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(Stroke. 2001;32:1890.)
© 2001 American Heart Association, Inc.

Original Contributions

Neurogenesis by Progenitor Cells in the Ischemic Adult Rat Hippocampus

Yoshiki Yagita, MD, PhD; Kazuo Kitagawa, MD, PhD; Toshiho Ohtsuki, MD, PhD; Ken-ichiro Takasawa, MD; Takaki Miyata, PhD; Hideyuki Okano, MD, PhD; Masatsugu Hori, MD, PhD Masayasu Matsumoto, MD, PhD

From the Division of Strokology, Department of Internal Medicine and Therapeutics (Y.Y., K.K., T.O., K-i.T., M.M.), Department of Neuroanatomy, Biomedical Research Center (T.M., H.O.), and Department of Clinical Neuroscience (M.M.), Osaka University Graduate School of Medicine, Osaka, Japan.

Correspondence to Dr Kazuo Kitagawa, Division of Strokology, Department of Internal Medicine and Therapeutics (A8), Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita City, Osaka 565-0871, Japan. E-mail kitagawa{at}medone.med.osaka-u.ac.jp

	Abstract

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Background and Purpose—— Recently, there has been great interest in adult neurogenesis. We investigated whether transient forebrain ischemia could influence the proliferation of neuronal progenitor in the subgranular zone (SGZ) of the rat hippocampus and whether aging could influence the neurogenesis after ischemia.

Methods—— Male Wistar rats were subjected to 4-vessel occlusion model. We used a bromodeoxyuridine (BrdU) labeling method to identify the postproliferation cells and double-immunostaining with confocal microscopy to determine the cell phenotype.

Results—— The number of BrdU-positive cells in the SGZ increased 5.7-fold 8 days after ischemia, compared with the control. BrdU-positive cells formed clusters, which suggested that these cells had divided from an original progenitor cell, and expressed Musashi1 (Msi1), a marker of neural stem/progenitor cells. Although astrocytes also expressed Msi1 in the adult brain, Msi1-positive cells that formed clusters in the SGZ did not express glial fibrillary acidic protein, an astrocyte marker. About 70% of all BrdU-positive cells in the SGZ represented the neuronal phenotype 4 weeks after the BrdU injection. Although proliferation of progenitor cells was stimulated in both young and older animals, aging accelerated the reduction in newborn cells after ischemia.

Conclusions—— Our results indicate that ischemic stress stimulated the proliferation of neuronal progenitor cells in the SGZ of both young and old rats but resulted in increased neurogenesis only in young animals. Our findings will be important in developing therapeutic intervention to enhance endogenous neurogenesis after brain injury.

Key Words: aging • cerebral ischemia, global • cerebral ischemia, transient • hippocampus • neurogenesis • rats

	Introduction

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Persistent neurogenesis occurs in the adult mammalian brain throughout life,^1–3 including in humans.⁴ Adult neurogenesis is observed in 2 limited areas: the subgranular zone (SGZ) in the hippocampus^1–4 and the rostral migratory stream from the subventricular zone (SVZ) to the olfactory bulb.^5,6 This finding suggests the potential ability of the brain to repair itself.⁷ This attractive hypothesis has facilitated research into adult neurogenesis, and it has been reported that several factors affect the activity of neurogenesis in the SGZ. Neurogenesis in the hippocampus is increased by learning, running, and an enriched environment.^8–10 Some pathological states, including adrenalectomy,¹¹ seizure,¹² mechanical dentate gyrus lesions,¹³ and ischemic insult,¹⁴ also increase hippocampal neurogenesis. Because ischemic brain injury frequently disrupts neural function in the central nervous system, it is very important to understand the response of neuronal progenitor cells after ischemia. Recently, Liu et al¹⁴ reported that neurogenesis increased in the adult gerbil brain after ischemia. However, there is no direct evidence of the increased proliferation of neuronal progenitor cells after ischemia. Because many glial cells are activated and proliferate in the hippocampus after transient forebrain ischemia, a suitable marker of neuronal progenitor cells is necessary to identify BrdU-labeled cells divided from neuronal progenitor cells.

In the present study, we evaluated the (1) activity of cell proliferation in the rat SGZ after ischemia, (2) proliferation of the neuronal progenitor cells using a Musashi-1 (Msi1) antibody, (3) neuronal phenotype expression in newborn cells, and (4) influences of aging on the survival rate of postproliferation cells after ischemia. Msi1 is a neural RNA-binding protein¹⁵ that is expressed in the neuronal progenitor cells and astrocytes in the adult mammalian brain.^16–18 The double-labeling technique of Msi1 and glial fibrillary acidic protein (GFAP) can demonstrate neuronal progenitor cells: neuronal progenitor cells express Msi1 without GFAP immunoreactivity. Aging influences neurogenesis in the SGZ,¹⁹ and ischemic stroke is so common in the aged brain that we also investigated birth from progenitor cells and subsequent reduction of newborn cells in the SGZ to evaluate the influences of aging on ischemia-induced neurogenesis.

	Materials and Methods

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Animal Model
Adult male Wistar rats (3 to 4 months or 1 year old) were used in the present study. The experimental protocol was approved by the Institutional Animal Care and Use Committee of Osaka University Graduate School of Medicine. Transient forebrain ischemia was induced through 4-vessel occlusion and reperfusion as previously described.^20,21 On the first day, rats were anesthetized with pentobarbital (45 mg/kg IP), and both vertebral arteries were cauterized through each alar foramen with an electrocautery needle. After recovery from anesthesia, rats had free access to water but were without food overnight. On the second day, rats were anesthetized with 1% inhaled halothane, and both common carotid arteries were occluded with miniature aneurysmal clips for 10 minutes. Only rats that showed loss of consciousness, pain reflex, and corneal reflex were chosen for the present study. Rats with respiratory distress were excluded. Rectal temperature was continuously monitored during ischemia and was maintained at 37°C to 37.5°C with an overhead heating lamp for up to 1 hour after removal of the clips. We also monitored the skull temperature with a needle thermometer probe placed on the parietal area of the skull and maintained it at 36.0°C during ischemia. Sham-operated animals went through only the procedure of the first day and carotid exposure without clipping.

Bromodeoxyuridine Labeling
We used bromodeoxyuridine (BrdU) (Boehringer Mannheim), a thymidine analog, to label proliferating cells. BrdU was incorporated into newly synthesized DNA.

We administered to the rats of the control, sham, and ischemia groups (each time point is 4, 7, 10, 14, or 28 days after ischemia; n=4 for each group) BrdU (50 mg/kg IP) 3 times every 4 hours over a period of 8 hours. The next day (therefore, each time point is 5, 8, 11, 15, or 29 days after ischemia, respectively), the rats were perfused transcardially with 4% paraformaldehyde (PFA) in 50 mmol/L concentration of a phosphate buffer while under deep anesthesia with pentobarbital. To evaluate the phenotype of postmitotic cells, we administered BrdU (50 mg/kg IP) 3 times 7 days after ischemia. Control and ischemic rats were perfused transcardially with 4% PFA 28 days after BrdU injection. Furthermore, we assessed the influences of aging on the birth and residual rate of newborn cells after ischemia; twenty-two 3- to 4-month-old rats and eighteen 1-year-old rats underwent this protocol. They were perfused 1 or 28 days after BrdU (50 mg/kg IP) 3 times at 7 days after ischemia.

Immunohistochemistry
After perfusion with 4% PFA, the brains were removed, cut into coronal blocks containing the hippocampus, and immediately immersion-fixed in 4% PFA. Coronal sections (30 µm thick) were cut on a vibratome.

The brain sections were incubated with a primary antibody diluted with TBS/0.1% Triton X-100 at 4°C overnight. After being washed in TBS/0.1% Triton X-100, the sections were incubated with a biotinylated secondary antibody for 1 hour at room temperature. They were washed and further incubated with a streptavidin-biotin-peroxidase complex (Vector Laboratories). The peroxidase reaction was carried out via incubation with diaminobenzidine and hydrogen peroxide. We used the following antibodies as primary antibodies: mouse monoclonal anti-BrdU antibody (1:100; Amersham), rat monoclonal anti-BrdU antibody (1:200; Harlan Sera-Labo), mouse monoclonal anti-NeuN antibody (1:200; Chemicon), rabbit polyclonal anti-GFAP antibody (1:200; Sigma Chemical Co), and rat monoclonal Msi1 antibody.¹⁸

For BrdU immunohistochemistry, DNA denaturing was required. Free floating sections were treated in 50% formamide and a 2x saline-sodium citrate buffer at 65°C for 2 hours. After washing in the 2x saline-sodium citrate buffer, sections were incubated in 2N HCl at 37°C for 30 minutes. Sections were rinsed in TBS/0.1% Triton X-100 for 20 minutes and incubated with an anti-BrdU antibody at 4°C overnight. The subsequent procedure was the same as that for other immunohistochemistry.

Double-immunostaining was performed with immunofluorescence and confocal microscopy (Zeiss). For double labeling of BrdU and cell markers (NeuN, neuron; GFAP, astrocyte; Msi1, neuronal progenitor cells and astrocytes), sections were incubated with an anti-BrdU antibody and antibodies for each cell marker at 4°C overnight after DNA denaturation. FITC- or rhodamine-labeled goat anti-IgG antibodies were used as the secondary antibodies. The combination of antibodies used in each double-immunostaining experiment was (1) rat anti-BrdU antibody and mouse anti-NeuN antibody as primary antibodies and rhodamine-labeled anti-rat IgG antibody and FITC-labeled anti-mouse IgG as secondary antibodies for BrdU-NeuN; (2) mouse anti-BrdU antibody and rabbit anti-GFAP antibody as primary antibodies and FITC-labeled anti-mouse IgG antibody and rhodamine-labeled anti-rabbit IgG antibody as secondary antibodies for BrdU-GFAP; (3) mouse anti-BrdU antibody and rat anti-Msi1 antibody as primary antibodies and FITC-labeled anti-mouse IgG antibody and rhodamine-labeled anti-rat IgG antibody as secondary antibodies for BrdU-Msi1; and (4) rat anti-Msi1 antibody and rabbit anti-GFAP antibody as primary antibodies and FITC-labeled anti-rat IgG antibody and rhodamine-labeled anti-rabbit IgG antibody as secondary antibodies for Msi1-GFAP.

Quantification
To count BrdU-positive cells colored by the peroxidase reaction, 5 sections from each hippocampus were obtained every 150 µm beginning at a section 1.5 mm caudal to the bregma. The granular cell layer (GCL) (60 µm) and SGZ, defined as a 2-cell body wide zone (10 µm) along the border of the GCL and the hilus, were always combined for quantification. The mean density of BrdU-labeled cells in each mouse was calculated as the number of labeled nuclei divided by the area. Statistical analysis was performed using ANOVA followed by Scheffé’s post hoc tests.

To assess what percentage of newborn cells acquire the neuronal phenotype after ischemia, we used a double-immunostaining technique. We detected BrdU-positive cells in the SGZ and GCL and determined whether they expressed NeuN signals with confocal microscopy. Double positive percentage was calculated as BrdU/NeuN-positive cells for total BrdU-positive cells. The mean values for data were obtained in 8 sections from 4 rats. The ischemic group rats were administered BrdU 7 days after ischemia, and both groups of rats were killed 28 days after BrdU labeling.

	Results

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Cell Mitotic Activity in the Hippocampus After Transient Forebrain Ischemia
A few BrdU-positive cells were observed in the SGZ of hippocampal dentate gyrus in the control (Figure 1A). The temporal and regional profiles of BrdU immunoreactivity did not differ significantly between control and sham-operated animals (data not shown). The number of BrdU-positive cells started to increase 5 days after ischemia in the SGZ. Only a few BrdU-positive cells were observed 8 days after ischemia in the dentate hilus. However, BrdU-positive cells in the SGZ continued to increase (Figure 1B). In addition, BrdU-positive cells formed clusters in this region (Figure 1C), which suggested that these cells had divided from an original progenitor cell. Semiquantitative analysis showed that cell mitotic activity in the SGZ reached a peak 8 days after ischemia (Figure 1D), and the number of BrdU-positive cells increased 5.7-fold compared with the control. Cell mitotic activity in the SGZ declined to the level of control rats 15 days after ischemia.

View larger version (65K): [in this window] [in a new window]   Figure 1. Postproliferation cells in the SGZ. All rats were administered BrdU (50 mg/kg IP) 3 times 1 day before they were killed. Note the marked increase in the BrdU-positive cells in the SGZ and GCL 8 days after ischemia (B), compared with the control (A). At this time point, few BrdU-positive cells were observed in the hilus and the molecular layer. BrdU-positive cells formed clusters in the SGZ (C). It seemed that these cells were divided from an original progenitor cell. Quantitative analysis showed that BrdU-positive cells increased 5.7-fold 8 days after ischemia compared with the control (D). In our experiments, the activity of cell proliferation was maximal at this time point. Five days after ischemia, BrdU-positive cells also increased significantly. Because many glial cells were still proliferating at this time point in the dentate gyrus, these BrdU-positive cells may have included not only proliferative neuronal progenitor cells but also glial cells. The significance of differences was determined using ANOVA followed by Scheffé’s post hoc test. Error bars indicate SD. *P<0.05 compared with the control group. D, C indicates control animal; 5, 8,11,15, and 29, 5, 8, 11, 15, and 29 days after ischemia. Bar (A and for B) 0.5 mm, bar (C) 30 µm.

Stimulation of Cell Division From the Msi1-Positive Neuronal Progenitor Cells
In the control and ischemic animals, many cells expressing Msi1 were observed in the hippocampus. Because both neuronal progenitor cells and astrocytes express Msi1,¹⁷ it seems that the majority of Msi1-positive cells in the hippocampus are astrocytes and that some cells expressing Msi1 in the SGZ are neuronal progenitor cells. We used the double-labeling technique to distinguish these 2 cell types. We demonstrated that BrdU-positive cells colocalized with Msi1 in the SGZ (Figures 2A to 2C), but not in the CA1 region (Figures 2D to 2F), 8 days after ischemia. Msi1-positive cells, which formed clusters in the SGZ, did not colocalize with GFAP (Figures 3A to 3C), whereas all of the Msi1-positive cells colocalized with GFAP in the CA1 region (Figures 3D to 3F).

View larger version (33K): [in this window] [in a new window]   Figure 2. Proliferation and cluster formation of Msi1-positive cells in the SGZ. To label newborn cells, we administered to the rats BrdU (50 mg/kg IP) 3 times 7 days after ischemia and were killed the next day after BrdU injection. In the SGZ (BrdU [A], Msi1 [B], BrdU/Msi1 [C]), BrdU-positive cells (green in A), which formed clusters, colocalized with Msi1 (red in B), as indicated with arrows (yellow in C). In contrast, in the CA1 region (BrdU [D], Msi1 [E], BrdU/Msi1 [F]), BrdU (arrows in D and F) and Msi1-positive cells (arrowheads in E and F) did not colocalize. Bar (for A to F) 30 µm.

View larger version (31K): [in this window] [in a new window]   Figure 3. Cluster-formed Msi1-positive cells did not colocalize with GFAP in the SGZ 8 days after ischemia. To confirm Msi1-positive cells were not astrocytes, we performed double-immunolabeling with Msi1 and GFAP. In the SGZ (Msi1 [A], GFAP [B], Msi1/GFAP [C]), Msi1-positive cells (green in A and C, an arrow), which formed clusters, did not colocalize with GFAP (red in B and C, arrowhead). In contrast, in the CA1 region (Msi1 [D], GFAP [E], Msi1/GFAP [F]), all Msi1-positive cells (green in D) colocalized with GFAP (red in E), as indicated with arrows in F. Bar (for A to F) 30 µm.

Enhanced Neurogenesis in the Dentate Gyrus After Ischemia
There were no BrdU-positive cells showing immunofluorescence for GFAP or NeuN in the SGZ or GCL 8 days after the BrdU injection (data not shown). We observed that BrdU-positive postmitotic cells derived from neuronal progenitor cells expressed the neuronal antigenic phenotype 28 days after BrdU injection (Figures 4A to 4C). In this region, >70% of all BrdU-positive cells represented the neuronal phenotype 28 days after BrdU injection in both the control and ischemia groups of young rats (Table 1). The number of newborn neurons, which could be demonstrated by BrdU-NeuN double-positive findings, on the single section clearly increased in the ischemia group compared with the control group (control 8.4±2.9, ischemia 39.5±8.2). On the other hand, BrdU-GFAP double-positive cells were rarely observed (Figure 4D). In this time point, the BrdU-positive cell nuclei were large and round (Figures 4E and 4F), which was in contrast to the immature cells immediately after proliferation, which were small and irregular (Figure 1C).

View larger version (55K): [in this window] [in a new window]   Figure 4. Neuronal differentiation of newborn cells after ischemia. We administered to the rats BrdU (50 mg/kg IP) 3 times 7 days after ischemia and were killed 28 days after BrdU injection. In the SGZ and GCL (NeuN [A], BrdU [B], NeuN/BrdU [C]), arrows indicate newborn neurons, which show NeuN (green in A) and BrdU (red in B) double-positive findings (yellow in C). In contrast, BrdU (green) and GFAP (red) immunofluorescence did not colocalize in the SGZ (BrdU/GFAP [D]). Newborn neurons that migrated into the GCL were large and round, similar to mature granular cells (E and F). Bar (A, for B and C, and D and F) 30 µm, bar (E) 0.5 mm.

View this table: [in this window] [in a new window]   Table 1. Rate of Differentiation Into the Neuron 28 Days After BrdU Labeling in the SGZ and GCL

Effects of Aging on the Proliferation of Neuronal Progenitor Cells and Reduction in Newborn Neurons After Ischemia
Transient forebrain ischemia for 10 minutes resulted in selective neuronal death in the CA1 and CA4 sector of the hippocampus in both young (3 to 4 months old) and older (1 year old) rats. The severity of neuronal damage was similar in both groups. In the SGZ of control animals, the larger number of BrdU-positive cells was observed in the young group than in the older group 1 day after BrdU administration; the number of newborn cells, on the other hand, increased significantly after ischemia in both the young group (5.7-fold) and the older group (10.6-fold) (Table 2). Residual rates 28 days after BrdU injection were almost the same (60% to 80%) in the control and ischemic rats of the young group. Therefore, there were significantly more BrdU-positive cells 28 days after BrdU injection in the ischemia group than in control group in the young SGZ (Table 2). In contrast, the residual rate was markedly lower in the ischemic rats of the older group (15.3%) than in the control group (43.7%). Therefore, BrdU-positive cells in the older SGZ after ischemia were markedly reduced 35 days after ischemia, and the number of BrdU-positive cells after ischemia did not differ from that of the control in the older group (P=0.069) (Table 2). These results indicated that the mitotic activity of neuronal progenitor cells, which was responsive to ischemic injury, remained preserved even in the hippocampus of older animals. However, the reduction in newborn cells from neuronal progenitor cells was accelerated in the older animals.

View this table: [in this window] [in a new window]   Table 2. Birth and Reduction in Newborn Cells After Ischemia

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In the present study, we demonstrated that neurogenesis in the SGZ increased after transient forebrain ischemia and that postmitotic cells labeled with BrdU formed clusters and expressed Msi1, a neural progenitor cell marker, including neural stem cells and neuronal progenitor cells.^15–18 Expression of the neural progenitor cell marker in the SGZ after ischemia has not been reported, and this is the first evidence of neuronal progenitor cells stimulated by ischemia. This response may be involved in the remodeling process of the hippocampus and functional recovery. Also, we showed that neurogenic cell proliferative activity was still preserved in the SGZ of 1-year-old rats. In the senior rats, however, newborn cells in the SGZ did not survive. Our findings concerned the hippocampus but also may be relevant to other brain areas because adult neurogenesis was recently reported in the cerebral cortex.²² In addition, the present findings might provide a clue for development of the new therapeutic approach not only to control the proliferation of progenitor cells but also to preserve newborn cells. In our rat model, mitotic activity of the neuronal progenitor cells was maximum 8 days after ischemia. Liu et al¹⁴ reported that neurogenesis was maximum 11 days after ischemia in the gerbil SGZ. This difference may be due to species differences. In addition, the gerbil is susceptible to seizure after ischemia.²³ Because seizure alone increases neurogenesis in the SGZ, seizure during and after ischemia may influence ischemia-induced neurogenesis in gerbils.

Evidence of Increased Proliferation of Neuronal Progenitor Cells After Ischemia
In this study, we used Msi1 as the marker of neuronal progenitor cells. Msi1, a neural RNA-binding protein, was cloned by Sakakibara et al.¹⁵ They reported that the level of Msi1 mRNA was very high in the embryonal brain and gradually decreased during the course of development. It is suggested that Msi1 may posttranscriptionally regulate the expression of common key genes that determine the fates of progenitor cells.²⁴ In the adult brain, Msi1 was expressed in the neural stem/neuronal progenitor cells and the astrocytes, but not in the microglia or oligodendroglia.¹⁶ Because astrocytes expressed GFAP, neuronal progenitor cells can be detected using the Msi1/GFAP double-immunostaining technique, and neuronal progenitor cells can be demonstrated as Msi1-positive and GFAP-negative cells. Because microglia and astrocytes were activated and proliferated in the whole hippocampus after ischemia, BrdU-positive cells in the SGZ after ischemia may divide not only from progenitor cells but also from glial cells. We showed for the first time proliferation stimulation of neuronal progenitor cells after ischemia using double immunostaining, including Msi1. Nestin, an intermediate filament protein, has also been used as a marker of neural progenitor cells, including neural stem cells.²⁵ In the adult brain, nestin is expressed not only in neural stem cells in the subependymal zone but also in reactive astrocyte, in a similar manner as Msi1.^5,16,17 In our preliminary experiment, we compared immunostaining with Msi1 and nestin in the control and ischemic hippocampus. In contrast to the results of Msi1 (Figures 2 and 3), immunoreaction for nestin was not observed in cluster-forming progenitor cells in the SGZ. Although immunoreaction for nestin after cerebral ischemia was previously investigated,^26,27 no studies with a commercially available antibody (Rat-401; PharMingen) demonstrated nestin localization in the progenitor cells in the SGZ. It is probable that the progenitor cells in the SGZ, in contrast to those in the SVZ, express Msi1 but not nestin; however, future studies will be required to clarify the precise character of these cells because a recent study with transgenic mice that express green fluorescence protein (GFP) under nestin promoter demonstrated GFP signals in the SGZ.²⁸ Furthermore, immunostaining with Msi1 may have an advantage for double-staining with BrdU because of its nuclear localization.

Aging Affects Neurogenesis After Ischemia Through Reduction of Newborn Cells
We observed that proliferation of neuronal progenitor cells increased after ischemia even in the 1-year-old rats. Although aging is a factor that decreases neurogenesis in the adult normal SGZ,¹⁹ our present data indicated that the decrease in neurogenesis due to aging was not due to the reduction in the neuronal progenitor cell population but rather to the disturbed survival of newborn cells. Recently, it was shown that the levels of corticosteroids were higher in the older brain and that the decreasing corticosteroid levels in aged rats increased proliferation of neuronal progenitor cells but not newborn neurons expressing polysialylated isoforms of neural cell adhesion molecule.^29,30 Therefore, it seems likely that aged brains contain factors that inhibit the survival of newborn cells. Alternatively, they lack factors that induce neuronal differentiation or support the survival of newborn cells. Because regulation of neurogenesis may be a target for therapeutic approaches for functional recovery, it is very important to explore factors that affect neurogenesis in in vivo animal models.

Is Modification of Endogenous Neurogenesis a Therapeutic Strategy for the Injured Brain?
The neuronal progenitor cells have been isolated from the adult rat and human brain and can generate new neurons under the presence of fibroblast growth factor-2 (FGF-2) or brain-derived neurotrophic factor in vitro, or both.^31,32 FGF-2 is a neurotrophic factor, and it can induce adult neurogenesis in vivo.^33,34 FGF-2 and FGF receptors are upregulated in the dentate gyrus 7 days after transient forebrain ischemia.³⁵ Increases in FGF-2 and FGF receptor expressions are concomitant with proliferation of neuronal progenitor cells. In addition, other neurotrophic factors (nerve growth factor and brain-derived neurotrophic factor) could be induced after cerebral ischemia.³⁶ These neurotrophic factors may stimulate the proliferation of neuronal progenitors, neuronal differentiation, or neuronal survival in the SGZ after ischemia. Recently, it was reported that subcutaneous injection of basic FGF could stimulate in vivo neurogenesis in the neonatal and adult rat brain.³³ Neurotrophin administration could be a new therapeutic approach to enhance endogenous neurogenesis in patients with stroke and other central nervous system diseases. It has been reported that several factors can affect the process of neurogenesis (proliferation of neuronal progenitor cells or survival of newborn neurons) in the SGZ. Learning hippocampus-dependent tasks,⁹ running,¹⁰ and an enriched environment⁸ are factors that increase neurogenesis. Interestingly, it was further demonstrated that running enhanced not only neurogenesis but also learning and synaptic plasticity.³⁷ Although it remains to be elucidated whether adult neurogenesis contributes to reorganization of the higher functions in the injured brain, newborn neurons and new neural circuits may lead to functional recovery with rehabilitation, which is commonly used for stroke patients.

	Acknowledgments

 
The present study was supported in part by a grant-in-aid from the Ministry of Education, Science and Culture. The authors wish to thank Dr Yanagihara (Osaka Neurological Disease Institute) for his constant interest and guidance in this investigation and Y. Imaeda and R. Morimoto for their secretarial assistance.

Received March 2, 2001; revision received May 8, 2001; accepted May 15, 2001.

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