Wu et al. Journal of Neuroinflammation(2024) 21:178
https://doi.org/10.1186/s12974-024-03171-y

Journal of Neuroinflammation

Open Access

RESEARCH

PEDF-34 attenuates neurological deficit
and suppresses astrocyte-dependentneuroinflammation by modulating astrocyte
polarization via 67LR/JNK/STAT1 signaling
pathway after subarachnoid hemorrhage
in rats
Lei Wu1,2,3†, Yanchao Liu1†, Qiuguang He2†, Guangnan Ao1, Ningbo Xu1,2,4, Wangqing He1, Xiao Liu1, Lei Huang2,5,
Qian Yu2, Hideki Kanamaru2, Siyuan Dong2, Shiyi Zhu2, Ye Yuan2, Mingyang Han2, Yeping Ling2, Lu Liu2, Chenyu Wu2,
You Zhou2, Prativa Sherchan2, Jerry J. Flores2, Jiping Tang2, Xionghui Chen2,6*, Xuying He1,3* and John H. Zhang2,5*
Abstract
Background Reactive astrocytes participate in various pathophysiology after subarachnoid hemorrhage (SAH),
including neuroinflammation, glymphatic–lymphatic system dysfunction, brain edema, BBB disruption, and cell
death. Astrocytes transform into two new reactive phenotypes with changed morphology, altered gene expression,and secretion profiles, termed detrimental A1 and beneficial A2. This study investigates the effect of 67LR activation
by PEDF-34, a PEDF peptide, on neuroinflammation and astrocyte polarization after the experimental SAH.
Methods A total of 318 male adult Sprague-Dawley rats were used in experiments in vivo, of which 272 rats were
subjected to the endovascular perforation model of SAH and 46 rats underwent sham surgery. 67LR agonist (PEDF34) was administrated intranasally 1 h after SAH. 67LR-specific inhibitor (NSC-47924) and STAT1 transcriptional
activator (2-NP) were injected intracerebroventricularly 48 h before SAH. Short- and long-term neurological tests,
brain water content, immunostaining, Nissl staining, western blot, and ELISA assay were performed. In experiments
in vitro, primary astrocyte culture with hemoglobin (Hb) stimulation was used to mimic SAH. The expression of the†Lei Wu, Yanchao Liu and Qiuguang He contributed equally to thiswork.
*Correspondence:
Xionghui Chenxhchen@suda.edu.cn
Xuying He
2517079319@qq.com
John H. Zhang
johnzhang3910@yahoo.com
Full list of author information is available at the end of the article© The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use,sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) andthe source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in thisarticle are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not includedin the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you willneed to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The
Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available
in this article, unless otherwise stated in a credit line to the data.

Wu et al. Journal of Neuroinflammation(2024) 21:178

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PEDF-34/67LR signaling pathway and neuro-inflammatory cytokines were assessed using Western blot, ELISA, andimmunohistochemistry assays both in vivo and in vitro.
Results Endogenous PEDF and 67LR expressions were significantly reduced at 6 h after SAH. 67LR was expressedin astrocytes and neurons. Intranasal administration of PEDF-34 significantly reduced brain water content, proinflammatory cytokines, and short-term and long-term neurological deficits after SAH. The ratio of p-JNK/JNK and
p-STAT1/STAT1 and the expression of CFB and C3 (A1 astrocytes marker), significantly decreased after PEDF-34
treatment, along with fewer expression of TNF-α and IL-1β at 24 h after SAH. However, 2-NP (STAT1 transcriptional
activator) and NSC-47924 (67LR inhibitor) reversed the protective effects of PEDF-34 in vivo and in vitro by promoting
A1 astrocyte polarization with increased inflammatory cytokines.
Conclusion PEDF-34 activated 67LR, attenuating neuroinflammation and inhibiting astrocyte A1 polarization partly
via the JNK/STAT1 pathway, suggesting that PEDF-34 might be a potential treatment for SAH patients.
Keywords Subarachnoid hemorrhage, Non-integrin 67-kDa laminin receptor, Pigment epithelial-derived factor,
Astrocyte polarization, Neuroinflammation
Graphical Abstract

Introduction
Subarachnoid hemorrhage (SAH) is a life-threatening
neurovascular disease with a 25–50% fatality rate andhigh disability [1–3]. Early brain injury (EBI) is the primary cause of mortality in SAH patients [4], which consists of acute pathological processes occurring in thebrain within the first 72 h after SAH. Neuroinflammation
is attributed significantly to EBI pathogenesis. Therefore,anti-neuroinflammatory approaches would be beneficial
in treating SAH.
Pigment epithelial-derived factor (PEDF), a 50-kDa
monomeric glycoprotein and member of the serine protease inhibitor superfamily [5], protects cells from oxidative stress [6]. Previous studies demonstrated theanti-inflammation properties of PEDF [7–12] which
effectively inhibited expressions of inflammatory

Wu et al. Journal of Neuroinflammation(2024) 21:178

cytokines TNF-α and IL-1β in dry eye disease [8, 13].
By activating the non-integrin 67-kDa laminin receptor (67LR), PEDF reduced myocardial infarct size andvascular leakage in rats [10]. 67LR is involved in cell
adherence to the basement membrane and regulates theinteractions between laminin and other receptors [14].
The laminin receptor activation inhibits endothelial tissue factor expression by inhibiting JNK phosphorylation[15], thus inhibiting the phosphorylation and transcription of STAT1 [3, 16, 17]. In addition, PEDF-peptide wasshown to upregulate AQP4 by activating 67LR in astrocytes, maintaining the integrity of the blood-brain barrier(BBB) [18, 19]. However, the effects of 67LR activation by
PEDF in EBI after SAH have never been reported.
Astrocytes are important in maintaining normal BBB
structures and functions in the central nervous system(CNS). Upon being stimulated by various inflammatory
cytokines after SAH, resting astrocytes become reactive and display two phenotypes, A1 and A2 [20, 21].
A1 astrocytes, marked with complement factor B (CFB)
or complement component 3 (C3), cause cell death byreleasing pro-inflammatory factors, while A2 astrocytes, marked with S100A10, perform neuroprotective
effects by secreting anti-inflammatory mediators [22]. A1
astrocytes were prevalent and persistent relative to A2
astrocytes in EBI after SAH, contributing to neuroinflammation and neuronal death [23–25]. Prokineticin 2 hasbeen shown to promote astrocytic polarization to an A2
phenotype and reduce SAH-induced neuronal injury andbehavior dysfunction [23]. Furthermore, inhibiting transformation into A1 astrocytes would attenuate neuronal
death by reducing the release of pro-inflammatory factors in EBI after SAH [25].
In the present study, the effects of PEDF/67LR signaling on astrocyte polarization and neuroinflammation
were investigated in experimental SAH. Considering thetherapeutic limitations of full-length PEDF, including
its short half-life, unstable pharmacological activity, anddelivery pathway [26], PEDF-34, a peptide fragment of
PEDF, was used [27]. The hypothesis was that activating
67LR by PEDF-34 would attenuate neuroinflammationand downregulate A1 astrocyte polarization via inhibiting the JNK/STAT1 pathway after SAH.

Materials and methods
Animals

All experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of
Loma Linda University (#23 − 004) following the National
Institutes of Health Guidelines for the Care and Use of
Animals in Neuroscience Research and ARRIVE guidelines. The study utilized 318 male Sprague-Dawley rats
(weighing 270–320 g, Envigo, Indianapolis, IN, USA),and the specific rat usage is presented (Additional file:

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Figure S1). Animals were housed in a controlled environment maintained at a constant temperature of 25 ± 1 °C
and relative humidity of 60 ± 5% and had access to foodand water on a 12-hour light-dark cycle.
SAH model

A previously published endovascular perforation SAH
model was used [28]. Briefly, rats were intubated andmechanically ventilated with 2–3% isoflurane in the air,maintaining a respiratory rate of 77 throughout the operation (isoflurane was reduced to 1.5% after puncture).
A sharp 4 − 0 monofilament polypropylene suture wasinserted from the external carotid artery and common
carotid bifurcation into the left internal carotid artery.
The nylon suture was advanced until encountering resistance at the bifurcation of the anterior and middle cerebral arteries, then pushed further to puncture the bloodvessels before being immediately withdrawn. The rats in
the Sham group underwent the same process above but
without vessel puncture. The skin incision was sutured
after surgery, and the rats were kept on a heating pad at
37.5 °C. Continuously monitor the vital signs of rats until
they fully recover.
Assessment of SAH grade

The SAH grade was evaluated according to the distribution of blood and blood clots in the six parts of the basal
brain cistern. Each part was considered by a score from0 to 3 in terms of the amount of subarachnoid blood andblood clots present [29, 30]. The mildly injured rats witha score of 8 or less at 24 h after SAH were excluded [30,31]. The assessment was done by a researcher who was
blind to the information of experimental groups.
Cell culture and SAH model in vitro

Primary astrocyte culture and hemoglobin stimulation
were used as an in vitro model of SAH. Primary astrocytes were extracted from P2 Sprague–Dawley rat pups.
Briefly, 10 ml of HBSS (14,175,095, Gibco, Carlsbad,
USA) was added to the culture dishes. After anesthesia
and sterilization, the pups’ heads were cut off and put
into HBSS. Whole brain tissues were removed from the
pup’s heads, and pia matter was carefully separated fromthe brains. Cortex tissues were collected, transferred toa centrifuge tube, and lysed with 0.25% EDTA-trypsin(25,200,072, Gibco, Carlsbad, USA) at 37 °C for 15 min.
Then, the lysing was terminated by a solution containing 79% high glucose DMEM (11,965,092, Gibco, Carlsbad, USA), 20% FBS (A5256701, Gibco, Carlsbad, USA),and 1% Antibiotic-Antimycotic (100×, 15,240,062, Gibco,
Carlsbad, USA). After centrifuging at 1000 rpm for
5 min, the cells were re-suspended with astrocyte culture medium, which contained 88% high glucose DMEM,
10% FBS, 1% Antibiotic-Antimycotic and 1% GlutaMAX™

Wu et al. Journal of Neuroinflammation(2024) 21:178

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Supplement (35,050,061, Gibco, Carlsbad, USA). The
cells were transferred to dishes and cultured in an incubator at 37 °C, with 5% CO2. The medium was halfchanged every 4 days. After 7–8 days of culture, astrocytes were subjected to in vitro experiments.
For Hemoglobin stimulation, rat primary astrocytes
were seeded onto 6-well plates at a density of 2 × 105,
and hemoglobin (9008-02-0, Sigma, USA) was dissolved
by PBS at a concentration of 250µM (stock solution)(161 mg Hemoglobin + 10 ml PBS). After culture maturations, the medium was completely replaced with a normal medium supplemented with a final concentrationof 25µM Hemoglobin (0.2 ml Hemoglobin stock solution + 1.98 ml normal medium).

(3 µg /kg). The Rotarod test was conducted on days 7, 14,
and 21 after SAH. Morris water maze was performed ondays 23–27 after SAH. The rats were euthanized on day
28, and Nissl staining was performed to assess brain neuronal degeneration.

Experimental design

Experiment 5: in vivo mechanism study (67LR/JNK/STAT1
signaling pathway)

The supplementary file (Additional file: Figures S1 andS2) shows the experimental design and the distribution ofanimals according to different groups.
Experiment 1: time course of PEDF, 67LR, p-JNK, JNK, p-STAT1,
STAT1, CFB, C3, S100A10 and cellular localization of 67LR in
SAH rat

Forty-two rats were randomly assigned to seven groups(n = 6/group): Sham group and six SAH groups (3 h, 6 h,
12 h, 24 h, 48 h, and 72 h after SAH). Western blot analysis was used to quantify expression changes of PEDF,
67LR, p-JNK, JNK, p-STAT1, STAT1, CFB, C3, and
S100A10. An additional 8 rats were randomly assigned to
the Sham (n = 4) and SAH group (24 h, n = 4) to evaluate
cellular localization of 67LR using double immunofluorescence staining.
Experiment 2: the effect of 67LR activation by PEDF-34 on
short-term neurological outcomes after SAH

Thirty rats were randomly assigned to five groups (n = 6/
group): Sham, SAH + Vehicle, SAH + PEDF-34 (1 µg/kg),
SAH + PEDF-34 (3 µg/kg), and SAH + PEDF-34 (9 µg/kg).
Modified Garcia score, beam balance, and brain watercontent assessments were conducted 24 h after SAH.
Based on the short-term neurological outcomes, the bestdose of PEDF-34 (3 µg /kg) was selected for the following
experiments, including long-term neurological outcomes
and anti-neuroinflammation mechanism. Additional
12 rats were randomly divided into three groups (n = 4/group), Sham, SAH + Vehicle, and SAH + PEDF-34 (3 µg/kg), to assess expressions of IL-1β and IL-10 in the ipsilateral hemisphere at 24 h after SAH using double-immunofluorescence staining.
Experiment 3: the effect of PEDF-34 on long-term
neurological outcomes after SAH

Thirty rats were randomly assigned to three groups(n = 10/group): Sham, SAH + Vehicle, SAH + PEDF-34

Experiment 4: dosage testing of NSC-47924 and 2-NP in SAH
rat model

For the dose test of the 67LR inhibitor, NSC-47924, andSTAT1 transcriptional activator (2-NP), 60 rats wererandomly divided into ten groups. Western blotting
was conducted to measure the expression of the 67LR
and its downstream p-JNK, JNK, p-STAT1, and STAT1
expression.

Thirty-six rats were randomly divided into six groups(n = 6/group): Sham, SAH + Vehicle, SAH + PEDF34 (3 µg/kg), SAH + PEDF-34 (3 µg/kg) + Vehicle,
SAH + PEDF-34 (3 µg/kg) + NSC-47924 (50 µg/kg), andSAH + PEDF-34 (3 µg/kg) + STAT1 transcriptional activator (2-NP, 15 µg/kg). The expressions of 67LR, p-JNK,
JNK, p-STAT1, STAT1, CFB, C3, and S100A10 were
evaluated by Western blot assay. 36 rats were randomly
divided into the same six groups for ELISA assay as theWestern blotting experiments group (n = 6/group). The
ipsilateral brain hemisphere was collected to measure
the cytokine level, including TNF-α, IL-1β, and IL-10.
Another set of 24 rats was divided randomly into thesame 6 groups (n = 4/group); immunofluorescence staining was performed to assess astrocyte polarization by costaining of C3 (A1 marker) or S100A10 (A2 marker) withGFAP (astrocyte marker).
Experiment 6: in vitro mechanism study (primary astrocyte
culture)In an in vitro experiment, primary astrocyte culture
was used, and SAH was mimicked by hemoglobin (Hb)stimulation. Knockdown efficacies were measured bywestern blot after transfection with different MOIs of sh67LR (MOI: 1:1, 3:1, 10:1). Cell viability was conductedby CCK-8 assay after treatment with different dosesof 2-NP (13.5µM, 45µM, 135µM). Cultured cells wererandomly divided into six groups: Sham, Hb + Vehicle,
Hb + PEDF-34, Hb + PEDF-34 + 67LR sh-control (MOI3:1), Hb + PEDF-34 + sh-67LR (MOI 3:1), Hb + PEDF34 + 2-NP (13.5 µΜ). Western blotting was performed tomeasure the expression of 67LR, p-JNK, JNK, p-STAT1,
STAT1, CFB, C3, and S100A10 levels (n = 6/group).
ELISA was performed to measure the cytokines level,
including TNF-α, IL-1β, and IL-10 (n = 6/group). Astrocyte polarization was assessed by co-localization of C3

Wu et al. Journal of Neuroinflammation(2024) 21:178(A1 marker) or S100A10 (A2 marker) with GFAP byimmunofluorescence staining (n = 4/group).
Drug administration

In in vivo experiments, a vehicle (10% DMSO) or PEDF34 (1 µg/kg, 3 µg/kg, or 9 µg/kg) was administrated intranasally 1 h after SAH. For the short-term neurologicaloutcomes and mechanism study, PEDF-34 was administered at 1 h post-SAH, once daily for 7 days after SAH
for long-term neurological tests. The 67LR inhibitor(NSC-47924, 50 µg/kg, Focus Biomolecules, USA) andthe STAT1 transcriptional activator (2-(1,8-naphthyridin-2-yl) phenol, 2-NP; 15 µg/kg, ab142704, Abcam,
Cambridge, USA) were administrated intracerebroventricularly (i.c.v.) 48 h before SAH. For i.c.v injection in
isoflurane-anesthetized rats, a 10 µl Hamilton syringe(Microliter 701, Hamilton Company, USA) was inserted
through a burr hole on the skull into the left lateral ventricle (0.9 mm posterior and 1.5 mm lateral to bregma,
3.2 mm depth). The rate of i.c.v injection was controlled
at 1 µl/min using an infusion pump. After injection,
the needle was kept in situ for 5 min and then slowly
retracted. The burr hole was sealed with bone wax immediately after removal of the needle. The rats were kept on
a warm blanket to recover from anesthesia.
In in vitro experiments, PEDF-34 (10.8 pM) [32] wasadministrated 1 h after Hb stimulation. STAT1 transcriptional activator (2-NP, 13.5 µM) was administrated 48 h
before Hb stimulation. 67LR shRNA was administrated
to culture medium 72 h before Hb stimulation. The vehicle and Hb groups were given the same dose of sh-control. 67LR shRNA and sh-control were customized andpurchased from Horizon Discovery Biosciences Limited(V3SR7594-237950775, Horizon Discovery Biosciences
Limited, Cambridge, UK). The sequence is listed as follows: sh-67LR: 5′- ​G​G​G​T​T​C​T​C​A​A​T​G​G​C​A​A​C​A​A-3′;
sh-control, 5′-​G​C​A​T​A​T​G​T​G​C​G​T​A​C​C​T​A​G​C​A​T-3′. The
virus titer of shRNA was 1.0 × 108 TU/ml. The western
blot result of the MOI test indicated that 3:1 is performing the best transfection ability (Additional file: Figure
S3B). The STAT1 transcriptional activator (2-NP) dosetest was performed [33] (Additional file: Figure S3E, F).
Short-term neurological performance evaluation

The modified Garcia scale consists of six parts with 0–3
points for each part: judgments of spontaneous activity,
symmetry in the movement of four limbs, forepaw outstretching, climbing, body proprioception, and response
to vibrissae touch. In the beam balance test, the walking
distance on a beam was evaluated within 1 min using ascore of 0–4 (a higher score indicates a longer walking
distance).

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Long-term neurological performance evaluation
Rotarod test

As described previously [14], the Rotarod test assessed
motor coordination and balance by measuring the time
staying on a rotating rod on days 7, 14, and 21 after SAH.
The rotating speed was started at 5 RPM or 10 RPM andincreased by 2 RPM every 5 s. A photo beam circuitrecorded how long rats could stay on the acceleratingrotating cylinder.
Morris water maze

The water maze test was initiated between days 22 and
27 to assess the spatial learning capacity and memory. As
previously demonstrated [34], a cue water maze test was
conducted on day 22 followed by spatial water maze teston 23–26 days after SAH. On day 27, the probe test was
performed, and the animals were placed in the pool without the escaping platform. The swim speed and path of
the animals were evaluated using the video tracking system SMART-2000 (Noldus Ethovision; Noldus, Tacoma,
USA).
Brain water content

The brain water content (BWC) assessment was consistent with 4 parts of the rat’s brain: the left brain,right brain, cerebellum, and brain stem. Each part wasweighed as its wet weight and then obtained its constant
dry weight by drying in a 105 °C oven for 72 h. The percentage of BWC was calculated using the formula: [(wetweight - dry weight)/wet weight] × 100%.
Immunofluorescence (IF)In in vivo experiments, anesthetized animals were euthanized with cold 10% formalin transcranial perfusion
at various time points after SAH or sham surgery (seestudy design). The brains were post-fixed in 10% formalin at 4°C for 24 h, followed by dehydration in 70%
sucrose for another 72 h After the brain was extracted
and embedded in Scigen Tissue-Plus™ O.C.T. Compound(23-730-571, Thermo Fisher Scientific, Carlsbad, USA),brain sections were cut into 10 µm coronal slices in acryostat (LM3050S, Leica Microsystems, Bannockburn,
Germany) and mounted on adhesive glass slides. For IF
staining, slides were washed by 1× PBS three times andincubated in 0.1% Triton-X-100 (Thermo Fisher Scientific, Carlsbad, USA) for 30 min at 4° C. After adding 5%
donkey serum to block for 1 h at a room temperature, theslides were incubated with the primary antibodies overnight at 4° C. The primary antibodies were C3 (1:100,
ab200999, Abcam, Cambridge, USA), S100A10 (1:100,
PA5-95505, Thermo Fisher Scientific, Carlsbad, USA)and 67LR (1:100, ab133645, Abcam, Cambridge, USA),GFAP (1:200, ab279289 Abcam, Cambridge, USA), NeuN
(1:200, ab177487, Abcam, Cambridge, USA), and Iba1.

Wu et al. Journal of Neuroinflammation(2024) 21:178(1:200, ab178846, Abcam, Cambridge, USA). On thesecond day, slides were washed with 1× PBS three times
and incubated with the second antibody for 1 h at room
temperature. The slides were washed with 1× PBS threetimes, and Aqueous Mounting Medium with DAPI (4’,6-Diamidino-2-Phenylindole, Dihydrochloride, Jackson
ImmunoResearch, West Grove, USA) was used before
coverslips were applied. A fluorescence microscope(DMi8, Leica Microsystems, Germany) observed andphotographed the slides.
In an in vitro experiment, the cultured primary astrocyte cells were fixed in 4% paraformaldehyde for 30 min
at 48 h after Hb stimulation. After being blocked in 1%
BSA for 1 h, the cells were incubated with primary antibodies against C3 (1:100, ab200999, Abcam, Cambridge,USA), S100A10 (1:100, PA5-95505, Thermo Fisher Scientific, Carlsbad, USA, ) or GFAP (1:500, ab279289
Abcam, Cambridge, USA) overnight at 4 °C. The nextday, the cells were incubated with appropriate secondary fluorescent antibodies. Finally, cell nuclei underwent
counterstaining of DAPI. The slides were observed andphotographed using a confocal microscope (Leica, Wetzlar, Germany).
ELISA

ELISA samples of brain tissue (left hemisphere) andcell cultures previously described [35]. TNF-α, IL-1β,
and IL-10 were quantified using TNF alpha ELISA Kit(ab236712, Abcam, Cambridge, USA), IL-1β ELISA Kit(ab255730, Abcam, Cambridge, USA), and IL-10 ELISA
Kit (ab214566, Abcam, Cambridge, USA) in accordancewith the manufacturer’s instructions.
Western blot analysis

The left ipsilateral hemisphere tissue was homogenizedin RIPA lysis buffer (sc-24,948, Santa Cruz Biotechnology, USA) with protease inhibitor cocktail for 15 min
and then centrifuged at 14,000 g at 4 °C for 30 min.
After collecting the supernatant, protein quantification was performed using a detergent-compatible assay(Bio-Rad, DC™ Protein Assay). Western blotting was
performed as described previously [36]. The primary
antibodies included the Anti-PEDF (1:1000, ab307083,
Abcam, Cambridge, USA), Anti-67LR antibody (1:1500,
ab133645, Abcam, Cambridge, USA), Anti-p-JNK antibody (1:2000, ab131499, Abcam, Cambridge, USA), AntiJNK antibody (ab179461, Abcam, Cambridge, USA),
Anti-p-STAT1 antibody (1:1000, sc-8394, Santa Cruz
Biotechnology Inc., USA), Anti-STAT1 antibody(1:1000,
sc-464, Santa Cruz Biotechnology Inc., USA), AntiCFB antibody (1:1500, ab231072, Abcam, Cambridge,USA), Anti-C3/C3b antibody (1:1000, ab200999, Abcam,
Cambridge, USA), Anti-S100A10 antibody (1:1000,

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PA5-95505, Thermo Fisher Scientific, Carlsbad, USA),
and Anti-β-actin (1:1000, ab8227, Abcam, Cambridge,
USA).
Statistical analysis

All analyses were performed and graphed using GraphPad Prism 10.0 software (GraphPad Software, La Jolla,
CA, USA). The data were presented as mean ± standard
deviation (means ± SD). A one-way analysis of variance(ANOVA) followed by Tukey’s post hoc test was used totest the differences among three or more groups. Longterm neurobehavioral tests were analyzed using Two-way
ANOVA. Differences were considered significant whenp < 0.05.
ResultMortality and SAH grade

A total of 318 rats were used, of which 46 rats underwent
sham surgery, and 272 rats underwent SAH induction. Of
the 272 SAH rats, 9 were excluded from this study due
to low SAH grading scores. The overall mortality of SAH
rats was 11.79% (31/263). No rats in the sham group died.
Subarachnoid blood clots were around the circle of Willis in the SAH group rats (Additional file: Figure S1A).
There was no statistical difference in SAH grading scoresamong the SAH groups. (Additional file: Figure S1B).
Time course of endogenous expressions of PEDF, 67LR, andcellular location of 67LR in the brain after SAH

There was a significant decrease in the PEDF level starting at 3 h after SAH compared to the Sham group, andthe level reached the lowest at 12 h after SAH (Fig. 1a, b).
The 67LR level significantly started decreasing at 6 h andreached the lowest at 24 h after SAH (Fig. 1a, c). Furthermore, immunofluorescence staining showed that 67LR
was expressed on astrocytes and neurons but was barely
observed on microglia in the ipsilateral basal cortex at
24 h after sham surgery and SAH (Fig. 1d).
Intranasal administration of PEDF-34 ameliorated short-term
neurological deficits at 24 h after SAH

There was no significant difference in SAH grades among
the SAH groups (Fig. 2a). The modified Garcia score andbeam balance score were lower in SAH + Vehicle andSAH + PEDF-34 (1 µg/kg) groups than in the Sham groupbut were significantly improved in SAH rats treated with
PEDF-34 at the dose of 3 µg/kg or 9 µg/kg (Fig. 2b, c).
The brain water content of both cerebral hemispheres
was significantly higher in the SAH + Vehicle andSAH + PEDF-34 (1 µg/kg) groups compared to the shamgroup (Fig. 2d), while the middle dose (3 µg/kg) andhigh dose (9 µg/kg) of PEDF-34 treatment significantly
reduced brain water contents (Fig. 2d) in SAH rats. However, the middle-dose group showed better performanceWu et al. Journal of Neuroinflammation(2024) 21:178

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Fig. 1 Time course of endogenous PEDF and 67LR expressions and cellular location of 67LR in the brain after SAH. (a-c) Representative western blot
images and quantitative analyses of endogenous PEDF, 67LR expression at 3 h, 6 h, 12 h, 24 h, 72 h, and 7 d after SAH. *p < 0.05 vs. Sham, n = 6 /group. (d)Representative microphotographs of immunofluorescence staining showing the colocalization of 67LR (red) with astrocyte (GFAP, green, left), neuron(NeuN, green, middle), or microglia (Iba1, green, right) in sham and SAH rats at 24 h after SAH. A small red box in the brain block indicated where themicrophotographs were taken. Scale bar = 50 μm. n = 4/groupthan the high-dose group. Based on the findings, theoptimal dose of PEDF-34 was determined as 3 µg/kg andused in the subsequent experiments.
Immunofluorescence staining showed IL-1β and IL10-positive astrocytes significantly increased in the
SAH + Vehicle group compared with the Sham group(Fig. 2e-h). However, PEDF-34 treatment significantly
reduced IL-1β-positive astrocytes (Fig. 2e, g) while
increasing IL-10-positive astrocytes (Fig. 2f, h).
PEDF-34 ameliorated long-term neurological dysfunction
after SAH

To evaluate the effect of PEDF-34 on long-term neurological function, Rotarod and Morris water maze tests
were performed after SAH. The Rotarod test showed thatthe latency to fall of SAH + Vehicle treated group was
significantly reduced in both 5RPM and 10RPM-accelerating velocity tests compared to the sham group at 1
and 2 weeks after SAH (Fig. 3a, b). However, treatment
of PEDF-34 significantly prolonged the fall latency timecompared with SAH + Vehicle group after SAH (Fig. 3a,b).

In the water maze test, there was no significant difference in the swimming distance among all the groups
in the block 1 and 2 tests. From block 3 to block 5 tests,
there were time-related reductions in swimming distancein the sham and SAH + PEDF-34 groups. However, no significant improvement was observed in the SAH + Vehicle
group (Fig. 3c). During the same period, the groups had
no significant difference in swimming velocity (Fig. 3d).
The probe trial showed that the SAH + PEDF-34 groupspent a significantly longer time in the quadrants than the
SAH + Vehicle group (Fig. 3e, f ). In addition, Nissl staining showed that in the SAH + Vehicle group, comparedwith the sham-operated group, neurons in the CA1, CA3,and dentate gyrus (DG) areas of the ipsilateral hippocampus were significantly lost 28 days after SAH (Fig. 3g-i).
However, neuronal damage was significantly reduced in
the PEDF-34-treated group compared with the Vehicle
group (Fig. 3i).

Wu et al. Journal of Neuroinflammation(2024) 21:178

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Fig. 2 The effects of PEDF-34 on short-term outcomes at 24 h after SAH. (a) SAH grading scores. (b-c) Modified Garcia and beam balance test. *p < 0.05
vs. Sham; #p < 0.05 vs. SAH + Vehicle. (d) Quantitative analysis of brain water content in the left/right hemisphere, cerebellum, and brain stem. n = 6 /group.
*p < 0.05 vs. Sham; #p < 0.05 vs. SAH + Vehicle. (e) Representative microphotographs of IL-1β (red) and GFAP (green) immunofluorescence staining in theipsilateral basal cortex. Nuclei were stained with DAPI (blue). Scale bar = 50 μm. (f) Representative immunofluorescence images of IL-10 (red) and GFAP
(green) expression in the ipsilateral basal cortex of different groups. Scale bar = 50 μm. (g) Quantitative analysis of IL-1β-positive astrocytes. (h) Quantitative analysis of IL-10-positive astrocytes. n = 4 /group. *p < 0.05 vs. Sham; #p < 0.05 vs. SAH + Vehicle

Wu et al. Journal of Neuroinflammation(2024) 21:178

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Fig. 3 PEDF-34 reduced neuronal loss in the hippocampus and improved long-term neurological function after SAH. (a, b) The falling latency of rotarod
test at 1 week, 2 weeks and 3 weeks after SAH. (c) Swimming distance of Morris water maze test on days 23 to 27 after SAH. (d) Quantification of swimming
velocities. (e-f) Representative thermal images of the probe trials and probe quadrant duration. n = 10/group, *p < 0.05 vs. Sham; #p < 0.05 vs. SAH + Vehicle.
(g-h) Representative microphotographs of Nissl staining in CA1, CA3, and DG hippocampal regions. Scale bar = 100 μm. Small red squares indicatedthe area for high magnification and quantification. (i) Quantitative analysis of surviving neuron number in different hippocampal regions. n = 10/group.
*p < 0.05 vs. Sham; #p < 0.05 vs. SAH + Vehicle

Wu et al. Journal of Neuroinflammation(2024) 21:178

Fig. 4 (See legend on next page.)

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Wu et al. Journal of Neuroinflammation(2024) 21:178

Page 11 of 16(See figure on previous page.)
Fig. 4 PEDF-34 promotes A2 astrocyte polarization via 67LR/JNK/STAT1 signaling pathway after SAH. (a) Representative western blotting bands. (b-h)
quantitively analysis of 67LR, p-JNK/JNK, p-STAT1/STAT1, CFB, C3, S100A10 expression. n = 6/ group. *p < 0.05 vs. Sham; #p < 0.05 vs. SAH + Vehicle, @p < 0.05
vs. SAH + PEDF-34 or SAH + PEDF-34 + DMSO. (i) Representative microphotographs of C3 (A1 Astrocyte marker, red) and GFAP (green) by immunofluorescent staining. Scale bar = 50 μm. (k) Quantification of C3-positive A1 astrocytes. n = 4/group. *p < 0.05 vs. Sham; #p < 0.05 vs. SAH + Vehicle, @p < 0.05 vs.
SAH + PEDF-34 or SAH + PEDF-34 + DMSO. (j) Representative microphotographs of S100A10 (A2 Astrocyte marker, red) and GFAP (green) immunofluorescence staining. Scale bar = 50 μm. (l) Quantification of S100A10-positive A2 astrocytes. n = 4/group. *p < 0.05 vs. Sham; #p < 0.05 vs. SAH + Vehicle, @p < 0.05
vs. SAH + PEDF-34 or SAH + PEDF-34 + DMSOPEDF-34 inhibit astrocyte A1 polarization via 67LR/JNK/
STAT1 signaling pathway

To explore the underlying mechanism of PEDF to protect
the brain against hemorrhage after SAH, 67LR specificinhibitor, NSC-47924, and STAT1 transcriptional activator, 2-NP was administrated intracerebroventricularly
48 h before SAH induction. Western blot results showedthat 67LR expression was significantly decreased. In contrast, the expression of p-JNK, p-STAT1, CFB (A1 astrocyte marker), C3 (A1 astrocyte marker), S100A10 (A2astrocyte marker) and the ratio of C3/S100A10 was significantly increased at 24 h after SAH compared with thesham group (Fig. 4a-h). PEDF-34 treatment decreased
expressions of p-JNK, p-STAT1, CFB, and the ratio of C3
to S100A10 (Fig. 4a-g). 67LR inhibitor, NSC-47924, did
not affect 67LR expressions (Fig. 4b-g) but reversed theeffects of PEDF-34 on expressions of p-JNK, p-STAT1,
CFB and C3 (Fig. 4b-g). Furthermore, the STAT1 transcriptional activator (2-NP) reversed the effects of PEDE34 treatment on p-STAT1, CFB, and C3 expression in
SAH rats (Fig. 4b-h).
The effects of 67LR on astrocytic polarization wereassessed by immunofluorescence staining. C3 (Fig. 4i)
and S100A10 (Fig. 4j) expressions on astrocytes wereincreased after SAH. While in PEDF-34 treated SAH rats,
there was a remarkable decrease in C3-positive astrocytes (Fig. 4i, k), but S100A10-positive astrocytes were
further significantly increased (Fig. 4j, l) compared to theSAH + Vehicle group. However, the effects of PEDF-34 on
astrocyte phenotype changes were reversed by treatment
of NSC-47924 or 2-NP (Fig. 4i-l).
Effects of PEDF-34 administration on JNK/STAT1 signaling
pathway and A1/A2 astrocyte polarization in Hb-stimulated
primary astrocytes

Primary astrocyte culture experiment was performed toverify whether the effects of PEDF was mediated by modulating astrocyte polarization through JNK/STAT1 signaling pathway. The results showed that Hb stimulation
significantly decreased 67LR expressions but increased
p-JNK, p-STAT1, CFB, C3, and S100A10 (Fig. 5a-h).
Compared with the Hb + Vehicle group, expressions of
p-JNK, p-STAT1, CFB, and C3 were decreased, while theexpression of S100A10 was increased in the Hb + PEDF34 treated group (Fig. 5a-h).
Knockdown of 67LR with 67LR shRNA significantly
decreased the 67LR expression and reversed the effectsof PEDF-34 on p-JNK, p-STAT1, CFB and C3 expressions compared with Hb + PEDF34 or Hb + PEDF-34 + shcontrol group (Fig. 5a-g). 2-NP reversed the effect of
PEDF-34 treatment on expressions of p-STAT1, CFB, C3and S100A10 compared to Hb + PEDF-34 or Hb + PEDF34 + sh-control group (Fig. 5a-g). Immunofluorescencestaining consistently showed that PEDF-34 decreased C3
positive astrocytes (Fig. 5i, k) but increased S100A10 positive astrocytes at 24 h after Hb stimulation compared to
Hb + Vehicle group or Hb + PEDF-34 + sh-control (Fig. 5j,l). These effects were reversed by knockdown of 67LR or
STAT1 transcriptional activator treatment (Fig. 5i-l).
PEDF-34 inhibits astrocyte-mediated inflammatory
cytokines secretion through 67LR/JNK/STAT1 signaling
pathway in vivo and in vitro

ELISA results showed that TNF-α, IL-1β, and IL-10 weresignificantly elevated at 24 h after SAH. PEDF-34 treatment reduced the level of pro-inflammatory cytokines
TNF-α (Fig. 6a) and IL-1β (Fig. 6b) but elevated the levelof anti-inflammatory cytokine IL-10 (Fig. 6c) in SAH
rats. Intraventricular administration of 67LR inhibitor,
NSC-47924 and the STAT1 transcriptional activator,
2-NP, reversed the effect of PEDF-34 on the secretion of
inflammatory relative cytokines (Fig. 6a-c).
In in vitro study, ELISA results showed that TNF-α,
IL-1β and IL-10 levels in the astrocytes culture medium
significantly increased at 24 h after Hb stimulation(Fig. 6d-f ). Incubation of PEDF-34 reduced the level ofpro-inflammatory cytokines of TNF-α (Fig. 6d) and IL-1β(Fig. 6e) but further elevated the level of anti-inflammatory cytokine IL-10 (Fig. 6f ). Knock-down of 67LR with
Sh-67LR or activation of STAT1 with 2-NP reverses theeffect of PEDF-34 on TNF-α, IL-1β and IL-10 secretion(Fig. 6d-f ).

Discussion
In the present study, we found that the endogenous
protein of PEDF and 67LR was decreased after SAH inrats. 67LR were expressed in astrocytes and neurons butbarely in microglia. The activation of 67LR with PEDF34 significantly improved the short-term neurobehavioral deficits and attenuated brain edema, accompanied
by decreased pro-inflammatory cytokines IL-1β and
IL-10 at 24 h after SAH. In addition, PEDF-34 improved
long-term neurobehavioral function 4 weeks after SAH.
Mechanistically, PEDF-34 treatment suppresses the ratio

Wu et al. Journal of Neuroinflammation(2024) 21:178

Fig. 5 (See legend on next page.)

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Wu et al. Journal of Neuroinflammation(2024) 21:178

Page 13 of 16(See figure on previous page.)
Fig. 5 PEDF-34 modulated astrocyte polarization by 67LR/JNK/STAT1 signaling pathway in vitro. (a) Representative western blotting and (b-h) quantitively analysis of 67LR, p-JNK/JNK, p-STAT1/STAT1, CFB, C3 and S100A10 expressions. n = 6/group. *p < 0.05 vs. PBS; #p < 0.05 vs. Hb + Vehicle, @p < 0.05 vs.
Hb + PEDF-34 or Hb + PEDF-34 + sh-control. (i) Representative microphotograph images of colocalization of C3 (A1 marker, red) and GFAP (green) immunofluorescence staining. Scale bar = 50 μm. (k) Quantification of C3-positive A1 astrocyte. n = 4/group. *p < 0.05 vs. PBS; #p < 0.05 vs. Hb + Vehicle, @p < 0.05
vs. Hb + PEDF-34 or Hb + PEDF-34 + sh-control. PEDF-34 effect on A2 astrocytes at 24 h after Hb stimulation. (j) Representative microphotographs of
S100A10 (A2 marker, red) and GFAP (green) immunofluorescence staining. Scale bar = 50 μm. (l) Quantification of S100A10 positive A2 astrocytes. n = 4
replicates/group. *p < 0.05 vs. PBS; #p < 0.05 vs. Hb + Vehicle, @p < 0.05 vs. Hb + PEDF-34 or Hb + PEDF-34 + sh-controlof p-JNK/JNK, p-STAT1/STAT1, CFB, and C3 expression
but increases the expression of S100A10. Furthermore,
the expression of p-JNK/JNK, p-STAT1/STAT1, CFB, C3,
and S100A10 was associated with SAH-induced PEDF
and 67LR expression changes (Additional file: Figure
S3A). 67LR specific inhibitor, NSC-47924/ knockdown of
67LR with Sh-67LR or STAT1 transcriptional activator,
2-NP abolished the beneficial effects of PEDF-34 on A1/A2 astrocyte polarization and astrocyte-mediated neuroinflammation in vivo and in vitro. Furthermore, we foundthat PEDF-34 alleviates delayed neuronal degeneration in
the hippocampus after SAH. Taken together, our findings
suggest that PEDF-34 could activate 67LR, promote A2
astrocyte polarization, and suppress astrocyte-mediatedneuroinflammation, partially by JNK/STAT1 signaling
pathway.
Pigment Epithelium-Derived Factor, PEDF, was firstfound to be secreted by the retinal pigment epithelium inthe eye, after which it was detected in both neurons andglia of the brain [5, 37]. Previous studies demonstrated
that PEDF was neuroprotective through its neurotrophic
effect, anti-inflammation, anti-oxidative stress, and preservation of BBB integrity [11, 38–40]. Upon activation of
67LR, PEDF mitigates the formation of status epilepticusinduced vasogenic edema by inhibiting the p38 MAPK–
PI3K/AKT/eNOS axis [18]. 67LR activations have beenfound to promote functional recovery after stroke andspinal cord injury [41, 42]. However, whether PEDF/67LR
signaling plays a role in SAH remains unclear.
To investigate the protective effect of PEDF in SAH,
PEDF 34-mer peptide (PEDF-34) was used in the experiments. PEDF-34, derived from amino acid positions
Asp44–Asn77 of the full-length PEDF, exhibits a higher
half-life, production yield, purity, and smaller size than
the full-length PEDF. These characteristics ensure thestable functioning of PEDF-34 within the brain [11,43, 44]. Our in vivo study demonstrated that SAH rats
treated with PEDF-34 at the dose of 3 µg/kg exhibited
significant improvements in short-term neurobehavioral
deficits 24 h after SAH. Moreover, PEDF-34 improved
long-term outcomes, including on the rotarod and water
maze tests in SAH rats. Better spatial learning function
was associated with less neuronal damage within ipsilateral hippocampal CA1, CA3, and dentate gyrus region.
67LR activations inhibit inflammatory mediators andcytokine levels in various cells, such as cerebral microvascular endothelial cells, macrophages, and adipocytes[45–48]. A recent study demonstrated that 67LR activation by Epigallocatechin gallate (EGCG) inhibited endotoxin-induced expression of inflammatory cytokines,
including TNF-α and IL-1β, in human cerebral microvascular endothelial cells [46]. After SAH, the level ofpro-inflammatory cytokines TNF-a and IL-1β increasedin the brain tissue [49, 50]. Consistently, pro-inflammatory cytokines were higher in the brains of SAH rats but
decreased after PEDF-34 treatment. However, inhibitionof 67LR reversed the anti-inflammatory effects of PEDF34, suggesting 67LR mediates the neuroprotective effectsof PEDF-34.
Reactive astrocytes play an essential role in regulatingneuroinflammation during EBI after SAH. In response toSAH, astrocytes undergo reactive changes and are further classified into harmful A1 phenotype and protective
A2 phenotype, contributing to neuronal death or upregulating specific neurotrophic factors for neuronal survival,
respectively [45]. Specifically, active A1 astrocytes induce
neuronal apoptosis by releasing pro-inflammatory factors after SAH [25]. Consistently in the current study,
the number of A1 phenotype astrocytes significantly
increased in SAH rats and primary astrocytes stimulatedby Hb. Both in vivo and in vitro experiments showed that
PEDF-34 reduced A1 phenotype astrocytes and increased
A2 phenotype astrocytes after SAH. However, the therapeutic effects of PEDF-34 were partially reversed byinhibiting 67LR with NSC-47924 or knocking down 67LR
with 67LR shRNA. These findings suggest that activationof PEDF-34/67LR signaling promotes astrocytic A1 to A2
phenotype changes, resulting in decreased secretion of
pro-inflammatory cytokines and increased anti-inflammatory cytokine secretion in the brain after SAH.
The phosphorylation of JNK and STAT1 can increase
A1 type astrocytes number and activation of the complement system, resulting in a solid inflammatory effect [3,51]. In the current study, the phosphorylation level of
JNK and STAT1 significantly increased in rat brains after
SAH and in Hb-stimulated primary astrocytes, while
PEDF-34 decreased the phosphorylation level of JNK and
STAT1. However, 67LR agonist, NSC-47924, or knockdown with 67LR shRNA reversed the effect of PEDF-34
in vivo and in vitro, respectively. Changes in the phosphorylation level of JNK and STAT1 are associated with
67LR activation and inhibition, suggesting that JNK and
STAT1 were the downstream molecules of this signaling
pathway.

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Fig. 6 PEDF-34 attenuated astrocytic pro-inflammatory cytokines in vivo and in vitro. (a-c) ELISA analysis of TNF-α, IL-1β, and IL-10 level. n = 6/group.
*p < 0.05 vs. Sham; #p < 0.05 vs. SAH + Vehicle, @p < 0.05 vs. SAH + PEDF-34 or SAH + PEDF-34 + DMSO. (d-f) ELISA analysis of TNF-α, IL-1β, and IL-10 levels in
astrocyte culture medium. n = 4/group. *p < 0.05 vs. PBS; #p < 0.05 vs. Hb + Vehicle, @p < 0.05 vs. Hb + PEDF-34 or Hb + PEDF-34 + sh-control

However, the study had limitations. Firstly, we focusedon PEDF/67LR’s effect on astrocyte-mediated neuroinflammation. However, 67LR is also expressed in neurons;
the PEDF-34/67LR-related neuronal protective mechanisms require further investigation. Secondly, patientswith clinically poor prognosis after SAH have significantly elevated PEDF levels in the acute phase of plasma[52]. In contrast, in our study, there was a significant
decrease in endogenous PEDF and 67LR expression in
the ipsilateral brain at the early stage after experimental
SAH, accompanied by increased p-JNK and p-STAT1
expressions and ratio of A1/A2 astrocytes phenotypes.
Further studies on the expression of PEDF in the brainsof SAH patients are needed, and the clinical role of PEDF
needs to be further explored. Lastly, gender differences
and age effects were not addressed in the current study.

Abbreviations
SAH	Subarachnoid hemorrhage
EBI	Early brain injury
PEDF	Pigment epithelium-derived factor
67LR	67-kDa non-integrate laminin receptor
BBB	Blood-brain barrier
2-	NP 2-(1,8-naphthyridin-2-yl) phenol
CNS	Central nervous system
ELISA	Enzyme-linked immunosorbent assay
Iba1	Ionized calcium-binding adapter molecule 1
NeuN	Neuronal nuclei
GFAP	Glial fibrillary acidic protein
IL	Interleukin IL
shRNA	Short hairpin RNA shRNA
TNF-α	α Tumor necrosis factor alpha
AQP4	Aquaporin 4
JNK	c-Jun N-terminal kinase
CFB	Complement factor B
C3	Complement component 3
STAT1	Signal transducer and activator of transcription 1

Conclusions
Our results demonstrated that 67LR activation by PEDF34 promoted astrocyte A2 polarization via the JNK/STAT1 pathway, thus attenuating neuroinflammationand improving neurological outcomes after experimental SAH. These findings suggested that early administration of PEDF-34 might serve as a promising therapeutic
approach for EBI and delayed brain injury following SAH.

The online version contains supplementary material available at https://doi.
org/10.1186/s12974-024-03171-y.

Supplementary Information

Supplementary Material 1
Supplementary Material 2
Supplementary Material 3
Supplementary Material 4

Wu et al. Journal of Neuroinflammation(2024) 21:178

Page 15 of 16

Acknowledgements
Not applicable.

8.

Author contributions
The work was designed by LW, YL, LH, NX, JJF, JT, XH, and JHZ. The experiments
were performed by LW, QH, SD, SZ, YY, MH, YL, LW, and YL. LL, CW, YZ, XC, GA,
WH, XL, QY, and XH collected and analyzed the data. LW, YL, NX, LH, HK, PS, XH,
and JHZ have drafted and substantively revised the work. All authors read andapproved the final manuscript.

9.

Funding
This work was supported by grants from the National Institutes of Health(NS117179 and NS082184), Guangdong Basic and Applied Basic Research
Fund (2021A1515010980), and Guangdong Second Provincial General
Hospital (WKZX2023CZ0214, TJGC-2023012).
Data availability
No datasets were generated or analysed during the current study.

10.
11.
12.
13.
14.

Declarations
Ethical approval
All animal experiments in this study have been approved by the Institutional
Animal Care and Use Committee of Loma Linda University in accordancewith the National Institutes of Health (NIH) Guide for the Care and Use ofLaboratory Animals.
Competing interests
The authors declare no competing interests.
Author details
1
Department of Cerebrovascular Surgery, Neurosurgery Center,
Engineering Technology Research Center of Education Ministry of
China on Diagnosis and Treatment of Cerebrovascular Disease, Zhujiang
Hospital, Southern Medical University, Guangzhou 510280, Guangdong,
China
2
Department of Physiology and Pharmacology, Basic Sciences, School of
Medicine, Loma Linda University, Loma Linda, CA 92354, USA
3
Department of Neurology, Guangdong Second Provincial General
Hospital, Guangzhou 510317, Guangdong, China
4
Department of Interventional Therapy, Zhujiang Hospital, Southern
Medical University, Guangzhou 510280, Guangdong, China
5
Department of Neurosurgery, Loma Linda University School of Medicine,
Loma Linda, CA 92354, USA
6
Department of Emergency Surgery, First Affiliated Hospital of Soochow
University, Suzhou 215000, Jiangsu, China
Received: 17 May 2024 / Accepted: 11 July 2024

15.

16.

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20.
21.
22.

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24.

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