Bone marrow mesenchymal stem cells decrease CHOP expression and neuronal apoptosis after spinal cord injury

Bone marrow mesenchymal stem cells decrease CHOP expression and neuronal apoptosis after spinal cord injury

G Model ARTICLE IN PRESS NSL-32434; No. of Pages 8 Neuroscience Letters xxx (2016) xxx–xxx Contents lists available at ScienceDirect Neuroscience...

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ARTICLE IN PRESS

NSL-32434; No. of Pages 8

Neuroscience Letters xxx (2016) xxx–xxx

Contents lists available at ScienceDirect

Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet

Research article

Bone marrow mesenchymal stem cells decrease CHOP expression and neuronal apoptosis after spinal cord injury Chuanlong Gu a , Heyangzi Li a , Chao Wang a , Xinghui Song b , Yuemin Ding c , Mingzhi Zheng d , Wei Liu e , Yingying Chen a , Xiaoming Zhang a , Linlin Wang a,∗ a

Department of Basic Medicine Sciences, School of Medicine, Zhejiang University, Hangzhou 310058, China Core Facilities, Zhejiang University School of Medicine, Hangzhou 310058, China c School of Medicine, Zhejiang University City College, Hangzhou 310015, China, d Department of Pharmacology, Hangzhou Medical College, Hangzhou 310053, China e Department of Prosthetics, Stomatology Hospital, School of Medicine, Zhejiang University, Hangzhou 310006, China b

h i g h l i g h t s • • • •

Traumatic injury increased CHOP expression and apoptosis in rat spinal cord. BMSCs transplantation decreased CHOP expression, apoptosis, and increased the locomotor function. Co-culture with BMSCs decreased OGD induced CHOP expression and apoptosis in motor neurons. Co-culture with BMSCs-CM restored the viability of post-OGD motor neurons.

a r t i c l e

i n f o

Article history: Received 14 August 2016 Received in revised form 13 November 2016 Accepted 15 November 2016 Available online xxx Keywords: Spinal cord injury Bone marrow mesenchymal stem cells C/EBP homology protein Endoplasmic reticulum stress Apoptosis

a b s t r a c t Spinal cord injury (SCI) leads to irreversible neuronal loss and ultimately leads to paralysis. Bone marrow derived mesenchymal stem cells (BMSCs) have been demonstrated to be an effective approach to treat SCI. The present study was designed to investigate the role of BMSCs in rats with spinal cord injury and in oxygen-glucose deprivation (OGD) treated motor neurons. The results demonstrated that BMSCs could improve locomotor function and decrease expression of pro-apoptotic transcription factor C/EBP homologous protein (CHOP) and apoptosis after SCI. Furthermore, co-culture with BMSCs or conditioned medium from BMSCs could also decrease the expression of CHOP and apoptosis in post-OGD motor neurons, supporting that BMSCs exerts protective effects by decreasing the expression of CHOP in injured motor neurons. Our findings provide a potential novel mechanism for BMSCs treatments in patients with SCI. © 2016 Published by Elsevier Ireland Ltd.

1. Introduction Spinal cord injury (SCI) leads to irreversible loss of neurons and impairments of motor and sensory function below the injury [1]. Recently, studies demonstrated that bone marrow mesenchymal stem cells (BMSCs) are an effective approach to treat SCI by supporting the cell survival, promoting axonal regeneration, and improving functional outcome [2,3]. BMSCs have the potential benefit for

Abbreviations: BMSCs, bone marrow mesenchymal stem cells; BMSCs-CM, conditioned medium of BMSCs; CHOP, C/EBP homology protein; ER, endoplasmic reticulum; H&E, hematoxylin and eosin; OGD, oxygen–glucose deprivation; SCI, spinal cord injury. ∗ Corresponding author at: Center for Stem Cell and Tissue Engineering, School of Medicine, Zhejiang University, Hangzhou 310058, China. E-mail address: [email protected] (L. Wang).

differentiating neurons, and related trophic factors can indirectly trigger endogenous survival signaling pathways to protect injured neurons and promote nerve regeneration after SCI [4]. However, the exact mechanism underlying BMSCs’ protective effects against SCI is still elusive. Endoplasmic reticulum (ER) is an intracellular organelle responsible for the synthesis and proper folding of proteins to maintain cellular homeostasis [5]. One of the main components of ER stressmediated apoptosis pathway is C/EBP (CCAAT enhancer binding protein) homologous protein (CHOP), a transcription factor [6]. Suppressing CHOP expression can reduce apoptosis [7]. Previous studies have proved that CHOP plays a direct functional role in the early stage of traumatic SCI and attenuating ER stress response could improve functional recovery after SCI [8]. In the present study, we used modified Allen’s weight-drop SCI rat model and oxygen glucose deprivation (OGD) treated VSC4.1

http://dx.doi.org/10.1016/j.neulet.2016.11.032 0304-3940/© 2016 Published by Elsevier Ireland Ltd.

Please cite this article in press as: C. Gu, et al., Bone marrow mesenchymal stem cells decrease CHOP expression and neuronal apoptosis after spinal cord injury, Neurosci. Lett. (2016), http://dx.doi.org/10.1016/j.neulet.2016.11.032

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motor neurons to investigate effects of BMSCs on CHOP expression and apoptosis in injured neurons after SCI. 2. Materials and methods 2.1. Primary BMSCs culture and characterization The experimental procedures were approved by the Animal Ethics Committee of Zhejiang University and were carried out in accordance with institutional guidelines. Rat primary BMSCs were isolated according to our previous study [9]. Cells were routinely characterized by flow cytometry analysis. Cells were labeled for 30 min using antibodies against CD34, CD44, CD90 and CD105, conjugated with FITC (1:200 v/v, Boster Biotechnology, China). To track the BMSCs in vivo after injection into the spinal cord, green fluorescent protein (GFP) tagged adenovirus vector was used. Some BMSCs at 80% confluence were transfected with a GFP-encoding adenovirus backbone vector, pHBAd-CMV-IRES-GFP. The transfection efficiency of GFP in BMSCs was >99%, which was confirmed under a fluorescent microscope (FluoView FV1000; Olympus Corporation, Tokyo, Japan). 2.2. Spinal cord injury mode Sprague-Dawley male rats (200–220 g) were divided into 3 groups: sham operation group, SCI group and BMSCs treatment SCI group (n = 20/group). After 40 mg/kg, i.p. sodium pentobarbital anesthesia, the vertebral column of the rats was exposed and a laminectomy was carried out at T10 level. A weight of 10 g was dropped from a height of 5 cm on the exposed spinal cord, and the impounder was left for 20 s before being withdrawn to produce a moderate contusion in SCI group. The sham operation animals received the same surgical procedure except injury. The 5 ␮l GFPpositive or GFP-negative BMSCs (5 × 105 ) were injected into the epicenter of the injured spinal cord using an electrode microneedle in BMSCs treatment rats while the same quantity PBS were injected into the sham rats after contusion immediately. Some rats were sacrificed for pathological, immunohistochemistry and RT-qPCR analysis 24 h after transplantation. The locomotor function was assessed by the Basso, Beattie and Bresnahan (BBB) locomotor rating scale within 7 days after transplantation (n = 10/group). The BBB scale is to evaluate the functional recovery of locomotor capacity in rats after spinal cord contusion [2]. The survival of BMSCs in the spinal cord was confimed by Western blot assay to detect the expression of GFP in our preliminary experiment. After 1 week transplantation, a spinal cord segment (4 mm length, n = 3) at the contusion epicenter was dissected and homogenized for protein assay. Spinal cord segment without BMSCs transplantation was set as control. The primary antibodies were as follows: GFP antibody (M1004; 1:300; Hua’an Biotechnology, China) and ␤-actin antibody (1:5000; Sigma, USA). See Supplementary Fig. 1. 2.3. H&E staining and in situ TUNEL staining Five rats per group were re-anesthetized with sodium pentobarbital (60 mg/kg, i.p.) and perfused with 4% paraformaldehyde in phosphate-buffered saline 24 h after transplantation. The lesion epicenter (4 mm length) of the spinal cord was removed and the transverse sections (8 ␮m) were used for H&E Staining and TUNEL staining. Some sections (20 ␮m) were stained with the DAPI staining solution (2-(4-Amidinophenyl)6-indolecarbamidine dihydrochloride, DAPI, Sigma) to show the nuclear and GFP-positive BMSCs were visualized under fluorescent microscopy.

TUNEL staining was used to detect the apoptotic cells in spinal cord according to the manufacturer’s protocol (Roche Diagnostics Corporation, Indianapolis, IN, USA) as previously reported [10]. Cells with blue granules in the nucleus were TUNEL-positive cells. Quantitative analysis was performed blindly by counting the number of TUNEL-positive cells in five microscopic fields from six cross sections of the injured spinal cord. 2.4. Immunohistochemistry The sections from rats (n = 5/group) were incubated overnight at 4 ◦ C with CHOP antibody (1:100, Santa Cruz Biotechnology, USA) followed by incubation with biotinylated horse-anti-mouse IgG (1:200, Boster Biotechnology, China) for 2 h at room temperature. Then, avidin–biotin–peroxidase complex solution (ABC, 1:100, Boster Biotechnology, China) was added and incubated for another 2 h. Subsequently, the sections were visualized with 3 3diaminobenzidine solution (DAB kit) (Gene Tech) under an optical microscope. 2.5. RT-qPCR The lesion epicenter of spinal cord was removed (about 4 mm), and total RNA was obtained using the RNA extraction kit (Qiagen, Hilden, Germany) (n = 5/group). Primers were used for the housekeeping gene glyceraldehydes-3phosphate dehydrogenase (GAPDH) in RT-PCR to amplify GAPDH (forward, 5 -AGTTCAACGGCACAGTCAAG-3 ; reverse, 5 -TACTCAGCACCAGCATCACC-3 ) as an internal control of CHOP (forward, 5 -CGGAGTGTACCCAGCACCATCA-3 ; reverse, 5 -CCCTCTCCTTTGGTCTACCCTCA-3 ). Fold changes in gene expression were estimated using the CT comparative method normalizing to GAPDH CT values and relative to control samples. CT = CT CHOP − CTGAPDH, CT = CT − CT control. Fold difference = 2 − (CT) and the expression of sham operation rats was set as 1. 2.6. Oxygen–glucose deprivation (OGD) The ventral spinal cord 4.1 (VSC4.1) motor neuron cells were grown in RPMI1640 media with fetal bovine serum (10%, v/v; Gibco, Invitrogen) and 1% penicillin and streptomycin at 37 ◦ C with 5% CO2 in a fully humidified incubator as previously reported [11]. Induction of cell death and apoptosis in vitro by OGD and reoxygenation model, was initiated as previously reported with slight modification [12]. Briefly, VSC4.1 motor neurons were incubated in glucose-free Hanks’ balanced salt solution in a sealed hypoxic GENbag fitted with a catalyst (BioMèrieux, Marcy I’Etoile, France) to scavenge free oxygen. Cells cultured in Hanks’ balanced salt solution containing normal concentration of glucose with 5% CO2 in a fully humidified incubator were used as the Non-OGD control. Cells were returned to original medium, and placed in a normoxic chamber (re-oxygenation, 37 ◦ C, 5% CO2 ) for 20 h. In preliminary experiments, a time-course study for VSC4.1 motor neurons involving in the time period of OGD for 2, 4, 8, 12, 16 or 20 h was carried out and a 8 h OGD period was determined as the optimum time point for co-culture of BMSCs and 8, 12, 16 h OGD periods were used for co-culture of conditioned medium of BMSCs. 2.7. Co-culture of post-OGD VSC4.1 motor neurons with BMSCs BMSCs (5 × 105 /well) were seeded to the insert chamber of the 6-well 0.4 ␮m transwell system and co-cultured with 5 × 105 /well VSC4.1 motor neurons for 48 h. Then the insert chambers were removed and VSC4.1 motor neurons were exposed to 8 h OGD and

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20 h re-oxygenation. At the end of re-oxygenation VSC4.1 motor neurons were collected. Morphological analysis of apoptosis in VSC4.1 motor neurons were detected by Wright staining. VSC4.1 motor neurons were fixed and stained as previously described [13]. Cells morphology was examined via optical microscopy. Cells were counted as apoptotic if they demonstrated reduction in cell volume, chromatin condensation, and/or cellular membrane blebbing. At least 500 cells were counted in each treatment and the percentage of apoptotic cells was calculated. The apoptosis of VSC4.1 motor neurons were also detected by flow cytometry. After treatment, VSC4.1 motor neurons were detached, washed and incubated in 500 ␮l binding buffer, 5 ␮l annexin V-FITC and 5 ␮l of propidium iodide (PI) (Invitrogen, Shanghai, China). Cells were then detected through fluorescence-activated cell sorting with a Becton-Dickinson FACScan (Immunocytochemistry Systems, San Jose, CA). Annexin V positive cells were labeled as apoptotic cells, and Annexin-V negative but PI positive cells were marked as necrotic cells. PI percentage was utilized as cell death percentage.

2.8. Western blot VSC4.1 motor neurons were directly lysed in homogenization buffer containing 1% Triton X-100. Primary antibody of CHOP (GADD 153 Antibody (F-168): sc-575; 1:500; Santa Cruz Biotechnology, USA) was incubated at 4 ◦ C in Tris-buffered saline and 0.1% Tween20 (TBST) supplemented with 3% BSA. After several washes with TBST, blots were incubated with infrared-labeled secondary antibody (Li-COR biosciences). Quantification of band intensity was performed using NIH Image J software after being normalized to references (␤-actin).

2.9. Preparation of conditioned medium from BMSCs (BMSCs-CM) and co-culture of post-OGD VSC4.1 motor neurons with BMSCs-CM BMSCs-CM was collected as previously reported [14]. In brief, BMSCs were cultured at 50,000 cells per cm2 with serum free DMEM to generate BMSCs-CM. The medium was collected after 48 h of culture and used as BMSCs-CM. BMSCs-CM was applied to OGD-injured VSC4.1 motor neurons in order to investigate the paracrine effects of BMSCs. VSC4.1 motor neurons (1.5 × 104 cells/well) were seeded into 96-well plates, and five parallel wells were used for each treatment. Four experimental groups were as below: No OGD group, No OGD + BMSCs-CM group, OGD group, OGD+ BMSCs-CM group. Neurons were subjected to OGD for 8 h, and then replaced the OGD medium with BMSCs-CM or control medium in a normoxic chamber (37 ◦ C, 5% CO2 ) for 20 h. To investigate the effects of different concentration of BMSCs-CM, neurons were subjected to OGD for 8, 12, 16 h and then replaced the OGD medium with different concentration of BMSCsCM (0, 25, 50, 75 and 100%). Subsequently, the survival of post-OGD VSC4.1 motor neurons was assayed using a CCK-8 Kit (Beyotime Institute of Biotechnology, Nantong, Jiangsu, China). Cell density was determined by measuring the absorbance at 450 nm using a TECAN-infinite M200 (TECAN, Switzerland) and is expressed as a percentage of control (Non-OGD group).

2.10. Statistical analysis Data is presented as mean ± SEM. Two-way ANOVA with Bonferroni post hoc test was used for BBB score and one-way analysis of variance (ANOVA) with Student’s-Newman-Keuls test was used

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for statistical comparison when appropriate. Differences were considered statistically significant at P < 0.05. 3. Results 3.1. Morphological and differentiation characteristics of BMSCs These cells expressed several markers that are considered to define adult BMSCs, including CD44, CD90 and CD105. The cells were negative for hematopoietic markers such as CD34 (Fig. 1A). The osteogenic differentiation assays showed that most of the cells had mineralized calcium deposits, as confirmed by Alizarin Red S staining (Fig. 1B). It was also found that the cells had the capacity to undergo adipogenic differentiation. This was evident through the accumulation of lipid vacuoles by Oil Red staining (Fig. 1C). 3.2. BMSCs improved functional recovery Fluorescent microscopy showed that BMSCs were localized in or around the injured parenchyma 24 h in the injured spinal cord after transplantation (Fig. 2A–F). BBB scale showed BMSCs group exhibited gradual improvement of locomotor function in comparison to the SCI group within 7 days. The improvement was significantly different from the SCI rats at day 6 and 7 after transplantation (P < 0.05, Fig. 2G). 3.3. H&E and TUNEL staining in the injured spinal cord Neurons were determined based on the morphology on H&E stained slides. The number of neurons was reduced considerably and blood cells invaded in the spinal cord in the SCI group. Treatment with BMSCs attenuated the injury remarkably. Numbers of neurons in BMSCs were increased compared with the SCI group (Fig. 3A–C). The number of TUNEL-positive cells in SCI groups was significantly increased as compared to the sham group (81.8 ± 9.5% vs 3.4 ± 1.7%). Treatment with BMSCs decreased the number of TUNEL-positive cells as compared to the SCI group (60.4 ± 9.5%) (P < 0.05, Fig. 3D–F). 3.4. Expression of CHOP in the injured spinal cord The expression level of CHOP mRNA in SCI rats was significantly increased to 5.9 ± 0.3 folds. In contrast, the expression levels of CHOP in BMSCs treatment rats were 2.3 ± 0.2 folds while in the sham operation rats were 1.0 fold (P < 0.05). The expression of CHOP protein in spinal cord slide was visualized in brown granular immunostain pattern. The number of CHOP positive cells were significantly increased in the lesions of the SCI group (10.8 ± 2.4/vision field) than that in the sham operation group (1.6 ± 0.9/vision field, P < 0.01) (Fig. 4A,B). Compared with SCI group, the number of CHOP positive cells was significantly decreased (4.6 ± 1.1/vision field, P < 0.05, Fig. 4B,C). 3.5. BMSCs decreased OGD-induced apoptosis and CHOP expression in motor neuron After co-culture with BMSCs for 48 h, VSC4.1 motor neurons in the lower chamber were exposed to OGD/re-oxygenation. Morphological features of apoptosis were detected by Wright staining OGD group showed more apoptotic morphological features and the percentage of apoptotic cells was significantly increased to 21.6 ± 3.3% (P < 0.01, vs control group [7.8 ± 2.5%]), which was restored by BMSCs and the percentage of apoptotic cells was dramatically decreased to 11.4 ± 2.4% (P < 0.01, Fig. 5A–C).

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Fig. 1. Characteristics of BMSCs. A. Surface markers of the BMSCs were analyzed by fluorescence automated cell sorting. B. Osteogenic induction for two weeks. Mineralized nodules were detected after Alizarin Red S staining. C. Adipogenic induction for 3 weeks. The cells were stained with oil Red O solution and showed a lipid laden adipocyte phenotype. Scale bars: 50 ␮M. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Results of Wright staining for apoptosis were further supported by flow cytometry. The number of apoptotic cells in OGD group was increased to about 3.9 ± 1.2 folds (P < 0.01), and co-culture with BMSCs significantly reduced the apoptotic cells to 2.5 ± 0.7 folds as compared to OGD group (P < 0.05, Fig. 5B,D). The expression level of CHOP in VSC4.1 motor neurons showed consistent results with apoptosis analysis. As compared to the NonOGD group, the expression level of CHOP protein was significantly increased in OGD group as evidenced by Western blot (0.84 ± 0.10 vs 0.13 ± 0.04, P < 0.01). The expression level of CHOP (0.49 ± 0.05)

in BMSCs treatment OGD group was less than that in the OGD group (P < 0.01, Fig. 5E).

3.6. BMSCs-CM restored the survival of post-OGD VSC4.1 motor neurons As shown in Fig. 6A, OGD exposure displayed obvious toxicity in cultured VSC4.1 motor neurons and showed a significant decrease in viability in a time-dependent manner. BMSCs-CM significantly reduced OGD (8 h)-induced neuronal injury and restored the viabil-

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Fig. 2. Localization of BMSCs in spinal cord and evaluation of locomotor function by BBB score. A. In the injured spinal cord, the blue fluorescence showed the cell’s nucleus with the DAPI staining. B. In the injured spinal cord, the green fluorescence showed the BMSCs were localized inside the injured parenchyma. C. Merged images of A and B. Magnification: 4×; Scale Bar: 500 ␮M; D, E and F. showed the amplified images. n = 5/group. Magnification: 20×; Scale bars: 100 ␮M. G. Evaluation of locomotor function by BBB scores within 7 days after transplantation. n = 10/group. **P < 0.01, vs control group. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3. H&E and TUNEL staining in injured spinal cord 24 h after transplantation. A. Sham operation group. B. SCI group. C. SCI + BMSCs group. Arrows indicate the body of survived neurons. D. Sham operation group. E. SCI group. F. SCI + BMSCs group. Arrows indicate the TUNEL-positive cells. Scale bars: 50 ␮M.

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Fig. 4. Immunohistochemistry staining of CHOP in injured spinal cord. Immunohistochemistry staining of CHOP expression in sections of segments in injured spinal cord. A. Sham operation group. B. SCI group. C. SCI + BMSCs group. Arrows indicate the CHOP positive cells in coronal sections of injured spinal cord. Scale bars: 50 ␮M.

Fig. 5. Co-cultured with BMSCs inhibited apoptosis and the expression of CHOP in post-OGD VSC4.1 motor neurons. Co-culture with BMSCs in a co-culture transwell system for 48 h inhibited subsequent OGD (8 h)-induced apoptosis in VSC4.1 motor neurons. Apoptosis was measured by (A) Wright Staining and (B) Flow Cytometry analysis. The arrows indicated apoptotic cells. Scale bars: 20 ␮m. (C) Data are mean ± SEM and calculated from three independent experiments of Wright Staining. **P < 0.01, vs the control group (Non-OGD and no BMSCs). ## P < 0.01, vs the OGD group. (D) Data are mean ± SEM and calculated from three independent experiments of Flow Cytometry analysis. **P < 0.01, vs the control group (Non-OGD and no BMSCs). # P < 0.05, vs the OGD group. (E) Analysis the expression of CHOP by Western Blot. **P < 0.01, vs the control group. ## P < 0.01, vs the OGD group without treatment of BMSCs.

ity from 62.8 ± 11.2% to 139.8 ± 13.7% (P < 0.01 vs Non-OGD), failed to restore the longer period time of OGD (12 h, 16 h) induced neuronal injury. To examine which concentration of BMSCs-CM could have the maximum protective effect, different concentrations of BMSCs-CM (0–100%) were added to neurons damaged by 12 h of exposure to OGD. As showed in Fig. 6B, BMSCs-CM reduced OGD-

induced neuronal injury in a concentration dependent manner with a maximum protective effect at 50% BMSCs-CM (P < 0.05) (Fig. 6). 4. Discussion The blood-brain barrier is disrupted after spinal cord injury and induces a reactive process of secondary injury [15]. This secondary

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Fig. 6. Co-cultured with BMSCs-CM restored the viability of OGD(8 h)-injured VSC4.1 motor neurons. (A) After cells were exposed to OGD for 8, 12, 16 h, and then replaced the OGD medium with BMSCs-CM or control medium in a normoxic chamber for 20 h. Cell viability was determined by CCK-8 assay. **p < 0.01, vs the control group(Non-OGD and no BMSCs-CM). ## p < 0.01, vs the OGD group without BMSCs-CM. (B) Co-culture with different concentration of BMSCs-CM with OGD(12 h)-injured VSC4.1 motor neurons. *p < 0.05, vs the control group (Non-OGD and no BMSCs-CM).

damage last for days and weeks after SCI, and leads to exacerbate the neurological dysfunction [16]. The apoptotic cells in the injured spinal cord play an important role in the early stage after SCI [17]. Ideal treatment of SCI should focus on preventing secondary injury and cells apoptosis in the injured spinal cord. BMSCs have great potential for the treatment of SCI by reducing lesion, supporting cell survival and neural differentiation potential [18]. Results from the present demonstrated that local delivery of BMSCs could significantly improve locomotor function of SCI rats on day 6 and 7 after transplantation. BMSCs were able to decrease the CHOP expression and apoptosis in the early stage. Consistent with the BBB values, pathological changes and neurons loss in the injured spinal cord were also improved after BMSCs transplantation. In addition, we found TUNEL-positive cells decreased and BMSCs treatment enhanced neuronal preservation in injured spinal cord. It is worth to note that, in present study, the apoptosis of neurons in damaged spinal cord tissue was detected by TUNEL staining. It is hard to distinguish the accurate portion of apoptotic neurons from the necrotic neurons, as well as from the apoptotic glial cells, astrocytes and endothelial cells. To avoid confusions, apoptosis of VSC4.1 motor neurons in vitro was further confirmed by Writhing staining and flow cytometry. The in vitro study revealed that co-culture with BMSCs could significantly decrease OGD-induced apoptosis and restored the viability of post-OGD VSC4.1 motor neurons. Inhibition of CHOP could promote the functional recovery after spinal cord injury [8]. CHOP-mediated apoptosis are believed to play an important role in ER stress-induced cell apoptosis and subsequent cell death [19]. CHOP activation might be a consequence of disturbed calcium stores, generation of free radicals, aberrant protein glycosylation, or increased membrane and protein turnover after injury [20]. The present results demonstrated that SCI triggered ER stress-induced apoptosis. BMSCs could attenuate the expression level of CHOP and neuronal apoptosis. It has been reported that CHOP is mobilized to the nucleus when the apoptotic signaling pathway is activated [21]. Therefore, it could be helpful to link CHOP activity with cellular apoptosis by analyzing the sub-cellular localization in our further experiment. Accumulating evidence showed that BMSCs act in a paracrine manner to protect cells from injuries. Interestingly, we revealed that the BMSCs-CM was effective to restore the viability of motor neurons after long period of time OGD (8 h). We then tested whether BMSCs-CM could restore the viability of motor neurons after extremely longer periods of OGD (12 h or 16 h). However, the damage in motor neurons was too severe to be restored by BMSCs-CM. Previous study suggested that BMSCs could provide an efficient route of therapeutic miRNA delivery to the brain in pathological conditions with clinical implications for regenerative medicine [22]. BMSCs can also transfer mitochondria to pulmonary alveoli to protect against acute lung injury [23]. Whether BMSCs

deliver miRNAs or mitochondria to these post-OGD neurons are going to be further investigated in our lab. 5. Conclusion The present results demonstrated that grafted BMSCs promoted the functional recover of SCI rats by decreasing CHOP expression and apoptosis after injury. The results might provide novel insights to understand the mechanisms underlying functional restoration in stem cell-based therapeutic approaches for the SCI. Conflict of interest The authors indicated no potential conflicts of interest. Author contributions Chuanlong Gu and Linlin Wang: conception and design; Xiaoming Zhang and Linlin Wang: financial support; Chuanlong Gu, Heyangzi Li, Chao Wang, Yuemin Ding, Xinghui Song and Mingzhi Zheng: experiments conduct, data acquisition and assembly of data; Yingying Chen, Wei Liu and Linlin Wang: data analysis and interpretation; Xiaoming Zhang and Linlin Wang: manuscript writing and final approval of manuscript. Funding The research was supported by the Science and Technology Department of Zhejiang Province (No. 2014C37027), the Health Department of Zhejiang province (No. 2013KYA120) and the National Natural Science Foundation of China (No. 81272158, 81371953 and 81572229). Acknowledgments The authors thank Dr. Faisal Rehman for critical proof reading of the manuscript. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.neulet.2016.11. 032. References [1] R.G. Grossman, R.F. Frankowski, K.D. Burau, E.G. Toups, J.W. Crommett, M.M. Johnson, M.G. Fehlings, C.H. Tator, C.I. Shaffrey, S.J. Harkema, J.E. Hodes, B.

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Please cite this article in press as: C. Gu, et al., Bone marrow mesenchymal stem cells decrease CHOP expression and neuronal apoptosis after spinal cord injury, Neurosci. Lett. (2016), http://dx.doi.org/10.1016/j.neulet.2016.11.032