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Induction of highly polysialylated neural cell adhesion molecule (PSA-NCAM) in postischemic gerbil hippocampus mainly dissociated with neural stem cell proliferation
Brain Research 902 (2001) 288–293 www.elsevier.com / locate / bres
Short communication
Induction of highly polysialylated neural cell adhesion molecule (PSA-NCAM) in postischemic gerbil hippocampus mainly dissociated with neural stem cell proliferation Masanori Iwai, Takeshi Hayashi, Wen Ri Zhang, Keiko Sato, Yasuhiro Manabe, Koji Abe* Department of Neurology, Okayama University Medical School, 2 -5 -1 Shikatacho, Okayama 700 -8558, Japan Accepted 13 March 2001
Abstract We investigated a possible expression of highly polysialylated neural cell adhesion molecule (PSA-NCAM) in gerbil hippocampus after 5 min of transient global ischemia in association to the proliferation of neural stem cell labeled with bromodeoxyuridine (BrdU). The number of PSA-NCAM positive cells increased in the granule cell layer (GCL) of dentate gyrus (DG) by 1.9 to 2.7-fold at 10 and 20 days after the reperfusion. The number of BrdU-labeled cells increased mainly in the subgranular zone of DG by 7.2 to 8.0-fold at 5 and 10 days after the reperfusion. Immunofluorescence for PSA-NCAM and BrdU showed that the majority of DG cells were not double labeled, while one or two cells per section were double labeled in the deepest portion of the GCL only at 10 days after the reperfusion. These results suggest different predominant spatial distribution and chronological change of PSA-NCAM positive and BrdU-labeled cells in DG after transient ischemia. 2001 Elsevier Science B.V. All rights reserved. Theme: Development and regeneration Topic: Cell differentiation and migration Keywords: Polysialylated acid; Neurogenesis; Transient global ischemia; Hippocampus
In most regions of the mammalian brain, the production of neurons is largely confined to the prenatal period. However, the dentate granule cells continue to be produced during the adult period in the innermost region of the granule cell layer (GCL) and the subgranular zone (SGZ) [3,4,14,15,23,24]. Recent studies have shown that adult neurogenesis is increased by an exogenous neurotrophic factor supplement [7], seizures [20], kindling [16], an enriched environment [22] and transient global ischemia
Abbreviations: BrdU, bromodeoxyuridine; CCAs, common carotid arteries; DG, dentate gyrus; D3V, dorsal third ventricle; FITC, fluorescein-isothiocyanate; GCL, granule cell layer; GFAP, glial fibrillary acidic protein; MAP-2, microtubule-associated protein 2; NeuN, neuronal nuclear antigen; PSA-NCAM, highly polysialylated neural cell adhesion molecule; SGZ, subgranular zone; SVZ, subventricular zone; TRITC, tetramethylrhodamine-isothiocyanate *Corresponding author. Tel.: 181-86-235-7365; fax: 181-86-2357368. E-mail address: [email protected] (K. Abe).
[17,26], and is decreased by stress [12], excitatory amino acids [5], and adrenal steroids [6]. PSA-NCAM is a well-known highly polysialylated form of glycoprotein that mediates cell–cell adhesion and recognition. The PSA-NCAM expression occurs during the period of development on the dendrites and cell bodies of the newly generated granule cells, and the expression decreases but maintains in adult brain [23]. Recent reports showed that the PSA-NCAM expression in normally developing cells was affected by spatial learning [10,19] and adrenal steroid [21]. The thymidine analog bromodeoxyuridine (BrdU) is incorporated into DNA of dividing cells in the S phase of the mitotic cycle and is used as an early marker of cell proliferation. In Mongolian gerbils, neural stem cell proliferation was detected with BrdU labeling in the dentate gyrus (DG) after 10 min of transient global ischemia, and became a maximum at 11 days with a 12-fold increase of BrdU-labeled nuclei [17]. Recent reports have shown a relation between PSANCAM and BrdU in normally developing rodent brain
0006-8993 / 01 / $ – see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 01 )02399-X
M. Iwai et al. / Brain Research 902 (2001) 288 – 293
[1,2,11,15,23,24]. However, it is not clear whether transient ischemia might affect PSA-NCAM expression of the granule cells in DG. Furthermore, the relation between PSA-NCAM and BrdU has not been investigated after transient global ischemia. In the present study, therefore, we examined a possible expression of PSA-NCAM in the DG after transient global ischemia of gerbils in association to the proliferation of neural stem cells labeled with BrdU. Adult male Mongolian gerbils (11 weeks of age) were used for this study. The animals were lightly anesthetized by inhalation of a nitrous oxide / oxygen / halothane (69%:30%:1%) mixture. After a neck incision, both common carotid arteries (CCAs) were exposed. When the animals began to awake after the stop of the anesthesia, both CCAs were occluded with aneurysm clips for 5 min, the clips were then removed to restore cerebral blood flow. The rectal temperature was maintained at 37.08C with a heating pad during the operation. Sham-control animals were treated identically, except for the occlusion of CCAs. The animals were then allowed free access to water and food at ambient temperature. At 5, 10 or 20 days after the blood flow restoration, the animals were killed by use of deep anesthesia with diethyl ether (n53 at each time point). At 24 h before each sampling, a single dose of BrdU (50 mg / kg; Sigma, MO) was intraperitoneally administered. The animal brains were quickly dissected and frozen in powdered dry ice. Three sham-operated animals were killed at 10 days. The brain was serially cut in 20-mm-thick coronal sections on a cryostat at 2208C. The brain slices were mounted on glass slides coated with polylysine, and then were stored at 2808C until use. For immunohistochemical detection of PSA-NCAM, the above sections were fixed in acetone, and were incubated in 0.3% H 2 O 2 for 30 min and blocked with 10% normal goat serum for 30 min. Sections were then incubated overnight at 48C with anti-PSA-NCAM mouse IgM monoclonal antibody (diluted 1:500, [24]). Then, the sections were incubated in biotinylated goat anti-mouse IgM (diluted 1:200; Vector Laboratories, CA) for 2 h at room temperature. For immunohistochemical detection of BrdU, the sections were treated in 2 N HCl for 15 min to denature DNA, and incubated in 2% H 2 O 2 for 20 min and with 10% normal horse serum for 30 min. The sections were incubated with anti-BrdU antibody (diluted 1:50, Calbiochem, Germany) for 75 min, and were then incubated with biotinylated horse anti-mouse IgG (diluted 1:200; Vector Laboratories) for 45 min. Then, both sections were placed in horseradish peroxidase / streptoavidin / biotin complex solution (Vectastain ABC Kit, Vector Laboratories) for 30 min, and were incubated for 2 min in a peroxidase reaction solution (0.02% diaminobenzidine, 0.02% H 2 O 2 ). For the immunofluorescence detection of PSA-NCAM and BrdU, sections were first incubated with the anti-PSANCAM antibody for 36 h at 48C, followed by the FITClabeled goat anti-mouse IgM (diluted 1:100, Sigma) for 2
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h. To denature DNA in the sections, the sections were then treated with 2 N HCl at room temperature for 10 min, that is the minimum period to detect BrdU while maintaining PSA-NCAM immunoreactivity. The sections were incubated with anti-BrdU antibody for 12 h at room temperature, followed by the TRITC-labeled goat anti-mouse IgG (diluted 1:200; Sigma) for 1 h. For detecting double labeling of FITC and TRITC, the sections were viewed under Olympus microscopy (BX50; Olympus, Japan) equipped with rhodamine and FITC filter sets. Images (102431024 pixels) were obtained by Hamamatsu Aquacosmos System (Hamamatsu Photonicus, Japan). PSA-NCAM positive cells and BrdU-labeled cells in the hippocampal DG, CA1 region and the sub-ventricular zone (SVZ) of the dorsal third ventricle (D3V) were counted in four sections per gerbil and averaged in each group. The statistical analysis was assessed by a Student’s t-test. Immunohistochemistry for PSA-NCAM showed that PSA-NCAM positive cells were detected in the GCL, but not in SGZ of DG in sham-control animals (Fig. 1a, b and f). The number of PSA-NCAM positive cells began to increase in the GCL at 5 days after the transient ischemia, and continued to increase at 10 (P,0.05) and 20 (P,0.01) days (Table 1). PSA-NCAM positive cells were mainly located in the deepest portion of GCL at the control (Fig. 1f, arrowhead) and 5 days of reperfusion (Fig. 1g, arrowhead), while outer cells of GCL became more positive at 10 to 20 days after the transient ischemia (Fig. 1d–i, arrows). Staining of the PSA-NCAM positive dendrite found in the control (Fig. 1f, open arrowhead) declined transiently at 5 days (Fig. 1g) after the ischemia. On the other hand, PSA-NCAM positive cell was not found in CA1 region or SVZ of the D3V in brains of sham-control and after ischemia (Table 1). BrdU-labeled cells were detected in DG and SVZ of D3V, but not in CA1 region and dentate hilus in shamcontrol animals (Fig. 1j and k). The BrdU-labeled cells in DG were mainly (90–95%) located in the SGZ and partially (5–10%) in the deepest portion of the GCL (Fig. 1k). The BrdU-labeled cell number increased after the transient global ischemia at 5 days (P,0.01, Fig. 1l), and reached the maximum at 10 days (P,0.01, Fig. 1m) with eightfold in number (Table 1). Thereafter, the number of BrdU-labeled cells decreased to the sham-control level by 20 days (Fig. 1n). In the SVZ of the D3V, BrdU-labeled cells were slightly increased at 5 days (P,0.05) and then gradually decreased by 20 days (Table 1). In CA1 region, BrdU-labeled cells began to be detected outside the CA1 pyramidal cell layer after the transient ischemia at 5 days, and reached the maximum at 10 days (Table 1). In the dentate hilus, BrdU-labeled cells were also increased at 5 and 10 days after the ischemia (Fig. 1l and m). On the spatial characteristics of PSA-NCAM and BrdU positive cells, PSA-NCAM positive cells were predominantly located in the GCL (Figs. 1b–e, 2a), while BrdUlabeled cells were mainly located in the SGZ and partially
M. Iwai et al. / Brain Research 902 (2001) 288 – 293
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Fig. 1. Immunohistochemistry for PSA-NCAM (panels a–i) and BrdU (panels j–n) in gerbil hippocampus of sham-control (a, b, f, j, k) and at 5 (c, g, l), 10 (d, h, m), and 20 (e, i, n) days after the transient ischemia. PSA-NCAM positive cells increased in the deepest portion of the granule cell layer (GCL, arrowheads in panels c–i) and in the outer cells of the GCL (arrows in panels d, e, h, i). Open arrowhead in panel f represents dendrites of granule cells positive for PSA-NCAM. BrdU-labeled cells increased in the SGZ of the DG after ischemia (arrowheads in panels l, m). Note delayed peak of PSA-NCAM positive cells (d, e) compared with that of BrdU-labeled cells (l, m). CA1 and CA3, fields CA1 and CA3 of Ammon’s horn; GCL, granule cell layer; H, hilus; ML, molecular layer. Scale bar5500 mm in a and j; 250 mm in b–e and k–n; 20 mm in f–i.
Table 1 Number of PSA-NCAM positive and BrdU-labeled cells in the hippocampal dentate gyrus (DG), CA1 region and the subventricular zone (SVZ) after transient global ischemia Time after ischemia Sham 5 days 10 days 20 days
PSA-NCAM positive cells
BrdU-labeled cells
DG
CA1
SVZ
DG
CA1
SVZ
129.8626.1 178.8639.9 247.2637.7* 349.5680.9**
0 0 0 0
0 0 0 0
12.165.2 86.7631.0** 97.0640.9** 12.368.0
0 4.666.9** 8.564.3** 1.461.4**
0.961.6 2.561.8* 1.661.3 0.861.5
Data are expressed as mean6S.D. of bilateral hemisphere. n53 at each time point. Significant differences from the sham *P,0.05, **P,0.01.
M. Iwai et al. / Brain Research 902 (2001) 288 – 293
Fig. 2. Double immunofluorescence labeling of PSA-NCAM (panel a, green) and BrdU (panel b, red) in the hippocampal first row of the granule cell layer at 10 days after the transient global ischemia. The merged image (panel c) shows a double-stained cell. Scale bar510 mm in c (also applies to a and b).
in the deepest portion of GCL (Figs. 1k–n, 2b). Immunofluorescence for PSA-NCAM with FITC and BrdU with TRITC showed that the majority of the DG cells were not double labeled, while one or two BrdU-labeled nuclei per section were found within the PSA-NCAM positive cell bodies in the deepest portion of the GCL only at 10 days after the reperfusion (Fig. 2c). PSA-NCAM is usually expressed during normal development in the dendrites and cell bodies of the newly generated dentate granule cells. However, it is also expressed in mossy fiber of granule cells in adult rat DG, playing an important role in the migration of neural stem cells [23,25]. The present results showed that the number of PSA-NCAM positive cells significantly increased in the GCL after the transient global ischemia. PSA-NCAM was
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first increased in the deepest cells of GCL, and then was gradually spread at the outer cells of GCL. These results suggest an activation of cell migration in GCL, or a reverting back of the mature granule cells to immature cells after the transient ischemia. In fact, differentiated cells such as ependymal cells or astrocytes can be neuronal progenitors in adult brain [8,13]. Transient reduction of the PSA-NCAM immunoreactivity in the dendrites of GCL (Fig. 1g) suggests a possibility that the polysialylic acid production or polysialyltransferase activity is depressed under the influence of ischemia. A recent report showed that PSA is involved in migration of neural precursors, lamination of mossy fibers, and longterm potentiation and long-term depression in CA1 [9]. Hippocampal neural activity and synaptic reorganization may be impaired while the reduction of the PSA-NCAM immunoreactivity and signaling mechanisms may regulate the change in polysialylation state. Dividing cells can be detected by the incorporation of BrdU after its intraperitoneal administration. A previous report showed a 12-fold increase of BrdU incorporation at the SGZ of DG between 1 and 2 weeks after 10 min of transient global ischemia in gerbils [17]. The present results showed an eightfold increase of BrdU incorporation at SGZ and GCL of DG between 5 and 10 days after 5 min of global ischemia. Because of a shorter period of transient ischemia in the present study, the level of increase in the BrdU-labeled cells was smaller and the peak time interval was shorter than the previous study. BrdU-labeled cells also increased in dentate hilus, CA1 region and SVZ of the D3V in the present study, while the change was only slight (Table 1). The stage of neurogenesis in DG of mature brain can be divided into three distinct steps. Firstly, neural precursor cells that reside at the border between the hilus and GCL undergo cell division. An early marker of this proliferation is incorporation of BrdU into DNA of dividing precursor cells. Secondly, newborn cells begin to migrate into the GCL and neuronal processes. This step is accompanied by an expression of PSA-NCAM. Thirdly, the cells are now integrated in GCL and begin to express the neuronal markers such as neuron-specific enolase, neuronal nuclear antigen (NeuN), glial fibrillary acidic protein (GFAP), microtubule-associated protein 2 (MAP-2) and calbindin. The present study showed that the increase of PSANCAM positive cells delayed (10–20 days) as compared with that of BrdU-labeled cells (5–10 days) in the DG (Table 1, Fig. 1), probably reflecting the second migrating and the first dividing steps of neural precursor cells, respectively. In fact, a previous study reported the double labeling of 60% of the BrdU-labeled cells in the GCL and neural markers such as NeuN, MAP-2, and calbindin at the later step 1 month after a transient global ischemia [17]. On the other hand, PSA-NCAM was not detected in the hilus, CA1 region and the SVZ of the D3V although BrdU-labeled cells were found in these regions (Table 1,
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Fig. 1). Therefore, BrdU-labeled cells may not always express PSA-NCAM. Small nuclei labeled with BrdU outside the CA1 pyramidal cell layer (data not shown) may represent microglia as was reported in the stratum radiatum, molecular layer, and the hilus of DG in ischemic gerbils [18]. This is the first report about the relationship between PSA-NCAM and BrdU of the granule cell after transient ischemia (Table 1, Figs. 1 and 2). With immunofluorescence study, the majority of DG cells were not double labeled, while only one or two cells per section were double labeled in the deepest portion of the GCL at 10 days after the ischemia. The results showed that PSANCAM was induced in DG, but was mostly dissociated with BrdU-labeling as a marker of the first step of neural stem cell proliferation. This could be explained by different predominant spatial distribution of PSA-NCAM positive (in GCL) and BrdU-labeled (in SGZ) cells, and chronological change of expression. Alternatively, a single administration of BrdU only detected cells when they were in the S phase of the mitotic cycle (Table 1). Further investigation with serial administration of BrdU may give detailed information of the relationship between the first and second steps of neural stem cell proliferation after ischemia.
Acknowledgements The authors thank Dr. T. Seki (Tokyo, Japan) for his generous gift of the antibody against PSA-NCAM, and Jin Zaishun (Department of Pathology, Okayama University) for her help in taking a photograph of immunofluorescence sections. This work was partly supported by Grant-in-Aid for Scientific Research (B) 12470141 from the Ministry of Education, Science, Culture and Sports of Japan, and by grants (Tashiro K, Itoyama Y) and Comprehensive Research on Aging and Health (H11-Choju-010, No.207, Koizumi A) from the Ministry of Health and Welfare of Japan.
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Short communication
Induction of highly polysialylated neural cell adhesion molecule (PSA-NCAM) in postischemic gerbil hippocampus mainly dissociated with neural stem cell proliferation Masanori Iwai, Takeshi Hayashi, Wen Ri Zhang, Keiko Sato, Yasuhiro Manabe, Koji Abe* Department of Neurology, Okayama University Medical School, 2 -5 -1 Shikatacho, Okayama 700 -8558, Japan Accepted 13 March 2001
Abstract We investigated a possible expression of highly polysialylated neural cell adhesion molecule (PSA-NCAM) in gerbil hippocampus after 5 min of transient global ischemia in association to the proliferation of neural stem cell labeled with bromodeoxyuridine (BrdU). The number of PSA-NCAM positive cells increased in the granule cell layer (GCL) of dentate gyrus (DG) by 1.9 to 2.7-fold at 10 and 20 days after the reperfusion. The number of BrdU-labeled cells increased mainly in the subgranular zone of DG by 7.2 to 8.0-fold at 5 and 10 days after the reperfusion. Immunofluorescence for PSA-NCAM and BrdU showed that the majority of DG cells were not double labeled, while one or two cells per section were double labeled in the deepest portion of the GCL only at 10 days after the reperfusion. These results suggest different predominant spatial distribution and chronological change of PSA-NCAM positive and BrdU-labeled cells in DG after transient ischemia. 2001 Elsevier Science B.V. All rights reserved. Theme: Development and regeneration Topic: Cell differentiation and migration Keywords: Polysialylated acid; Neurogenesis; Transient global ischemia; Hippocampus
In most regions of the mammalian brain, the production of neurons is largely confined to the prenatal period. However, the dentate granule cells continue to be produced during the adult period in the innermost region of the granule cell layer (GCL) and the subgranular zone (SGZ) [3,4,14,15,23,24]. Recent studies have shown that adult neurogenesis is increased by an exogenous neurotrophic factor supplement [7], seizures [20], kindling [16], an enriched environment [22] and transient global ischemia
Abbreviations: BrdU, bromodeoxyuridine; CCAs, common carotid arteries; DG, dentate gyrus; D3V, dorsal third ventricle; FITC, fluorescein-isothiocyanate; GCL, granule cell layer; GFAP, glial fibrillary acidic protein; MAP-2, microtubule-associated protein 2; NeuN, neuronal nuclear antigen; PSA-NCAM, highly polysialylated neural cell adhesion molecule; SGZ, subgranular zone; SVZ, subventricular zone; TRITC, tetramethylrhodamine-isothiocyanate *Corresponding author. Tel.: 181-86-235-7365; fax: 181-86-2357368. E-mail address: [email protected] (K. Abe).
[17,26], and is decreased by stress [12], excitatory amino acids [5], and adrenal steroids [6]. PSA-NCAM is a well-known highly polysialylated form of glycoprotein that mediates cell–cell adhesion and recognition. The PSA-NCAM expression occurs during the period of development on the dendrites and cell bodies of the newly generated granule cells, and the expression decreases but maintains in adult brain [23]. Recent reports showed that the PSA-NCAM expression in normally developing cells was affected by spatial learning [10,19] and adrenal steroid [21]. The thymidine analog bromodeoxyuridine (BrdU) is incorporated into DNA of dividing cells in the S phase of the mitotic cycle and is used as an early marker of cell proliferation. In Mongolian gerbils, neural stem cell proliferation was detected with BrdU labeling in the dentate gyrus (DG) after 10 min of transient global ischemia, and became a maximum at 11 days with a 12-fold increase of BrdU-labeled nuclei [17]. Recent reports have shown a relation between PSANCAM and BrdU in normally developing rodent brain
0006-8993 / 01 / $ – see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 01 )02399-X
M. Iwai et al. / Brain Research 902 (2001) 288 – 293
[1,2,11,15,23,24]. However, it is not clear whether transient ischemia might affect PSA-NCAM expression of the granule cells in DG. Furthermore, the relation between PSA-NCAM and BrdU has not been investigated after transient global ischemia. In the present study, therefore, we examined a possible expression of PSA-NCAM in the DG after transient global ischemia of gerbils in association to the proliferation of neural stem cells labeled with BrdU. Adult male Mongolian gerbils (11 weeks of age) were used for this study. The animals were lightly anesthetized by inhalation of a nitrous oxide / oxygen / halothane (69%:30%:1%) mixture. After a neck incision, both common carotid arteries (CCAs) were exposed. When the animals began to awake after the stop of the anesthesia, both CCAs were occluded with aneurysm clips for 5 min, the clips were then removed to restore cerebral blood flow. The rectal temperature was maintained at 37.08C with a heating pad during the operation. Sham-control animals were treated identically, except for the occlusion of CCAs. The animals were then allowed free access to water and food at ambient temperature. At 5, 10 or 20 days after the blood flow restoration, the animals were killed by use of deep anesthesia with diethyl ether (n53 at each time point). At 24 h before each sampling, a single dose of BrdU (50 mg / kg; Sigma, MO) was intraperitoneally administered. The animal brains were quickly dissected and frozen in powdered dry ice. Three sham-operated animals were killed at 10 days. The brain was serially cut in 20-mm-thick coronal sections on a cryostat at 2208C. The brain slices were mounted on glass slides coated with polylysine, and then were stored at 2808C until use. For immunohistochemical detection of PSA-NCAM, the above sections were fixed in acetone, and were incubated in 0.3% H 2 O 2 for 30 min and blocked with 10% normal goat serum for 30 min. Sections were then incubated overnight at 48C with anti-PSA-NCAM mouse IgM monoclonal antibody (diluted 1:500, [24]). Then, the sections were incubated in biotinylated goat anti-mouse IgM (diluted 1:200; Vector Laboratories, CA) for 2 h at room temperature. For immunohistochemical detection of BrdU, the sections were treated in 2 N HCl for 15 min to denature DNA, and incubated in 2% H 2 O 2 for 20 min and with 10% normal horse serum for 30 min. The sections were incubated with anti-BrdU antibody (diluted 1:50, Calbiochem, Germany) for 75 min, and were then incubated with biotinylated horse anti-mouse IgG (diluted 1:200; Vector Laboratories) for 45 min. Then, both sections were placed in horseradish peroxidase / streptoavidin / biotin complex solution (Vectastain ABC Kit, Vector Laboratories) for 30 min, and were incubated for 2 min in a peroxidase reaction solution (0.02% diaminobenzidine, 0.02% H 2 O 2 ). For the immunofluorescence detection of PSA-NCAM and BrdU, sections were first incubated with the anti-PSANCAM antibody for 36 h at 48C, followed by the FITClabeled goat anti-mouse IgM (diluted 1:100, Sigma) for 2
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h. To denature DNA in the sections, the sections were then treated with 2 N HCl at room temperature for 10 min, that is the minimum period to detect BrdU while maintaining PSA-NCAM immunoreactivity. The sections were incubated with anti-BrdU antibody for 12 h at room temperature, followed by the TRITC-labeled goat anti-mouse IgG (diluted 1:200; Sigma) for 1 h. For detecting double labeling of FITC and TRITC, the sections were viewed under Olympus microscopy (BX50; Olympus, Japan) equipped with rhodamine and FITC filter sets. Images (102431024 pixels) were obtained by Hamamatsu Aquacosmos System (Hamamatsu Photonicus, Japan). PSA-NCAM positive cells and BrdU-labeled cells in the hippocampal DG, CA1 region and the sub-ventricular zone (SVZ) of the dorsal third ventricle (D3V) were counted in four sections per gerbil and averaged in each group. The statistical analysis was assessed by a Student’s t-test. Immunohistochemistry for PSA-NCAM showed that PSA-NCAM positive cells were detected in the GCL, but not in SGZ of DG in sham-control animals (Fig. 1a, b and f). The number of PSA-NCAM positive cells began to increase in the GCL at 5 days after the transient ischemia, and continued to increase at 10 (P,0.05) and 20 (P,0.01) days (Table 1). PSA-NCAM positive cells were mainly located in the deepest portion of GCL at the control (Fig. 1f, arrowhead) and 5 days of reperfusion (Fig. 1g, arrowhead), while outer cells of GCL became more positive at 10 to 20 days after the transient ischemia (Fig. 1d–i, arrows). Staining of the PSA-NCAM positive dendrite found in the control (Fig. 1f, open arrowhead) declined transiently at 5 days (Fig. 1g) after the ischemia. On the other hand, PSA-NCAM positive cell was not found in CA1 region or SVZ of the D3V in brains of sham-control and after ischemia (Table 1). BrdU-labeled cells were detected in DG and SVZ of D3V, but not in CA1 region and dentate hilus in shamcontrol animals (Fig. 1j and k). The BrdU-labeled cells in DG were mainly (90–95%) located in the SGZ and partially (5–10%) in the deepest portion of the GCL (Fig. 1k). The BrdU-labeled cell number increased after the transient global ischemia at 5 days (P,0.01, Fig. 1l), and reached the maximum at 10 days (P,0.01, Fig. 1m) with eightfold in number (Table 1). Thereafter, the number of BrdU-labeled cells decreased to the sham-control level by 20 days (Fig. 1n). In the SVZ of the D3V, BrdU-labeled cells were slightly increased at 5 days (P,0.05) and then gradually decreased by 20 days (Table 1). In CA1 region, BrdU-labeled cells began to be detected outside the CA1 pyramidal cell layer after the transient ischemia at 5 days, and reached the maximum at 10 days (Table 1). In the dentate hilus, BrdU-labeled cells were also increased at 5 and 10 days after the ischemia (Fig. 1l and m). On the spatial characteristics of PSA-NCAM and BrdU positive cells, PSA-NCAM positive cells were predominantly located in the GCL (Figs. 1b–e, 2a), while BrdUlabeled cells were mainly located in the SGZ and partially
M. Iwai et al. / Brain Research 902 (2001) 288 – 293
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Fig. 1. Immunohistochemistry for PSA-NCAM (panels a–i) and BrdU (panels j–n) in gerbil hippocampus of sham-control (a, b, f, j, k) and at 5 (c, g, l), 10 (d, h, m), and 20 (e, i, n) days after the transient ischemia. PSA-NCAM positive cells increased in the deepest portion of the granule cell layer (GCL, arrowheads in panels c–i) and in the outer cells of the GCL (arrows in panels d, e, h, i). Open arrowhead in panel f represents dendrites of granule cells positive for PSA-NCAM. BrdU-labeled cells increased in the SGZ of the DG after ischemia (arrowheads in panels l, m). Note delayed peak of PSA-NCAM positive cells (d, e) compared with that of BrdU-labeled cells (l, m). CA1 and CA3, fields CA1 and CA3 of Ammon’s horn; GCL, granule cell layer; H, hilus; ML, molecular layer. Scale bar5500 mm in a and j; 250 mm in b–e and k–n; 20 mm in f–i.
Table 1 Number of PSA-NCAM positive and BrdU-labeled cells in the hippocampal dentate gyrus (DG), CA1 region and the subventricular zone (SVZ) after transient global ischemia Time after ischemia Sham 5 days 10 days 20 days
PSA-NCAM positive cells
BrdU-labeled cells
DG
CA1
SVZ
DG
CA1
SVZ
129.8626.1 178.8639.9 247.2637.7* 349.5680.9**
0 0 0 0
0 0 0 0
12.165.2 86.7631.0** 97.0640.9** 12.368.0
0 4.666.9** 8.564.3** 1.461.4**
0.961.6 2.561.8* 1.661.3 0.861.5
Data are expressed as mean6S.D. of bilateral hemisphere. n53 at each time point. Significant differences from the sham *P,0.05, **P,0.01.
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Fig. 2. Double immunofluorescence labeling of PSA-NCAM (panel a, green) and BrdU (panel b, red) in the hippocampal first row of the granule cell layer at 10 days after the transient global ischemia. The merged image (panel c) shows a double-stained cell. Scale bar510 mm in c (also applies to a and b).
in the deepest portion of GCL (Figs. 1k–n, 2b). Immunofluorescence for PSA-NCAM with FITC and BrdU with TRITC showed that the majority of the DG cells were not double labeled, while one or two BrdU-labeled nuclei per section were found within the PSA-NCAM positive cell bodies in the deepest portion of the GCL only at 10 days after the reperfusion (Fig. 2c). PSA-NCAM is usually expressed during normal development in the dendrites and cell bodies of the newly generated dentate granule cells. However, it is also expressed in mossy fiber of granule cells in adult rat DG, playing an important role in the migration of neural stem cells [23,25]. The present results showed that the number of PSA-NCAM positive cells significantly increased in the GCL after the transient global ischemia. PSA-NCAM was
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first increased in the deepest cells of GCL, and then was gradually spread at the outer cells of GCL. These results suggest an activation of cell migration in GCL, or a reverting back of the mature granule cells to immature cells after the transient ischemia. In fact, differentiated cells such as ependymal cells or astrocytes can be neuronal progenitors in adult brain [8,13]. Transient reduction of the PSA-NCAM immunoreactivity in the dendrites of GCL (Fig. 1g) suggests a possibility that the polysialylic acid production or polysialyltransferase activity is depressed under the influence of ischemia. A recent report showed that PSA is involved in migration of neural precursors, lamination of mossy fibers, and longterm potentiation and long-term depression in CA1 [9]. Hippocampal neural activity and synaptic reorganization may be impaired while the reduction of the PSA-NCAM immunoreactivity and signaling mechanisms may regulate the change in polysialylation state. Dividing cells can be detected by the incorporation of BrdU after its intraperitoneal administration. A previous report showed a 12-fold increase of BrdU incorporation at the SGZ of DG between 1 and 2 weeks after 10 min of transient global ischemia in gerbils [17]. The present results showed an eightfold increase of BrdU incorporation at SGZ and GCL of DG between 5 and 10 days after 5 min of global ischemia. Because of a shorter period of transient ischemia in the present study, the level of increase in the BrdU-labeled cells was smaller and the peak time interval was shorter than the previous study. BrdU-labeled cells also increased in dentate hilus, CA1 region and SVZ of the D3V in the present study, while the change was only slight (Table 1). The stage of neurogenesis in DG of mature brain can be divided into three distinct steps. Firstly, neural precursor cells that reside at the border between the hilus and GCL undergo cell division. An early marker of this proliferation is incorporation of BrdU into DNA of dividing precursor cells. Secondly, newborn cells begin to migrate into the GCL and neuronal processes. This step is accompanied by an expression of PSA-NCAM. Thirdly, the cells are now integrated in GCL and begin to express the neuronal markers such as neuron-specific enolase, neuronal nuclear antigen (NeuN), glial fibrillary acidic protein (GFAP), microtubule-associated protein 2 (MAP-2) and calbindin. The present study showed that the increase of PSANCAM positive cells delayed (10–20 days) as compared with that of BrdU-labeled cells (5–10 days) in the DG (Table 1, Fig. 1), probably reflecting the second migrating and the first dividing steps of neural precursor cells, respectively. In fact, a previous study reported the double labeling of 60% of the BrdU-labeled cells in the GCL and neural markers such as NeuN, MAP-2, and calbindin at the later step 1 month after a transient global ischemia [17]. On the other hand, PSA-NCAM was not detected in the hilus, CA1 region and the SVZ of the D3V although BrdU-labeled cells were found in these regions (Table 1,
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Fig. 1). Therefore, BrdU-labeled cells may not always express PSA-NCAM. Small nuclei labeled with BrdU outside the CA1 pyramidal cell layer (data not shown) may represent microglia as was reported in the stratum radiatum, molecular layer, and the hilus of DG in ischemic gerbils [18]. This is the first report about the relationship between PSA-NCAM and BrdU of the granule cell after transient ischemia (Table 1, Figs. 1 and 2). With immunofluorescence study, the majority of DG cells were not double labeled, while only one or two cells per section were double labeled in the deepest portion of the GCL at 10 days after the ischemia. The results showed that PSANCAM was induced in DG, but was mostly dissociated with BrdU-labeling as a marker of the first step of neural stem cell proliferation. This could be explained by different predominant spatial distribution of PSA-NCAM positive (in GCL) and BrdU-labeled (in SGZ) cells, and chronological change of expression. Alternatively, a single administration of BrdU only detected cells when they were in the S phase of the mitotic cycle (Table 1). Further investigation with serial administration of BrdU may give detailed information of the relationship between the first and second steps of neural stem cell proliferation after ischemia.
Acknowledgements The authors thank Dr. T. Seki (Tokyo, Japan) for his generous gift of the antibody against PSA-NCAM, and Jin Zaishun (Department of Pathology, Okayama University) for her help in taking a photograph of immunofluorescence sections. This work was partly supported by Grant-in-Aid for Scientific Research (B) 12470141 from the Ministry of Education, Science, Culture and Sports of Japan, and by grants (Tashiro K, Itoyama Y) and Comprehensive Research on Aging and Health (H11-Choju-010, No.207, Koizumi A) from the Ministry of Health and Welfare of Japan.
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