- Email: [email protected]
Distribution of inducible nitric oxide synthase and cell proliferation in rat brain after transient middle cerebral artery occlusion
BR A I N R ES E A RC H 1 0 9 3 ( 2 00 6 ) 1 9 0 –19 7
a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s
Research Report
Distribution of inducible nitric oxide synthase and cell proliferation in rat brain after transient middle cerebral artery occlusion Yoshihide Sehara, Takeshi Hayashi, Kentaro Deguchi, Shoko Nagotani, Hanzhe Zhang, Mikio Shoji, Koji Abe ⁎ Department of Neurology Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Okayama 700-8558, Japan
A R T I C LE I N FO
AB S T R A C T
Article history:
Nitric oxide (NO) can be neuroprotective or neurotoxic during cerebral ischemia, depending
Accepted 16 March 2006
on the NO synthase (NOS) isoform involved. In addition to neurotoxic effect in ischemic
Available online 15 May 2006
brain, inducible NOS (iNOS) also adversely affect ischemic outcome by blocking neurogenesis. In the present study, therefore, we studied the chronological and spatial
Keywords:
change of the distribution of iNOS and cell proliferation in subventricular zone (SVZ) after
Cell proliferation
transient focal cerebral ischemia. After 90 min of transient middle cerebral artery occlusion
Inducible nitric oxide synthase
(tMCAO), iNOS-positive cells decreased in the ischemic core at 1 to 21 days, and increased in
Middle cerebral artery occlusion
the ipsilateral periischemic area at 1 and 3 days. 5-Bromodeoxyuridine (BrdU)-positive cells
Rat
appeared in the ischemic core at 3 to 21 days, appeared in the periischemic area at 3 and 7 days, and increased in the ipsilateral SVZ at 7 days. ED-1-positive cells appeared in the
Abbreviations:
ischemic core at 3 to 21 days, and some of them were double positive with BrdU or iNOS, but
BrdU, 5-bromodeoxyuridine
the majority were BrdU-negative. The present study suggests that astrocytes are born within
CNS, central nervous system
the periischemic area at early stage after tMCAO and migrate from SVZ into periischemic
DG, dentate gyrus
area at later stage, and that time-dependent and spatial changes of iNOS expression may be
eNOS, endothelial NOS
involved in the proliferation and differentiation of adult neurogenesis after focal cerebral
FITC, fluorescein-isothiocyanate
ischemia.
GFAP, glial fibrillary acidic protein iNOS, inducible NOS IL-1, interleukin-1 IL-6, interleukin-6 L-NAME,
Nω-nitro-L-arginine methyl
ester MCA, middle cerebral artery mt NOS, mitochondrial NOS NeuN, neuronal nuclear antigen nNOS, neuronal NOS NO, nitric oxide NOS, nitric oxide synthase ⁎ Corresponding author. Fax: +81 86 235 7368. E-mail address: [email protected] (K. Abe). 0006-8993/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2006.03.092
© 2006 Elsevier B.V. All rights reserved.
BR A I N R ES E A RC H 1 0 9 3 ( 2 00 6 ) 1 9 0 –1 97
191
PBS, phosphate-buffered saline SC, sham control SVZ, subventricular zone tMCAO, transient middle cerebral artery occlusion TNF-α, tumor necrosis factor-α
1.
Introduction
Nitric oxide (NO) is a physiological messenger synthesized by a family of enzymes, the NO synthase (NOS), including neuronal nNOS, endothelial eNOS, mitochondrial mtNOS, and an inducible isoform, iNOS, originally isolated from macrophages (Alderton et al., 2001; Förstermann et al., 1991). All CNS cell types synthesize NO by oxidation of L-arginine, in a reaction catalyzed by NOS. Various isoforms of NOS can be found in almost all the tissues, and they can even coexist in the same tissue. Exclusive inhibitors for isoformspecific NOS are not yet available. NO can be neuroprotective or neurotoxic during cerebral ischemia, depending on the NOS isoform involved. Deleterious effects of NO produced by iNOS were demonstrated using relatively selective iNOS inhibitors. Aminoguanidine, a partially selective iNOS inhibitor, reduced infarct volume in models of transient (Iadecola et al., 1996; Park et al., 2004; Zhu et al., 2002) and permanent ischemia (Cockroft et al., 1996; Iadecola et al., 1995; Sugimoto and Iadecola, 2002). 1400W, a highly selective iNOS inhibitor, exhibited neuroprotective effect in transient and permanent ischemia (Armengou et al., 2003; Parmentier et al., 1999; Pérez-Asensio et al., 2005). These results suggest a neurotoxic effect of iNOS. It is well established that specific regions of the mammalian brain retain the capacity to generate new neurons throughout the entire life in many animal species (Eriksson et al., 1998; Gage et al., 1995; Lois and Alvarez-Buylla, 1993). The generation of new neurons in the adult brain is largely restricted to two regions: the SVZ lining the lateral ventricles and the subgranular zone of the dentate gyrus (DG) in the hippocampus. Ischemia-induced neurogenesis produces cells that may have the capacity to replace dead neurons because these new cells migrate into sites of ischemic brain lesions (Arvidsson et al., 2002; Jin et al., 2003; Parent et al., 2002). Thus, investigation of the factor correlated with neurogenesis may attribute to therapeutic potential of cerebral ischemia. NO donors decreased proliferation of stem cells in murine SVZ (Cheng et al., 2003; Matarredona et al., 2004). Administration of a NOS inhibitor, Nω-nitro-L-arginine methyl ester (LNAME), increased BrdU labeling in cells of SVZ (Moreno-López et al., 2004; Packer et al., 2003). On the other hand, in iNOSknockout compared with wild-type mice, the number of BrdUpositive cells in DG was reduced ipsilateral, to an ischemic region, pointing to iNOS as a positive mediator of ischemiainduced neurogenesis in DG (Zhu et al., 2003). Thus, the role of NO is still controversial. Although iNOS plays a neurotoxic effect in ischemic brain, but it could also adversely affect outcome by blocking ischemia-induced neurogenesis. However, time-dependent spatial changes of iNOS have not been reported in relation to stem cells after ischemia. In the present
study, therefore, we studied the chronological and spatial changes of the distribution of iNOS and cell proliferation in SVZ after transient focal cerebral ischemia.
2.
Results
2.1. Chronological change in the distribution of iNOS-positive cells In the sham control brain, the basal level of iNOS-positive cells was found in the bilateral cerebral cortex, caudoputamen and SVZ, where the number of iNOS-positive cells was not different between the identical areas of hemisphere (Fig. 3). After tMCAO, the number of iNOS-positive cells significantly decreased in the ischemic core compared with sham control by 70% at 1 days, by 66% at 3 days, by 38% at 7 days and by 40% at 21 days, while significantly increased in the periischemic area by 39% at 1 day and by 34% at 3 days. The number of iNOSpositive cells in the periischemic area returned to the basal level by 7 days after ischemia. Although not significant, the number of iNOS-positive cells increased in the ipsilateral SVZ by 16% at 1 day and by 18% at 3 days. The number of iNOSpositive cells remained unchanged throughout 1 to 21 days in the cerebral cortex, caudoputamen, and SVZ of contralateral hemisphere.
2.2. Chronological change in the distribution of BrdU-positive cells In the sham control brain, the basal level of BrdU-positive cells was found only in the SVZ (Fig. 4), but not in other areas of the brain including cerebral cortex and caudoputamen. After tMCAO, BrdU-positive cells became to be found at 3 days in the ischemic core, lasting 3 to 21 days. In the periischemic area, BrdU-positive cells were found at 3 and 7 days. BrdU-positive cells were not found in the identical area of the contralateral cerebral cortex and the caudoputamen. The number of BrdUpositive cells significantly increased approximately twofold at 7 days in the ipsilateral SVZ. In contrast, in the contralateral SVZ, the number of BrdU-positive cells remained unchanged from 1 to 21 days.
2.3.
Immunophenotype of BrdU-positive cells
In the sham control brain, ED-1-positive cells were not found. After tMCAO, ED-1-positive cells appeared only in the ischemic core at 3 to 21 days (Fig. 1). In such an ischemic core, some ED-1-positive cells were double positive with BrdU or iNOS (Figs. 5a–f), but many ED-1-positive cells were BrdU negative. BrdU plus iNOS double positive cells were not observed in this study.
192
BR A I N R ES E A RC H 1 0 9 3 ( 2 00 6 ) 1 9 0 –19 7
Fig. 1 – Chronological change of the number of ED-1-positive cells in the ischemic core after tMCAO. In the sham control (SC) brain, ED-1-positive cells were not found. After tMCAO, ED-1-positive cells appeared only in the ischemic core at 3 to 21 days. Each column and bar denotes mean ± SD. Significant differences from the sham control group at **P < 0.01.
The staining of NeuN disappeared in the ischemic core after 1 day of tMCAO. However, in other areas of the brain, the expression of NeuN remained unchanged throughout 1 to 21 days including bilateral SVZ, contralateral cerebral cortex, periischemic area and identical area of contralateral hemisphere (data not shown). The staining of NeuN disappeared in the ischemic core after 1 day of tMCAO. In this series of experiments, BrdU plus NeuN double positive cells were not
observed. iNOS plus NeuN double positive cells were observed in the bilateral cerebral cortex, caudoputamen, and SVZ (Figs. 5g–i), except in the ischemic core. In the sham control brain, the basal level of GFAPpositive cells was found in the bilateral cerebral cortex, caudoputamen, and SVZ, and the number was not different between the identical areas of hemisphere (data not shown). After tMCAO, GFAP-positive cells increased in the periischemic area but not in the identical area of contralateral hemisphere at 3 days and reached a peak at 7 days. The number of GFAP-positive cells then decreased by 21 days, which was still greater than sham control. The number of GFAP-positive cells remained unchanged throughout 1 to 21 days in cerebral cortex of contralateral hemisphere and SVZ of bilateral hemisphere. The staining of GFAP disappeared in the ischemic core after 1 day of tMCAO. In the periischemic area, the majority of BrdU-positive cells expressed GFAP (Figs. 5j–l). In this series of experiments, on the other hand, iNOS plus GFAP double positive cells were not confirmed (not shown). To determine the fate of the BrdU-positive cells, double immunofluorescence of 21(6)-day group was examined. A very small number of BrdU-positive cells in the periischemic area were double labeled with NeuN (Figs. 2a–c), but the majority of BrdU-positive cells again expressed GFAP (Figs. 2d–f).
3.
Discussion
This work shows chronological and spatial changes of distribution of iNOS and cell proliferation in the ischemic core, periischemic area, and SVZ of adult rats. In this study, iNOS-positive cells decreased in the ischemic core at 1 to
Fig. 2 – Representative double immunofluorescent pictures of 21(6)-day group. Confocal microphotographs show double labeled cells for BrdU (red, a) + NeuN (green, b) and BrdU (red, d) + GFAP (green, e) with merged images in panels c and f, respectively, in the periischemic area. Scale bar = 20 μm.
BR A I N R ES E A RC H 1 0 9 3 ( 2 00 6 ) 1 9 0 –1 97
Fig. 3 – Chronological change of the number of iNOS-positive cells after tMCAO. Each column corresponds to the number of iNOS-positive cells of each area in Fig. 2 from left in order. In I, II, V, and VI, the number of iNOS-positive cells was represented per millimeter square, but in SVZ (III and IV), the total numbers of the boxed areas were represented. iNOS-positive cells decreased in the ischemic core (checked bar) at 1 to 21 days, and increased in the periischemic area (filled bar) at 1 and 3 days after tMCAO. Each column and bar denotes mean ± SD. Significant differences from the sham control group at **P < 0.01. SC: sham control.
21 days and increased in the ipsilateral periischemic area at 1 and 3 days after tMCAO. BrdU-positive cells appeared in the ischemic core at 3 to 21 days, appeared in the periischemic area at 3 and 7 days, and increased in the ipsilateral SVZ at 7 days. iNOS-mediated NO generation aggravates neuronal damage after cerebral ischemia (Iadecola et al., 1995; Iadecola and Ross, 1997; Nagayama et al., 1998; Parmentier et al., 1999), and NO acts as a negative regulator of cell proliferation in adult mammalian brain (Moreno-López et al., 2004; Packer et al., 2003). However, there have been few articles that examined distribution of iNOS in relation to cell proliferation in rat brain after focal ischemia. The present study showed a strong expression of iNOS in the periischemic area (Figs. 3, 4), followed by an increase of BrdU-positive cells after tMCAO in the ipsilateral SVZ. NO is a hydrophobic gas which has high diffusibility and easily pass cell membrane. In water, 50% of NO diffuse 45 μm at 25 °C in 1 s. The diffusion coefficient of NO is approximately 1.4-fold higher than oxygen and carbon monoxide. Thus, NO has the highest diffusibility in vivo. However, NO has a too short half-time to measure quantitatively, resulting in somewhat controversial data (Lapshin et al., 1995; McQuillan et al., 1994; Shaul et al., 1995; Xue et al., 1994). Instead, presence of NOS represents NO production. Because neurons branch very fine dendrite reticularly in CNS, neurons are estimated to be not micrometers away from the occurrence origin of NO (Bredt et al., 1990). The timedependent and spatial changes of iNOS and BrdU-positive cells in the present study (Figs. 3, 4) suggest that iNOS expression in the periischemic area suppresses cell proliferation of SVZ.
193
Although glial cells (astrocytes and microglia) and macrophages synthesize NO through iNOS activation by different stimuli (Murphy et al., 1993), neurons and endothelial cells can also synthesize NO through iNOS activation (Kilbourn and Belloni, 1990; Minc-Golomb et al., 1996; Moro et al., 1998; Olivenza et al., 2000). ED-1-positive cells (representing macrophage) were scarcely double positive with iNOS in this study (Figs. 5a–f). In the ischemic core, most BrdU-positive cells were double positive with ED-1 but not with GFAP or NeuN. In the periischemic area, iNOS-positive cells significantly increased at 1 and 3 days. Although not significant, iNOS-positive cells increased in ipsilateral SVZ at 1 and 3 days. In consideration of chronological profile and spatial distance, iNOS expressed in the ischemic core would not affect cell proliferation of SVZ, and the role of iNOS in the ischemic core has to wait further research. On the other hand, NO generated in the periischemic area could negatively influence the cell proliferation in SVZ. In this study, NeuN-positive cells were also labeled for iNOS in the bilateral cerebral cortices, caudoputamen, and SVZ (Figs. 5g–i) except for those at the ischemic core. On the other hand, we did not find GFAP plus iNOS-positive cells. It may be because immunofluoreactivity for iNOS in astrocytes was so weak that it could not be detected. In the 3- or 7-day group, rats received BrdU injection 2 or 6 days after ischemia, respectively, and were sacrificed on the following day. In this series of experiments, BrdU plus NeuN-
Fig. 4 – Chronological change of the number of BrdU-positive cells after tMCAO. From left in order, each column corresponds to the number of BrdU-positive cells of each area of I to IV in Fig. 2. In I and II, the number of BrdU-positive cells was represented per millimeter square, but in SVZ (III and IV), the total numbers of the boxed areas were represented. Because BrdU-positive cells were not observed in sham control (SC), and data of V and VI are not shown here. BrdU-positive cells appeared in the ischemic core (checked bar) at 3 to 21 days, appeared in the periischemic area (shaded bar) at 3 and 7 days, increased in the ipsilateral SVZ (blank bar) at 7 days, but did not change in contralateral SVZ (filled bar). Each column and bar denotes mean ± SD. Significant differences from the sham control group at **P < 0.01.
194
BR A I N R ES E A RC H 1 0 9 3 ( 2 00 6 ) 1 9 0 –19 7
Fig. 5 – Representative double immunofluorescent pictures of 7-day group. Confocal microphotographs show double labeled cells for BrdU (red, a) + ED-1 (green, b) and ED-1 (red, d) + iNOS (green, e) with merged images in panels c and f, respectively, in the ischemic core. Confocal microphotographs show double labeled cells for NeuN (red, g) + iNOS (green, h) and BrdU (red, j) + GFAP (green, k) with merged image in panels i and l, respectively, in the periischemic area. Scale bar = 20 μm.
positive cells were not found in the periischemic area, and most of the BrdU-positive cells in the periischemic area were double labeled with GFAP, suggesting that reactive astrocytosis was actively and quickly occurring within the periischemic area (Figs. 5j–l). Previous reports (Arvidsson et al., 2002; Jin et al., 2003; Parent et al., 2002) showed that newborn cells in the SVZ migrated into ischemic regions of the caudoputamen and cerebral cortex after focal ischemia. Our previous study showed a temporal profile of stem cell proliferation, migration, and differentiation from SVZ to olfactory bulb (Iwai et al., 2003). In the present study, a significant increase of the
number of BrdU-positive cells was found in the ipsilateral SVZ at 7 days (Fig. 3). iNOS may suppress cell proliferation of SVZ, but other factors which show similar expression profile in focal cerebral ischemia such as interleukin-1 (IL-1), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6), may also be implicated. Neuroinflammation precedes the progressive expansion of the infarct volume, suggesting that the inflammation is causal rather than a consequence of the brain damage. IL-1 and TNF-α act as a deleterious factor in focal cerebral ischemia (Basu et al., 2005; Hosomi et al., 2005), but IL6 plays a protective role in cerebral ischemia (Yamashita et al.,
BR A I N R ES E A RC H 1 0 9 3 ( 2 00 6 ) 1 9 0 –1 97
2005). A more detailed understanding of these cytokines in the CNS could bring more efficacious treatments of cerebral ischemia. To determine the subsequent differentiation of BrdUpositive cells, rats received BrdU injection 6 days after ischemia and were sacrificed 3 weeks later (21(6)-day group). Even in this series of experiments, most BrdU-positive cells in the periischemic area did not express NeuN, but again expressed GFAP. This finding suggests that proliferating cells of SVZ migrate to the periischemic region differentiating mainly into astrocytes but not neurons. Although it is not yet elucidated how long it takes the neural precursor cells differentiate into mature neurons or astrocytes in vivo, the present study suggests that astrocytes are born within the periischemic area at early stage after tMCAO and migrate from SVZ into periischemic area at later stage. Taken together, the present results indicate that iNOS, which is expressed in the proximity of neuronal precursors in the SVZ, may participate in the proliferation and differentiation of adult neurogenesis after focal cerebral ischemia.
4.
Experimental procedures
4.1.
Animals and surgery
Adult male Wistar rats (12 weeks old; 270 ± 15 g; Japan Charles River) were used in this study. The rats were kept under diurnal lighting conditions and were allowed for food and water ad libitum. The rats were anesthetized by inhalation of a nitrous oxide–oxygen–halothane (69%:30%:1%) mixture throughout the operation. Body temperature was maintained at 37 ± 0.5 °C with heating pads until the animals had recovered from surgery. A middle neck incision was made, and the right common carotid artery was exposed. With use of a nylon thread, the right middle cerebral artery (MCA) was transiently occluded through the common carotid artery for 90 min (tMCAO) and then withdrawn as described previously (Abe et al., 1988). Sham control (SC) animals were treated identically, except that the MCAs were not occluded.
4.2.
195
Bromodeoxyuridine labeling
In order to estimate the number of proliferating cells, the rats were treated intraperitoneally with 50 mg/kg BrdU (Roche) two consecutive times at 12-h intervals 24 h before sampling. The rats were sacrificed at 1, 3, 7, and 21 days after tMCAO (n = 6, each). Sham-operated rats were sacrificed at 7 days after the sham operations (n = 6). To determine the phenotypes of BrdUlabeled cells, the rats were treated with BrdU at 6 days after tMCAO and sacrificed at 3 weeks after the BrdU injection. The experimental paradigm is summarized in Fig. 6.
4.3.
Brain sampling and histology
Under deep anesthesia with an overdose of pentobarbital, the rats were decapitated, and the brains were quickly removed, frozen in powdered dry ice, and stored at −80 °C. Coronal cryostat sections of 10-μm thickness were processed. These sections were obtained from caudoputamen level and were identical among the investigated animals. To determine the area of the ischemic lesions, the sections were stained with Nissl and observed with a light microscope. The area for quantification was as follows (Fig. 7): the ischemic core (ipsilateral cerebral cortex), periischemic area and SVZ in the ipsilateral hemisphere, and the identical area of contralateral hemisphere.
4.4.
Double fluorescent immunohistochemistry
For fluorescent immunohistochemistry, the sections were incubated with a primary antibody diluted with PBS at 4 °C overnight. After being washed in PBS, the sections were incubated with a secondary antibody for 2 h at room temperature. For BrdU fluorescent immunohistochemistry, the sections were first treated with HCl and boric acid. The sections were first treated with 2 M HCl at 37 °C for 10 min, rinsed in 0.1M boric acid (pH 8.5) at room temperature for 3 min, and incubated with rat monoclonal anti-BrdU (1:200; Oxford Biotechnologies) at 4 °C overnight. The subsequent procedure was the same as that for other immunohistochemistry.
Fig. 6 – Experimental groups with sham operated (q) or tMCAO (▼). BrdU was intraperitoneally injected (50 mg/kg) twice before the day of sacrifice in each group (n = 6, each). In 21(6)-day group, rats received BrdU injection twice at 6 days and were sacrificed at 21 days after tMCAO.
196
BR A I N R ES E A RC H 1 0 9 3 ( 2 00 6 ) 1 9 0 –19 7
4.5.
Cell counting
To evaluate the results of fluorescent immunohistochemical analysis quantitatively, positively stained cells were counted from 5 consecutive coronal sections. Statistical analysis was performed using Tukey-Kramer method, with P < 0.01 considered statistically significant. Data are presented as mean ± SD.
Acknowledgments Fig. 7 – Schematic representation of the brain sections in the rat brain after tMCAO. Infarct area was shown as checked pattern. Number of iNOS and BrdU-positive cells was quantified in six areas as follows: I: ischemic core (ipsilateral cerebral cortex); II: periischemic area; III: ipsilateral SVZ; IV: contralateral SVZ; V: contralateral caudoputamen; VI: contralateral cerebral cortex.
This work was partly supported by Grant-in-aid for Scientific Research (B) 15390273 (Hoga) 17659445 and (Wakate B) 17790583 and National Project on Protein Structural and Functional Analyses from the Ministry of Education, Science, Culture and Sports of Japan, grants (Itoyama Y, Imai T and Kuzuhara S) from the Ministry of Health and Welfare of Japan and grants from Mitsubishi Pharma Research Foundation.
The other primary antibodies used in this study were as follows: rabbit polyclonal anti-iNOS antibody (1:50; BD transduction laboratories), mouse monoclonal anti-rat ED-1 antibody (1:100; Serotec), mouse anti-GFAP monoclonal antibody (1:200; Chemicon), and mouse anti-NeuN monoclonal antibody (1:200; Chemicon). The combinations of antibodies used in each double immunostaining experiment were (1) mouse anti-rat ED-1 antibody and rabbit anti-iNOS antibody as primary antibodies and rhodamine labeled antimouse IgG antibody and FITC-labeled anti-rabbit IgG as secondary antibodies for ED-1+ iNOS; (2) rat anti-BrdU antibody and mouse anti-rat ED-1 antibody as primary antibodies and rhodamine-labeled anti-rat IgG antibody and FITC-labeled anti-mouse IgG as secondary antibodies for BrdU+ ED-1; (3) mouse anti-NeuN antibody and rabbit anti-iNOS antibody as primary antibodies and rhodaminelabeled anti-mouse IgG antibody and FITC-labeled anti-rabbit IgG as secondary antibodies for NeuN+ iNOS; (4) mouse antiGFAP antibody and rabbit anti-iNOS antibody as primary antibodies and rhodamine-labeled anti-mouse IgG antibody and FITC-labeled anti-rabbit IgG as secondary antibodies for GFAP+ iNOS; (5) rat anti-BrdU antibody and mouse anti-GFAP antibody as primary antibodies and rhodamine-labeled antirat IgG antibody and FITC-labeled anti-mouse IgG as secondary antibodies for BrdU+ GFAP; (6) 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. These stained sections were then covered with VECTASHIELD mounting medium (Vector Laboratories) and then scanned with confocal microscope equipped with argon and HeNe1 laser (LSM-510, Zeiss, Jena, Germany). Sets of fluorescent images were equipped sequentially for the red and green channels to prevent crossover of signals from green to red or red to green channels. As negative control sections, all procedures were processed in the same manner without the primary antibodies.
REFERENCES
Abe, K., Araki, T., Kogure, K., 1988. Recovery from edema and of protein synthesis differs between in the cortex and caudate following transient focal cerebral ischemia in rats. J. Neurochem. 51, 1470–1476. Alderton, W.K., Cooper, C.E., Knowles, R.G., 2001. Nitric oxide synthases: structure, function and inhibition. Biochem. J. 357, 593–615. Armengou, A., Hurtado, O., Leira, R., Obón, M., Pascual, C., Moro, M.A., Lizasoain, I., Castillo, J., Dávalos, A., 2003. L-Arginine levels in blood as a marker of nitric oxide-mediated brain damage in acute stroke: a clinical and experimental study. J. Cereb. Blood Flow Metab. 23, 978–984. Arvidsson, A., Collin, T., Kirik, D., Kokaia, Z., Lindvall, O., 2002. Neuronal replacement from endogenous precursors in the adult brain after stroke. Nat. Med. 8, 963–970. Basu, A., Lazovic, J., Krady, J.K., Mauger, D.T., Rothstein, R.P., Smith, M.B., Levison, S.W., 2005. Interleukin-1 and the interleukin-1 type 1 receptor are essential for the progressive neurodegeneration that ensues subsequent to a mild hypoxic/ischemic injury. J. Cereb. Blood Flow Metab. 25, 17–29. Bredt, D.S., Hwang, P.M., Snyder, S.H., 1990. Localization of nitric oxide synthase indicating a neural role for nitric oxide. Nature 347, 768–770. Cheng, A., Wang, S., Cai, J., Rao, M.S., Mattson, M.P., 2003. Nitric oxide acts as in a positive feedback loop with BDNF to regulate neural progenitor cell proliferation and differentiation in the mammalian brain. Dev. Biol. 258, 319–333. Cockroft, K.M., Meistrell III, M., Zimmerman, G.A., Risucci, D., Bloom, O., Cerami, A., Tracey, K.J., 1996. Cerebroprotective effects of aminoguanidine in a rodent model of stroke. Stroke 27, 1393–1398. Eriksson, P.S., Perfilieva, E., Björk-Eriksson, T., Alborn, A.M., Nordborg, C., Peterson, D.A., Gage, F.H., 1998. Neurogenesis in the adult human hippocampus. Nat. Med. 4, 1313–1317. Förstermann, U., Schmidt, H.H., Pollock, J.S., Sheng, H., Mitchell, J.A., Warner, T.D., Nakane, M., Murad, F., 1991. Isoforms of nitric oxide synthase. Biochem. Pharmacol. 42, 1849–1857. Gage, F.H., Ray, J., Fisher, L.J., 1995. Isolation, characterization, and use of stem cells from the CNS. Annu. Rev. Neurosci. 18, 159–192.
BR A I N R ES E A RC H 1 0 9 3 ( 2 00 6 ) 1 9 0 –1 97
Hosomi, N., Ban, C.R., Naya, T., Takahashi, T., Guo, P., Song, X.R., Kohno, M., 2005. Tumor necrosis factor-α neutralization reduced cerebral edema through inhibition of matrix metalloproteinase production after transient focal cerebral ischemia. J. Cereb. Blood Flow Metab. 25, 959–967. Iadecola, C., Ross, M.E., 1997. Molecular pathology of cerebral ischemia: delayed gene expression and strategies for neuroprotection. Ann. N. Y. Acad. Sci. 835, 203–217. Iadecola, C., Zhang, F., Xu, X., 1995. Inhibition of inducible nitric oxide synthase ameliorates cerebral ischemic damage. Am. J. Physiol. 268, R286–R292. Iadecola, C., Zhang, F., Casey, R., Clark, H.B., Ross, E., 1996. Inducible nitric oxide synthase gene expression in vascular cells after transient focal cerebral ischemia. Stroke 27, 1373–1380. Iwai, M., Sato, K., Kamada, H., Omori, N., Nagano, I., Shoji, M., Abe, K., 2003. Temporal profile of stem cell division, migration, and differentiation from subventricular zone to olfactory bulb after transient forebrain ischemia in gerbils. J. Cereb. Blood Flow Metab. 23, 331–341. Jin, K., Sun, Y., Xie, L., Peel, A., Mao, X.O., Batteur, S., Greenberg, D.A., 2003. Directed migration of neuronal precursors into the ischemic cerebral cortex and striatum. Mol. Cell. Neurosci. 24, 171–189. Kilbourn, R.G., Belloni, P., 1990. Endothelial cell production of nitrogen oxides in response to interferon gamma in combination with tumor necrosis factor, interleukin-1, or endotoxin. J. Natl. Cancer Inst. 82, 772–776. Lapshin, A.V., Manukhina, E.B., Meerson, F.Z., Kikoyan, V.D., Kubrina, L.N., Vanin, A.F., 1995. Decreased content of nitric oxide in rat organs after adaptation to intermittent hypoxia. Hypoxia Med. 1, 3–5. Lois, C., Alvarez-Buylla, A., 1993. Proliferating subventricular zone cells in the adult mammalian forebrain can differentiate into neurons and glia. Proc. Natl. Acad. Sci. U. S. A. 90, 2074–2077. Matarredona, E.R., Murillo-Carretero, M., Moreno-López, B., Estrada, C., 2004. Nitric oxide synthesis inhibition increases proliferation of neural precursors isolated from the postnatal mouse subventricular zone. Brain Res. 995, 274–284. McQuillan, L.P., Leung, G.K., Marsden, P.A., Kostyk, S.K., Kourembanas, S., 1994. Hypoxia inhibits expression of eNOS via transcriptional and posttranslational mechanisms. Am. J. Physiol. 267, H1921–H1927. Minc-Golomb, D., Yadid, G., Tsarfaty, I., Resau, J.H., Schwartz, J.P., 1996. In vivo expression of inducible nitric oxide synthase in cerebellar neurons. J. Neurochem. 66, 1504–1509. Moreno-López, B., Romero-Grimaldi, C., Noval, J.A., Murillo-Carretero, M., Matarredona, E.R., Estrada, C., 2004. Nitric oxide is a physiological inhibitor of neurogenesis in the adult mouse subventricular zone and olfactory bulb. J. Neurosci. 24, 85–95. Moro, M.A., De Alba, J., Leza, J.C., Lorenzo, P., Fernández, A.P., Bentura, M.L., Boscá, L., Rodrigo, J., Lizasoain, I., 1998. Neuronal expression of inducible nitric oxide synthase after oxygen and glucose deprivation in rat forebrain slices. Eur. J. Neurosci. 10, 445–456. Murphy, S., Simmons, M.L., Agullo, L., Garcia, A., Feinstein, D.L., Galea, E., Reis, D.J., Minc-Golomb, D., Schwartz, J.P., 1993.
197
Synthesis of nitric oxide in CNS glial cells. Trends Neurosci. 16, 323–328. Nagayama, M., Zhang, F., Iadecola, C., 1998. Delayed treatment with aminoguanidine decreases focal cerebral ischemic damage and enhances neurologic recovery in rats. J. Cereb. Blood Flow Metab. 18, 1107–1113. Olivenza, R., Moro, M.A., Lizasoain, I., Lorenzo, P., Fernández, A.P., Rodrigo, J., Boscá, L., Leza, J.C., 2000. Chronic stress induces the expression of inducible nitric oxide synthase in rat brain cortex. J. Neurochem. 74, 785–791. Packer, M.A., Stasiv, Y., Benraiss, A., Chmielnicki, E., Grinberg, A., Westphal, H., Goldman, S.A., Enikolopov, G., 2003. Nitric oxide negatively regulates mammalian adult neurogenesis. Proc. Natl. Acad. Sci. U. S. A. 100, 9566–9571. Parent, J.M., Vexler, Z.S., Gong, C., Derugin, N., Ferriero, D.M., 2002. Rat forebrain neurogenesis and striatal neuron replacement after focal stroke. Ann. Neurol. 52, 802–813. Park, E.M., Cho, S., Frys, K., Racchumi, G., Zhou, P., Anrather, J., Iadecola, C., 2004. Interaction between inducible nitric oxide synthase and poly (ADP-ribose) polymerase in focal ischemic brain injury. Stroke 2896–2901. Parmentier, S., Böhme, G.A., Lerouet, D., Damour, D., Stutzmann, J.M., Margaill, I., Plotkine, M., 1999. Selective inhibition of inducible nitric oxide synthase prevents ischaemic brain injury. Br. J. Pharmacol. 127, 546–552. Pérez-Asensio, F.J., Hurtado, O., Burguete, M.C., Moro, M.A., Salom, J.B., Lizasoain, I., Torregrosa, G., Leza, J.C., Alborch, E., Castillo, J., Knowles, R.G., 2005. Inhibition of iNOS activity by 1400W decreases glutamate release and ameliorates stroke outcome after experimental ischemia. Neurobiol. Dis. 18, 375–384. Shaul, P.W., North, A.J., Brannon, T.S., Ujiie, K., Wells, L.B., Nisen, P.A., Lowenstein, C.J., Snyder, S.H., Star, R.A., 1995. Prolonged in vivo hypoxia enhances nitric oxide synthase type I and III gene expression in adult rat lung. Am. J. Respir. Cell Mol. Biol. 13, 167–174. Sugimoto, K., Iadecola, C., 2002. Effects of aminoguanidine on cerebral ischemia in mice: comparison between mice with and without inducible nitric oxide synthase gene. Neurosci. Lett. 331, 25–28. Xue, C., Rengasamy, A., Le Cras, T.D., Koberna, P.A., Dailey, G.C., Johns, R.A., 1994. Distribution of NOS in normoxic vs. hypoxic rat lung: upregulation of NOS by chronic hypoxia. Am. J. Physiol. 267, L667–L668. Yamashita, T., Sawamoto, K., Suzuki, S., Suzuki, N., Adachi, K., Kawase, T., Mihara, M., Ohsugi, Y., Abe, K., Okano, H., 2005. Blockade of interleulin-6 signaling aggravates ischemic cerebral damage in mice: possible involvement of Stat3 activation in the protection of neurons. J. Neurochem. 94, 459–468. Zhu, D.Y., Deng, Q., Yao, H.H., Wang, D.C., Deng, Y., Liu, G.Q., 2002. Inducible nitric oxide synthase expression in the ischemic core and penumbra after transient cerebral ischemia in mice. Life Sci. 17, 1985–1996. Zhu, D.Y., Liu, S.H., Sun, H.S., Lu, Y.M., 2003. Expression of inducible nitric oxide synthase after focal cerebral ischemia stimulates neurogenesis in the adult rodent dentate gyrus. J. Neurosci. 23, 223–229.
a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s
Research Report
Distribution of inducible nitric oxide synthase and cell proliferation in rat brain after transient middle cerebral artery occlusion Yoshihide Sehara, Takeshi Hayashi, Kentaro Deguchi, Shoko Nagotani, Hanzhe Zhang, Mikio Shoji, Koji Abe ⁎ Department of Neurology Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Okayama 700-8558, Japan
A R T I C LE I N FO
AB S T R A C T
Article history:
Nitric oxide (NO) can be neuroprotective or neurotoxic during cerebral ischemia, depending
Accepted 16 March 2006
on the NO synthase (NOS) isoform involved. In addition to neurotoxic effect in ischemic
Available online 15 May 2006
brain, inducible NOS (iNOS) also adversely affect ischemic outcome by blocking neurogenesis. In the present study, therefore, we studied the chronological and spatial
Keywords:
change of the distribution of iNOS and cell proliferation in subventricular zone (SVZ) after
Cell proliferation
transient focal cerebral ischemia. After 90 min of transient middle cerebral artery occlusion
Inducible nitric oxide synthase
(tMCAO), iNOS-positive cells decreased in the ischemic core at 1 to 21 days, and increased in
Middle cerebral artery occlusion
the ipsilateral periischemic area at 1 and 3 days. 5-Bromodeoxyuridine (BrdU)-positive cells
Rat
appeared in the ischemic core at 3 to 21 days, appeared in the periischemic area at 3 and 7 days, and increased in the ipsilateral SVZ at 7 days. ED-1-positive cells appeared in the
Abbreviations:
ischemic core at 3 to 21 days, and some of them were double positive with BrdU or iNOS, but
BrdU, 5-bromodeoxyuridine
the majority were BrdU-negative. The present study suggests that astrocytes are born within
CNS, central nervous system
the periischemic area at early stage after tMCAO and migrate from SVZ into periischemic
DG, dentate gyrus
area at later stage, and that time-dependent and spatial changes of iNOS expression may be
eNOS, endothelial NOS
involved in the proliferation and differentiation of adult neurogenesis after focal cerebral
FITC, fluorescein-isothiocyanate
ischemia.
GFAP, glial fibrillary acidic protein iNOS, inducible NOS IL-1, interleukin-1 IL-6, interleukin-6 L-NAME,
Nω-nitro-L-arginine methyl
ester MCA, middle cerebral artery mt NOS, mitochondrial NOS NeuN, neuronal nuclear antigen nNOS, neuronal NOS NO, nitric oxide NOS, nitric oxide synthase ⁎ Corresponding author. Fax: +81 86 235 7368. E-mail address: [email protected] (K. Abe). 0006-8993/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2006.03.092
© 2006 Elsevier B.V. All rights reserved.
BR A I N R ES E A RC H 1 0 9 3 ( 2 00 6 ) 1 9 0 –1 97
191
PBS, phosphate-buffered saline SC, sham control SVZ, subventricular zone tMCAO, transient middle cerebral artery occlusion TNF-α, tumor necrosis factor-α
1.
Introduction
Nitric oxide (NO) is a physiological messenger synthesized by a family of enzymes, the NO synthase (NOS), including neuronal nNOS, endothelial eNOS, mitochondrial mtNOS, and an inducible isoform, iNOS, originally isolated from macrophages (Alderton et al., 2001; Förstermann et al., 1991). All CNS cell types synthesize NO by oxidation of L-arginine, in a reaction catalyzed by NOS. Various isoforms of NOS can be found in almost all the tissues, and they can even coexist in the same tissue. Exclusive inhibitors for isoformspecific NOS are not yet available. NO can be neuroprotective or neurotoxic during cerebral ischemia, depending on the NOS isoform involved. Deleterious effects of NO produced by iNOS were demonstrated using relatively selective iNOS inhibitors. Aminoguanidine, a partially selective iNOS inhibitor, reduced infarct volume in models of transient (Iadecola et al., 1996; Park et al., 2004; Zhu et al., 2002) and permanent ischemia (Cockroft et al., 1996; Iadecola et al., 1995; Sugimoto and Iadecola, 2002). 1400W, a highly selective iNOS inhibitor, exhibited neuroprotective effect in transient and permanent ischemia (Armengou et al., 2003; Parmentier et al., 1999; Pérez-Asensio et al., 2005). These results suggest a neurotoxic effect of iNOS. It is well established that specific regions of the mammalian brain retain the capacity to generate new neurons throughout the entire life in many animal species (Eriksson et al., 1998; Gage et al., 1995; Lois and Alvarez-Buylla, 1993). The generation of new neurons in the adult brain is largely restricted to two regions: the SVZ lining the lateral ventricles and the subgranular zone of the dentate gyrus (DG) in the hippocampus. Ischemia-induced neurogenesis produces cells that may have the capacity to replace dead neurons because these new cells migrate into sites of ischemic brain lesions (Arvidsson et al., 2002; Jin et al., 2003; Parent et al., 2002). Thus, investigation of the factor correlated with neurogenesis may attribute to therapeutic potential of cerebral ischemia. NO donors decreased proliferation of stem cells in murine SVZ (Cheng et al., 2003; Matarredona et al., 2004). Administration of a NOS inhibitor, Nω-nitro-L-arginine methyl ester (LNAME), increased BrdU labeling in cells of SVZ (Moreno-López et al., 2004; Packer et al., 2003). On the other hand, in iNOSknockout compared with wild-type mice, the number of BrdUpositive cells in DG was reduced ipsilateral, to an ischemic region, pointing to iNOS as a positive mediator of ischemiainduced neurogenesis in DG (Zhu et al., 2003). Thus, the role of NO is still controversial. Although iNOS plays a neurotoxic effect in ischemic brain, but it could also adversely affect outcome by blocking ischemia-induced neurogenesis. However, time-dependent spatial changes of iNOS have not been reported in relation to stem cells after ischemia. In the present
study, therefore, we studied the chronological and spatial changes of the distribution of iNOS and cell proliferation in SVZ after transient focal cerebral ischemia.
2.
Results
2.1. Chronological change in the distribution of iNOS-positive cells In the sham control brain, the basal level of iNOS-positive cells was found in the bilateral cerebral cortex, caudoputamen and SVZ, where the number of iNOS-positive cells was not different between the identical areas of hemisphere (Fig. 3). After tMCAO, the number of iNOS-positive cells significantly decreased in the ischemic core compared with sham control by 70% at 1 days, by 66% at 3 days, by 38% at 7 days and by 40% at 21 days, while significantly increased in the periischemic area by 39% at 1 day and by 34% at 3 days. The number of iNOSpositive cells in the periischemic area returned to the basal level by 7 days after ischemia. Although not significant, the number of iNOS-positive cells increased in the ipsilateral SVZ by 16% at 1 day and by 18% at 3 days. The number of iNOSpositive cells remained unchanged throughout 1 to 21 days in the cerebral cortex, caudoputamen, and SVZ of contralateral hemisphere.
2.2. Chronological change in the distribution of BrdU-positive cells In the sham control brain, the basal level of BrdU-positive cells was found only in the SVZ (Fig. 4), but not in other areas of the brain including cerebral cortex and caudoputamen. After tMCAO, BrdU-positive cells became to be found at 3 days in the ischemic core, lasting 3 to 21 days. In the periischemic area, BrdU-positive cells were found at 3 and 7 days. BrdU-positive cells were not found in the identical area of the contralateral cerebral cortex and the caudoputamen. The number of BrdUpositive cells significantly increased approximately twofold at 7 days in the ipsilateral SVZ. In contrast, in the contralateral SVZ, the number of BrdU-positive cells remained unchanged from 1 to 21 days.
2.3.
Immunophenotype of BrdU-positive cells
In the sham control brain, ED-1-positive cells were not found. After tMCAO, ED-1-positive cells appeared only in the ischemic core at 3 to 21 days (Fig. 1). In such an ischemic core, some ED-1-positive cells were double positive with BrdU or iNOS (Figs. 5a–f), but many ED-1-positive cells were BrdU negative. BrdU plus iNOS double positive cells were not observed in this study.
192
BR A I N R ES E A RC H 1 0 9 3 ( 2 00 6 ) 1 9 0 –19 7
Fig. 1 – Chronological change of the number of ED-1-positive cells in the ischemic core after tMCAO. In the sham control (SC) brain, ED-1-positive cells were not found. After tMCAO, ED-1-positive cells appeared only in the ischemic core at 3 to 21 days. Each column and bar denotes mean ± SD. Significant differences from the sham control group at **P < 0.01.
The staining of NeuN disappeared in the ischemic core after 1 day of tMCAO. However, in other areas of the brain, the expression of NeuN remained unchanged throughout 1 to 21 days including bilateral SVZ, contralateral cerebral cortex, periischemic area and identical area of contralateral hemisphere (data not shown). The staining of NeuN disappeared in the ischemic core after 1 day of tMCAO. In this series of experiments, BrdU plus NeuN double positive cells were not
observed. iNOS plus NeuN double positive cells were observed in the bilateral cerebral cortex, caudoputamen, and SVZ (Figs. 5g–i), except in the ischemic core. In the sham control brain, the basal level of GFAPpositive cells was found in the bilateral cerebral cortex, caudoputamen, and SVZ, and the number was not different between the identical areas of hemisphere (data not shown). After tMCAO, GFAP-positive cells increased in the periischemic area but not in the identical area of contralateral hemisphere at 3 days and reached a peak at 7 days. The number of GFAP-positive cells then decreased by 21 days, which was still greater than sham control. The number of GFAP-positive cells remained unchanged throughout 1 to 21 days in cerebral cortex of contralateral hemisphere and SVZ of bilateral hemisphere. The staining of GFAP disappeared in the ischemic core after 1 day of tMCAO. In the periischemic area, the majority of BrdU-positive cells expressed GFAP (Figs. 5j–l). In this series of experiments, on the other hand, iNOS plus GFAP double positive cells were not confirmed (not shown). To determine the fate of the BrdU-positive cells, double immunofluorescence of 21(6)-day group was examined. A very small number of BrdU-positive cells in the periischemic area were double labeled with NeuN (Figs. 2a–c), but the majority of BrdU-positive cells again expressed GFAP (Figs. 2d–f).
3.
Discussion
This work shows chronological and spatial changes of distribution of iNOS and cell proliferation in the ischemic core, periischemic area, and SVZ of adult rats. In this study, iNOS-positive cells decreased in the ischemic core at 1 to
Fig. 2 – Representative double immunofluorescent pictures of 21(6)-day group. Confocal microphotographs show double labeled cells for BrdU (red, a) + NeuN (green, b) and BrdU (red, d) + GFAP (green, e) with merged images in panels c and f, respectively, in the periischemic area. Scale bar = 20 μm.
BR A I N R ES E A RC H 1 0 9 3 ( 2 00 6 ) 1 9 0 –1 97
Fig. 3 – Chronological change of the number of iNOS-positive cells after tMCAO. Each column corresponds to the number of iNOS-positive cells of each area in Fig. 2 from left in order. In I, II, V, and VI, the number of iNOS-positive cells was represented per millimeter square, but in SVZ (III and IV), the total numbers of the boxed areas were represented. iNOS-positive cells decreased in the ischemic core (checked bar) at 1 to 21 days, and increased in the periischemic area (filled bar) at 1 and 3 days after tMCAO. Each column and bar denotes mean ± SD. Significant differences from the sham control group at **P < 0.01. SC: sham control.
21 days and increased in the ipsilateral periischemic area at 1 and 3 days after tMCAO. BrdU-positive cells appeared in the ischemic core at 3 to 21 days, appeared in the periischemic area at 3 and 7 days, and increased in the ipsilateral SVZ at 7 days. iNOS-mediated NO generation aggravates neuronal damage after cerebral ischemia (Iadecola et al., 1995; Iadecola and Ross, 1997; Nagayama et al., 1998; Parmentier et al., 1999), and NO acts as a negative regulator of cell proliferation in adult mammalian brain (Moreno-López et al., 2004; Packer et al., 2003). However, there have been few articles that examined distribution of iNOS in relation to cell proliferation in rat brain after focal ischemia. The present study showed a strong expression of iNOS in the periischemic area (Figs. 3, 4), followed by an increase of BrdU-positive cells after tMCAO in the ipsilateral SVZ. NO is a hydrophobic gas which has high diffusibility and easily pass cell membrane. In water, 50% of NO diffuse 45 μm at 25 °C in 1 s. The diffusion coefficient of NO is approximately 1.4-fold higher than oxygen and carbon monoxide. Thus, NO has the highest diffusibility in vivo. However, NO has a too short half-time to measure quantitatively, resulting in somewhat controversial data (Lapshin et al., 1995; McQuillan et al., 1994; Shaul et al., 1995; Xue et al., 1994). Instead, presence of NOS represents NO production. Because neurons branch very fine dendrite reticularly in CNS, neurons are estimated to be not micrometers away from the occurrence origin of NO (Bredt et al., 1990). The timedependent and spatial changes of iNOS and BrdU-positive cells in the present study (Figs. 3, 4) suggest that iNOS expression in the periischemic area suppresses cell proliferation of SVZ.
193
Although glial cells (astrocytes and microglia) and macrophages synthesize NO through iNOS activation by different stimuli (Murphy et al., 1993), neurons and endothelial cells can also synthesize NO through iNOS activation (Kilbourn and Belloni, 1990; Minc-Golomb et al., 1996; Moro et al., 1998; Olivenza et al., 2000). ED-1-positive cells (representing macrophage) were scarcely double positive with iNOS in this study (Figs. 5a–f). In the ischemic core, most BrdU-positive cells were double positive with ED-1 but not with GFAP or NeuN. In the periischemic area, iNOS-positive cells significantly increased at 1 and 3 days. Although not significant, iNOS-positive cells increased in ipsilateral SVZ at 1 and 3 days. In consideration of chronological profile and spatial distance, iNOS expressed in the ischemic core would not affect cell proliferation of SVZ, and the role of iNOS in the ischemic core has to wait further research. On the other hand, NO generated in the periischemic area could negatively influence the cell proliferation in SVZ. In this study, NeuN-positive cells were also labeled for iNOS in the bilateral cerebral cortices, caudoputamen, and SVZ (Figs. 5g–i) except for those at the ischemic core. On the other hand, we did not find GFAP plus iNOS-positive cells. It may be because immunofluoreactivity for iNOS in astrocytes was so weak that it could not be detected. In the 3- or 7-day group, rats received BrdU injection 2 or 6 days after ischemia, respectively, and were sacrificed on the following day. In this series of experiments, BrdU plus NeuN-
Fig. 4 – Chronological change of the number of BrdU-positive cells after tMCAO. From left in order, each column corresponds to the number of BrdU-positive cells of each area of I to IV in Fig. 2. In I and II, the number of BrdU-positive cells was represented per millimeter square, but in SVZ (III and IV), the total numbers of the boxed areas were represented. Because BrdU-positive cells were not observed in sham control (SC), and data of V and VI are not shown here. BrdU-positive cells appeared in the ischemic core (checked bar) at 3 to 21 days, appeared in the periischemic area (shaded bar) at 3 and 7 days, increased in the ipsilateral SVZ (blank bar) at 7 days, but did not change in contralateral SVZ (filled bar). Each column and bar denotes mean ± SD. Significant differences from the sham control group at **P < 0.01.
194
BR A I N R ES E A RC H 1 0 9 3 ( 2 00 6 ) 1 9 0 –19 7
Fig. 5 – Representative double immunofluorescent pictures of 7-day group. Confocal microphotographs show double labeled cells for BrdU (red, a) + ED-1 (green, b) and ED-1 (red, d) + iNOS (green, e) with merged images in panels c and f, respectively, in the ischemic core. Confocal microphotographs show double labeled cells for NeuN (red, g) + iNOS (green, h) and BrdU (red, j) + GFAP (green, k) with merged image in panels i and l, respectively, in the periischemic area. Scale bar = 20 μm.
positive cells were not found in the periischemic area, and most of the BrdU-positive cells in the periischemic area were double labeled with GFAP, suggesting that reactive astrocytosis was actively and quickly occurring within the periischemic area (Figs. 5j–l). Previous reports (Arvidsson et al., 2002; Jin et al., 2003; Parent et al., 2002) showed that newborn cells in the SVZ migrated into ischemic regions of the caudoputamen and cerebral cortex after focal ischemia. Our previous study showed a temporal profile of stem cell proliferation, migration, and differentiation from SVZ to olfactory bulb (Iwai et al., 2003). In the present study, a significant increase of the
number of BrdU-positive cells was found in the ipsilateral SVZ at 7 days (Fig. 3). iNOS may suppress cell proliferation of SVZ, but other factors which show similar expression profile in focal cerebral ischemia such as interleukin-1 (IL-1), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6), may also be implicated. Neuroinflammation precedes the progressive expansion of the infarct volume, suggesting that the inflammation is causal rather than a consequence of the brain damage. IL-1 and TNF-α act as a deleterious factor in focal cerebral ischemia (Basu et al., 2005; Hosomi et al., 2005), but IL6 plays a protective role in cerebral ischemia (Yamashita et al.,
BR A I N R ES E A RC H 1 0 9 3 ( 2 00 6 ) 1 9 0 –1 97
2005). A more detailed understanding of these cytokines in the CNS could bring more efficacious treatments of cerebral ischemia. To determine the subsequent differentiation of BrdUpositive cells, rats received BrdU injection 6 days after ischemia and were sacrificed 3 weeks later (21(6)-day group). Even in this series of experiments, most BrdU-positive cells in the periischemic area did not express NeuN, but again expressed GFAP. This finding suggests that proliferating cells of SVZ migrate to the periischemic region differentiating mainly into astrocytes but not neurons. Although it is not yet elucidated how long it takes the neural precursor cells differentiate into mature neurons or astrocytes in vivo, the present study suggests that astrocytes are born within the periischemic area at early stage after tMCAO and migrate from SVZ into periischemic area at later stage. Taken together, the present results indicate that iNOS, which is expressed in the proximity of neuronal precursors in the SVZ, may participate in the proliferation and differentiation of adult neurogenesis after focal cerebral ischemia.
4.
Experimental procedures
4.1.
Animals and surgery
Adult male Wistar rats (12 weeks old; 270 ± 15 g; Japan Charles River) were used in this study. The rats were kept under diurnal lighting conditions and were allowed for food and water ad libitum. The rats were anesthetized by inhalation of a nitrous oxide–oxygen–halothane (69%:30%:1%) mixture throughout the operation. Body temperature was maintained at 37 ± 0.5 °C with heating pads until the animals had recovered from surgery. A middle neck incision was made, and the right common carotid artery was exposed. With use of a nylon thread, the right middle cerebral artery (MCA) was transiently occluded through the common carotid artery for 90 min (tMCAO) and then withdrawn as described previously (Abe et al., 1988). Sham control (SC) animals were treated identically, except that the MCAs were not occluded.
4.2.
195
Bromodeoxyuridine labeling
In order to estimate the number of proliferating cells, the rats were treated intraperitoneally with 50 mg/kg BrdU (Roche) two consecutive times at 12-h intervals 24 h before sampling. The rats were sacrificed at 1, 3, 7, and 21 days after tMCAO (n = 6, each). Sham-operated rats were sacrificed at 7 days after the sham operations (n = 6). To determine the phenotypes of BrdUlabeled cells, the rats were treated with BrdU at 6 days after tMCAO and sacrificed at 3 weeks after the BrdU injection. The experimental paradigm is summarized in Fig. 6.
4.3.
Brain sampling and histology
Under deep anesthesia with an overdose of pentobarbital, the rats were decapitated, and the brains were quickly removed, frozen in powdered dry ice, and stored at −80 °C. Coronal cryostat sections of 10-μm thickness were processed. These sections were obtained from caudoputamen level and were identical among the investigated animals. To determine the area of the ischemic lesions, the sections were stained with Nissl and observed with a light microscope. The area for quantification was as follows (Fig. 7): the ischemic core (ipsilateral cerebral cortex), periischemic area and SVZ in the ipsilateral hemisphere, and the identical area of contralateral hemisphere.
4.4.
Double fluorescent immunohistochemistry
For fluorescent immunohistochemistry, the sections were incubated with a primary antibody diluted with PBS at 4 °C overnight. After being washed in PBS, the sections were incubated with a secondary antibody for 2 h at room temperature. For BrdU fluorescent immunohistochemistry, the sections were first treated with HCl and boric acid. The sections were first treated with 2 M HCl at 37 °C for 10 min, rinsed in 0.1M boric acid (pH 8.5) at room temperature for 3 min, and incubated with rat monoclonal anti-BrdU (1:200; Oxford Biotechnologies) at 4 °C overnight. The subsequent procedure was the same as that for other immunohistochemistry.
Fig. 6 – Experimental groups with sham operated (q) or tMCAO (▼). BrdU was intraperitoneally injected (50 mg/kg) twice before the day of sacrifice in each group (n = 6, each). In 21(6)-day group, rats received BrdU injection twice at 6 days and were sacrificed at 21 days after tMCAO.
196
BR A I N R ES E A RC H 1 0 9 3 ( 2 00 6 ) 1 9 0 –19 7
4.5.
Cell counting
To evaluate the results of fluorescent immunohistochemical analysis quantitatively, positively stained cells were counted from 5 consecutive coronal sections. Statistical analysis was performed using Tukey-Kramer method, with P < 0.01 considered statistically significant. Data are presented as mean ± SD.
Acknowledgments Fig. 7 – Schematic representation of the brain sections in the rat brain after tMCAO. Infarct area was shown as checked pattern. Number of iNOS and BrdU-positive cells was quantified in six areas as follows: I: ischemic core (ipsilateral cerebral cortex); II: periischemic area; III: ipsilateral SVZ; IV: contralateral SVZ; V: contralateral caudoputamen; VI: contralateral cerebral cortex.
This work was partly supported by Grant-in-aid for Scientific Research (B) 15390273 (Hoga) 17659445 and (Wakate B) 17790583 and National Project on Protein Structural and Functional Analyses from the Ministry of Education, Science, Culture and Sports of Japan, grants (Itoyama Y, Imai T and Kuzuhara S) from the Ministry of Health and Welfare of Japan and grants from Mitsubishi Pharma Research Foundation.
The other primary antibodies used in this study were as follows: rabbit polyclonal anti-iNOS antibody (1:50; BD transduction laboratories), mouse monoclonal anti-rat ED-1 antibody (1:100; Serotec), mouse anti-GFAP monoclonal antibody (1:200; Chemicon), and mouse anti-NeuN monoclonal antibody (1:200; Chemicon). The combinations of antibodies used in each double immunostaining experiment were (1) mouse anti-rat ED-1 antibody and rabbit anti-iNOS antibody as primary antibodies and rhodamine labeled antimouse IgG antibody and FITC-labeled anti-rabbit IgG as secondary antibodies for ED-1+ iNOS; (2) rat anti-BrdU antibody and mouse anti-rat ED-1 antibody as primary antibodies and rhodamine-labeled anti-rat IgG antibody and FITC-labeled anti-mouse IgG as secondary antibodies for BrdU+ ED-1; (3) mouse anti-NeuN antibody and rabbit anti-iNOS antibody as primary antibodies and rhodaminelabeled anti-mouse IgG antibody and FITC-labeled anti-rabbit IgG as secondary antibodies for NeuN+ iNOS; (4) mouse antiGFAP antibody and rabbit anti-iNOS antibody as primary antibodies and rhodamine-labeled anti-mouse IgG antibody and FITC-labeled anti-rabbit IgG as secondary antibodies for GFAP+ iNOS; (5) rat anti-BrdU antibody and mouse anti-GFAP antibody as primary antibodies and rhodamine-labeled antirat IgG antibody and FITC-labeled anti-mouse IgG as secondary antibodies for BrdU+ GFAP; (6) 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. These stained sections were then covered with VECTASHIELD mounting medium (Vector Laboratories) and then scanned with confocal microscope equipped with argon and HeNe1 laser (LSM-510, Zeiss, Jena, Germany). Sets of fluorescent images were equipped sequentially for the red and green channels to prevent crossover of signals from green to red or red to green channels. As negative control sections, all procedures were processed in the same manner without the primary antibodies.
REFERENCES
Abe, K., Araki, T., Kogure, K., 1988. Recovery from edema and of protein synthesis differs between in the cortex and caudate following transient focal cerebral ischemia in rats. J. Neurochem. 51, 1470–1476. Alderton, W.K., Cooper, C.E., Knowles, R.G., 2001. Nitric oxide synthases: structure, function and inhibition. Biochem. J. 357, 593–615. Armengou, A., Hurtado, O., Leira, R., Obón, M., Pascual, C., Moro, M.A., Lizasoain, I., Castillo, J., Dávalos, A., 2003. L-Arginine levels in blood as a marker of nitric oxide-mediated brain damage in acute stroke: a clinical and experimental study. J. Cereb. Blood Flow Metab. 23, 978–984. Arvidsson, A., Collin, T., Kirik, D., Kokaia, Z., Lindvall, O., 2002. Neuronal replacement from endogenous precursors in the adult brain after stroke. Nat. Med. 8, 963–970. Basu, A., Lazovic, J., Krady, J.K., Mauger, D.T., Rothstein, R.P., Smith, M.B., Levison, S.W., 2005. Interleukin-1 and the interleukin-1 type 1 receptor are essential for the progressive neurodegeneration that ensues subsequent to a mild hypoxic/ischemic injury. J. Cereb. Blood Flow Metab. 25, 17–29. Bredt, D.S., Hwang, P.M., Snyder, S.H., 1990. Localization of nitric oxide synthase indicating a neural role for nitric oxide. Nature 347, 768–770. Cheng, A., Wang, S., Cai, J., Rao, M.S., Mattson, M.P., 2003. Nitric oxide acts as in a positive feedback loop with BDNF to regulate neural progenitor cell proliferation and differentiation in the mammalian brain. Dev. Biol. 258, 319–333. Cockroft, K.M., Meistrell III, M., Zimmerman, G.A., Risucci, D., Bloom, O., Cerami, A., Tracey, K.J., 1996. Cerebroprotective effects of aminoguanidine in a rodent model of stroke. Stroke 27, 1393–1398. Eriksson, P.S., Perfilieva, E., Björk-Eriksson, T., Alborn, A.M., Nordborg, C., Peterson, D.A., Gage, F.H., 1998. Neurogenesis in the adult human hippocampus. Nat. Med. 4, 1313–1317. Förstermann, U., Schmidt, H.H., Pollock, J.S., Sheng, H., Mitchell, J.A., Warner, T.D., Nakane, M., Murad, F., 1991. Isoforms of nitric oxide synthase. Biochem. Pharmacol. 42, 1849–1857. Gage, F.H., Ray, J., Fisher, L.J., 1995. Isolation, characterization, and use of stem cells from the CNS. Annu. Rev. Neurosci. 18, 159–192.
BR A I N R ES E A RC H 1 0 9 3 ( 2 00 6 ) 1 9 0 –1 97
Hosomi, N., Ban, C.R., Naya, T., Takahashi, T., Guo, P., Song, X.R., Kohno, M., 2005. Tumor necrosis factor-α neutralization reduced cerebral edema through inhibition of matrix metalloproteinase production after transient focal cerebral ischemia. J. Cereb. Blood Flow Metab. 25, 959–967. Iadecola, C., Ross, M.E., 1997. Molecular pathology of cerebral ischemia: delayed gene expression and strategies for neuroprotection. Ann. N. Y. Acad. Sci. 835, 203–217. Iadecola, C., Zhang, F., Xu, X., 1995. Inhibition of inducible nitric oxide synthase ameliorates cerebral ischemic damage. Am. J. Physiol. 268, R286–R292. Iadecola, C., Zhang, F., Casey, R., Clark, H.B., Ross, E., 1996. Inducible nitric oxide synthase gene expression in vascular cells after transient focal cerebral ischemia. Stroke 27, 1373–1380. Iwai, M., Sato, K., Kamada, H., Omori, N., Nagano, I., Shoji, M., Abe, K., 2003. Temporal profile of stem cell division, migration, and differentiation from subventricular zone to olfactory bulb after transient forebrain ischemia in gerbils. J. Cereb. Blood Flow Metab. 23, 331–341. Jin, K., Sun, Y., Xie, L., Peel, A., Mao, X.O., Batteur, S., Greenberg, D.A., 2003. Directed migration of neuronal precursors into the ischemic cerebral cortex and striatum. Mol. Cell. Neurosci. 24, 171–189. Kilbourn, R.G., Belloni, P., 1990. Endothelial cell production of nitrogen oxides in response to interferon gamma in combination with tumor necrosis factor, interleukin-1, or endotoxin. J. Natl. Cancer Inst. 82, 772–776. Lapshin, A.V., Manukhina, E.B., Meerson, F.Z., Kikoyan, V.D., Kubrina, L.N., Vanin, A.F., 1995. Decreased content of nitric oxide in rat organs after adaptation to intermittent hypoxia. Hypoxia Med. 1, 3–5. Lois, C., Alvarez-Buylla, A., 1993. Proliferating subventricular zone cells in the adult mammalian forebrain can differentiate into neurons and glia. Proc. Natl. Acad. Sci. U. S. A. 90, 2074–2077. Matarredona, E.R., Murillo-Carretero, M., Moreno-López, B., Estrada, C., 2004. Nitric oxide synthesis inhibition increases proliferation of neural precursors isolated from the postnatal mouse subventricular zone. Brain Res. 995, 274–284. McQuillan, L.P., Leung, G.K., Marsden, P.A., Kostyk, S.K., Kourembanas, S., 1994. Hypoxia inhibits expression of eNOS via transcriptional and posttranslational mechanisms. Am. J. Physiol. 267, H1921–H1927. Minc-Golomb, D., Yadid, G., Tsarfaty, I., Resau, J.H., Schwartz, J.P., 1996. In vivo expression of inducible nitric oxide synthase in cerebellar neurons. J. Neurochem. 66, 1504–1509. Moreno-López, B., Romero-Grimaldi, C., Noval, J.A., Murillo-Carretero, M., Matarredona, E.R., Estrada, C., 2004. Nitric oxide is a physiological inhibitor of neurogenesis in the adult mouse subventricular zone and olfactory bulb. J. Neurosci. 24, 85–95. Moro, M.A., De Alba, J., Leza, J.C., Lorenzo, P., Fernández, A.P., Bentura, M.L., Boscá, L., Rodrigo, J., Lizasoain, I., 1998. Neuronal expression of inducible nitric oxide synthase after oxygen and glucose deprivation in rat forebrain slices. Eur. J. Neurosci. 10, 445–456. Murphy, S., Simmons, M.L., Agullo, L., Garcia, A., Feinstein, D.L., Galea, E., Reis, D.J., Minc-Golomb, D., Schwartz, J.P., 1993.
197
Synthesis of nitric oxide in CNS glial cells. Trends Neurosci. 16, 323–328. Nagayama, M., Zhang, F., Iadecola, C., 1998. Delayed treatment with aminoguanidine decreases focal cerebral ischemic damage and enhances neurologic recovery in rats. J. Cereb. Blood Flow Metab. 18, 1107–1113. Olivenza, R., Moro, M.A., Lizasoain, I., Lorenzo, P., Fernández, A.P., Rodrigo, J., Boscá, L., Leza, J.C., 2000. Chronic stress induces the expression of inducible nitric oxide synthase in rat brain cortex. J. Neurochem. 74, 785–791. Packer, M.A., Stasiv, Y., Benraiss, A., Chmielnicki, E., Grinberg, A., Westphal, H., Goldman, S.A., Enikolopov, G., 2003. Nitric oxide negatively regulates mammalian adult neurogenesis. Proc. Natl. Acad. Sci. U. S. A. 100, 9566–9571. Parent, J.M., Vexler, Z.S., Gong, C., Derugin, N., Ferriero, D.M., 2002. Rat forebrain neurogenesis and striatal neuron replacement after focal stroke. Ann. Neurol. 52, 802–813. Park, E.M., Cho, S., Frys, K., Racchumi, G., Zhou, P., Anrather, J., Iadecola, C., 2004. Interaction between inducible nitric oxide synthase and poly (ADP-ribose) polymerase in focal ischemic brain injury. Stroke 2896–2901. Parmentier, S., Böhme, G.A., Lerouet, D., Damour, D., Stutzmann, J.M., Margaill, I., Plotkine, M., 1999. Selective inhibition of inducible nitric oxide synthase prevents ischaemic brain injury. Br. J. Pharmacol. 127, 546–552. Pérez-Asensio, F.J., Hurtado, O., Burguete, M.C., Moro, M.A., Salom, J.B., Lizasoain, I., Torregrosa, G., Leza, J.C., Alborch, E., Castillo, J., Knowles, R.G., 2005. Inhibition of iNOS activity by 1400W decreases glutamate release and ameliorates stroke outcome after experimental ischemia. Neurobiol. Dis. 18, 375–384. Shaul, P.W., North, A.J., Brannon, T.S., Ujiie, K., Wells, L.B., Nisen, P.A., Lowenstein, C.J., Snyder, S.H., Star, R.A., 1995. Prolonged in vivo hypoxia enhances nitric oxide synthase type I and III gene expression in adult rat lung. Am. J. Respir. Cell Mol. Biol. 13, 167–174. Sugimoto, K., Iadecola, C., 2002. Effects of aminoguanidine on cerebral ischemia in mice: comparison between mice with and without inducible nitric oxide synthase gene. Neurosci. Lett. 331, 25–28. Xue, C., Rengasamy, A., Le Cras, T.D., Koberna, P.A., Dailey, G.C., Johns, R.A., 1994. Distribution of NOS in normoxic vs. hypoxic rat lung: upregulation of NOS by chronic hypoxia. Am. J. Physiol. 267, L667–L668. Yamashita, T., Sawamoto, K., Suzuki, S., Suzuki, N., Adachi, K., Kawase, T., Mihara, M., Ohsugi, Y., Abe, K., Okano, H., 2005. Blockade of interleulin-6 signaling aggravates ischemic cerebral damage in mice: possible involvement of Stat3 activation in the protection of neurons. J. Neurochem. 94, 459–468. Zhu, D.Y., Deng, Q., Yao, H.H., Wang, D.C., Deng, Y., Liu, G.Q., 2002. Inducible nitric oxide synthase expression in the ischemic core and penumbra after transient cerebral ischemia in mice. Life Sci. 17, 1985–1996. Zhu, D.Y., Liu, S.H., Sun, H.S., Lu, Y.M., 2003. Expression of inducible nitric oxide synthase after focal cerebral ischemia stimulates neurogenesis in the adult rodent dentate gyrus. J. Neurosci. 23, 223–229.