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P088. NO and neurogenesis—a question of the NOS isotype and of animal age
Nitric Oxide 14 (2006) A45 www.elsevier.com/locate/yniox
Posters
NO Differentiation/Stem Cells P088. NO and neurogenesis—a question of the NOS isotype and of animal age Gerburg Keilhoff Institute of Medical Neurobiology, University of Magdeburg, Germany It is well accepted that, in the adult brain of many animal species, there are two main neurogenic areas: the subventriculare zone (SVZ) and the subgranular zone of the dentate gyrus (DG) of the hippocampus. Particular attention has been focused on the factors involved in the regulation of adult neurogenesis. From a close proximity between nitrergic neurons and neuronal precursors described in the SVZ and DG of adult mice it was suggested that NO plays a role in adult neurogenesis. Logical, it has been shown that reduction of neuronal NOS activity by NOS inhibitors or by knocking out this NOS isoform increases neurogenesis in both the SVZ and the DG. On the other hand, mice deficient in endothelial NOS show a decreased SVZ cell proliferation and migration, and enhanced expression of inducible NOS after focal cerebral ischemia stimulates neurogenesis in the DG. Moreover, it has long been accepted that another critical factor modulating cell proliferation is the animal age. With increasing age, the proliferation rate and ratio of cells differentiating into neurons rapidly decline. The current study assessed the neurogenic pattern in juvenile, adult (10 weeks old) and aged (18 month) mice in which the different types of NOS isoforms were knocked out. The proliferative response was determined using BrdU labeling. The differentiation of newly generated cells was investigated using cell-specific markers. After 5 days of BrdU exposure, in adult nNOS-KO mice we found an increase of about 28% in the number of BrdU-labeled cells in the DG and SVZ compared to the respective wild type mice. In adult eNOS-KO mice a decrease of about 30% was evident in the DG and SVZ, whereas in adult iNOS-KO mice cell proliferation was reduced by 30% in the DG and by 60% in the SVZ. In the DG of juvenile mice, there is no difference in cell proliferation between wild type and nNOS-KO mice, whereas in iNOS- and eNOSKO mice the number of BrdU-labeled cell is slightly reduced. In the SVZ of juvenile mice, however, a significant reduction of BrdU-labeled cell can be found in all three types of NOS-KO mice compared to the wild type. In the DG of aged mice, the number of BrdU-labeled cells is about five times lower as in adult animals with lowest numbers in nNOS- and iNOSKO mice. Cell proliferation pattern in the SVZ of nNOS-KO mice shows an unexpected significant reduction of BrdU-labeled cells of about 90%. The proliferation rats of wild type, eNOS- and iNOS-KO mice reach
doi:10.1016/j.niox.2006.04.151
nearly equal levels, which in case of wild type mice is about five times lower as in adults. In eNOS- and iNOS-KO mice the reduction is of about two times as compared to adults. Our findings suggest that influencing cell proliferation NO is unlike NO, and that the NO-mediated changes in cell proliferative potency seem to be equalized over the live span. doi:10.1016/j.niox.2006.04.152
P089. Nitric oxide enhances osteoclast formation possibly by mediating fusion Dorrin Nilforoushan a, Morris Frank Manolson b Faculty of Dentistry, University of Toronto, Canada b University of Toronto, Canada a
Nitric oxide (NO) plays an important role in bone turnover and bone cell function. Osteoclasts are multinucleated bone resorbing cells which form by fusion of preosteoclasts. We investigated the involvement of NO in osteoclast formation by mediating fusion. Time lapse photography, using NO indicator fluorescent dye (DAF-2 DA) in RAW cell derived osteoclast as well as authentic rabbit osteoclast cultures, revealed increased NO signal in preosteoclasts prior to fusion and formation of multinucleated osteoclasts. Osteoclasts were differentiated from RAW 264.7 cells in presence of nitric oxide synthase (NOS) inhibitor (LNMMA) and NO donors (NOC-12 and NOC-18). L-NMMA significantly decreased osteoclast formation in a dose dependant manner with the most effect observed at 0.25 lM L-NMMA. On the other hand, NO donors significantly increased osteoclast formation dose dependently with the most effect seen at 15 lM NOC-12 and 5 lM NOC-18. The effect of L-NMMA on reducing osteoclast formation was abolished when receptor activator of nuclear factor kappa B ligand (RANKL) concentration was increased. Measuring nitrite (NO end product) in 24 h media of RAW cells undergoing differentiation and osteoclast formation, showed significantly increased NO production coinciding with fusion of preosteoclasts. Preventing preosteoclast fusion by plating RAW cells on poly-(L-lysine) coated wells significantly decreased NO production. These data suggests a role for NO in osteoclast formation possibly by mediating fusion of mononuclear preosteoclasts to form multinucleated osteoclasts. doi:10.1016/j.niox.2006.04.153
Posters
NO Differentiation/Stem Cells P088. NO and neurogenesis—a question of the NOS isotype and of animal age Gerburg Keilhoff Institute of Medical Neurobiology, University of Magdeburg, Germany It is well accepted that, in the adult brain of many animal species, there are two main neurogenic areas: the subventriculare zone (SVZ) and the subgranular zone of the dentate gyrus (DG) of the hippocampus. Particular attention has been focused on the factors involved in the regulation of adult neurogenesis. From a close proximity between nitrergic neurons and neuronal precursors described in the SVZ and DG of adult mice it was suggested that NO plays a role in adult neurogenesis. Logical, it has been shown that reduction of neuronal NOS activity by NOS inhibitors or by knocking out this NOS isoform increases neurogenesis in both the SVZ and the DG. On the other hand, mice deficient in endothelial NOS show a decreased SVZ cell proliferation and migration, and enhanced expression of inducible NOS after focal cerebral ischemia stimulates neurogenesis in the DG. Moreover, it has long been accepted that another critical factor modulating cell proliferation is the animal age. With increasing age, the proliferation rate and ratio of cells differentiating into neurons rapidly decline. The current study assessed the neurogenic pattern in juvenile, adult (10 weeks old) and aged (18 month) mice in which the different types of NOS isoforms were knocked out. The proliferative response was determined using BrdU labeling. The differentiation of newly generated cells was investigated using cell-specific markers. After 5 days of BrdU exposure, in adult nNOS-KO mice we found an increase of about 28% in the number of BrdU-labeled cells in the DG and SVZ compared to the respective wild type mice. In adult eNOS-KO mice a decrease of about 30% was evident in the DG and SVZ, whereas in adult iNOS-KO mice cell proliferation was reduced by 30% in the DG and by 60% in the SVZ. In the DG of juvenile mice, there is no difference in cell proliferation between wild type and nNOS-KO mice, whereas in iNOS- and eNOSKO mice the number of BrdU-labeled cell is slightly reduced. In the SVZ of juvenile mice, however, a significant reduction of BrdU-labeled cell can be found in all three types of NOS-KO mice compared to the wild type. In the DG of aged mice, the number of BrdU-labeled cells is about five times lower as in adult animals with lowest numbers in nNOS- and iNOSKO mice. Cell proliferation pattern in the SVZ of nNOS-KO mice shows an unexpected significant reduction of BrdU-labeled cells of about 90%. The proliferation rats of wild type, eNOS- and iNOS-KO mice reach
doi:10.1016/j.niox.2006.04.151
nearly equal levels, which in case of wild type mice is about five times lower as in adults. In eNOS- and iNOS-KO mice the reduction is of about two times as compared to adults. Our findings suggest that influencing cell proliferation NO is unlike NO, and that the NO-mediated changes in cell proliferative potency seem to be equalized over the live span. doi:10.1016/j.niox.2006.04.152
P089. Nitric oxide enhances osteoclast formation possibly by mediating fusion Dorrin Nilforoushan a, Morris Frank Manolson b Faculty of Dentistry, University of Toronto, Canada b University of Toronto, Canada a
Nitric oxide (NO) plays an important role in bone turnover and bone cell function. Osteoclasts are multinucleated bone resorbing cells which form by fusion of preosteoclasts. We investigated the involvement of NO in osteoclast formation by mediating fusion. Time lapse photography, using NO indicator fluorescent dye (DAF-2 DA) in RAW cell derived osteoclast as well as authentic rabbit osteoclast cultures, revealed increased NO signal in preosteoclasts prior to fusion and formation of multinucleated osteoclasts. Osteoclasts were differentiated from RAW 264.7 cells in presence of nitric oxide synthase (NOS) inhibitor (LNMMA) and NO donors (NOC-12 and NOC-18). L-NMMA significantly decreased osteoclast formation in a dose dependant manner with the most effect observed at 0.25 lM L-NMMA. On the other hand, NO donors significantly increased osteoclast formation dose dependently with the most effect seen at 15 lM NOC-12 and 5 lM NOC-18. The effect of L-NMMA on reducing osteoclast formation was abolished when receptor activator of nuclear factor kappa B ligand (RANKL) concentration was increased. Measuring nitrite (NO end product) in 24 h media of RAW cells undergoing differentiation and osteoclast formation, showed significantly increased NO production coinciding with fusion of preosteoclasts. Preventing preosteoclast fusion by plating RAW cells on poly-(L-lysine) coated wells significantly decreased NO production. These data suggests a role for NO in osteoclast formation possibly by mediating fusion of mononuclear preosteoclasts to form multinucleated osteoclasts. doi:10.1016/j.niox.2006.04.153