. Tumor
necrosis factor receptor-associated factor 5 (Traf5) is anadaptor molecule associated with the tumor necrosis
factor (TNF) receptor superfamily and the interleukin-1
receptor/Toll-like receptor superfamily, playing a pivotal
role in the modulation of multiple signaling cascades[53]. Previous research has indicated that Traf5 upregulation occurs in glial cells and neurons post-spinal cord
injury [54], and that a deficiency in Traf5 confers protection against cerebral ischemia-reperfusion injury bydampening inflammation and preserving the integrity ofthe blood-brain barrier (BBB) [53]. In the current study,
the effects of LPS-IFN-γ in promoting pro-inflammatory polarization were significantly diminished following Traf5 inhibition via lentiviral transduction in BV2cells. Correspondingly, mice administered with Traf5shRNA exhibited a marked decrease in pro-inflammatory
microglia phenotype on the seventh day post-TBI, relative to mice that underwent TBI alone. These findings
lend further support to the anti-inflammatory propertiesof TRAF5 inhibition.
Mitogen-activated protein kinases (MAPKs), encompassing p38, ERK1/2, and JNK, belong to the serinethreonine protein kinase family and are implicated in
the upregulation of pro-inflammatory cytokines [55,56]. The MAPK signaling pathway has been reported tobe significantly activated in conditions such as TBI [57],stroke [16], and spinal cord injury [58], and the inhibition

Page 18 of 20

of this pathway has been shown to provide neuroprotection in murine models of TBI [59, 60]. Our investigation into the role of p38 in the regulation of microglial
activation, neuroinflammation, and white matter injury
was facilitated by the use of a specific p-p38 inhibitor(SB242235). Aligning with the findings from previous
studies, our results indicate that the suppression of p38
phosphorylation inhibits the pro-inflammatory polarization of microglia, thereby alleviating WMI induced by
TBI. Collectively, these findings suggest that the inhibition of CD36 can prevent white matter injury by modulating microglial polarization through the Traf5-MAPK
signaling pathway.
While our study pioneers in demonstrating that inhibiting CD36 can alleviate white matter injury post-TBI, it
presents certain limitations. Primarily, the applicabilityof conclusions drawn from murine models to humans
remains uncertain. Furthermore, despite extensive
research on the CD36 receptor inhibitor SSO, its clinicalsafety and effectiveness remain unverified. Additionally,
considering that pharmacological analgesia might interfere with the results of some experiments, no analgesic
drugs were administered to the mice, which, however,may carry the inherent risk of introducing biases into
the neurobehavioral test results. And the data collection
and analysis in neurobehavioral tests rely on evaluators,
who might overlook subtle yet relevant changes. Future
research should, therefore, focus on expanding animalstudies to gather substantial data, developing CD36-specific inhibitors, and conducting clinical trials to assess thesafety and effectiveness of CD36 inhibitors, offering new
therapeutic avenues for TBI patients.

Conclusions
In summary, the findings of our investigation offer robust
support for the premise that the inhibition of CD36 is
instrumental in averting white matter injury. This protective effect is achieved by the modulation of microglial
polarization, steering cells away from the pro-inflammatory phenotype via the downregulation of the Traf5MAPK signaling pathway, as depicted in the graphicalabstract. The data advocate for the targeting of CD36
expression in microglia as a viable therapeutic strategy
to mitigate TBI-induced neuroinflammation and theensuing white matter injury. This research enriches ourunderstanding of the pathophysiological mechanisms
underlying white matter injury post-TBI and introduces a potential therapeutic avenue for TBI clinicalinterventions.
Abbreviations
Arg1	Arginase 1
bFGF	Basic fibroblast growth factor
BP	Biological processes
BSA	Bovine serum albumin

Hou et al. Journal of Neuroinflammation(2024) 21:148

CC	Corpus callosum
CCI	Controlled cortical impact
CNS	Central nervous system
CNTF	Ciliary neurotrophic factor
DEGs	Differentially expressed genes
DMEM	Dulbecco’s modified eagle medium
GFAP	Glial fibrillary acidic protein
GSEA	Gene Set Enrichment Analysis
Iba-1	Ionized calcium-binding adapter molecule-1
IFN-γ	Interferon gamma
IL-4	Interleukin 4
iNOS	Inducible nitric oxide synthase
KD	KnockdownKO	Knockout
LDH	Lactate dehydrogenase
LFB	Luxol fast blue
LPS	Lipopolysaccharides
MAPK	Mitogen-activated protein kinase
MBP	Myelin basic protein
NeuN	Neuron-specific nuclear protein
OE	Overexpress
OGD	Oxygen glucose deprivation
Olig2	Oligodendrocyte Lineage Transcription Factor 2
OLs	Oligodendrocytes
OPCs	Oligodendrocyte precursor cells
PDGF	Platelet-derived growth factor
p-p38	Phosphorylated p38
SMI-32	Neurofilament H (NF-H)
SSO	Sulfosuccinimidyl oleate
T3	Triiodothyronine
TBI	Traumatic brain injury
TNF	Tumor necrosis factor
Traf5	Tumor necrosis factor receptor-associated factor 5
WMI	White matter injury

Supplementary Information

The online version contains supplementary material available at https://doi.
org/10.1186/s12974-024-03143-2.
Supplementary Material 1
Supplementary Material 2
Acknowledgements
Not applicable.
Author contributions
XH, XQ, and WC contributed to the study conception and design. XS, YY,
WC, CW, and YG contributed to data acquisition. XH, XQ, XS, CY, and HS
contributed to the analysis and interpretation of data and drafting of themanuscript. KZ, YZ, HX, LL, DZ, and LH contributed to the critical revision of
the manuscript. All authors read and approved the final manuscript.
Funding
This work was supported by the Key Clinical Specialty of PLA (Neurosurgery)[Lijun Hou, grand numbers JDLCZDZK-HLJ], National Natural Science
Foundation of China (NSFC) [Danfeng Zhang, grant numbers 82271396,
Liquan Lv, grant numbers 82071363 and Wen Chen, grant numbers
82201519], Chenguang Project of Shanghai [Danfeng Zhang, grant numbers
19CG39], The “Three Hang” Talent Development Programme [Danfeng Zhang]and Shanghai Sailing Program (Chunhui Wang, grant numbers 21YF1457200).
Data availability
All data used during the current study available from the corresponding
author on reasonable request.

Page 19 of 20

Declarations
Ethics approval and consent to participate
All experimental operations were approved by the Animal Care and Use
Committee of the Naval Medical University (No. 82201519).
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Author details
1
Department of Neurosurgery, Changzheng Hospital, Naval Medical
University, 415 Fengyang Rd, Shanghai, China
2
Department of Neurosurgery, The First Affiliated Hospital of Wenzhou
Medical University, Wenzhou Medical University, Wenzhou, China
3
Department of Neurosurgery, The First Naval Hospital of Southern
Theater Command, Zhanjiang, China
4
Department of Neurosurgery, Xishan People’s Hospital of Wuxi City, Wuxi,
China
Received: 9 November 2023 / Accepted: 27 May 2024

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