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作者：Pr Thoreda,c, Andreas Arvidssona,c, Emanuele Caccib,c, Henrik Ahleniusb,c, Therse Kallurb,c, Vladimer Darsaliab,c, Christine T. Ekdahla,c, Zaal Kokaiab,c, Olle Lindvalla,c作者单位：a Laboratory of Neurogenesis and Cell Therapy, Section of Restorative Neurology, Wallenberg Neuroscience Center, University Hospital, Lund, Sweden;b Laboratory of Neural Stem Cell Biology, Section of Restorative Neurology, Stem Cell Institute, University Hospital, Lund, Sweden;c Lund Strategic Resea
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9 y7 K# A; T0 n8 M7 h! t$ H 【摘要】* t6 d" E8 D4 q! ^! W
Neural stem cells in the subventricular zone of adult rodents produce new striatal neurons that may replace those that have died after stroke; however, the neurogenic response has been considered acute and transient, yielding only small numbers of neurons. In contrast, we show herein that striatal neuroblasts are generated without decline at least for 4 months after stroke in adult rats. Neuroblasts formed early or late after stroke either differentiate into mature neurons, which survive for several months, or die through caspase-mediated apoptosis. The directed migration of the new neurons toward the ischemic damage is regulated by stromal cell-derived factor-1 and its receptor CXCR4. These results show that endogenous neural stem cells continuously supply the injured adult brain with new neurons, which suggests novel self-repair strategies to improve recovery after stroke.
' X8 ~ E P$ p+ `0 { 【关键词】 Stem cells Neurogenesis Striatum Stroke! f4 u" w0 {+ a4 O+ e
INTRODUCTION% n% v2 ]4 K' J: M# h8 F% V6 {2 ~
5 W: c, d" h* iStroke is a leading cause of chronic disability in humans for which effective treatment is lacking. Recent experimental findings raise the possibility that functional improvement after stroke may be induced through neuronal replacement by endogenous neural stem cells (NSCs) in the subventricular zone (SVZ). Ischemic stroke caused by middle cerebral artery occlusion (MCAO) triggers increased cell proliferation in the rat SVZ .
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X" Q% t& R8 A* cWe show herein that stroke-induced neurogenesis is extensive and long-lasting, with continuous production of mature striatal neurons for several months after the insult. We also find that stromal cell¨C derived factor-1 (SDF-1)/CXCR4 signaling regulates the directed migration of new neurons to the injured area.
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MATERIALS AND METHODS
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3 e" W/ u% K, R# _! E+ w: T* EInduction of Stroke and 5-Bromo-2'-Deoxyuridine Labeling/ T$ s& w7 V5 J5 | e X
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Under halothane anesthesia, the middle cerebral artery of artificially ventilated male Wistar rats was occluded with a filament inserted through the common carotid artery . After 30 minutes or 2 hours, the filament was withdrawn. For sham surgery, the filament was advanced a few millimeters inside the internal carotid artery. Physiological parameters were kept within a predetermined range. Intraperitoneal injections of 5-bromo-2'-deoxyuridine (BrdU; 50 mg/kg, Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) were given twice daily during weeks 1¨C2, 5¨C6, or 7¨C8 after surgery. In one experiment, three BrdU injections with 2-hour intervals were given 4 days or 6 weeks after MCAO, and the animals were killed 2 hours thereafter.# Z1 u' ?7 |# E7 w8 @8 ^1 [. `+ w
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Caspase Inhibitor and CXCR4 Antagonist Treatments* s% g, `$ o2 L7 m. P& }4 v
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Daily intraventricular infusions of a caspase inhibitor cocktail, comprising equal portions of a multicaspase, a caspase 3, and a caspase 9 inhibitor, were given over 12 days, starting on day 3 after MCAO ) or PBS for 2 weeks into the lateral ventricle.
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In Vitro Assays# }0 b: [" Q: D+ h+ {
3 X% W3 r/ _* c) ?Neurospheres were grown in Dulbecco¡¯s modified Eagle¡¯s medium(DMEM)/F12(Gibco-BRL,Gaithersburg,MD,http://www.gibcobrl.com) supplemented with B27 containing 20 ng/ml epidermal growth factor and 10 ng/ml basic fibroblast growth factor (R&D Systems Inc., Minneapolis, http://www.rndsystems.com) . After trituration of SVZ tissue or primary neurospheres, cells were plated in 24-well plates for 7 days, and the number and diameter of spheres were quantified.5 N- X2 F4 U j! n9 j
& L, F/ J9 P8 F2 W* C) vFor studies of phosphorylation, adult rat SVZ cells were plated in 96-well plates. After 5 days in vitro, serum and growth factors were withdrawn for 18 hours. Cells were treated with the basal medium either alone or containing 500 ng/ml SDF-1 (R&D Systems) for 10 minutes. Phosphorylation of extracellular signal-related kinase (ERK)-1/2 was assessed using FACE enzyme-linked immunosorbent assay kits (Active Motif, Rixensart, Belgium, http://www.activemotif.com).* r- v+ D% f t5 t) {
6 O& T& @1 ?6 _3 B9 W4 N3 p; hFor the migration assay, neurospheres from the SVZ were distributed on a poly-L-lysine coated polyvinylpyrrolidine-free polycarbonate filter in the upper section of a 96-well microchemotaxis chamber (Neuro Probe, Gaithersburg, MD, http://www.neuroprobe.com). SDF-1 (100 or 500 ng/ml) was added to either the lower wells, upper wells, or both. Basal medium without SDF-1 served as control. Neurospheres were allowed to migrate for 12 hours. The filter was fixed in 4% phosphate-buffered paraformaldehyde (PFA) and stained with Hoechst 33342 (Molecular Probes Inc., Eugene, OR, http://probes.invitrogen.com), and migrated cells were counted using an epifluorescence microscope., E; d6 }4 n( E/ d
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Immunohistochemistry# s3 K: [7 }6 C4 Z' r
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After transcardial perfusion with 4% ice-cold PFA, brains were postfixed overnight in PFA and sectioned coronally at 30 µm on dry ice. Prior to staining using diaminobenzidine (DAB), free-floating sections were quenched for 20 minutes in 3% hydrogen peroxide and 10% methanol, and prior to staining for BrdU, sections were incubated in 1 M HCl at 65¡ãC for 10 minutes and at 37¡ãC for 20 minutes. Following preincubation with the appropriate normal sera, sections were incubated for 36 hours at 4¡ãC with either two of the following antibodies¡ªrat anti-BrdU (1:100; Harlan Sera-Lab, Loughborough, U.K., http://www.harlaneurope.com), goat anti-doublecortin (Dcx) antibody (1:400; Santa Cruz Biotechnology Inc., Santa Cruz, CA, http://www.scbt.com), guinea-pig anti-Dcx (1:3,000; Abcam, Cambridge, U.K., http://www.abcam.com), mouse anti-neuronal nuclei (NeuN; 1:100; Chemicon, Temecula, CA, http://www.chemicon.com), goat anti-CXCR4 (recognizes SDF-1 and SDF-1ß, 1:100; Santa Cruz Biotechnology), goat anti-SDF-1 (recognizes SDF-1 and SDF-1ß, 1:100; Santa Cruz Biotechnology), mouse anti-ED1 (1:200; SeroTec, Ltd., Oslo, Norway, http://www.serotec.com), or mouse glial fibrillary acidic protein (GFAP; 1:1,000; Sigma-Aldrich)¡ªor overnight with mouse anti-NeuN only, in normal sera. Stainings were visualized by incubation for 2 hours with Cy3-conjugated (1:200; Jackson ImmunoResearch Laboratories, West Grove, PA, http://www.jacksonimmuno.com) and biotinylated secondary antibodies (1:200; Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com) followed by Alexa 488-conjugated streptavidin (1:200; Molecular Probes) for 2 hours for double stainings, or avidin-biotin-peroxidase complex for 1 hour followed by treatment with DAB (0.5 mg/ml) and hydrogen peroxide for single staining. Sections were then mounted on glass slides and coverslipped. For caspase-cleaved poly(ADP-ribose) polymerase (cPARP)/Dcx double staining, sections were stained for Dcx as above and mounted on glass slides before incubation overnight with rabbit anti-rat cPARP antibody (1:20; Cell Signaling Technology, Beverly, MA, http://www.cellsignal.com) in 3% bovine serum albumin at 4¡ãC. For in vitro stainings, neurospheres were plated on poly-L-lysine-coated four-well chamber slides and differentiated for 7 days in basal medium containing 1% fetal bovine serum (differentiation medium). Cells were fixed in 4% PFA at room temperature and stained using mouse anti-ßIII-tubulin antibody (1:350; Sigma-Aldrich). For CXCR4 staining, SVZ-derived neurospheres or primary cells were plated overnight in expansion medium, fixed, and stained with goat anti-CXCR4 antibody (1:100). Both stainings were counterstained with Hoechst 33342.
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Microscopical Analysis
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Cells double-labeled with BrdU and Dcx or NeuN were counted using an epifluorescence microscope with 40x objective (Olympus A/S, Glostrup, Denmark, http://www2.olympus.dk). The accuracy of double labeling was validated using a confocal laser-scanning microscope (Leica, Heerbrugg, Switzerland, http://www.leica.com). Dcx , Dcx /BrdU , and NeuN /BrdU cells were counted, using a 0.25-mm-wide quadratic grid, in the entire striatum in four sections, at 1.60, 1.00, 0.48, and ¨C0.26 mm from bregma, the most medial column lining the SVZ. Striatal volumes were measured using stereological equipment in the same sections. The size of the SVZ was measured in sections from the same rostrocaudal levels stained with NeuN and cresyl violet. The SVZ was delineated by cells stained with cresyl violet only. BrdU cells in the SVZ were counted in three sections ( 1.60, 0.70, and ¨C0.26 mm from bregma) with the optical fractionator method on stereological equipment.) ]6 l, Q6 S; |$ S3 X$ I
* j8 l0 _: p0 u7 q- Y4 ], K% L( d, JBehavioral Tests* b/ a% Z( G6 x0 F- X
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Behavioral tests were performed 24 hours before (control) and 48 hours, 4 weeks, and 16 weeks after 30-minute MCAO. In the modified horizontal ladder walking test .
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z/ n& h& [' l, ]Animal Groups and Statistical Analysis7 }4 V% c0 s# \5 F& |/ i
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Quantification of the number of Dcx , Dcx /BrdU , and NeuN /BrdU cells 2, 6, and 16 weeks after stroke, and cell migration and SVZ volume, was performed on the same groups of animals, whereas cell numbers at 8 and 12 weeks were taken from the vehicle-treated animals in the AMD 3100 experiment. Data in the caspase inhibitor and AMD3100 experiments, following BrdU pulses 4 days and 6 weeks after stroke as well as in all behavioral tests, were obtained from separate animal groups. Values are reported as mean ¡À standard error of the mean. Group differences were assessed with unpaired t tests and side differences were assessed with paired t tests. A one-way analysis of variance with Fischer¡¯s post hoc test was used to assess differences in the number of migrated cells and level of phosphorylation in vitro. For studies of correlation, Pearson¡¯s correlation coefficients were generated and analyzed for significance using Fisher¡¯s r to z transformation.1 j3 u! y) h( u: }
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4 i. g. |* H9 Z& e/ iStriatal Neuroblasts Are Generated from SVZ Precursors for Several Months after Stroke
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We first studied the occurrence of Dcx neuroblasts in the adult rat striatum at various time points after 2-hour MCAO. Contralateral to the lesion, as well as in sham-operated animals, Dcx immunoreactivity was confined to the SVZ and a few striatal cells (Fig. 1A, 1B). In contrast, in the damaged striatum, the number of Dcx cells was already elevated at 1 week, and was stable thereafter for up to 16 weeks following the insult (Fig. 1A). The majority of the Dcx cells had an immature neuronal morphology. At all time points, Dcx cells had similar distributions within the striatum (Fig. 1B, 1C) and appeared to migrate from the SVZ into the damaged area. Stroke in humans varies in severity, influencing both the magnitude and time course of functional recovery . We, therefore, determined whether the long-lasting increase in Dcx neuroblasts after the 2-hour MCAO also occurred in response to a less severe insult. We analyzed rats subjected to a 30-minute MCAO, which gave rise to a lesion restricted to the striatum (Fig. 2A). Following 2-hour MCAO, there was more extensive striatal damage and, in addition, injury to the overlying parietal cortex (Fig. 2A, 2B). Dcx cells were markedly fewer in the damaged striatum in the 30-minute than in the 2-hour MCAO group both 2 and 6 weeks after the insults (Fig. 2C). The number of Dcx cells correlated significantly with the volume of striatal injury (Fig 2E). Thus, the extent of the ischemic lesion influences the degree of activation of the molecular and cellular mechanisms that promote recruitment of new striatal neurons after stroke.
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Figure 1. Neuroblasts are continuously produced and migrate into damaged striatum for more than 4 months after stroke. (A): Number of Dcx neuroblasts in striatum 1¨C16 weeks after 2-hour MCAO or sham surgery. n = 6¨C10 rats for each time point except at 1 week, in which cell numbers from 5 and 9 days after 2-hour MCAO are pooled (n = 23). (B): Dcx immunoreactivity in the contralateral striatum 2 weeks after 2-hour MCAO and in the ipsilateral striatum 2, 8, or 16 weeks after 2-hour MCAO. At all time points, abundant Dcx cells are distributed from the SVZ laterally and ventrally toward the damaged area. Scale bar = 100 µm. (C): Distribution of Dcx neuroblasts within the ipsilateral striatum 2, 6, and 16 weeks after 2-hour MCAO, shown as a percentage of the total number of Dcx cells at different distances from the SVZ. n = 8¨C10 for each time point. (D): Number of Dcx /BrdU and Dcx /BrdU¨C neuroblasts following 2 weeks of daily BrdU injections performed either early (weeks 1 and 2) or late (weeks 7 and 8) after 2-hour MCAO. n = 6¨C10 for each time point. Data are shown as the mean ¡À standard error of the mean. (E): Confocal images of Dcx /BrdU cells 8 weeks after 2-hour MCAO with daily BrdU injections during weeks 7 and 8. Shown are orthogonal reconstructions from confocal z-series presented as viewed in the x-z (bottom) and y-z (right) planes. Abbreviations: BrdU, 5-bromo-2'-deoxyuridine; Dcx, doublecortin; MCAO, middle cerebral artery occlusion; SVZ, subventricular zone.
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Figure 2. Stroke severity influences the magnitude of the neurogenic response. (A, B): Schematic illustration of the distribution of ischemic damage (shaded area) (A) and volume of remaining striatal tissue (B) 6 weeks following a 30-minute or 2-hour MCAO. n = 8 and 7 for the 30-minute and 2-hour MCAO, respectively. (C): Number of all Dcx neuroblasts and Dcx /BrdU cells in the damaged striatum 2 weeks and all Dcx neuroblasts and mature NeuN /BrdU neurons 6 weeks after 30-minute and 2-hour MCAO with daily BrdU injections during weeks 1 and 2. (D): Distribution of Dcx neuroblasts within damaged striatum 2 weeks after 30-minute and 2-hour MCAO, shown as the percentage of the total number of Dcx cells at different distances from the SVZ. n = 7 and 9 for 30-minute and 2-hour MCAO, respectively. Data are presented as the mean ¡À standard error of the mean. *, p . q; p: P- M/ J, B- a
0 v5 B- G/ Z2 kWe then explored when the Dcx cells observed at early and late time points following 2-hour MCAO had been formed. In animals given BrdU during the first 2 weeks after ischemia and sacrificed directly thereafter, about 46% of the Dcx cells were BrdU , indicating that they had been formed during this time period (Fig. 1D). Virtually no Dcx /BrdU double-labeled cells were detected in animals that survived for 6 or 16 weeks after stroke, despite the total number of Dcx cells at these time points being similar to that at 2 weeks (Fig. 1D). These findings indicated that the production of new striatal neuroblasts continued beyond the first 2 weeks, and we therefore injected BrdU during weeks 7 and 8 after MCAO. Supporting the occurrence of neurogenesis late after stroke, the majority of Dcx cells (about 71%) in the damaged striatum were BrdU at 8 weeks (Fig. 1D, 1E), but at 12 weeks hardly any Dcx /BrdU cells remained.. `- |$ Y$ F# p; T& T
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We asked whether the dramatic difference between ischemic and nonischemic striatum in the number of neuroblasts produced at late time points after stroke could depend on a corresponding difference in SVZ cell proliferation. Consistent with increased proliferation early after stroke .
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" }. U9 s% q5 O pFigure 3. Stroke induces a long-term increase in the size of the sub-ventricular zone (SVZ) and its content of neural stem/progenitor cells. (A): Number of BrdU cells in the ipsilateral and contralateral SVZ 4 days and 6 weeks following 2-hour MCAO with BrdU given three times just prior to sacrifice. n = 7 and 6 for 4 days and 6 weeks, respectively. (B): Size of the ipsilateral and contralateral SVZ 2 and 6 weeks after 2-hour MCAO as observed in neuronal nuclei (NeuN)¨Cstained sections counterstained with cresyl violet. n = 6 and 7 for 2 and 6 weeks, respectively. (C, D): Number (C) and size (D) of primary and secondary neurospheres generated in vitro from ipsilateral and contralateral SVZ 6 weeks after 2-hour MCAO. Note that after growing the neurospheres in vitro for 1 week and subsequent passage, the differences found in the primary neurospheres were no longer observed in the secondary spheres. Data are presented as the mean ¡À standard error of the mean. *, p
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Neuroblasts Generated Early and Late after Stroke Differentiate into Mature Neurons or Die Through Caspase-Mediated Apoptosis
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Following both the 30-minute and 2-hour insults, the number of new cells expressing mature neuronal markers (NeuN /BrdU ) after 6 weeks corresponded to 37% of the number of Dcx /BrdU neuroblasts observed at 2 weeks (Fig. 4A, 4B, and Fig. 2). One third of the NeuN /BrdU cells 6 weeks after 2-hour MCAO survived 4 months postischemia (Fig. 4A). When we injected BrdU during weeks 7 and 8, the number of NeuN /BrdU neurons at 12 weeks constituted 23% of the number of Dcx /BrdU neuroblasts at 8 weeks. Thus, similar to neuroblasts produced early after stroke, those produced a long time after the insult developed into mature neurons. In accordance, neurosphere cultures from ipsilateral and contralateral SVZ 6 weeks following MCAO generated similar numbers of mature neurons (Fig. 4C).8 o+ X: s7 H- i# W- J O
# A& h! b& v3 u* L5 J" FFigure 4. Striatal neuroblasts generated early and late after stroke form mature neurons or die through caspase-mediated apoptosis. (A): Number of neuroblasts (Dcx /BrdU ) and mature neurons (NeuN /BrdU ) generated during periods of daily BrdU injections at weeks 1 and 2 or weeks 7 and 8 after 2-hour MCAO. n = 6¨C10 for each time point. (B, D): Confocal images of NeuN /BrdU cells 4 months (B) and cPARP /Dcx cells 2 weeks (D) after 2-hour MCAO. Orthogonal reconstructions of double-labeled cells are presented as viewed in the x-z (bottom) and y-z (right) planes. (C): Photomicrograph of ßIII-tubulin¨Cpositive cells from subventricular zone (SVZ)¨Cderived secondary neurospheres isolated 6 weeks after 2-hour MCAO and maintained under differentiation conditions for 7 days. Cells from both sides have morphological characteristics of mature neurons. Scale bar = 50 µm. (E): Number of Dcx and Dcx /BrdU cells in damaged striatum 14 days after 2-hour MCAO with daily BrdU injections from day 1 and infusion of a caspase inhibitor cocktail from day 3. n = 6 and 7 for vehicle and caspase inhibitors, respectively. Data are presented as the mean ¡À standard error of the mean. *, p + [$ L! r+ c/ u/ C& }8 o: g# y8 j
& u P I5 D7 k) ?/ vApparently, only a portion of stroke-generated striatal neuroblasts formed mature neurons. The virtual absence of Dcx /BrdU cells 4 weeks after BrdU injections provided strong evidence that the new neuroblasts had not remained in an undifferentiated state. A non-neuronal fate was also unlikely because neither we nor others have observed any Dcx cells coexpressing glial markers . Both 2 and 8 weeks after MCAO, scattered Dcx cells in the ipsilateral striatum coexpressed cPARP (Fig. 4D).
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# d( o, C8 p9 q/ i6 u& j- c7 sWe wanted to confirm the involvement of caspases in compromising the survival of the newly formed striatal neurons, and to explore whether the inhibition of caspases could rescue these neurons. Caspase inhibitors were infused from day 3¨C14 after 2-hour MCAO, and striatal neurogenesis was analyzed directly thereafter. Infusions began when striatal neuronal death was virtually complete and, in accordance, no difference in ischemic damage was observed compared with the control group (data not shown). Caspase inhibitor-treated rats showed a markedly higher number of Dcx cells in the damaged striatum (Fig. 4E).
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: B7 P v. t0 H/ n. r: ?Migration of Neuroblasts Generated from SVZ Precursors to the Damaged Striatum Is Regulated by SDF-1/CXCR4 Signaling+ w5 M4 y) }6 U6 B9 k4 C
: E& z( g+ K" j4 d/ xWe hypothesized that the long-lasting directed migration of the new neurons toward the ischemic injury was dependent on the interaction between the chemokine SDF-1 and its receptor CXCR4. We first demonstrated SDF-1 immunoreactivity in the periphery of the damaged striatal area, extending toward the SVZ 2, 6, and 16 weeks after 2-hour MCAO (Fig. 5A, 5B). The majority of SDF-1 cells and processes coexpressed GFAP and were probably reactive astrocytes (Fig. 5B). In addition, large ED1 /SDF-1 cells, presumably activated microglia, were found in the core of the damage. The 30-minute MCAO induced a similar pattern of SDF-1 expression, but the number of processes and staining intensity were lower than after 2-hour MCAO (Fig. 5A). No specific SDF-1 staining was detected in the intact striatum (Fig. 5A). X# y0 I/ _9 k# m& E; }3 v
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Figure 5. SDF-1/CXCR4 signaling regulates migration of neuroblasts in damaged striatum. (A): Confocal images of SDF-1 expression (green) in striatum contralateral to the 2-hour middle cerebral artery occlusion (MCAO) and ipsilateral to the 30-minute or 2-hour MCAO at 6 weeks; Dcx (red) expression in relation to SDF-1 expression following 2-hour MCAO in right panels. Dcx cells are surrounded by SDF-1 processes but do not express SDF-1. Scale bar = 40 µm. (B): Confocal images showing SDF-1-expressing and GFAP-expressing cells and processes in the ipsilateral striatum 6 weeks after 2-hour MCAO. Most SDF-1 cells coexpress GFAP. Scale bar = 20 µm. (C): SVZ-derived neurosphere stained against CXCR4 (green) and counterstained with Hoechst (blue). Scale bar = 50 µm. (D, E): Effect of SDF-1 on the number of SVZ neurosphere cells migrating from upper to lower wells of a microchemotactic chamber (D) and on extracellular signal-related kinase (ERK)-1/2 phosphorylation in primary cultures of SVZ cells (E). *, p
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3 }* F5 z8 H5 }: W J1 v8 TWe then analyzed whether SDF-1/CXCR4 signaling influenced the migration of cells derived from adult rat SVZ in vitro. Cells in neurospheres (Fig. 5C), as well as in primary SVZ cultures, expressed the CXCR4 receptor. In a chemotactic chamber assay, neurosphere cells migrated toward a gradient of SDF-1 (Fig. 5D). SDF-1 also increased phosphorylation of ERK-1/2 in primary SVZ cultures (Fig. 5E). Both effects were blocked by the CXCR4 receptor antagonist AMD3100 .
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Finally, we explored if SDF-1/CXCR4 signaling is involved in the in vivo migration of striatal neuroblasts after stroke. We infused AMD3100 during weeks 5 and 6 after 2-hour MCAO, and rats were killed directly thereafter. The AMD3100 infusion did not change the total number of striatal Dcx cells, but suppressed the migration of the new neurons in the striatal parenchyma (Fig. 5F). In the vehicle-treated rats, about 50% of the Dcx and Dcx /BrdU neuroblasts were localized within 0.25 mm from the border of the SVZ. Following administration of the CXCR4-receptor blocker, about 70% of the cells remained in this area (Fig. 5F).
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Behavioral Recovery Continues for Several Months after Stroke-Induced Striatal Damage. J7 ~) z* T$ r# ~ u1 M. m, v
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We finally wanted to explore whether spontaneous functional recovery after stroke occurs concomitantly with the long-lasting production of new striatal neurons. We used 30-minute MCAO, which triggers continuous striatal neurogenesis, albeit of low magnitude, and, importantly, induces selective damage of the striatum. Thus, we avoided the confounding effects of the cortical lesion induced by the 2-hour insult. In in vivo behavioral tests, the stepping and cylinder tests, improvement had already plateaued within the first month. In contrast, there was a significant improvement between 1 and 4 months in the grid-walking test (Fig 6). Taken together, these findings indicate a progressive recovery of certain behavioral deficits that continues beyond 1 month after the stroke.
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Figure 6. Spontaneous functional recovery after stroke-induced striatal injury occurs gradually over several months concomitantly with striatal neurogenesis. The graph shows the degree of behavioral deficits over time in the horizontal ladder-walking test. Note the significant improvement between 1 and 4 months after stroke. In contrast, the improvement in two other tests, the stepping and cylinder tests, had already plateaued within the first month (data not shown). Photograph shows rat walking on the horizontal ladder. n = 6. Data are presented as the mean ¡À standard error of the mean. *, p
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DISCUSSION* f. A; z4 h& ]1 {) q I
+ F/ O* w* Y4 u; `$ }The present data provide the first evidence that the adult brain responds to a stroke by long-lasting generation of neurons from its own NSCs. An increased number of Dcx cells was observed in the damaged striatum for at least 4 months after stroke. Dcx is a reliable marker of neurogenesis, which is transiently expressed (for about 2¨C3 weeks) in newly formed neuroblasts . BrdU injections performed early and late after stroke confirmed that Dcx cells were continuously produced. The striatal neuroblasts formed several months following stroke retained the capacity for differentiation into mature neurons.( T) }- `5 o, L8 J3 u0 n
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Maximum cell proliferation in the ipsilateral SVZ occurs 1 to 2 weeks after MCAO . We found a greater number and size of neurospheres isolated from the ipsilateral SVZ 6 weeks post-MCAO, and expansion of the ipsilateral SVZ at both 2 and 6 weeks. BrdU labeling was greater in the ipsilateral SVZ 4 days but not 6 weeks after stroke, which argues against a long-term increase in the pool of rapidly dividing progenitors. Taken together, our findings provide evidence that stroke leads to long-term alterations in the stem cell niche in the SVZ.0 m2 M m8 e$ T3 o! W9 v) r
" z1 K9 x" y O( x. y. c& K* uOur data indicate that caspase-mediated apoptotic death caused the loss of the stroke-generated neuroblasts, and that caspase inhibitors can rescue these neuroblasts. Neurons formed in the dentate gyrus after an epileptic insult also undergo caspase-mediated apoptotic death . Also, the new striatal neurons will most likely die if they do not establish synaptic connections and receive trophic signals. Our finding that a population of new neurons survived for 4 months after stroke suggests that integration and trophic support can be achieved in the stroke-damaged striatum.
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We provide the first evidence that SDF-1/CXCR4 signaling regulates the directed migration of new striatal neurons generated from endogenous NSCs toward the ischemic damage. Interaction between SDF-1 and CXCR4 mediates homing of transplanted bone marrow¨Cderived cells to sites of brain injury . A direct action on these cells is supported by our finding that SDF-1 activated mitogen-activated protein kinases and induced CXCR4-mediated chemotaxis in cells derived from adult rat SVZ. However, AMD3100 only partially suppressed migration in vivo, which suggests that other mechanisms also direct the new neurons to the damaged area.$ y5 H7 [& Q6 ^* G2 `$ e# z% s
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Similar to observations after extensive ischemic lesions , is associated with superior recovery. However, with available methods, it is not possible to prove a causal relationship between neurogenesis and behavioral improvement after stroke. Suppression or enhancement of stroke-induced neurogenesis through delivery of mitosis inhibitors or trophic factors, respectively, will also affect other processes. Generation of conditional transgenic mice, in which newly formed neuroblasts carry a gene for selective ablation, could solve this problem.) T8 f% X# B$ o
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Our finding that stroke-induced neurogenesis is persistent has several implications. The number of new striatal neurons that potentially could replace the dead neurons is much larger than suggested by previous calculations . Furthermore, experimental studies to explore mechanisms of neuronal replacement from endogenous NSCs in the damaged brain, as well as various interventions to promote neurogenesis, are not restricted to the acute postischemic phase but can be applied over an extended time period. Most importantly, we now know that adult NSCs continuously produce a cellular raw material that may be used for self-repair in the brain during the recovery phase after stroke.; q0 O' `% A$ K4 l7 K5 l
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7 C. x/ U: @. N& f* c0 G4 MThe authors indicate no potential conflicts of interest.2 ]3 z6 l' B2 N- N# a& J2 t+ B
" n( A8 u/ ?+ ^4 W) n3 N, FACKNOWLEDGMENTS
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, O: T% H0 c" x% `This work was supported by the Swedish Research Council, EuroStemCell (European Union Framework 6 project LSHB-CT-2003-503005), and the King Gustav V and Queen Victoria, Söderberg, Crafoord, and Kock Foundations. The Lund Stem Cell Center is supported by a Center of Excellence grant in Life Sciences from the Swedish Foundation for Strategic Research. Z.K. and O.L. contributed equally to this study.
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发表于 2015-6-14 18:02
