Journal of Molecular Neuroscience

https://doi.org/10.1007/s12031-021-01887-7

Zeb2/Axin2‑Enriched BMSC‑Derived Exosomes Promote Post‑Stroke

Functional Recovery by Enhancing Neurogenesis and Neural Plasticity
Rui Wei1 · Lin Zhang2 · Wei Hu3 · Xinying Shang1 · Yuyan He4 · Wei Zhang1 

Received: 19 May 2021 / Accepted: 5 July 2021

© The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2021

Abstract
Exosomes harvested from bone marrow-derived mesenchymal stromal cells (BMSCs) have shown treatment potential in
many diseases. In vitro, Zeb2/Axin2 stimulated endogenous neurogenesis, which induced functional recovery after stroke.
Here, we investigated whether the Zeb2/Axin2-enriched exosomes harvested from BMSCs transfected with a Zeb2/Axin2
overexpression plasmid would enhance neurological recovery. Compared with the control, both exosome treatments sig-
nificantly improved functional recovery, and Zeb2/Axin2-enriched exosomes had significantly more improved effects on
neurological function, neurogenesis, and neurite remodeling/neuronal dendrite plasticity than the control BMSC exosome
treatment in a middle cerebral artery occlusion MCAO rat model. After stimulation with Zeb2/Axin2-enriched BMSC
exosomes, the spatial memory and nerve function of MCAO rats showed marked recovery. The number of neurons was
increased in the subventricular zone (SVZ), hippocampus, and cortex area, while the expression of nerve growth factors
(NGF, BDNF, etc.) was upregulated. In the ischemic boundary zone, Zeb2/Axin2-enriched exosomes promoted synaptic
remodeling by increasing the number of synapses and reversed the axonal loss of phosphorylated neurofilament (SMI-31)
and synaptophysin (SYN) caused by ischemic injury, thus alleviating axonal demise and promoting synaptic proliferation.
In vitro, Zeb2/Axin2-enriched exosomes significantly increased neurite branching and elongation of cultured cortical embry-
onic rat neurons under oxygen- and glucose-deprived (OGD) conditions. Moreover, Ex-Zeb2/Axin2-enriched exosomes
downregulated the protein level of SOX10, endothelin-3/EDNRB, and Wnt/β-catenin expression. In conclusion, exosomes
harvested from Ex-Zeb2/Axin2 BMSC could improve post-stroke neuroplasticity and functional recovery in MCAO rats by
promoting proliferation and differentiation of neural stem cells. The mechanism may be related to the SOX10, Wnt/β-catenin,
and endothelin-3/EDNRB pathways.

Keywords  Zeb2 · Axin2 · Exosomes · BMSCs · MCAO · Neurological recovery

Introduction

Stroke is one of the three major diseases that seriously

endanger human health worldwide and has a mortality rate
* Wei Zhang of up to 56.6% (Brouns et al. 2019). Even surviving patients
zhangwei190725@163.com
are left with serious sequelae, such as long-term motor dis-
1
Department of Emergency, The First Affiliated Hospital abilities, which seriously affect their quality of life (Ferro
of Kunming Medical University, No. 295 Xichang Rd, et al. 2016). Ischemic stroke (cerebral infarction) is the
Kunming 650032, Yunnan, China main type of stroke and is caused by middle cerebral artery
2
Department of Rehabilitation, The First Affiliated Hospital occlusion (MCAO) (Vahidy et al. 2016). Currently, there
of Kunming Medical University, No. 295 Xichang Rd, is no effective therapy to improve functional recovery in
Kunming 650032, Yunnan, China
the post-ischemic stage. However, accumulating evidence
3
Department of Cardiology, The First Affiliated Hospital shows that exosome therapy may provide a novel thera-
of Kunming Medical University, No. 295 Xichang Rd,
Kunming 650032, Yunnan, China peutic approach for ischemic stroke (Tian et al. 2018a, b;
4 Manuel et al. 2017; Ling et al. 2018).
Kunming Medical University, No. 1168 West Chunrong
Road, Kunming 650504, Yunnan, China

13
Vol.:(0123456789)

Neurogenesis comprises two processes: neural progeni-
tor cell (NPC) proliferation/maturation in regions of neural
proliferation and migration of these cells into the lesion area
(Khodanovich and Nemirovich-Danchenko 2019; Aimone
et al. 2014). Therefore, the regulation of these two processes
is favorable for promoting functional recovery after stroke.
Although human embryonic stem cell (hESC)-derived NPCs
have shown good effects in treating stroke, ethical issues
limit their wide use as a source for stem cell therapy (Chang
et al. 2013). Recent studies have reported that autologous
bone marrow-derived mesenchymal stem cells (BMSC)
transdifferentiate into NPCs and improve ischemic injury in
different animal models (Tang et al. 2012; Oki et al. 2012;
Qu et al. 2007; Ji et al. 2013; Chen et al. 2008). However,
BMSCs could not restore brain function to healthy control
levels (Mahmood et al. 2006). Thus, BMSCs may require
an additional push to promote NPC proliferation/maturation
and migration and restore the brain to a healthy state.
The zinc finger E-box binding homeobox 2 protein (Zeb2)
is a transcriptional regulator and is associated with the trans-
forming growth factor β (TGF-β) signaling pathway, which
is essential during early neurodevelopment (Comijn et al.
2001). Axin2 is a scaffolding protein that participates in at
least two signaling pathways, the Wnt/β-catenin pathway
and the TGF-β pathway, related to endothelial cell angiogen-
esis (Dao et al. 2007). This relationship implies that Zeb2
and Axin2 may have therapeutic potential in treating stroke.
Zeb2 can affect microglia, an essential type of immunocom-
petent cell that responds to environmental and physiologi-
cal changes during ischemic stroke (Rexach et al. 2019).
Fancy et al. reported that Axin2 is an essential regulator of
remyelination in the setting of newborn brain injury (Fancy
et al. 2011). In addition, MCAO model rats showed low
expression of AXIN2, severe neurological deficits, acceler-
ated apoptosis, and reduced angiogenesis (Wu et al. 2020;
Fancy et al. 2011). These findings suggest that Zeb2/Axin2
may improve the benefits of treatments for cerebral ischemic
injury.
Exosomes are described as small membrane-derived vesi-
cles secreted by various cells. Exosomes have been found
to contain multiple kinds of host-derived proteins, RNAs
including mRNAs and miRNAs, and lipids (Théry et al.
2002). Evidence has revealed that exosomes are involved
in intercellular communication by delivering mRNA car-
goes, thereby affecting protein alterations in the recipient
cells (Valadi et al. 2007). In the nervous system, exosomes
are released by neurons in a manner dependent on synaptic
activity. These exosomes mediate intercellular communi-
cation by the transport of synaptic proteins, mRNAs, and
microRNAs. They are locally directed to the presynaptic
terminal or transported back to the presynaptic neuronal cell
body. Exosomes could thus constitute an ideal mechanism
for the interneuronal transfer of information, enabling the
13
Journal of Molecular Neuroscience
anterograde and retrograde signaling across synapses neces-
sary for neural plasticity (Braccioli et al. 2014; Chivet et al.
2012). A recent study showed that exosomes have therapeu-
tic potential for cerebral ischemic injury, as evidenced by the
strong suppression of the inflammatory response and cellular
apoptosis observed in the lesion area (Tian et al. 2018a, b).
Therefore, the use of exosomes to efficiently transport repair
factors to the ischemic area may have overlapping benefits
for ischemic stroke.
In this study, we administered Zeb2/Axin2-enriched
exosomes derived from BMSCs transfected with a Zeb2/
Axin2 overexpression plasmid to MCAO rats and investi-
gated their effect on post-stroke functional recovery, neuro-
genesis, and neurological plasticity. In addition, we explored
the downstream pathways underlying neurogenesis.
Material and Methods
Ethics Statement
The experiment was conducted in accordance with the stand-
ards and procedures of the National Institutes of Health
(NIH) Guide for the Care and Use of Laboratory Animals
and was approved by the Laboratory Animal Ethics Review
Committee of Kunming Medical University.
Exosome Isolation and Quantification
The BMSCs were extracted from the femur and tibia of SD
rats according to the previous research (Jin et al. 2018). The Zeb2/
Axin2 over-expressed lentivirus was constructed with the
lentivirus vectors (pLV-ORFSpark; Sino Biological Inc.,
China) and then infected to BMSC according to the manu-
facturer’s suggested protocol. The BMSCs were selected by
puromycin for constant gene expression.
Exosome-free Dulbecco’s modified Eagle’s medium
(DMEM) containing 10% fetal bovine serum, 1% strep-
tomycin, and penicillin mixture was used to culture
BMSCs, and the medium was collected after 48 h. An
aliquot of 10 mL medium was centrifuged at 3000 × g
for 15  min to remove cell debris. Then ExoQuick-TC
Exosome Precipitation Solution (System Biosciences,
Pu Mai Technology, Beijing, China) was used to isolate
exosomes from the BMSC medium. BMSCs were infected
with the lentivirus of Zeb2 and Axin2, and the Zeb2/
Axin2-enriched exosomes were extracted. Exosome pro-
tein content was measured using the BCA protein assay
kit (Thermo Fisher Scientific, USA). The ultrastructure
was analyzed by transmission electron microscopy. Pro-
tein markers CD9, CD63, CD81, Alix, TSG101, Zeb2,
and Axin2 were determined by immunoblotting.
Journal of Molecular Neuroscience
MCAO Model and Treatment
Male Sprague–Dawley (SD) rats (280–320 g) were purchased
from Kunming Medical University Experiment Animal Center
(SCXK [Yunnan] 2011–0004). The animals were given ade-
quate clean drinking water and food. The middle cerebral
artery occlusion (MCAO) model was established according
to the method described by Longa et al. (1989) and Nagasawa and
Kogure (1989). In brief, rats were anesthetized with an intra-
peritoneal injection of a gas flow (1.0 L/min) of isoflurane
(1.5%) in a 2:1 mixture of N ­ 2O and O ­ 2, and then fixed on
the operating table. The right common carotid artery (CCA),
internal carotid artery (ICA), and external carotid artery (ECA)
were exposed and wired for reserve. ECA and CCA were
ligated with a silk suture. ECA and ICA were quickly split after
the distal end of the ICA was clamped. Nylon thread with one
end heated into a smooth sphere (0.25 mm in diameter, marked
at 2.0 cm from the sphere) was inserted into the ICA through a
small puncture in the CCA, and advanced 18 ± 0.5 mm into
the ICA until encountering gentle resistance, to occlude the
middle cerebral artery and cause cerebral ischemia. Two hours
after the operation, reperfusion was performed by withdrawing
the remaining thread until the blood flow was restored. Rats
in the control group were not treated with any surgery. In the
sham operation group, only ECA and ICA ligation was per-
formed. The rats were housed one per cage with an enriched
environment of small toys in each cage. Room temperature
and humidity were controlled at 70 °F and 50%, respectively.
Rats were observed once per day to make sure that they were
not in pain or discomfort, and that they were able to function
normally and had free to access food and water.
After surgery, all rats were randomly divided into three
groups: MCAO group (n = 6), MCAO + BMSC-exosome
group (n = 6), and MCAO + Zeb2/Axin2-enriched exo-
some group (n = 6). Briefly, the rats were anesthetized by
10% chloral hydrate (3 mg/kg body weight) and mounted
on the stereotaxic apparatus. The hair on the head was
shaved. After sterilization, a 1–2 cm cut was made on the
middle part of the scalp tissue and the skull was exposed.
The front fontanelle point was marked with a marker pen.
A small hole (1 mm in diameter) was made using an elec-
tric drill at a point 1 mm behind the anterior fontanelle
and 1.5 mm to the right of the sagittal suture. A 10 μl
microinjection needle was slowly inserted 3.5 mm. For
the MCAO group, 4 μL phosphate-buffered saline (PBS)
was injected from the lateral ventricles. For the exo-
some control (MCAO + exo-ctrl) group, 5 μL of BMSC-
extracted exosomes at a density of 1­ 011 exosome particles
per milliliter was injected from the lateral ventricles. For
the Zeb2/Axin2-enriched exosome (MCAO + exo-Zeb2/
Axin2) group, 5 μL of Zeb2/Axin2-enriched exosomes was
injected at a density of 1­ 011 exosome particles per milli-
liter from the lateral ventricles. The density of exosomes
was detected using the FluoroCet Exosome Quantitation
Kit (SBI, CA, USA) according to the manufacturer’s
instructions. 5-Bromodeoxyuridine (BrdU) injections were
started 24 h after stroke onset and were continued twice
daily during the first and second weeks, and then once a
day during the third and fourth weeks, ending on day 28
after stroke induction. Within the first 7 days after surgery,
five of the 24 rats subjected to MCAO died (two in the
PBS-treated group, two in the exo-ctrl-treated group, and
one in the exo-Zeb2/Axin2-treated group).
Behavioral Tests
Morris Water Maze
The rats were put into the water on the opposite side of the
quadrant containing the platform. The navigation training
lasted 4 days, after which the platform was removed and the
Morris water maze space probe experiment was performed
to assess spatial memory. The rats were put into the water
on the opposite side of the quadrant where the platform had
been. The latency to the platform was counted.
Modified Neurological Severity Score
(mNSS)
The mNSS was performed after surgery for 4 weeks. The
mNSS comprised various tests including motor, sensory, and
reflex function. The scores were graded on a scale of 0 to 14:
the higher the score, the more severe the neurological deficit.
Foot Fault Test
The foot fault test is a tool for assessing locomotor function
in rodent models of central nervous system (CNS) disorders.
Subjects are placed on an elevated horizontal ladder and
trained to cross the device. Video scoring or sensors record
paws slipping along the steps of the ladder as the animal
crosses. Paw misplacements and the ambulatory time are
recorded for each run of the test; a total of four runs were
recorded for each animal.
Sample Collection
After behavioral tests, the rats were deeply anesthetized with
isoflurane and then fixed on the operating table, coated, and
sterilized. The heart was exposed and perfused with pre-
cooled saline. After the liver turned white, the saline was
replaced with 4% paraformaldehyde. Perfusion stopped
13

when the liver became hard and the limbs and tail were
stiff. Brain tissue was obtained and put in 30% sucrose at
4 °C overnight and then frozen at − 80 °C. Two-micrometer
sections were prepared by a sliding freezing microtome
at − 20 °C and stored at 4 °C for subsequent experiments.
5‑Triphenyltetrazolium Chloride (TTC)
Staining
For TTC (Sigma-Aldrich) staining, brains were rapidly har-
vested and cut into 2-mm-thick coronal sections on day 3
after reperfusion. Then slices were immersed in 2% TTC
solution at 37 °C for 15 min in the dark and fixed in 10. The
infarct tissues in brain slices appeared white. The percent-
age of infarct volume was analyzed using ImageJ software.
Immunofluorescence
The brain standard paraffin block was obtained from the
center of the lesion (bregma − 1 to + 1 mm). A series of
14-mm-thick sections were cut from the block. Coronal sec-
tions were rinsed three times with PBS and then immersed in
an antigen retrieval solution (sodium citrate). Then the sec-
tions were boiled and cooled, washed with PBS, and treated
with 2 M hydrochloric acid. After that, the sections were
blocked in 5% goat serum for 1 h and incubated with sheep
anti-rat BrdU antibodies labeling fluorescein isothiocyanate
(FITC) (1:300, Abcam, China) and rabbit anti-rat NeuN anti-
bodies labeling PE (1:400, Abcam, China) or rabbit anti-rat
brain-derived neurotrophic factor (BDNF) antibodies labe-
ling PE (1:200, Abcam, China) at 4 °C overnight. Images
were acquired by laser scanning confocal microscopy (LSM
710, Carl Zeiss, Germany).
Bielschowsky Silver Staining
Axons were stained using Bielschowsky silver staining.
Briefly, slides were placed in 20% silver nitrate in the dark,
ammonium hydroxide was added, and the slides were then
treated with NaOH and sodium thiosulfate. Images were
acquired by a BX53 microscope (Olympus, Japan).
HE Staining and Immunohistochemistry
For hematoxylin and eosin (HE) staining, 6-μm-thick brain
bregma sections were deparaffinized by xylene and hydra-
tion, stained for 5 min with hematoxylin, differentiated for
30 s by hydrochloric acid ethanol, soaked for 15 min in
water, and stained for 2 min with eosin.
For immunohistochemistry, the free-floating slices were
washed in Tris-buffered saline (TBS) and incubated in
13
Journal of Molecular Neuroscience
anti-SMI-31 (1:100, Sigma, USA) or anti-synaptophysin
(SYN) (1:100, Sigma, USA) for 24 h, followed by stain-
ing with the appropriate secondary antibodies at 37 °C for
30 min. Data are representative of four to five animals per
group. Images were acquired by a BX53 microscope (Olym-
pus, Japan).
Primary Cultured Neuron (PCN) Culture and Neurite
Outgrowth Measurement In Vitro
To investigate the effect of exo-Zeb2/Axin2 treatment on den-
drite or neurite outgrowth, an in vitro oxygen- and glucose-
deprived (OGD) culture on PCN was used, followed by the
method described by Chavez et al. (2006). Embryonic day 17
(E17) cortical cells were isolated from the embryonic brains
of Wistar rats using mechanical digestion. Then PCNs were
cultured in DMEM supplemented with DMEM/high glucose
(1 ×) (HyClone) containing 20% fetal bovine serum, 1% strep-
tomycin, and penicillin mixed with 5% ­CO2 at 37 °C. The
OGD model was used to mimic the in vivo ischemic condi-
tion. Briefly, the original culture medium was discarded. The
culture plate was rinsed twice with PBS. The culture medium
with deoxygenated glucose-free Hanks’ balanced salt solution
(KCl 3 mM, NaCl 156 mM, MgSO4 2 mM, CaCl2 2 mM,
KH2PO4 1.25 mM, HEPES 10 mM, pH = 7.35) was added
and then placed in an airtight container filled with the anaero-
bic mixture for 15 min. The airtight container was placed in a
37 °C incubator for 2 h, after which the culture medium was
replaced by DMEM medium, and PCNs were treated with
exo-ctrl or exo-Zeb2/Axin2.
After 24 h exosome treatment, PCN cultures were per-
formed for microtubule-associated protein 2 (MAP2, a phe-
notypic marker of neural cells) immunofluorescence staining
using a monoclonal anti-MAP2 antibody (1:200, Sigma-
Aldrich, USA) with PE. Slides were then gently washed
three times in PBS before incubation in secondary antibodies
for 30 min at 37 °C. Coverslips were sealed using Prolong
Gold antifade reagent with DAPI (Invitrogen, Carlsbad, CA).
Images were acquired by laser scanning confocal microscopy
(LSM 710, Carl Zeiss, Germany), and PCN counting and
analysis of neurite outgrowth were performed using Image-
Pro Plus 6.0.
RT‑qPCR
Total RNA was extracted from the cells using TRIzol rea-
gent (Invitrogen, USA) according to the manufacturer’s
instructions. Then extracted RNA was used as the reverse
transcription template to synthesize cDNA. Quantitative
real-time polymerase chain reaction (PCR) was performed
with SYBR® Premix Ex Taq™ (Life Technologies, USA).
Journal of Molecular Neuroscience
The samples were processed using an ABI StepOne system
(Applied Biosystems, Foster City, CA, USA). The PCR
amplification included an initial denaturation at 94 °C for
30 s, 32 cycles of denaturation at 94 °C for 30 s, annealing
at 55 °C for 30 s, and extension at 72 °C for 1 min, with
a final extension at 72 °C for 10 min. GAPDH was used
as the loading control. Data were collected and analyzed
using the ­2−△△Ct method. The primers used are listed in
Table 1.
Western Blot
Protein was extracted from cells in the hippocampus,
subventricular  zone (SVZ), and cortex, respectively.
Twenty micrograms of protein was added to 15% sodium
dodecyl sulfate–polyacrylamide gel in each lane for elec-
trophoresis and transferred to a polyvinylidene fluoride
(PVDF) membrane in a semi-wet apparatus at 160 mA
for 1 h 30 min. Tris-buffered solution (0.1%) was used to
wash the membrane three times. Then the membrane was
exposed to appropriate primary antibodies overnight at
4 °C, washed, and incubated with secondary antibodies
for 1 h. Finally, the blots were detected using an enhanced
chemiluminescence kit (Millipore, Billerica, MA, USA)
and the bands were quantified using a Bio-Rad imaging
system (Hercules, CA). Anti-proBDNF (1:2000, Santa
Cruz, USA), anti-SOX10 (1:500, Santa Cruz, USA),
anti-endothelin-3 (1:500, Santa Cruz, USA), anti-EDNRB
(1:500, Santa Cruz, USA), anti-Wnt3a (1:500, Santa Cruz,
USA), and anti-β-catenin (1:1000, Santa Cruz, USA) were
used as the primary antibodies, and anti-mouse horse-
radish peroxidase (HRP) (1:5000, Santa Cruz, USA) was
Table 1  Primer sequences in this study
Gene Sequence
ZEB2 Forward: 5′-ACC​GAC​TCA​AGG​AGA​CAG​AT-3′
Reverse: 5′-GAG​TGG​ATA​TGC​TGT​GGT​TCTC-3′
Axin2 Forward: 5′-GGC​TGC​GCT​TTG​ATA AGGTC-3′
Reverse: 5′-CCC​GAA​ ATC​CAT​CGC​TCT​GA-3′
BDNF Forward: 5′-GCC​CAA​CGA​AGA​AAA​CCA​TAAG-3′
Reverse: 5′-GTT​TGC​GGC​ATC​CAG​GTA​ATT-3′
VEGF Forward: 5′-CAG​CTA​TTG​CCG​TCC​AAT​TGA-3′
Reverse: 5′-CCA​GGG​CTT​CAT​CAT​TGC​A-3′
SDF-1 Forward: 5′-TTT​CAC​TCT​CCG​TCC​ACC​TC-3′
Reverse: 5′-ATC​TGA​AGG​GCA​CAG​TTT​GG-3′
TGF-β Forward: 5′-TAC​AGG​GCT​TTC​GCT​TCA​GT-3′
Reverse: 5′-TGG​TTG​TAG​AGG​GCA​AGG​AC-3′
β-actin Forward 5′- ATC​TGG​CAC​CAC​ACC​TTC​-3′
Reverse 5′- AGC​CAG​GTC​CAG​ACGCA-3′
used as the secondary antibody. β-Actin (1:2000, Santa
Cruz, USA) was used as the loading control.
Statistics
Experiments were performed in triplicate, with a minimum
of three independent experiments. Results of all experi-
ments are depicted as mean ± SD. Comparisons between two
groups were analyzed using Student's t-test. Comparisons
between three or more groups were analyzed using one-way
ANOVA with a Bonferroni test. Differences with values of
p < 0.05 were considered statistically significant.
Results
Expression of Zeb2/Axin2 Is Elevated in Tissues
and Exosomes
To elucidate the relationship between the expression of
Zeb2/Axin2 and ischemia, the mRNA expression levels
of Zeb2/Axin2 in both the cortex and corpus striatum of
MCAO model rats were measured by qPCR. The expression
of Zeb2/Axin2 in the ipsilateral cortex and corpus striatum
in the MCAO group was significantly higher than that in the
sham group (P < 0.05, Fig. 1A–D).
BMSCs were infected with control lentivirus and Zeb2/
Axin2 overexpression lentivirus, and cells were harvested
and exosomes isolated after 48 h. Electron microscopic anal-
ysis revealed a typical exosome diameter of 30 to 100 nm
(Fig. 1E). Expression of exosome biomarkers CD63, CD81,
CD9, ALIX, and TSG101 was confirmed by immunoblotting
(Fig. 1F). The expression of these molecules was increased
in both the exo-ctrl and exo-Zeb2/Axin2 groups (Fig. 1F).
In addition, the levels of Zeb2 and Axin2 in BMSCs and
exosomes were significantly increased compared with
those in the control groups (Fig. 1F). These data indicate
that Zeb2/Axin2-enriched exosomes were successfully
established.
Effects of Treatment with Zeb2/Axin2‑Enriched
Exosomes on Post‑Stroke Behavioral Outcomes
To elucidate the effect of Zeb2/Axin2-enr iched
exosomes on nerve function recovery in MCAO rats,
neuromotor and cognitive dysfunction were evaluated
by the Morris water maze and foot fault tests. Com-
pared with rats in the MCAO group, rats in both the
exo-ctrl and exo-Zeb2/Axin2 groups exhibited a sig-
nificant reduction in neurological deficits (Fig. 2A, B).
Compared with the exo-ctrl group, rats in the exo-Zeb2/
13

Fig. 1  The expression of Zeb2 and Axin2 in brain tissue and
exosomes. The expression of Axin2 in (A) corpus striatum and (B)
cortex; the expression of Axin2 in (C) corpus striatum and (D) cor-
tex of the MCAO model. (E) The exosome ultrastructure by transmis-
sion electron microscopy. Scale bar, 100  nm. (F) The expression of
Axin2 group exhibited a significant improved neu-
rological function, as indicated by the results of the
mNSS test (P < 0.05 after day 21; Fig.  2C) and foot
fault test (P < 0.05 after day 14; Fig. 2D). In addition,
tissue loss was significantly lower in exo-Zeb2/Axin2-
treated rats compared with controls (11.48 ± 2.51% vs.
37.87 ± 4.74%, P < 0.01; Fig. 2E).
Effects of Treatment with Zeb2/Axin2‑Enriched
Exosomes on Neurogenesis
To evaluate the effects of Zeb2/Axin2-enriched exosomes
on nerve function recovery, endogenous neural stem cells
(NSCs) were identified by BrdU/NeuN labeling at day 28
after MCAO. Immunofluorescence staining of BrdU/NeuN
showed a greater number of newly generated neurons in the
13
Journal of Molecular Neuroscience
exosomes biomarker protein and Zeb2/Axin2 was detected in both
cell lysis and exosomes lysis by western blot. Error bars denote SD.
n = 6. *P < 0.05, compared to the sham group, analyzed by one-way
ANOVA followed by Bonferroni test. WBL, whole-cell lysis; Exo,
exosome
exosome-treated group than in the MCAO group. Compared
with the exo-ctrl group, the exo-Zeb2/Axin2 group exhibited
a significant increase in the total number of BrdU- and NeuN-
positive cells in the SVZ, hippocampus, and cortex (Fig. 3A,
B). These data indicate that Zeb2/Axin2-enriched exosomes
promoted neuronal division after induction of ischemia.
Zeb2/Axin2‑Enriched Exosomes Promote
the Expression of Nerve Growth Factors
Generally, growth and neurotrophic factors are potent
regulators of neurogenesis (Schmidt and Duman 2007).
Therefore, we screened relevant factors including BDNF,
vascular endothelial growth factor (VEGF), stromal cell-
derived factor-1 (SDF-1), and TGF-β1 by RT-qPCR.
The mRNA levels of BDNF were significantly increased
Journal of Molecular Neuroscience
Fig. 2  Zeb2/Axin2–enriched exosomes treatment improved neuro-
logical behavioral outcome. (A) Representative track images of each
group of rats in the day 7 probe trial test. (B) Latency to the plat-
form of each group of rats in the day 7 probe trial test. (C) Modified
neurological severity score (mNSS) test and (D) foot fault test were
after exosome treatment (Fig.  3C), and this increase
was more pronounced in the exo-Zeb2/Axin2 group
(Fig. 3C). Additionally, increased BDNF protein levels
examined. (E) Whole-brain infarct volume was assessed by TTC stain
at 21  days post-injury. n = 6/group. MCAO, middle cerebral artery
occlusion. *P < 0.05, compared to MCAO group. #P < 0.05, com-
pared to the MCAO + exo-ctrl group, analyzed by one-way ANOVA
followed by the Bonferroni test
were observed in hippocampal tissues in both exosome-
treated groups, but no difference was found between the
exo-ctrl and exo-Zeb2/Axin2 groups (Fig. 3D). Moreo-
ver, the changes in BDNF expression were confirmed by
13

Fig. 3  Zeb2/Axin2–enriched exosomes enhanced neurogenesis and
expression of nerve growth factors in the ischemic brain. (A) Rep-
resentative micrographs show the double-stained cells with BrdU
(green) and NeuN (red) in the subventricular zone (SVZ), hippocam-
pus and cortex, and (B) quantitative data. (C) RT-PCR analysis for
growth and neurotrophic factors at 28  days after MCAO. (D) West-
ern blot and quantitative data were assessed for BDNF expression
13
Journal of Molecular Neuroscience
in different groups. (E) Immunofluorescence stain and (F) mean
fluorescence intensity of BDNF (red) and BrdU (green). n = 6/
group. MCAO, middle cerebral artery occlusion. Scale bar, 100 μm.
*P < 0.05, compared to MCAO group. #P < 0.05, compared to the
MCAO + exo-ctrl group, analyzed by one-way ANOVA followed by
the Bonferroni test
Journal of Molecular Neuroscience

immunofluorescence staining in the hippocampus, SVZ,
and cortex (Fig. 3E, F).
Zeb2/Axin2‑Enriched Exosomes Promote Neurite
Remodeling in the Ischemic Boundary Zone (IBZ)
To elucidate the effect of Zeb2/Axin2-enriched exosomes on
functional recovery, we assessed dendritic, axonal, synaptic,
and myelin remodeling. First, HE staining was performed
on coronal sections. Compared to control rats, MCAO rats
exhibited massive vacuoles and demyelination in nuclei. The
vacuoles and demyelination were alleviated after treatment
with control exosomes and Zeb2/Axin2-enriched exosomes,
although treatment with Zeb2/Axin2-enriched exosomes
resulted in more prominent effects (Fig. 4A). Second, Biels-
chowsky silver immunostaining was performed to detect the

Fig. 4  Zeb2/Axin2–enriched
exosomes promote synaptic
remodeling in the ischemic
boundary zone. (A) HE stain-
ing on coronal sections. (B)
Bielschowsky silver stain and
(E) histogram analysis in the
ischemic boundary zone. (C)
Immunohistochemistry of
synaptophysin and (F) histo-
gram analysis in the ischemic
boundary zone. (D) Immuno-
histochemistry of SMI-31 and
(G) histogram analysis in the
ischemic boundary zone. n = 6/
group. MCAO, middle cerebral
artery occlusion. Scale bar,
100 μm. *P < 0.05, compared
to MCAO group. #P < 0.05,
compared to the MCAO + exo-
ctrl group, analyzed by two-way
ANOVA or one-way ANOVA
followed by Bonferroni test

13

axons in the IBZ. The positive staining area was increased
in the exo-ctrl and exo-Zeb2/Axin2 groups compared with
the MCAO group, but the increase was more significant in
the exo-Zeb2/Axin2 group (Fig. 4B, E). Third, immunohis-
tochemical staining was performed to assess post-stroke SYN
expression in axons and dendrites as a measure of synaptic
plasticity in the peri-infarct region (Fig. 4C, F). Compared
with untreated MCAO rats, exosome-treated rats exhibited
significantly increased positive SYN immunoreactivity in the
IBZ. Finally, we detected myelin remodeling by staining for
phosphorylated neurofilament (SMI-31). The immunostaining
data showed that exosome treatment significantly enhanced the
positive staining for SMI-31 compared with that in the MCAO
group, while treatment with Zeb2/Axin2-enriched exosomes
further promoted remyelination in the IBZ (Fig. 4D, G).
Zeb2/Axin2‑Enriched Exosomes Increase Neurite
Outgrowth In Vitro
To elucidate the mechanisms underlying the effect of Zeb2/
Axin2-enriched exosomes on MCAO, primary cultured
neurons (PCNs) were cultured under OGD conditions as an
in vitro model to mimic in vivo ischemic conditions. Then,
PCNs were treated with control and Zeb2/Axin2-enriched
exosomes, and outgrowth was examined by morphological
observation and immunofluorescence staining. Compared
Fig. 5  Zeb2/Axin2–enriched exosomes increased neurite outgrowth
and regulated the downstream pathway in primary cultured neurons
(PCN) after oxygen–glucose deprivation (OGD). (A) The morphol-
ogy of cultured neurons in this assay. (B) Neurite outgrowth in the
PCN immunofluorescence-stained by MAP2. (C) Western blot
13
Journal of Molecular Neuroscience
to the control group, the neurite number and total neur-
ite length per neuron were increased after exosome treat-
ment. Zeb2/Axin2-enriched exosomes showed an enhanced
effect. Furthermore, compared with the exo-ctrl group, the
exo-Zeb2/Axin2 group exhibited enhanced promotion of
glial neuronal branching and elongation under OGD con-
ditions (Fig. 5A, B). These data suggest that Zeb2/Axin2-
enriched exosomes increased neurite outgrowth in vitro.
Zeb2/Axin2‑Enriched Exosomes Regulate SOX10,
Endothelin‑3/EDNRB, and Wnt/β‑Catenin Pathways
Existing evidence indicates that Zeb2 and AXIN2 can
regulate the endothelin-3/EDNRB and Wnt/β-catenin
signaling pathways, respectively, via the SOX10 gene
(Watanabe et al. 2017). Thus, we investigated the protein
expression of SOX10, endothelin-3/EDNRB, and Wnt/β-
catenin via western blot analysis. Compared with those
in the control group, the expression levels of SOX10,
Wnt, and β-catenin were decreased in the OGD group
(Fig. 5C, D). Exosome treatment reversed this reduc-
tion, and treatment with Zeb2/Axin2-enriched exosomes
enhanced this reversal. Similarly, treatment with Zeb2/
Axin2-enriched exosomes reversed the reductions in
the endothelin-3 and EDNRB levels induced by OGD
in primary cortical neurons (PCNs) (Fig. 5C, D). Taken
together, these data suggest that treatment with Zeb2/
quantification and (D) histogram analysis of SOX10, endothelin-3,
EDNRB, Wnt3a, and β-catenin. Scale bar, 100  μm. *P < 0.05, com-
pared to control group. #P < 0.05, compared to the OGD + exo-ctrl
group, analyzed by one-way ANOVA followed by the Bonferroni test
Journal of Molecular Neuroscience
Axin2-enriched exosomes may improve neural plastic-
ity and functional recovery through activation of the
SOX10-mediated downstream endothelin-3/EDNRB and
Wnt/β-catenin pathways.
Discussion
Ischemia can cause irreversible damage to nerve function.
Evidence has shown that the CNS, especially the cerebral
cortex, has enormous capacity for physiological and anatomi-
cal reorganization, a property termed plasticity (Nudo 2001).
Studies have reported that cell-free exosomes derived from
MSCs restore post-stroke function in rats, and this effect is
similar to that of MSC treatment (Xin et al. 2014, 2013a). In
this study, we generated exosomes from BMSCs overexpress-
ing both Zeb2 and Axin2, which may amplify the therapeu-
tic benefit of the exosomes. After stimulation with BMSC-
derived exosomes, MCAO rats exhibited marked restoration
of spatial memory and nerve function, and these effects were
more obvious after long-term exposure. Thus, Zeb2/Axin2-
enriched BMSC-derived exosomes enhanced recovery.
Although neuronal damage is irreversible, the brain can
participate in its repair and functional recovery after stroke
through two main processes: proliferation/maturation of
NSCs and migration of these cells to the damaged stria-
tum (Darsalia et al. 2007). Transient forebrain ischemia is
generally believed to increase neurogenesis, which leads
to peak proliferation levels approximately 1–2 weeks after
ischemic injury (Felling and Levison 2003). Therefore,
we evaluated the expression of Zeb2 and Axin2 on the 7th
and 14th days. The results showed that the mRNA levels
of both genes were higher at 7 and 14 days. This trend is
consistent with the above-mentioned neuronal cell pro-
liferation, and indicates that Zeb2 and Axin2 may play a
certain role in promoting proliferation/maturation of NSCs
in brain injury.
We next investigated the effect of exo-Zeb2/Axin2 on
NSC proliferation 28 days after MCAO. We stained the
neuronal marker NeuN and the proliferation marker BrdU
in the SVZ, hippocampus, and cortical areas. Treatment
with exosomes increased the number of BrdU-positive
cells in the SVZ, hippocampus, and cortex areas. Although
the cortex is not traditionally considered a "true" neuro-
genic area, various studies have shown that neural precur-
sor cells and mature neurons can be observed in the cortex
area (Magavi et al. 2000; Chen et al. 2004). In this study,
proliferating neurons can be observed in the cortex after
exosome treatment, which also confirms the above view.
Interestingly, the number of BrdU/NeuN double-positive
cells in the SVZ or cortical areas did not differ between
the exo-ctrl and exo-Zeb2/Axin2 groups, while the number
of differentiated cells was increased in the hippocampus
of rats in the exo-Zeb2/Axin2 group. Studies indicate the
number of neuronal progenitors and immature neurons in
the hippocampus could be drastically affected by patho-
logical conditions (Moreno-Jiménez et al. 2019; Seki et al.
2019). Thus, exo-Zeb2/Axin2 shows better promotion of
NPC proliferation/maturation. These results indicate that
treatment with Zeb2/Axin2-enriched exosomes promotes
the enhancement of NSC proliferation and maturation.
Growth factors and neurotrophic factors are impor-
tant regulators of neurogenesis (Lee and Son 2009). We
used qPCR to screen possible regulators involved in the
therapeutic mechanism of exosomes. The results showed
that exosome treatment increased the BDNF and VEGF
mRNA levels and that treatment with Zeb2/Axin2-enriched
exosomes further increased the level of BDNF compared to
treatment with control exosomes. BDNF and VEGF stimu-
late neurogenesis and enhance the appearance and migra-
tion of new neurons in the SVZ and dentate gyrus (Kim
et al. 2014). Moreover, intravenous BDNF therapy after
ischemia can improve the long-term functional neurological
prognosis of induced neurogenesis (Scharfman et al. 2005;
Quesseveur et al. 2013).
In addition, in the IBZ, Zeb2/Axin2-enriched exosomes
promoted synaptic remodeling by increasing the number
of synapses and reversed axonal loss of SMI-31 and SYN
caused by ischemic injury, thus alleviating axonal death
and promoting synaptic proliferation. Accumulated studies
have revealed that the enhancement of plastic reorganization
can result in superior behavioral recovery (Théry et al. 2006). In
addition, the in vitro data also showed that treatment with
Zeb2/Axin2-enriched exosomes significantly enhanced the
neurite growth from PCNs, consistent with the effect of
previously tested exosomes. Several studies have reported
that exosomes derived from MSCs mediate the transfer of
miRNAs to astrocytes and neurons, which regulates gene
expression and subsequently benefits neurite remodeling and
functional recovery after stroke (Xin et al. 2013a, b, 2017a,
b). Therefore, combination therapy with Zeb2 and Axin2
enhanced neurogenesis, synaptic plasticity, and axon growth,
which mediated functional improvement.
SOX10 is a transcription factor that plays an impor-
tant role in the growth of spinal neurons and can maintain
their multidirectional differentiation potential (Shin et al.
1999). Additionally, EDNRB plays an important role in
the development of intestinal nerves (Sidebotham et al. 2002).
The endothelin-3/EDNRB pathway has been reported to
be regulated by SOX10 and Zeb2 (Watanabe et al. 2017)
and the Wnt/β-catenin pathway by SOX10 and Axin2
(Dai et al. 2014). Thus, we evaluated the expression of
these proteins. As expected, treatment with Zeb2/Axin2-
enriched exosomes upregulated the expression of SOX10
13

and Wnt/β-catenin pathway components, but downregu-
lated endothelin-3/EDNRB pathway components.
In conclusion, our data demonstrate that Zeb2/Axin2-
enriched BMSC-derived exosomes can upregulate BDNF,
increase neurogenesis, and promote synaptic plasticity and
neurite remodeling in the ischemic brain. These effects
improved neuroplasticity and functional recovery after
stroke in MCAO rats. Our findings suggest that engineering
exosomes could be a useful strategy to enhance functional
restoration processes after stroke.
Authors’ Contributions  RW and WZ contributed to the conception and
design of the study. LZ, WH, XS and YH performed the experiments,
and analyzed and interpreted data. RW and WZ drafted and revised
the manuscript. All authors read and approved the final manuscript.
Funding  This work was supported by National Natural Science Foun-
dation of China (Grant 81760349, 81560319) and Applied Basic
Research Foundation of Yunnan Province (Grant 2016FB130).
Availability of Data and Materials  The datasets used and/or analyzed
during the current study are available from the corresponding author
on reasonable request.
Declarations  Jin C, Tian H, Li J et al (2018) Stem cell education for medical students
Ethics Approval and Consent to Participate  The present study was
approved by the Laboratory Animal Ethics Review Committee of
Kunming Medical University (Kunming, China).
Competing Interests  The authors declare that they have no competing
interests.
References
Aimone JB, Li Y, Lee SW, Clemenson GD, Deng W, Gage FH (2014)
Regulation and function of adult neurogenesis: from genes to cog-
nition. Physiol Rev 94(4):991–1026
Braccioli L, Velthoven C, Heijnen CJ (2014) Exosomes: A New
Weapon to Treat the Central Nervous System. Mol Neurobiol
49(1):113–119
Brouns R, Espinoza AV, Goudman L, Moens M, Verlooy J (2019)
Interventions to promote work participation after ischaemic
stroke: A systematic review. Clin Neurol Neurosurg 105458
Chang D-J, Oh S-H, Lee N, Choi C, Jeon I, Kim HS et al (2013)
Contralaterally transplanted human embryonic stem cell-derived
neural precursor cells (ENStem-A) migrate and improve brain
functions in stroke-damaged rats. Exp Mol Med 45(11):e53–e53
Chavez JC, Baranova O, Lin J, Pichiule P (2006) The transcriptional
activator hypoxia inducible factor 2 (HIF-2/EPAS-1) regulates the
oxygen-dependent expression of erythropoietin in cortical astro-
cytes. 26(37):9471–9481
Chen J, Magavi SS, Macklis JD (2004) Neurogenesis of corticospinal
motor neurons extending spinal projections in adult mice. Proc
Natl Acad Sci 101(46):16357–16362
Chen Z-Z, Jiang X-D, Zhang L-L, Shang J-H, Du M-X, Xu G et al
(2008) Beneficial effect of autologous transplantation of bone
13
Journal of Molecular Neuroscience
marrow stromal cells and endothelial progenitor cells on cerebral
ischemia in rabbits. Neurosci Lett 445(1):36–41
Chivet M, Hemming F, Pernet-Gallay K, Fraboulet S, Sadoul R (2012)
Emerging Role of Neuronal Exosomes in the Central Nervous
System. Front Physiol 3:145
Comijn J, Berx G, Vermassen P, Verschueren K, van Grunsven L,
Bruyneel E et al (2001) The two-handed E box binding zinc fin-
ger protein SIP1 downregulates E-cadherin and induces invasion.
Mol Cell 7(6):1267–1278
Dai Z-M, Sun S, Wang C, Huang H, Hu X, Zhang Z et al (2014) Stage-
specific regulation of oligodendrocyte development by Wnt/β-
catenin signaling. J Neurosci 34(25):8467–8473
Dao DY, Yang X, Di C, Zuscik M, O’Keefe, RJ (2007) Axin1 and
Axin2 are regulated by TGF-β and mediate cross-talk between
TGF-β and Wnt signaling pathways. Ann N Y Acad Sci 1116:82
Darsalia V, Kallur T, Kokaia Z (2007) Survival, migration and neu-
ronal differentiation of human fetal striatal and cortical neural
stem cells grafted in stroke-damaged rat striatum. Eur J Neurosci
26(3):605–614
Fancy SP, Harrington EP, Yuen TJ, Silbereis JC, Zhao C, Baranzini SE
et al (2011) Axin2 as regulatory and therapeutic target in newborn
brain injury and remyelination. Nat Neurosci 14(8):1009
Felling RJ, Levison SW (2003) Enhanced neurogenesis following
stroke. J Neurosci Res 73(3):277–283
Ferro JM, Caeiro L, Figueira ML (2016) Neuropsychiatric sequelae of
stroke. Nat Rev Neurol 12(5):269
Ji L-L, Long X-F, Tian H, Liu Y-F (2013) Effect of transplantation of
bone marrow stem cells on myocardial infarction size in a rabbit
model. World J Emerg Med 4(4):304
at Tongji University: Primary cell culture and directional differ-
entiation of rat bone marrow mesenchymal stem cells. Biochem
Mol Biol Educ 46:151–154
Khodanovich M, Nemirovich-Danchenko NM (2019) New neurons in
the post-ischemic and injured brain: migrating or resident? Front
Neurosci 13:588
Kim YR, Kim HN, Ahn SM, Choi YH, Shin HK, Choi BT (2014)
Electroacupuncture promotes post-stroke functional recovery
via enhancing endogenous neurogenesis in mouse focal cerebral
ischemia. PLoS One 9(2)
Lee E-G, Son H (2009) Adult hippocampal neurogenesis and related
neurotrophic factors. BMB Rep 42(5):239–244
Ling X, Zhang G, Zhu Q, Zhang J, Li Q, Niu X et al (2018) Exosomes
from Human Urine-Derived Stem Cells Promote Neurogenesis
Via Histone Deacetylase6 Regulation in Ischemic Stroke. Avail-
able at SSRN 3288885
Longa EZ, Weinstein P, Carlson S, Cummins R (1989) Reversible mid-
dle cerebral artery occlusion without craniectomy in rats. Stroke
20:84–91
Magavi SS, Leavitt BR, Macklis JD (2000) Induction of neurogenesis
in the neocortex of adult mice. Nature 405(6789):951–955
Mahmood A, Lu D, Qu C, Goussev A, Chopp M (2006) Long-term
recovery after bone marrow stromal cell treatment of traumatic
brain injury in rats. J Neurosurg 104(2):272–277
Manuel GE, Johnson T, Liu D (2017) Therapeutic angiogenesis of
exosomes for ischemic stroke. International Journal of Physiology,
Pathophysiology and Pharmacology 9(6):188
Moreno-Jiménez EP, Flor-García M, Terreros-Roncal J, Rábano A,
Cafini F, Pallas-Bazarra N et al (2019) Adult hippocampal neu-
rogenesis is abundant in neurologically healthy subjects and
drops sharply in patients with Alzheimer’s disease. Nat Med
25(4):554–560
Nagasawa H, Kogure K (1989) Correlation between cerebral blood
flow and histologic changes in a new rat model of middle cerebral
artery occlusion. Stroke 20(8):1037–1043
Journal of Molecular Neuroscience
Nudo RJ (2001) Role of adaptive plasticity in recovery of function after
damage to motor cortex. Muscle Nerve 24
Oki K, Tatarishvili J, Wood J, Koch P, Wattananit S, Mine Y et al
(2012) Human-induced pluripotent stem cells form functional
neurons and improve recovery after grafting in stroke-damaged
brain. STEM CELLS 30(6):1120–1133
Qu R, Li Y, Gao Q, Shen L, Zhang J, Liu Z et al (2007) Neurotrophic
and growth factor gene expression profiling of mouse bone mar-
row stromal cells induced by ischemic brain extracts. Neuropa-
thology 27(4):355–363
Quesseveur G, David D, Gaillard M, Pla P, Wu M, Nguyen H et al
(2013) BDNF overexpression in mouse hippocampal astrocytes
promotes local neurogenesis and elicits anxiolytic-like activities.
Transl Psychiatry 3(4):e253–e253
Rexach J, Swarup V, Chang T, Geschwind D (2019) Dementia risk
genes engage gene networks poised to tune the immune response
towards chronic inflammatory states. bioRxiv 597542
Scharfman H, Goodman J, Macleod A, Phani S, Antonelli C, Croll
S (2005) Increased neurogenesis and the ectopic granule cells
after intrahippocampal BDNF infusion in adult rats. Exp Neurol
192(2):348–356
Schmidt HD, Duman RS (2007) The role of neurotrophic factors in
adult hippocampal neurogenesis, antidepressant treatments and
animal models of depressive-like behavior. Behav Pharmacol
18(5–6):391–418
Seki T, Hori T, Miyata H, Maehara M, Namba T (2019) Analysis of
proliferating neuronal progenitors and immature neurons in the
human hippocampus surgically removed from control and epilep-
tic patients. Sci Rep 9(1):1–14
Shin MK, Levorse J, Ingram RS, Tilghman SM (1999) The temporal
requirement for endothelin receptor-B signalling during neural
crest development. Nature 402:496–501
Sidebotham EL, Woodward MN, Kenny SE et al (2002) Localization
and endothelin-3 dependence of stem cells of the enteric nervous
system in the embryonic colon. J Pediatr Surg 37(2):145–150
Tang Y, Cui Y-C, Wang X-J, Wu A-L, Hu G-F, Luo F-L et al (2012)
Neural progenitor cells derived from adult bone marrow mes-
enchymal stem cells promote neuronal regeneration. Life Sci
91(19–20):951–958
Théry C, Amigorena S, Raposo G (2006) Isolation and characterization
of exosomes from cell culture supernatants and biological fluids.
Curr Protoc Cell Biol 3(3):22
Théry C, Zitvogel L, Amigorena S (2002) Exosomes: composition,
biogenesis and function. Nat Rev Immunol 2(8):569–579
Tian T, Zhang H-X, He C-P, Fan S, Zhu Y-L, Qi C et al (2018a) Surface
functionalized exosomes as targeted drug delivery vehicles for
cerebral ischemia therapy. Biomaterials 150:137–149
Tian T, Zhang H, He C, Fan S, Zhu Y, Qi C et al (2018b) Surface
Functionalized Exosomes as Targeted Drug Delivery Vehicles for
Cerebral Ischemia Therapy 150:137–149
Vahidy FS, Rahbar MH, Zhu H, Rowan PJ, Bambhroliya AB, Savitz
SI (2016) Systematic review and meta-analysis of bone marrow–
derived mononuclear cells in animal models of ischemic stroke.
Stroke 47(6):1632–1639
Valadi H, Ekström K, Bossios A, Sjöstrand M (2007) Exosome-mediated
transfer of mRNAs and microRNAs is a novel mechanism of
genetic exchange between cells. Nat Cell Biol 9(6):654–659
Watanabe Y, Stanchina L, Lecerf L, Gacem N, Conidi A, Baral V
et al (2017) Differentiation of mouse enteric nervous system pro-
genitor cells is controlled by endothelin 3 and requires regula-
tion of Ednrb by SOX10 and ZEB2. Gastroenterology 152(5):
1139–1150. e1134
Wu Z, Liang Y, Yu S (2020) Downregulation of microRNA-103a
reduces microvascular endothelial cell injury in a rat model
of cerebral ischemia by targeting AXIN2. J Cell Physiol
235(5):4720–4733
Xin H, Katakowski M, Wang F, Qian J-Y, Liu XS, Ali MM et al (2017a)
MicroRNA-17–92 cluster in exosomes enhance neuroplasticity
and functional recovery after stroke in rats. Stroke 48(3):747–753
Xin H, Li Y, Chopp M (2014) Exosomes/miRNAs as mediating cell-
based therapy of stroke. Front Cell Neurosci 8:377
Xin H, Li Y, Cui Y, Yang JJ, Zhang ZG, Chopp M (2013a) Systemic
administration of exosomes released from mesenchymal stromal
cells promote functional recovery and neurovascular plasticity
after stroke in rats. J Cereb Blood Flow Metab 33(11):1711–1715
Xin H, Li Y, Liu Z, Wang X, Shang X, Cui Y et al (2013b) MiR-133b
Promotes Neural Plasticity and Functional Recovery After Treat-
ment of Stroke with Multipotent Mesenchymal Stromal Cells in
Rats Via Transfer of Exosome-Enriched Extracellular Particles.
STEM CELLS 31(12):2737–2746
Xin H, Wang F, Li Y, Lu Q-E, Cheung WL, Zhang Y et al (2017b)
Secondary release of exosomes from astrocytes contributes to
the increase in neural plasticity and improvement of functional
recovery after stroke in rats treated with exosomes harvested from
microRNA 133b-overexpressing multipotent mesenchymal stro-
mal cells. Cell Transplant 26(2):243–257
Publisher's Note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
13