2 against stroke in rats

Brain Research Bulletin 149 (2019) 42–52

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Protective effects of mesenchymal stem cells overexpressing extracellular regulating kinase 1/2 against stroke in rats

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Xiaoqing Gaoa,b, Dandan Wua, Ling Doua, Haibo Zhanga, Liang Huanga, Jiaqi Zenga, Yiiie Zhanga, ⁎ Chaoxian Yangb, Huanhuan Lia, Lifen Liua, Bin Mac, Qionglan Yuana, a

Department of Neurology, Shanghai Tongji hospital, Tongji University School of Medicine, Shanghai, 200065, China Department of Anatomy and Neurobiology, Southwest Medical University, Luzhou, 646000, China c Department of Molecular and Biomedical Sciences, School of Veterinary and Life Sciences, Murdoch University, Perth, WA, Australia b

A R T I C LE I N FO

A B S T R A C T

Keywords: Bone mesenchymal stem cells (MSCs) Extracellular regulating kinase 1/2 (ERK1/2) Neuronal de-differentiation Neuroinflammation Intrinsic proliferation Stroke

Objective: Although transplantation of bone marrow-derived mesenchymal stem cells (MSCs) has shown beneficial effects on stroke, lower survival of MSCs limits effects. Extracellular regulating kinase 1/2 signaling (ERK1/2) is crucial for cell survival, differentiation, and proliferation. This study was designed to explore whether MSCs modified by over-expressing ERK1/2 may reinforce beneficial effects on stroke in rats. Methods: rat MSCs transfected with ERK1/2 and empty lentivirus to generate MSCs overexpressing ERK1/2 (ERK/MSCs) and MSCs (as a control), respectively. In vitro, ERK/MSCs were plated and exposed to glutamateinduced condition, and viability of ERK/MSCs was measured. Furthermore, neural induction of ERK/MSCs was investigated in vitro. Cerebral ischemic rats were induced by occluding middle cerebral artery, and then were stereotaxically injected into ipsilateral right lateral ventricle with ERK/MSCs or MSCs 3 days after stroke and survived for 7 or 14 days after injection. Results: ERK/MSCs showed better viability in physiological and glutamate-induced neurotoxic conditions compared to MSCs. After neural induction, more neurons were be differentiated from ERK/MSCs than from MSCs. After transplantation, more numbers of grafted cells and improved functional recovery were observed in ERK/MSCs-treated rats compared with MSCs-treated rats. Compared with MSCs treatment, ERK/MSCs treatment significantly increased proliferation of neural stem cells in the subventricle zone (SVZ) and the MAP2/nestin double-labeled cells adjacent to the SVZ, enhanced the numbers of reactive astrocytes while suppressed microglial activation. Besides, TNF-α level was elevated in ERK/MSCs-treated rats. Conclusion:ERK/MSCs transplantation showed better functional recovery after stroke in rats, likely in part through enhancing survival of MSCs and possibly by modulating the proliferation, neuronal de-differentiation and neuroinflammation.

1. Introduction Stroke is one of the leading causes of death and disability worldwide and has no effective clinical therapies with the exception of tissue-type plasminogen activator (tPA). Cell-based transplantation has shown a promise in improving neurological sequelae in animal models. Bone marrow-derived mesenchymal stem cells (MSCs) are strongly preferred among candidates of stem cells because they are available and immune privileged for allogeneic cells (Tse et al., 2003; Li et al., 2006). Compelling evidence has shown that MSCs transplantation significantly improves functional recovery after stroke in rats (Chen et al., 2001; Rempe and Kent, 2002; Iihoshi et al., 2004) and neurodegenerative disease such as Alzheimer’s diseases (Babaei et al., 2012; Yun et al.,

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2013). MSCs-based therapy was initially considered for the replacement of neuronal loss. However, in the past two decades, very few MSCs expressing neural markers have been confirmed (Hess and Borlongan, 2008; Kondo et al., 2011; Tu et al., 2014). More importantly, by using ultrastructural and electrophysiological analyses, no neuronal transdifferentiation of MSCs as demonstrated by the presence of specific synapses or typical neuronal action potentials has been observed (Li and Chopp, 2009). Currently, it is believed that MSCs exert effects through multiple mechanisms. For example, growing evidence suggests that the benefts of MSCs are attributable to the neurotrophins, growth factors, and other supportive substances secreted by MSCs after damage to the brain in rodents (Chen et al., 2002; Li et al., 2002; Caplan and Dennis, 2006; Qu et al., 2007; Hess and Borlongan, 2008; Wakabayashi

Corresponding author. E-mail address: [email protected] (Q. Yuan).

https://doi.org/10.1016/j.brainresbull.2019.04.006 Received 22 March 2018; Received in revised form 28 February 2019; Accepted 9 April 2019 Available online 16 April 2019 0361-9230/ © 2019 Published by Elsevier Inc.

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et al., 2010). Thus, MSCs are thought to work as “small molecular factories” that reduce apoptosis and promote the survival of damaged neurons, leading to functional benefits. Moreover, neuroinflammation modulated by MSCs is regarded as another important mechanism (Li et al., 2005; Lee et al., 2010; Sheikh et al., 2011; Wei et al., 2012). Activated microglia and astrocytes secret neurotrophic factors, such as transforming growth factor beta (TGF-β), brain-derived neurotrophic factors (BDNF), and glial cell line-derived neurotrophic factors (GDNF), which induce intrinsic neurogenesis, angiogenesis, neuroprotection and neurite outgrowth (Zhang and Chopp, 2002; Wakabayashi et al., 2010; Wei et al., 2012). In addition, the activated glial cells produce proinflammatory cytokines and anti-inflammatory cytokines (Sheikh et al., 2011; Wei et al., 2012; Wang et al., 2013), resulting in neuroprotective or neurotoxic effects depending on the niches (vonBernhardi et al., 2015). Taken together, the multiple effects of MSCs transplantation on stroke may lead to neurobiological recovery. However, recent studies have indicated that only a small subset of MSCs homed in on the injured tissues, and disappeared quickly (Gao et al., 2001; Munoz et al., 2005; Prockop, 2007). Therefore, fewer numbers of MSCs in the recipient may limit the beneficial effects of MSCs. One potential approach promoting cell survival is to modulate the properties of MSCs. Extracellular regulating kinase 1/2 (ERK1/2), a member of mitogen-activated protein kinase (MAPK) family, is wellknown for its improvement of cell survival and exhibits anti-apoptotic effects against a variety of paradigms including oxidative and ischemichock(Wen et al., 2006; Chao et al., 2010; Seo et al., 2010; Tang et al., 2011). Therefore, in this study, we assessed whether MSCs overexpressing ERK1/2 (ERK/MSCs) increase the viability of MSCs after transplantation and exert more efficient roles in rats post stroke.

transfecting 293FT cells with Lipofectamine 2000 (Invitrogen, Tokyo, Japan). The virus solutions were kept at -80 °C until transfection. 2.1.2. Viral infection of MSCs Rat MSCs were prepared as previously described (Yang et al., 2011). After third passage MSCs were seeded in 6-wells plate and reached 80% confluence, ERK1/2 virus and empty virus solution (viral titer: 1.1 × 108 TU/ml) were added to each well and incubated for 48 h at 37 °C in an incubator supplied with 5% CO2. The expression of ERK1/2 in MSCs was analyzed using reverse transcriptase PCR (RT-PCR, Fig.1B) and western blot analysis (Fig. 1C). 2.2. Glutamate treatment in ERK/MSCs culture MSCs (infected with the empty virus) and ERK/MSCs were plated in 96 well-plates for 24 h. The cells were then exposed to a final concentration 500 μM glutamate in DMEM at 37 °C for 15 min, and the medium was then refreshed. After further incubated for 24 h, the cells and culture media were collected for the following experiments. 2.2.1. MTT assay A MTT kit (Beyotime, China) was used to identify cell viability according to the manufacturer’s instructions. Briefly, after exposure to glutamate for 24 h, 20 μl of 0.5% MTT solution was added to 200 μl medium in each well, and the cells were incubated for 4 h at 37 °C in the dark. The resulting blue-colored formazan crystals were dissolved in dimethyl sulphoxide (DMSO, Sigma) and mixed by shaking. Absorbance at 490 nm was recorded by using a fluorescence plate reader. 2.2.2. LDH assay The level of LDH release in the cell culture supernatant was analyzed by using a LDH cytotoxicity assay detection kit (Beyotime, China) according to the manufacturer’s instructions. Briefly, 50 μl supernatant from each well was collected and mixed with 50 μl reaction solution from the kit for 15 min at 37 °C in the dark. 50 μl stopping solution (1 N HCl) per well was then added to stop the reaction. The absorbance at 490 nm was read by using a microplate reader.

2. Materials and methods 2.1. Construction of recombinant ERK1/2 lentivirus and transfected into MSCs 2.1.1. Construction of pLV.EX2d.P/puro-EF1 A > ERK1/T2 A/ERK2/ P2 A/eGFP To construct the full-length ERK1 and ERK2 vectors, the interim vector pDown-ERK1/T2 A/ERK2/P2 A/eGFP was firstly generated. Using ERK1/T2 A /ERK2/P2 A and eGFP (previously constructed in our lab) as templates, ERK1 front end, ERK2 rear end, and enhanced green fluorescent protein (eGFP) were generated by using polymerase chain reaction (PCR). The resultant PCR products were fused and amplified by PCR to generate the attB1-Kozak-ERK1 front end /ERK2 rear end /P2 A/eGFP/attB2. Finally, NdeI-ERK1 rear end /T2 A/ERK2 front endEcoRI was generated by PCR. By using gateway technology, pDown-ERK1 front end /ERK2 rear end /P2 A/eGFP were generated by means of attB1-Kozak-ERK1 front end /ERK2 rear end /P2 A/eGFP/attB2 and pDONR221 as templates. The reaction products were transformed into E.coli, and the positive clones were screened by colony PCR and selected for DNA sequencing. After pDown-ERK1 front end /ERK2 rear end /P2 A/eGFP and NdeIERK1 rear end /T2 A/ERK2 front end-EcoRI were digested with NdeI and EcoRI, the products were ligated by T4 DNA ligase to form the pDown-ERK1/T2 A/ERK2/P2 A/eGFP, which was then transformed into E.coli. The positive clones were screened by colony PCR and selected for DNA sequencing. By using gateway technology, pLV.EX2d.P/ puro-EF1 A > ERK1 /T2 A /ERK2 /P2 A/eGFP was generated by means of pLV.Des2d.P/ puro and pDown-ERK1/T2 A/ERK2/P2 A/eGFP as templates. The reaction products were transformed into E.coli, and the positive clones were screened by colony PCR and selected for DNA sequencing. The recombinant lentivirus containing ERK1and ERK2 was shown in Fig. 1A. The recombinant ERK1/2 lentivirus expressing ERK1 and ERK2 (ERK1/2) and empty viruses (as a control) were prepared by

2.2.3. Hoechst 3325833,258 staining Hoechst 33358 staining was performed to classify viable and apoptotic cells as previously described (Yuan et al., 2007; Chen et al., 2009). After exposure to glutamate, the cells were fixed in 4% paraformaldehyde (PFA) for 30 min at room temperature (RT). 0.5 ml of Hoechst 33258 solutions (Beyotime, China) was added to each well, and the cells were incubated at 37 °C for 15 min in the dark. Then, apoptotic/viable cells were counted from duplicate wells. 2.2.4. In situ detection of DNA fragmentation TUNEL staining was used to identify the number of cells exhibiting DNA fragmentation under light microscope by using TUNEL-peroxidase combined with nonisotopic digoxigenin-11 dUTP and terminal transferase according to the manufacturer’s instructions (Roche, Basel, Switzerland). After exposure to glutamate, the cells were fixed in 4% PFA at RT for 30 min, and then treated with newly made 3% H2O2 for 15 min to inactivate endogenous peroxidases. These cells were followed to be incubated with 50 ml TUNEL reaction mixture (5 ml enzyme solution and 45 ml label solution) at 37 °C for 60 min and then with converter-peroxidase at 37 °C for 30 min and developed with 3, 3′diaminobenzidine for 3 to 5 min. Finally, these cells were slightly counterstained with hematoxylin. The apoptotic/viable cells were counted from three separate experiments. 2.3. Neural induction in vitro For the neural differentiation induction of MSCs in vitro, cells were reseeded at a density of 1 × 104/cm2 onto coverslips pre-coated with 43

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Fig. 1. MSCs are efficiently infected by the ERK1/2 recombinant lentivirus. (A) lentivirus vector encoding ERK1 and ERK2. (B–C) Increased ERK mRNA levels (B) and their protein levels (C) in ERK/MSCs were shown compared with MSCs measured by RT-PCR and western blot analysis, respectively.

was allowed by withdrawal of the suture. After surgery, rats exhibiting neurological deficits characterized by failure to extend the left forepaw were used to perform cell transplantation.

poly-L-lysine in 6-well plates in DMEM. When the cells reached 80% confluence, neural differentiation was induced as previously described (Yang et al., 2011). Briefly, MSCs and ERK/MSCs were pre-induced with DMEM/10% FBS/1 mM β-mercaptoethanol (BME) for 24 h, followed by induction with DMEM/100 mM all-trans retinoic acid (Sigma) for 6 h. Cells were then collected for immunocytochemical staining and RT-PCR.

2.4.2. Cell transplantation Rats with neurological deficits were randomly divided into experimental groups. All transplantation procedures were performed under aseptic conditions as described in our previous studies (Swanson et al., 1990; Yang et al., 2011). At 3 days after MCAO, animals were anesthetized with pentobarbital sodium and then placed in a stereotaxic frame (Angle Two™ Stereotaxic Instrument w/Rat Atlas Product: #464601, USA). An incision was made to expose Bregma. Burr holes were drilled for the ipsilateral right lateral ventricle according to the following coordinates: -0.8 to 1.0 mm anterior/posterior, -1.8 to 2.0 mm medial/lateral, -4.0–5.0 mm dorsal/ventral. 20 μl cell suspension containing MSCs or ERK/MSCs (5 × 105 cells) was injected at a speed of 5 μl /min into the rat brain. The control group received 20 μl PBS. None of the animals received immunosuppressants and antibiotics. Animals survived for 1 week or 2 weeks after transplantation.

2.3.1. Immunocytochemical staining Cells on coverslips were treated with 0.2% Triton X-100 for 5 min after fixation with PFA and then blocked with 5% goat serum at room temperature for 30 min in a moisture chamber. Cells were then incubated with primary antibodies against cell-specific markers, including mouse anti- microtubule-associated protein 2 (MAP2, 1:200, Cell Signaling Technology) for neurons, rabbit anti-glial fibrillary acidic protein (GFAP, 1:500, Abcam) for astrocytes, and rabbit anti-galactosecerebroside (Galc, 1:200, Chemicon) for oligodendrocytes at 4 °C overnight, followed by incubation with Cy3-conjugated AffiniPure goat anti-rabbit IgG or goat anti-mouse IgG (1:200) at room temperature for 1.5 h. Cells were rinsed with phosphate buffered saline (PBS) and then stained with diamidino-phenyl-indole (DAPI) followed by mounting with Permount. Coverslips were imaged with a laser scanning confocal microscope (Olympus, Tokyo, Japan) at a 20× objective lens. Each experimental group included three wells, and five non-overlapped fields in each well were counted.

2.4.3. Behavioral tests Behavioral tests were performed just before transplantation (on 3 days after MCAO), 1 and 2 weeks after transplantation. Modified Neurological Severity Scale (mNSS) scores were used to evaluate neurological function according to previous methods (Horita et al., 2006).The mNSS was a composite of motor, sensory, reflex, and balance tests. Neurological function was graded on a scale of 0–18 (normal: 0; maximal deficit score: 18). Higher total points indicate a more severe injury.

2.3.2. RT-PCR analysis To further identify the differentiation after neural induction of MSCs in vitro, total RNA was isolated from cultured cells. To prepare first strand cDNA, 2 μg total RNA was reverse-transcribed with reverse transcriptase (RiverTraAce, Toyobo) in 20 μl reaction mixture. To analyze the mRNA level, PCR was performed with the cDNA and genespecific primers for MAP2, Galc, and Ki67. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control. The mRNA level was normalized to corresponding GAPDH mRNA and quantified by using the relative quantification method.

2.4.4. TTC staining At 1 and 2 weeks after transplantation, lesion volume was measured by 2, 3, 5-triphenyltetrazoliumchloride (TTC, Sigma) staining as previously described (Wislet-Gendebien et al., 2005). In brief, rats (n = 4 per group) were deeply anesthetized, and brains were carefully removed and dissected into 6 coronal blocks (1.5 mm thick each). The fresh blocks were immersed in 2% TTC in saline at 37 °C for 30 min and were then imaged by a digital camera (Canon, EOS 40D). The lesion volume was calculated indirectly by subtracting the intact area of the ipsilateral hemisphere from the area of the contralateral hemisphere (Bederson et al., 1986; Yang et al., 2011). The results were expressed as a percentage of lesion volume compared with contralateral hemisphere area (regarded as 100%).

2.4. Transient ischemia rat model and ERK/MSCs transplantation 2.4.1. Transient ischemia rat model All protocols were approved by the Animal Study Committee at the Tongji University School of Medicine, and the animal care complied with the Guide for the Care and Use of Laboratory Animals. Adult male Wistar rats (270 to 300 g) were used for ischemic stroke experiments. Transient (2 h) middle cerebral artery occlusion (MCAO) was induced as previously described (Horita et al., 2006). In brief, rats were anesthetized with 1% pentobarbital sodium in 0.9% NaCl (30 mg/kg, i.p.). The right common carotid artery, external carotid artery, and internal carotid artery were then exposed. A length of 4-0 monofilament nylon suture (19 to 20 mm long) with its tip rounded was advanced from the common carotid artery into the lumen of the internal carotid artery until it blocked the origin of the middle cerebral artery. At 2 h after MCAO, animals were re-anesthetized with ethyl ether, and reperfusion

2.4.5. Brain tissue preparation and immunohistochemistry All rats (n = 4 per group) were killed at 1 or 2 weeks after transplantation and transcardially perfused with PBS followed by 4% PFA. Brains were removed, post-fixed in 4% PFA for 10–16 h, and kept in 20% sucrose for cryoprotection before being sectioned with a cryostat (CM 1950, Leica, Heidelberg, Germany). Coronal sections (25 μm thick) were then cut and processed for the following staining. To observe the MAP-2 positive cells and GFAP positive cells, the sections were permeabilized with 0.3% Triton X-100 and then blocked 44

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with 5% fetal bovine serum (FBS). Primary antibodies used in this ex periment were mouse-anti MAP2 and rabbit anti-GFAP. The slices were then incubated with Cy3-conjugated AffiniPure goat anti-rabbit IgG or goat anti-mouse IgG (1:200) at room temperature for 2 h, stained with Hoechst 33258, and mounted with Permount. To directly observe MSC or ERK/MSCs under fluorescence microscope, brain slides were kept in the dark to prevent quenching throughout the whole processes. To observe endogenous neural progenitor cells (NPCs) and possible dedifferentiation in rats after stroke, double-immunofluorescent staining was performed. To quench fluorescence of MSC or ERK/MSCs, these brain slides were exposed to light for 5 days in the room temperature before double-immunostaining. Sections were incubated with the following two primary antibodies: rabbit anti-nestin (1:200, Abcam) and mouse-anti GFAP, or rabbit anti-nestin and mouse anti-MAP 2 at 4 °C overnight. After washing with PBS, the slice were incubated with appropriate secondary antibodies Cy3-, FITC-conjugated AffiniPure goat anti-rabbit or anti-mouse IgG (1:200, Jackson ImmunoResearch Laboratories) for 2 h at room temperature. Finally, the slices were stained with Hoechst 33258 followed by mounting with Permount.

ANOVA followed by Tukey's post hoc test for multiple pair-wise examinations in vivo experiments. The difference was considered significant if P < 0.05. 3. Results 3.1. ERK1/2 overexpression in ERK/MSCs In order to generate MSCs overexpressing ERK1/2, isolated MSCs were infected with ERK 1/2 recombination virus and then further cultured for 5–7 days. For control experiments, empty virus solution was used for the infection to generate mock transduced MSCs (named hereafter MSCs). We observed that ERK/MSCs and MSCs displayed a spindle or triangular phenotype under phase contrast microscopy and fluorescent microscopy (data not shown). RT-PCR and Western blot analysis revealed that the protein levels of ERK1/2 were apparently increased in ERK/MSCs compared to MSCs (Fig.1 B–C). These results suggest that ERK1 and ERK2 are stably overexpressed in ERK/MSCs. 3.2. Enhanced viability of ERK/MSCs after exposure to glutamate

2.4.6. Quantification of cell counting in brain sections To estimate the number of MSCs and neural differentiation in ischemic rats, serial coronal sections through the ischemic region (Bregma -0.2 -1.8 mm) were collected and examined in the dark to avoid bleaching of the fluorescence. Three sections with five intervals per animal were evaluated. The grafted cells were counted in each section with five areas in the ischemic striatum and cortex. Numbers of GFAP-positive cells (red), MAP-2-positive cells (red), grafted cells (GFP, green), and graft-derived neurons (i.e. MAP 2/GFP-double-positive cells, yellow) or astrocytes (GFAP/GFP-double-positive cells, yellow) in each slice were counted by using a confocal microscope with a 20× objective lens. To measure endogenous NPCs and any dedifferentiation in rats, nestin-positive cells, nestin/MAP2-double-positive cells, and nestin/ GFAP-double-positive cells were counted. Images were analyzed by using Image Tool Software (Pro Plus v 6.0).

To detect the viability of ERK/MSCs in vitro, ERK/MSCs and MSCs were plated in 24-well plates at the same density and grown for 48 h, 72 h and 96 h. At 96 h, ERK/MSCs grew much better than MSCs (Fig. 2A), and the MTT analysis showed that the viability of ERK/MSCs was significantly increased compared with MSCs (P < 0.05) (Fig. 2B). After exposure to glutamate-induced neurotoxicity, the viability of ERK/MSCs was determined by a series of methods. Firstly, Hoechst 33258 staining is commonly used to evaluate the viable and apoptotic cells. The viable cells are characterized by regular and round nuclei with a pallid blue fluorescence; apoptotic cells are condensed and fragmented (Fig. 2C). In our study, the percentage of apoptosis was significantly decreased in ERK/MSCs compared with MSCs (P < 0.05) (Fig. 2 C–D). Second, the MTT analysis showed that the viability of ERK/MSCs was significantly increased compared to MSCs (P < 0.05) (Fig. 2E). Third, the amount of LDH release into the medium indicates the damage of the cell membrane. The results showed that ERK/MSCs released fewer LDH activity into the cultures (P < 0.05) (Fig. 2F), suggesting more viable cells in ERK/MSCs. Finally, TUNEL staining was used to detect apoptotic cells. TUNEL staining showed that ERK/MSCs had fewer apoptotic cells than MSCs (Fig. 2G-H). Taken together, these results suggest that ERK/MSCs have better viability than MSCs in both physiological and glutamate-induced condition

2.4.7. Western blot analysis Rats (n = 3 per group) were killed under deep anesthesia with pentobarbital sodium. The right forebrain was collected, rapidly frozen in liquid nitrogen, and then stored at −80 °C until homogenization. The protein concentration of each sample was determined. After sodium dodecylsulfate polyacrylamide gel electrophoresis, the proteins were then transferred to a nitrocellulose membrane, followed to be incubated with primary antibodies overnight at 4 °C. The primary antibodies used were rabbit anti-platelet endothelial cell adhesion molecule-1 (PECAM1, 1:500, Santa Cruz Biotechnology), mouse anti-GAPDH (1:1000, Santa Cruz Biotechnology), rabbit anti-CD68 (1:500, Abcam), rabbit antiTGF-β (1:500, Abcam), rabbit tumor necrosis factor-α (TNF-α, 1:500, Abcam), mouse anti-vascular endothelial growth factor (VEGF, 1:500, R &D systems), and rabbit anti-ERK1/2 (1:1000, Cell Signaling Technology). Blots were then washed three times with 0.1 M TBS and incubated with appropriate sheep anti-rabbit IgG-horseradish peroxidase (HRP) or sheep anti-mouse IgG-HRP (1:5000, Santa Cruz Biotechnology) for 1 h at room temperature. The immunoreactive bands were detected by using an ECL Western blot detection kit (Millipore, USA) and then visualized by using the FluorChemE System. The images were analyzed quantitatively by means of ImageJ image analysis software. The final results were expressed as a ratio of the expression level of the protein of interest to that of GAPDH.

3.3. Increased percentage of MAP2-Positive cells in ERK/MSCs cultures Although neural differentiation of MSCs has been reported in previous studies (Wislet-Gendebien et al., 2005), the lower neuronal differentiation rate limits therapeutic potentials in vivo (Chen et al., 2001). Therefore, whether ERK1/2 over-expression can influence the potential neural differentiation of MSCs was investigated. After induction, neural differentiation of MSCs was measured by expressing cell specific markers as our previously described (Yang et al., 2011). Primary antibodies against MAP2, GFAP, and galactosecerebroside (Galc) were used to identify neuron, astrocyte and oligodendrocytes, respectively. As shown in Fig. 3, the immunocytochemical staining analysis demonstrated that the percentage of MAP2 positive cells in ERK/MSCs was significantly higher than that in MSCs. Meanwhile, the percentage of GFAP-positive cells and Galc-positive cells in ERK/MSCs was significantly lower than that in MSCs (P < 0.05) (Fig. 3A-D). In addition, a RT-PCR analysis of these genes (MAP2, GFAP, Galc) and Ki67 (a marker for prolifernation) confirmed that ERK/MSCs had an enhanced mRNA expression of Ki67 and MAP2 genes compared with MSCs, but had a reduced mRNA expression of Galc gene (P < 0.05) (Fig. 3E-F). The higher Ki67 mRNA expression levels, suggesting higher capability of proliferation in ERK/ MSCs, were in agreement with the observation that ERK/MSCs grow

2.5. Statistical analysis Data are expressed as means ± standard error. Student’s unpaired t-tests were used to assess the differences between groups in culture experiments. Multiple comparisons were performed by using one-way 45

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Fig. 2. ERK/MSCs show better viability in normal and neurotoxic condition. (A) Representative microphotographs of MSCs and ERK/MSCs cultured for 96 h at the same plating density under phase contrast microscopy. Scale bar: 100 μm. (B) Viability of ERK/MSCs and MSCs by MTT analysis. (C-F) After exposure to glutamate, cell viability was determined by Hoechst 33258 staining, MTT, and LDH assay. (C) Representative Hoechst 33258 staining showed apoptotic (solid arrow) and viable cells (dotted arrow). Scale bar: 50 μm. (D) Quantitative analysis of Hoechst 33258 staining showed a lower percentage of apoptotic cells in ERK/MSCs compared with MSCs. (E) MTT assay showed a higher absorbance of ERK/MSCs than that of MSCs. (F) LDH release analysis revealed a lower LDH release in ERK/MSCs compared to MSCs. (G) Representative TUNEL staining showed apoptotic cells (solid arrow, brown) and viable cells (dotted arrow, light blue) stained by hematoxylin. (H) Quantitative analysis of TUNEL staining showed fewer apoptotic cells in ERK/MSCs compared with MSCs. *P < 0 0.05 compared to MSCs.

astrocyte differentiation from the grafted cells was analyzed by immunostaining with GFAP and MAP2 antibodies. No double-fluorescent cells were observed (data not shown). All these results suggest more viable ERK/MSCs compared with MSCs in rats after transplantation and no neural differentiation of grafted cells in rats after stroke. Next, ERK protein levels in the ipsilateral forebrain were detected by Western blot analysis. Compared with MSCs- or PBS- treated rats, the ERK protein levels in ERK/MSCs-treated rats were increased but no significant difference at 7 days; and significantly increased at 14 days (P < 0.05) (Fig. 4F-G), suggesting ERK/MSCs over-express ERK in rats. Notably, no differences in ERK protein levels were observed between MSCs- or PBS- treated rats (Fig. 4F-G).

faster than MSCs in vitro (Fig. 2A). All together, these results suggest the possibility of enhanced neuronal differentiation of ERK/MSCs in vitro. 3.4. Enhanced functional recovery after ERK/MSCs treatment After ERK/MSCs transplantation on stroke in rats, ischemic volume was measured using TTC staining. The remaining normal brain tissues were typically stained with TTC (red), but ischemic tissues showed no or reduced staining (white) (Bederson et al., 1986). At 7 and 14 days after stroke, PBS-treated rats showed apparent atrophy (Fig. 4A). The ischemic volume of MSCs- or ERK/MSCs-treated rats was significantly smaller compared with PBS-treated rats at 7 and 14 days (P < 0.05) (Fig. 4B). However, no significant differences were observed between MSCs and ERK/MSCs groups (Fig. 4B). Next, neurological performance was evaluated by a modified neurological severity score (Yang et al., 2011). No significant differences were observed at 3 days after stroke in rats (time point of transplantation, Fig. 4C). However, MSCs- or ERK/MSCs- treated rats showed significant improvements in behaviors compared with PBS-treated rats (* P < 0.05 and ** P < 0.01) (Fig. 4C). Notably, ERK/MSCs-treated rats displayed more improvements than MSCs-treated rats (# P < 0.05 and ## P < 0.01) (Fig. 4C).

3.6. Enhanced intrinsic proliferation and neuronal de-differentiation in rats To detect whether ERK/MSCs transplantation affects intrinsic proliferation in rats after stroke, neural progenitor cells (NPCs) were measured by using a specific cellular nestin antibody. NPCs in rodents are located in subventricular zone (SVZ) of lateral ventricle and subgranular zone (SGZ) of the hippocampus, and are activated in stroke (Arvidsson et al., 2002; Parent et al., 2002; Komitova et al., 2005). As shown in Fig. 5, nestin-positive cells appeared in the ipsilateral SVZ, the peri-infarct cortex and striatum after stroke in all rats, and showed morphological diversity in different regions. For example, nestin-positive cells in the SVZ and peri-infarct striatum had larger round cell bodies with a larger nucleus with/without one or two processes, whereas had smaller cell bodies with more than one longer processes in the peri-infarct cortex (Fig. 5A-B). The number of nestin-positive cells in MSCs- or ERK/MSCs- treated rats was significantly increased compared to PBS-treated rats at 7 and 14 days (P < 0.05) (Fig. 5C). Notably, more nestin-positive cells were observed in ERK/MSCs-treated rats than in MSCs-treated rats at 7 days (P < 0.05) (Fig. 5C). Together, our

3.5. Enhanced survival of ERK/MSCs and ERK over-expression in rats At 7 days after transplantation, a few of the grafted cells migrated toward the ischemic striatum and cortex, and some stayed in the ipsilateral ventricle. However, a majority of grafted cells were located in the ischemic striatum and cortex at 14 days (Fig. 4D). The grafted cells in the ischemic boundary zone (IBZ) of the striatum were measured and they did not differ in the two groups at 7 days, but more ERK/MSCs were observed at 14 days (P < 0.05) (Fig. 4E). In addition, neuron and 46

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Fig. 3. Increased percentage of MAP2-positive cells in ERK/MSCs cultures (A-C) Representative confocal images showing neurons, astrocytes, and oligodendrocytes derived from MSCs or ERK/MSCs after neural induction in vitro. For lentivirus carrying eGFP, MSCs or ERK/BMSCs appeared green fluorescence under fluorescence microscope. MAP2-, GFAP- and Galc-positive cells were stained in red fluorescence. Nuclei were shown by diamidino-phenyl-indole (DAPI) staining and appeared blue. The mergers showed colocalization of green and red by laser scanning confocal microscope. Scale bar: 100 μm. (D) Quantitative analyses of the immunocytochemical data showed a higher percentage of MAP2+ cells and lower percentage of GFAP + cells or Galc + cells in ERK/MSCs compared to MSCs. (E-F) Representative RT-PCR results of the mRNA level of Ki67, MAP2, and Galc genes from MSCs or ERK/MSCs (E) and Quantitative analyses of RT-PCR showed a significantly increased mRNA level of MAP2 and Ki67 genes in ERK/MSCs compared to MSCs after neural induction in vitro (F). * P < 0 0.05 compared with MSCs.

positive cells. The MAP2/nestin double-labeled cells were located in the ipsilateral striatum adjacent to SVZ (Fig. 5A). The number of these double-labeled cells in MSCs- or ERK/MSCs- treated rats was significantly increased compared with PBS-treated rats (* P < 0.05 and **

results indicate that ERK/MSCs transplantation may enhance intrinsic proliferation in rat after stroke. Next, double-immunostaining with cellular specific antibodies against MAP2 and GFAP was used to explore the identity of nestin47

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Fig. 4. ERK/MSCs transplantation enhances functional recovery in rats after stroke. (A) Representative brain slices stained by TTC (2, 3, 5-triphenyltetrazolium) staining showed lesion regions at 7 days after stroke. Notably, the brain atrophy was apparent in PBS-treated rats. (B) Ischemic lesion volume evaluated by TTC staining. * P < 0.05 compared to PBS group. (C) Neurological function of rats after stroke was determined by modified neurological severity scores (mNSS). ERK/MSCs or MSCs treatment significantly enhanced neurological functional recovery at 7 and 14 days after transplantation compared to PBS treatment. * P < 0.05;** P < 0.01 compared to PBS group, and ERK/MSCs treatment showed stronger roles in functional recovery than MSCs treatment. # P < 0.05, ## P < 0.01 compared to MSCs group. (D-E) ERK/MSCs and MSCs at the ischemic boundary zone of the striatum at 2 weeks after transplantation (D). Scale bar: 50 μm. And quantification of the grafted cells showed a significant increase in ERK/MSCstreated rats compared to MSCs treated-rats at 2 weeks after transplantation (E). * P < 0.05 vs MSCs group. (F-G) Representative ERK immunoreactive bands from ischemic forebrain in rats as shown by Western blot analysis (F), and higher ERK protein levels in rats treated with ERK/MSCs compared with rats treated with MSCs and PBS at 2 weeks after transplantation (G). * P < 0.05 vs MSCs group or PBS group.

cytokine) and TGF-β expression (an anti-inflammatory cytokine) in the ipsilateral hemisphere of rats were then examined. No significant difference of TGF-β expression was observed between groups (Fig. 6A and 6C). Surprisingly, TNF-α levels were significantly increased in ERK/ MSCs-treated rats compared with MSCs- or PBS- treated rats (P < 0.05) (Fig. 6A and 6D). Previous studies have shown that MSCs transplantation benefits the recipient by enhancing angiogenesis after stroke (Wei et al., 2012). The expression level of PECAM-1 and VEGF (markers for endothelial cells of blood vessels) was also observed and was no different in all groups (Fig. 6E-G).

P < 0.01) (Fig. 5D). Notably, more double-labeled cells were observed in ERK/MSCs-treated rats than MSCs-treated rats at 7 days (P < 0.05) (Fig. 5D). In contrast, GFAP/nestin double-labeled cells were not observed in all groups (Fig. 5E). These results may indicate reinforced neuronal de-differentiation in ERK/MSCs-treated rats.

3.7. Modulating glial activation and neuroinflammation after ERK/MSCs transplantation To understand the neuroprotective mechanisms of ERK/MSCs transplantation, glial activation, neuroinflammation, and angiogenesis in the ipsilateral forebrain were assessed. The activation of astrocyte was assessed by GFAP immunostaining. We found that more GFAPpositive cells in IBZ of striatum of ERK/MSCs- or MSCs- treated rats than PBS-treated rats (P < 0.05)(Fig. 5E-F), and more GFAP-positive cells in ERK/MSCs-treated rats compared with MSCs-treated rats (P < 0.05) (Fig. 5E-F), indicative of enhanced astrocyte proliferation. Activated microglia was evaluated using a specific marker, CD68, which can label ameoid-like microglia. Using western blot analysis, the results showed that the CD68 expression was significantly decreased in ERK/MSCs-treated rats on 14 days compared with MSCs- or PBStreated rats (P < 0.05) (Fig. 6A-B), suggesting inhibition of microglia activation. Together, these data indicate that ERK/MSCs treatment modulates glial activation in rats after stroke. Microglia and astrocyte display diverse functions including the secretion of pro-inflammatory cytokines to anti-inflammatory cytokines depending on specific settings (Hanisch, 2002; Ohtaki et al., 2008; vonBernhardi et al., 2015). TNF-α expression (an inflammatory

4. Discussion In the present study, we explored the therapeutic effects of ERK/ MSCs after stroke in rats. Previous studies have shown that only a small subset of grafted MSCs can survive over time in recipient rats (Kopen et al., 1999; Chen et al., 2001), which heavily limits the effects of MSCs transplantation. Our results showed that cultured ERK/MSCs displayed better viability, and more cells were observed after transplantation in rats post-stroke. Notably, ERK/MSCs treatment showed more efficiency in behavioral recovery at 7 and 14 days. Taken together, these results firstly confirm that ERK1/2 over-expression in MSCs enhances viability of MSCs, which may underlie the more beneficial effects of ERK/MSCs in rats post-stroke. Given that upregulation of ERK1/2 signaling pathway occurs in a large fraction of tumours, and MEK inhibitors have the potential to target all tumours dependent on ERK1/2 pathway signaling (Hatzivassiliou et al., 2013), it may be questioned that 48

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Fig. 5. ERK/MSCs transplantation increased intrinsic proliferation and neuronal dedifferentiation after stroke in rats. (A) Representative confocal images showing nestin + cells (green arrow), MAP2+ cells (red arrow), and nestin+/MAP2+ cells (yellow allow) in ischemic striatum adjacent to the SVZ. Blue represents Hoechst 33258 nuclear staining. Lower panel is the zoom in upper panel. Scale bar: 50 μm for upper panel, 25 μm for lower panel. (B) The morphology of nestin + cells in the SVZ, peri-infarct striatum and the cortex was shown. Scale bar: 10 μm. (C) Nestin + cells in rats treated with MSCs and ERK/MSCs were significantly increased compared with rats treated with PBS. * P < 0.05 vs PBS group; more nestin + cells in rats-treated with ERK/MSCs were observed than rats treated with MSCs at 1 week. # P < 0.05 vs MSCs group.(D) Nestin+/MAP2+ cells in ERK/MSCs-treated rats and MSCstreated rats were significantly increased compared with those in PBS-treated rats. * P < 0.05, ** P < 0.01 vs PBS group. ERK/ MSCs-treated rats had a significant increase in nestin+/MAP2+ cells compared with MSCstreated rats at 7 days. # P < 0.05 vs MSCs group. (E) Representative merged confocal images of nestin (red arrow) and GFAP (green arrow) double-immunostaining. Blue represents Hoechst 33258 nuclear staining. No double-stained cells were observed. Scale bar: 50 μm. (F) Increased GFAP + cells were observed in ERK/MSCs- and MSCs-treated rats compare with PBS-treated rats. * P < 0.05 vs PBS group, and significantly increased GFAP + cells were found in ERK/MSCs-treated rats compared to MSCs-treated rats. # P < 0.05 vs MSCs group.

vitro after neural induction, we did not find that ERK/MSCs and MSCs expressed neural markers, such as MAP2 and GFAP, indicating no neural differentiation of MSCs. Our previous study observed this neural differentiation when MSCs overerpressing glial cell line-derived neurotrophic factor in rats with hemorrhage (Yang et al., 2011). This disagreement might be ascribed to the large differences in environment in vitro and in vivo. Taken together, our results further confirm that ERK/ MSCs play beneficial roles probably not via cell replacement of MSCs (Kopen et al., 1999; Chen et al., 2002; Caplanand Dennis, 2006; Hess and Borlongan, 2008). It is well accepted that intrinsic neural progenitor cell (NPC) proliferation correlates with functional recovery (Chen et al., 2003; Horita et al., 2006; Chopp et al., 2008; Kernie and Parent, 2010). In our study, ERK/MSCs significantly endogeneous NPC proliferation determined by more nestin-positive cells of SVZ. Interestingly, we found that nestin/

transplanted ERK/MSCs could proliferate to generate brain tumors. This concern may not exist because of great differences in environment in vivo. The grafted ERK/MSCs were suffered to ischemic injury. Anyway, their safety can be evaluated after observing longer time after transplantation in next research. Although increasing evidence suggests that transplantation of MSCs in rodent post-stroke improves functional recovery (Swanson et al., 1990; Li et al., 2000; Chopp and Li, 2002; Li and Chopp, 2009; Joyce et al., 2010), the underlying mechanisms are still controversial. Multiple mechanisms may be involved in functional recovery, such as neural differentiation, neurogenesis, angiogenesis, and neuroinflammation (Li et al., 2005; Joyce et al., 2010; Shen et al., 2010; Alvarez and Banzan, 2011; Sheikhet al., 2011; Wei et al., 2012; Aizman et al., 2013; Gutiérrez-Fernández et al., 2013). Firstly, neural differentiation of MSCs was investigated in rats. Contrary to the results in 49

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Fig. 6. ERK/MSCs transplantation modulates glial activation and neuroinflammation. (A) Representative immunoreactive bands of CD68, TNFα, and TGF-β in ipsilateral hemisphere homogenates. (B) A significant decrease in CD68 level in ERK/MSCstreated rats on 14 days compared with MSCs- or PBS- treated rats. *P < 0.05 vs PBS group or MSCs group. (C) No difference in TGF-β expression between groups. (D) An increased TNF-α level in rats treated with ERK/MSCs compared with rats with MSCs or PBS on 7 and 14 days. *P < 0.05 vs PBS group or MSCs group. (E-G) Representative immunoreactive bands of PECAM-1 and VEGF in ipsilateral hemisphere homogenates (E). No significant differences of PECAM (F) and VEGF (G) expression were detected between groups.

MSCs treatment post-stroke (Gao et al., 2005; Li et al., 2005; Zhang et al., 2006; Gao et al., 2008; Sheikh et al., 2011). To further determine the beneficial or detrimental roles of activated glial cells, the expression of TNF-α and TGF-β in the ipsilateral brain was measured, and we found an increased level of TNF-α and unchanged level of TGF-β in ERK/MSCs-treated rats compared with MSCs-treated or PBS-treated rats. For both activated microglia and reactive astrocytes can produce TNF-α and TGF-β (Lehrmann et al., 1998; Dhandapani and Brann, 2003; Gordon, 2003; Martinez et al., 2008; Cherry et al., 2014; Fang et al., 2015). Our study has some limitations because cell types of secreting TNF-α and TGF-β were not determined. Considering that the functional patterns of activated glial cells are highly complex in response to diverse stimuli (Lee et al., 2010; vonBernhardi et al., 2015), the anti-inflammatory or pro-inflammatory roles of activated glial cells after ERK/MSCs treatment remain unclear. Collectively, neuroinflammation modulation may partially explain the mechanisms of ERK/ MSCs treatment in rats post-stroke. Although we observed some interesting results, obvious limitations existed at this present stage. For example, given that failure to observe the neural differentiation of ERK/MSC in vivo, it is better to observe the expressions of more neurotropic factors, such as transforming growth factor (TGF-β), nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) to confirm the trophic effects of ERK/MSC in ischemic rats. In addition, it needs to observe longer time (such as 3 months) after grafting to further clarify the protective effects in stroke rats and observe their safety. In near future research, these issues will be focused and explored.

MAP2 double-positive cells were localized to striatum adjacent to SVZ, and ERK/MSCs significantly enhanced the numbers of these doublepositive cells compared with MSCs at 7 days after transplantation. It has been reported that nestin-positive cells link to the proliferation and "dedifferentiation" of reactive astrocytes in rats with stroke (Fang et al., 2015). In addition, cortical reactive astrocytes have been reported to form neural spheres that can differentiate into neurons, astrocytes, and oligodendrocytes in vitro (Buffo et al., 2008; Shimada et al., 2010, 2012). In our study, the nestin/MAP2 double-positive cells may represent the de-differentiation of neurons after stroke. Li and Chopp also reported that nestin/MAP-2 double-positive cells in rats after stroke (Li and Chopp, 1999). To our knowledge, the phenomenon that de-differentiation of mature neurons has never been paid attention. If these mature neurons can de-differentiate into NPCs, brain remodeling might be facilitated after stroke. The neurogenesis and de-differentiation in rats after stroke might simultaneously occur and contribute to brain remodeling. Taken together, these results provide evidence that the beneficial effects of ERK/MSCs after stroke might be, at least partially, related to the enhanced endogeneous proliferation and de-differentiation of neurons. Neuroinflammation has been shown to be modulated by MSCs treatment post-stroke (Leker et al., 2007; Ohtaki et al., 2008; Sheikh et al., 2011; Wei et al., 2012). Neuroinflammation is choreographed by microglia and astrocytes and is defined by increased levels of a complex arrangement of mediators, including interleukin-1 beta (IL-1β), TNF-α, and TGF-β (Von Bernhardi, 2007; Kuwabara et al.,2017). Our study showed that ERK/MSCs treatment inhibited microglia activation and enhanced reactive astrocytes measured by decreased CD68 levels and more GFAP-positive cells. However, the disagreements exist about the effects of MSCs administration on glial activation post-stroke. Ohtaki et al. have reported that human MSCs activate microglia and enhance the expression of neuroprotective gene YM-1 in microglia (Ohtaki et al., 2008). In contrast, other reports show that MSCs treatment suppresses the activation of microglia and reduces OX-42- or Iba-1-positive cells in ischemic boundary zone (Sheikh et al., 2011; Wei et al., 2012). Meanwhile, many pro-inflammatory cytokines/chemokines are down-regulated (Zhang and Chopp, 2002; Wei et al., 2012). Microglia are well known for displaying diverse functional properties, including classical M1 activation (associated with cytotoxicity), alternative phagocytic/ neuroprotective M2 activation, or regulatory activation (Gordon, 2003; Martinez et al., 2008; Mosser and Edwards, 2008). Similarly, an increase or decrease of reactive astrocytes has also been reported after

5. Conclusions In conclusion, we demonstrated the potentially protective effect of ERK/MSCs against stroke-affected rats, which may depend on enhanced viability of ERK/MSCs in recipient. The underlying mechanisms may be multifaceted including enhanced endogeneous proliferation, neuronal de-differentiation, and neuroinflammation modulation. Our study may provide a potential new insight for the therapeutic application of MSCs in neurological diseases. Funding The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This 50

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research received funding from National Natural Science Foundation of China (No. 81371213, 81070987, and 30971531) and by grants from the Ministry of Science and Technology of China (2010CB945600 and 2010CB945601).

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