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NEUROPHARMACOLOGY
Department of Neurosurgery, Kumamoto University School of Medicine, Kumamoto, Japan (J.M., M.M., Y.H., T.K., Y.Y., T.M., S.Y., Y.K., J.K.); and Department of Pharmacology, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan (K.F.)
Received for publication
March 15, 2006
Accepted
June 14, 2006.
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Abstract |
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; Brichard and Henquin, 1995). Because vanadate, the +5 oxidation state of vanadium, seems to act as a phosphate analog (Bevan et al., 1995; Huyer et al., 1997) mimicking the transition state (Zhang et al., 1997), it behaves as a competitive inhibitor of protein tyrosine phosphatases, including intracellular signal transduction systems. We previously reported that in gerbils with transient forebrain ischemia, sodium orthovanadate (SOV) and insulin-like growth factor (IGF)-1 lead to Akt activation through phosphatidylinositol 3-kinase (PI3K) activation and to extracellular signal-regulated kinase (ERK) activation. Since both Akt and ERK underlie survival in various cells, SOV treatments indeed rescue hippocampal CA1 neurons from delayed neuronal cell death (Kawano et al., 2001, 2002). Similarly, SOV had neuroprotective effects on rat middle cerebral artery (MCA) occlusion model (Hasegawa et al., 2003). However, the time window of SOV-induced neuroprotective effects was limited only in the early phase (within few hours) after stroke (Hasegawa et al., 2006). Since the Akt pathway is believed to be a key component in proliferation or differentiation of neuronal progenitor cells, it is valuable to use SOV in promotion of neuronal self-repair mechanisms after acute ischemic phase. In stroke patients, it is difficult to administer SOV during the acute ischemic phase, and their progression to cerebral infarction indicates that the opportunity to rescue neurons from cell death has been missed. Our rat experiments were designed to examine the possibility of reducing the infarct area during, and of promoting central nervous system (CNS) self-repair after, the acute phase, because CNS regeneration would improve neural function in stroke patients. We focused on the effect on CNS self-repair of SOV delivered after the acute ischemic phase.
Although the adult mammalian CNS was previously considered to be incapable of significant self-repair or regeneration (Bjorklund and Lindvall, 2000), progenitor cells with the ability to differentiate new neuron and glia have been found to persist in the adult CNS. Temple (2001) reported that neural stem cells, characterized by long-term self-renewal and multipotentiality, are present throughout life in various mammalian species, including humans and that the adult CNS contains a range of progenitors with limited growth- and differentiation potential. These adult progenitors are abundant in the periventricular areas, including the subventricular zone (SVZ), and they are distributed throughout the parenchyma of various CNS regions (Palmer et al., 1999). They proliferate in vivo in response to growth factor stimulation by fibroblast growth factor-2, epidermal growth factor, IGF-1, and vascular endothelial growth factor. The proliferation of neural progenitor cells is up-regulated in a variety of pathological conditions, including ischemia (Dempsey et al., 2003). In adult rats, exogenous IGF-1 increased the proliferation of cells induced by focal ischemia (Dempsey et al., 2003).
Most growth factors bind to their receptors and activate ERK in the cytosol. ERK are members of the mitogen-activated protein kinase (MAPK) family involved in the transduction of extracellular signals into intracellular responses, and the stimulation of ERK phosphorylation by growth factors mediates cell proliferation (Harada et al., 2004; Zhou et al., 2004). We reported elsewhere that SOV activated down-stream signals of IGF-1 receptor and that SOV activated ERK and Akt by receptor activation (Kawano et al., 2001). Because many growth factor receptors are activated by autophosphorylation of tyrosine residue, SOV can activate or maintain the activity of these receptors by inhibiting the dephosphorylation of tyrosine residue.
We hypothesized that the administration of SOV after brain ischemia may induce progenitor cell activation and adult CNS regeneration. To assess neuronal regeneration after cerebral infarction, we produced transient MCA occlusion in rats and investigated the effect of SOV on progenitor cell activation in the ipsilateral SVZ. SOV delivered during the acute phase is neuroprotective (Hasegawa et al., 2003); in the current study, we injected SOV after the acute phase. We found that consecutive i.p. administrations of SOV after acute ischemic phase promoted the proliferation of progenitor cells in the SVZ. Our findings suggest that SOV, delivered after the acute ischemic phase, has the potential to promote neural self-repair or regeneration via the induction of SVZ progenitor cell proliferation.
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Materials and Methods |
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Induction of Focal Ischemia (MCA Occlusion). We induced reversible focal ischemia by a modification of the method of Koizumi et al. (1986) as described by Hasegawa et al. (2003). After exposing the left common, external, and internal carotid arteries through a midline incision in the neck, 20 mm of a 3-0 monofilament nylon suture coated with poly-L-lysine (Belayev et al., 1996) were gently advanced from the left common carotid artery into the lumen of the internal carotid artery. After 1.5 h, the suture was carefully with-drawn to allow MCA reperfusion. Neurological findings were scored 10 min before reperfusion using a modification of the neurological score of Bederson et al. (1986); grade 0 was recorded in the absence of observable deficits; grade 1 was scored if there was forelimb flexion; grades 2 and 3 were scored if there was decreased resistance to a lateral push in the absence or presence of circling, respectively; and grade 4 was assigned to comatose animals. Rats with grade 0 and 4 were excluded from further experiments.
Administration of SOV. Starting at 24 h after MCA occlusion, the rats received daily i.p. injections (2 ml/kg) of 12.5 or 25 mM SOV (Na3VO4; Wako Pure Chemicals, Osaka, Japan) in saline or saline alone for seven consecutive days (Fig. 1A).
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Measurement of the Area of Ischemic Brain Injury. On the eighth day after transient MCA occlusion, the rat brains (15/group) were fixed by transcardial perfusion with 4% ice-cold PFA, immersed overnight in fixative, postfixed in 4% PFA, and 30-µm-thick sections were cut in the coronal plane between bregma levels +3 mm and –1 mm on a vibratome. These sections were H&E- and Nissel-stained (Fig. 1B, showing only Nissel staining). The area of ischemic brain injury was measured as reported previously (Hasegawa et al., 2003) using the image analysis software (Scion Image Beta 4.02; Scion Corporation, Frederick, MD). The area of infarction was corrected to compensate for edema formation by subtracting the area of the intact ipsilateral hemisphere from that of the intact contralateral hemisphere. The area of the cortex and caudate putamen on each slice was calculated separately, and the total infarct area and the area of the cortex and caudate putamen on each slice were added and multiplied by the slice thickness to obtain the infarction area.
Immunohistochemistry. The sections were then double-stained for BrdU and double cortin (Dcx) or various neuronal makers. Dcx is a microtubule-associated protein expressed by migrating neuroblasts in the developing and adult nervous system (Francis et al., 1999; Gleeson et al., 1999). Free-floating coronal sections (30 µm) were washed three times (10 min each) in PBS at room temperature (RT), and denatured by incubation in 2 mol/l hydrochloric acid (90 min at RT) before BrdU staining. They were washed for 30 s with PBS, pH 8.5, and then three more times (5 min each at RT) in PBS. Nonspecific binding sites were blocked for 1 h in blocking solution (PBS in 3% bovine serum albumin) containing 1% Triton X-100. The sections were then incubated at 4°C for 24 h in PBS blocking solution containing 0.5% Triton X-100 in the presence of primary mouse monoclonal antibody against BrdU (1:250; Chemicon International, Temecula, CA) and goat polyclonal anti-Dcx (N-18) antibody (1:200; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), or either of these antibodies, and one of the following antibodies: mouse monoclonal anti-neuronal nuclei (NeuN) antibody (1:1000; Chemicon International), mouse monoclonal anti-Nestin antibody (1:1000; BD Biosciences, San Jose, CA), rabbit polyclonal anti-glial fibrillary acidic protein (GFAP) antibody (1:2000; Dako North America, Inc., Carpinteria, CA), mouse monoclonal anti-GFAP antibody (1:2000; Chemicon International), rabbit polyclonal anti-phosphorylated-Akt (Ser473) (p-Akt) antibody (1:100; Cell Signaling Technology Inc., Beverly, MA), mouse monoclonal anti-MAPK [activated (phosphorylated)-ERK 1/2] (p-ERK) antibody (1:500; Sigma-Aldrich), or sheep polyclonal anti-BrdU (1:200; Exalpha Biologicals, Inc., Watertown, MA). After three more 5-min washes in PBS, the sections were labeled for 2 h with fluorescein-labeled anti-mouse secondary antibody (1:200; Invitrogen, Eugene, OR) and rhodamine-labeled antigoat secondary antibody (1:200; Chemicon International), or either of these antibodies, and Texas Red-labeled anti-mouse secondary antibody (1:200; Invitrogen), fluorescein-labeled anti-sheep secondary antibody (1:200; Vector Laboratories, Burlingame, CA), or Texas Red-labeled anti-rabbit secondary antibody (1:200; Invitrogen). After several washes with PBS, the sections were mounted on glass slides, coverslipped, and analyzed by using a Fluoview FV300 laser confocal microscope (Olympus Optical Co., Tokyo, Japan).
Image Analysis and Quantification. BrdU-positive nuclei were displayed on a computer monitor for better visualization and counted in one focal plane to avoid oversampling. Only cells with uniform nuclear BrdU immunostaining were counted. Sampling of the structures and counting of the BrdU-positive cells were by a modification of the method of Kuhn et al. (1996).
We cut the brain with a vibratome and obtained many serial 30-µm thick coronal sections at a level of bregma +3 to –1 mm. From these sections, we have selected the serial sections in which SVZ was clearly observed, based on an anatomical structure, and we have performed immunostaining. All BrdU-positive nuclei in the SVZ were counted. BrdU-positive nuclei in these areas are presented in terms of cell density per squared millimeter. The cell density in seven examined sections per rat was averaged to obtain a mean density value for each animal.
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Terminal Deoxynucleotidyl Transferase dUTP Nick-End Labeling Staining. DNA fragmentation and apoptotic bodies were detected with the terminal deoxynucleotidyl transferase dUTP nickend labeling (TUNEL) method using an in situ apoptosis detection kit (Takara Bio Inc., Shiga, Japan). TUNEL signals were amplified with streptavidin Alexa Fluor-488 conjugate (1:200; Invitrogen) using the TSA Biotin System (PerkinElmer Life and Analytical Sciences, Boston, MA).
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) that SOV injected more than 24 h after ischemia induction did not inhibit ischemic neuronal injury. As shown in Fig. 2, A, B, and E, in rats exposed to 90-min MCA occlusion and injected for the next 7 days with BrdU, the ipsilateral SVZ clearly contained BrdU-positive cells. The density of these cells per squared millimeter was 5564 ± 131, 3569 ± 249, and 1734 ± 55 in rats treated with 25 mM SOV, 12.5 mM SOV, or saline, respectively. The density of BrdU-positive cells in the ipsilateral SVZ was significantly greater than that in SOV-treated animals (p < 0.05) and was dose-related. At 25 and 12.5 mM SOV, the density of BrdU-positive cells in the contralateral SVZ was 1156 ± 40 and 1143 ± 22; it was 1134 ± 43 in rats injected with saline. The differences were not statistically significant (Fig. 2E). But in each group, the density of BrdU-positive cells in the ipsilateral SVZ was significantly greater than that in the contralateral SVZ (Fig. 2E). We also found no significant differences when we examined the density of BrdU-positive cells in the ipsilateral caudate putamen and parietal cortex of rats treated with 25 or 12.5 mM SOV or injected with saline (Fig. 2, G and H). Our data indicate that SOV, delivered in the subacute phase after MCA occlusion, dose dependently enhanced cell proliferation in the ipsilateral SVZ of adult rats. There was no significant difference in the density of BrdU-positive cells in the SVZ of nonischemic rats treated with SOV or saline (Fig. 2, C, D, and F). The caudate putamen and parietal cortex of these animals did not contain BrdU-positive cells (data not shown).
To characterize the proliferating cells, we stained tissue sections for Dcx, a marker of migrating neuroblasts (Fig. 3). According to Nacher et al. (2001), in the normal adult rats, Dcx is expressed in the SVZ and rostral migratory stream (RMS), but only a few Dcx-expressing cells were detected in the striatum. We found a similar Dcx expression in nonischemic rats (Fig. 4, D and F). Conversely, Dcx-labeled cells were abundant in rats exposed to transient MCA occlusion (Fig. 3, B, E, H, and T). As shown in Fig. 3T, the density (cells per millimeter2) of BrdU-Dcx double-positive cells in the SVZ after MCA occlusion was 3201 ± 28 and 2061 ± 24 in rats treated with 25 and 12.5 mM SOV, respectively; it was 1175 ± 47 in rats injected with saline. The density of BrdU-Dcx double-positive cells was significantly higher in SOV-treated ischemic rats (p < 0.05), and the increase was SOV dose-dependent. In contrast, the ratio of BrdU-Dcx double-positive cells among BrdU-positive cells was not significantly different; it was 57.5, 57.8, and 67.8% in rats treated with 25 mM SOV, 12.5 mM SOV, or saline (Fig. 3U).
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Next, we performed immunostaining of Nestin, which is a class VI intermediate filament protein, that has been used as a biological marker to identify neural stem cells (Lendahl et al., 1990; Reynolds et al., 1992). Nestin-labeled cells were abundant in rats exposed to transient MCA occlusion (Fig. 5, B, E, G, and J). As shown in Fig. 5M, the density (cells per millimeter2) of BrdU-Nestin double-positive cells in the SVZ after MCA occlusion was 2990 ± 339 in rats treated with 25 mM SOV, and it was 1133 ± 44 in rats injected with saline. The density of BrdU-Nestin double-positive cells was significantly higher in SOV-treated ischemic rats (p < 0.05). Furthermore, the density (cells per millimeter2) of Nestin-Dcx double-positive cells in the SVZ after MCA occlusion was 1923 ± 107 in rats treated with 25 mM SOV, and it was 1525 ± 88 in rats injected with saline. The density of Nestin-Dcx double-positive cells was significantly higher in SOV-treated ischemic rats (p < 0.05) (Fig. 5N). Alternatively, the ratio of BrdU-Nestin double-positive cells among BrdU-positive cells was not significantly different; it was 60.6 and 64.5% in rats treated with 25 mM SOV and saline, respectively (Fig. 5O).
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These data indicate that SOV does not enhance the proliferation of only neuroblasts. Rather, it may enhance the proliferation of total progenitor cells in the SVZ ipsilateral to the site of MCA occlusion.
To estimate apoptotic change in the SVZ, we used TUNEL staining (Fig. 6, A and B). The density (cells per millimeter2) of TUNEL-positive cells in the SVZ after MCA occlusion was 52 ± 12 in rats treated with 25 mM SOV, and it was 49 ± 3 in rats injected with saline. The density of TUNEL-positive cells was not significantly different in rats treated with 25 mM SOV or saline (Fig. 6E). According to these data, it was suggested that the major effect of SOV is activation of SVZ cells, rather than inhibition of apoptosis.
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Because we previously found that SOV exerts its neuroprotective effect via activation of Akt and ERK (Kawano et al., 2001; Hasegawa et al., 2003), we investigated their phosphorylation in BrdU-positive cells using anti p-Akt and p-ERK antibodies. We compared the density of immunoreactive cells in the SVZ of rats injected with 25 mM SOV or saline and found that most of the BrdU-positive cells were immunostained for p-Akt and p-ERK antibodies (Figs. 7 and 8). The density (cells per millimeter2) of BrdU/p-Akt double-positive cells in the SVZ of ischemic rats treated with 25 mM SOV (3409 ± 63) was significantly higher than that in rats injected with saline (1620 ± 67; p < 0.05) (Fig. 7, A–D, and I). In contrast, the ratio of BrdU-p-Akt double-positive cells among BrdU-positive cells was not significantly different; it was 68.2 or 69.2% in rats treated with 25 mM SOV or saline, respectively (Fig. 7J). In nonischemic rats treated with 25 mM SOV or saline, the difference was not significant (Fig. 7, E–H, and K). The density of BrdU/p-ERK double-positive cells in the SVZ of ischemic rats treated with 25 mM SOV or saline was 2494 ± 167 and 1611 ± 101 cells/mm2 (p < 0.05) (Fig. 8, A–D, and I). As for BrdU/p-Akt, the ratio of BrdU-p-ERK double-positive cells among BrdU-positive cells was not significantly different; it was 51.0 or 69.2% in rats treated with 25 mM SOV or saline, respectively (Fig. 8J), and there was no difference between nonishcemic rats treated with 25 mM SOV or saline (Fig. 8, E–H, and K). Few GFAP-positive cells were immunostained for p-Akt and p-ERK. We have observed many double-positive cells of p-Akt and p-ERK, p-Akt and Dcx, and p-ERK and Dcx. Thus, we considered that Akt and ERK were activated in Dcx-positive cells after ischemia (see Supplemental Figure). It was concluded that activation of Akt and ERK correlated with activation of SVZ cells by SOV.
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; Garcia-Verdugo et al., 1998), and neuroepithelial SVZ cell populations contain multipotent progenitor cells that give rise to neurons and glial cells throughout life (Garcia-Verdugo et al., 1998). In the normal mouse brain, proliferating cells constitute an average of 10% of the cell population in the SVZ (Morshead and van der Kooy, 1992). We identified BrdU-positive cells in the adult rat SVZ; their characteristics were similar to those reported for SVZ progenitor cells (Morshead and van der Kooy, 1992; Gage et al., 1998; Garcia-Verdugo et al., 1998).
BrdU, an analog of thymidine, is incorporated into DNA of cells during the S phase; it has been used to investigate cell proliferation (Dolbeare, 1996; Kuhn et al., 1996). Zhang et al. (2001) reported that proliferating progenitor cells in the adult rodent brain increase after MCA occlusion. We found that progenitor cells in the ipsilateral SVZ of rats increased after 90-min MCA occlusion; their proliferating pattern was similar to that reported by Zhang et al. (2001). On the basis of our findings, we concluded that in adult rats, SVZ cells proliferate in response to an ischemic insult.
SOV Enhances Proliferating Progenitor Cells in the SVZ after Ischemia. In ischemic rats treated with SOV, the density of BrdU-positive cells in the SVZ was dramatically increased. Interestingly, SOV enhanced only the proliferation of progenitor cells in the ipsilateral SVZ; it had no effect on cells in the contralateral SVZ or in the normal rat brain. Thus, because the effect of SOV was restricted to the ischemic hemisphere, we suggest that the ischemic insult triggered-off the proliferation of SVZ cells and that SOV treatment induced the proliferation of these cells in a dose-dependent manner. SOV behaves as a competitive inhibitor of protein tyrosine phosphatase (Bevan et al., 1995; Huyer et al., 1997; Zhang et al., 1997) and increases the tyrosine phosphorylation levels of intracellular proteins (Morinville et al., 1998). Following ligand binding, some growth factor receptors are activated by tyrosine residue autophosphorylation, and SOV can activate or prolong the activity of these receptors. Indeed, we reported that the administration of SOV and IGF-1 induced Akt activation via IGF-receptor activation and that SOV is neuroprotective by delayed neuronal cell death of gerbil hippocampal CA1 neurons (Kawano et al., 2001). We also found that it exerts antiapoptotic effects via Akt and ERK activation after transient MCA occlusion (Hasegawa et al., 2003). The observed increase in BrdU-positive cells may be reflection of decreased apoptosis. However, because we found that a few cells in the SVZ of SOV-treated ischemic rats were TUNEL-positive, we postulate that SOV administered during the subacute phase affected cell proliferation rather than apoptosis.
Dempsey et al. (2003) showed that in adult rats, exogenous IGF-1 increased the proliferation of cells induced by focal ischemia. We postulated that SOV activated the proliferation of progenitor cells via the activation of growth factor receptors such as the IGF receptor. This hypothesis explains why SOV had an effect only on the ipsilateral side and only after ischemic insult. Ischemia may result in an increase in some growth factors in a restricted area (ipsilateral side), which, in turn, may lead to proliferation of progenitor cells in the SVZ, and SOV may enhance the effect of these growth factors.
Zhang et al. (2001) reported that in untreated ischemic rats, the number of BrdU-positive cells in the SVZ significantly increased during the first 14 days; the increase peaked on day 7, and the number of dividing cells returned to the control level at 3 to 5 weeks after ischemia induction. Thus, we examined the eighth day after ischemic insult, and the peak cell density in SOV-treated rats was approximately 3 times greater than in our saline-treated controls. We found that the effects of SOV were dose-dependent. Our observations suggest that the effect of SOV is ascribable to its enhancement of progenitor cell proliferation.
Characteristics of Proliferating Cells in SVZ. Because cells in the SVZ possess proliferative potential and neural stem cell characteristics (Chiasson et al., 1999), we stained sections for Dcx, a marker for migrating neuroblasts. Nacher et al. (2001) reported that in the normal adult rat brain, Dcx was expressed in the SVZ and RMS; however, only a few Dcx-positive cells were detected in the striatum. We found a similar Dcx expression pattern in our sham-operated rats (Figs. 2 and 4). Although the number of Dcx-positive cells was significantly higher in SOV-treated rats, the ratio of BrdU/Dcx double-positive cells among BrdU-positive cells in SOV-treated and saline-treated rats was not significantly different from our ischemic rats (Fig. 3).
None of the BrdU-positive cells exhibited NeuN immunoreactivity in the ipsilateral SVZ, cortex, and subcortex in the present study (data not shown). About 5% of BrdU-positive cells were immunoreactive for GFAP, and the ratio of BrdU/GFAP double-positive cells was not significantly different between SOV-treated and control rats. Although the number of BrdU/Nestin double-positive cells significantly higher in SOV-treated rats, the ratio of BrdU/Nestin double-positive cells among BrdU-positive cells in SOV-treated and saline-treated rats was not significantly different in our ischemic rats (Fig. 5). We found a similar expression pattern as that observed with Dcx. On the basis of these observations, we concluded that SOV has the potential to increase the total number of progenitor cells in the SVZ after MCA occlusion and that its effect is not cell type-specific.
SOV Effect Is Correlated to Activation of Akt and ERK in the SVZ. Mammalian neurogenesis is regulated by an interaction between intrinsic genetic mechanisms and extrinsic cues, such as growth factors and their downstream signaling pathways. PI3K/Akt signaling has been shown to play an important role in neurogenesis (Li et al., 2001). In addition, Akt is critical mediators of cellular responses to growth factors. The MAPK cascade is a highly conserved signaling system through which cells respond to a variety of extracellular stimuli (Marshall, 1995), and MAPK signaling promotes neurogenesis (Li et al., 2001; Zhao et al., 2003). Both PI3K and MAPK pathways are implicated in the proliferation of progenitor cells (Jin et al., 2005).
We previously reported that SOV induced the activation of Akt and ERK, which resulted in an antiapoptotic effect (Kawano et al., 2001, 2002); however, it remained unclear whether their activation contributes to cell proliferation. To investigate the mechanisms underlying the actions of SOV, we performed immunohistochemical studies using antibodies against p-Akt and p-ERK. In BrdU-positive cells of SOV-treated rats, the immunoreactivity of p-Akt and p-ERK was significantly increased. Thus, the activation of Akt and ERK correlates to SVZ cell proliferation by SOV. It is possible that activated Akt and ERK may enhance cell proliferation. However, the causal relationship of SOV, Akt/ERK activation, and SVZ cell activation is still unclear.
CNS Self-Renewal Potential after Ischemia. Under physiological conditions, cells originating in the SVZ migrate along the RMS via chain migrations to the olfactory bulb (OB) where they differentiate into granule and periglomerular neurons (Garcia-Verdugo et al., 1998). Progenitor migration from the SVZ to the forebrain ceases by the second postnatal week (Levison and Goldman, 1993). However, after focal cerebral ischemia, an increase in the number of BrdU-labeled cells in the SVZ results in an increase in the number of BrdU-labeled cells in the OB (Zhang et al., 2001). Therefore, some BrdU-labeled cells detected in the ipsilateral cortex and subcortex after an ischemic insult may derive from cells in the SVZ.
The RMS, along which progenitor cells migrate from the SVZ to the OB, is located near the peri-infarct zone. Gu et al. (2000) suggested that the BrdU/microtubule-associated protein-positive cells they observed in the ischemic cortex of adult rats may have been SVZ cells that escaped the RMS. Parent et al. (2002) showed that some newly proliferated SVZ cells migrated into the striatum and differentiated into a region-specific phenotype, and Arvidsson et al. (2002) reported that after stroke, some newly formed neurons and neuroblasts migrated from the SVZ into damaged striatal areas where they matured into medium-sized spiny neurons. These observations suggest that after ischemia, neuronal cells are regenerated from cells migrating from the SVZ, although their density may be low.
Indeed, region-specific neuronal regeneration has been observed several weeks after ischemia (Gu et al., 2000; Arvidsson et al., 2002; Gritti et al., 2002). Because we studied the effect of SOV on the eighth day after transient MCA occlusion, we were unable to assess the presence of newly formed neurons in the damaged area. Studies are underway in our laboratory to determine whether SOV treatment of rats with transient MCA occlusion results in the presence of newly formed neurons in the chronic phase.
In summary, we demonstrated that in rats exposed to transient MCA occlusion, SOV induces an increase in the proliferation of progenitor cells in the ipsilateral SVZ. SOV had no effect on the contralateral side or in nonischemic rats. We postulate that the delivery of SOV after an ischemic insult may enhance subsequent CNS self-repair.
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Acknowledgements |
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Footnotes |
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: SOV, sodium orthovanadate; IGF, insulin-like growth factor; PI3K, phosphatidylinositol 3-kinase; ERK, extracellular signal-regulated kinase; MCA, middle cerebral artery; CNS, central nervous system; SVZ, subventricular zone; MAPK, mitogen-activated protein kinase; BrdU, 5-bromo-2'-deoxyuridine; PBS, phosphate-buffered saline; PFA, paraformaldehyde; Dcx, double cortin; RT, room temperature; NeuN, neuronal nuclei; GFAP, glial fibrillary acidic protein; p-, phosphorylated; TUNEL, terminal deoxynucleotidyl transferase dUTP nick-end labeling; RMS, rostral migratory stream; OB, olfactory bulb; i.p., intraperitoneal.
The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material. 
Address correspondence to: Dr. Motohiro Morioka, Department of Neurosurgery, Kumamoto University School of Medicine, 1-1-1 Honjo, Kumamoto 860-8556, Japan. E-mail: morioka{at}kaiju.medic.kumamoto-u.ac.jp
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