(2023) 20:178
Han et al. Journal of Neuroinflammation
https://doi.org/10.1186/s12974-023-02860-4

Journal of Neuroinflammation

Open Access

RESEARCH

Targeted ablation of signal transducer
and activator of transduction 1 alleviates
inflammation by microglia/macrophages
and promotes long‑term recovery after ischemic
strokeWenxuan Han1†, Hongjian Pu1†, Sicheng Li1, Yaan Liu1, Yongfang Zhao1, Mingyue Xu1, Caixia Chen1, Yun Wu1,
Tuo Yang1, Qing Ye1, Hong Wang2, R. Anne Stetler1,3, Jun Chen1,3 and Yejie Shi1,3*

Abstract
Background Brain microglia and macrophages (Mi/MΦ) can shift to a harmful or advantageous phenotype following an ischemic stroke. Identification of key molecules that regulate the transformation of resting Mi/MΦ could aid
in the development of innovative therapies for ischemic stroke. The transcription factor signal transducer and activator of transduction 1 (STAT1) has been found to contribute to acute neuronal death (in the first 24 h) following ischemic stroke, but its effects on Mi/MΦ and influence on long-term stroke outcomes have yet to be determined.
Methods We generated mice with tamoxifen-induced, Mi/MΦ-specific knockout (mKO) of STAT1 driven
by ­Cx3cr1CreER. Expression of STAT1 was examined in the brain by flow cytometry and RNA sequencing after ischemic
stroke induced by transient middle cerebral artery occlusion (MCAO). The impact of STAT1 mKO on neuronal cell
death, Mi/MΦ phenotype, and brain inflammation profiles were examined 3–5 days after MCAO. Neurological deficits
and the integrity of gray and white matter were assessed for 5 weeks after MCAO by various neurobehavioral tests
and immunohistochemistry.
Results STAT1 was activated in Mi/MΦ at the subacute stage (3 days) after MCAO. Selective deletion of STAT1 in Mi/
MΦ did not alter neuronal cell death or infarct size at 24 h after MCAO, but attenuated Mi/MΦ release of high mobility
group box 1 and increased arginase 1-producing Mi/MΦ 3d after MCAO, suggesting boosted inflammation-resolving
responses of Mi/MΦ. As a result, STAT1 mKO mice had mitigated brain inflammation at the subacute stage after MCAO
and less white matter injury in the long term. Importantly, STAT1 mKO was sufficient to improve functional recovery
for at least 5 weeks after MCAO in both male and female mice.
Conclusions Mi/MΦ-targeted STAT1 KO does not provide immediate neuroprotection but augments inflammation-resolving actions of Mi/MΦ, thereby facilitating long-term functional recovery after stroke. STAT1 is,†Wenxuan Han and Hongjian Pu contributed equally to this work.

*Correspondence:
Yejie Shi
y.shi@pitt.edu
Full list of author information is available at the end of the article© The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
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Han et al. Journal of Neuroinflammation(2023) 20:178

Page 2 of 21

therefore, a promising therapeutic target to harness beneficial Mi/MΦ responses and improve long-term outcomes
after ischemic stroke.
Keywords Behavior test, Conditional gene knockout, Neuroinflammation, STAT1, White matter injury

Background
Ischemic stroke continues to be a significant health concern, even though it has dropped to the fifth leading
cause of death in the United States. Currently, the only
treatment option remains recanalization, but it only benefits a limited number of patients. Extensive efforts havebeen focused on developing neuroprotective agents overthe past 30 years; however, all of the clinical trials stemming from these attempts ended in disappointment.
This lack of success may be attributed to several factors,
including a narrow focus on neurons without consideringother cell types, the need to extend the treatment time
window, and the absence of long-term functional outcomes in assessments [1]. Solely concentrating on protecting neurons from acute death may not be sufficient
to prevent progressive injury and mitigate the subsequent
deterioration of neurological functions after stroke.
Therefore, there is a pressing need to develop treatmentsthat provide longer-lasting effects to address these challenges effectively.
In the context of ischemic stroke, damaged neurons
release danger-associated molecular patterns (DAMPs),
which trigger inflammatory responses [2]. Inflammation
is a process that is activated during the subacute stageof stroke and can persist for an extended period of time.
This inflammatory response affects both the injury andrepair processes in the post-stroke brain [2, 3]. Microglia
and monocyte-derived macrophages, the innate immune
cells in the brain, play critical roles in determining the
inflammatory response and exert significant influence
over the extent of injury and subsequent repair processes.
Upon activation, microglia and macrophages (Mi/MΦ)
can adopt detrimental or beneficial phenotypes that isproinflammatory or inflammation-resolving/pro-repair[4–6]. While this classification is well-described, only
limited options are available to modulate the phenotypeof Mi/MΦ to alter the inflammatory response and longterm functions in stroke. In this regard, one promisingavenue for intervention is targeting the signal transducer
and activator of transcription (STAT) family of transcription factors.
STATs can be phosphorylated by the Janus kinases following ligand–receptor interactions, translocate to the
nucleus, and bind to specific DNA sequence motifs [7].
As transcription regulators, STATs have been implicatedin a wide range of cellular processes, including apoptosis and inflammation [8]. Specifically, STAT1 is potently

activated in canonical interferon signaling and directly
induces proinflammatory factors, such as inducible
nitric oxide synthase (iNOS) and interleukin (IL)-12 [7].
Despite its critical role in immune and inflammatory
responses, studies on STAT1 in ischemic stroke have primarily focused on its pro-apoptotic action on neurons
at the acute injury stage (the first 24 h), whereby STAT1
knockout mice were reported to develop smaller brain
infarct and have less neuronal apoptosis at 24 h after
brain ischemia [9]. The use of the global STAT1 knockout
mice in these studies, however, does not allow for the distinction between the contributions of neurons and nonneuronal cells to the observed effects. Furthermore, there
have been no studies conducted to assess the impact
of STAT1 knockout on long-term stroke outcomes. It
remains unknown whether STAT1 regulates Mi/MΦ
function and affects the evolving long-term neurological recovery—the primary endpoint of clinical stroke. On
this backdrop, the present study aimed to directly investigate the specific role of STAT1 in the modulation of Mi/
MΦ phenotype, inflammation, and long-term functional
recovery after ischemic stroke, using a Mi/MΦ-targeted
STAT1 knockout mouse model.

Methods
Key resources that are essential to reproduce the results
are provided in Additional file 1: Table S1.
Animals

C57BL/6J (RRID: IMSR_JAX:000664), ­STAT1LoxP (RRID:MMRRC_032054-JAX) [10], and ­Cx3cr1CreER (RRID:IMSR_JAX:021160) [11] mice were purchased from theJackson Laboratory (Additional file 1: Table S1). The­STAT1LoxP mice harbor a conditional null allele of theStat1 gene, where LoxP sites flank its first two untranslated exons and the first translated exon. Mi/MΦ-specific
STAT1 knockout (STAT1 mKO) mice were obtainedby crossing the ­Cx3cr1CreER mice and ­STAT1LoxP micefor two generations. The STAT1 mKO mice (genotype:Cx3cr1CreER/wt; Stat1flox/flox) were viable, fertile, and did
not exhibit any gross physical or behavioral abnormalities. To induce gene deletion, STAT1 mKO mice received
intraperitoneal injections of tamoxifen (75 mg/kg dailyfor 4 days). Hemizygous C­ x3cr1CreER mice (genotype:
CreER/wt
wt/wt
Cx3cr1; Stat1) served as age- and sex-matchedwild type (WT) control mice for the STAT1 mKO miceand received the same tamoxifen treatments. Mice wereHan et al. Journal of Neuroinflammation(2023) 20:178

subjected to experimental stroke 10 days after tamoxifen
treatments.
Mice were housed in a specific pathogen-free facility
with a 12-h light/dark cycle. Food and water were available ad libitum. All animal experiments were performed inaccordance with the NIH Guide for the Care and Use ofLaboratory Animals, approved by the University of Pittsburgh Institutional Animal Care and Use Committee,
and reported in accordance with the ARRIVE guidelines[12]. Experimental procedures were performed following
criteria derived from Stroke Therapy Academic Industry Roundtable (STAIR) group guidelines for preclinical evaluation of stroke therapeutics [13]. Accordingly,
experimental group assignments were randomized witha lottery-drawing box, and surgeries and all outcome
assessments were performed by investigators blinded
to mouse genotype and experimental group assignment
whenever possible.
Transient focal cerebral ischemia modelTransient focal cerebral ischemia was induced in adultmale and female mice (10–14 weeks, 22–28 g) by intraluminal occlusion of the left middle cerebral artery (MCA)
for 1 h, as we previously described [14]. Three weeks
before MCA occlusion (MCAO), female mice were subjected to bilateral ovariectomy as we described previously[15], which depletes endogenous estrogen and partially
reproduces the systemic conditions in postmenopausal
women. Mice were anesthetized with 1.5% isoflurane in
67%:30% ­N2O/O2. A 7–0 nylon monofilament with silicone–rubber coated tip (Doccol Corporation 702,134; tip
diameter 0.21 mm, coating length 3–4 mm) was introduced into the internal carotid artery, advanced to theorigin of the MCA, and left in place for 1 h. Rectal temperature was maintained at 37 ± 0.5 °C with a heating pad
during surgical procedures. Regional cerebral blood flow(rCBF) was monitored in all stroke animals using laser
Doppler flowmetry or a two-dimensional laser speckle
imaging system (PeriCam PSI-NR; Perimed) as we previously described [14]. Only animals with an rCBF reduction of > 70% of pre-MCAO baseline levels were included
for further experimentation. Animals that died during or
immediately after surgery were excluded from the studies (less than 2%). Sham-operated animals underwent thesame anesthesia and surgical procedures with the exception of the MCA occlusion.
Neurobehavioral tests

The rotarod test, cylinder test, foot fault test, and Morris water maze test were performed to assess neurological functions before and up to 35 days after MCAO. The
mice that died before 35 days after MCAO were excluded
from the final behavioral analyses. On the first day after

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MCAO, neurological functions were evaluated by a sixpoint scale [16] as follows: 0, no observable deficit; 1,
torso flexion to right; 2, spontaneous circling to right; 3,leaning/falling to right; 4, no spontaneous movement; 5,
death.
Rotarod test

The rotarod test was performed to assess post-stroke
motor deficits as we described previously [17]. Briefly,
mice were placed on a rotating drum with a speed accelerating from 4 to 40 rpm within 5 min. The time at which
the mouse fell off the drum (latency to fall) was recorded.
On the day of MCAO surgery (but before the surgery),5 trials were performed on each mouse and the meanof trial numbers 3, 4, and 5 was used as the pre-MCAO
baseline value. After MCAO, mice were tested for 5 trials
on each testing day with intervals of 15 min, and the data
for trial numbers 3–5 were used to calculate the meanlatency to fall on that day.
Cylinder test

The cylinder test was performed to assess forepaw use
asymmetry after stroke as we described previously [16].
The mouse was placed in a transparent cylinder (diameter: 9 cm; height: 15 cm), and videotaped from the top
for 8 min. Videotapes were analyzed in slow motion,and forepaw (left/right/both) use during the first contact
against the cylinder wall after rearing and during lateral
exploration was recorded. Uninjured mice typically do
not exhibit any inclination toward one forepaw over theother, whereas mice that have suffered a stroke exhibit
reduced utilization of their right forepaw (impaired side;
contralateral to ischemic lesion), depending on the extent
of the injury. Forepaw use asymmetry was calculated withthe following equation: contralateral paw use = right/(left + right + both) × 100%.
Foot fault test

The foot fault test was performed as we described previously [18] to assess sensorimotor coordination. Mice
were placed on an elevated grid surface with a grid opening of 2.25 ­cm2 and videotaped for 2 min from below thegrid. The videotapes were analyzed by a blinded investigator to count the number of total steps and the numberof foot faults made by the right limbs (impaired side; contralateral to ischemic lesion). Foot faults were recorded
when the mouse misplaced its left forepaw or hindpaw,
such that the paw fell through the grid, and expressed asa percentage of total steps.
Morris water maze test

The Morris water maze test was performed as we
described previously [14] to assess cognitive functions.

Han et al. Journal of Neuroinflammation(2023) 20:178

Briefly, a round platform (diameter: 11 cm) was submerged 1.5 cm under the water surface in the center of
one quadrant of a circular pool (diameter: 109 cm; depth:
33 ± 0.5 cm). White tempera paint was added to the waterfor opacity and the temperature of the pool was maintained at 20 ± 1 °C. The test was composed of a spatialacquisition phase to assess learning capacity and a spatial retention phase to assess memory function. Spatial
learning was assessed 22–26 days after MCAO, where
the mouse was released from one of the four quadrants
and was allowed to swim for 60 s to find the hidden
platform. Mice were pre-trained for 3 consecutive days
before MCAO (4 trials on each day). After MCAO, four
trails were performed on each testing day. At the end ofeach trial, the mouse was placed on the platform or wasallowed to remain on the platform for 30 s with prominent spatial cues displayed around the room. The timespent finding the hidden platform was recorded as thelatency to escape from the forced swimming task, and themean latency of four trials was quantified as a measureof spatial learning. Spatial memory was evaluated on day
27 after MCAO by removing the hidden platform. Eachmouse was placed in the pool for a single 60-s probe trial.
The time the mouse spent in the target quadrant wererecorded as spatial memory and expressed as percentageof the total trial time (60 s).
Flow cytometry and fluorescence‑activated single cellsorting (FACS)

Mice were deeply anesthetized and transcardially perfused with ice-cold Hank’s balanced salt solution, andthe ipsilesional and non-injured contralesional cerebral
hemispheres were harvested. Single-cell suspensions
were prepared from the mouse brain using the Neural
Tissue Dissociation Kit and gentleMACS Octo Dissociator with Heaters (Miltenyi Biotec) according to the
manufacturer’s instructions and as we described previously [19]. Suspensions were passed through a 70 μm
cell strainer, and fractionated on a 30% and 70% Percoll
gradient at 800 g for 30 min to remove myelin and celldebris. Mononuclear cells at the interface were collected
and resuspended at 1 × ­107 cells per mL. The cells werefirst stained with the Zombie NIR Fixable Viability Dyefor 20 min at 4 °C for assessment of viability. The cells
were then stained with fluorophore-conjugated antibodies (Additional file 1: Table S1) recognizing surface antigens. After washing, cells were fixed and permeabilizedwith an eBioscience Foxp3/Transcription Factor Staining Buffer Set or 4% formaldehyde followed by ice-coldmethanol, and then incubated with antibodies recognizing intracellular antigens. Flow cytometry was performed
using a BD LSRFortessa Cell Analyzer driven by the
FACSDiva software or a Cytek Aurora spectral analyzer

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driven by the SpectroFlo software. FACS was performed
using a BD FACSAria cell sorter driven by the FACSDiva
software. Fluorescence compensation was performed
using single-stained OneComp eBeads according to the
manufacturer’s instructions.
Data were analyzed using the FlowJo software to quantify positively stained cells. Furthermore, individual
immune cells (­CD45+ gated) were plotted using tSNE
function in FlowJo to map high-dimensional cytometry
data onto two dimensions based on the fast interpolation-based t-distributed stochastic neighbor embedding(FIt-SNE) algorithm [20]. t-SNE plots were generated
with 1000 iterations of 6000 cells randomly selected fromeach mouse. Major cell types were identified according totheir expression of prototypic markers.
Real‑time polymerase chain reaction (PCR)

Quantitative PCR was performed on FACS-sorted cells
to verify the deletion of STAT1. Total RNA was extracted
from the sorted cells using a Qiagen RNeasy Plus Micro
Kit, and genomic DNA was eliminated. First-standcDNA was generated using a Qiagen RT2 First Strand Kit
according to the manufacturer’s instructions. Real-time
PCR was performed using the Qiagen RT2 SYBR GreenqPCR Mastermix on a Bio-Rad Opticon 2 Real-TimePCR Detection System. All reactions were performed intriplicate, and the amount of mRNA was normalized tointernal controls (Gapdh). Data were summarized as foldchanges relative to WT control mice.
Immunohistochemistry and data analyses

Mice were deeply anesthetized and transcardially perfused with 0.9% NaCl, followed by 4% paraformaldehyde
in PBS. Brains were harvested and cryoprotected in 30%
sucrose in PBS, and frozen serial coronal brain sections(25-μm thick) were prepared using a ThermoFisher
HM525 NX cryostat. Sections were blocked with 5%
donkey serum in PBS for 1 h, followed by overnightincubation (at 4 °C) with primary antibodies (Additional
file 1: Table S1). For immunostaining involving mouseprimary antibodies, a Mouse on Mouse Immunodetection Kit was used following the manufacturer’s instructions. After washing, sections were incubated for 1 h
at 20 °C with donkey secondary antibodies conjugatedwith DyLight 488 or Cy3 fluorophores (1:1000, Jackson
ImmunoResearch Laboratories). Alternate sections fromeach experimental condition were incubated in all solutions except the primary antibodies to assess nonspecific secondary antibody staining. To visualize apoptotic
cells, TUNEL staining was performed using a Roche
In Situ Cell Death Detection Kit following manufacturer’s
instructions. Sections were then mounted and coverslipped with Fluoromount-G containing DAPI (Southern

Han et al. Journal of Neuroinflammation(2023) 20:178

Biotech). Fluorescence images were captured with aninverted Nikon Diaphot-300 fluorescence microscope
equipped with a SPOT RT slider camera and Meta Series
Software 5.0 (Molecular Devices), or with an Olympus
Fluoview FV1000 confocal microscope and FV10-ASW
2.0 software.
Quantitative analysis of immunofluorescence staining
images was performed manually by a blinded investigatorusing ImageJ. Brain infarct and tissue loss after MCAO
were measured on six equally spaced coronal brain sections encompassing the MCA territory immunostained
for microtubule-associated protein 2 (MAP2). Volumesof infarct or tissue loss were calculated as the volume of
the MAP2-immunopositive contralateral hemisphereminus that of the ipsilateral hemisphere. Positively
stained cells were manually counted from 1 to 2 randomly
selected 40 × microscopic fields (317.4 μm × 317.4 μm)
in the cortex and striatum for each brain section, and 2
brain sections were processed for each mouse. Nodesof Ranvier (NORs) were counted from two 60 × microscopic fields with 2.5 × digital magnification (final area
83.97 μm × 83.97 μm) in the external capsule. A threshold
was set to differentiate the target signal from background.
The image-processing software Imaris was used toreconstruct three-dimensional images of Iba1, CD16/32,
and arginase 1 (Arg1) immunofluorescence and quantify morphological parameters of cells as we previously
described [15]. Briefly, a z-stack of 5 images spanning
10 μm were imported into Imaris, and the immunosignalof each channel was remodeled to 3D images by the surface operation. Smoothing was set at 0.6 μm for all channels and images. A threshold was set to differentiate thetarget signal from background. Non-specific signals weremanually removed, and the 3D-rendered images wereconstructed. All images were processed with the same
adjustments. Morphological analysis of resting microglia
was performed on 3 randomly selected regions of interest
per brain. Cell volume and sphericity were automaticallycalculated by Imaris.
RNA sequencing (RNA‑seq)

Microglia were gated as ­CD11c–Ly6G–CD11b+CD45low
cells and enriched from the mouse brain by FACS as
described above, and subjected to bulk RNA-seq. RNA
extraction, library preparation, and sequencing were performed at the University of Pittsburgh HSCRF Genomics
Research Core as we previously described [18]. Briefly,
total RNA was extracted from FACS-sorted cells using
a Qiagen RNeasy Plus Micro Kit, and RNA integrity wasassessed using the High Sensitivity RNA ScreenTape
system on an Agilent 2200 TapeStation. The SMARTSeq HT Kit was used to generate cDNA from 10 ng of
total RNA, and the cDNA product was checked by anPage 5 of 21

Agilent Fragment Analyzer system for quality control.
The sequencing library was constructed by following theIllumina Nextera XT Sample Preparation Guide. One
nanogram of input cDNA was tagmented (tagged andfragmented) and amplified using the Illumina Nextrera
XT kit. Sequence libraries of each sample were finallyequimolarly pooled and sequenced on an Illumina Nextseq 500 system, using a paired-end 75-bp strategy.
RNA‑seq data analysis

RNA-seq data were analyzed using R/Bioconductor [21],following our published pipeline [18, 22]. Preprocessingof the RNA-seq data was completed using Chipster [23].
Fastq files were quality controlled using FastQC [24], andall samples passed quality control criteria. Reads were
mapped to the GRCm38 mouse genome using HISAT2[25] and counted by HTSeq [26]. Genes were identified
by Ensembl ID [27]. The R package DESeq2 [28] wasused to normalize the counts and to perform differential expression analysis. Differentially expressed genes(DEGs) were defined as genes with a ­log2(fold change) > 1
or < − 1, and with a false discovery rate (FDR) adjusted pvalue < 0.05 (Benjamini–Hochberg method).
DEGs identified by DESeq2 were submitted to Ingenuity Pathway Analysis (IPA) for pathway analysis using
the Ingenuity Knowledge Base (Qiagen Bioinformatics).
The fold change and adjusted p value for each gene wereused to perform the core analysis. Diseases and functions
were considered significantly enriched with a p value ofoverlap < 0.01 and an activation z-score > 2 (predicted
to be activated) or < − 2 (predicted to be inhibited). The
Upstream Regulator analysis was used to identify thecascade of upstream transcriptional regulators that can
explain the observed gene expression changes in the data
set. An upstream regulator was predicted to be strongly
activated or inhibited if its activation z-score was > 2or < − 2, respectively. The cutoff p value was set at 0.01.
Proteomic array analysis

Mice were deeply anesthetized and transcardially perfused with 0.9% NaCl, and fresh brain tissues were harvested. Protein was extracted from the left cerebral
hemisphere using cell lysis buffer with protease andphosphatase inhibitor cocktail. The concentration ofprotein was measured using the Bradford protein assay.
The content of 40 inflammatory factors was measured
using a RayBiotech Mouse Inflammation Array kit following manufacturer’s instructions and as we described
previously [19]. The signal intensity for each antigenspecific antibody spot was measured using ImageJ, andbackground signal averaged from 3 blank spots was subtracted. The signal intensity for each inflammatory factor was then normalized to the average intensity of theHan et al. Journal of Neuroinflammation(2023) 20:178

3 positive control spots on each membrane. The concentrations of various factors were expressed as fold changes
relative to sham controls.
Statistical analyses

Data are presented as mean ± standard deviation (SD).
Individual data points are plotted where applicable. Statistical comparison between two groups was accomplished by the student’s t test (for normally distributeddata) or Mann–Whitney U test (for non-normally distributed data). Differences in means among multiple
groups were analyzed by one- or two-way ANOVA followed by the Bonferroni-adjusted multiple comparisons.
Data that do not follow a normal distribution were analyzed by Kruskal–Wallis test followed by the Dunn’s test.
Data from behavior tests which consist of repeated testing on different days were analyzed by two-way repeatedmeasures ANOVA. Linear regression analysis and Pearson’s correlation coefficients were used to correlate thehistological parameters with neurobehavioral functions.
A p value less than 0.05 was deemed statistically significant, and all testing was two-tailed.

Results
STAT1 is activated in brain Mi/MΦ after ischemic stroke

It has been reported that ischemic stroke can induce theactivation of STAT1 in the brain [9], yet the cell type(s)accountable for the increased activity of STAT1 have notbeen investigated thus far. We performed flow cytometry
to assess the expression of STAT1 in various types of cells
in the mouse brain after transient focal cerebral ischemia
induced by 1 h of MCAO (Fig. 1A). Robust upregulationof STAT1 was observed in ­CD11b+ Mi/MΦ at 24 and72 h after MCAO compared to control mice subjected tosham injury, whereby the percentages of Mi/MΦ expressing STAT1 increased by 3 and 3.8 folds, respectively(Fig. 1B). In contrast, a larger portion of astrocytes, oligodendrocytes and other CNS cells expressed STAT1 underbaseline conditions compared to Mi/MΦ, but these cell

Page 6 of 21

types did not exhibit substantial alterations in response
to ischemic stroke (Fig. 1B). On the other hand, phosphorylation of STAT1 at Y701 (reflecting its activity) markedly increased in Mi/MΦ at 24 and 74 h after MCAO,
whereas astrocytes, oligodendrocytes and other CNS
cells had low levels of phospho-STAT1 from 2 to 72 h
after MCAO (Fig. 1C). We conducted further classification of all ­CD11b+ cells expressing STAT1 based on theirexpression of CD45 (Fig. 1D), and found that more than
60% of STAT1-expressing cells were ­CD45low (microglia)
at all timepoints examined (Fig. 1E). The proportion of­CD45high cells (macrophages) in STAT1-immunopositive
cells gradually increased from 2 to 72 h after MCAO
(Fig. 1E), likely reflecting the infiltration of blood monocytes/macrophages into the post-stroke brain. Together,
these data suggest that STAT1 is elevated and activatedin post-stroke Mi/MΦ at the subacute stage (24 and 72 h)
and may influence the function of these cells.
To confirm the activation of STAT1 in Mi/MΦ after
stroke, we conducted RNA-seq analyses on microglia
enriched from the post-stroke brain 3 days after MCAO
and from non-injury sham controls using FACS (Fig. 1F).
Differential expression analysis identified 2183 genesthat were upregulated or downregulated with a L­ og2(foldchange) > 1 or < − 1 and an FDR-adjusted p value < 0.05
in post-MCAO microglia compared to sham microglia(Additional file 2: Table S2), suggesting that microglia
undergo substantial transcriptome reprogramming in
response to ischemic brain injury. To gain further insight
into the biological functions implicated by these DEGs,
we performed IPA and examined the diseases and functions overrepresented by the DEGs. Functions related
to myeloid cells and inflammatory responses were predicted to be strongly activated (z-score > 2 and p < 0.01)
in post-MCAO microglia (Fig. 1G). Furthermore,
Upstream Regulator analysis predicted several members of the STAT family to be activated in post-stroke
microglia, with STAT1 being the most activated (Fig. 1H;z-score = 5.85, p = 9.84 × ­10–19). A total of 81 DEGs were(See figure on next page.)
Fig. 1 STAT1 is activated in brain microglia/macrophages after ischemic stroke. A–E Mice were subjected to 1-h MCAO or sham injury. Expressionof STAT1 in various types of cells in the brain was examined at 2, 6, 24, and 72 h after MCAO using flow cytometry. A Gating strategy for microglia/
macrophages (Mi/MΦ; ­Ly6G–CD11b+CD45+), astrocytes (­ GLAST+), oligodendrocytes ­(O4+), and other CNS cells ­(CD11b–CD45–GLAST–O4–).
STAT1-immunopositive cells were gated based on the Fluorescence Minus One (FMO) control. B, C The numbers of cells immunopositive for STAT1
B or phosphorylated STAT1 C were quantified in Mi/MΦ, astrocytes, oligodendrocytes, and other CNS cells, and were expressed as percentagesof all cells of that type. D Cells co-expressing CD11b and STAT1 were examined for their expression of CD45. C­ D45+ cells were classified as microglia­(CD45low) and macrophages (­ CD45high). E Percentages of microglia and macrophages in all ­CD11b+STAT1+ cells at different times after MCAO.
More than 60% of ­STAT1+ cells were microglia in all groups. n = 4–6 mice/group. #p < 0.05, ###p < 0.001 vs. sham. NS, no significant difference. F
RNA-seq experimental design and data analysis pipeline. Mice were subjected to MCAO or sham injury. Three days later, microglia were purified
from the brain by fluorescence-activated cell sorting (FACS) and subjected to RNA-seq. n = 2–3 biological replicates/group. Ingenuity pathway
analysis (IPA) was performed on the differentially expressed genes (DEGs) induced by MCAO compared to sham controls. G IPA predicted several
functions of myeloid cells to be significantly activated (z-score > 2, p value of overlap < 0.01) in post-MCAO microglia. H The upstream regulator
analyses in IPA calculated the activation z-score and p value of overlap for each STAT as a potential upstream regulator in post-MCAO microglia.
The cutoff values for predicated activation were z-score > 2 and p value < 0.01. I DEGs that are regulated by STAT1 are shown in a network view,
with annotations on the subcellular localization and types of their products

Han et al. Journal of Neuroinflammation

Fig. 1 (See legend on previous page.)(2023) 20:178

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Han et al. Journal of Neuroinflammation(2023) 20:178

in the molecular network of STAT1 targets (Fig. 1I),
including genes encoding the proinflammatory cytokines
IL-1β, tumor necrosis factor alpha (TNF-α), and IL-12,
all of which were upregulated in microglia after ischemic
stroke. The collective findings from our flow cytometry and RNA-seq analyses provide strong evidence that
STAT1 is activated in brain Mi/MΦ after ischemic stroke,and may dictate the proinflammatory responses of Mi/
MΦ at the subacute injury stage.
In vivo selective deletion of STAT1 does not inducechanges to homeostatic microglia

To date, there have been no studies that directly address
the role of STAT1 in Mi/MΦ due to the lack of specificgenetic models targeting these cell types. To bridge thisknowledge gap, we generated mice with tamoxifeninduced, Mi/MΦ-targeted knockout of STAT1 (STAT1
mKO) by crossing the C­ x3cr1CreER mice with S­ TAT1LoxP
mice (Fig. 2A). To confirm microglia-targeted deletion of
STAT1, we isolated microglia ­(CD11b+CD45+ cells) andother CNS cells (­ CD11b–CD45– cells) from the brains of
STAT1 mKO mice and age-matched WT control mice
10 days after tamoxifen treatment and performed quantitative PCR (Fig. 2B, C). mRNA of the Stat1 gene was
reduced by 92% in microglia from STAT1 mKO micecompared to WT controls, whereas its levels were similar in non-microglial CNS cells between STAT1 mKO
and WT mice (Fig. 2D), confirming selective deletion of
STAT1 in microglia.
The STAT1 mKO mice did not display any noticeable
physical or behavioral abnormalities when compared to
WT control mice. To assess whether STAT1deletion in
microglia had any discernible effects on microglia abundance or morphology under physiological conditions,
we performed immunostaining of Iba1 on brain sections
from naïve STAT1 mKO mice and WT mice (Fig. 2E).
STAT1 mKO did not cause changes in the number of­Iba1+ cells (Fig. 2F) or the volume and sphericity of thesecells (Fig. 2G), suggesting that loss of STAT1 did not
result in overt phenotypic changes of homeostatic microglia. Furthermore, when subjected to transient focal cerebral ischemia induced by MCAO, STAT1 mKO miceand WT mice had comparable rCBF reduction (Fig. 2H,I), suggesting that they had similar hemodynamics in
response to ischemic injury.
Mi/MΦ‑targeted STAT1 deletion does not reduce acuteneuronal cell death after ischemic stroke

Previous studies have found that constitutive STAT1
knockout reduces brain infarct volume 24 h after
ischemic stroke, mainly through protection of neurons
against acute apoptosis [9]. We, therefore, examined
whether selective deletion of STAT1 only in Mi/MΦ had

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an impact on acute brain injury after ischemic stroke.
Male STAT1 mKO mice and WT control mice were subjected to 1-h MCAO followed by 24 h of reperfusion. At
24 h after MCAO, STAT1 mKO mice had similar neurological deficit scores with WT controls (Fig. 3A), andtheir infarct volumes were not different as measured byimmunostaining of the neuronal marker MAP2 (Fig. 3B,
C). On a microscopic level, the numbers of apoptotic
neurons identified by double-staining of TUNEL and
NeuN (Fig. 3D) were also similar between STAT1 mKO
mice and WT mice in both the peri-infarct cortex andstriatum (Fig. 3E).
Dead/dying neurons in the post-stroke brain release
DAMPs to elicit proinflammatory responses [2, 29]. We
examined the expression of high mobility group box 1(HMGB1), a major DAMP in ischemic brain [30, 31], inneurons at 24 h after MCAO. In the non-injured contralateral brain hemisphere, most N­ euN+ cells (> 95%)
were also immunopositive for HMGB1 (Fig. 3F), indicating healthy neurons retaining endogenous HMGB1. On
the contrary, 32.0% and 30.5% of ­NeuN+ cells became
HMGB1-negative in the ipsilateral cortex and striatumof WT mice, respectively (Fig. 3G), suggesting that theseneurons were damaged and released HMGB1 into theextracellular space. The numbers of ­HMGB1+ neurons
were similar in STAT1 mKO mice and WT mice 24 hafter MCAO (Fig. 3 F and G), suggesting similar neuronal injury and the initial trigger of proinflammatory
responses at the acute stage after ischemic stroke. Collectively, these data suggest that targeted deletion of STAT1
only in Mi/MΦ does not affect neuronal death or stroke
outcome at the acute injury stage (24 h after MCAO).
STAT1 deficiency ameliorates neurotoxic Mi/MΦ responses
at the subacute stage after ischemic stroke

Despite lack of influence on acute neuronal death after
brain ischemia, targeted deletion of STAT1 effectively
reduced neurotoxic Mi/MΦ responses at the subacute injury stage. When we extended the examinationof HMGB1 release from neurons to 3 and 5 days after
MCAO (Fig. 4A), we found that the total number of­NeuN+HMGB1+ cells remained significantly less than
non-injured sham controls (Fig. 4B). Interestingly, thenumber of ­NeuN+HMGB1+ cells were higher in STAT1mKO mice than WT mice 3 days after MCAO in boththe peri-infarct cortex and striatum (Fig. 4B), possibly due to a decrease in neuronal stress signaling andretention of intracellular HMGB1 by neurons in STAT1mKO mice. Since the STAT1 mKO mice had selective
deletion of STAT1 in Mi/MΦ, we speculated that theseneuroprotective effects of STAT1 mKO was secondary
to reduced inflammatory signaling of Mi/MΦ. Therefore, we examined the expression of HMGB1 in Mi/MΦHan et al. Journal of Neuroinflammation(2023) 20:178

Page 9 of 21

Fig. 2 Generation and characterization of microglia/macrophage-targeted STAT1 knockout (STAT1 mKO) mice. A Generation of STAT1 mKO mice.
LoxP sites were engineered flanking the first two untranslated exons and the first translated exon of the Stat1 gene. When crossed to ­Cx3cr1CreER
mice, the LoxP sites recombine and delete the floxed region of Stat1, knocking out the protein in CX3CR1-expressing cells in the presenceof tamoxifen. B–D Microglia ­(CD11b+CD45+ cells) and other CNS cells (­ CD11b–CD45– cells) were purified from the brains of STAT1 mKO miceand WT mice by FACS, and subjected to quantitative PCR to measure the mRNA levels of Stat1. n = 7 mice/group. E The morphology of microglia
was assessed by Iba1 immunostaining in the striatum and cortex of STAT1 mKO mice and WT mice. Cells were counterstained with DAPI for nuclear
labeling. Rectangle: the region enlarged and 3D-rendered in the 3rd row. F, G STAT1 mKO did not alter the number F or morphology G of microglia
in the homeostatic brain. n = 6 mice/group F or 38 cells/group (G). H, I Focal cerebral ischemia was induced in STAT1 mKO mice and WT control
mice by 1 h of MCAO. H Two-dimensional laser speckle images show cortical CBF before MCAO (baseline) and 10 min after the onset of MCAO. I
Summarized data show that STAT1 mKO mice had comparable cortical CBF changes during MCAO with WT mice. n = 7 mice/group. ###p < 0.001
MCAO vs. baseline. NS no significant differenceHan et al. Journal of Neuroinflammation(2023) 20:178

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Fig. 3 Selective STAT1 deletion in microglia/macrophages does not reduce acute neuronal death after ischemic stroke. STAT1 mKO mice and WT
control mice were subjected to 1-h MCAO followed by 24 h of reperfusion. A Neurological deficit scores. B, C Brain infarct volume was measured
on six coronal sections encompassing the MCA territory immunostained for MAP2 (green). D TUNEL/NeuN double-staining was performed
to assess the apoptosis of neurons. Dashed line: infarct border. Arrows: apoptotic neurons (­ TUNEL+NeuN+ cells) enlarged in the insets. E The
numbers of apoptotic neurons in the inner border zone (rectangles in D) in the cortex and striatum. F Neuronal expression of HMGB1 was examinedin the ipsilateral (IL) inner border zone or corresponding areas in the non-injured contralateral (CL) side by double-label immunostaining of NeuN
and HMGB1. Arrow: NeuN and HMGB1 double-immunopositive cells. Arrowhead: ­NeuN+ cells that are negative for HMGB1. G The numbersof ­NeuN+HMGB1+ cells were counted in the cortex and striatum, and expressed as numbers per ­mm2 (upper panels) or as percentages of total­NeuN+ cells (lower panels). n = 7 mice/group. ###p < 0.001 IL vs. CL. NS no significant difference

as a marker for the healthiness of these cells. Throughdouble-label immunostaining of Iba1 and HMGB1, wewere able to quantify Mi/MΦ retention and release of
HMGB1 at 3 and 5 days after MCAO (Fig. 4C). Compared to sham controls, MCAO increased the numbersof ­Iba1+ cells, reflecting infiltration of peripheral ­Iba1+

monocyte-derived macrophages (Fig. 4D). To compensate for such changes in the total number of ­Iba1+ cells,
we quantified the percentages of I­ba1+ cells that were
immunopositive for HMGB1. The results showed that
MCAO-induced release of HMGB1 from I­ba1+ Mi/MΦ were significantly ameliorated in STAT1 mKO miceHan et al. Journal of Neuroinflammation(2023) 20:178

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Fig. 4 Microglia/macrophage-targeted STAT1 knockout alleviates subacute neurotoxic microglial/macrophage responses after ischemic
stroke. STAT1 mKO mice and WT control mice were subjected to 1-h MCAO followed by 3 or 5 days of reperfusion. A, B Neuronal expressionof HMGB1 was examined in the ipsilateral peri-infarct areas or corresponding areas in sham-operated brains by double-label immunostainingof NeuN and HMGB1. A Representative images taken from the cortex. Arrowhead: ­NeuN+ cells that are negative for HMGB1. B The numbersof ­NeuN+HMGB1+ cells were counted in the cortex and striatum 3 and 5 days after MCAO, and expressed as numbers per ­mm2 (top panels)or as percentages of total ­NeuN+ cells (bottom panels). C, D Microglia/macrophage expression of HMGB1 was examined by double-label
immunostaining of Iba1 and HMGB1. C Representative images taken from the cortex. Arrowhead: ­Iba1+ cells that are negative for HMGB1. Dashedrectangles: Cells that are enlarged and 3D rendered in the 3rd and 4th rows, respectively. C The numbers of ­Iba1+HMGB1+ cells were counted
in the cortex and striatum 3 and 5 days after MCAO, and expressed as numbers per ­mm2 (top panels) or as percentages of total ­Iba1+ cells (bottom
panels). n = 4 (sham) or 6–7 (MCAO) mice/group. #p < 0.05, ##p < 0.01, ###p < 0.001 MCAO vs. sham. *p < 0.05, **p < 0.001, ***p < 0.001 STAT1 mKO vs.
WT. NS no significant differenceHan et al. Journal of Neuroinflammation(2023) 20:178

compared to WT mice in both the peri-infarct cortex andstriatum, at both 3 days and 5 days after MCAO (Fig. 4D).
In summary, these data suggest that STAT1-deficient Mi/
MΦ have less propensity for DAMP inflammatory activity in the ischemic brain than their counterpart in WT
mice at the subacute stage after ischemic stroke, which
may lead to reduced secondary neuronal death.
Deletion of STAT1 drives post‑stroke Mi/MΦ
toward an inflammation‑resolving phenotype

We further assessed the functional phenotype of Mi/
MΦ upon STAT1 deletion by examining a panel ofmarkers indicative of their proinflammatory or inflammation-resolving functions at the subacute stage after
MCAO. Using multicolor flow cytometry, we identified
major subsets of immune cells in the post-MCAO brain(Fig. 5A) based on their expression of prototypic markers(Fig. 5B). The macrophage population (­CD11b+CD45highcells) was smaller in STAT1 mKO mice than WT mice
3 days after MCAO (Fig. 5C), likely reflecting a general
reduction of inflammatory responses in the brain. As aresult, the number of microglia (­CD11b+CD45low cells)
per 1000 single cells examined was relatively larger in
STAT1 mKO mice than WT mice (Fig. 5C). To accountfor the variation in total numbers of microglia and macrophages, we quantified the cells expressing a specificphenotypic marker both as the number per 1000 single cells and as a percentage of total microglia or macrophage numbers. This approach enabled us to accurately
assess and compare the presence of cells with the desired
phenotypic characteristics, irrespective of the differences in overall microglia and macrophage counts. A
panel of 6 phenotypic markers was examined, including
the proinflammatory markers CD16/32, TNF-α, CD86,
and markers indicative of inflammation-resolving activities CD206, Arg1, and IL-10 (Fig. 5D). The percentage of
microglia expressing all 6 markers were similar between
STAT1 mKO mice and WT mice (Fig. 5E, F), suggestingnegligible changes in microglia phenotype induced by
STAT1 mKO. In contrast, macrophages in STAT1 mKO

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mice exhibited a substantial alleviation in their proinflammatory profile. The percentage of proinflammatory
macrophages expressing CD86 was significantly reduced,
whereas the inflammation-resolving macrophages
expressing Arg1 was markedly elevated, in STAT1 mKO
mice compared to WT mice 3 days after MCAO (Fig. 5E,
F).
We further verified the findings from flow cytometry using immunostaining of Iba1, double-labeled withthe proinflammatory phenotypic marker CD16/32 andthe inflammation-resolving marker Arg1 (Fig. 5G).
Consistent with flow cytometry findings, the numberof CD16/32+ Mi/MΦ was significantly less in STAT1mKO mice than WT mice, and the number of ­Arg1+
Mi/MΦ were increased in STAT1 mKO mice, in boththe peri-infarct cortex and striatum 3 days after MCAO
(Fig. 5H). In summary, these data suggest that STAT1
mKO potently shifts the phenotype of Mi/MΦ toward an
inflammation-resolving phenotype after ischemic stroke.
Targeted knockout of STAT1 Mi/MΦ mitigates brain
inflammation after ischemic stroke

Mi/MΦ are important mediators of inflammatory
responses in the post-stroke brain, which greatly influences stroke outcomes [2, 3]. Next, we examined whether
alteration of Mi/MΦ phenotype by STAT1 deletion led tosecondary changes in the brain inflammation profiles atthe subacute stage after stroke. We first determined theinflammation burden of the post-stroke brain by measuring the presence of infiltrating blood immune cells
3 days after MCAO (Fig. 6A). Flow cytometry analysis
showed that MCAO-induced peripheral immune cell
infiltration into the brain was markedly reduced 3 days
after MCAO in STAT1 mKO mice compared to WT
mice (Fig. 6B). There were significantly less neutrophils,
dendritic cells, and ­CD11b+CD45high macrophages
and activated microglia in the ipsilateral hemisphere ofischemic STAT1 mKO mice, whereas the numbers ofT cells and B cells were comparable between ischemic
STAT1 mKO mice and WT mice (Fig. 6B). As a result,(See figure on next page.)
Fig. 5 Loss of STAT1 ameliorates proinflammatory microglial/macrophage responses after ischemic stroke. A–F MCAO was induced in STAT1mKO mice and WT mice. Three days after MCAO, flow cytometry was performed to assess the phenotype of microglia and macrophages
in the ipsilesional brain hemisphere. A Representative t-SNE plots of 12,000 C­ D45+ cells pooled from one WT and one STAT1 mKO mouse brains.
Each dot represents one cell. Color corresponds to cell type. B t-SNE plots showing the expression levels of prototypic cell markers in ­CD45+ cells
that identified major cell types: microglia (­ CD11b+CD45low), macrophage (­ CD11b+CD45high), neutrophil (­ Ly6G+), and lymphocytes ­(CD11b–CD45high).
Color represents fluorescence intensity on a logarithmic scale. C The numbers of microglia (­ Ly6G–CD11b+CD45low cells) and macrophages­(Ly6G–CD11b+CD45high cells) in STAT1 mKO and WT mouse brains. D t-SNE plots of all microglia and macrophages demonstrate cells from WTand STAT1 mKO mice that were immunopositive for a panel of proinflammatory (CD16/32, TNF-α, CD86) and anti-inflammatory (CD206, arginase
1, IL-10) markers. E, F The numbers of microglia and macrophages positive for each marker were quantified after manual gating and expressed
as cell number per 1000 single cells E or percentages of total microglia or macrophages (F). G, H The phenotype of microglia/macrophages
was assessed 3 days after MCAO using double-label immunostaining of Iba1 and CD16/32 or arginase 1. Shown are representative images takenfrom the peri-infarct cortex G and summarized data on the numbers of double-immunopositive cells in the peri-infarct cortex and striatum (H).
n = 5–6 mice/group. *p < 0.05, **p < 0.01, ***p < 0.001 STAT1 mKO vs. WTHan et al. Journal of Neuroinflammation(2023) 20:178

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Fig. 5 (See legend on previous page.)

the relative portion of C
­ D11b+CD45low cells (mostly
quiescent microglia) per 1000 single cells examined wasincreased in the ischemic hemisphere of STAT1 mKO
mice compared to WT mice (Fig. 6B). In parallel, we
determined the content of 40 inflammatory cytokines
in the brain 5 days after MCAO using an antibody array(Fig. 6C). Compared to the baseline inflammation level insham controls, MCAO elevated 19 inflammation markers, including the proinflammatory cytokines IL-6, IL-12,
and the chemokines attracting migrating immune cells
MIP-1α/CCL3, and MIP-1γ/CCL9 (Fig. 6D). The expression of 11 out of these 19 markers which may exacerbate

Han et al. Journal of Neuroinflammation(2023) 20:178

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Fig. 6 Selective deletion of STAT1 in microglia/macrophages mitigates brain inflammation after ischemic stroke. STAT1 mKO mice and WT mice
were subjected to 1-h MCAO followed by 3 or 5 days of reperfusion. A, B Flow cytometry was performed 3 days after MCAO to assess the infiltrationof peripheral immune cells into the post-MCAO brain. A Gating strategy for various immune cells in the brain. B The numbers of various immune
cells in the ipsilesional (IL) and non-injured contralesional (CL) brain hemispheres. C, D A panel of 40 inflammatory cytokines was measured
in the ipsilesional brain hemisphere 5 days after MCAO using an antibody array. C Representative blots with significantly altered markers labeled.
D Heat map showing the mean expression levels of 17 markers that were significantly different among groups. Sham controls were pooled
from both WT and STAT1 mKO mice, between which there was no significant difference. n = 6 mice/group. ##p < 0.01, ###p < 0.001 IL vs. CL. *p < 0.05,**p < 0.01, ***p < 0.001 STAT1 mKO vs. WT after MCAO. NS no significant difference

brain injury by potentiating neuroinflammation was significantly reduced in STAT1 mKO mice compared to
WT mice 5 days after MCAO (Fig. 6D). Interestingly,

the expression of the anti-inflammatory cytokine IL-10,
which was downregulated by MCAO, was relatively
increased in ischemic STAT1 mKO mice compared toHan et al. Journal of Neuroinflammation(2023) 20:178

ischemic WT mice (Fig. 6D). These data consistently
demonstrated mitigation of inflammation in the poststroke brain by Mi/MΦ-targeted STAT1 deletion.
STAT1 mKO is sufficient to improve long‑term functional
recovery after stroke in both male and female mice

To determine the extent to which the alterations in Mi/
MΦ phenotype resulting from STAT1 mKO impact theoverall outcome following ischemic stroke, the recoveryof neurological functions was evaluated in STAT1 mKO
mice over a 35-day period. We employed a panel of three
behavioral tests, namely, the rotarod test, cylinder test,
and foot fault test, to assess the sensorimotor functionsof adult male mice (Fig. 7A–C). The results obtained
from all three tests consistently demonstrated significant
sensorimotor deficits in the mice following MCAO whencompared to sham-operated mice (Fig. 7A–C). Importantly, STAT1 mKO mice demonstrated significantly better sensorimotor functions in all three tests (Fig. 7A–C;
p < 0.01 or p < 0.001). We also carried out the Morris
water maze test to assess the animals’ spatial learning andspatial memory at 22–26 days after MCAO (Fig. 7D), but

Page 15 of 21

STAT1 mKO did not cause significant difference compared to WT mice in either the learning phase or the
memory phase of this test (Fig. 7E–G). Collectively, thesedata suggest that by promoting inflammation-resolving
Mi/MΦ responses, STAT1 mKO is effective in inducing long-term improvements in functional recovery following MCAO. These findings highlight the potential of
STAT1 as a promising therapeutic target for the treatment of ischemic stroke.
Sex differences in the outcome after stroke andresponsiveness to therapeutics are well-noted. To
enhance the translatability of stroke therapeutics,
it is crucial to conduct testing of interventions thathave been proven effective in male subjects on female
subjects as well [32]. To this end, we tested whether
STAT1 mKO had similar beneficial effect in improving long-term outcome after stroke in female mice. To
minimize any potential confounding effects associatedwith gonadal hormones and to better reflect the conditions observed in aged female stroke patients [33–35],
we performed ovariectomy prior to MCAO (Fig. 8A).
Ovariectomy leads to the depletion of endogenous

Fig. 7 Microglia/macrophage-targeted STAT1 knockout improves long-term neurological functions in male mice after ischemic stroke. Adult male
STAT1 mKO mice and WT mice were subjected to 1-h MCAO or sham operation. A–C Sensorimotor functions were assessed before (Pre) and up to
35 days after injury by the rotarod test (A), cylinder test (B), and foot fault test (C). D–G Cognitive functions were assessed by the Morris water maze
22–27 days after injury. D Representative swim paths in the learning and memory phases of the Morris water maze test. E Spatial learning assessed
22–26 days after injury demonstrated no difference among all groups. F Spatial memory assessed 27 days after injury. G All groups had similar
swim speeds, indicating comparable gross locomotor functions. n = 10–11 mice/group. *p < 0.05, **p < 0.01, ***p < 0.001 mKO MCAO vs. WT MCAO
by two-way repeated measures ANOVA (bracket in A, B, C, E) or one-way ANOVA (individual timepoint in A, B, C, E; F and G). In B, data on days 5–35
were analyzed. NS no significant differenceHan et al. Journal of Neuroinflammation(2023) 20:178

Page 16 of 21

Fig. 8 Microglia/macrophage-targeted STAT1 knockout ameliorates long-term neurological deficits after ischemic stroke in female mice. A
Experimental timeline. Female STAT1 mKO mice and WT mice were subjected to ovariectomy (OVX). One week after ovariectomy, mice received
intraperitoneal injections of tamoxifen (75 mg/kg daily for 4 days) to induce the deletion of STAT1 in microglia/macrophages. Mice were subjected
to 1-h MCAO or sham operation 3 weeks after ovariectomy. B, C Sensorimotor deficits were examined in ovariectomized female STAT1 mKO and WT
mice up to 35 days after injury by the rotarod test B and cylinder test (C). D–F Spatial learning and memory were assessed in female mice 22–27
days after injury by the Morris water maze. n = 5–7 mice per sham group. n = 11 mice per MCAO group. *p < 0.05, **p < 0.01, ***p < 0.001 mKO MCAO
vs. WT MCAO by two-way repeated measures ANOVA (bracket in B–D) or one-way ANOVA (individual timepoint in B–F). In B, data on days 10–35
were analyzed. NS, no significant difference

estrogen levels and models surgical menopause to
replicate the hormonal milieu of postmenopausal
women to a certain extent. Consistent with the observations in male mice, female STAT1 mKO had better
sensorimotor function than age-matched female WT
mice for at least 35 days after MCAO, as tested by therotarod (Fig. 8B) and cylinder (Fig. 8C) tests. Genotype did not cause differences in the animals’ performance in the Morris water maze test at 22–27 days
after MCAO (Fig. 8D–F). These data suggest that thelong-term beneficial effect of STAT1 mKO toward
improving post-stroke sensorimotor functions is not
sex-dependent.

Selective deletion of STAT1 in Mi/MΦ promotes
the integrity white matter after ischemic stroke

We observed that ischemic brain tissue loss at the acutestage after MCAO was not different between STAT1
mKO and WT mice (Fig. 3B, C). However, given thelong-lasting functional improvement elicited by STAT1
mKO after MCAO, we sought to determine if STAT1
mKO affected the long-term integrity of the post-stroke
brain. Chronic tissue loss in the gray matter measured
on coronal brain sections immunostained with the neuronal marker MAP2 at 35 days after MCAO (Fig. 9A)
was not significantly different between STAT1 mKO
mice and WT mice, regardless of their sex (Fig. 9B),

Han et al. Journal of Neuroinflammation(2023) 20:178

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Fig. 9 Microglia/macrophage-targeted STAT1 knockout improves long-term white matter integrity after ischemic stroke. A, B Chronic
neuronal tissue loss was assessed in male and ovariectomized female STAT1 mKO mice and WT mice 35 days after MCAO on coronal brain
sections immunostained for MAP2 (green). Dash line shows the relative area of the contralesional hemisphere to illustrate ipsilesional tissue
atrophy. n = 10–11 mice/group. C The integrity of nodes of Ranvier (NORs) was assessed by double-label immunostaining of Caspr and Nav1.6
in the peri-lesion external capsule 35 days after MCAO or sham operation. Arrow: intact NOR. Arrowhead: damaged NOR. Rectangle: the region
enlarged in the right columns. D Summarized data on the numbers of NORs, paranode lengths, and lengths of paranode gaps. n = 6–7 mice/group.
##p < 0.01, ###p < 0.001 vs. sham. *p < 0.05, ***p < 0.001 STAT1 mKO vs. WT. NS, no significant difference. E, F Pearson correlation between the animals’
performance in the rotarod test and foot fault test, and various parameters representing gray matter E and white mater F integrity 35 days
after MCAO. Dashed lines: 95% confidence intervals

consistent with observations at more acute stages following MCAO (Fig. 3B, C). However, STAT1 mKO
markedly improved the integrity of white matter. Welabeled the nodes of Ranvier (NORs) in the ipsilateralexternal capsule—a white matter-enriched region—
with contactin-associated protein (Caspr) and sodium
channel Nav1.6 [16]. Caspr represents an axonal membrane protein found at the nodes, while Nav1.6 is predominantly located at the paranodes (Fig. 9C). An
intact NOR was defined as the presence of a gap positive for Nav1.6 between a pair of paranodal Caspr staining. We observed significant loss of morphologicallyintact NORs in the external capsule at 35 days after
MCAO, with a concomitant reduction in the lengthof paranodal Caspr immunosignal of individual NORs
(Fig. 9C, D). Such NOR damage induced by MCAO waseffectively ameliorated in STAT1 mKO mice compared
to WT mice at 35 days after MCAO (Fig. 9C, D), suggesting improved white matter integrity in the STAT1mKO mice. The lengths of the NORs (paranodal gaps)
were not different among all groups (Fig. 9D).

To test whether the improved white matter integrity
underlies better sensorimotor functions in the STAT1mKO mice, we performed Pearson correlation analyses on the same cohort of mice subjected to both neurobehavioral tests and immunostaining. While the graymatter tissue loss at 35 days after MCAO did not demonstrate strong correlation with the animals’ sensorimotor deficits detected by the rotarod test and foot fault test
(Fig. 9E), the animals’ performance in both behavioral
tests was significantly correlated with the parameters forwhite matter integrity (number of NORs and paranodelength; Fig. 9F). In conclusion, these data suggest that
STAT1 mKO-elicited long-term functional improvement
after MCAO was attributable, at least partially, to the rescued white matter integrity.

Discussion
In this study, we utilized a conditional knockout mousemodel to delineate the role of STAT1 in modulating thefunction of Mi/MΦ following an ischemic stroke. Ourfindings provide compelling evidence that STAT1 plays

Han et al. Journal of Neuroinflammation(2023) 20:178

a significant role in promoting proinflammatory Mi/MΦ responses. Importantly, in vivo selective deletion of
STAT1 drives Mi/MΦ toward an inflammation-resolving
phenotype, resulting in mitigation of neuroinflammationand improvement of long-term stroke outcomes.
STAT1 is a transcription factor that is potently activated in canonical interferon signaling, and is defined
as an important mediator of macrophage proinflammatory M1 polarization [36]. Independent of its role
downstream of interferon gamma, STAT1 also promotes
apoptosis through regulating caspases [37, 38]. While
the pro-apoptotic role of STAT1 had been examined in
the context of ischemic stroke in previous studies [9, 39,40], its proinflammatory function had so far only been
implicated in an indirect and correlative way in existingstudies. Reduced activity of STAT1 was found by several studies to accompany less neuroinflammation upontreatment of anti-inflammatory drugs [41, 42]; however,a direct role of STAT1 in regulating inflammation, especially through modulating the phenotype of Mi/MΦ, isunknown. Our study addressed several crucial questions
that were previously unanswered. First, we revealed thetemporal profile of STAT1 expression in different typesof brain cells during the acute to subacute stages following brain ischemia and reperfusion. Second, by utilizing
Mi/MΦ-targeted conditional knockout of STAT1, wewere able to specifically investigate the role of STAT1
in Mi/MΦ, separate from its effects in other cell types,
such as neurons. While the deletion of STAT1 in Mi/MΦ
did not directly impact acute neuronal death, it resulted
in decreased proinflammatory and neurotoxic Mi/MΦ
responses. Third, our study established a direct role for
Mi/MΦ STAT1 in determining long-term stroke outcomes. Although compensatory changes in Mi/MΦ orother cell types may occur, they are secondary to the primary effects of Mi/MΦ STAT1 deletion.
As a transcription factor, STAT1 has a lot of target
genes. In our study, we observed changes in several predefined phenotype markers upon STAT1 deletion in Mi/
MΦ, one of which is Arg1. Arg1 metabolizes L-arginine
into urea and ornithine and is a countermeasure to iNOS,
which produces nitric oxide from arginine [43]. Arg1 isboth a prototypic marker and a strong inducer of M2
macrophages [43]. The functional implication of Arg1
is twofold: first, by competing with iNOS for substrates,
Arg1 has potent anti-inflammatory effect; second, Arg1
was previously reported to enhance phagocytic function of Mi/MΦ after ischemic stroke [22, 44]. It is, therefore, an important question for future studies to assess
whether STAT1 deletion promotes phagocytic clearance of debris or dead cells by Mi/MΦ. The mechanism
underlying the increase in Arg1 upon STAT1 knockout is not yet fully understood. Transcription of Arg1 isPage 18 of 21

prominently induced by IL-4, which requires the directbinding of both STAT6 and cAMP-response elementbinding protein (CREB)-binding protein (CBP) to a special DNA response element in the Arg1 promoter [45,46]. We are currently testing the possibility that STAT1
suppresses Arg1 expression by inhibiting the bindingof CBP to the Arg1 promoter. On the other hand, proinflammatory (“M1”-like) and inflammation-resolving(“M2”-like) markers were largely derived from in vitro
polarization studies and may not be as informative about
the activation and functional states of Mi/MΦ in vivo.
In this regard, our ongoing studies are also exploringchanges in gene expression of STAT1-null Mi/MΦ using
unbiased RNA-seq at the whole transcriptome level. This
comprehensive approach will enable us to gain deeper
insights into the functional states of Mi/MΦ and downstream targets of STAT1, going beyond the pre-defined
phenotypic markers examined in the current study. By
analyzing the entire transcriptome, we aim to identify
novel targets and pathways that are influenced by STAT1
deletion in Mi/MΦ. It is important to acknowledge that
our current method of standard enzymatic digestionof brain tissues at 37 °C to prepare cell suspensions forRNA-seq could potentially induce ex vivo transcriptionalchanges in microglia [47]. However, we took measures
to mitigate the impact of these artifacts by subjecting
all groups to identical cell isolation procedures for a faircomparison. Future studies should consider optimizing
the cell isolation protocols, e.g., by adding transcriptional
and translational inhibitors or using a mechanical dissociation protocol conducted at low temperatures [47] tominimizing the activation of microglia ex vivo.
One limitation of our study is that we did not clearly
separate microglia from macrophages in most of ourassessment parameters due to the lack of definitive markers that can reliably distinguish between these two cell
types. Traditionally, microglia have been considered as­CD45low, while macrophages are ­CD45high. However,activated microglia can upregulate CD45 expression andbecome indistinguishable from macrophages in terms ofCD45 expression. In addition, recently identified markers, such as Tmem119, which were thought to be "microglia-specific," may have altered expression under disease
conditions [48], raising concerns about their reliability as
markers for microglia in the context of ischemic injury.
In our study, we utilized Mi/MΦ-targeted STAT1 knockout mice generated under the Cx3cr1 promoter, which isstrongly expressed by both microglia and macrophages,
including those macrophages residing at the CNS borders (i.e., meningeal, perivascular and choroid-plexus
macrophages) [49]. Using these mice, we found STAT1
in both microglia and macrophages could be important.
Future studies can employ strategies to more definitively

Han et al. Journal of Neuroinflammation(2023) 20:178

separate microglia and macrophages to determine if
microglia and macrophage STAT1 have redundant functions or if one dominates over the other in driving thephenotypic changes observed. For example, a tamoxifen
pulse knockout strategy employed in our recent study[50] could be used to restrict the deletion of STAT1 to
only occur in long-living cells, such as microglia. Recent
advancements in identifying new markers may also leadto the development of Cre strains with improved targeting specificity, particularly in distinguishing microglia
from CNS border-associated macrophages [48]. Notably,the ­Cx3cr1CreER mice used in this study did not target all
monocyte-derived macrophages due to the existence ofthe ­CX3CR1−Ly6Chi monocyte subset [51]. Therefore, in
our STAT1 mKO mice, STAT1 deletion occurred in mostof the microglia but only part of the macrophages in thebrain. To further reveal the role of macrophage STAT1,
future studies utilizing monocyte-targeting tools (e.g., therecently developed C­ CR2CreER mice [52]) are warranted.
Another limitation of the ­Cx3cr1CreER mice used in thecurrent study is that these mice lack one copy of the fulllength endogenous Cx3cr1 allele [11]. To compensate forthe potential impact of CX3CR1 haploinsufficiency on
the phenotype of Mi/MΦ, we used C­ x3cr1CreER hemizygous mice as WT controls to the STAT1 mKO mice, sothat both groups of mice had identical Cx3cr1 alleles.
We found that the deletion of STAT1 in Mi/MΦ
resulted in less brain inflammation at the subacute stage
after ischemic stroke, manifested by less infiltrationof blood-borne immune cells and reduced content ofcytokines in the post-stroke brain. However, it is important to note that immune cells and cytokines may also
play a beneficial role in the chronic stage of stroke byaiding in tissue repair and remodeling. While the deletion of STAT1 in Mi/MΦ did not affect chronic neuronaltissue loss at 35 days after MCAO, it had a significant
positive impact on white matter integrity. The improvement in white matter integrity is believed to contributeto the observed enhancement in sensorimotor functions.
We observed improvements in neurological functions
in both male and female STAT1 mKO mice, suggestingthat the therapeutic targeting of STAT1 may be effective
regardless of sex. It should be noted that in our study,
ovariectomized female mice were used to partially mimic
the systemic conditions of aged women, who make up
the majority of stroke patients. However, it is worth considering that there may be other hormone-independent
mechanisms that contribute to sex differences in treatment efficacy. Considering the known differences in
microglia physiology during aging [53, 54], future studies
should aim to test the conclusions of our research in aged
subjects to provide valuable insights for potential therapeutic interventions in elderly stroke patients.

Page 19 of 21

The identification of therapeutic targets for stroke treatment remains an urgent need. In our current study, we
employed genetically engineered mice and demonstrated
that STAT1 is a promising target for reducing inflammation and secondary ischemic injury by modulating Mi/
MΦ phenotype. Future studies are warranted to investigate the translatability of the conclusions drawn from thepresent study to clinical stroke treatment. To this end,
STAT1 inhibitors are promising agents to be tested fortheir therapeutic efficacy against ischemic stroke, e.g., thepurine analog fludarabine. Fludarabine is approved by the
FDA to treat hematologic malignancies for its ability toinhibit DNA synthesis [55]. It can also specifically deplete
STAT1 mRNA and protein but not other STATs [56],thereby exerting anti-inflammatory and immunosuppressive effects. As a nucleoside analog, fludarabine can easily
cross the BBB, and oral administration of this drug has
similar tolerability profile and clinical efficacy comparedto the intravenous route [57, 58]. We recently reported
that systemically administered fludarabine exert potent
anti-inflammatory effects in a mouse model of traumaticbrain injury [19]. We are currently conducting investigations to assess the therapeutic potential of this drug forthe treatment of ischemic stroke.

Conclusions
STAT1 plays a significant role in promoting proinflammatory responses of Mi/MΦ following ischemic stroke.
By selectively deleting STAT1 in Mi/MΦ, the post-stroke
neuroinflammation can be effectively mitigated, leading to improved long-term functional recovery. Thesefindings highlight the potential of targeting STAT1 as apromising therapeutic approach for stroke treatment.
Such intervention has the potential to enhance beneficial Mi/MΦ responses and ultimately improve long-term
outcomes in stroke patients.
Abbreviations
Arg1	Arginase 1
Caspr	Contactin-associated protein
DAMP	Damage-associated molecular pattern
DEG	Differentially expressed gene
FACS	Fluorescence-activated cell sorting
FDR	False discovery rate
HMGB1	High mobility group box 1
IL	Interleukin
iNOS	Inducible nitric oxide synthase
IPA	Ingenuity pathway analysis
MAP2	Microtubule-associated protein 2
MCA	Middle cerebral artery
MCAO	Middle cerebral artery occlusion
Mi/MΦ	Microglia and macrophagesmKO	Microglia/macrophage-specific knockout
NOR	Node of Ranvier
PCR	Polymerase chain reaction
rCBF	Regional cerebral blood flow
RNA-seq	RNA sequencing

Han et al. Journal of Neuroinflammation(2023) 20:178

STAT​	Signal transducer and activator of transduction
TNF-α	Tumor necrosis factor alpha
WT	Wild type

Supplementary Information
The online version contains supplementary material available at https://​doi.​
org/​10.​1186/​s12974-​023-​02860-4.
Additional file 1: Table S1. Key resources.
Additional file 2: Table S2. List of the 2183 differentially expressed genes(DEGs) induced by MCAO in microglia compared to sham controls (related
to Fig. 1). Genes are ranked according to fold changes (MCAO vs. sham)from smallest to largest.
Acknowledgements
This project used the University of Pittsburgh Health Sciences Core Research
Facilities (HSCRF) Genomics Research Core RNA sequencing service. Data
analysis was performed using Ingenuity Pathway Analysis software licensed
through the Molecular Biology Information Service of the Health Sciences
Library System, University of Pittsburgh.
Author contributions
YS and JC designed the project. WH, HP, SL, YL, YZ, MX, CC, YW, TY and QY
performed the experiments. WH, HW and YS analyzed the data. WH prepared
the figures and wrote the manuscript. RAS, JC and YS critically revised themanuscript. All authors read and approved the final manuscript.
Funding
This work was supported in part by the National Institutes of Health grant
R01NS117550 and the American Heart Association grant 17SDG33630130 (toY.S.). J.C. is the Richard King Mellon Professor of Neurology and a recipient of
the Senior Research Career Scientist Award (IK6 BX006298) and Merit Reviews(BX002495 and BX003377) from the U.S. Department of Veterans Affairs.
Availability of data and materials
RNA sequencing data are deposited in the Gene Expression Omnibus database at the National Center for Biotechnology Information (GSE141972 andGSE234865). Other data sets used and/or analyzed in this study are available
from the corresponding author upon reasonable request.

Declarations
Ethics approval and consent to participate
All animal experiments were approved by the University of Pittsburgh Institutional Animal Care and Use Committee and were performed in accordancewith the NIH Guide for the Care and Use of Laboratory Animals.
Consent for publication
Not applicable.
Competing interests
The authors declares that they have no competing interests.
Author details
1
Pittsburgh Institute of Brain Disorders and Recovery and Department of Neurology, University of Pittsburgh, 3500 Terrace Street, S‑510 BST, Pittsburgh, PA
15213, USA. 2 Department of Biostatistics, University of Pittsburgh, Pittsburgh,
PA 15213, USA. 3 Geriatric Research, Education and Clinical Center, Veterans
Affairs Pittsburgh Health Care System, Pittsburgh, PA 15261, USA.
Received: 9 June 2023 Accepted: 25 July 2023

Page 20 of 21

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