(2024) 21:193
Hu et al. Journal of Neuroinflammation
https://doi.org/10.1186/s12974-024-03186-5

Journal of Neuroinflammation

Open Access

RESEARCH

Lactate promotes microglial scar formationand facilitates locomotor function recovery
by enhancing histone H4 lysine 12 lactylation
after spinal cord injury
Xuyang Hu1,2†, Jinxin Huang1,2†, Ziyu Li1,2†, Jianjian Li1,2, Fangru Ouyang1,2, Zeqiang Chen1,2, Yiteng Li1,2,
Yuanzhe Zhao1,2, Jingwen Wang1,2, Shuisheng Yu1,2*, Juehua Jing1,2* and Li Cheng1,2*

Abstract
Lactate-derived histone lactylation is involved in multiple pathological processes through transcriptional regulation.
The role of lactate-derived histone lactylation in the repair of spinal cord injury (SCI) remains unclear. Here we reportthat overall lactate levels and lactylation are upregulated in the spinal cord after SCI. Notably, H4K12la was significantly elevated in the microglia of the injured spinal cord, whereas exogenous lactate treatment further elevatedH4K12la in microglia after SCI. Functionally, lactate treatment promoted microglial proliferation, scar formation, axon
regeneration, and locomotor function recovery after SCI. Mechanically, lactate-mediated H4K12la elevation promoted
PD-1 transcription in microglia, thereby facilitating SCI repair. Furthermore, a series of rescue experiments confirmedthat a PD-1 inhibitor or microglia-specific AAV-sh-PD-1 significantly reversed the therapeutic effects of lactate following SCI. This study illustrates the function and mechanism of lactate/H4K12la/PD-1 signaling in microglia-mediatedtissue repair and provides a novel target for SCI therapy.
Keywords Spinal cord injury, Microglia, Lactate, PD-1, H4K12la†

Xuyang Hu, Jinxin Huang and Ziyu Li contributed equally.

*Correspondence:
Shuisheng Yu
ysspaper@sina.com
Juehua Jing
jjhhu@sina.com
Li Cheng
chengli7788@163.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-NonCommercial-NoDerivatives 4.0
International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as longas 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 the licensed material. You do not have permission under this licence to share adapted material derived from this article orparts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicatedotherwise in a credit line to the material. 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/.

Hu et al. Journal of Neuroinflammation(2024) 21:193

Page 2 of 23

Graphical Abstract

BackgroundSpinal cord injury (SCI) is a serious disorder of thecentral nervous system (CNS), causing autonomic,
sensory, and motor dysfunction [1, 2]. SCI triggers acomplicated range of inflammatory processes that culminate in scar formation involving astrocytic, fibrotic,
and microglial scarring [3–5]. Previous research hassuggested that the secretion of chondroitin sulfate proteoglycans (CSPGs) by astrocytic scars is responsible
for failure of axon regeneration [6]. However, anotherstudy has challenged this belief by proposing that astrocytic scars support axon regeneration [7]. The fibrotic
scar clearly leads to failure of axon regeneration, as
axon terminals generate retraction bulbs upon contact
with fibroblasts and cannot pass through the scar [8].
Microglial scars have received limited attention until

recently. In the first week after SCI, microglia are activated into a proliferative state and quickly migrate to
the lesion core, where they phagocytose myelin debris
and participate in microglial scar formation [9, 10].
However, the mechanism underlying microglial scarformation remains unclear.
SCI is known to cause microvascular damage andinflammation, resulting in an ischemic and hypoxic
microenvironment in the metabolically active spinal
cord, thereby disrupting the coupling of glycolysis andoxidative phosphorylation [11, 12]. Lactate, produced
as a byproduct of glycolytic metabolism under hypoxia
plays a crucial role in the flexibility of metabolism andis emerging as a potential bioenergetic fuel and cellularfunction regulator [13]. Lactate accumulation has beensuggested to result from an initial inflammatory response
or insufficient blood perfusion [14]. Both exogenous andHu et al. Journal of Neuroinflammation(2024) 21:193

endogenous lactate can contribute to histone lactylationand subsequently activate downstream gene transcription [15, 16]. The pivotal roles of histone lactylation incell fate determination, embryonic development, andneuropsychiatric disorders have been confirmed [17].
Nevertheless, the role of lactate and histone lactylation
after SCI remains unclear.
In this study, we report a significant elevation in lactate
and lactylation levels in the spinal cord following SCI.
Notably, lactylation and H4K12la levels were significantly
increased in microglia after SCI. Functionally, lactate
treatment was found to promote microglial proliferation,scar formation, axon regeneration, and locomotor function recovery after SCI. Mechanically, lactate-mediatedH4K12la elevation was found to promote the transcription of PD-1 in microglia. Our findings elucidate thefunction and mechanism of lactate/H4K12la/PD-1 signaling in microglia after SCI, providing a novel approachfor treating SCI.

Materials and methods
Animals and SCI model

The relevant animal procedures complied with the Animal Committee of Anhui Medical University (Approval
No. LLSC20220381). Eight-week-old C57BL/6 J mice
(18–22 g) were purchased from the Experimental Animal
Center of Anhui Medical University and, and 8-week-old
CCR2RFPCX3CR1GFP dual-reporter mice (Catalog No.
032127) were purchased from Shanghai Model Organism. All mice were housed in an environment with controlled temperature and humidity and a 12/12 h light/
dark cycle.
Establishment of the spinal cord compression injury
model has been described in detail in our previous
study [18]. In brief, mice were injected intraperitoneallywith pentobarbitone (50 mg/kg). After laminectomy,
the exposed thoracic level 10 (T10) spinal cord was
exposed. A 5-s moderate crush injury was performed
using Dumont forceps No. 5 (Fine Science Tools, 11251–
20, Germany) with a spacer resulting in a tip width of
0.2 mm and a spacing of 0.25 mm upon closure [19]. In
addition, a forcep imprint can be observed after immediate crush injury and is not accompanied by hemorrhagic
erythema of the adjacent spinal cord. Auxiliary urination
nursing was performed twice a day, and a daily subcutaneous injection of Baytril (10 mg/kg) was used for antiinfection for 1 week.
Primary microglial culture

Primary microglia were extracted from the brains ofmice (postnatal 0–3 days old). Briefly, brain tissues weredivided into pieces and digested with 0.25% trypsin (Biological Industries) for 25 min following removal of the

Page 3 of 23

blood arteries and meninges, before adding 3 ml of culture medium to stop digestion. After gently pipetting
the cells up and down ten times using a 1-ml pipette,the cell suspension was filtered with a 40-μm-mesh filterand centrifuged at 1200 rpm for 5 min to obtain mixed
glial cells. For primary microglia, the mixture of cells was
inoculated into poly-D-lysine-precoated T25 flasks andcultured in DMEM/F12 supplemented with fetal bovine
serum (FBS; Thermo-Fisher Scientific), penicillin, andstreptomycin. After 14 days of in vitro culture, the flasks
were shaken at 250 rpm for 6 h to remove mature microglia. The collected microglia were seeded in culture plates
and maintained at 37 °C with 5% ­CO2. For all assays,
microglia were seeded at a density of approximately
1 × ­106/ml for 24 h before each treatment.
Western blotting

Spinal cord tissues were lysed using lysis buffer containing RIPA, protease inhibitor and phosphatase inhibitor. The protein concentration of the lysate was tested
using the BCA assay and was set to the same final concentration. After heat denaturation for 10 min, identical proteins were separated by SDS-PAGE transferredto PVDF membranes, and incubated in blocking buffer(5% non-fat dry milk in TBST) for 2 h. The membranes
were incubated overnight in the following primary antibodies: rabbit anti-Pan Kla (1:1000, PTM-1401, PTM
BIO), rabbit anti-H4K5la (1:1000, PTM-1409, PTM BIO),
rabbit anti-H4K8la (1:1000, PTM-1405, PTM BIO), rabbit anti-H4K12la (1:1000, PTM-1411, PTM BIO), rabbit
anti-H3K18la (1:1000, PTM-1406RM, PTM BIO), rabbit anti-H4 (1:1000, 16047–1-AP, Proteintech), rabbit
anti-β-Tubulin (1:1000, 10094–1-AP, Proteintech), rabbit
anti-PD-1 (1:1000, ab214421, Abcam), and mouse antiGAPDH (1:1000, 60004–1-Ig, Proteintech). After threerounds of washing, the membranes were incubated withthe corresponding secondary antibodies β-Tubulin, H4,
and GAPDH used as loading controls. Immunoreactive
bands were detected using enhanced chemiluminescence(ECL) and quantified using ImageJ software.
Immunofluorescence

For in vivo analysis, 16 μm-thick sagittal spinal cord sections encompassing the lesion core from mice at preoperation (Pre), and 7, 14, and 28 days post-injury (dpi)were applied for immunofluorescence staining followed
procedures described previously [20]. For in vitro analysis, primary microglia cultured on coverslips were fixed
in 4% PFA for 30 min at room temperature. For BrdU
staining, the sections and coverslips were pretreatedwith 2 N hydrochloric acid (HCl, GEMIC, China) for
30 min at 37 °C, followed by 0.1 M boric acid buffer(KGR0101, Key-GEN BioTECH, China) for 15 min atHu et al. Journal of Neuroinflammation(2024) 21:193

room temperature. The sections and coverslips were thensubjected to immunofluorescence staining.
The sections and coverslips were cleaned thrice withphosphate buffered saline (PBS) and incubated withblocking buffer (10% donkey serum containing 0.3%
Triton X-100) for 2 h, before incubating overnight at
4 °C with the following primary antibodies: rabbit antiPan-lysine lactylation (Pan Kla, 1:100, PTM-1401, PTM
BIO), rabbit anti-H4K12la (1:100, PTM-1411, PTM BIO),
goat anti-Iba1 (1:500, NB100-1028, Novus Biologicals),
chicken anti-GFP (1:500, GFP1010, Aves Labs), goat antiRFP (1:200, 200-101-379, Rockland), rabbit anti-PD-1(1:100, ab214421, Abcam), mouse anti-CX3CR1 (1:100,
sc-377227, Santa Cruz), rabbit anti-CX3CR1 (1:100,
Abcam, ab8021), goat anti-PDGFRβ (1:100, AF1042-SP,
R&D Systems) rat anti-GFAP (1:400, 13–0300, Invitrogen), goat anti-5-hydroxytryptamine (5-HT) (1:100,
20079, Immunostar), rat anti-BrdU (1:100, ab6326,
Abcam), and mouse anti-NeuN (1:100, MAB377, Millipore). After washing, sections and cell coverslips wereincubated with secondary antibodies conjugated with
Alexa Fluor 488 and Alexa Fluor 555, or Alexa Fluor
594 (1:500, Invitrogen). The sections and cell coverslips
were stained with 4′, 6-diamidino-2-phenylindole (DAPI;
D9542, Sigma) to label the cell nuclei. Zen software(Zeiss) was used to capture the images.
Pharmacological agents

For the in vivo experiments, mice received daily intraperitoneal injections of the following drugs: Sodium
lactate (lactate, 1 g/kg, L7022, Sigma), with a final concentration selected according to previous reports [16];alpha-cyano-4-hydroxycinnamate (4-CIN, 200 mg/g,
C2020, Sigma) [21]; and sodium oxamate (OX, 300 mg/
kg, O2751, Sigma) [22]. To block PD-1/PD-L signalingin vivo, InVivoPlus anti-mouse PD-1 (CD279) (P0146, Bio
X Cell) was administered intraperitoneally at 5 mg/kg at
1 and 14 dpi, and mice were sacrificed at 28 dpi [23].
For in vitro experiments, microglia were treated withlactate (20 mM), PBS, AAV-sh-PD-1 (1–2 × ­1010 vg/ml),or a combination of both as appropriate for another 24 h,
before collecting microglia for subsequent experiments.
To assess microglial proliferation in vivo or in vitro, themice were intraperitoneally injected with BrdU (50 mg/
kg, Biosharp) daily from 1 to 7 dpi, and the primary
microglia were cultured with BrdU (l0 μg/ml) for 24 h.
Image acquisition and quantitative analysis

Representative images of the sections were acquired
using a Zeiss LSM 900 confocal microscope and a Zeiss
Axio Scope A1 fluorescence microscope. Immunofluorescent images were quantified using ImageJ version 2.0(NIH, United States) by observers who were blinded toPage 4 of 23

the experimental conditions. For each individual analysis, three sagittal sections (spaced 160 μm apart) encompassing the lesion core in each sample were selected,
with three animals per group. To quantify the intensityof Pan ­Kla+, ­H4K12la+, and PD-1+ in microglia ­(Iba1+,­GFP+RFP−, or ­CX3CR1+), three random 40 × images
encompassing the lesion core per section were analyzed, and the average was used as the final data for eachsample. The data were expressed as relative fluorescence intensities between different groups, as previously
described [16].
To identify the spatial expression pattern of H4K12laafter SCI, three sections in each sample were stained for
H4K12la and CX3CR1, PDGFRβ (pericytes), and GFAP
(astrocytes). Three random 40 × images were counted
per section, and the average was used as the final data
for each sample. The data were expressed as a percentageof ­H4K12la+CX3CR1+ cells, ­H4K12la+PDGFRβ+ cells,++or ­H4K12la GFAP cells relative to the total number of­H4K12la+ cells.
For the quantification of the proliferating microglia­(BrdU+ ­CX3CR1+) in vivo, three sections in each sample
were stained with BrdU and CX3CR1. And the numberof ­BrdU+ ­CX3CR1+ cells per ­mm2 was counted on threerandom 40 × images around the lesion core. To quantify
proliferating microglia in vitro, the number of ­BrdU+
cells per ­mm2 was counted on three random 40 × images,
with three coverslips per culture, using the average as the
final data for each culture.
To quantify the microglial scar ­(CX3CR1+) [5].
100 μm-square grids were generated over the 10 × images
encompassing the lesion core. The number of ­CX3CR1+
cells was counted on every six squares, and only ­DAPI+
cells were counted, using the average as the final data foreach sample.
To quantify the number of ­NeuN+ cells, three sagittal sections of each sample were stained with GFAP and
NeuN. The sections were divided into three zones basedon their proximity to the lesion core. These zones werecomposed of Z1, Z2, and Z3 as previously described [24].
The number of ­NeuN+ cells was counted on 10 × images,
with the average used as the final data for each sample.
To quantify the regenerated 5-HT axons, three sagittal
sections of each sample were stained for GFAP and 5-HT.
The immunoreactivity of 5-HT was normalized to thearea of the ­GFAP− region on 10 × images, with average
used as the final data for each sample.
Adeno‑associated virus infection

AAV-CX3C R1-eGFP-5′ miR30-shRNA-3′ miR30WPRE(AAV-sh-PD-1)(Genomeditech,
Shanghai, China) was used to target ­CX3CR1+ cells
and interrupt PD-1 expression in microglia, with

Hu et al. Journal of Neuroinflammation(2024) 21:193

AAV-CX3CR1-eGFP-5′miR30-MCS-3′miR30-WPRE
used as a control vector (AAV-Con). AAV9 vectors
expressing a short hairpin RNA (shRNA) directed at
PD-1 (AAV-sh-PD-1) or a control hairpin (AAV-Con)
were generated to induce local depletion of PD-1 in vivo.
Following SCI, the mice were placed in a stereotaxic
frame, and a 10 μl Hamilton Neuros syringe connected
to a micro-syringe pump controller (neMESYS OEM,
Cetoni, Germany) was inserted to inject the viral vector(5 × ­109 vg; 1 μl) into the spinal cord at a speed of 200 nl/
min into the region of the injured core at a depth of 1 mm
per corner.
Cleavage under targets and tagmentation (CUT&Tag)

The CUT&Tag assay was conducted using the­Hyperactive™ In-Situ Chromatin immunoprecipitation(ChIP) Library Prep Kit for Illumina (TD901-TD902,
Vazyme Biotech, China) according to the manufacturer’s
instructions [25]. Briefly, Concanavalin A-attached magnetic beads were used to bind the Pre and 14 dpi tissue
samples, followed by resuspension in antibody buffer
and incubation with rabbit anti-H4K12la primary antibody at 4 °C overnight with slow rotation. After incubation with goat anti-rabbit IgG H&L (AB206-01-AA) atroom temperature for 1 h, the samples were incubatedwith protein-A-Tn5 (pA-Tn5) transposase before resuspending in tagmentation buffer for 1 h. Following transposon activation and tagmentation, fragmented DNA
was isolated, expanded, and purified to build a library,which was subsequently sequenced on the Illumina
NovaSeq6000 platform to produce 150-bp paired-end
reads for further analysis. DiffBind was employed todetect differential peaks from peak sets. The statisticallysignificant differential binding sites were identified basedon binding affinity, with a threshold of p-value < 0.05, and|log2(Foldchange)|> 1. Benjamini and Hochberg FDR
procedure was used for multiple testing of significance.
The raw sequence data were analyzed by (OE Biotech,
Shanghai China), and the target genes were studied. Furthermore, differential peaks in promoter regions werescreened using Gene Ontology (GO) enrichment andKyoto Encyclopedia of Genes and Genomes (KEGG)
pathway enrichment analyses based on the hypergeometric distribution.
The ChIP assay was performed using a ChIP-IT® Express
Enzymatic Magnetic Chromatin Immunoprecipitation
Kit & Enzymatic Shearing Kit (Active Motif ), as previously described [26]. Briefly, 1% formaldehyde solution
was used to crosslink isolated Pre and 14 dpi tissues orcultured primary microglia for 30 min at 37 °C, followedby the addition of glycine for 10 min. Tissues or cells
ChIP‑qPCR

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were cleaned three times with PBS and centrifuged at2000 rpm for 10 min, followed by lysis, sonication, andimmunoprecipitation in accordance with the manufacturer’s guidelines. Diluted chromatin was immunoprecipitated with H4K12la or PD-1 antibody, while normal
rabbit IgG was used as a nonspecific control. The mixture was continuously rotated while incubating at 4 °C
overnight. Subsequently, 50 μl protein A/G magnetic
beads were added and incubated at 4 °C for 2 h. Wash
buffer was used to clean the beads, and crosslinking wasreversed by incubating them for 12 h at 50 °C in 1% SDS
and 0.1 M N­ aHCO3, as provided in the kit. DNA fragments were then purified using a QIAquick PCR purification Kit (QIAGEN). DNA collected via ChIP was
detected via qPCR. The primer sequences for PD-1 in
ChIP were as follows: forward primer (TGC​ATG​TGT​
GTT​GTG​GGA​TA); reverse primer (TGC​ATG​TGT​GTT​
GTG​
GGA​
TA). The primer sequences for GAPDH in
ChIP were as follows: forward primer (AGC​CTC​CTC​
CAA​TTC​AAC​CCTT); reverse primer (TGC​ATG​TGT​
GTT​GTGG GATA).
qPCR

Total RNA was extracted from tissues or cells using TRIzol Reagent (Invitrogen) according to the manufacturer’s
protocol. Then, cDNA was synthesized using a one-step
first-strand cDNA synthesis kit (Transgen). Gene expression was detected using SYBR Green-based qPCR. The
primer sequences for PD-1 in qPCR were as follows:forward primer (TGC​CCT​AGT​GGG​TATC CCTG);
reverse primer (GAA​GGC​TCC​TCC​TTC​AGA​GTGT).
The primer sequences for GAPDH in qPCR were as follows: forward primer (AAG​AGG​GAT​GCT​GCC​CTT​
ACC); reverse primer (CCC​AAT​ACG​GCC​AAA​TCC​
GT). Finally, the mRNA expression were standardized tothose of GAPDH.
Detection of lactate levels

The lactate levels in spinal cord tissues were detected aspreviously described [27]. Briefly, spinal cord tissues weresonicated and homogenized using lysis buffer at 500 W(4 s on and 8 s off ) for 4 min, followed by centrifugation at 15,000 rpm for 15 min to collect the supernatant.
According to the manufacturer’s guidelines, the tissue
lactate levels were tested using the ab65330 Lactate Assay
Kit (Colorimetric/Fluorometric), with the lactate concentration shown in mM.
Basso mouse scale (BMS) score assessment

According to the protocol developed by Basso and colleagues, each mouse received a BMS score at 0, 3, 7, 14
and 28 dpi to evaluate the recovery of locomotor function [28]. Three experienced evaluators scored the mice

Hu et al. Journal of Neuroinflammation(2024) 21:193

in a double-blind manner. Utilizing the criteria for scoring (mobility of the posterior ankle joint, coordination, paw posture, trunk stability, and tail posture), thescore ranged from 0 to 9, with a higher score indicating
improved hindlimb function.
Footprint analysis

Footprint analysis was used to assess motor function
recovery at 28 dpi. The mice walked along a paper-paved
track, as previously mentioned [29]. The front paws andhindpaws are colored green, and red respectively. Qualitative analysis considered the stride length, width, andpaw rotation.
Quantification and statistical analysis

All experimental data were expressed as themean ± standard error of the mean (SEM) unless statedotherwise. Data analysis was performed using GraphPad
Prism software (version 8.0). For multiple comparisons,one- or two-way analysis of variance (ANOVA) was performed using Tukey’s post hoc test. Student’s t-test wasused for pairwise comparisons between the two groups.
A p-value ≤ 0.05 was considered statistically significant
and expressed as *p < 0.05, **p < 0.01, ***p < 0.001, and****p < 0.0001.

Results
Lactate and lactylation levels elevate after SCI

After SCI, the hypoxic microenvironment regulates lactate levels at the injury site [12]. We detected changes inlactate levels in the injured spinal cord. The colorimetricassay revealed a significant increase in lactate levels at thelesion site from 7 to 28 dpi (Fig. 1A). Because lactate, asa precursor factor, may cause histone lactylation [30], we
detected changes of in lactylation after SCI using western
blotting. The results showed an increase in Pan Kla levels at 7, 14, and 28 dpi (Fig. 1B). It has been shown thatmicroglia play a key role in the regulation of SCI pathobiology and recovery. Given that microglia are metabolically flexible and can be controlled by lactylation [31], we
hypothesized that lactylation may be altered in microglia after SCI. Compared to Pre levels, the Pan Kla signal
intensity was considerably increased in microglia from 7
to 28 dpi (Fig. 1C, D). These results revealed that overall
lactate level was elevated at the lesion site, while Pan Kla
was upregulated in the entire spinal cord and microglia
after SCI.
H4K12la levels are elevated in microglia after SCI

Previous studies have highlighted the importance ofsite-specific histone lactylation in regulating macrophage polarization, tumor immunity, and antiviral
responses [30, 32–34]. To investigate site-specific histone

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lactylation after SCI, we performed western blotting
using antibodies against distinct types of histone lactylation. The results revealed an increase of H3K18la andH4K12la levels in the injured spinal cord (Fig. 2A–J).
Importantly, the H4K12la level increased early at 7 dpi.
To study the spatial expression pattern of H4K12la after
SCI, we used co-immunostaining with H4K12la andspinal cord cell markers including CX3CR1 (microglia),
PDGFRβ (pericyte), and GFAP (astrocyte) at 7 dpi. The
results indicated that H4K12la was highly expressed in­CX3CR1+ microglia and slightly expressed in G
­ FAP++
astrocytes or PDGFRβ pericytes around the lesionsite. H4K12la protein exhibited nuclear distribution in­CX3CR1+ microglia at 7 dpi (Fig. 2K–N). Pearson’s correlation coefficient was used to validate the co-localization (Fig. S1). Microglia can undergo rapid proliferation
with a peak at 7 dpi, before forming a protective microglial scar at 14 dpi [5]. Interestingly, H4K12la expression
was notably elevated at 7 and 14 dpi, and was temporally
correlated with microglial proliferation and scar formation. To verify whether H4K12la modification was alteredin microglia after SCI, we performed immunofluorescence co-staining of microglial markers (CX3CR1 andIba1) and H4K12la. Our results suggested that H4K12lasignal intensity was significantly increased in microglia
after SCI (Fig. 2O–R). Consistently, using CCR2RFPCX3CR1GFP dual-reporter mice, in which macrophages werelabeled with RFP and microglia were labeled with GFP,
we found that the intensity of H4K12la was significantly
increased in microglia after SCI (Fig. S2). These results
demonstrate the upregulation of H4K12la in microglia
after SCI.
Exogenous lactate treatment upregulates H4K12la levels
in microglia after SCI

It has been reported that exogenous lactate stimulates histone lactylation in cells [30]. Hence, we next
investigated whether exogenous lactate could elevate
H4K12la levels in microglia after SCI. The injured mice
were treated with lactate, 4-CIN (a monocarboxylatetransporter 2 inhibitor that can obstruct lactate transport into cells), and OX (an inhibitor of lactate dehydrogenase that can inhibit lactate production) after SCI(Fig. 3A). We found that lactate treatment increased
lactate levels in spinal cord, whereas 4-CIN treatment
showed no significant changes in lactate levels, while
OX treatment reduced lactate levels at 14 and 28 dpi(Fig. 3B). Western blotting analysis indicated that the
H4K12la levels were significantly increased after lactate treatment at 14 and 28 dpi (Fig. 3C, D), whereas
there were insignificant changes in H4K12la levels after
4-CIN treatment (Fig. 3E, F). Notably, OX treatment
reduced H4K12la levels at 14 and 28 dpi (Fig. 3G, H).

Hu et al. Journal of Neuroinflammation(2024) 21:193

Page 7 of 23

Fig. 1 Changes in lactate and lactylation modification levels after SCI in vivo. A Lactate levels of the spinal cord at Pre, and 3, 7, 14, 28 dpi in vivo.
n = 3 mice per group, *p < 0.05; ****p < 0.0001; N.S., not significant. B Western blot analysis of the Pan histone lactylation levels in the spinal cord
at Pre, and 3, 7, 14, and 28 dpi. C Representative images of Pan Kla (green) co-stained with microglia (Iba1, magenta) at Pre, and 7, 14, and 28 dpi
in vivo. White arrowheads indicate the colocalization of Pan Kla and microglia. Asterisks indicate the lesion site. Scale bars: low magnification,
100 μm; high magnification, 20 μm. D Quantification of Pan Kla intensity in ­Iba1+ microglia in (C). All data were presented as the mean ± SEM,n > 100 cells per group, ****p < 0.0001

Furthermore, immunofluorescence assays confirmedthat the H4K12la signal intensity in microglia was significantly increased after lactate treatment, whereas
an insignificant difference was observed after 4-CIN
treatment. OX treatment suppressed H4K12la signal

intensity at 14 and 28 dpi (Fig. 3I, J). Collectively, theseresults demonstrated that exogenous lactate treatment
increased the overall lactate level in the spinal cord andelevated H4K12la levels in the spinal cord and microglia after SCI.

Hu et al. Journal of Neuroinflammation(2024) 21:193

Lactate treatment promotes microglial proliferationand scar formation after SCI

Microglia proliferate rapidly at 7 dpi, which contributes to microglial scar formation after SCI [5]. Considering the upregulation of H4K12la levels in microglia
after SCI and the temporal and spatial correlation of thischange with microglial proliferation and scar formation, we further investigated the effect of exogenous lactate on microglial proliferation and scarring. Our results
showed that the number of B
­ rdU+CX3CR1+ cells wasconsiderably augmented following lactate treatment,
did not change significantly following 4-CIN treatment,
and reduced following OX treatment at 7 dpi (Fig. 4A,
B). Immunofluorescence analysis revealed an increasein the C­ X3CR1+ microglial scar after lactate treatment,a non-significant difference after 4-CIN treatment, anda decrease after OX treatment at 28 dpi (Fig. 4C, D).
These results confirmed that lactate treatment promoted
microglial proliferation, thereby contributing to microglial scar formation after SCI.
Lactate treatment promotes axon regenerationand locomotor function recovery after SCI

Previous studies have demonstrated that fibrotic scars
hinder axon regeneration, astrocytic scars may either
hinder or aid axon regeneration depending on their
physical orientation, and microglial scars promote axon
regeneration after SCI [3, 4, 7, 35, 36]. In addition, proliferated microglia prevent the diffusion of inflammatory
cells and maintain residual viable neurons away from thelesion site [5, 9, 37]. After confirming that lactate treatment facilitated microglial scarring following SCI, we
focused on the effects of lactate on residual neurons. We
used the neuron marker NeuN to detect residual neurons
in specific regions (Z1_Z3) of viable neurons located in
the epicenter and edges after SCI [24]. The results illustrated that lactate treatment significantly increased
the number of residual N­ euN+ neurons in the Z1_Z3

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regions, whereas 4-CIN treatment slightly reduced thecounts of residual neurons. In contrast, OX treatment
significantly reduced the counts of residual neurons
in the Z1_Z3 regions at 28 dpi (Fig. 5A, B). We thendetected descending serotonergic (5-HT) axon regeneration via immunofluorescence at 28 dpi. Lactate treatment
obviously increased the number of 5-HT axons penetrating into the lesion site compared to 4-CIN and OX treatment (Fig. 5C, D).
To examine whether exogenous lactate-mediated axon
regeneration promoted locomotor function recovery, we
conducted behavioral tests following SCI. In a chronic
experiment, mice were administered daily intraperitoneal lactate, 4-CIN, and OX injections for 28 consecutive
days after SCI (Fig. 5E). Lactate treatment significantly
improved locomotor function recovery, whereas 4-CIN
treatment had no visible effect. Conversely, OX treatment
hindered functional recovery as indicated by the BMS
scores for behavioral analysis from 0 to 28 dpi (Fig. 5F).
The footprint analysis yielded similar outcomes (Fig. 5G–
J). According to the aforementioned results, lactate treatment could promote axon regeneration and locomotor
function recovery after SCI.
H4K12la modifications activate PD‑1 transcription
in microglia

It has been demonstrated that histone lactylation modification contributes to the transcriptional regulation ofdownstream genes [30]. To identify the H4K12la-target
gene after SCI, we used genome-wide CUT&Tag analysis to identify candidate genes regulated by H4K12la.
CUT&Tag analysis utilizing anti-H4K12la antibodies showed that H4K12la peaks were enriched at 14 dpi(Fig. 6A). H4K12la was enriched in promoter domains,
and the Pre sample showed 14,463 H4K12la binding
peaks, with 60.51% of the peaks situated within promoter
sequences (≤ 3 Kb). At 14 dpi, 16043 H4K12la binding peaks were detected, with 50.32% situated within(See figure on next page.)
Fig. 2 The changes of site-specific histones lactylation after SCI in vivo. A–J Western blotting analysis of site-specific histones lactylation in spinal
cord in vivo at Pre, and 3, 7, 14, and 28 dpi with quantification of protein levels. All data were presented as the mean ± SEM, n = 3 mice per group,*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; N.S., not significant. K–M Representative immunofluorescence images for expression of H4K12la(green) and CX3CR1 (microglia, magenta) (K), H4K12la and PDGFRβ (pericyte, magenta) (L), or H4K12la and GFAP (astrocyte, magenta) (M) in injured
spinal cords at 7 dpi in vivo. White arrowheads indicated the colocalization of H4K12la and CX3CR1. Asterisks indicate the lesion site, scale bars:
low magnification, 100 μm; higher magnification, 20 μm. N Quantitative analysis of the percentage of ­H4K12la+CX3CR1+, ­H4K12la+PDGFRβ+and ­H4K12la+GFAP+ cells in total ­H4K12la+ cells in injured spinal cords. All data are mean ± SEM, n = 3 mice per group, ****p < 0.0001­(H4K12la+PDGFRβ+ or ­H4K12la+GFAP+ vs. H­ 4K12la+ ­CX3CR1+). O Representative images of H4K12la (green) co-stained with microglia (CX3CR1,
magenta) at Pre, and 7, 14, and 28 dpi in vivo. White arrowheads indicated the colocalization of H4K12la and CX3CR1. Asterisks indicate the lesion
site, scale bars: low magnification, 100 μm; high magnification, 20 μm. P Quantification of H4K12la intensity in (O). All data were presented
as the mean ± SEM, n > 100 cells per group, ****p < 0.0001. Q Representative images of H4K12la (green) co-stained with microglia (Iba1, magenta)
at Pre, and 7, 14, and 28 dpi in vivo. White arrowheads indicated the colocalization of H4K12la and Iba1. Asterisks indicate the lesion site, scale bars:
low magnification, 100 μm; high magnification, 20 μm. R Quantification of H4K12la intensity in (Q). All data were presented as the mean ± SEM,n > 100 cells per group, ****p < 0.0001

Hu et al. Journal of Neuroinflammation(2024) 21:193

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Fig. 2 (See legend on previous page.)the promoter sequences (Fig. 6B). To further identify
potential downstream targets of H4K12la, we screened
37 genes with upregulated H4K12la expression in the
promoter region for 37 genes at 14 dpi. Among these
upregulated genes, PD-1 was upregulated after SCI
and predominantly expressed in ­Iba1+ cells [38]. PD-1
has also been shown to promote microglial proliferation in the tumor microenvironment [39]. Importantly,

a previous study also preliminarily confirmed the prorepair effect of PD-1 on SCI [40, 41]. These findings suggested that lactate-mediated lactylation partly exerts abeneficial effect by regulating PD-1 gene transcription in­Iba1+ microglia after SCI. Further analysis showed thatthe H4K12la level at the PD-1 promoter region was significantly elevated in the spinal cord at 14 dpi (Fig. 6C).
To further confirm that PD-1 transcription was activatedHu et al. Journal of Neuroinflammation(2024) 21:193

by H4K12la, we performed ChIP analysis using antibodies against H4K12la and found that the H4K12la levelon the PD-1 promoter was significantly elevated in the
spinal cord at 14 dpi (Fig. 6D). Consistently, qPCR analysis showed that PD-1 expression was elevated at 14
dpi (Fig. 6E). Taken together, these results indicate that
H4K12la modifications in promoter areas activate PD-1
transcription following SCI.
Lactate promotes H4K12la expression in microglia,
and H4K12la activates the transcription of PD-1 after
SCI. We further explored the impact of H4K12la on thetranscription of PD-1 in microglia. The results of ChIP
analysis illustrated that the H4K12la level on the PD-1
promoter was increased in microglia treated with lactate (Fig. 6F). Consistent with H4K12la-mediated transcriptional activation in the SCI model, qPCR revealed
that the expression of PD-1 was significantly elevatedin microglia treated with lactate (Fig. 6G). Collectively,
these results demonstrated that H4K12la modification
activated PD-1 transcription in microglia. Additional
analyses of genes with differential promoter lactylation
are presented in Fig. S3.
Intraperitoneal injection of a PD‑1 inhibitor suppresses
microglial proliferation and scar formation, hinders axon
regeneration, and prevents locomotor function recovery
after SCI

Our results showed that lactate promoted H4K12la
microglial proliferation and scar formation after SCI.
Considering PD-1 activation by H4K12la, we next
explored the influence of PD-1 on microglial proliferationand scar formation after SCI. The western blotting results
showed that PD-1 expression was elevated at 7, 14 and 28
dpi (Additional file 1: Fig. S4A, B), while the immunofluorescence assays revealed that the fluorescence intensityof PD-1 was increased in microglia after SCI (Additional
file 1: Fig. S4C–F). These results are consistent with previous findings [40]. We then explored the effect of PD-1
on microglial proliferation and scar formation after SCI

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by inhibiting PD-1/PD-L signaling with a PD-1 inhibitor [23]. Mice were intraperitoneally injected with PD-1
inhibitor at 1 and 14 dpi and sacrificed at 28 dpi (Fig. 7A).
The effect of PD-1 inhibitors on microglial proliferation
was assessed at 7 dpi, when the peak microglial proliferation was observed [5]. CX3CR1 cells were co-stainedwith BrdU to identify microglial proliferation. The results
showed that the number of B
­ rdU+CX3CR1+ cells was
lower in PD-1 inhibitor group than in control group(Fig. 7B, C). Moreover, there was a significant decrease in
microglial scar formation in PD-1 inhibitor group at 28
dpi (Fig. 7D, E).
To investigate the influence of PD-1 inhibition on
residual neurons, we next used NeuN immunostaining
to assess neuronal survival in particular zones (Z1_Z3)
surrounding the lesion core at 28 dpi. The number ofresidual ­NeuN+ neurons in the Z1_Z3 region was considerably lower in the PD-1 inhibitor group than in thecontrol group (Fig. 7F, G). We then detected descending
5-HT axon regeneration at 28 dpi. In the lesion site, significantly fewer 5-HT axons penetrating intothe lesion
site in the PD-1 inhibitor group than in the control group(Fig. 7H, I). Next, BMS score and footprint analyses were
performed to identify locomotor function recovery following SCI. The mice injected with the PD-1 inhibitor exhibited worse hindlimb locomotor function thanthose in the control group, corresponding to the resultsof BMS score (Fig. 7J). Accordingly, footprint analysis
showed that mice in the PD-1 inhibitor group had worse
locomotor function than those in the control group after
SCI (Fig. 7K–N). These results confirmed positive roleof PD-1 in microglial proliferation, scar formation, axon
regeneration, and locomotor functional recovery after
SCI.
PD‑1 inhibitor reverses the advantageous effects of lactate
on SCI repair

To further verify whether PD-1 participates in the lactate/H4K12la signaling pathway after SCI, rescue assays(See figure on next page.)
Fig. 3 Exogenous lactate treatment upregulates H4K12la level in microglia after SCI in vivo. A Schedule described that mice were injected
intraperitoneally (i. p.) with lactate, 4-CIN, or OX daily until 28 dpi. B Lactate levels of the spinal cord at 14 and 28 dpi in PBS (control), lactate, 4-CIN
and OX groups in vivo. All data were presented as the mean ± SEM, n = 3 mice per group, ****p < 0.0001; N.S., not significant. C Western blotting
analysis detected H4K12la expression levels of the spinal cord at 14 and 28 dpi in lactate group in vivo. D Quantification of H4K12la expression levelsin (C). All data were presented as the mean ± SEM, n = 3 mice per group, **p < 0.01; ***p < 0.001. E Western blotting analysis detected the H4K12laexpression levels of the spinal cord at 14 and 28 dpi in 4-CIN group in vivo. F Quantification of H4K12la expression levels in (E). All data were
presented as the mean ± SEM, n = 3 mice per group, N.S. meant control compared with 4-CIN, not significant. G Western blotting analysis detected
H4K12la expression levels of the spinal cord at 14 and 28 dpi in OX group in vivo. H Quantification of H4K12la expression levels in (G). All data were
presented as the mean ± SEM, n = 3 mice per group, *p < 0.05; ***p < 0.001. I Representative images of H4K12la (green) co-stained with microglia(Iba1, magenta) at 14 and 28 dpi in lactate, 4-CIN, and OX groups in vivo. White arrowheads indicated the colocalization of H4K12la and microglia.
Asterisks indicate the lesion site, scale bars: low magnification, 100 μm; high magnification, 20 μm. J Quantification of H4K12la intensity in (I). Alldata were presented as the mean ± SEM, n > 100 cells per group, ****p < 0.0001; N.S., not significant

Hu et al. Journal of Neuroinflammation(2024) 21:193

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Fig. 3 (See legend on previous page.)

were performed. We first detected microglial proliferation with BrdU via immunofluorescence staining. The
results showed that lactate-mediated microglial proliferation was dramatically reversed by PD-1 inhibitor
at 7 dpi (Fig. 8A, B). The PD-1 inhibitor abolished thepositive effect of lactate on microglial scar formation

at 28 dpi (Fig. 8C, D). The number of residual N­ euN+
neurons in the Z1_Z3 region of the spinal cord wasincreased in the lactate group, whereas the PD-1 inhibitor reversed the lactate-mediated increase in residual
neurons (Fig. 8E, F). Additionally, the PD-1 inhibitor
reversed the lactate-induced increase in 5-HT axons

Hu et al. Journal of Neuroinflammation(2024) 21:193

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Fig. 4 Lactate treatment promotes microglia proliferation and scar formation after SCI in vivo. A Representative immunofluorescence imagesof microglia proliferation by double-staining BrdU (magenta) and CX3CR1 (green) in control, lactate, 4-CIN, and OX groups at 7 dpi in vivo. Scale bar:
20 μm. B Quantification of the density of ­BrdU+CX3CR1+ cells in (A). All data were presented as the mean ± SEM, n = 3 mice per group, ****p < 0.0001;
N.S., not significant. C Immunofluorescence staining of CX3CR1 (red) in control, lactate, 4-CIN, and OX groups at 28 dpi in vivo. Asterisks indicate
the lesion site, scale bar: 100 μm. D Quantification of the density of ­CX3CR1+ cells in (C). All data were presented as the mean ± SEM, n = 3 mice
per group, ***p < 0.001; N.S., not significant

penetrating into the lesion site (Fig. 8G, H). Behavioral analysis experiments were conducted to observe
improvement in locomotor function after SCI. Lactate
treatment promoted locomotor function recovery, andPD-1 inhibition reversed lactate-mediated locomotor function recovery as shown in the BMS score andfootprint analysis after SCI (Fig. 8I–M). These results
confirmed that PD-1 inhibitors reversed the promotionof lactate on microglial proliferation, microglial scarformation, axon regeneration, and locomotor function
recovery, supporting the role of PD-1 as a downstream
regulator of lactate/H4K12la signaling after SCI.

PD‑1 suppression in microglia hinders axon regeneration,and inhibits functional recovery after SCI

To investigate the effects of microglial PD-1 inhibition
on axonal regeneration and function recovery after SCI,
we injected microglial-specific AAV-sh-PD-1 into theinjured spinal cord immediately after SCI [42]. Western blotting revealed that the expression of PD-1 in the
AAV-sh-PD-1 group was significantly lower than that in
the AAV-Con group at 14 and 28 dpi (Additional file 1:
Fig. S5A, B). Immunofluorescence staining was also performed to measure the intensity of PD-1 in microglia inmice administered AAV-sh-PD-1. The results illustrated

Hu et al. Journal of Neuroinflammation(2024) 21:193

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Fig. 5 Lactate treatment promotes axon regeneration and locomotor function recovery after SCI in vivo. A Images of astrocytes (GFAP, green)stained with neurons (NeuN, magenta) in Zone1-Zone3 (Z1_Z3) at 28 dpi in control, lactate, 4-CIN, and OX groups in vivo. Z1 (0–250 μm) was in thelesion site, Z2 (250–500 μm) was started immediately adjacent to Z1, and Z3 (500–750 μm) was adjacent to Z2. Scale bars: magnification, 100 μm.
Compass: D, dorsal; V, ventral; R, rostral; C, caudal. B Quantification of ­NeuN+ neurons in three zones (Z1_Z3) in control, lactate, 4-CIN, and OX
groups. All data were presented as the mean ± SEM, n = 3 mice per group, *p < 0.05; **p < 0.01. C Images of GFAP (green) co-stained with axons (5-HT,
magenta) at 28 dpi in control, lactate, 4-CIN, and OX groups in vivo, and the boxed region indicated the lesion center magnified in right panels.
Asterisks indicate the lesion site, scale bars: low magnification, 100 μm; high magnification, 20 μm. C Quantification of the percentage of 5-HT+
area in the area of the spinal cord segment penetrating into the lesion core at 28 dpi in vivo. All data are presented as the mean ± SEM, n = 3 mice
per group, *p < 0.05; ***p < 0.001; N.S., not significant. D Schedule of locomotor function analysis and the intraperitoneal injection with lactate, 4-CIN
or OX daily until 28 dpi. E Locomotor function was evaluated by BMS at the indicated time points. All data are presented as the mean ± SEM, n = 8
mice per group, *p < 0.05; ***p < 0.001; ****p < 0.0001. F Representative images of footprint analysis in control, lactate, 4-CIN, and OX groups at 28
dpi. H–J Quantification of the stride length and width and paw rotation at 28 dpi. All data are presented as the mean ± SEM, n = 8 mice per group;*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; N.S., not significant

that the intensity of PD-1 in microglia in mice injectedwith AAV-sh-PD-1 was significantly lower than that inmice injected with AAV-Con at 28 dpi (Additional file 1:
Fig. S5C–F). Importantly, we observed reduced penetration of 5-HT axons into the lesion site in mice injectedwith AAV-sh-PD-1 compared to those injected with
AAV-Con at 28 dpi (Fig. 9A, B). To investigate the influence of microglial PD-1 suppression on viable neurons,
we stained the neuronal marker NeuN in specific regions(Z1_Z3) around the lesion core. The results revealed thatmice injected with AAV-sh-PD-1 substantially reduced
viable neurons compared to those injected with AAVCon at 28 dpi (Fig. 9C, D). Footprint analysis and BMS

scoring showed that the mice injected with AAV-shPD-1 exhibited reduced locomotor function recovery(Fig. 9E–I). These results indicate that interruption of thelactate/H4K12la/PD-1 signaling pathway via suppressionof PD-1 in microglia hinders axon regeneration and functional recovery after SCI.
Suppression of PD‑1 expression in microglia reverses
the advantageous effects of lactate on SCI repair

To further explore the effect of microglial PD-1 suppression on lactate/H4K12la signaling, a series of rescue
experiments were designed. We performed immunofluorescence staining with BrdU to detect proliferation,

Hu et al. Journal of Neuroinflammation(2024) 21:193

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Fig. 6 H4K12la activates PD-1 transcription in the injured spinal cord in vivo and microglia in vitro. A The binding density of H4K12la was visualized
by deep Tools: the heatmap presents the CUT&Tag tag counted on the H4K12la binding peaks in the spinal cord at Pre and 14 dpi in vivo, orderedby signal strength. B Genome-wide distribution of H4K12la-binding peaks in the spinal cord at Pre and 14 dpi in vivo. C Genome browser tracksof CUT&Tag signal at the PD-1 loci. D ChIP analysis of the indicated promoters was performed using antibodies against H4K12la in the spinal
cord at Pre and 14 dpi in vivo. All data are presented as the mean ± SEM, n = 3 mice per group, ****p < 0.0001. E qPCR assays monitoring PD-1
expression in the spinal cord at Pre and 14 dpi in vivo. All data are presented as the mean ± SEM, n = 3 mice per group, **p < 0.01. F ChIP analysisof the indicated promoters was performed using antibodies against H4K12la in primary microglia treated with PBS (control) and lactate in vitro. Alldata are presented as the mean ± SEM, n = 3 per group, ***p < 0.001. G qPCR assays monitoring expression of the PD-1 in primary microglia treatedwith PBS (control) and lactate in vitro. All data are presented as the mean ± SEM, n = 3 per group, **p < 0.01

and the results showed that microglial proliferation wasincreased in mice injected with lactate, whereas AAVsh-PD-1 obviously reversed the promotion of microglial
proliferation induced by lactate at 7 dpi (Fig. 10A, B).

We next assessed whether AAV-sh-PD-1 could abolishthe effect of lactate on microglial proliferation in vitro.
The lactate-mediated primary microglial proliferation
was reversed by AAV-sh-PD-1 infection (Fig. 10C, D).

Hu et al. Journal of Neuroinflammation(2024) 21:193

Additionally, the microglial scarring in mice injectedwith lactate was significantly reversed by AAV-sh-PD-1
administration at 28 dpi (Fig. 10E, F). The results ofimmunofluorescence staining showed that the lactatemediated increase in residual neurons was reversed by
AAV-sh-PD-1 administration at 28 dpi (Fig. 10G, H).
AAV-sh-PD-1 administration reversed the lactate-mediated increase in 5-HT axon at 28 dpi (Fig. 10I, J). The
results of footprint analysis and BMS scoring showedthat AAV-sh-PD-1 administration significantly reversed
lactate-mediated locomotor functional recovery after SCI(Fig. 10K–O). Taken together, these results demonstrated
that AAV-sh-PD-1 reversed the beneficial effects of lactate on microglial proliferation, scar formation, axon
regeneration, and locomotor function recovery after SCI,
supporting PD-1 as a target of lactate/H4K12la signalingin microglia after SCI.

Discussion
Histone lactylation has been widely reported to be closely
associated with disease onset and development, such as
AD, lung fibrosis, and post-myocardial infarction [16, 33,
42, 43]. SCI generates a hypoxic microenvironment in
the injured area, resulting in active glycolysis and thegeneration of substantial amounts of lactate as a substrate for histone lactylation [44, 45]. We therefore proposed that histone lactylation was aberrant in the injured
spinal cord and focused on the role and mechanisms ofhistone lactylation after SCI. In the present study, we
discovered an elevation in the overall lactate level in the
spinal cord together with an increase in globle lactylation in both the spinal cord and microglia after SCI. Our
results demonstrated that H4K12la levels were significantly upregulated in microglia after SCI. Lactate treatment increased overall lactate level in the spinal cord andupregulated H4K12la expression in both the spinal cord
and microglia. Functionally, lactate promoted microglial

Page 15 of 23

proliferation, scar formation, axon regeneration, andlocomotor function recovery after SCI. Mechanically,lactate-mediated H4K12la promoted transcription of
PD-1 in microglia, which was further demonstrated toinduce microglial proliferation and thereby contribute to
microglial scar formation and functional recovery after
SCI. Our study illustrates the function and mechanismof the lactate/H4K12la/PD-1 signaling pathway in microglia-mediated SCI repair and identifies a novel target for
SCI treatment.
Scars play complex roles in injury repair and axon
regeneration after SCI. Fibrotic scars serve as barriers
that inhibit axon regeneration and can result in permanent functional deficits [40]. Astrocytic scars have beena much-debated topic for SCI and repair. Traditionally, astrocytic scars were regarded as obstacles to axon
regeneration [46], yet they can also act as bridges topromote axon regeneration. Indeed, regenerating 5-HTor corticospinal axons often closely connect with astrocyte processes when crossing the lesion core after SCI[47–50]. A previous study reported that microglia proliferate and gather in large numbers around the lesion
site, forming a protective microglial scar between the
fibrotic and astrocytic scars at the lesion edge [5]. The
microglia depletion results in enhanced immune cell
infiltration, decreased neuronal survival, and impaired
functional recovery after SCI [5, 37]. In contrast, local
application of colony-stimulating factor at the lesion site
to promote microglial proliferation also facilitated axon
regeneration and functional recovery after SCI [51, 52].
Consistent with these findings, our results showed that
lactate-mediated lactylation promoted microglial proliferation and accumulation leading to microglial scarring,
thereby facilitating axon regeneration and functional
recovery following SCI. However, it remains unclear
whether this injury repair was a direct effect of lactate oran indirect effect via microglia. Additionally, each type of(See figure on next page.)
Fig. 7 Intraperitoneal injection of PD-1 inhibitor suppresses microglial proliferation and scar formation, hinders axon regeneration, and prevents
locomotor function recovery after SCI in vivo. A The schedule described that the mice were treated with PD-1 inhibitor at 1 and 14 dpi
via intraperitoneal (i. p.) injection and were sacrificed at 28 dpi. B Immunofluorescence staining of BrdU (magenta), CX3CR1 (green) in sagittal
sections of the PBS (control) and PD-1 inhibitor groups at 7 dpi in vivo. Scale bar: 20 μm. C Quantification of the density of ­BrdU+CX3CR1+ cellsin (B). All data are presented as the mean ± SEM, n = 3 mice per group, **p < 0.01. D Immunofluorescence staining of CX3CR1 (red) in control
and PD-1 inhibitor groups at 28 dpi in vivo. Scale bar: 100 μm. E Quantification of the density of ­CX3CR1+ cells in (D). All data are presented
as the mean ± SEM, n = 3 mice per group, **p < 0.01. F Images of astrocytes ­(GFAP+, green) stained with neurons ­(NeuN+, magenta) in Zone1-Zone3(Z1_Z3) at 28 dpi in PD-1 inhibitor and control groups in vivo. Scale bar: 100 μm. G Quantification of ­NeuN+ neurons in three zones (Z1_Z3) in (F).
All data are presented as the mean ± SEM, n = 3 mice per group, **p < 0.01. H Images of GFAP (green) co-stained with axons (5-HT, magenta) at 28
dpi in PD-1 inhibitor and control groups in vivo. Asterisks indicate the lesion site, scale bars: low magnification, 100 μm; high magnification, 20 μm.
I Quantification of the percentage of 5-HT+ area in the area of the spinal cord segment penetrating into the lesion core at 28 dpi in (H). All data arepresented as the mean ± SEM, n = 3 mice per group, **p < 0.01. J Locomotor function was evaluated by BMS at the indicated time points in PD-1
inhibitor and control groups. All data are presented as the mean ± SEM, n = 8 mice per group, *p < 0.05; ***p < 0.001; ****p < 0.0001. K Representative
images of footprint analysis in PD-1 inhibitor and control groups at 28 dpi. L, N Quantification of the paw rotation, stride width and stride length
at 28 dpi. All data are presented as the mean ± SEM, n = 8 mice per group, **p < 0.01; ****p < 0.0001

Hu et al. Journal of Neuroinflammation(2024) 21:193

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Fig. 7 (See legend on previous page.)fibrotic, astrocytic, and microglial scar cell interacts witheach other, contributing to permanently remodeled tissue[3]. Particularly, microglia are considered the vital cell

type orchestrating intercellular crosstalk and have been
demonstrated to regulate border formation of astrocytic
scars, potentially by controlling astrocyte proliferation,

Hu et al. Journal of Neuroinflammation(2024) 21:193

adhesion, and gliosis [51]. Microglial depletion disrupts
the density and contiguous boundary of fibrotic andastrocytic scars, leading to widespread inflammation in
the lesion core [5]. Moreover, microglia can contributeto the absence of fibrotic and astrocytic scars, thereby
coordinating scar-free healing after SCI in neonates [53].
In this context, lactate-induced regulation of microglial
scar formation may be involved in the interconnection
among different scar types. To clarify changes in lactate
expression in microglia, the widely reported markers
Iba1 and CX3CR1, as well as CCR2RFPCX3CR1GFP dualreporter mice, were used to distinguish microglia frommacrophages. The differences in the series of cellularfunctional changes induced by variations in lactylation
between microglia and macrophages need to be elucidated using transgenic methods in subsequent studies.
Nevertheless, our results supported the positive role oflactate in SCI repair.
Growing evidence suggests that lactate is a neuroprotective energy substrate with a protective effect against
excitotoxicity [54, 55]. Exogenous lactate injection also
improved neurological function in CNS injury [56–58].
In the CNS, microglia are crucial early responders toinjury, constantly monitor the microenvironment forchanges resulting from injury or disease, and are essential
in tissue remodeling [59, 60]. Lactate-induced regulationof microglial clearance ability may expedite the elimination of cellular debris and enhance tissue repair while
reducing inflammation in the ischemic area [13]. Lactate
treatment may reduce neuroinflammation by altering
the polarization of microglia [61]. Our results suggested
that lactate promoted microglial proliferation after SCI;
however, we did not explore the effects of lactate on
phagocytosis and polarization of microglia, pending further testing in subsequent studies, which represents a
shortcoming of the present study. Lactate production isdetermined by the equilibrium between glycolysis andmitochondrial metabolism; enzymes activity in these

Page 17 of 23

pathways can regulate lactate levels, which modulate histone lactylation [62]. It has been reported that a lactate
dehydrogenase inhibitor (OX) reduced lactate production
and histone lactylation induced by hypoxia [30]. Similarly, we found that OX treatment reduced lactate levels
and histone lactylation after SCI. Furthermore, monocarboxylate transporters (MCTs) are known to mediate lactate release and uptake and are widely expressedin various tissues, including the liver, brain, and heart[63]. A previous study revealed that 4-CIN blocked lactate transport into neurons [34]. However, treatment
with 4-CIN (an MCT2 inhibitor) had no obvious effects
on microglia, which is to be expected given that MCT2
is almost exclusively present in neurons [13]. Moreover,the expression of lactate transporter proteins MCT1 and
MCT4 was specifically increased in LPS-stimulated primary and BV2 microglia. We speculated that the 4-CIN
target MCT2 might not be expressed in microglia after
SCI. Thus, the key role of MCTs in microglia following
SCI requires further investigation.
Lactate-derived lysine lactylation in histones serves
as a novel histone mark, and at least 28 lactylation
spots on core histones have been identified, including
H4K12, H3K18, H3K23, H4K5, and H4K8 [30]. Lactate
affects gene transcription through histone lactylation,aunique posttranslational modification [64]. For example, in the late phase of proinflammatory macrophage
polarization, H3K18la in promoter areas triggered theexpression of homeostatic genes involved in woundhealing [30]. H3K18la in promoter areas hastened
tumorigenesis by activating YTHDF2 transcription,
thereby representing potential histone lactylation targets for ocular melanoma treatment [33]. In this study,
we detected enhanced H3K18la expression at 14 and 28
dpi; however, we did not investigate the function andmechanism of H3K18la after SCI, which can be further
explored in subsequent studies. Furthermore, H4K12lain promoter areas activated PKM transcription in AD(See figure on next page.)
Fig. 8 PD-1 inhibitor reverses the advantageous effects of lactate on SCI repair in vivo. A Immunofluorescence staining of BrdU (green)
and CX3CR1 (magenta) in sagittal sections of the PBS (control), lactate, and lactate + PD-1 inhibitor groups at 7 dpi in vivo. Scale bar: 20 μm. B
Quantification of the density of ­BrdU+CX3CR1+ cells in (A). All data are presented as the mean ± SEM, n = 3 mice per group, **p < 0.01; ***p < 0.001. C
Immunofluorescence staining of CX3CR1 (red) in sagittal sections of the control, lactate, and lactate + PD-1 inhibitor groups at 28 dpi in vivo. Scalebar: 100 μm. D Quantification of the density of ­CX3CR1+ cells in (C). All data are presented as the mean ± SEM, n = 3 mice per group, ***p < 0.001;****p < 0.0001. E Images of astrocytes (GFAP, green) stained with neurons (NeuN, magenta) in Z1_Z3 at 28 dpi in control, lactate, and lactate + PD-1
inhibitor groups in vivo. Scale bar: 100 μm. F Quantification of ­NeuN+ neurons in Z1_Z3 in (E). All data are presented as the mean ± SEM, n = 3 mice
per group, **p < 0.01; ***p < 0.001; ****p < 0.0001. G Images of GFAP (green) co-stained with axons (5-HT, magenta) at 28 dpi in control, lactate,
and lactate + PD-1 inhibitor groups in vivo. Asterisks indicate the lesion site, scale bars: low magnification, 100 μm; high magnification, 20 μm. H
Quantification of the percentage of 5-HT+ area in the area of the spinal cord segment penetrating into the lesion core at 28 dpi in (G). All data arepresented as the mean ± SEM, n = 3 mice per group, ****p < 0.0001. I The locomotor function was evaluated by BMS at the indicated time points.
All data are presented as the mean ± SEM, n = 8 mice per group, **p < 0.01; ****p < 0.0001. J Representative images of footprint analysis in micein control, lactate, and lactate + PD-1 inhibitor groups at 28 dpi. K–M Quantification of the stride length and width and paw rotation at 28 dpi. Alldata are presented as the mean ± SEM, n = 8 mice per group, **p < 0.01; ***p < 0.001; ****p < 0.0001

Hu et al. Journal of Neuroinflammation(2024) 21:193

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Fig. 8 (See legend on previous page.)microglia, demonstrating that the positive feedback
loop of glycolysis/H4K12la/PKM2 in microglia accelerates the onset of AD and illustrated that inhibiting
this loop may provide a novel therapeutic strategy for
AD treatment [16]. Our study represents the first to

demonstrate that H4K12la in promoter areas induces
PD-1 expression in microglia and promotes SCI repair.
However, additional research is required to determine
whether other histone modifications contributed to
PD-1 transcription.

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Fig. 9 PD-1 suppression in microglia hinders axon regeneration and inhibits functional recovery after SCI in vivo. A Images of GFAP (green)
co-stained with axons (5-HT, magenta) in AAV-Con and AAV-sh-PD-1 groups at 28 dpi in vivo. Asterisks indicate the lesion site, scale bars: low
magnification, 100 μm; high magnification, 20 μm. B Quantification of the percentage of 5-HT+ area in the area of the spinal cord segment spanningthe lesion site in (A). All data are presented as the mean ± SEM, n = 3 mice per group, **p < 0.01. C Representative immunofluorescence images
of GFAP (green) and NeuN (magenta) neurons in Z1-Z3 zones adjacent to central lesion site in AAV-Con and AAV-sh-PD-1 mice at 28 dpi in vivo.
Scale bar: magnification, 100 μm. D Quantification of ­NeuN+ neurons in Z1_Z3 zones adjacent to lesion site in AAV-Con and AAV-sh-PD-1 groupsin (C). All data are presented as the mean ± SEM, n = 3 mice per group, **p < 0.01; ***p < 0.001; ****p < 0.0001. E Representative images of footprint
analysis in mice treated with AAV-Con or AAV-sh-PD-1 at 28 dpi in vivo. F–H Quantification of the stride length and width and paw rotation. Alldata are presented as the mean ± SEM, n = 8 mice per group, **p < 0.01; ***p < 0.001. I Locomotor function was evaluated by BMS at the indicatedtime points in mice treated with AAV-Con or AAV-sh-PD-1. All data were presented as the mean ± SEM, n = 8 mice per group, *p < 0.05; **p < 0.01;****p < 0.0001(See figure on next page.)
Fig. 10 Inhibition of PD-1 expression in microglia rescues the beneficial effects of lactate on microglial proliferation, scar formation, axon
regeneration, and locomotor function recovery after SCI in vivo. A Immunofluorescence staining of BrdU (magenta) and CX3CR1 (green) in sagittal
sections of the control, lactate, and lactate + AAV-sh-PD-1 groups at 7 dpi in vivo. Scale bar: 20 μm. B Quantification of the density of ­BrdU+CX3CR1+
cells in (A). All data are presented as the mean ± SEM, n = 3 mice per group, **p < 0.01; N.S., not significant. C Immunofluorescence stainingof BrdU (red) in microglia treated with PBS (control), lactate, and lactate + AAV-sh-PD-1 in vitro. Scale bar: 20 μm. D Quantification of the densityof ­BrdU+CX3CR1+ cells in (C). n = 3 per group, **p < 0.01; N.S., not significant. E Immunofluorescence staining of CX3CR1 (red) in sagittal sections
from control, lactate, and lactate + AAV-sh-PD-1 groups at 28 dpi in vivo. Scale bar: 100 μm. F Quantification of the density of ­CX3CR1+ cells in (E).
All data are presented as the mean ± SEM, n = 3 mice per group, **p < 0.01; ***p < 0.001; N.S., not significant. G Representative immunofluorescence
images of GFAP (green) NeuN (magenta) neurons in Z1_Z3 zones adjacent to central lesion core in the control, lactate, and lactate + AAV-sh-PD-1
groups at 28 dpi in vivo. Scale bar: 100 μm. H Quantification of ­NeuN+ neurons in Z1_Z3 in (G). All data were presented as the mean ± SEM, n = 3
mice per group, *p < 0.05; **p < 0.01; ****p < 0.0001; N.S., not significant. I Images of GFAP (green) co-stained with axons (5-HT, magenta) at 28
dpi in control, lactate, and lactate + AAV-sh-PD-1 groups in vivo. Asterisks indicate the lesion site, scale bars: low magnification, 100 μm; highmagnification, 20 μm. J Quantification of the percentage of 5-HT+ area in the area of the spinal cord segment spanning the lesion core at 28
dpi in (I). All data are presented as the mean ± SEM, n = 3 mice per group, **p < 0.01; N.S., not significant. K Representative images of footprint
analysis in control, lactate, and lactate + AAV-sh-PD-1 groups at 28 dpi. (L–N Quantification of the stride length and width and paw rotation
at 28 dpi. All data are presented as the mean ± SEM, n = 8 mice per group, *p < 0.05; **p < 0.01; ***p < 0.001; N.S., not significant. O BMS scores
during 28 days of recovery after SCI demonstrate better functional recovery in control, lactate and lactate + AAV-sh-PD-1 groups. All data arepresented as the mean ± SEM, n = 8 mice per group, ###meant lactate compared to lactate + AAV-sh-PD-1, ###P < 0.001; ####meant lactate compared
to lactate + AAV-sh-PD-1, ####p < 0.0001; ***meant lactate compared to the control, *p < 0.05; ***p < 0.001

Hu et al. Journal of Neuroinflammation

Fig. 10 (See legend on previous page.)(2024) 21:193

Page 20 of 23

Hu et al. Journal of Neuroinflammation(2024) 21:193

PD-1, a 288-amino acid type-I transmembrane protein belonging to the CD28 superfamily, is expressed
in immune cells, glial cells, and neurons, enabling several levels of immunomodulatory and neuromodulatory activity in the CNS [65, 66]. PD-1 signaling may
be crucial in microglia, peripherally recruited immune
cells, and neurons within the CNS under both normal
and pathological conditions [65]. Previous study showedthat PD-1 protein was expressed mainly on macrophages/
microglia after SCI, and PD-1 modulated macrophageand microglial phenotypes after SCI [67]. Moreover, dexmedetomidine has been shown to mitigate microgliamediated neuroinflammation through PD-1 upregulationin an SCI model [41]. Furthermore, PD-1 inhibitors
have been shown to inhibit microglial proliferation andinduce glioma apoptosis, suggesting that PD-1 promotes
microglial proliferation [68]. Consistently, our research
revealed that PD-1 expression was elevated in microglia
after SCI, and PD-1 inhibition suppressed microglial proliferation, scar formation, axon regeneration, and functional recovery. Certainly, we cannot completely rule out
the potential role of the lactate/H4K12la/PD-1 signaling
pathway in other cell types in the SCI microenvironment.
Because of its expression in many cell types, the effectsof the PD-1 inhibitor observed in this study cannot be
specifically attributed to microglia alone. Therefore, we
next used AAV-sh-PD-1 to specifically suppress PD-1 inmicroglia, which blocked axon regeneration and function recovery after SCI. Importantly, we designed andconducted rescue assays, and the results indicated thatboth PD-1 inhibitor and AAV-sh-PD-1 administration
reversed the beneficial effects of lactate on SCI, supporting that lactate-mediated SCI repair may be involved in
the regulation of H4K12la/PD-1 in microglia. However,
our AAV transduction faces possible neuronal trophism
and the potential of increasing immune activation [69]. In
addition, the specific mechanisms by which PD-1 affects
microglial proliferation were not further clarified inthis study. We speculate that PD-1 promotes microglial
proliferation by directly activating proliferation-related
pathways, as previous studies have shown that directly
blocking PD-1 in microglia in vitro can inhibit their proliferation [70].
In this study, we used female mice for all experiments
to avoid increasing the risk of urinary tract infections
due to difficulty with urination, which does represent alimitation of our study. Although previous research on
SCI, including studies on microglia, has commonly usedfemale animal models [71–73], it is well known thatmicroglia are sexually dimorphic [74]. Moreover, Andrew
N. Stewart et al. found differences in the number of macrophages and individual gene expression levels in macrophages/microglia between sexes after SCI [75], while

Page 21 of 23

another clinical study reported that the numbers of maleand female patients with SCI is comparable [76]. Thesefindings suggest that the role of sex in pathology andtreatment should be considered in future research. In the
future, we will use PD-1-deficient mice to further confirm our findings. Given the mature clinical applicationof targeting PD-1, future studies should investigate therelationship between targeting PD-1 and the functional
recovery in patients with SCI.

Conclusion
We describe an unrecognized lactate/H4K12la/PD-1
signaling pathway involved in microglia-mediated SCI
repair. Lactate induces H4K12la levels, thereby promoting PD-1 transcription in microglia, further resulting inenhanced microglial scar formation and functional recovery after SCI. Our study provides a previously unknown
insight by demonstrating that post-translational protein
modifications derived from lactate exert vital functions
and contribute to microglia-mediated tissue repair after
CNS injury.
Abbreviations
SCI	Spinal cord injury
CNS	Central nervous system
CSPGs	Chondroitin sulfate proteoglycans
T10	Thoracic level 10
ECL	Enhanced chemiluminescencePre	Pre-operation
dpi	Days post-injury
Pan Kla	Pan-lysine lactylation
5-HT	5-Hydroxytryptamine
DAPI	4′, 6-Diamidino-2-phenylindole
lactate	Sodium lactate
4-CIN	A-cyano-4-hydroxycinnamate
OX	Odium oxamate
BrdU	Bromodeoxyuridine
PFA	ParaformaldehydeshRNA	Short hairpin RNA
FBS	Fetal bovine serum
PBS	Phosphate-bufered saline
CUT&Tag	Cleavage under targets and tagmentation
ChIP	Chromatin immunoprecipitation
GO	Gene Ontology
KEGG	Kyoto Encyclopedia of Genes and Genomes
pA-Tn5	Protein-A-Tn5
BMS	Basso mouse scale
SEM	Standard error of the mean
ANOVA	Analysis of varianceAD	Alzheimer’s disease
MCTs	Monocarboxylate transporters
i.p.	IntraperitoneallySupplementary Information
The online version contains supplementary material available at https://​doi.​
org/​10.​1186/​s12974-​024-​03186-5.
Supplementary file 1.

Hu et al. Journal of Neuroinflammation(2024) 21:193

Acknowledgements
We are grateful to all of the study staff for their support and cooperation
during the study. We acknowledge the experimental platform provided by the
Scientific Research and Experiment Center of the Second Affiliated Hospital of
Anhui Medical University.
Author contributions
X.H., J.H., and Z.L. designed the research. J.L., F.O., and Z.C. carried out theexperiments, Y.L., Y.Z., and J.W. analyzed the data. X.H. wrote the manuscript.
Z.L., S.Y., and C.L., edited the final manuscript. J.J. and C.L. provided funding. All
authors discussed and approved the final manuscript.
Funding
This study was supported by National Natural Science Foundation of China(Grant number: 82271413), Anhui Provincial Clinical Research Transformation
Project (Grant number: 202304295107020013, 202304295107020009), andNatural Science Research Key Project of Colleges and Universities of Anhui
Province (Grant number: KJ2021A0310).

Page 22 of 23

9.
10.

11.
12.
13.
14.
15.

Availability of data and materials
CUT&Tag data have been deposited in the NCBI SRA under the accession
number PRJNA970050. Additional data are available from the corresponding
authors upon request. No datasets were generated or analysed during thecurrent study.

16.

Declarations

18.

17.

Ethics approval and consent to participate
The studies involving animals were reviewed and approved by the Animal Ethics Committee of Anhui Medical University.

19.

Consent for publication
Not applicable.

20.

Competing interests
The authors declare that no competing interests exists.
Author details
1
Department of Orthopaedics, The Second Affiliated Hospital of Anhui Medical University, 678 Furong Road, Hefei 230601, China. 2 Institute of Orthopaedics, Research Center for Translational Medicine, The Second Affiliated Hospitalof Anhui Medical University, Hefei 230601, Anhui, China.
Received: 4 May 2024 Accepted: 26 July 2024

21.
22.

23.

24.
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