Do Routine Laboratory Parameters have Predictive Ability to Differentiate Subjects with Fibromyalgia from Healthy Subjects?

The aim of this study was to evaluate laboratory parameters for investigating their potential predictive ability to differentiate patients with fibromyalgia (FM) from healthy subjects. We carried out a case-control study with 79 FM patients and 20 controls to analyze complete blood count, serum chemistry profile, glycosylated hemoglobin (HbA1c), and erythrocyte sedimentation rate (ESR). The predictive value of these parameters was determined by receiver operating characteristic (ROC) analysis. We also examined the relationships with clinical parameters (functional capacity, pain, and physical and mental health status). Results showed significant differences in red blood cell count, hematocrit, mean corpuscular hemoglobin concentration, platelet count, creatinine, HbA1c, and ESR between groups. According to ROC analysis, all these parameters may assist in making FM diagnosis. Hematocrit and ESR values were correlated with FM clinical parameters. The determination of these routine laboratory parameters may be an uncomplicated means of facilitating FM diagnosis, together with the clinical data of the patient.

Biological Implications of a Stroke Therapy Based in Neuroglobin Hyaluronate Nanoparticles. Neuroprotective Role and Molecular Bases.

Exogenous neuroprotective protein neuroglobin (Ngb) cannot cross the blood-brain barrier. To overcome this difficulty, we synthesized hyaluronate nanoparticles (NPs), able to deliver Ngb into the brain in an animal model of stroke (MCAO). These NPs effectively reached neurons, and were microscopically identified after 24 h of reperfusion. Compared to MCAO non-treated animals, those treated with Ngb-NPs showed survival rates up to 50% higher, and better neurological scores. Tissue damage improved with the treatment, but no changes in the infarct volume or in the oxidative/nitrosative values were detected. A proteomics approach (p-value < 0.02; fold change = 0.05) in the infarcted areas showed a total of 219 proteins that significantly changed their expression after stroke and treatment with Ngb-NPs. Of special interest, are proteins such as FBXO7 and NTRK2, which were downexpressed in stroke, but overexpressed after treatment with Ngb-NPs; and ATX2L, which was overexpressed only under the effect of Ngb. Interestingly, the proteins affected by the treatment with Ngb were involved in mitochondrial function and cell death, endocytosis, protein metabolism, cytoskeletal remodeling, or synaptic function, and in regenerative processes, such as dendritogenesis, neuritogenesis, or sinaptogenesis. Consequently, our pharmaceutical preparation may open new therapeutic scopes for stroke and possibly for other neurodegenerative pathologies.

Hydroxytyrosol as a Promising Ally in the Treatment of Fibromyalgia.

Fibromyalgia (FM) is a chronic and highly disabling syndrome, which is still underdiagnosed, with controversial treatment. Although its aetiology is unknown, a number of studies have pointed to the involvement of altered mitochondrial metabolism, increased oxidative stress and inflammation. The intake of extra virgin olive oil, and particularly of one of its phenolic compounds, hydroxytyrosol (HT), has proven to be protective in terms of redox homeostatic balance and the reduction of inflammation. In this context, using a proteomic approach with nanoscale liquid chromatography coupled to tandem mass spectrometry, the present study analysed: (i) Changes in the proteome of dermal fibroblasts from a patient with FM versus a healthy control, and (ii) the effect of the treatment with a nutritional relevant dose of HT. Our results unveiled that fibroblast from FM show a differential expression in proteins involved in the turnover of extracellular matrix and oxidative metabolism that could explain the inflammatory status of these patients. Moreover, a number of these proteins results normalized by the treatment with HT. In conclusion, our results support that an HT-enriched diet could be highly beneficial in the management of FM.

Effects of Olive Oil Consumption on Cardiovascular Risk Factors in Patients with Fibromyalgia.

We have recently reported that patients with fibromyalgia (FM) may be at increased risk for cardiovascular disease. Olive oil reportedly has cardioprotective effects. We examined the influence of olive oil consumption on cardiovascular risk factors in FM. This preliminary study was performed on blood samples of women with FM who consumed 50 mL of organic olive oil daily for 3 weeks. Patients were randomized into two groups: 15 women ingested extra virgin olive oil (EVOO) and 15 refined olive oil (ROO). Cardiovascular risk markers were measured at baseline (pre measure) and after consumption of olive oil (post measure). Red blood cell count and erythrocyte sedimentation rate (ESR; both p < 0.05) declined significantly post-treatment in the EVOO group. Consumption of ROO increased mean platelet volume and reduced platelet distribution width (PDW), neutrophil-to-lymphocyte ratio, ESR and fibrinogen (all p < 0.05). Significant differences were found in pre-post change between the EVOO and ROO groups for cortisol and PDW (both p < 0.05). Our results have shown that consumption of olive oil may have antithrombotic and antiinflammatory properties in patients with FM, thereby improving a number of cardiovascular risk markers. Both EVOO and ROO may be useful as adjuvants for the prevention and/or treatment of cardiovascular disorders in these patients.

Are Patients With Fibromyalgia in a Prothrombotic State?

OBJECTIVES: The aim of this study was to investigate thrombosis-related parameters (blood coagulation parameters, platelet indices, red blood cell [RBC] count, and inflammatory markers) in patients with fibromyalgia (FM). METHOD: We carried out a case-control study with 35 women with FM and 12 age-matched healthy volunteers to analyze fibrinogen levels, prothrombin time, cephaline time, platelet count, platelet distribution width (PDW), mean platelet volume (MPV), RBC count, neutrophil-to-lymphocyte ratio, and platelet-to-lymphocyte ratio (PLR). RESULTS: The results showed significantly increased fibrinogen levels ( p < .05), platelet count ( p < .05), PDW ( p = .059), RBC count ( p < .05), and PLR ( p < .05) in women with FM versus the healthy volunteers. Prothrombin time ( p < .05) and MPV ( p < .05) were significantly lower in patients with FM than in the controls. CONCLUSIONS: Elevated platelet and RBC counts, PDW values, and fibrinogen levels as well as decreased prothrombin time are all indicative of a prothrombotic state in FM patients, which may be enhanced by an increased inflammatory tone. This prothrombotic state may increase the risk of thrombosis-related cardiovascular disease in patients with FM.

Catecholamine and Indolamine Pathway: A Case-Control Study in Fibromyalgia.

OBJECTIVES: Fibromyalgia (FM) is a complex syndrome characterized by widespread pain. Its etiology is unclear, and diagnosis is difficult. The aim of this study was to assess plasma levels of monoamine neurotransmitters (catecholamines, indolamines, and intermediate metabolites) in patients with FM and healthy controls to investigate possible alterations in the metabolism of these molecules in FM. We also examined potential relationships between monoamine neurotransmitters and clinical features of FM. The predictive value of these molecules in FM was determined by receiver operating characteristic analysis. METHOD: We measured plasma catecholamines (epinephrine, norepinephrine, and dopamine), as well as indolamines and intermediary metabolites (serotonin or 5-hydroxytryptamine [5-HT], 5-hydroxyindolacetic acid [5-HIAA], 5-hydroxytryptophan [5-HTP], and N-acetyl-5-hydroxytryptamine [Nac-5-HT]) in 35 women with FM and 12 age-matched healthy women. RESULTS: Higher levels of norepinephrine and lower levels of dopamine, 5-HT, 5-HIAA, and 5-HTP were found in women with FM in comparison with controls. Epinephrine and Nac-5-HT levels did not differ significantly between groups. Higher norepinephrine levels were associated with worse physical health status in FM patients. Also, plasma norepinephrine levels > 694.69 pg/ml might be an accurate predictor of FM. CONCLUSIONS: These findings show evidence of the dysregulation of the catecholamine and indolamine pathway in patients with FM, which may contribute to the physiopathology of this syndrome. In addition, the determination of plasma norepinephrine levels could help in the FM diagnosis.

Insight into the biological pathways underlying fibromyalgia by a proteomic approach.

Fibromyalgia (FM) is a form of non-articular rheumatism difficult to diagnose and treat because its etiology remains still elusive. Proteomics makes possible the systematic analysis of hundreds of proteins in clinical samples. Consequently, it has become a key tool for finding altered molecular pathways in different diseases. In this context, the present study analyzes changes in the plasma proteome of patients with FM by nanoscale liquid chromatography coupled to tandem mass spectrometry. Deregulated proteins were studied using Ingenuity Pathways Analysis (IPA) and Kyoto Encyclopedia of Genes and Genomes. Conventional analytical methods were used to validate selected proteins. We found a total of 33 proteins differentially expressed in patients with FM. Haptoglobin and fibrinogen showed the highest FM/control ratio. IPA analysis revealed that the top enriched canonical pathways were acute-phase response signaling, Liver-X Receptor/Retinoid-X Receptor activation, Farnesoid-X Receptor/Retinoid-X Receptor activation, and coagulation and complement systems. The importance of inflammation in FM was corroborated by the increase in erythrocyte sedimentation rate. In conclusion, our results support the existence of a plasma protein signature of FM that involves different biological pathways all of them related to inflammation, and point to haptoglobin and fibrinogen as plausible biomarker-candidates for future studies. SIGNIFICANCE: The etiology of fibromyalgia (FM) remains elusive making its diagnosis and treatment difficult. The characterization of the proteome signature of this syndrome will improve its understanding. However, to date proteomic analyses in FM are scarce. The goal of the present work is to analyse, for the first time, changes in plasma protein profiles of patients with FM in comparison to control subjects, using label free relative protein quantification by nanoscale liquid chromatography coupled to tandem mass spectrometry. Our data demonstrate the existence of a common protein signature in the plasma of patients with FM that could explain some of the symptoms associated to this syndrome. The analysis of the 33 proteins differentially expressed corroborates the crucial role of inflammation in the pathogenesis of this syndrome. The interplay of the complement and coagulation cascades contributes to the inflammatory process, while the activation of Liver-X Receptor/Retinoid-X Receptor and Farnesoid-X Receptor/Retinoid-X Receptor could attempt to alleviate it. Finally, we have identified two proteins, haptoglobin and fibrinogen, as potential biomarker-candidates of FM for future studies.

Response of the Nitric Oxide System to Hypobaric Hypoxia in the Aged Striatum.

BACKGROUND: Nitric oxide (NO) appears to play a key role in the hypoxic injury to the brain. We have previously reported that hypoxia/reoxygenation downregulated NO synthases (NOS) in the adult striatum. Until now, no data were available concerning the influence of aging in conjunction with hypoxia/reoxygenation on the NO system in the striatum. OBJECTIVE: The aim of this study was to assess the role of the NO pathway in the hypoxic aged striatum. METHODS: Wistar rats 24-25 months old were submitted to hypobaric hypoxia (20 min)/reoxygenation (0 h, 24 h, 5 days). Expression (PCR, immunohistochemistry/image analysis) and activity (NADPH-diaphorase/image analysis) of NOS isoforms (neuronal NOS or nNOS, endothelial NOS or eNOS, inducible NOS or iNOS) were analyzed together with nitrated protein expression (immunohistochemistry/image analysis). NO levels were indirectly quantified as nitrates/nitrites (NOx). RESULTS: The mRNA levels of NOS isoforms were undetectable at 0 h after hypoxia in the striatum compared to the control. At later reoxygenation times, nNOS mRNA decreased, while eNOS mRNA augmented. Protein levels of nNOS and eNOS rose at 24 h after hypoxia, and iNOS protein increased at 5 days. NOx levels remained unchanged, whereas in situ NOS activity and protein nitration diminished during reoxygenation in the aged striatum. CONCLUSION: The aged striatum may overexpress NOS isoforms as a neuroprotective-adaptive mechanism to hypoxia. However, this mechanism may not work properly in the aged striatum, since no changes in NO levels were detected after hypoxia. This may be related to the low activity of NOS isoforms in the hypoxic striatum.

Extra Virgin Olive Oil Improves Oxidative Stress, Functional Capacity, and Health-Related Psychological Status in Patients With Fibromyalgia: A Preliminary Study.

OBJECTIVES: Fibromyalgia (FM) is a chronic disease that imposes physical, psychological, and social limitations. We have reported that oxidative stress may play a role in the pathophysiology of FM. Olive oil has been shown to be effective treatment against the oxidative stress associated with several diseases. The aim of this study was to investigate the effect of olive oil on oxidative stress and health-related parameters in FM. METHODS: This preliminary study was performed on blood samples of 23 women diagnosed with FM who consumed 50 ml of organic olive oil daily for 3 weeks. Subjects were randomized into two groups: one ingested extra virgin olive oil (EVOO) and the other refined olive oil (ROO), which have different antioxidant content. The patients' oxidative (lipid, protein, and DNA oxidation) and antioxidative (antioxidant enzyme activities and compounds) profiles were examined before and after the treatment period. Functional capacity and physical and mental health status were assessed using the Fibromyalgia Impact Questionnaire (FIQ) and the Physical Component (PCS-12) and Mental Component Summaries (MCS-12) of the Short Form-12 Health Survey, respectively. RESULTS: Significant differences were found in pre-post change between the EVOO and ROO groups for protein carbonyls, lipid peroxidation, and FIQ and MCS-12 scores. Differences between groups approached statistical significance for oxidative DNA damage and levels of the antioxidant compound zinc. CONCLUSIONS: EVOO may protect women with FM against oxidative stress in addition to improving functional capacity and health-related psychological status. Findings suggest that olive oil may be a valuable therapeutic support in FM.

Nitric Oxide, Inflammation, Lipid Profile, and Cortisol in Normal- and Overweight Women With Fibromyalgia.

OBJECTIVES: Research has identified many factors associated with fibromyalgia (FM), but findings have been inconsistent. This study aimed to investigate changes in levels of nitric oxide (NO), inflammatory markers, lipid profile, and cortisol in normal- and overweight patients with FM and controls. Since most patients with FM are overweight, we explored possible changes in these markers according to body mass index (BMI). METHODS: This preliminary study was performed on serum samples of women with FM and age-matched controls, grouped according to their BMI: 12 normal-weight patients and 12 controls and 13 overweight patients and 8 controls. Ozone-based chemiluminescence assay was used to measure NO. Inflammatory mediators and cortisol were determined by immunoassay. Lipid profile was measured by a spectrophotometric procedure. Functional capacity was assessed by the fibromyalgia impact questionnaire (FIQ). RESULTS: Normal-weight patients showed higher levels of C-reactive protein (CRP) and apolipoprotein B compared to controls (both p < .05). CRP, apolipoprotein B, and triglycerides were higher in overweight patients versus overweight controls (all p < .05) and in overweight versus normal-weight patients (CRP p < .01; apolipoprotein B, triglycerides p < .05). The other markers were unaffected. Apolipoprotein B (r = .762; p < .05) and NO (r = -.921; p < .05) levels correlated with FIQ score in normal-weight patients. CRP level correlated with FIQ (r = .912; p < .05) in overweight patients. CONCLUSIONS: CRP and apolipoprotein B, biomarkers linked to cardiovascular events, may be associated with FM-related dysfunction in normal- and overweight women with FM. Their increased levels in these patients may indicate an increased risk of cardiovascular disease.

Acute hypoxia-induced depletion of striatal nitric oxide synthase pathway.

Hypoxia-induced alteration of nitric oxide (NO) production may lead to brain disease, especially in the areas most sensitive to oxygen deficiency, such as the striatum. To date, the behaviour of the striatal NO pathway under hypoxia/reoxygenation remains unknown and its elucidation constitutes the aim of this work. Wistar rats were submitted to hypoxia (20min) and analyzed after 0h, 24h, and 5 days of reoxygenation. Expression, activity, and location of the NO synthase (NOS) isoforms (neuronal, endothelial, and inducible) as well as nitrated protein expression were analyzed in the rat striatum. NO levels were indirectly quantified as nitrates and nitrites (NO(x)), which act as NO-generating molecules. NOS isoform mRNA levels remained unaltered in hypoxic groups vs. normoxic control. However, quantification of immunoreaction showed a significant decrease in eNOS and nNOS after hypoxia. While in situ NOS activity and NO(x) levels fell, levels of nitrotyrosine-modified proteins rose throughout the reoxygenation period. Our data revealed the great complexity of the NO pathway, showing that both acute hypoxia and the successive recovery period down-regulated the NOS system in the rat striatum. However, under hypoxia/reoxygenation NO may be produced in a NOS-independent way from the NO-storage molecules, compensating for the hypoxia-reduced NOS activity.

Does inducible NOS have a protective role against hypoxia/reoxygenation injury in rat heart?

PURPOSE: The present study analyzes the role of the nitric oxide (NO) derived from inducible NO synthase (iNOS) under cardiac hypoxia/reoxygenation situations. METHODS: For this, we have designed a follow-up study of different parameters of cell and tissue damage in the heart of Wistar rats submitted for 30 min to acute hypobaric hypoxia, with or without prior treatment with the selective iNOS inhibitor N-(3-(aminomethyl)benzyl) acetamidine or 1400W (10 mg/kg). The rats were studied at 0 h, 12 h, and 5 days of reoxygenation, analyzing NO production (NOx), lipid peroxidation, apoptosis, and protein nitration expression and location. This is the first time-course study which analyzes the effects of the iNOS inhibition by 1400W during hypoxia/reoxygenation in the adult rat heart. RESULTS: The results show that when 1400W was administered before the hypoxic episode, NOx levels fell, while both the lipid peroxidation level and the percentage of apoptotic cells rose throughout the reoxygenation period. Levels of nitrated proteins expression fell only at 12 h post-hypoxia. CONCLUSIONS: The inhibition of iNOS raises the peroxidative and apoptotic level in the hypoxic heart indicating that this isoform may have a protective effect on this organ against hypoxia/reoxygenation injuries, and challenging the conventional wisdom that iNOS is deleterious under these conditions. These findings could help in the design of new treatments based on NO pharmacology against hypoxia/reoxygenation dysfunctions.

Inducible NOS inhibitor 1400W reduces hypoxia/re-oxygenation injury in rat lung.

Nitric oxide (NO(*)) from inducible NO(*) synthase (iNOS) has been reported to either protect against, or contribute to, hypoxia/re-oxygenation lung injury. The present work aimed to clarify this double role in the hypoxic lung. With this objective, a follow-up study was made in Wistar rats submitted to hypoxia/re-oxygenation (hypoxia for 30 min; re-oxygenation of 0 h, 48 h, and 5 days), with or without prior treatment with the selective iNOS inhibitor 1400W (10 mg/kg). NO(*) levels (NOx), lipid peroxidation, apoptosis, and protein nitration were analysed. This is the first time-course study which investigates the effects of 1400W during hypoxia/re-oxygenation in the rat lung. The results showed that the administration of 1400W lowered NOx levels in all the experimental groups. In addition, lipid peroxidation, the percentage of apoptotic cells, and nitrated protein expression fell in the late post-hypoxia period (48 h and 5 days). Our results reveal that the inhibition of iNOS in the hypoxic lung reduced the damage observed before the treatment with 1400W, suggesting that iNOS-derived NO(*) may exert a negative effect on this organ during hypoxia/re-oxygenation. These findings are notable, since they indicate that any therapeutic strategy aimed at controlling excess generation of NO(*) from iNOS may be useful in alleviating NO(*)-mediated adverse effects in hypoxic lungs.

Lung eNOS and iNOS are reoxygenation time-dependent upregulated after acute hypoxia.

Nitric oxide plays a critical role in many physiological and physiopathological processes in the lung. Changes in the NO/NOS (Nitric Oxide/Nitric Oxide Synthase) system after hypoxia situations remain controversial in this organ, so that the aim of this work is to perform a complete study of this system in the hypoxic lung after different reoxygenation times ranging from 0 h to 5 days posthypoxia. This is a novel follow-up study carried out in Wistar rats submitted for 30 min to acute hypobaric hypoxia. We measured endothelial and inducible NOS (eNOS, iNOS) mRNA and protein expression, location, and in situ NOS activity as well as nitrated protein expression and location. In addition, NO levels were indirectly quantified (NOx) as well as the apoptosis level. Results showed an increase in eNOS mRNA, protein, activity as well as eNOS positive immunostaining at 0 h posthypoxia, coinciding with raised NOx levels. Contrary, iNOS, nitrated protein expression and apoptosis level augmented during the final reoxygenation times. The lung NO/NOS system provokes two responses to the hypoxia/reoxygenation processes: (i) eNOS is responsible of the immediate response, producing NO, which causes vasodilation and bronchodilation, and (ii) iNOS is related to the second late response, which seems to be involved in some of the deleterious consequences that hypoxia induces in the lung.

Age modulates the nitric oxide system response in the ischemic cerebellum.

To determine whether age influences the nitric oxide system response to ischemia in the cerebellum, we have analyzed the levels of nitrogen oxides (NOx) and the expression of the different nitric oxide synthase isoforms (NOS) in mature adult (4-5 months old) and aged rats (24-27 months old) subjected to a transient global ischemia/reperfusion (I/R) model. We also analyzed the nitrated proteins and the glial fibrillary acidic protein (GFAP) expression. NOx concentration in adult rats, which more than doubled the values found in the aged rats, decreased after the ischemia and reperfusion. However, in the aged animals, these NOx levels did not significantly change after I/R. Constitutive isoforms were first down-regulated in the ischemic period, in both adult and aged animals. However, after 6 h of reperfusion, these isoforms were up-regulated, but only in aged rats. After I/R, iNOS was up-regulated in adults but down-regulated in the aged rats. Hence, after an episode of transient global ischemia and reperfusion, the aged cerebellum maintains a balanced NO production, silencing the iNOS isoform and inducing a weak expression of nNOS and eNOS; this allows NO physiological functions while avoiding possible undesirable effects such as the nitrative damage or astrocyte activation.

Steatosis recovery after treatment with a balanced sunflower or olive oil-based diet: involvement of perisinusoidal stellate cells.

AIM: To analyze the relationship between perisinusoidal stellate cell (PSC) activation and the dietary fat quantity and composition in the treatment of hepatic steatosis. METHODS: Using an experimental rat model of steatosis based on the intake of a hyperlipidic diet (14% fat as olive oil or sunflower oil, HL-O and HL-S, respectively), we analyzed the liver's capability of recovery after the treatment with a normal-lipidic diet (5% fat as olive oil or sunflower oil, NL-O and NL-S, respectively) by immunocytochemical and Western blot analysis of glial fibrillary acidic protein (GFAP) expression in PSCs, collagen quantification and serum aminotransferase determination. RESULTS: The fatty infiltration in the steatotic livers decreased after the treatment with both NL diets, indicating liver recovery. This decrease was accompanied with a lower collagen deposition and aminotransferase level as well as changes in the PSC population that increased the GFAP expression. The above-mentioned effects were more pronounced in animals fed on NL-O based diet. CONCLUSION: Treatment with a balanced diet enriched in olive oil contributes to the liver recovery from a steatotic process. The PSC phenotype is a marker of this hepatic-recovery model.

Immunohistochemistry of neuronal nitric oxide synthase and protein nitration in the striatum of the aged rat.

To ascertain the possible implications of the nitric oxide (NO*) producing system in striatal senescence, and by using immunohistochemistry and image-processing approaches, we describe the presence of the enzyme nitric oxide synthase (NOS), the NADPH-diaphorase (NADPH-d) histochemical marker, and nitrotyrosine-derived complexes (N-Tyr) in the striatum of adult and aged rats. The results showed neuronal NOS immunoreactive (nNOS-IR) aspiny medium-sized neurons and nervous fibres in both age groups, with no variation in the percentage of immunoreactive area but a significant decrease in the intensity and in the number of somata with age, which were not related to the observed increase with age of the striatal bundles of the white matter. In addition, NADPH-d activity was detected in neurons with morphology similar to that of the nNOS-IR cells; a decrease in the percentage of area per field and in the number of cells, but an increase in the intensity of staining for the NADPH-d histochemical marker, were detected with age. The number of neuronal NADPH-d somata was higher than for the nNOS-IR ones in both age groups. Moreover, N-Tyr-IR complexes were observed in cells (neurons and glia) and fibres, with a significant increase in the percentage of the area of immunoreaction, related to the increase of white matter, but a decrease in intensity for the aged group. On the other hand, we did not detect the inducible isoform (iNOS) either in adult or in aged rats. Taken together, these results support the contention that NADPH-d staining is not such an unambiguous marker for nNOS, and that increased protein nitration may participate in striatal aging.

Age-related changes of the nitric oxide system in the rat brain.

This work examines the age-related changes of the NO pathway in the central nervous system (CNS), analyzing nitric oxide synthase (NOS) isoform expression, the level of nitrotyrosine-modified proteins, and the NOS activity in the cerebral cortex, decorticated brain (basal ganglia, thalamus, hypothalamus, tegtum and tegmentum) and cerebellum of young, adult and aged rats. Our data demonstrate that the different NOS isoforms are not uniformly expressed across the CNS. In this sense, the nNOS and eNOS isoenzymes are expressed mainly in the cerebellum and decorticated brain, respectively, while the iNOS isoenzyme shows the highest level in cerebellum. Concerning age, in the cerebral cortex nNOS significantly increased its expression only in adult animals; meanwhile, in the cerebellum the eNOS expression decreased whereas iNOS increased in adult and aged rats. No age-related changes in any isoform were found in decorticated brain. NOS activity, determined by nitrate plus nitrite quantification, registered the highest levels in the cerebellum, where the significant increase detected with aging was probably related to iNOS activity. The number of nitrotyrosine-modified immunoreactive bands differed among regions; thus, the highest number was detected in the decorticated brain while the cerebellum showed the least number of bands. Finally, bulk protein nitration increased in cerebral cortex only in adult animal. No changes were found in the decorticated brain, and the decrease detected in the cerebellum of aged animals was not significant. According to these results, the NO pathway is differently modified with age in the three CNS regions analyzed.