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CARDIOVASCULAR

Stromal Cell-Derived Factor-1 Promotes Neuroprotection, Angiogenesis, and Mobilization/Homing of Bone Marrow-Derived Cells in Stroke Rats

Department of Neurology, Center for Neuropsychiatry, China Medical University and Hospital, Taichung, Taiwan (W.-C.S., S.-Z.L., D.-C.C., H.-J.W.); Department of Radiology, Tzu-Chi Buddhist General Hospital, Tzu-Chi University, Hualien, Taiwan (P.-S.Y.); and Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan (C.-Y.S., H.L.)

Received June 25, 2007; accepted November 19, 2007.

	Abstract

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Stromal cell-derived factor (SDF)-1 is involved in the trafficking of hematopoietic stem cells from bone marrow to peripheral blood, and its expression is increased in the penumbra of the ischemic brain. In this study, SDF-1 was found to exert neuroprotective effects that rescued primary cortical cultures from H₂O₂ neurotoxicity, and to modulate neurotrophic factor expression. Rats receiving intracerebral administration of SDF-1 showed less cerebral infarction due to up-regulation of antiapoptotic proteins, and they had improved motor performance. SDF-1 injection enhanced the targeting of bone marrow (BM)-derived cells to the injured brain, as demonstrated in green fluorescent protein-chimeric mice with cerebral ischemia. In addition, increased vascular density in the ischemic cortex of SDF-1-treated rats enhanced functional local cerebral blood flow. In summary, intracerebral administration of SDF-1 resulted in neuroprotection against neurotoxic insult, and it induced increased BM-derived cell targeting to the ischemic brain, thereby reducing the volume of cerebral infarction and improving neural plasticity.

Expression of CXCR4 and SDF-1 after focal cerebral ischemia (Stumm et al., 2002) led us to speculate that this chemokine may also signal adhesion and migration of HSCs to ischemic tissue. On this basis, we hypothesized that locally overexpressed SDF-1 in cerebral ischemia may enhance HSC plasticity and provide an environment that enhances differentiation of HSCs into original lineage cell types of the damaged brain, such as endothelial cells and neurons.

In the present study, we examined the neuroprotective effects of SDF-1 against H₂O₂-induced neurotoxicity in primary cortical neurons. In addition, we analyzed the results of intracerebral administration of SDF-1 on cerebral ischemic rats by measuring the extent of induced cerebral infarction and neurological behavior before and after cerebral ischemia. SDF-1 was found to play a role not only in BM-derived cell differentiation but also in ischemia-induced trafficking of BM-derived cells from peripheral blood to the damaged brain. If a sufficient number of BM-derived cells could home in on cerebral ischemic injuries to promote neuronal repair and recovery of function, this would provide a novel insight into the mechanisms driving BM-derived cell differentiation and recruitment into damaged tissues. Our findings should have important implications for stem cell-based therapy of other ischemic disorders.

	Materials and Methods

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In Vitro Primary Cortical Culture Preparation. Primary cortical cells (PCC) were prepared from the cerebral cortex of gestation day 17 embryos from Sprague-Dawley rats as described previously (Murphy et al., 1990). In brief, pooled cortical tissues isolated from the brain were dissociated by mechanical trituration in ice-cold calcium- and magnesium-free Hanks' balanced salt solution, pH 7.4. Then, cells were counted and plated at a density of 5 x 10⁵ cells in 24-well culture plates precoated with 0.02 g/l poly-lysine. Cells were grown in Dulbecco's modified Eagle's medium (Invitrogen Life Technologies, Carlsbad, CA) with 10% heat-inactivated fetal bovine serum, 1 x 10^-3 M pyruvate, 4.2 x 10^-3 M sodium bicarbonate, 20 x 10^-3 M HEPES, and antibiotics penicillin (100 U/ml) and streptomycin (100 µg/ml). Cultures were maintained at 37°C in a humidified atmosphere of 5% CO₂ and 95% air. Four days after isolation, the cultures were replenished with minimum essential medium (Invitrogen Life Technologies) containing 0.5 g/l BSA and N-2 supplement, 0.5 x 10^-3 M pyruvate, and antibiotics. In conclusion, the culture medium was changed to serum-free minimum essential medium containing 1 x 10^-3 M pyruvate, 1 x 10^-3 M glutamate, 0.5 g/l BSA, 0.3 x 10^-3 M KCl, and antibiotics on the seventh day of incubation.

Measurement of LDH Activity and Immunocytochemistry of MAP-2. PCC were prepared in 24-well plates and pretreated with SDF-1 (ProSpec-Tany TechnoGene, Rehovot, Israel). After 20 min, H₂O₂ (10^-5 or 10^-4 M as used previously; Wang et al., 2001) was added to the medium for 24 h, and culture media were collected for lactate dehydrogenase (LDH) activity assays as described previously (Koh and Choi, 1987). In brief, LDH activity (U/10^-3 liters) was calculated from the slope of the decrease in optical density at 340 nm over a 3-min time period. One unit of LDH activity is defined as the amount of enzyme that catalyzes the consumption of 1 x 10^-3 mol of NADH per minute. For microtubule-associated protein (MAP)-2 immunostaining, primary cortical cell cultures were washed with PBS and fixed with 1% paraformaldehyde. Then, the fixed cells were treated for 20 min with blocking solution (10 g/l BSA and 0.03% Triton X-100). Cultures were incubated overnight at 4°C with a monoclonal antibody against MAP-2 (1:1000; BM). The bound primary antibody was visualized by the labeled streptavidin-biotin method (Dako LASB-2 kit, peroxidase; Dako, Mississauga, ON, Canada). The immunostaining procedure and the method for quantification of MAP-2⁺ cell density have been described previously (Wang et al., 2001).

Measurement of Caspase-3 Activity, Immunofluorescent Study of Caspase-3, and Expression of Antiapoptotic Proteins. To evaluate the antiapoptotic effect of SDF-1, caspase-3 activity, capase-3 immunofluorescent analysis and antiapoptotic protein expression were measured in primary cortical neurons. Primary cortical neuron cultures were pretreated with 1 µg/ml SDF-1 and 10^-5 or 10^-4 MH₂O₂ for 24 h. Fluorometric assays of caspase-3 activity were performed on the above-mentioned treated cells using commercial kits (Bio-Rad, Hercules, CA) following the manufacturer's instructions. For immunofluorescent study of activated caspase-3, primary cortical neuron cultures were treated as described above and incubated with primary antibody against active fragment of caspase-3 (R&D Systems, Minneapolis, MN) as described previously (Niquet et al., 2003). Caspase-3-positive cells were also quantified as described previously (Niquet et al., 2003). In addition, Western blot analyses of Bcl-2, Bcl-xL, Bax, and Bad expression from primary cortical culture were performed after treatment with 1 µg/ml SDF-1 and 10^-5 MH₂O₂ for 24 h. Then, the cells were lysed in a buffer containing 320 mM sucrose, 5 mM HEPES, 1 µg/ml leupeptin, and 1 µg/ml aprotinin. Lysates were centrifuged at 13,000g for 15 min. The resulting pellet was resuspended in sample buffer (62.5 mM Tris-HCl, 10% glycerol, 2% SDS, 0.1% bromphenol blue, and 50 mM dithiothreitol) and subjected to SDS-polyacrylamide gel (4–12%) electrophoresis. The gel was then transferred to a Hybond-P nylon membrane (GE Healthcare, Chalfont St. Giles, UK). This was followed by incubation with appropriately diluted antibodies to Bcl-2 (dilution 1:200; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), Bcl-xL (dilution 1:200; BD Biosciences Transduction Laboratories, Lexington, KY), Bax (dilution 1:200; Santa-Cruz Biotechnology, Inc.), Bad (dilution 1:200; BD Biosciences Transduction Laboratories), and β-actin (dilution 1:2000, Santa Cruz Biotechnology, Inc.). Membrane blocking, primary and secondary antibody incubations, and chemiluminescence reactions were conducted for each antibody individually, according to the manufacturer's protocols. The intensity of each band was measured using a Kodak Digital Science 1D Image Analysis System (Eastman Kodak, Rochester, NY).

Quantitative RT-PCR for Trophic Factor Synthesis. Primary cortical neuron cultures were treated with SDF-1 at different doses (0.01, 0.1, 1, and 10 µg/ml) for 24 h. Total RNA was isolated using RNeasy (QIAGEN, Dorking, Surrey, UK). The relative amount of target mRNA was determined by quantitative (Q)PCR using SYBR Green following the manufacturer's instructions (Roche Diagnostics, Mannheim, Germany), and specific primers were used as summarized in Table 1 (GenBank accession numbers AY057901 [GenBank] for BDNF, L19062 [GenBank] for GDNF, AY162414 [GenBank] for nerve growth factor, AF118263 [GenBank] for transforming growth factor-β, NM002006 for fibroblast growth factor-II, and AY750957 [GenBank] for VEGF). The relative expression levels of target mRNA were normalized against the control. GAPDH was used as an internal standard. The whole procedure of QRT-PCR using the ABI Prism 7900 Sequence Detection System (Applied Biosystems, Foster City, CA) has been described previously (Luo et al., 2004).

In Vivo Brain Ischemia/Reperfusion. Adult male Sprague-Dawley rats (weight <350 g; 8 weeks old) were used for this study. The rats were anesthetized with 0.4 g/kg i.p. chloral hydrate, and then they were subjected to right middle cerebral artery (MCA) ligation and bilateral common carotid artery (CCAs) clamping as described previously (Chen et al., 1986). In brief, the bilateral CCAs were clamped with nontraumatic arterial clips. Using a surgical microscope, a 2-x 2-mm craniotomy was drilled at the point where the zygoma fuses to the squamosal bone, and the right MCA was then ligated with a 10-0 nylon suture. Cortical blood flow was measured continuously with a laser Doppler flowmeter (PF-5010, Periflux system; Perimed AB, Stockholm, Sweden) in anesthetized animals. A photodetector probe (0.45 mm in diameter) was stereotaxically placed through a skull burr hole (1 mm in diameter) in the frontoparietal cortex (l.3 mm posterior, 2.8 mm lateral to the bregma, and l.0 mm below the dura). Then, experimental rats received injections stereotaxically with recombinant human SDF-1 (4 µgin4 µl of PBS per one cortical area) (ProSpec-Tany TechnoGene), vehicle (4 µl of PBS per one cortical area), or control protein (BSA, 4 µgin4 µl of PBS per one cortical area) at 30 min after MCA ligation through a 26-gauge Hamilton syringe (Hamilton Co., Reno, NV) into three cortical areas adjacent to the right MCA, 3.0 to 5.0 mm below the dura. The coordinates for these sites were l.0 to 2.0 mm anterior to the bregma and 2.5 to 3.0 mm lateral to the midline, 0.5 to l.5 mm posterior to the bregma and 3.5 to 4.0 mm lateral to the midline, and 3.0 to 4.0 mm posterior to the bregma and 4.5 to 5.0 mm lateral to the midline. The needle was retained for 5 min after each injection, and a small piece of bone wax was applied to the skull defect to prevent leakage of the injected solution. After 90 min of ischemia, the 10-0 suture on the MCA and arterial clips on CCAs were removed to allow for reperfusion. During recovery from anesthesia, body temperature was maintained at 37°C with a heat lamp.

Neurological Behavioral Measurements. Behavioral assessments (n = 16) were performed 3 days before cerebral ischemia, and 3 days after cerebral ischemia. The tests measured body asymmetry and locomotor activity as described previously (Chang et al., 2003). In brief, the baseline-test scores were recorded to normalize those taken after cerebral ischemia. The elevated body swing test was used to assess body asymmetry after MCA ligation, and the data were evaluated quantitatively as described previously (Borlongan et al., 1998). At first, animals were examined for lateral movement, upon being suspended by their tails. The frequency of initial head swing contra-lateral to the ischemic side was counted in 20 continuous tests, and it was normalized as follows: percentage of recovery = [1 - (lateral swings in 20 tests - 10)/10 x 100%]. For locomotor activity measurements, rats were subjected to VersaMax Animal Activity monitoring (Accuscan Instruments, Inc., Columbus, OH) for behavioral recording at night. The VersaMax Animal Activity monitoring contained 16 horizontal and eight vertical infrared sensors. The vertical sensors were situated 10 cm from the floor of the chamber. Motor activity was counted as the number of beams broken by a rat movement in the chamber. Some of data in the 42 movement parameters were calculated and were just picked up over 1 h for significance between SDF-1-treated and control group.

Triphenyltetrazolium Chloride Staining. Three days after reperfusion, rats (n = 16) were perfused intracardially with saline. The overall TTC staining procedure has been described previously (Wang et al., 2001). In brief, the brain tissue was removed, immersed in ice-cold saline for 5 min, and sliced into 2.0-mm-thick sections (seven slices per rat). The brain slices were incubated in 20g/l TTC (Research Organics, Cleveland, OH) dissolved in saline for 30 min at 37°C, and then transferred into a 5% formaldehyde solution for fixation. The area of infarction in each slice was measured with a digital scanner. The volume of infarction was obtained from the product of average slice thickness (2 mm) and the sum of infarcted areas in all brain slices examined. To minimize any artifacts induced by postischemic edema in the infarcted tissue, the volume of infarction was also calculated using a modified method, described by Lin et al. (1993). To measure the infarcted area in the right cortex, we subtracted the noninfarcted area in the right cortex from the total cortical area of the left hemisphere.

Western Blot Analysis of Antiapoptotic Protein. Anti- and proapoptotic protein expression (Bcl-2, BCL-xL, Bax, and Bad) in the right cortex and striatum region (n = 12) was also examined in the SDF-1-treated and control rats using Western blot analysis as described previously (Shyu et al., 2005). In brief, experimental animals were decapitated at 24 h after reperfusion with 90-min MCA ligation. Samples of ischemic cerebral cortex were taken from the peripheral region of infarcted brains (penumbra area) and striatum. Western blot analysis was performed on these samples. As a result, ischemic brain tissue was homogenized and lysed in a buffer containing 320 mM sucrose, 5 mM HEPES, 1 µg/ml leupeptin, and 1 µg/ml aprotinin. Lysates were centrifuged at 13,000g for 15 min. The resulting pellet was resuspended in sample buffer (62.5 mM Tris-HCl, 10% glycerol, 2% SDS, 0.1% bromphenol blue, and 50 mM DTT), and then it was subjected to SDS-polyacrylamide gel (4–12%) electrophoresis. Proteins were then transferred to a Hybond-P nylon membrane for incubation with appropriately diluted antibodies of Bcl-2 (dilution 1:200; Santa Cruz Biotechnology, Inc.), Bcl-xL (dilution 1:200; BD Biosciences Transduction Laboratories), Bax (dilution 1:200; Santa Cruz Biotechnology, Inc.), Bad (dilution 1:200; BD Biosciences Transduction Laboratories), and β-actin (dilution 1:2000; Santa Cruz Biotechnology, Inc.). Membrane blocking, primary and secondary antibody incubations, and chemiluminescence reactions were conducted for each antibody individually according to the manufacturer's protocol. The intensity of each band was measured using a Kodak Digital Science 1D Image Analysis System (Eastman Kodak). The ratio of band intensity in Western blots in comparison with the internal control was calculated.

Bromodeoxyuridine Labeling and BrdU Immunohistochemistry. BrdU, a thymidine analog that is incorporated into the DNA of dividing cells during S phase, was used for mitotic labeling (Sigma-Aldrich, St. Louis, MO). The labeling protocol has been described previously (Zhang et al., 2001). A pulse-labeling method (n = 16) was used to observe the time course of changes in proliferative cells in the brain after cerebral ischemia: experimental rats received i.p. injection with 50 mg/kg BrdU every 4 h for 12 h before sacrifice. A cumulative labeling method (n = 16) was used to examine the population of proliferative cells during 14 days of cerebral ischemia. In brief, rats received daily injections of 50 mg/kg i.p. BrdU for 14 consecutive days, starting the day after MCA ligation. These rats were sacrificed 14 days after the last injection. The BrdU-immunostaining procedure with a specific antibody against BrdU (1:400; Roche Diagnostics, Basel, Switzerland). In brief, experimental rats' brains were fixed by transcardial perfusion with saline, followed by perfusion and immersion in 4% paraformaldehyde. As a result, the brain samples were dehydrated in 30% sucrose. After brains had been frozen on dry ice, a series of adjacent 6-µm-thick sections were cut in the coronal plane with a cryostat, the sections were stained with H&E, and then they were observed by light microscopy (Nikon E600; Nikon, Tokyo, Japan). For BrdU immunostaining, DNA was first denatured by incubating each section in 1 N HCl at 55°C for 10 min and then in 5 µg/ml proteinase K Tris-HCl buffer at 37°C for 10 min. The immunostaining procedure was performed using the labeled streptavidin-biotin method (Dako LASB-2 kit, peroxidase; Dako). After deparaffinization, tissue slides were incubated with diluted antibodies to BrdU (for nuclear identification, 1:400; Roche Diagnostics) at room temperature for 1 h. After washing with Tris-buffered saline containing 0.1% Tween 20, the specimens were sequentially incubated for 10 to 30 min with biotinylated anti-rabbit and anti-mouse (1:200; R&D Systems) immunoglobulins and peroxidase-labeled streptavidin. Quantification of BrdU-immunoreactive cells was performed on cryostat sections, and immunoreactivity was counted digitally with a laser-scanning confocal microscope (LSM510; Carl Zeiss, Jena, Germany) via a computer imaging analysis system (Imaging Research, St. Catharines, ON, Canada). Cerebral cells with uniform nuclear BrdU immunostaining were counted as described previously (Kuhn et al., 1996).

Analysis of the Cellular Mechanism of BM-Derived Cell Mobilization and Homing. To verify the enhancement of the BM-derived cell mobilization and homing into brain by SDF-1 administration, a sample of bone marrow was removed from the long bones of adult male donor mice [green fluorescence protein (GFP) transgenic mice] as reported previously (Hill et al., 2004). Bilateral ends of the femur and tibia were penetrated using a syringe with a 25-gauge needle, allowing the marrow to be flushed out with sterile saline. Total marrow from one femur was diluted to 1 ml, and then it was strained through 30-µm SpectraMesh (Fisher Scientific, Suwanee, GA). Before bone marrow transplantation, the female recipient mice underwent whole body -irradiation with ¹³⁷Cs using a Gammacell 40 irradiator (MDS Nordion, Ottawa, ON, Canada). A total dose of 9 Gy (900 rads) was administered to ablate the whole bone marrow. Because high levels of radiation might cause significantly increased death rates during and immediately after cerebral ischemic surgery, the mice received rescuing bone marrow transplantations within 24 h of irradiation. Donor bone marrow was injected into the recipient animal's tail as a 100-µl cell suspension containing 1 to 1.5 x 10⁶ cells. At 4 to 8 weeks after transplantation, the mice (GFP-chimeric mice) were anesthetized with 0.3 g/kg i.p. chloral hydrate and subjected to right MCA ligation and bilateral CCA clamping for 60 min, as described above, and previously (Chen et al., 1986). Then, 30 min after MCA ligation, experimental mice were received injections stereotaxically with SDF-1 (1 µgin3 µl of saline per one cortical area) (ProSpec-Tany TechnoGene) or vehicle (3 µlof saline) through a 27-gauge Hamilton syringe as described above. Furthermore, BrdU labeling was also performed for each mouse using the above-mentioned procedure. In addition, measurement of the infarcted volume of each mouse was calculated by H&E staining using the method described previously (Giffard et al., 2005). In quantitative analysis of GFP⁺ cells of each group, the number of GFP⁺ cells located in the penumbric area of the right hemisphere was scored and summed per square millimeter using sample slides from five different positions based on the bregma (+1.6 mm, +0.5 mm, -1.3 mm, -3.5 mm, and -5.6 mm) as described previously (Kawada et al., 2006).

Laser-Scanning Confocal Microscopy for Double Immunofluorescence Analysis. To identify the expression of cell type-specific markers in BrdU⁺ cells, double immunofluorescence labeling was performed for glial fibrillary acidic protein (GFAP), a marker for astrocytes; von Willebrand factor (vWF), a marker for endothelial cells; and MAP-2 and neuronal nuclei (Neu-N), markers for neural cells. For BrdU immunostaining, DNA was first denatured by incubating brain sections in 1 N HCl at 55°C for 10 min and then in 5 µg/ml proteinase K Tris-HCl buffer at 37°C for 10 min. Sections were then rinsed with phosphate-buffered saline and incubated in 5% normal goat serum to block the background. Double immunofluorescence was conducted with a mouse monoclonal antibody against BrdU (1:200; Chemicon International, Temecula, CA) or the other specific markers: rabbit anti-GFAP (1:300; Chemicon International), rabbit anti-MAP-2 (1:300; Chemicon International), mouse anti-Nestin (1:50; Chemicon International), mouse anti-Neu-N (1:200; Chemicon International) conjugated with Alexa Fluor 488, rabbit anti-vWF (1:200; Dako North America Inc., Carpinteria, CA), CD11b (1:200; eBioscience, San Diego, CA) conjugated with phosphatidylethanolamine, CD45 (1:200; eBioscience) conjugated with phosphatidylethanolamine, CXCR4 (CD184, 1:100; Chemokine Therapeutic Corp., Vancouver, BC, Canada), Doublecortin (Dcx, 1:100; Santa Cruz Biotechnology, Inc.), and rat anti-mouse F4/80 (1:50; Serotec, Oxford, UK), left overnight at 4°C and incubated with Alexa Fluor-conjugated donkey anti-mouse 488, or anti-rabbit 555, or anti-rat 555 or 647 (1:500; Invitrogen Life Technologies) for 3 h at room temperature (Li et al., 2002). The tissue sections were analyzed with a LSM510 laser-scanning confocal microscope.

Evaluation of SDF-1-Induced Angiogenesis. To examine the blood vessels, cerebral microcirculation was analyzed by administering the fluorescent plasma marker (FITC-dextran; Sigma-Aldrich) intravenously to rats (n = 12), and then observing them with fluorescent microscopy (Axiovert 200M; Carl Zeiss), as described previously (Morris et al., 1999). In addition, to quantify the cerebral blood vessel density, rats (n = 12) were anesthetized with chloral hydrate and perfused with saline. Histological brain sections (6 µm) were stained with specific antibody to CD31 (1:100; BD Biosciences PharMingen, San Diego, CA), conjugated with cyanine-3 (1:500; Jackson ImmunoResearch Laboratories Inc., West Grove, PA), and the number of blood vessels determined as described previously (Tanaka et al., 2003).

Measurement of Reactive Cerebral Blood Flow. Experimental rats (n = 12) were positioned in a stereotaxic frame, and baseline local cortical blood flow (bCBF) was measured every day for 5 days after cerebral ischemia with a laser Doppler flowmeter (LDF monitor; Moore Instrument, Axminster, Devon, UK) in an anesthetized state (chloral hydrate) as described previously with modification (Démolis et al., 2000). In brief, the probe of the LDF monitor was positioned unilaterally over the frontoparietal cortex (l.3 mm posterior and 2.8 mm lateral to the bregma, and l.0 mm below the dura). The reactive cerebral blood flow (rCBF) was examined after i.p. injection of 50 mg/kg acetazolamide (Diamox; Lederle, Wayne, NJ), and rCBF was defined as percentage of changes of bCBF.

Blood Pressure, Heart Rate, Blood Glucose, and Blood Gas Measurement. Physiological parameters were measured at 1 h after SDF-1 or vehicle treatment in 14 rats, following a procedure described previously (Lin et al., 1999).

Statistical Analysis. All measurements in this study were performed blindly. Results were expressed as mean ± S.E.M. The behavioral scores have been evaluated for normality. Student's t tests were used to evaluate mean differences between the control and the treated group. The data lacking normal distribution were analyzed by a by a nonparametric analysis of variance (Kruskal-Wallis test). A value of P < 0.01 was taken as significant.

	Results

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Neuroprotective Effect of SDF-1 in PCC. To evaluate the neuroprotectivity of SDF-1 in vitro, LDH activity and neuronal survival (positive MAP-2 immunoreactivity) under H₂O₂-induced neurotoxic conditions were measured in PCC with or without SDF-1 treatment (Fig. 1, A–D). Treatment with 0.1 µg/ml SDF-1 before H₂O₂ administration significantly reduced LDH activity in cultures exposed to 10^-4 or 10^-5 MH₂O₂ in comparison with the control (P < 0.01) (Fig. 1E).

View larger version (86K): [in this window] [in a new window]   Fig. 1. Pretreatment with SDF-1 attenuates H2O2-induced toxicity in PCC. A to D, representative morphology (MAP-2 immunostaining) of PCC under various conditions (control, SDF-1,H2O2 alone, and H2O2 + SDF-1). E, degree of cell death assessed by LDH activity in media of PCC with or without SDF-1 (0.1 µg/ml), before exposure to various doses of H2O2 (0, 10-5, and 10-4 M). H2O2 dose-dependently increased LDH activity. SDF-1 pretreatment attenuated H2O2-induced cell death significantly. F, density of MAP-2-immunoreactive cells of PCC was significantly reduced by H2O2, but loss of MAP-2+ cells was attenuated by pretreatment with SDF-1. G, in the caspase-3 activity measurement, had primary cortical cells treated with H2O2 and/or SDF-1 for 24 h significantly reduced caspase-3 activity compared with the control cells. H, significant reduction of caspase-3-positive immunofluorescent cells in the SDF-1-treated group compared with controls. I, in Western blot analysis and quantitative measurement, increased expression of antiapoptotic proteins such as Bcl-2 was found after SDF-1 pretreatment. J, BDNF, VEGF, and GDNF mRNA related to GAPDH mRNA, in response to SDF-1, as determined by QRT-PCR. Data are expressed as mean ± S.E.M. *, P < 0.01 versus control. Scale bar, 40 µm.

To examine the neuroprotective effect of SDF-1 by blocking apoptotic pathways, caspase-3 activity and caspase-3 immunofluorescence was measures under H₂O₂-induced neurotoxic conditions in PCC with or without SDF-1 treatment. The results showed that PCC treated with H₂O₂ and/or SDF-1 for 24 h had significantly reduced caspase-3 activity as measured by the fluorometric method in comparison with the control cells (P < 0.01) (Fig. 1G). In addition, there was a significant reduction of caspase-3-positive immunofluorescent cells in the SDF-1-treated group compared with the control group (Fig. 1H).

The molecular mechanism of this neuroprotective antiapoptotic effect of SDF-1 was further examined by analyzing the expression of a variety of apoptosis-linked proteins using Western blot analysis. Expression of antiapoptotic protein Bcl-2 was significantly up-regulated when PCC were exposed to H₂O₂ and SDF-1 pretreatment (compared with an internal protein control such as actin) (P < 0.01) (Fig. 1I).

The possible effect of SDF-1 on neurotrophic factor synthesis was studied in PCC by comparing the expression of GDNF, VEGF, and BDNF relative to GAPDH. SDF-1 led to a significant and dose-dependent increase at 6 h in all three of the tested neurotrophins, by QRT-PCR. Relative expression compared with GADPH increased to a maximum of approximately 2-fold (Fig. 1J).

Intracerebral SDF-1 Administration Improves Neurological Behavior after Cerebral Ischemia. To evaluate the neuroprotective effect of intracerebral administration of SDF-1 in rats receiving cerebral ischemia, body asymmetry trials and locomotor activity tests were carried out to assess the neurological deficit in SDF-1-treated (n = 10) and control rats (n = 10). Rats intracerebrally treated with SDF-1 at 30 min after cerebral ischemia exhibited significantly less body asymmetry 3 days after cerebral ischemia compared with the controls (P < 0.01) (Fig. 2B). The measured locomotor activities such as vertical activity, vertical movement time, and number of vertical movements significantly increased 3 days after cerebral ischemia in rats receiving SDF-1 treatment compared with control animals (P < 0.01) (Fig. 2, C–E).

View larger version (54K): [in this window] [in a new window]   Fig. 2. Intracerebral administration of SDF-1 in cerebral ischemic rats improves neurological dysfunction and markedly reduces cortical infarction induced by MCA ligation in rats. A, schematic representation of the investigation of chronic cerebral ischemia with three therapeutic protocols. Cerebral ischemia was induced by three-vessel ligation in each experimental rat on day 0. B, rats receiving intracerebral SDF-1 (12 µg) at 30 min after cerebral ischemia showed significantly reduced body asymmetry examined by the elevated body swing test after MCA ligation, compared with control rats 3 days after cerebral ischemia. C to E, rats receiving SDF-1 treatment showed significantly increased locomotor activity (including vertical activity, number of vertical movements, and vertical movement time), compared with the controls. F, representative TTC staining of the infracted brain. The white area of TTC staining indicates the infarcted zone in the right cerebral cortex sequential (brain slices of SDF-1-treated, vehicle control, and BSA-treated rats are arranged in vertical rows). G, volume of infarction was significantly reduced in rats treated with SDF-1 30 min after MCA ligation compared with the BSA-treated and control group. Data are expressed as mean ± S.E.M. *, P < 0.01 versus control. I, ischemic; SO, sham-operated.

Intracerebral Administration of SDF-1 Reduces Infarct Volume after Cerebral Infarction.

SDF-1 Protects Neurons from Cerebral Ischemic Damage. To verify the neuroprotective effect of SDF-1 in repairing neuronal injury after cerebral ischemia (Fig. 3, A and B), we analyzed ischemic brain tissues for neuronal survival using specific antibodies that recognized neuron-specific proteins (Neu-N conjugated with FITC and MAP-2 conjugated with cyanine-3) (Fig. 3, C–N). In the penumbric area surrounding the ischemic core, the number of cells positive for Neu-N and MAP-2 were significantly increased in SDF-1-treated rats (n = 10) compared with the controls (n = 10) (P < 0.01) (Fig. 3, O and P). Ischemic brain tissue from animals that received injections with the vehicle (n = 8) contained fewer cells positive for Neu-N (Fig. 3, G and H) or MAP-2 (Fig. 3, M and N) in both the penumbric area and the ischemic core compared with the SDF-1-treated rats (Fig. 3, D and E and J and K, respectively).

View larger version (79K): [in this window] [in a new window]   Fig. 3. Intracerebral administration of SDF-1 at 30 min after cerebral ischemia rescues neuronal cells positive for MAP-2 and Neu-N from ischemic injury and diminishes activated caspase-3 immunoreactivity. A and B, representative coronary slice taken 3 days after cerebral ischemia from animals that received injections with SDF-1 and vehicle control, respectively, and stained with TTC. C and E, sections derived from the brain slice shown in A were immunostained to identify Neu-N-containing neurons (circle in C, square in D, and triangle in E). F to H, sections derived from the brain slice shown in B were immunostained to identify Neu-N-containing neurons (circle in F, square in G, triangle in H). I to K, sections from the brain slice shown in A were processed to identify MAP-2-containing neurons (circle in I, square in J, and triangle in K). L to N, sections from the same brain slice shown in B were immunostained to identify MAP-2-containing neurons (circle in L, square in M, and triangle in N). O and P, quantitative analysis revealed that the number of Neu-N and MAP-2-immunoreactive cells in the penumbra area (D and J) was significantly increased in SDF-1-treated rats compared with control rats (G and M). Q, Western blot analysis revealed significantly increased expression of Bcl-2 and Bcl-xL in the SDF-1 (I) and SDF-1 (SO) rats at 24 h after cerebral ischemia compared with control (I) and control (SO) rats. Data are expressed as mean ± S.E.M. *, P < 0.01. Scale bar, 20 µm.

SDF-1 Rescues Neural Tissue by Up-Regulation of Antiapoptotic Proteins.

SDF-1 Stimulates Stem Cell Mobilization and Homing to the Ischemic Brain. To determine whether stem cells homed in on the injured brain tissue of SDF-1-treated rats, BrdU labeling was used to follow the engraftment of BM-derived cells to the brain 14 days after cerebral ischemia. A cumulative labeling method was used to examine the population of proliferative cells during 14 days of cerebral ischemia. In the SDF-1-treated rats (n = 10), cumulative BrdU labeling revealed a few BrdU-immunoreactive cells in the ipsilateral cortex near the infarcted boundary (Fig. 4, A–C) and subventricular region of the ischemic hemisphere (Fig. 4, D and F). BrdU-immunoreactive cells were also found around the lumen of blood vessels of varying calibers in the perivascular portion of the ischemic hemisphere (Fig. 4, G–I). A pulse-labeling method was used to observe the time course of changes in proliferative cells in the brain after cerebral ischemia. The result of pulse-labeling experiments showed that BrdU-immunoreactive cells significantly increased in SDF-1-treated rats (n = 10) compared with those of saline control rats (n = 10) (P < 0.01) (Fig. 4J).

View larger version (70K): [in this window] [in a new window]   Fig. 4. Immunohistochemical staining for BrdU and analysis of the cellular mechanism of stem cell homing. Rats treated with SDF-1 30 min after cerebral ischemia, few BrdU-immunoreactive cells were detected in the ipsilateral cortex near the infarction boundary (A–C; black arrows) and subventricular area (D–F; black arrows). G to I, BrdU-immunoreactive cells were also found around the blood vessels in the ipsilateral cortex (black arrows). J, quantitative analysis revealed that the number of BrdU-immunoreactive cells in the ipsilateral hemisphere of SDF-1 (I) and SDF-1 (SO) rats significantly increased 3 days after MCA ligation compared with control (I) and control (SO) rats. K, representative gross anatomy of the infarcted area (black arrowheads) of a GFP-chimeric mouse brain is shown for each of the SDF-1-treated and control mouse (top left). Prominent homing stemcells (green) were found in the SDF-1-treated and untreated mice (bottom left), and quantitative measurement of the GFP-positive cells showed there were significantly more in SDF-1-treated than control mice (right). L, in colocalization studies, GFP+ cells coexpressed with specific markers antibodies against CD11b, CD45, GFAP, and vWF. M, histology (H&E) of an infarcted mouse brain (black arrow shows infarcted area). Infarct volume calculated in H&E staining is shown for SDF-1-treated and control mice. Data are expressed as mean ± S.E.M. *, P < 0.01 versus control. Scale bar, 50 µm.

SDF-1 and Ischemic Tissue Promote BM-Derived Cell Mobilization/Homing Demonstrated by GFP-Chimeric Mice Model.

SDF-1 Enhances Neurogenesis in Vivo. To determine whether mobilized BM-derived cells differentiated into neuronal, glial, or endothelial cells at ischemic sites in the brains of SDF-1-treated rats 14 days after cerebral ischemia, double staining immunohistochemistry was performed on each brain slice from SDF-1-treated and control rats. Some BrdU⁺ cells colocalized with antibodies for Nestin, Neu-N, MAP-2, and GFAP in SDF-1-treated brains (Fig. 5). Ischemic cortical areas of SDF-1-treated rats (n = 8) revealed an increase in BrdU⁺ cells coexpressing the neuronal phenotypes of Neu-N⁺, Nestin⁺, and MAP-2⁺ cells (Fig. 5, A–I) and the glial phenotype of GFAP⁺ cells (Fig. 5, J–L) compared with saline-treated rats (n = 8). In total, the number of BrdU⁺ cells costained with Nestin, Neu-N, MAP-2, and GFAP were significantly increased in SDF-1-treated rat brains compared with controls (Fig. 5M).

View larger version (31K): [in this window] [in a new window]   Fig. 5. Double immunofluorescent laser-scanning confocal microscopy. A to I, ischemic cortical area () of SDF-1-treated rats revealed an increase in BrdU+ cells coexpressing the neuronal phenotypes of Neu-N+, Nestin+, and MAP-2+ cells (white arrows) (J–L) and the glial phenotype of GFAP+ cells (white arrows). M, quantitative results showed the number of BrdU with Neu-N+-, Nestin+-, MAP-2+-, and GFAP-coimmunoreactive cells significantly increased in SDF-1-treated rats compared with controls. N to P, compared with saline-treated rats, some BrdU+ cells showing specific markers of stem cell homing (CXCR4; white arrow) and migration (Dcx; white arrow) (Q–S) were also found around the penumbric area of the ischemic hemispheres in SDF-1-treated rats. Data are expressed as mean ± S.E.M. *, P < 0.01 versus control. Scale bar, 50 µm.

SDF-1 Induces Angiogenesis in Vivo. To determine whether SDF-1 could induce angiogenesis through homing BM-derived cells that differentiated into vascular-endothelial cells at ischemic sites, double-staining immunohistochemistry, FITC-dextran perfusion studies, and blood vessel density assays were performed on each brain slice from SDF-1-treated and control rats. We found several BrdU⁺ cells showing vascular phenotypes (vWF⁺ cells) around the perivascular and endothelial regions (Fig. 6, A–C) of the ischemic hemispheres of SDF-1-treated rats. Visual inspection indicated that treatment with SDF-1 (n = 8) considerably enhanced cerebral microvascular perfusion with FITC-dextran compared with the controls (n = 8) (Fig. 6, D and E). Quantitative measurement of blood vessel density examined by CD31 immunoreactivity (Fig. 6, F and G) showed that in ischemic rats treated with SDF-1 (n = 8) the amount of neovasculature in the penumbric area and hippocampus significantly increased compared with that of controls (n = 8) (P < 0.01) (Fig. 6H).

View larger version (25K): [in this window] [in a new window]   Fig. 6. SDF-1 treatment enhanced angiogenesis in the ipsilateral hemisphere. A to C, double-immunofluorescent studies show some BrdU+ cells with vascular phenotypes (vWF+ cells) (white arrows) around the perivascular and endothelial regions of the ischemic hemispheres of SDF-1-treated rats. D and E, to assess cerebral microvascular perfusion, FITC-dextran was administrated intravenously to evaluate the microvascular pattern of SDF-1-treated and control rats. F and G, calculating the cerebral blood vessel density stained by CD31 immunoreactivity (white arrows), SDF-1 (H; I) and SDF-1 (H; SO) showed a significant increase in the amount of neovasculature in the penumbric area and hippocampus in comparison with that of controls (I) and controls (SO). I, analysis of rCBF using LDF monitoring with Diamox injection showed a significant increase in rCBF of the brain in the SDF-1 (I) and SDF-1 (SO) rats compared with controls (I) and controls (SO). Data are expressed as mean ± S.E.M. *, P < 0.01 versus control. Scale bar, 50 µm.

SDF-1 Facilitates rCBF in the Ischemic Brain.

SDF-1 Treatment Does Not Influence Physiological Parameters. To demonstrate that the neuroprotective effect of SDF-1 did not occur as a result of changes to other physiological parameters, systemic physiological parameters were analyzed in 14 experimental rats. Compared with vehicle control (n = 7), intracerebral administration of SDF-1 (n = 7) did not alter systemic blood pressure, blood gases, blood glucose, or serum electrolyte levels. These data are summarized in Table 2.

View this table: [in this window] [in a new window]   TABLE 2 Physiological parameters were not altered by SDF-1 treatment

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
This study indicates that SDF-1 may play a neuroprotective role in the brain of cerebral ischemic rats by diminishing LDH activity and enhancing neurotrophic factor synthesis, as shown in primary cortical cultures under H₂O₂ stress. In addition, we also demonstrated that SDF-1 treatment 30 min after cerebral ischemia significantly enhanced neural repair by reducing infarct volume and improving recovery of neurological dysfunction in rats suffering from cerebral ischemia. We found that SDF-1 increased the mobilization of BM-derived cells to damaged areas of the brain, resulting in stimulation of cell repair in the penumbra of the ischemic brain. In addition, the increased preservation of cells immunoreactive for MAP-2 and Neu-N and the promotion of BM-derived cell differentiation in the penumbra of the ischemic brain suggest that SDF-1 could exert a neuroplastic effect on neurons in adult rat brains after stroke.

The SDF-1 chemokines constitute a family of small secreted proteins (8–12 kDa) that are known to cause activation and migration of leukocytes (Murdoch and Finn, 2000). The SDF-1 receptor CXCR4 is expressed in a variety of developmental neuronal tissues, including sympathetic ganglia, dorsal root sensory ganglia, midbrain, and the granular cell layer of the cerebellum (McGrath et al., 1999). In addition, there is also some evidence suggesting that increased expression of SDF-1 and CXCR4 is found in cases of neuropathogenesis induced by many forms of injury, including trauma, stroke, and inflammation (Hill et al., 2004). Therefore, SDF-1 might not only provide generalized trophic support to both embryonic and mature neurons but also help support neurons damaged by injury or inflammation. Furthermore, in a recent report, SDF-1 was demonstrated to play a critical role as a guidance molecule regulating axon outgrowth in cultured cerebellar granular neurons (Arakawa et al., 2003). In this study, we demonstrate that SDF-1 reduces LDH activity and increases the number of positive MAP-2-immunoreactive neurons after H₂O₂ toxicity in primary cortical neuron cultures. Moreover, SDF-1 exerted its neuronal survival effect by increasing the synthesis or release of many neurotrophic factors to protect the neurons from neurotoxicity.

In addition to up-regulation of neurotrophic factors, our study also demonstrates that SDF-1 could induce an increase in expression of VEGF in PCC. SDF-1 has previously been shown to induce increased VEGF expression in microvascular endothelial cells. Neuhaus et al. (2003) have also demonstrated that SDF-1 up-regulated the expression of VEGF and Erg-1 through induction of extracellular signal-regulated kinase 1/2 signaling, and SDF-1 could further enhance VEGF-induced proliferation in human umbilical vein endothelial cell cells. In addition, because an interaction of SDF-1/CXCR4 plays a significant role in extracellular matrix-dependent endothelial tube formation in vitro, SDF-1 and CXCR4 are both essential regulators of endothelial cell morphogenesis and angiogenesis (Salvucci et al., 2002). In this study, homing BM-derived cells were seen to replace some parts of injured blood vessels in the penumbric area of cerebral ischemic rats by double immunofluorescence analysis. In concert with the findings of FITC-dextran perfused cerebral vessels and CD31 staining, significantly increased vascular density was found to enhance the functional local cerebral blood flow (using laser Doppler study) in the ischemic cortex of SDF-1-treated rats. This result indicated that functional "new" blood vessels were formed that improved tissue perfusion. Therefore, we speculate that the improved neurological function in cerebral ischemic rats after early intracerebral administration of SDF-1 might be partially caused by an angiogenic effect.

In a previous report, the pathogenic mechanism of neurotoxicity induced by HIV-1 was demonstrated to be mediated by intracellular CXCR4 signaling. Although interaction of SDF-1 and gp-120 with microglia and astrocytes have been demonstrated to be indirectly cytotoxic to neurons of rat hippocampal cultures through tumor necrosis factor- and glutamate released from astrocytes (Bezzi et al., 2001), it has widely been suggested that SDF-1 may promote neuronal survival through CXCR4 signaling, thereby inducing both a G_i protein-linked decrease in cAMP and also a down-regulation of caspase-3 activation. These data suggest that SDF-1 could protect neurons from HIV-1 neurotoxicity via inhibition of caspase-3 activation. However, in cerebral ischemic models proapoptotic mechanisms are activated during ischemic-reperfusion to facilitate caspase-3-mediated cell death (Sasaki et al., 2000). In this study, we found that SDF-1 disrupts the downstream caspase-3 apoptotic signal in the ischemic penumbra of cerebral ischemic rats, resulting in cortical neuronal protection and a diminished infarct volume. In summary, an antiapoptotic effect through inhibition of caspase-3 activation might be another mechanism exerted by SDF-1 to rescue ischemic neurons.

However, because SDF-1 and VEGF would increase the vascular permeability causing tissue edema and possible aberrant neovascularization, in clinical applications therapeutic dosages of SDF-1 need to be adjusted carefully (Brooks et al., 2004). In summary, we have shown that SDF-1 administered to cerebral ischemic rats can instigate new neuronal and vascular formation within infarcted brain regions, attenuating tissue damage and leading to a reduction in infarct volume and improved neurological function. We propose that SDF-1 administration mobilizes autologous BM-derived cells into circulation and that their translocation into the ischemic brain to reduce the numbers of injured neurons over the peri-infarcted region. We believe that the SDF-1 therapeutic protocol presented here holds great promise for the development of new therapeutic strategies, including gene therapy and small molecule drugs, which can improve tissue regeneration and repair postcerebral ischemia to clinically significant levels.

Stumm et al. (2002) recently demonstrated that focal cerebral ischemia causes an increase in SDF-1 expression in regions adjacent to the infarcted area. This lesion-induced up-regulation of endothelial SDF-1 (Stumm et al., 2002) together with the appearance of increased CXCR4 expression in the ischemic hemisphere 4 h after ischemia, observed in our previous study (Shyu et al., 2004) indicates that cerebroendothelial SDF-1 could be a chemoattractant for peripheral blood cells. To further verify the homing effect of SDF-1, we injected recombinant SDF-1 protein into the ischemic brain of GFP-chimeric mice in this study. Our results, which revealed BM-derived cell homing to the injured brain in cerebral ischemic rats, support the hypothesis of a potential role for SDF-1/CXCR4 in adaptive early localized postischemic inflammation and later reorganization of the infarcted area. By attracting HSCs to the ischemic region, an SDF-1/CXCR4 interaction may be directly involved in vascular remodeling, angiogenesis, and neurogenesis, thereby alleviating stroke symptoms. This chemotaxis may take place in a manner similar to the migration of leukocytes into damaged or inflamed tissues. In addition, HSCs migrating to the ischemic hemisphere could create local chemical gradients and/or localized chemokine accumulation, dictating a directional response in endothelial, neuronal, and glial progenitor cells (Yamaguchi et al., 2003). In addition to inducing HSC migration to ischemic regions, SDF-1 has also been shown to extend the survival of cultured CD34⁺ cells (Yamaguchi et al., 2003) and to regulate endothelial cell branching morphogenesis. Taken together, we hypothesize that plasma levels of SDF-1, released from damaged tissues, may provide a host defense signal, which may in turn attract mobilizing HSCs to repair the damaged tissue. This and other studies (Kucia et al., 2004) also provide evidence that the ultimate degree of physiological improvement of specific organ function in any kind of injury is dependent on the recruitment of sufficient HSCs to the damaged area at an early stage after tissue injury.

	Acknowledgements

 
We thank Dr. Harry Wilson and M. Loney of Academia Sinica for critical reading of this manuscript.

	Footnotes

 
This work was supported in part by Chen-Han Foundation for Education, Academia Sinica Grant 94M003 and National Science Council Grant NSC95-2745-B-001-001-MY3.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.107.127746.

ABBREVIATIONS: SDF, stromal cell-derived factor; HSC, hematopoietic stem cell; CXCR4, CXC receptor 4; BM, bone marrow; PCC, primary cortical cells; BSA, bovine serum albumin; LDH, lactate dehydrogenase; MAP, microtubule-associated protein; PBS, phosphate-buffered saline; QRT-PCR, quantitative reverse transcription-polymerase chain reaction; BDNF, brain-derived neurotrophic factor; GDNF, glial cell-derived neurotrophic factor; VEGF, vascular endothelial growth factor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MCA, middle cerebral artery; CCA, common carotid artery; TCC, triphenyltetrazolium chloride; BrdU, bromodeoxyuridine; H&E, hematoxylin and eosin; GFP, green fluorescent protein; GFAP, glial fibrillary acidic protein; vWF, von Willebrand factor; Neu-N, neuronal nuclei; Dcx, Doublecortin; rCBF, reactive cerebral blood flow; bCBF, baseline local cortical blood flow; I, ischemic; SO, sham-operated.

Address correspondence to: Dr. Hung Li, Institute of Molecular Biology, Academia Sinica, 128 Sec. 2, Academia Rd., Nankang, Taipei 11529, Taiwan. E-mail: hungli{at}ccvax.sinica.edu.tw

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