Postacute Stromal Cell–Derived Factor-1α Expression Promotes Neurovascular Recovery in Ischemic Mice
Background and Purpose—Acute interventions of stroke are often challenged by a narrow treatment window. In this study, we explore treatments in the postacute phase of stroke with wider windows of opportunity. We investigated the effects of stromal cell–derived factor (SDF-1α) in neurovascular recovery during the postacute phase and downstream signaling pathways, underlying SDF-1α–mediated neurovascular recovery.
Methods—Adult male Institute of Cancer Research (ICR) mice underwent middle cerebral artery occlusion. One week after middle cerebral artery occlusion, the animals received stereotactic injection of adenoassociated virus (AAV) carrying SDF-1α gene as treatment or AAV-green fluorescent protein as control and were monitored for 5 weeks. Neurobehavioral outcomes were evaluated, and brain atrophy was measured. Neurogenesis and angiogenesis were examined. The proliferation and migration of neural progenitor cells were evaluated. Downstream pathways of SDF-1α were investigated. Inflammatory response was monitored.
Results—Neurobehavioral outcomes were improved, and brain atrophy was greatly reduced for ≤5 weeks in AAV-SDF-1α groups when compared with the control. SDF-1 receptor CXCR4 was upregulated and colocalized with neural and endothelial progenitor cells. The number of nestin+ and doublecortin+/bromodeoxyuridine+ cells in the subventricular zone, doublecortin+ and neuron+/bromodeoxyuridine+ cells in the perifocal region, and cluster of differentiation (CD)31+ and bromodeoxyuridine+/CD31+ microvessels are also significantly increased in AAV-SDF-1α groups. Administration of CXCR4 antagonist AMD3100 eliminated the beneficial effects of SDF-1α. SDF-1α/CXCR4 interaction activated AKT, extracellular signal-regulated kinases (ERK), and P38 mitogen-activated protein kinase (MAPK) signaling pathways but not the c-Jun N-terminal kinase (JNK) pathway.
Conclusions—SDF-1α promoted neurogenesis and angiogenesis during the postacute phase of ischemia without eliciting an inflammatory response. AAV-SDF-1α expression represents a promising avenue for ischemic stroke therapy with a wider treatment window.
Although the majority of patients with ischemic stroke survive with timely treatment in the acute phase, many still experience various long-term neurological deficits.1 One of the obstacles to neurobehavioral recovery is limited spontaneous neurogenesis and angiogenesis in the postacute phase. Therapeutics that target the postacute phase with a wider treatment window would help improve functional recovery after stroke.
Stromal cell–derived factor-1 (SDF-1), also called C-X-C motif chemokine ligand 12, functions by interacting with its receptor CXCR4, which is found on the cell surface of leucocytes and various kinds of stem cells.2–7 Previously, we have shown that blocking SDF-1/CXCR4 interaction suppresses inflammatory responses and reduces brain infarction in the acute phase of ischemic stroke.8,9 In addition, CXCR4 gene transfer into the heart before myocardial ischemia enhances ischemia-reperfusion injury and increases the influx of inflammatory cells.10 These results suggest that SDF-1 functions mainly as an inflammatory initiator during the acute phase of ischemia. However, the function of SDF-1 during the postacute phase of ischemia is still unknown.
It is noteworthy that SDF-1 plays an important role during the development of the central nervous system and has a strong modulation effect on neurons, interneurons, and granule cells.11–15 It is now recognized that SDF-1 regulates the development of nervous tissue, particularly because of its effects on cell migration and axon guidance.16 SDF-1 is also essential to the development of vasculature by recruiting circulating endothelial progenitor cells (EPCs) involved in angiogenesis.17–22 It is possible that SDF-1 can help recruit progenitor cells in the brain and consequently benefit stroke recovery. In this work, we examined this hypothesis.
To investigate the function of SDF-1 during the postacute phase of ischemia, we applied adenoassociated virus (AAV)–mediated SDF-1α gene transfer to the peri-infarct area 1 week after permanent middle cerebral artery occlusion (MCAO) surgery. The results show that SDF-1α was successfully expressed in a broad area around the virus injection site. Overexpression of SDF-1α during the postacute phase of ischemia significantly reduced brain atrophy and improved neurological outcomes. SDF-1α gene transfer promoted both neurogenesis and angiogenesis but did not elicit a focal inflammatory response. The results from the CXCR4 antagonist group suggest that SDF-1α mainly functions through CXCR4-mediated downstream AKT, extracellular signal-regulated kinases (ERK), and P38 mitogen-activated protein kinase (MAPK) signaling pathways but not the c-Jun N-terminal kinase (JNK) signaling pathway.
Materials and Methods
Animal procedures were performed according to a protocol approved by the Institutional Animal Care and Use Committee of Shanghai Jiao Tong University, Shanghai, China. The experimental design is illustrated in Figure 1A. Bromodeoxyuridine (BrdU) powder (Sigma-Aldrich, St Louis, MO) was dissolved in normal saline in a concentration of 10 mg/mL. BrdU solution was injected intraperitoneally at 50 mg/kg once a day for 7 consecutive days 3 and 5 weeks after MCAO. AMD3100 powder (Sigma-Aldrich) was used 1 mg/kg per day, which is sufficient to block CXCR4 without causing stem cell mobilization.23,24 The same amount of normal saline was used in the control group.
Permanent MCAO in Mice
Sixty-two adult male Institute of Cancer Research (ICR) mice (Sippr-BK, Shanghai, China) weighing 30±2 g were anesthetized by ketamine/xylazine (100/10 mg/kg; Sigma-Aldrich). MCAO was performed as described previously.8 Briefly, after isolation of the common carotid artery, external and internal carotid arteries, left MCA was occluded by inserting a 6-0 nylon suture coated with silica gel. Body temperature was maintained at 37°C throughout the surgery using a thermal blanket. Successful occlusion was verified by Laser Doppler Flowmetry (Moor Instruments; Axminster, Devon, United Kingdom).
AAV-SDF-1α Viral Vector Injection
One week after MCAO, the mice were anesthetized and immobilized on a stereotaxic frame (RWD Life Science, Shenzhen, China). A total volume of 5 μL of PBS containing 5×108 AAV-SDF-1α or AAV-green fluorescent protein (GFP) viral particles were injected stereotactically at a rate of 200 nL/min at 2 mm lateral to the bregma and 3 mm under the dura.
An investigator who was blinded to the experimental design and treatment performed rotarod test and neurological evaluations using modified neurological severity scores (mNSS). Baseline values were established by averaging 6 trials before the surgery. Mice were examined for ≤5 weeks after MCAO. Rotarod test required mice to balance on a rotating rod. Mice were allowed 1-minute adaption on the rod, after which the rod was accelerated to 40 rpm ≥2 minutes; and the time spent on the rod was recorded. mNSSs of the animals were graded on a scale of 0 to 14, which is a composite of motor, reflex, and balance tests.25
Brain Atrophy Measurement
Brains were removed and frozen immediately after euthanizing the mice. A series of 20-μm coronal sections 1.3 to −2.7 mm from the bregma was cut and mounted on slides. The atrophic area was calculated by subtracting the cresyl violet stained area in the ipsilateral hemisphere from the whole area of the contralateral hemisphere using ImageJ software (National Institutes of Health, Bethesda, MD).
Immunohistochemistry was performed according to the protocol previously described.8 Care was taken to sample sections with similar anatomic features. The primary antibodies were SDF-1α and CXCR4 (1:100; Abcam, Cambridge, MA); glial fibrillary acidic protein (GFAP; 1:100; Beyotime, Hangzhou, China), neuron (NeuN), and nestin (1:100; Millipore, Billerica, MA); BrdU, cluster of differentiation (CD)31, and doublecortin (DCX; 1:200; Santa Cruz Biotechnology, Santa Cruz, CA). For biotinylated immunostaining, the brain sections were incubated in the same primary antibodies and developed for the same amount of time.
DCX/BrdU, NeuN/BrdU, and CD31/BrdU Double Immunostaining
Brains were postfixed for 4 to 5 hours followed by 24 hours of immersion in 30% sucrose in PBS and immediately frozen, and then sectioned using a cryostat (Leica, Solms, Germany). A thickness of 20-μm coronal sections was cut. Floating coronal sections were collected in antigen protective solution, which involves 50% PBS, 20% glycol, and 30% glycerol. Sections were first treated with 2 mol/L HCl for 30 minutes at 37°C and then neutralized with sodium borate twice each for 10 minutes. Sections were then treated with 0.3% triton-100 in PBS for 30 minutes, blocked by 5% normal donkey serum, incubated with anti-BrdU and anti-DCX, NeuN, or CD31 antibody at 4°C overnight. Finally, the sections were incubated with proper secondary antibodies for 60 minutes at room temperature. Stained sections were mounted after rinsing.
Cell and Vessel Counting
Six fields were randomly selected from the perifocal region at ×20 objective. Nestin+ and DCX+/BrdU+ cells in the subventricular zone (SVZ) were counted for each image (DM2500; Leica Microsystems, Wetzlar, Germany). Sections were incubated with the same primary antibodies and imaged under the same conditions by a blinded investigator. The quantity of nestin+, DCX+, NeuN+/BrdU+ cells, and CD31+ and CD31+/BrdU+ microvessels in the perifocal region was counted and quantified by an investigator who was blinded to the experimental groups in the same manner. Four serial sections, spaced 400-μm apart (1.10 to −0.1 mm from the bregma), were selected from each animal. Positive cells were counted and averaged from 6 optical fields for each mouse.
Western Blot Analysis
Mice were anesthetized by ketamine/xylazine intraperitoneally. After anesthesia, brains were quickly removed to a cooled brain mold and then cut into 4 sections by 3 blades that are 2-mm apart; the second rostral section, including ischemic core, was collected. The protein extracted from ipsilateral striatum was used for further Western blot analysis. Western blot protocol was performed as previously described.8 The primary antibodies were CXCR4 (1:1000; Abcam); pAKT, AKT, pERK, ERK, p-P38 MAPK, P38 MAPK, phosphorylated JNK (pJNK) and JNK (1:1000; Cell Signaling Technology, Danvers, MA); β-actin; and GAPDH (1:1000; Santa Cruz Biotechnology).
Protein levels of SDF-1α were quantified using an ELISA kit (Mouse SDF-1α ELISA Kit; RayBiotech, Norcross, GA) according to the manufacturer’s protocol. Readings from each sample were normalized for protein concentration.
Parametric data from different groups were compared using 1-way ANOVA followed by Student–Newman–Keuls tests using GraphPad Prism version 3.05 (GraphPad Software, Inc, La Jolla, CA). All data were presented as mean±SD. A value of P<0.05 was considered statistically significant.
Postacute SDF-1α Gene Expression Improved Neurobehavioral Outcomes and Reduced Brain Atrophy
To induce exogenous expression of SDF-1α in vivo, we constructed pAAV-SDF-1α-internal ribosome entry site (IRES)-GFP plasmid by inserting SDF-1α cDNA into a pAAV-IRES-GFP backbone. AAV-SDF-1α mediated successful expression of SDF-1α in vitro and in vivo for ≤5 weeks (Figure I in the online-only Data Supplement). To evaluate the effect of postacute SDF-1α gene expression on neurological outcomes, neurological assessments using mNSS and rotarod test were performed for ≤5 weeks after MCAO. mNSS severity was greatly reduced (Figure 1B; 12% [3 weeks] and 25% [5 weeks] reduced, AAV-SDF-1α versus AAV-GFP; P<0.05), and rotarod maintaining time was significantly prolonged (Figure 1C; 1.7-fold [3 weeks; P<0.001] and 3.2-fold [5 weeks; P<0.05] increase, AAV-SDF-1α versus AAV-GFP) in SDF-1α gene-transferred mice when compared with AAV-GFP control mice at 3 and 5 weeks after ischemia. To characterize whether SDF-1α gene expression in the postacute period influenced brain recovery further, we measured brain atrophy and found that it was significantly reduced 3 weeks after ischemia in SDF-1α gene-transferred mice (Figure 1D; 49.6% [3 weeks] and 74.3% [5 weeks] reduced, AAV-SDF-1α versus AAV-GFP; P<0.05).To identify the receptors through which SDF-1α functions, CXCR4 antagonist AMD3100 was used in the third experimental group in addition to AAV-SDF-1α administration. Results showed that all the improvements observed in the AAV-SDF-1α group, including the mNSS assessment (Figure 1B; 17.1% [3 weeks] and 32.8% [5 weeks] reduced, AAV-SDF-1α versus AAV-SDF-1α/AMD3100; P<0.001), rotarod test (Figure 1C; 2.2-fold [3 weeks] and 3.4-fold [5 weeks] increase, AAV-SDF-1α versus AAV-SDF-1α/AMD3100; P<0.001), and brain atrophy (Figure 1D; 53.3% [3 weeks; P<0.05] and 85.0% [5 weeks; P<0.001] reduced, AAV-SDF-1α versus AAV-SDF-1α/AMD3100), were eliminated in AMD3100-treated mice. This indicates that the SDF-1/CXCR4 signaling pathway plays a major role in SDF-1α–mediated neurobehavioral recovery during the postacute phase of ischemic stroke.
To investigate potentially unfavorable effects of SDF-1α, observed previously during the acute phase,8 we examined the focal inflammatory response by measuring myeloperoxidase expression and activity. Results showed that there were no differences in myeloperoxidase+ cells and myeloperoxidase activity in the AAV-GFP and AAV-SDF-1 groups (Figure II in the online-only Data Supplement), suggesting that SDF-1 gene expression in the postacute phase of ischemic stroke did not elicit a focal inflammatory response.
SDF-1α Expression Was Increased and Was Mainly Located on Neurons and Astrocytes in SDF-1α Gene-Transferred Mice
To define what types of cells in the brain were infected by AAV-SDF-1α, immunohistochemical staining was performed for NeuN, GFAP, and CD31. The GFP expressed by AAV-SDF-1α transfected cells was colocalized with NeuN+ and GFAP+ cells, but not with CD31+ cells, indicating that AAV-SDF-1α was capable of transfecting neurons and astrocytes but inefficient at transfecting endothelial cells (Figure III in the online-only Data Supplement).
To examine whether the stroke recovery resulted from the increased SDF-1α expression after SDF-1α gene transfer, real-time polymerase chain reaction and ELISA studies were performed. The results showed that SDF-1α mRNA and protein expression were significantly increased for ≤5 weeks after ischemia in the perifocal region in both the SDF-1α gene-transferred group and the AAV-SDF-1α/AMD3100 cotreated group (Figure 2).
CXCR4 Expression Was Upregulated After SDF-1α Gene Expression and Was Colocalized With Neural Progenitor Cells, Neuroblasts, Neurons, and EPCs but Not by Mature Endothelial Cells
AMD3100 eliminates the beneficial effects of SDF-1, indicating that the SDF-1/CXCR4 signaling pathway is vitally important in improving the neurobehavioral outcomes of ischemic mice, so studies were performed to detect the expression of CXCR4. Results showed that CXCR4 was significantly upregulated for ≤5 weeks after ischemia in both AAV-SDF-1α gene-transferred mice and SDF-1α/AMD3100 cotreated mice (Figure 3A). To explore how SDF-1α affects neurovascular recovery and neurobehavioral recovery, immunohistochemical double staining for CXCR4 in the peri-infarct area was performed with nestin, DCX, NeuN, CD34, fetal liver kinase (Flk)-1, and CD31. CXCR4 (Figure 3B, red) was discovered on nestin+, DCX+, NeuN+, CD34+ (cell-like) and Flk-1+ (green, arrows) cells, whereas it could not be found on CD34+ (vessel-like) and CD31+ (green, asterisk) cells, or GFAP+ cells (Figure IV in the online-only Data Supplement), suggesting that CXCR4 can be expressed by neural progenitor cells (NPCs), neuroblasts, neurons, and EPCs but not by mature endothelial cells or astrocytes. These findings suggest that the SDF-1α/CXCR4 signaling pathway may be involved in promoting neurovascular recovery.
Postacute SDF-1α Gene Expression Promoted Neurogenesis by Enhancing NPC Proliferation and Differentiation in Ischemic Mice
To analyze whether SDF-1α gene expression promotes focal neurogenesis, which facilitates the functional recovery after ischemia,26 the immunostaining for nestin was performed. Figure 4Aa shows the 3,3'-diaminobenzidine (DAB) stained coronal sections, depicting nestin+ cells in the ipsilateral hemisphere and enlarged sections from SVZ (Figure 4Aa1) and perifocal area (Figure 4Aa2). The number of nestin+ NPCs significantly increased in the SVZ in SDF-1α gene-transferred mice when compared with the control, whereas it decreased in mice treated with AMD3100 (Figure 4Ab; 5 weeks: 1.5-fold increase, AAV-SDF-1α versus AAV-GFP; P<0.01 and 2.3-fold increase, AAV-SDF-1α versus AAV-SDF-1α/AMD3100; P<0.001), suggesting that SDF-1α promotes NPC proliferation during the postischemia recovery stage. Similar results also obtained by quantification of sex determining region Y-box 2 (Sox2)+ cells in SVZ 5 weeks after MCAO in ipsilateral hemisphere (Figure V in the online-only Data Supplement).The number of nestin+ cells in the perifocal region was also greatly expanded when compared with the AAV-GFP control and AAV-SDF-1α/AMD3100 cotreated mice (Figure 4Ac; 1.8-fold [3 weeks; P<0.05] and 2.6-fold [5 weeks; P<0.001] increase, AAV-SDF-1α versus AAV-GFP and 1.8-fold [3 weeks; P<0.01] and 3.3-fold [5 weeks; P<0.001] increase, AAV-SDF-1α versus AAV-SDF-1α/AMD3100) at 3 and 5 weeks after stroke during the postischemic recovery stage.
To analyze whether SDF-1α gene expression promotes NPC differentiation further, immunohistochemical double staining of DCX and BrdU was performed. DCX is upregulated exclusively in postmitotic neurons and principally expressed in active cells, including proliferating NPCs, newly generated neuroblasts, as well as migrating and differentiating neurons.27–29 DCX can be used as an estimation of the rate and degree of adult neurogenesis.30 The number of DCX+/BrdU+ cells SDF-1α gene-transferred mice was profoundly augmented at 5 weeks after ischemia in SVZ when compared with AAV-GFP control and SDF-1α/AMD3100 cotreated mice (Figure 4B; 1.8-fold [P<0.001] increase, AAV-SDF-1α versus AAV-GFP and 1.7-fold [P<0.001] increase, AAV-SDF-1α versus AAV-SDF-1α/AMD3100). The number of DCX+ cells in the perifocal region of SDF-1α gene-transferred mice was profoundly augmented at 3 and 5 weeks after ischemia when compared with AAV-GFP control and SDF-1α/AMD3100 cotreated mice (Figure 4C; 2.4-fold [3 weeks; P<0.01] and 2.3-fold [5 weeks; P<0.001] increase, AAV-SDF-1α versus AAV-GFP and 2.6-fold [3 weeks; P<0.01] and 2.8-fold [5 weeks; P<0.001] increase, AAV-SDF-1α versus AAV-SDF-1α/AMD3100). Moreover, to determine the production of mature neurons, which is related to functional recovery, double immunostaining for NeuN and BrdU was performed. The results showed that the number of NeuN+/BrdU+ cells in the perifocal region of SDF-1α gene-transferred mice was profoundly augmented at 5 weeks after ischemia when compared with AAV-GFP control and SDF-1α/AMD3100 cotreated mice (Figure 4D; 2.8-fold [P<0.001] increase, AAV-SDF-1α versus AAV-GFP and 2.5-fold [P<0.001] increase, AAV-SDF-1α versus AAV-SDF-1α/AMD3100). The number of nestin+, DCX+/BrdU+, DCX+, and NeuN+/BrdU+ cells were all greatly increased in the AAV-SDF-1 group, suggesting that postacute expression of SDF-1α augmented neurogenesis, possibly through promoting NPCs migration and maturation.
Postacute SDF-1α Gene Expression Promoted Angiogenesis
Because neurogenesis is always correlated with angiogenesis during the development and disease and it shown to promote neuronal function recovery by forming neurovascular unit,31 we counted CD31+ microvessels to evaluate angiogenesis after SDF-1α gene transfer. The results demonstrated that the vascular density in the perifocal region of the ipsilateral hemisphere was significantly higher in SDF-1α gene-transferred mice than in AAV-GFP control and AAV-SDF-1α/AMD3100 cotreated mice (Figure 5Aa,b; 1.2-fold [3 weeks; P<0.01] and 1.3-fold [5 weeks; P<0.05] increase, AAV-SDF-1α versus AAV-GFP and 1.8-fold [3 weeks] and 1.8 [5 weeks] increase, AAV-SDF-1α versus AAV-SDF-1α/AMD3100; P<0.001). The ratio of perifocal/contralateral CD31+ microvessels was higher in SDF-1α gene-transferred mice than in AAV-GFP control and AAV-SDF-1α/AMD3100 cotreated mice at 3 and 5 weeks after permanent MCAO (Figure 5Ad). The number of BrdU+/CD31+ cells in the perifocal region of SDF-1α gene-transferred mice increased to a modest but significant extent versus control animals at 5 weeks after ischemia (Figure 5B; 1.9-fold increase, AAV-SDF-1α versus AAV-GFP and 2.3-fold increase, AAV-SDF-1α versus AAV-SDF-1α/AMD3100; P<0.05), indicating that SDF-1α gene expression can promote angiogenesis during the stroke recovery phase.
SDF-1α/CXCR4 Interaction Activated AKT, ERK, and P38 MAPK Signaling Pathways but Not the JNK Signaling Pathway in Postacute Ischemic Mice
To investigate which signaling pathway was activated by SDF-1/CXCR4, major signaling pathways activated by classical G-protein–coupled receptors were examined. Western blot analysis of the ipsilateral striatum lysates showed that SDF-1/CXCR4 interaction activated AKT, ERK, and P38 MAPK signaling pathways, which can be blocked by AMD3100, but had no effect on the JNK pathway (Figure 6).
Promoting neurogenesis and angiogenesis requires the appropriate proliferation, migration, and maturation of NPCs and EPCs. SDF-1, which regulates neural-vascular system development,11–13,17–19 is therefore a critical element of neural-vascular remodeling after ischemia-induced brain injury. Our data indicate that SDF-1α gene expression in the perifocal area 1 week after stroke is beneficial to neurogenesis and angiogenesis in the recovery phase. SDF-1α expression promoted NPC proliferation in SVZ, migration to the perifocal area and maturation. SDF-1α may also recruit EPCs to take part in angiogenesis in the perifocal region. These processes may couple together to promote neurovascular recovery. Blocking SDF-1/CXCR4 interaction with AMD3100 decreased the number of NPCs in the SVZ and the perifocal area, neuroblasts and microvessels in the perifocal area, and consequently abrogated the beneficial effects of SDF-1 gene expression in the neurobehavioral outcomes of mice. These data identify SDF-1 as a critical regulator of neurogenesis and angiogenesis within the damaged ischemic brain.
Genetic inactivation of SDF-1 signaling disturbs nerve-vessel alignment and abolishes arteriogenesis,32 which suggests that nerve-vessel alignment depends on SDF-1 signaling. The number of microvessels per field assessed by CD31 staining and the ratio of ipsilateral/contralateral of CD31 microvessels in SDF-1α gene-transferred group is significantly higher than the control group. Corroborating this result, the number of newly formed microvessels assessed by BrdU and CD31 double immunostaining in SDF-1α gene-transferred mice is twice that in AAV-GFP control mice and AAV-SDF-1α/AMD3100 cotreated mice. BrdU+/CD31+ microvessels may come from 2 sources: proliferating endothelial cells or newly matured EPCs. In our experiment, the number of BrdU+/CD31+ microvessels increases at a modest level in SDF-1α gene-transferred mice in assessed sections ranging between 1.1 to −0.1 mm from the bregma. Given that the matured endothelial cells in the brain do not express CXCR4 receptor, this limited neovascularization may result from the limited number of EPCs, which is not sufficient to form abundant angiogenesis. We have reported previously that an intravenous injection of 106 EPCs given to ischemic mice results in significantly increased angiogenesis in the perifocal region.33 Knowing now that the postacute expression of SDF-1α does not elicit a strong inflammatory response, combining AAV-mediated SDF-1α expression with EPCs transplantation during the postacute phase may be a promising strategy for promoting angiogenesis.
SDF-1α gene expression not only promoted angiogenesis, as reflected by the increased number of BrdU+/CD31+ microvessels, but also protected the existing vessels as indicated by the much higher density of CD31+ microvessels and higher ratio of ipsilateral/contralateral of CD31+ microvessels in the SDF-1α gene-transferred group. It is known that nestin+ and DCX+ cells can protect endothelial cells from cell death during ischemia.34 It is possible that SDF-1α indirectly protects endothelial cells and stabilizes brain vasculature by promoting the proliferation and migration of NPCs.
In conclusion, we demonstrated that SDF-1α gene expression in the postacute phase after brain ischemia improved neurobehavioral outcomes and reduced brain atrophy, which is related to active focal neurogenesis and angiogenesis. It is important that we established that SDF-1α gene expression in the postacute phase does not elicit a focal inflammatory response. This study indicates the timing of expression is crucial for designing treatments using SDF-1. Using the benefits of SDF-1 during the postacute phase after stroke may represent an effective approach for developing stroke therapies with larger treatment windows.
We thank members of the Neuroscience and Neuroengineering Research Center for their collective support.
Sources of Funding
This work was supported by the National Natural Science Foundation of China 81100868 (Dr Wang) and the Major State Basic Research Development Program of China (973 Program) 2011CB504405 (Drs Wang and Yang).
The online-only Data Supplement is available with this article at http://stroke.ahajournals.org/lookup/suppl/doi:10.1161/STROKEAHA.114.005078/-/DC1.
- Received February 7, 2014.
- Revision received March 19, 2014.
- Accepted April 8, 2014.
- © 2014 American Heart Association, Inc.
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