YEXNR-11969; No. of pages: 10; 4C: Experimental Neurology xxx (2015) xxx–xxx

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Experimental Neurology journal homepage: www.elsevier.com/locate/yexnr

Intranasal delivery of hypoxia-preconditioned bone marrow-derived mesenchymal stem cells enhanced regenerative effects after intracerebral hemorrhagic stroke in mice Jinmei Sun a,b,c, Zheng Zachory Wei a,b,c, Xiaohuan Gu c, James Ya Zhang c, Yongbo Zhang a,b, Jimei Li a,b,⁎, Ling Wei a,b,c,d,⁎⁎ a

Department of Neurology, Beijing Friendship Hospital, Capital Medical University, Beijing 100050, China Laboratories of Stem Cell Biology and Neural Regeneration and Function Recovery, Beijing Friendship Hospital, Capital Medical University, Beijing 100050, China c Department of Anesthesiology, Emory University School of Medicine, Atlanta, GA 30322, USA d Department of Neurology, Emory University School of Medicine, Atlanta, GA 30322, USA b

a r t i c l e

i n f o

Article history: Received 16 January 2015 Revised 10 March 2015 Accepted 13 March 2015 Available online xxxx Keywords: Intracerebral hemorrhage Stroke Hypoxic preconditioning Neurogenesis Intranasal transplantation

a b s t r a c t Intracerebral hemorrhagic stroke (ICH) causes high mortality and morbidity with very limited treatment options. Cell-based therapy has emerged as a novel approach to replace damaged brain tissues and promote regenerative processes. In this study we tested the hypothesis that intranasally delivered hypoxia-preconditioned BMSCs could reach the brain, promote tissue repair and improve functional recovery after ICH. Hemorrhagic stroke was induced in adult C57/B6 mice by injection of collagenase IV into the striatum. Animals were randomly divided into three groups: sham group, intranasal BMSC treatment group, and vehicle treatment group. BMSCs were pre-treated with hypoxic preconditioning (HP) and pre-labeled with Hoechst before transplantation. Behavior tests, including the mNSS score, rotarod test, adhesive removal test, and locomotor function evaluation were performed at varying days, up to 21 days, after ICH to evaluate the therapeutic effects of BMSC transplantation. Western blots and immunohistochemistry were performed to analyze the neurotrophic effects. Intranasally delivered HP-BMSCs were identified in peri-injury regions. NeuN +/BrdU + co-labeled cells were markedly increased around the hematoma region, and growth factors, including BDNF, GDNF, and VEGF were significantly upregulated in the ICH brain after BMSC treatment. The BMSC treatment group showed significant improvement in behavioral performance compared with the vehicle group. Our data also showed that intranasally delivered HP-BMSCs migrated to peri-injury regions and provided growth factors to increase neurogenesis after ICH. We conclude that intranasal administration of BMSC is an effective treatment for ICH, and that it enhanced neuroregenerative effects and promoted neurological functional recovery after ICH. Overall, the investigation supports the potential therapeutic strategy for BMSC transplantation therapy against hemorrhagic stroke. © 2015 Published by Elsevier Inc.

Introduction Intracerebral hemorrhage (ICH) is a devastating stroke subtype associated with high mortality and disability in the adult population (Keep et al., 2012; Yang et al., 2011). Latest report about primary ICH patients indicates that the 1-year survival rate is only 46%, and that the 5-year survival rate decreases to 29.2% (Poon et al., 2013). Compared with ischemic stroke, ICH has received less research

⁎ Correspondence to: J. Li, Department of Neurology, Beijing Friendship Hospital, Capital Medical University, Beijing 100050, China. ⁎⁎ Correspondence to: L. Wei, 101 Woodruff Circle WMRB 617, Department of Anesthesiology, Emory University School of Medicine, Atlanta, GA 30322, USA. Fax: +1 404 727 6300. E-mail addresses: [email protected] (J. Li), [email protected] (L. Wei).

attention (Kleinig and Vink, 2009; Fagan et al., 2013; Mohan et al., 2012). There is no effective clinical treatment for hemorrhagic stroke, and so the development of effective therapies for ICH is urgently needed. Cell-based therapy has emerged as a promising strategy for treatment of neurological disorders including ischemic stroke, hemorrhagic stroke, traumatic brain injury (TBI), spinal cord injury and many others (Andres et al., 2008; Kan et al., 2010; Otero et al., 2012; Liu et al., 2014; Yu et al., 2013). Transplantation therapy using bone marrow-derived mesenchymal stem cells (BMSC) and other stem cells/neural progenitors has been tested in ischemic stroke models. Transplanted cells show beneficial effects of increasing trophic supports, neuroprotection and functional recovery in experimental stroke models (Yu et al., 2013). Increasing evidence also suggest that transplanted BMSCs can differentiate to neuronal and non-neuronal cells in the host brain.

http://dx.doi.org/10.1016/j.expneurol.2015.03.011 0014-4886/© 2015 Published by Elsevier Inc.

Please cite this article as: Sun, J., et al., Intranasal delivery of hypoxia-preconditioned bone marrow-derived mesenchymal stem cells enhanced regenerative effects after intra..., Exp. Neurol. (2015), http://dx.doi.org/10.1016/j.expneurol.2015.03.011

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Among different stem cells, BMSCs have received a greater attention for their advantages such as possible autologous transplantation, amenability to manipulation and expansion in vitro, and versatile terminal differentiation products (Yang et al., 2011; Seyfried et al., 2010). Therefore, BMSCs hold a great potential in regenerative medicine for ischemic as well as hemorrhagic stroke. Transplanted cells generally survive poorly in the injured brain. We recently developed the hypoxic preconditioning strategy to promote the survival and regenerative properties of transplanted cells (Yu et al., 2013; Theus et al., 2008; Wei et al., 2013a,b; Francis and Wei, 2010). We showed that BMSCs pre-treated under non-lethal hypoxia before transplantation gained enhanced expression of some key pro-survival and regenerative factors such as Bcl-2, BDNF, VEGF, and SDF-1. After transplantation into the ischemic cortex, they survived much better and exhibited greater ability to migrate to the lesion sites and promote functional recovery (Yu et al., 2013; Theus et al., 2008; Wei et al., 2013a,b; Francis and Wei, 2010; Hu et al., 2011). It was suggested that hypoxic preconditioning (HP) should be applied as an indispensable priming preparation in stem cell transplantation therapy. In the present investigation, hypoxic preconditioned BMSCs (HP-BMSCs) were used to demonstrate therapeutic benefits. In stem cell transplantation therapy, cells are injected directly into the brain or via the intravenous or intra-arterial routes. Intracerebral injection is an invasive method, while systemic administration results in very few cells homing to the brain. We recently reported the use of intranasal route in stem cell therapy for non-invasive brain specific delivery. We showed the distribution of intranasal delivered cells in the ischemic cortex several hours after cell administration. Hypoxic preconditioning greatly promoted the efficiency of the intranasal method by promoting brain distribution of the cells, increased cell survival, migration to the ischemic cortex and increased trophic supports (Yu et al., 2013; Hu et al., 2011). This approach, however, has not been tested after hemorrhagic stroke. The present investigation specifically examined the therapeutic benefits of non-invasive intranasal transplantation after hemorrhagic stroke using hypoxic preconditioned BMSCs.

Materials and methods Induction of hemorrhagic stroke in the mouse The hemorrhagic stroke model was induced by injection of collagenase IV stereotaxically based on previously described methodology (Krafft et al., 2012). We decided to use the collagenase IV model, rather than autologous whole blood, because the former results in more prolonged injury, making it more suitable for our long-term analysis of functional recovery. Adult male C57BL/6 mice (25–28 g, 8– 10 weeks) were used in all experiments. Mice were anesthetized with 4% chloral hydrate. A scalp incision was made along the midline and a 1 mm burr hole was drilled on the right side of skull. Next, 0.15 U collagenase type IV was injected using a Hamilton syringe (Hamilton Company USA, Reno, NV). A 26G needle was orthogonally inserted into the brain through the burr hole (relative to bregma: anterior: 0.4 mm, lateral: 2.5 mm, depth: 3.7 mm). The syringe needle was left in place for an additional 10 min to prevent backflow. During the surgery and collagenase injection, animal temperature was kept at 37 °C using a homeothermic blanket control unit (Harvard Apparatus Limited, UK). Incisions were then closed using surgical glue (3 M Corporate, St. Paul, MN). Sham-operated mice were treated similarly with a needle puncture into brain, except without collagenase injection. After surgery, mice were housed in an incubator until they were fully awake. Procedures involving animals were performed in accordance with current protocols approved by the Emory University IACUC. Surgery was performed by the same animal surgeon in order to minimize variations in the experiments.

BMSC isolation and hypoxic preconditioning BMSCs were isolated and harvested as previously described (Wei et al., 2013a). In brief, BMSCs were flushed from the tibias of postnatal day 21 Wistar rats (Charles River, Wilmington, MA, USA) using a 25-gauge needle. Mononuclear cells were suspended in Dulbecco's modified Eagle's medium (Cellgro, Manassas, VA, USA) supplemented with 15% fetal bovine serum (Sigma, St. Louis, MO, USA) and plated into dishes. Cultures were maintained at 37 °C in a humidified atmosphere containing 5% carbon dioxide. After 24 h, nonadherent cells were discarded, and adherent cells were washed four times with phosphate-buffered saline solution (PBS; Sigma). Fresh complete medium was added and replaced every 2 days. Each primary culture was subcultured 1:3 when the BMSCs grew to approximately 80% confluence. All cells used in this study were freshly isolated within five passages and harvested for analysis when they were around 80–90% confluent. Hypoxic preconditioning of BMSCs Cells were incubated under normoxic conditions or in a finely controlled ProOx C-chamber system (Biospherix, Redfield, NY, USA). For hypoxia preconditioning, the oxygen concentration in the chamber was maintained at 0.1–0.3% with a residual gas mixture composed of 5% carbon dioxide balanced with nitrogen for 24 h, followed by 1 h reoxygenation before transplantation. Intranasal delivery of HP-BMSCs At 3 and 7 days after ICH, HP-BMSCs or vehicle were administered by intranasal injections. Intranasal delivery was performed as previously described (Wei et al., 2013a) with minor modifications. Prior to the injection, the mice were anesthetized with 4% chloral hydrate (100 mg/kg, IP) and placed on a heating pad, with the head in the upright position. All animals received 100 U hyaluronidase (Sigma) dissolved in sterile PBS 30 min prior to the administration of cells. By catalyzing the hydrolysis of hyaluronan, hyaluronidase can increase tissue permeability. Hyaluronidase was used here to disrupt the barrier function of the nasopharyngeal mucosa and facilitate cell entry to the brain. Five microliter drops containing either cell suspension or vehicle were carefully placed on one nostril and then allowed to be fully inhaled. Droplets were administered onto alternating nostrils in 1 min intervals. A total volume of 100 μl of cell suspension (1 × 106 cells) or vehicle was used. This volume and cell number were optimized in our previous study, and shown to be effective in delivering BMSCs into the brain through intranasal administration.(Wei et al., 2013a, 2015; Song et al., 2013). Western blot analysis 14 days after ICH, one cohort of mice was sacrificed for western blotting, in order to compare the expression of growth factors between control and treatment group. Fresh frozen brain tissues collected from the peri-hematoma region were homogenized with western blot lysis buffer on ice at 4 °C. After centrifuging at 17,000 rpm for 15 min, protein concentration was determined by a bicinchoninic acid (BCA) assay, and then quantified by a UV spectrophotometer. Samples were preserved at −80 °C before use. For immunoblotting, A 30 μg portion of proteins from each sample was separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to PVDF membrane. 0.2% Tween in Tris-buffered saline (TBS-T) containing 5% BSA was used to block the membrane. The specific primary antibodies (VEGF, BDNF, GDNF, 1:1000, Santa Cruz Biotechnology) diluted in TBS-T with 5% BSA were added as probes to incubate with the membrane at 4 °C overnight. The secondary antibody (1:2500, alkaline phophastase-conjugated goat anti mouse/rabbit, Promega, Madison,

Please cite this article as: Sun, J., et al., Intranasal delivery of hypoxia-preconditioned bone marrow-derived mesenchymal stem cells enhanced regenerative effects after intra..., Exp. Neurol. (2015), http://dx.doi.org/10.1016/j.expneurol.2015.03.011

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WI, USA) was added for 1 h. After that, the membrane was washed using TBST for three times, and then BCIP/NBT solution (Sigma-Aldrich) was added to develop the bands. Image J program 5.0 (NIH, Bethesda, MD) was used to compare the relative densities of the bands. Immunohistochemistry staining Immunohistochemistry staining was used to test the neurogenesis markers after ICH. Preparation of brain sections was performed as previously described (Li et al., 2008). Briefly, 10-μm thick coronal fresh frozen sections or 14-μm of 4% PFA perfusion brain sections were prepared using a cryostat (Ultapro 5000; St. Louis, MO). The slides were completely air-dried and then fixed in 10% buffered formalin phosphate for 10 min. After incubation in a −20 °C ethanol/acetic acid (2:1) solution for 12 min and 0.2% Triton-100 for 5 min, slides were blocked by 1% gelatin from cold water fish (Sigma), diluted in PBS at room temperature for 1 h, following incubation with primary antibody

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(mouse anti-NeuN, 1:400, MAB377, Chemicon; goat anti-DCX, 1:100, Santa-Cruz Biotechnology, rat anti-BrdU, 1:400, Abcam, Cambridge, MA) diluted in PBS overnight at 4 °C. Slides were washed with PBS and incubated with specific secondary antibody (CY3, Alexa Fluor488, goat/donkey anti-mouse, rat, goat 1:200, Santa-Cruz Biotechnology). Finally, slides were washed again with PBS before being mounted with ProLong AntiFade (Invitrogen, Grand Island, NY). Visualized fluorescence was detected by confocal microscopy (BX61; Olympus, Tokyo, Japan). Hoechst 33342 (Molecular Probes, Eugene, OR) was used to counterstain all nuclei. Stereological images were taken and cells were counted using the ImageJ 5.0 (NIH, Bethesda, MD). Neurological deficit scoring A 10-grade modified neurological stroke scale (NSS) was applied in evaluation of behavioral changes among treated and untreated animals as previously described (Stahel et al., 2000). Briefly, this test consists of

Fig. 1. Tracking transplanted cells in the brain after ICH. Animals were sacrificed at 6 h after transplantation. PI (red) counterstains all the nuclei of the brain section. HP-BMSCs were prelabeled with Hoechst 33342 (blue) for 1 h prior to the transplantation. At 6 h post-transplantation, HP-BMSCs were identified throughout the brain, including the olfactory bulb (A), ipsilateral cortex (B), peri-vascular spaces (C) and peri-hematoma regions (D). The white curved line demarcates the border of hematoma. (E) High magnification picture show the presence of HP-BMSCs in cortex. (F) 3D confocal image of transplanted cell. Scale bar = 10 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Please cite this article as: Sun, J., et al., Intranasal delivery of hypoxia-preconditioned bone marrow-derived mesenchymal stem cells enhanced regenerative effects after intra..., Exp. Neurol. (2015), http://dx.doi.org/10.1016/j.expneurol.2015.03.011

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motor tests, sensory tests, beam balance tests, reflex evaluation and observation of abnormal movements. The behavioral test started at 3 days after stroke and then was re-evaluated on days 7, 14, and 21 post-stroke.

Rotarod assessment The rotarod test was performed using an accelerating rotarod (UGO Basile, Collegeville, PA). The rotarod starts at a speed of 4 rotations per minute (rpm) to 40 rpm over the course of 5 min. Prior to the surgery, mice were trained for 3 trials/day for 3 days, and each trial lasted for 5 min with a 2-h interval between each trial. At days 3, 7, 14, and 21 after ICH, we measured the mice's time of maintaining its balance on the beam.

Adhesive removal test As a sensitive method for detecting and monitoring sensorimotor deficits after CNS injury, the adhesive removal test also can be used in the hemorrhagic stroke model (Beray-Berthat et al., 2010). Mice were gently scruffed and inverted, and then a small quarter-circle adhesive was placed onto either the left or the right forepaw. The time of contact and remove the dot was measured separately, with the adhesive placed onto alternating forepaws. The test was repeated 3 times for both the right and left forepaws (ie. 6 trials in total for each animal). Before each test, animals were placed into the experimental home cage for 2 min for acclimation. Prior to the surgery, animals were trained each day for 3 days in order to familiarize the animals with the test, as well as exclude any outliers from subsequent testing. The adhesive removal test was performed on days 3, 7, 14, and 21 after ICH.

Locomotion assessment An open field system (TopScan, CleverSys Inc., Reston, VA) was used to test locomotor activity 14 days after ICH. Each mouse was individually placed in the open field apparatus (40 × 40 × 30 cm; Accuscan Instruments, Columbus, OH), with cameras on the top. The specific parameters: total distance traveled (mm), velocity (mm/s), duration of grooming (s), and turns (bouts) were selected in the program before each test. The data were collected for 1 h. All trials were performed in the same time and in the same behavior room in order to achieve optimally consistent results with minimal confounding variables. Brain atrophy measurement We used the volume of the caudate as a marker of brain tissue loss after ICH, and brain atrophy was measured by the enlargement of ipsilateral ventricle. Coronal frozen sections (10 μm thick, taken every 200 μm from anterior to posterior of the hematoma) were stained with 0.1% cresyl violet. Following a previously described protocol (Sun et al., 2011) Image J was used to quantify the caudate and ventricle size. The tissue loss was represented by the ratio of the volume of the ipsilateral caudate to that of the contralateral caudate, and ventricle enlargement was expressed as a percentage of the ipsilateral ventricle size relative to the contralateral ventricle size (ipsilateral designates the side of the injury). Statistical analysis All data are presented as mean ± standard deviation in the Results section, and as mean ± standard error of the mean (SEM) in the figures. Statistical analysis was performed using GraphPad Prism 5 (GraphPad

Fig. 2. HP-BMSC transplantation upregulated growth factors in the brain. Protein levels of GDNF, VEGF, and BDNF were analyzed by Western blot. (A) Representative Western bands show levels of GDNF, VEGF, and BDNF in the peri-hematoma regions 14 days after ICH. Relative density was normalized with β-actin and quantified by Image J. The ICH significantly attenuated the expression of growth factors GDNF (B), VEGF (C), and BDNF (C), as compared to the sham group. HP-BMSC treatment significantly increased the expression of GDNF (B), VEGF (C) and BDNF (D), as compared to the ICH-vehicle group. Data is represented as mean ± SEM. *: p b 0.05, ICH-BMSC vs. ICH-vehicle. #: p b 0.05, ICH-vehicle vs. Sham. N = 6–8 each group. One way ANOVA followed by Tukey post-hoc test was used.

Please cite this article as: Sun, J., et al., Intranasal delivery of hypoxia-preconditioned bone marrow-derived mesenchymal stem cells enhanced regenerative effects after intra..., Exp. Neurol. (2015), http://dx.doi.org/10.1016/j.expneurol.2015.03.011

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Software, Inc., San Diego, CA). If data are normally distributed, the student unpaired-t test was used to compare two groups and the oneway ANOVA followed by the Tukey post-hoc test was used to compare more than 2 groups. For non-parametric data like the mNSS score, the Mann–Whitney U test was used to compare two groups. Comparisons in which p b 0.05 were considered significantly different. Results Detection of transplanted HP-BMSCs in the brain HP-BMSCs were pre-labeled with Hoechst 33342 and detected by PI staining after transplantation. We located the HP-BMSCs throughout the ICH brain at 6 h after intranasal administration (Fig. 1). Hoechstpositive cells can be found in the olfactory bulb (Fig. 1A), ipsilateral cortex (Fig. 1B), and perivascular spaces (Fig. 2C), and cells were detected in and around the hematoma (Fig. 1D). The double labeling of Hoechst and PI was confirmed by capturing 3D images (Fig. 1F). Our data demonstrate that HP-BMSCs reached the damaged area after ICH. Neurotrophic supports by HP-BMSC delivery To investigate the mechanisms of the effects of HP-BMSC on ICH, we used Western blot to analyze the neurotrophic factors in the perihematoma regions at 14 days after stroke (7 days after the second HPBMSC delivery). Levels of glial cell-derived neurotrophic factor (GDNF), vascular endothelial growth factor (VEGF), and brain-derived neurotrophic factor (BDNF) were measured in the peri-hematoma regions (Fig. 2A). After ICH, the protein level of VEGF transiently increased at 7 days after stroke, and then decreased back to normal (data not

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shown). Interestingly, the levels of both neurogenic and angiogenic growth factors were reduced following ICH, which was unexpected because the compensatory response of endothelial and glial cells due to the injury should increase growth factor levels. One possible explanation is that the pathophysiology of the ICH was so severe and rapid that the loss of cells overwhelmed the native compensatory response, resulting in a depletion in growth factors. However, at 14 days following the injury, HP-BMSC transplantation was able to rescue the levels of GDNF, VEGF, and BDNF that were downregulated as a result of ICH (Figs. 2B–D). HP-BMSC treatment significantly increased neurotrophic factors in the peri-hematoma regions as compared to the vehicle treatment group. This data suggests a strong regenerative potential for HP-BMSC transplantation, and that this regeneration is at least in part due to the trophic support provided by BMSCs, possibly through either direct secretion of factors such as GDNF and VEGF, or through stimulation of endogenous release by host glial cells, such as astrocytes, and endothelial cells. HP-BMSC transplantation stimulates neurogenesis To confirm the HP-BMSC's neurotrophic effects in the SVZ and perihematoma region, immunohistochemistry staining for BrdU and DCX, as well as BrdU and NeuN was performed. BrdU-positive cells were significantly increased in the SVZ region in the HP-BMSC treatment group, as compared to PBS treatment group (Figs. 3A–H, K). The number of BrdU +/DCX + co-labeled cells in the SVZ was higher in HP-BMSCtreated mice (34.46 ± 13.12 vs. 52.50 ± 17.06) (Fig. 3J). These showed that HP-BMSCs greatly enhanced neuroblast proliferation and migration at 14 days after ICH. In peri-hematoma region, BrdU/NeuN double staining was performed and colabeled cells were counted (Figs. 4A–K). The

Fig. 3. Effect of transplanted HP-BMSC on endogenous neurogenesis after ICH. In the SVZ region, immunohistochemistry staining was performed 14 days after ICH. The proliferation of neuroprogenitor cells was labeled with BrdU (red; C,G) and DCX (green; B,F). A–D show the vehicle (PBS) treatment group. E–H show the HP-BMSC treatment group. (I) 3D confocal imaging confirmed the colocalization of DCX and BrdU within the same neuroblast. More BrdU positive cells (J), DCX positive cells (K), and BrdU/DCX colabeled cells (L) were found in the HP-BMSC treatment group. Data is represented as mean ± SEM. *: p b 0.05. N = 6–8 each group. Student unpaired t-test was used. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Please cite this article as: Sun, J., et al., Intranasal delivery of hypoxia-preconditioned bone marrow-derived mesenchymal stem cells enhanced regenerative effects after intra..., Exp. Neurol. (2015), http://dx.doi.org/10.1016/j.expneurol.2015.03.011

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Fig. 4. Intranasally transplanted HP-BMSC stimulated neurogenesis after ICH. At 14 days after ICH, in the peri-hematoma region, neurogenesis was detected by the colocalization of the neuronal marker NeuN (green, B, F), the proliferation marker BrdU (red, C, G), and the nuclei marker Hoechst (blue, A, E). A–D show BrdU/NeuN colabeled cells in the peri-hematoma region of vehicle (PBS) treatment group, and E–H show HP-BMSC treatment group. (I) Lower power image of NeuN and BrdU cells in peri-hematoma regions. (J) 3D confocal imaging captures the triple labeling of a neuron. (K) Cell counting results indicated that HP-BMSC significantly increased the numbers of BrdU+ neurons, suggesting enhanced neural regeneration due to treatment. Data is represented as mean ± SEM. *: p b 0.05, N = 6–8 each group. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

results showed a significant increase in BrdU/NeuN-colabeled cells in the peri-hematoma region after HP-BMSC treatment (47.13 ± 9.61 vs. 62.88 ± 16.88) (Fig. 4K). HP-BMSC transplantation enhanced functional recovery In order to assess the therapeutic benefits of HP-BMSC on ICH, a series of behavior tests, including the adhesive-removal test, rotarod test, modified neurological severity score (mNSS) assessment, and open field test were performed. Locomotor activity of ICH mice was measured at 14 days after ICH by a TopScan monitoring system. After ICH, the travel distance, velocity, bouts of turns, and grooming time were all significantly reduced as compared with sham animals. However, HP-BMSC treatment significantly improved the neurological functions (Figs. 5A–E). From the TopScan motion traces, sham animals move much more randomly in the arena, which reflects normal exploratory behavior in a new environment. ICH + PBS animals exhibited avoidance behavior into the center of the arena, which is suggestive of increased anxiety following injury. HP-BMSC-treated mice showed similar movement behavior as the sham mice (Fig. 5F). At 3 days post-ICH, we carried out the adhesive-removal test, and we observed increased contact and removal times in ICH animals. After HP-BMSC transplantation, a significant improvement was observed in the HP-BMSC treatment group as compared to PBS control group (Figs. 6A, B), indicating a sensorimotor functional recovery promoted by transplanted HPBMSCs. In the rotarod test, at 14 and 21 days after ICH, animals receiving HP-BMSC transplantation were able to maintain balance on the beam for a longer time as compared to control group animals. Finally,

transplantation therapy was able to significantly lower severity scores in the mNSS assessment, which suggests greater functional recovery (Fig. 6D). Our battery of behavior tests collectively demonstrated that transplantation of HP-BMSCs was able to provide a comprehensively improvement in functional recovery and suggests that this therapeutic strategy could ameliorate a wide range of symptoms following hemorrhagic stroke. Brain atrophy changes after HP-BMSC injections Brain tissue loss and ventricle enlargement were analyzed at 21 days after ICH (Fig. 7A). After ICH, there was a significant reduction in the caudate and an increase in the size of the left ventricle, which is an indirect measure of brain atrophy. We showed that HP-BMSC transplantation, as compared to vehicle treatment group, showed a reduction in tissue loss and a similar reduction in the enlargement of ventricle cavity size (Fig. 7B). These data suggest that HP-BMSC transplantations might protect against tissue loss in the ipsilateral striatum (Bliss et al., 2007; Lee et al., 2009; Wei et al., 2012; Donega et al., 2013). Discussion Intracerebral hemorrhagic stroke is a major cause of morbidity and mortality worldwide (Seyfried et al., 2010; Xiong et al., 2013). Stem cell therapy potentially provides a therapeutic approach for ICH. In a previous study, we have demonstrated that at 24 h after ischemic stroke, intranasal delivery of HP-BMSCs can reduce the neurological deficits associated with the stroke. In the present investigation, we

Please cite this article as: Sun, J., et al., Intranasal delivery of hypoxia-preconditioned bone marrow-derived mesenchymal stem cells enhanced regenerative effects after intra..., Exp. Neurol. (2015), http://dx.doi.org/10.1016/j.expneurol.2015.03.011

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Fig. 5. Effect of intranasally transplanted HP-BMSC on functional recovery. Functional recovery was assessed by 1-h open field behavioral monitoring using Topscan system at 14 days after ICH. (A) Total travel distance, (B) velocity, (C) total slow-moving time, (D) total bouts of turn, (E) grooming, (F) and representative map traces of each group are shown. The ICH injury resulted in a significant deficit in all the measured behavior modalities, as compared to the sham control. BMSC treatment was able to restore the behavior back to baseline (as determined by sham performance), suggesting enhanced functional recovery conferred by BMSC transplantation. Data is represented as mean ± SEM. #: p b 0.05, ICH-vehicle vs. Sham. *: p b 0.05, ICHBMSC vs. ICH-vehicle. N = 8 each group. One way ANOVA followed by Tukey post-hoc test was used to compare groups.

successfully demonstrated that intranasal administration of HP-BMSCs in the mouse brain after ICH leads to survival of the exogenous cells and migration into the peri-hematoma regions. The transplantation therapy effectively alleviated functional deficits, in part by enhancing neuroregenerative effects, including endogenous neurogenesis after ICH.

Hypoxic preconditioning (HP) induces endogenous protective responses against subsequent lethal hypoxia. Under HP (0.5% O2) treatment, more BMSCs survived and migrated, and there was greater upregulation of growth factor expression, as compared to normal conditions. We have previously demonstrated that hypoxic preconditioned BMSCs (HP-BMSCs) can reduce cell death, suppress inflammatory response,

Please cite this article as: Sun, J., et al., Intranasal delivery of hypoxia-preconditioned bone marrow-derived mesenchymal stem cells enhanced regenerative effects after intra..., Exp. Neurol. (2015), http://dx.doi.org/10.1016/j.expneurol.2015.03.011

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Fig. 6. Intranasally transplanted HP-BMSC promoted functional recovery. Adhesive-removal test was performed to assess sensorimotor function. Animals receiving HP-BMSCs performed faster in time to contact (A) and time to remove the adhesive attached to the left paw (B). (C) Rotarod test was performed to assess motor function and balance ability. (D) mNSS score was used to assess neurological deficit. Data is represented as mean ± SEM. *: p b 0.05. N = 15–18 each group. One way ANOVA with the Tukey post-hoc test was used in the adhesive-removal test and rotarod tests. For the nonparametric data of mNSS, Mann–Whitney U-test was applied.

upregulate the release of HIFα, BDNF, VEGF, and GDNF, and enhance cell migration (Wei et al., 2012, 2013a; Hu et al., 2011; Das et al., 2010). Adipose-derived stem cells, neural stem/progenitor cells, human adipose-derived mesenchymal stem cells and embryonic stem cells have all shown that HP is capable of producing similar therapeutic benefits across different models (Theus et al., 2008; Kang et al., 2014; Ara and De Montpellier, 2013; Liu et al., 2013).

Previous studies have shown that BMSC may be a viable option for treating CNS disorders (Otero et al., 2011, 2012; Mahmood et al., 2007). Transplanted BMSCs protect and repair injured brain through multiple mechanisms: cell replacement via survival and migration to the lesion and differentiation into astrocyte and mature neurons (Otero et al., 2010); attenuation of the native inflammatory response; alleviation of cell death; enhancement of endogenous recovery

Fig. 7. Effect of intranasally delivered HP-BMSC on brain atrophy. Ventricle enlargement is a marker of brain atrophy. (A) Representative samples of Nissl stains of brain sections showing the changes in caudate volume and ventricle size in sham, ICH with vehicle, and ICH with HP-BMSC treatment animals at 21 days after ICH. (B) Significant reduction of ventricle enlargement was observed in the HP-BMSC group. Data is represented as mean ± SEM. *: p b 0.05, ICH-BMSC vs. ICH-vehicle. N = 12 each group. One way ANOVA was used, followed by the Tukey post-hoc test.

Please cite this article as: Sun, J., et al., Intranasal delivery of hypoxia-preconditioned bone marrow-derived mesenchymal stem cells enhanced regenerative effects after intra..., Exp. Neurol. (2015), http://dx.doi.org/10.1016/j.expneurol.2015.03.011

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processes; (Onteniente, 2013) and augmentation of neurotrophic factor release (Qin et al., 2013). Furthermore, a recent study showed that BMSCs contribute towards the formation of neuronal circuitry. After ICH, BMSC can integrate with the host brain and enhance the axonal sprouting (Liang et al., 2013). Aside from enhancement of angiogenesis, neurogenesis, and synaptogenesis, Vanessa and colleagues reported that intranasally delivered BMSCs can migrate to the injury region and improve cognitive function after neonatal hypoxia–ischemia in rats (Donega et al., 2013). Endogenous neurogenesis has been identified following CNS injury, such as ischemic stroke (Jin et al., 2001), ICH (Masuda et al., 2007), and TBI (Ruan et al., 2013). After collagenase-induced ICH, neural stem/progenitor cells can differentiate and migrate from the SVZ to the peri-hemorrhage area, and their levels are prominently detected at 14 and 28 days after ICH (Masuda et al., 2007). The effect of HPBMSC on endogenous neurogenesis could be an option for ICH treatment (Onteniente, 2013). In this study, HP-BMSC transplantation significantly increased the number of BrdU + (proliferating cells), DCX + cells, and BrdU+/DCX+ co-label cells (neuroblasts) in the SVZ as well as in peri-hematoma regions. HP-BMSC also enhances the release of various trophic factors, including BDNF, VEGF, and GDNF, which all play important roles in rebuilding the neurovascular unit (Song et al., 2013). BDNF can increase the number and migration of neuroblasts, augment neuronal differentiation of endogenous progenitor cells, and decrease brain tissue loss after experimental ICH (Chen et al., 2012). VEGF levels increase during most pathological events in the brain and are essential for brain repair after ICH (Lei et al., 2013). GDNF also plays important roles in brain repair after CNS disorders (Yang et al., 2011, 2012). These factors possess neurotrophic and neuroprotective activities in the CNS. We confirmed the increase of VEGF, BDNF and GDNF in peri-hematoma regions by Western blot, which supports the neuroregenerative effects of HP-BMSC treatment as an ICH therapy. These regenerative activities ultimately lead to marked functional recovery after ICH in mice. In addition, we found that HP-BMSC can reduce the tissue loss, which was also confirmed in another study (Ishizaka et al., 2013). Intracerebral/intracerebroventricular/intrathecal administration may bring additional injury to the normal brain/nerve tissues, which limits their clinical applications (Wang et al., 2013; Fischer et al., 2009). Intranasal strategy seems to be a promising route for delivery of stem cells into brain. Daniel Yan and colleagues administered MSC and human glioma cells intranasally to normal mice and rats (Danielyan et al., 2009). The exact migration pathway from the olfactory epithelium to the damaged brain has still not been fully elucidated (Chapman et al., 2013). Olfactory nerve cells connect the nasal mucosa to the olfactory bulb and frontal cortex of the brain. Peripheral trigeminal nerves also directly connect nose passages with the brain stem and spinal cord. This anatomic feature plays vital roles in stem cell migration from the nose to the brain. Through the many pathways, transplanted cells can be detected in the brain as early as 1 h following intranasal administration (Jiang et al., 2011; Lochhead and Thorne, 2012). Furthermore, it has been shown that intranasally transplanted cells may be able to hone in more efficiently to the injured area (Wu et al., 2013). In our model, we report that intranasally administered HP-BMSCs were detected in brain sections after ICH. More specifically, the transplanted cells were observed in the olfactory bulb, cortex and peri-hematoma regions (Osanai et al., 2010; Banerjee et al., 2012). The appropriate time frame for stem cell transplantation after ICH is still controversial (Miao et al., 2013). The endogenous inflammatory response is highly active during the acute phase of ICH. Inflammatory factors, combined with dead blood cell debris, iron, thrombin, other chemokines, cytokines, and proteases generate a hostile environment for grafted cells (Li et al., 2007; Xue and Del Bigio, 2000). Previous studies have shown that transplantation of BMSCs modulates endogenous repair activities such as neurogenesis and promotes better neurological recovery (Vaquero et al., 2013). Li and colleagues found that intracarotid

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administration of neural stem cells at 7 days after ICH leads to significantly better functional recovery and the highest survival of transplanted cells. In the present study, we designed the administration of HP-BMSCs at 3 and 7 days after ICH in order to evaluate whether bypassing the acute phase will promote grafted cell survival and therapeutic efficacy. One important consideration is that in order to track the cells, we pre-labeled the HP-BMSCs with Hoechst prior to transplantation. However, if the cells die following transplantation, the dead cells will release the Hoechst, which can be taken up through phagocytosis by microglia, leading to misinterpretation of cell viability (Kircher et al., 2011). In order to resolve this issue, we used transgenic cells that also contained a green fluorescence protein (GFP) fluorescent tag. Overall, our results indicate that HP-BMSC transplantation dramatically increases the expression of neurotrophic factors, enhances endogenous neurogenesis, and improves functional deficits. Taken together, HP-BMSC therapy may provide a potential clinical therapeutic option for ICH. Furthermore, our data corroborates the efficacy of a noninvasive method of delivery of stem cells into the brain. We have demonstrated that intranasal administration of HP-BMSCs can effectively deliver HP-BMSCs into the injury regions, resulting in neuroregenerative effects and behavioral recovery. Conflict of interest The authors have no disclosure of conflicts of interest related to this investigation. Acknowledgment This work was supported by the NIH grants NS062097, NS073378, NS075338, an American Heart Association Established Investigator Award, and a grant of National Science Foundation of China (81350012) to LJ. References Andres, R.H., et al., 2008. Cell replacement therapy for intracerebral hemorrhage. Neurosurg. Focus. 24 (3–4), E16. Ara, J., De Montpellier, S., 2013. Hypoxic-preconditioning enhances the regenerative capacity of neural stem/progenitors in subventricular zone of newborn piglet brain. Stem Cell Res. 11 (2), 669–686. Banerjee, S., et al., 2012. The potential benefit of stem cell therapy after stroke: an update. Vasc. Health Risk Manag. 8, 569–580. Beray-Berthat, V., et al., 2010. Long-term histological and behavioural characterisation of a collagenase-induced model of intracerebral haemorrhage in rats. J. Neurosci. Methods 191 (2), 180–190. Bliss, T., et al., 2007. Cell transplantation therapy for stroke. Stroke 38 (2 Suppl.), 817–826. Chapman, C.D., et al., 2013. Intranasal treatment of central nervous system dysfunction in humans. Pharm. Res. 30 (10), 2475–2484. Chen, S.J., et al., 2012. Brain-derived neurotrophic factor-transfected and nontransfected 3T3 fibroblasts enhance migratory neuroblasts and functional restoration in mice with intracerebral hemorrhage. J. Neuropathol. Exp. Neurol. 71 (12), 1123–1136. Danielyan, L., et al., 2009. Intranasal delivery of cells to the brain. Eur. J. Cell Biol. 88 (6), 315–324. Das, R., et al., 2010. The role of hypoxia in bone marrow-derived mesenchymal stem cells: considerations for regenerative medicine approaches. Tissue Eng. Part B Rev. 16 (2), 159–168. Donega, V., et al., 2013. Intranasal mesenchymal stem cell treatment for neonatal brain damage: long-term cognitive and sensorimotor improvement. PLoS One 8 (1), e51253. Fagan, S.C., et al., 2013. Recommendations for preclinical research in hemorrhagic transformation. Transl. Stroke Res. 4 (3), 322–327. Fischer, U.M., et al., 2009. Pulmonary passage is a major obstacle for intravenous stem cell delivery: the pulmonary first-pass effect. Stem Cells Dev. 18 (5), 683–692. Francis, K.R., Wei, L., 2010. Human embryonic stem cell neural differentiation and enhanced cell survival promoted by hypoxic preconditioning. Cell Death Dis. 1, e22. Hu, X., et al., 2011. Hypoxic preconditioning enhances bone marrow mesenchymal stem cell migration via Kv2.1 channel and FAK activation. Am. J. Physiol. Cell Physiol. 301 (2), C362–C372. Ishizaka, S., et al., 2013. Intra-arterial cell transplantation provides timing-dependent cell distribution and functional recovery after stroke. Stroke 44 (3), 720–726. Jiang, Y., et al., 2011. Intranasal delivery of stem cells to the brain. Expert Opin. Drug Deliv. 8 (5), 623–632.

Please cite this article as: Sun, J., et al., Intranasal delivery of hypoxia-preconditioned bone marrow-derived mesenchymal stem cells enhanced regenerative effects after intra..., Exp. Neurol. (2015), http://dx.doi.org/10.1016/j.expneurol.2015.03.011

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Jin, K., et al., 2001. Neurogenesis in dentate subgranular zone and rostral subventricular zone after focal cerebral ischemia in the rat. Proc. Natl. Acad. Sci. U. S. A. 98 (8), 4710–4715. Kan, E.M., Ling, E.A., Lu, J., 2010. Stem cell therapy for spinal cord injury. Curr. Med. Chem. 17 (36), 4492–4510. Kang, S., Kim, S.M., Sung, J.H., 2014. Cellular and molecular stimulation of adipose-derived stem cells under hypoxia. Cell Biol. Int. 38 (5), 553–562. Keep, R.F., Hua, Y., Xi, G., 2012. Intracerebral haemorrhage: mechanisms of injury and therapeutic targets. Lancet Neurol. 11 (8), 720–731. Kircher, M.F., Gambhir, S.S., Grimm, J., 2011. Noninvasive cell-tracking methods. Nat. Rev. Clin. Oncol. 8 (11), 677–688. Kleinig, T.J., Vink, R., 2009. Suppression of inflammation in ischemic and hemorrhagic stroke: therapeutic options. Curr. Opin. Neurol. 22 (3), 294–301. Krafft, P.R., et al., 2012. Modeling intracerebral hemorrhage in mice: injection of autologous blood or bacterial collagenase. J. Vis. Exp. 67, e4289. Lee, H.J., et al., 2009. Human neural stem cells overexpressing glial cell line-derived neurotrophic factor in experimental cerebral hemorrhage. Gene Ther. 16 (9), 1066–1076. Lei, C., et al., 2013. Effects of high-mobility group box1 on cerebral angiogenesis and neurogenesis after intracerebral hemorrhage. Neuroscience 229, 12–19. Li, F., et al., 2007. Therapeutic time window and effect of intracarotid neural stem cells transplantation for intracerebral hemorrhage. Neuroreport 18 (10), 1019–1023. Li, W.L., et al., 2008. Enhanced neurogenesis and cell migration following focal ischemia and peripheral stimulation in mice. Dev. Neurobiol. 68 (13), 1474–1486. Liang, H., et al., 2013. Transplantation of bone marrow stromal cells enhances nerve regeneration of the corticospinal tract and improves recovery of neurological functions in a collagenase-induced rat model of intracerebral hemorrhage. Mol. Cells 36 (1), 17–24. Liu, L., et al., 2013. Hypoxia preconditioned human adipose derived mesenchymal stem cells enhance angiogenic potential via secretion of increased VEGF and bFGF. Cell Biol. Int. 37 (6), 551–560. Liu, X., et al., 2014. Cell based therapies for ischemic stroke: from basic science to bedside. Prog. Neurobiol. 115, 92–115. Lochhead, J.J., Thorne, R.G., 2012. Intranasal delivery of biologics to the central nervous system. Adv. Drug Deliv. Rev. 64 (7), 614–628. Mahmood, A., et al., 2007. Treatment of traumatic brain injury with a combination therapy of marrow stromal cells and atorvastatin in rats. Neurosurgery 60 (3), 546–553 (discussion 553–554). Masuda, T., et al., 2007. Increase in neurogenesis and neuroblast migration after a small intracerebral hemorrhage in rats. Neurosci. Lett. 425 (2), 114–119. Miao, Z., et al., 2013. Hypothermia induced by adenosine 5'-monophosphate attenuates early stage injury in an acute gouty arthritis rat model. Rheumatol. Int. 33 (8), 2085–2092. Mohan, S., et al., 2012. Putative role of prostaglandin receptor in intracerebral hemorrhage. Front. Neurol. 3, 145. Onteniente, B., 2013. The multiple aspects of stroke and stem cell therapy. Curr. Mol. Med. 13 (5), 821–831. Osanai, T., et al., 2010. Noninvasive transplantation of bone marrow stromal cells for ischemic stroke: preliminary study with a thermoreversible gelation polymer hydrogel. Neurosurgery 66 (6), 1140–1147 (discussion 1147). Otero, L., et al., 2010. Intralesional administration of allogeneic bone marrow stromal cells reduces functional deficits after intracerebral hemorrhage. Histol. Histopathol. 25 (4), 453–461. Otero, L., et al., 2011. Late transplantation of allogeneic bone marrow stromal cells improves neurologic deficits subsequent to intracerebral hemorrhage. Cytotherapy 13 (5), 562–571.

Otero, L., et al., 2012. Allogeneic bone marrow stromal cell transplantation after cerebral hemorrhage achieves cell transdifferentiation and modulates endogenous neurogenesis. Cytotherapy 14 (1), 34–44. Poon, M.T., Fonville, A.F., Al-Shahi Salman, R., 2013. Long-term prognosis after intracerebral haemorrhage: systematic review and meta-analysis. J. Neurol. Neurosurg. Psychiatry 85 (6), 660–667. Qin, J., et al., 2013. Functional recovery after transplantation of induced pluripotent stem cells in a rat hemorrhagic stroke model. Neurosci. Lett. 554, 70–75. Ruan, L., et al., 2013. Neurogenesis in neurological and psychiatric diseases and brain injury: from bench to bedside. Prog. Neurobiol. 115, 116–137. Seyfried, D.M., et al., 2010. Localization of bone marrow stromal cells to the injury site after intracerebral hemorrhage in rats. J. Neurosurg. 112 (2), 329–335. Song, M., et al., 2013. Restoration of intracortical and thalamocortical circuits after transplantation of bone marrow mesenchymal stem cells into the ischemic brain of mice. Cell Transplant. 22 (11), 2001–2015. Stahel, P.F., et al., 2000. Experimental closed head injury: analysis of neurological outcome, blood–brain barrier dysfunction, intracranial neutrophil infiltration, and neuronal cell death in mice deficient in genes for pro-inflammatory cytokines. J. Cereb. Blood Flow Metab. 20 (2), 369–380. Sun, Y., et al., 2011. Neuroprotection and sensorimotor functional improvement by curcumin after intracerebral hemorrhage in mice. J. Neurotrauma 28 (12), 2513–2521. Theus, M.H., et al., 2008. In vitro hypoxic preconditioning of embryonic stem cells as a strategy of promoting cell survival and functional benefits after transplantation into the ischemic rat brain. Exp. Neurol. 210 (2), 656–670. Vaquero, J., et al., 2013. Cell therapy with bone marrow stromal cells after intracerebral hemorrhage: impact of platelet-rich plasma scaffolds. Cytotherapy 15 (1), 33–43. Wang, L., et al., 2013. Therapeutic applications of bone marrow-derived stem cells in ischemic stroke. Neurol. Res. 35 (5), 470–478. Wei, L., et al., 2012. Transplantation of hypoxia preconditioned bone marrow mesenchymal stem cells enhances angiogenesis and neurogenesis after cerebral ischemia in rats. Neurobiol. Dis. 46 (3), 635–645. Wei, N., et al., 2013a. Delayed intranasal delivery of hypoxic-preconditioned bone marrow mesenchymal stem cells enhanced cell homing and therapeutic benefits after ischemic stroke in mice. Cell Transplant. 22 (6), 977–991. Wei, S., et al., 2013b. Acute and delayed protective effects of pharmacologically induced hypothermia in an intracerebral hemorrhage stroke model of mice. Neuroscience 252, 489–500. Wei, Z.Z., et al., 2015. Intranasal delivery of bone marrow mesenchymal stem cells improved neurovascular regeneration and rescued neuropsychiatric deficits after neonatal stroke in rats. Cell Transplant. [Epub ahead of print]. Wu, S., et al., 2013. Intranasal delivery of neural stem cells: a CNS-specific, non-invasive cell-based therapy for experimental autoimmune encephalomyelitis. J. Clin. Cell Immunol. 4 (3). Xiong, X.Y., et al., 2013. Iron and intracerebral hemorrhage: from mechanism to translation. Transl. Stroke Res. 5 (4), 429–441. Xue, M., Del Bigio, M.R., 2000. Intracerebral injection of autologous whole blood in rats: time course of inflammation and cell death. Neurosci. Lett. 283 (3), 230–232. Yang, C., et al., 2011. Neuroprotective effects of bone marrow stem cells overexpressing glial cell line-derived neurotrophic factor on rats with intracerebral hemorrhage and neurons exposed to hypoxia/reoxygenation. Neurosurgery 68 (3), 691–704. Yang, K.L., et al., 2012. Human adipose-derived stem cells for the treatment of intracerebral hemorrhage in rats via femoral intravenous injection. Cell. Mol. Biol. Lett. 17 (3), 376–392. Yu, S.P., Wei, Z., Wei, L., 2013. Preconditioning strategy in stem cell transplantation therapy. Transl. Stroke Res. 4 (1), 76–88.

Please cite this article as: Sun, J., et al., Intranasal delivery of hypoxia-preconditioned bone marrow-derived mesenchymal stem cells enhanced regenerative effects after intra..., Exp. Neurol. (2015), http://dx.doi.org/10.1016/j.expneurol.2015.03.011