Study of the nitric oxide system in the rat cerebellum during aging
© Blanco et al; licensee BioMed Central Ltd. 2010
Received: 15 October 2009
Accepted: 24 June 2010
Published: 24 June 2010
The cerebellum is the neural structure with the highest levels of nitric oxide, a neurotransmitter that has been proposed to play a key role in the brain aging, although knowledge concerning its contribution to cerebellar senescence is still unclear, due mainly to absence of integrative studies that jointly evaluate the main factors involved in its cell production and function. Consequently, in the present study, we investigate the expression, location, and activity of nitric oxide synthase isoenzymes; the protein nitration; and the production of nitric oxide in the cerebellum of adult and old rats.
Our results show no variation in the expression of nitric oxide synthase isoforms with aging, although, we have detected some changes in the cellular distribution pattern of the inducible isoform particularly in the cerebellar nuclei. There is also an increase in nitric oxide synthase activity, as well as greater protein-nitration levels, and maintenance of nitrogen oxides (NOx) levels in the senescent cerebellum.
The nitric oxide/nitric oxide syntahses system suffers from a number of changes, mainly in the inducible nitric oxide synthase distribution and in overall nitric oxide synthases activity in the senescent cerebellum, which result in an increase of the protein nitration. These changes might be related to the oxidative damage detected with aging in the cerebellum.
The cerebellum is the neural structure that produces the highest levels of nitric oxide (NO) within the central nervous system (CNS) . This high levels of NO may make cerebellum more susceptible to oxidative and nitrosative stress, particularly in the senescence . However, only a few preliminary studies from our research group [3, 4] and others  have tried to set a basic approach concerning the involvement of NO in the senescent cerebellum.
Although many studies have pointed to the gaseous neuromodulator nitric oxide (NO) as a key molecule in the whole brain-aging process , knowledge about its involvement in the senescent cerebellum is still unclear, due to absence of studies that analyse both the NOS expression and location within the cerebellum during aging. In this sense it is necessary to carry out a comprehensive study that covers all these as yet unexplained facts.
Considering all these antecedents, we hypothesize that aging could develop an overall key role in the modulation of the NO/NOS system in the cerebellum. Consequently, we analysed the response of the NO/NOS system during aging in the cerebellum of the rat. For this, we examined the expression, location and activity of the three nitric oxide synthase isoforms (nNOS, eNOS and iNOS) on mature adults (4 months old) and aged rats (26 months old). The study was completed with the analysis of the nitrotyrosine expression and location in the different experimental groups, and the measurement of nitrogen oxides (NOx) levels.
2.1. NOS isoform expression and location
Nitrated protein expression and location
Total nitrogen oxides (NOx)
In this study, we analyse the NO/NOS system and its involvement in the aging process in rat cerebellum. More specifically, the three NOS isoforms (nNOS, eNOS and iNOS) expression analysed by western-blot in the cerebellum of adult and aged individuals did not show any change with aging. This result confirms previous studies of our group , which only found changes in the expression of these enzymes when comparing young rats (sexually immature) with adult and old ones, but not between these two last groups. However, our results regarding NOS cellular distribution and activity, show new data that make it possible to appreciate age-related changes not detected by means of protein-expression studies solely.
Regarding the location of the three NOS isoforms within the different cell populations of the cerebellum, our data on adult animals were generally consistent with those previously reported by other authors [19–23]. In older animals, however, we found some changes related mostly to iNOS. These age-dependent changes suggest the possibility that the NO pathways involved in the cerebellar functions, previously established by other authors [24–26], might suffer certain changes during senescence. Although further analyses will be necessary to elucidate the positive/negative role of such changes, previous studies of our group  have suggested that aging appears to induce a type of preconditioning towards ischemic hypoxia in the cerebellum, possibly due to high baseline nitration levels, a feature also found in this study as we discuss later.
In relation to the immunohistochemical results, we have observed that the basket-cell processes surrounding Purkinje cell soma were nNOS-positive; this suggests that Purkinje cells may act as NO targets, a fact consistent both with the presence of nitrated proteins in Purkinje cells. These findings regarding nNOS location coincide with those described in adult rats [20, 22], and also indicate that there are no significant changes associated to aging in relation to the nitrergic system involved in Purkinje cells functions.
As previously mentioned, eNOS expression in the cerebellum shows no age-related changes when analysed by means of western-blot. This result coincides with those found by other authors, both in cerebral cortex  and cerebellum [4, 18]. Few works show eNOS location in the cerebellum; all these studies agree on the eNOS vascular location ; in addition, studies by our group  and others , have also found this isoform in Purkinje cells and neurons of the cerebellar nuclei as well.
According to our data, iNOS immunoreactivity in the cerebellar cortex is restricted to a faint mark in Purkinje cells in 4 and 26-month-old rats. On the contrary, while in adult individuals no iNOS-immunoreactive (iNOS-IR) neurons were observed in the cerebellar nuclei, interestingly, in the aged rats intensely marked neurons appeared. However, although cerebellar iNOS location pattern differs between both age groups, the overall iNOS expression in the cerebellum does not show significant differences with aging. In this sense, previous studies of our group  and other authors  have shown no changes in the expression of iNOS during aging as well, but only as a result of sexual maturation.
After treatment with aldehydes, only NOS-related NADPH-diaphorase (NADPH-d) activity remains, so that NADPH-d histochemical staining is usually used as a complementary method for the indirect demonstration of NOS activity by light microscopy [32, 33]. Further, some authors  have demonstrated a patchy expression pattern in the cerebellum of adult mice, but only in the granular layer. However, in the cerebellum of both adult and old rats, we detected a diffuse and uniformly distributed NADPH-diaphorase histochemical staining. Thus, we found similar location patterns between the two experimental groups, although the intensity of the histochemical mark is higher in the 26-month-old rat group. In addition, some studies have demonstrated an association of NADPH-d activity with NOS activity [35–38]. Considering all these facts, we could postulate that although overall NOS expression does not vary with age, NOS activity or at least that of one of its isoforms seems to be increased in older animals.
Our results also indicate an increase in protein nitration with aging, and the presence of n-Tyr-immunoreactive (n-Tyr-IR) Purkinje cells in both age groups. These data are consistent with those reported by Chung and collaborators . In addition, in the aged cerebellum we have found some Purkinje cells that show retraction phenomena affecting its apical processes, which could in turn affect the functionality of the cerebellar cortex. This phenomenon has been previously described in the ischemic rat cerebellum by Rodrigo and collaborators . We have also detected n-Tyr-IR glial cells, which could imply a mismatch in their defence and trophic support functions, thus influencing cerebellar neurons life expectancy as previously described by other studies  during aging.
The NOx levels analysed in this study do not vary significantly with the age of the rat; this fact is consistent with the stability in NOS expression, but not with the increased diaphorase activity previously described. Nevertheless, If we consider the protein nitration boost detected during aging, it could be interpreted that NO produced during senescence is assigned to a greater extent in the aged animals to protein nitration. The experimental procedure used for NOx determination detects only NO measured as nitrate plus nitrite, but not the NO bound to tyrosine residues. This fact could explain the gap between the increased protein nitration data and the invariability in NOx levels detected during aging. Moreover, Jesko and collaborators  stated that nNOS is the main responsible, by means of phosphorylation, for NOS activity during aging. NADPH-d activity can also be related to eNOS  as well, but some studies indicate a decline in the catalytic activity of eNOS in the aged cerebellum . Taking into account these data and that we have not found any differences between nNOS and eNOS expression or location with aging, we could hypothesise that the increased NADPH-d activity, and the subsequent increase in protein nitration detected during aging may not be due to eNOS, but further analysis are required.
In summary, the NO/NOS system suffers from a number of changes with aging in the cerebellum: 1) A change in the cellular distribution of iNOS but not in its expression. 2) Greater NADPH-d activity, and 3) An increased protein nitration. These results suggest that there are changes affecting the NO/NOS system and the destiny of the NO produced during aging in the cerebellum, such as variations in the production of reactive oxygen or decreases in anti-oxidation protection mechanisms.
The study was performed on 10 adult (Ad) (4-5 months old) and 10 aged (Ag) (26-27 months old) male albino Wistar rats kept under standard conditions of light and temperature and allowed ad libitum access to food and water. All procedures were carried out in accordance with the European Communities Council Directive (86/609/EEC), reviewed by the Ethics Committee of the Spanish Council for Scientific Research and approved by the Committee of Bioethics of the University of Jaén.
For western-blot analysis, the cerebella from 5 animals from each group were dissected and stored at -80°C until used. Tissues were homogenized in 1/3 (w/v) of 30 mM Tris-HCl, pH 7.4 containing 0.5 mM DTT, 1 mM EDTA, 1% SDS and protease inhibitors. The resulting homogenates were centrifuged for 1 h at 100,000xg. All the procedures were performed at 0 - 4°C. Protein concentrations in the supernatants were determined by the Bradford method . Equal amounts of the denatured proteins per lane were loaded and separated on a 7.5% (NOS) and 10% (Nitrotyrosine) SDS-polyacrylamide gel (Mini Protean 3. Bio-Rad, Hercules, CA, EE.UU.), as described by Laemmli . Afterwards, proteins were transferred to a PVDF membrane (Immobilon-P. Ref.: IPVH00010. Millipore, Billerica, MA, USA). The membranes were blocked with 5% powdered non-fat milk in 25 mM Tris-HCl, pH 7.6, 137 mM NaCl, 2.6 mM KCl, 0.1% Tween-20 and incubated overnight at 4°C with diluted, rabbit polyclonal anti-nNOS (1:3000, gift from V. Riveros-Moreno of Welcome Research Laboratories, Berkenhem, UK), monoclonal anti-iNOS and anti-eNOS (1:800, Transduction, Lexington, KY, USA), and rabbit polyclonal anti-nitrotyrosine (1:2000, produced by our group)  in blocking solution. Bound antibody was revealed by means of an enhanced chemiluminescence kit (Amersham, Little Chalfont, UK) according to the manufacturer's instructions. After immunodetection, membranes were probed with anti-α-tubulin (Sigma, St. Louis, MO, USA) as a loading control. The relative amount of the proteins in each sample was quantified by densitometric scanning and expressed as arbitrary units (A.U.).
For immunohistochemical procedures, cerebella from 5 adult (Ad) and 5 aged (Ag) rats were extracted and processed as follows. Deeply anaesthetized animals (15 mg/100 g B.W. i.p.; Ketolar. Parke Davis, Madrid, Spain) were perfused through the left ventricle with 50 ml of carbogenated 0.01 M phosphate-buffered saline (PBS; pH 7.4), and then with 300 ml of 4% paraformaldehyde in 0.1 M phosphate buffer (PB). The cerebella were removed and then postfixed for a further 4 h in the same fixative at room temperature. Samples were then cryoprotected by immersion overnight at 4°C in 0.1 M PB containing 30% sucrose. After that, the cerebella were embedded in O.C.T medium and frozen in 2-methylbutane pre-chilled in liquid nitrogen. Serial rostro-caudal sections (40 μm) were cut using a cryostat (Cryocut 1800. Reichert Jung, Wetzlar, Germany). Free-floating sections were incubated for 4 h in PBS containing 0.1% Triton X-100, and then in: nNOS, 1:900 (this antiserum was a gift from Dr. V. Riveros Moreno of Welcome Research Laboratories, Beckenham, UK; ); eNOS and iNOS, 1:150 (Transduction, Lexington, KY, USA); or in nitrotyrosine, 1:1,000  antisera diluted in PBS containing 0.2% Triton X-100, overnight at 4°C. After several rinses in PBS, and depending on the origin of the primary antisera, the sections were incubated with biotinylated goat anti-rabbit IgG or biotinylated rabbit anti-mouse IgG, 1:100 (Pierce, Rockford, IL, USA), and processed by the avidin-biotin peroxidase complex (ABC) procedure (Pierce, Rockford, IL, USA). The peroxidase activity was demonstrated following the nickel-enhanced diaminobenzidine assay . Control procedures were carried out on adjacent sections of the same tissues. No immunolabelling was detected when the primary antibody was either omitted or replaced with an equivalent concentration of pre-immune serum. Sections were then mounted on slides, dehydrated, and covered using DPX (Fluka, Madrid, Spain).
Free-floating sections were incubated for 4 h in PBS containing 0.1% Triton X-100. After several washes in 0.1 M Tris-HCl, pH 7.4 buffer, they were incubated in the dark, for 45 min at 37°C, in 0.1 M Tris-HCl, pH 7.4, containing 1 mM β-NADPH and 2 mM NBT (in 70% dimethylformamide). The sections were then washed twice with 0.1 M Tris-HCl, pH 7.4, quickly dehydrated in a graded ethanol series, cleared and mounted in DPX (Fluka, Madrid, Spain).
NO production was determined by tissue accumulation of nitrite and nitrate in the cerebellum of the same animals used for western-blot analysis. Briefly, a portion of tissue specimens from 5 animals of each group was homogenized in 3 volumes (w/v) of PBS (pH 7.6) at 4°C. Homogenates were then sonicated and centrifuged at 100,000xg for 60 min at 4°C. The nitrate plus nitrite (NOx) was determined in the supernatants using a colorimetric kit according to the manufacturer's instructions (Nitrate/Nitrite colorimetric Assay Kit. Cayman Chemical, Ann Arbor, MI, USA).
Data are expressed as means ± S.D. Student's t test was performed to evaluate the significance of differences between groups, accepting p < 0.05 as the level of significance.
List of abbreviations
endothelial nitric oxide synthase
inducible nitric oxide synthase
neuronal nitric oxide synthase
nitric oxide synthase
We wish to thank to Mr. David Nesbitt for his editorial help with the English version of the manuscript. Supported by "MCyT" (BIO 2000-0405-P4-05; SAF2003-04398-C02-02), the "Instituto de Salud Carlos III" (FIS-P1021240) and Junta de Andalucia (CVI-0184).
- Forstermann U, Gorsky LD, Pollock JS, Schmidt HH, Heller M, Murad F: Regional distribution of EDRF/NO-synthesizing enzyme(s) in rat brain. Biochem Biophys Res Commun. 1990, 168: 727-732. 10.1016/0006-291X(90)92382-A.View ArticlePubMedGoogle Scholar
- Peinado MA, del Moral ML, Esteban FJ, Martinez-Lara E, Siles E, Jimenez A, Hernandez-Cobo R, Blanco S, Rodrigo J, Pedrosa JA: [Aging and neurodegeneration: molecular and cellular bases]. Rev Neurol. 2000, 31: 1054-1065.PubMedGoogle Scholar
- Martinez-Romero R, Canuelo A, Martinez-Lara E, Hernandez R, Del Moral ML, Pedrosa JA, Peinado MA, Siles E: Aging affects but does not eliminate the enzymatic antioxidative response to hypoxia/reoxygenation in cerebral cortex. Exp Gerontol. 2006, 41: 25-31. 10.1016/j.exger.2005.09.009.View ArticlePubMedGoogle Scholar
- Siles E, Martinez-Lara E, Canuelo A, Sanchez M, Hernandez R, Lopez-Ramos JC, Del Moral ML, Esteban FJ, Blanco S, Pedrosa JA, et al.: Age-related changes of the nitric oxide system in the rat brain. Brain Res. 2002, 956: 385-392. 10.1016/S0006-8993(02)03575-8.View ArticlePubMedGoogle Scholar
- Chung YH, Shin CM, Joo KM, Kim MJ, Cha CI: Immunohistochemical study on the distribution of nitrotyrosine and neuronal nitric oxide synthase in aged rat cerebellum. Brain Res. 2002, 951: 316-321. 10.1016/S0006-8993(02)03261-4.View ArticlePubMedGoogle Scholar
- Harman D: Free radical theory of aging: effect of free radical reaction inhibitors on the mortality rate of male LAF mice. J Gerontol. 1968, 23: 476-482.View ArticlePubMedGoogle Scholar
- McCann SM: The nitric oxide hypothesis of brain aging. Exp Gerontol. 1997, 32: 431-440. 10.1016/S0531-5565(96)00154-4.View ArticlePubMedGoogle Scholar
- McCann SM, Licinio J, Wong ML, Yu WH, Karanth S, Rettorri V: The nitric oxide hypothesis of aging. Exp Gerontol. 1998, 33: 813-826. 10.1016/S0531-5565(98)00050-3.View ArticlePubMedGoogle Scholar
- Daniel H, Hemart N, Jaillard D, Crepel F: Long-term depression requires nitric oxide and guanosine 3':5' cyclic monophosphate production in rat cerebellar Purkinje cells. Eur J Neurosci. 1993, 5: 1079-1082. 10.1111/j.1460-9568.1993.tb00961.x.View ArticlePubMedGoogle Scholar
- Hartell NA: cGMP acts within cerebellar Purkinje cells to produce long term depression via mechanisms involving PKC and PKG. Neuroreport. 1994, 5: 833-836. 10.1097/00001756-199403000-00024.View ArticlePubMedGoogle Scholar
- Lev-Ram V, Makings LR, Keitz PF, Kao JP, Tsien RY: Long-term depression in cerebellar Purkinje neurons results from coincidence of nitric oxide and depolarization-induced Ca2+ transients. Neuron. 1995, 15: 407-415. 10.1016/0896-6273(95)90044-6.View ArticlePubMedGoogle Scholar
- Beckman JS, Koppenol WH: Nitric oxide, superoxide, and peroxynitrite: the good, the bad, and ugly. Am J Physiol. 1996, 271: C1424-1437.PubMedGoogle Scholar
- Eliasson MJ, Huang Z, Ferrante RJ, Sasamata M, Molliver ME, Snyder SH, Moskowitz MA: Neuronal nitric oxide synthase activation and peroxynitrite formation in ischemic stroke linked to neural damage. J Neurosci. 1999, 19: 5910-5918.PubMedGoogle Scholar
- Hanafy KA, Krumenacker JS, Murad F: NO, nitrotyrosine, and cyclic GMP in signal transduction. Med Sci Monit. 2001, 7: 801-819.PubMedGoogle Scholar
- Ischiropoulos H, Beckman JS: Oxidative stress and nitration in neurodegeneration: cause, effect, or association?. J Clin Invest. 2003, 111: 163-169.PubMed CentralView ArticlePubMedGoogle Scholar
- Crepel F, Jaillard D: Protein kinases, nitric oxide and long-term depression of synapses in the cerebellum. Neuroreport. 1990, 1: 133-136. 10.1097/00001756-199010000-00013.View ArticlePubMedGoogle Scholar
- Shimizu-Albergine M, Rybalkin SD, Rybalkina IG, Feil R, Wolfsgruber W, Hofmann F, Beavo JA: Individual cerebellar Purkinje cells express different cGMP phosphodiesterases (PDEs): in vivo phosphorylation of cGMP-specific PDE (PDE5) as an indicator of cGMP-dependent protein kinase (PKG) activation. J Neurosci. 2003, 23: 6452-6459.PubMedGoogle Scholar
- Strosznajder JB, Jesko H, Zambrzycka A, Eckert A, Chalimoniuk M: Age-related alteration of activity and gene expression of endothelial nitric oxide synthase in different parts of the brain in rats. Neurosci Lett. 2004, 370: 175-179. 10.1016/j.neulet.2004.08.013.View ArticlePubMedGoogle Scholar
- Leon-Chavez BA, Aguilar-Alonso P, Gonzalez-Barrios JA, Eguibar JR, Ugarte A, Brambila E, Ruiz-Arguelles A, Martinez-Fong D: Increased nitric oxide levels and nitric oxide synthase isoform expression in the cerebellum of the taiep rat during its severe demyelination stage. Brain Res. 2006, 1121: 221-230. 10.1016/j.brainres.2006.08.097.View ArticlePubMedGoogle Scholar
- Rodrigo J, Alonso D, Fernandez AP, Serrano J, Richart A, Lopez JC, Santacana M, Martinez-Murillo R, Bentura ML, Ghiglione M, Uttenthal LO: Neuronal and inducible nitric oxide synthase expression and protein nitration in rat cerebellum after oxygen and glucose deprivation. Brain Res. 2001, 909: 20-45. 10.1016/S0006-8993(01)02613-0.View ArticlePubMedGoogle Scholar
- Rodrigo J, Springall DR, Uttenthal O, Bentura ML, Abadia-Molina F, Riveros-Moreno V, Martinez-Murillo R, Polak JM, Moncada S: Localization of nitric oxide synthase in the adult rat brain. Philos Trans R Soc Lond B Biol Sci. 1994, 345: 175-221. 10.1098/rstb.1994.0096.View ArticlePubMedGoogle Scholar
- Serrano J, Encinas JM, Salas E, Fernandez AP, Castro-Blanco S, Fernandez-Vizarra P, Bentura ML, Rodrigo J: Hypobaric hypoxia modifies constitutive nitric oxide synthase activity and protein nitration in the rat cerebellum. Brain Res. 2003, 976: 109-119. 10.1016/S0006-8993(03)02691-X.View ArticlePubMedGoogle Scholar
- Southam E, Morris R, Garthwaite J: Sources and targets of nitric oxide in rat cerebellum. Neurosci Lett. 1992, 137: 241-244. 10.1016/0304-3940(92)90413-2.View ArticlePubMedGoogle Scholar
- Hartell NA: Receptors, second messengers and protein kinases required for heterosynaptic cerebellar long-term depression. Neuropharmacology. 2001, 40: 148-161. 10.1016/S0028-3908(00)00107-6.View ArticlePubMedGoogle Scholar
- Reynolds T, Hartell NA: Roles for nitric oxide and arachidonic acid in the induction of heterosynaptic cerebellar LTD. Neuroreport. 2001, 12: 133-136. 10.1097/00001756-200101220-00034.View ArticlePubMedGoogle Scholar
- Schweighofer N, Ferriol G: Diffusion of nitric oxide can facilitate cerebellar learning: A simulation study. Proc Natl Acad Sci USA. 2000, 97: 10661-10665. 10.1073/pnas.97.19.10661.PubMed CentralView ArticlePubMedGoogle Scholar
- Blanco S, Castro L, Hernandez R, Del Moral ML, Pedrosa JA, Martinez-Lara E, Siles E, Peinado MA: Age modulates the nitric oxide system response in the ischemic cerebellum. Brain Res. 2007, 1157: 66-73. 10.1016/j.brainres.2007.01.141.View ArticlePubMedGoogle Scholar
- Liu P, Smith PF, Appleton I, Darlington CL, Bilkey DK: Age-related changes in nitric oxide synthase and arginase in the rat prefrontal cortex. Neurobiol Aging. 2004, 25: 547-552. 10.1016/j.neurobiolaging.2003.07.003.View ArticlePubMedGoogle Scholar
- Hernandez R, Martinez-Lara E, Del Moral ML, Blanco S, Canuelo A, Siles E, Esteban FJ, Pedrosa JA, Peinado MA: Upregulation of endothelial nitric oxide synthase maintains nitric oxide production in the cerebellum of thioacetamide cirrhotic rats. Neuroscience. 2004, 126: 879-887. 10.1016/j.neuroscience.2004.04.010.View ArticlePubMedGoogle Scholar
- Shin T, Weinstock D, Castro MD, Hamir AN, Wampler T, Walter M, Kim HY, Acland H: Immunohistochemical localization of endothelial and inducible nitric oxide synthase within neurons of cattle with rabies. J Vet Med Sci. 2004, 66: 539-541. 10.1292/jvms.66.539.View ArticlePubMedGoogle Scholar
- Vernet D, Bonavera JJ, Swerdloff RS, Gonzalez-Cadavid NF, Wang C: Spontaneous expression of inducible nitric oxide synthase in the hypothalamus and other brain regions of aging rats. Endocrinology. 1998, 139: 3254-3261. 10.1210/en.139.7.3254.PubMedGoogle Scholar
- Kugler P, Hofer D, Mayer B, Drenckhahn D: Nitric oxide synthase and NADP-linked glucose-6-phosphate dehydrogenase are co-localized in brush cells of rat stomach and pancreas. J Histochem Cytochem. 1994, 42: 1317-1321.View ArticlePubMedGoogle Scholar
- Roufail E, Stringer M, Rees S: Nitric oxide synthase immunoreactivity and NADPH diaphorase staining are co-localised in neurons closely associated with the vasculature in rat and human retina. Brain Res. 1995, 684: 36-46. 10.1016/0006-8993(95)00394-6.View ArticlePubMedGoogle Scholar
- Schilling K, Schmidt HH, Baader SL: Nitric oxide synthase expression reveals compartments of cerebellar granule cells and suggests a role for mossy fibers in their development. Neuroscience. 1994, 59: 893-903. 10.1016/0306-4522(94)90293-3.View ArticlePubMedGoogle Scholar
- Andronowska A, Wasowska B, Calka J, Doboszynska T: Localization and correlation between NADPH-diaphorase and nitric oxide synthase isoforms in the porcine uterus during the estrous cycle. Cell Tissue Res. 2005, 321: 243-250. 10.1007/s00441-005-1085-9.View ArticlePubMedGoogle Scholar
- Necchi D, Virgili M, Monti B, Contestabile A, Scherini E: Regional alterations of the NO/NOS system in the aging brain: a biochemical, histochemical and immunochemical study in the rat. Brain Res. 2002, 933: 31-41. 10.1016/S0006-8993(02)02302-8.View ArticlePubMedGoogle Scholar
- Paakkari I, Lindsberg P: Nitric oxide in the central nervous system. Ann Med. 1995, 27: 369-377. 10.3109/07853899509002590.View ArticlePubMedGoogle Scholar
- Page GK, Morton AJ: Correlation of neuronal loss with increased expression of NADPH diaphorase in cultured rat cerebellum and cerebral cortex. Brain Res. 1995, 697: 157-168. 10.1016/0006-8993(95)00801-V.View ArticlePubMedGoogle Scholar
- Sloane JA, Hollander W, Moss MB, Rosene DL, Abraham CR: Increased microglial activation and protein nitration in white matter of the aging monkey. Neurobiol Aging. 1999, 20: 395-405. 10.1016/S0197-4580(99)00066-4.View ArticlePubMedGoogle Scholar
- Jesko H, Chalimoniuk M, Strosznajder JB: Activation of constitutive nitric oxide synthase(s) and absence of inducible isoform in aged rat brain. Neurochem Int. 2003, 42: 315-322. 10.1016/S0197-0186(02)00098-0.View ArticlePubMedGoogle Scholar
- Del Moral ML, Esteban FJ, Hernandez R, Blanco S, Molina FJ, Martinez-Lara E, Siles E, Viedma G, Ruiz A, Pedrosa JA, Peinado MA: Immunohistochemistry of neuronal nitric oxide synthase and protein nitration in the striatum of the aged rat. Microsc Res Tech. 2004, 64: 304-311. 10.1002/jemt.20081.View ArticlePubMedGoogle Scholar
- Martinez-Lara E, Canuelo AR, Siles E, Hernandez R, Del Moral ML, Blanco S, Pedrosa JA, Rodrigo J, Peinado MA: Constitutive nitric oxide synthases are responsible for the nitric oxide production in the ischemic aged cerebral cortex. Brain Res. 2005, 1054: 88-94. 10.1016/j.brainres.2005.06.060.View ArticlePubMedGoogle Scholar
- Riveros-Moreno V, Heffernan B, Torres B, Chubb A, Charles I, Moncada S: Purification to homogeneity and characterisation of rat brain recombinant nitric oxide synthase. Eur J Biochem. 1995, 230: 52-57. 10.1111/j.1432-1033.1995.tb20533.x.View ArticlePubMedGoogle Scholar
- Bradford MM: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976, 72: 248-254. 10.1016/0003-2697(76)90527-3.View ArticlePubMedGoogle Scholar
- Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970, 227: 680-685. 10.1038/227680a0.View ArticlePubMedGoogle Scholar
- Uttenthal LO, Alonso D, Fernandez AP, Campbell RO, Moro MA, Leza JC, Lizasoain I, Esteban FJ, Barroso JB, Valderrama R: Neuronal and inducible nitric oxide synthase and nitrotyrosine immunoreactivities in the cerebral cortex of the aging rat. Microsc Res Tech. 1998, 43: 75-88. 10.1002/(SICI)1097-0029(19981001)43:1<75::AID-JEMT11>3.0.CO;2-0.View ArticlePubMedGoogle Scholar
- Shu SY, Ju G, Fan LZ: The glucose oxidase-DAB-nickel method in peroxidase histochemistry of the nervous system. Neurosci Lett. 1988, 85: 169-171. 10.1016/0304-3940(88)90346-1.View ArticlePubMedGoogle Scholar
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