Cerebrospinal Fluid Nitrite/Nitrate Levels in Neurologic Diseases

Abstract: Nitric oxide has been proposed to mediate cytotoxic effects in inflammatory diseases. To investigate the possibility that overproduction of nitric oxide might play a role in the neuropathology of inflammatory and noninflammatory neurological diseases, we compared levels of the markers of nitric oxide, nitrite plus nitrate, in the CSF of controls with those in patients with various neurologic diseases, including Huntington's and Alzheimer's disease, amyotrophic lateral sclerosis, and HIV infection. We found that there were no significant increases in the CSF levels of these nitric oxide metabolites, even in patients infected with HIV or in monkeys infected with poliovirus, both of which have significantly elevated levels of the neurotoxin quinolinic acid and the marker of macrophage activation, neopterin. However, CSF quinolinic acid, neopterin, and nitrite/nitrate levels were significantly increased in a small group of patients with bacterial and viral meningitis.     

Effect of alpha-phenyl-N-tert-butylnitrone on brain cell membrane function and energy metabolism in experimental Escherichia coli meningitis in the newborn piglet

We evaluated the efficacy of alpha-phenyl-N-tertbutylnitrone as an adjunctive therapy in experimental bacterial meningitis in the newborn piglet. Meningitis was induced by intracisternal injection of 10(8) colony-forming units of Escherichia coli in 100 microl of saline. Alpha-Phenyl-N-tert-butylnitrone 100 mg/kg was given as a bolus intravenous injection 30 min before induction of meningitis. Although it completely abolished the elevated CSF tumor necrosis factor-a level observed in the meningitis group, alpha-phenyl-N-tert-butylnitrone did not down-modulate parameters of inflammatory responses such as increased intracranial pressure, hypoglycorrhachia, elevated CSF lactate level, and CSF leukocytosis observed in this group. However, alpha-phenyl-N-tert-butylnitrone treatment mitigated alterations in brain cell membrane structure and function during meningitis, evidenced by amelioration of increased brain cell membrane lipid peroxidation products (conjugated dienes) and decreased Na+, K+-ATPase activity. Reduced mean arterial blood pressure, cerebral perfusion pressure, brain glucose concentration, and cerebral energy stores and marginally increased brain lactate level observed in the meningitis group were also ameliorated. These results suggest that although it failed to attenuate the inflammatory responses, alpha-phenyl-N-tert-butylnitrone was effective in ameliorating brain injury in neonatal bacterial meningitis.     

Extraordinary Changes in Excitatory Amino Acid Levels in Cerebrospinal Fluid of Influenza-Associated Encephalopathy of Children

The correlation between the glutamate-glutamine cycle and nitric oxide (NO) production in the central nervous system (CNS) of a new type of influenza-associated encephalopathy in children is discussed. When measurements of several amino acids and NOx (nitrite/nitrate) levels in the cerebrospinal fluid (CSF) using HPLC-fluorescence and -UV methods, respectively, were made. the CSF glutamate levels of patients with the new type of encephalitis were significantly lower, and both glutamine and NOx levels were significantly higher than those of the control group and the patients of the meningitis group. Results indicate that the turnover rate of glutamate in CNS, particularly in the brain, increases in the influenza-associated encephalopathy. The high mortality in the disease may correlate with the hyperactivity of supra-spinal glutamate neurons and the subsequent high activity levels of NOx in CNS.     

Oral administration of Crataegus flavonoids protects against ischemia/reperfusion brain damage in gerbils

Stroke is the third leading cause of death as dementia is a main symptom of Alzheimer's disease. One of the important mechanisms in the pathogeny of stroke is free radical production during the reperfusion period, therefore the effects of a type of natural antioxidant, i.e. Crataegus flavonoids (CF), on brain ischemic insults were investigated in Mongolian gerbil stroke model. Results showed that pretreatment of the animals with CF decreased reactive oxygen species (ROS) production, thiobarbituric acid reactive substances content, and nitrite/nitrate concentration in brain homogenate, increased the brain homogenate-associated antioxidant level in a dose-dependent manner. CF pretreatment increased the amount of biologically available NO by scavenging of superoxide anion produced during reperfusion. At same time, in the process of ischemia/reperfusion brain damage, the content of nitrite/nitrate (the end product of NO) increased, and of NO detected by ESR decreased. Oral pretreatment with CF decreased the nitrite/nitrate content in the brain homogenate and increased the biologically available NO concentration in a dose-dependent manner. The increasing effect of antioxidant on NO might be due to its scavenging effect on superoxide anion, which could react with NO into peroxynitrite. iNOS was implied in delayed neuron death after brain ischemic damage and it was found that pretreatment with CF could decrease the protein level of tumor necrosis factor (TNF)-alpha and nuclear factor-kappa B (NF-kappaB), and increase the mRNA level of NOS estimated by western blotting and RT-PCR. More neurons survived and fewer cells suffered apoptosis in the hippocampal CA1 region of CF treated animal brain. These results suggest that oral administration of this antioxidant increases the antioxidant level in the brain and protects the brain against delayed cell death caused by ischemia/reperfusion injury.     

Spin trapping agent, phenyl N-tert-butylnitrone, reduces nitric oxide production in the rat brain during experimental meningitis

Phenyl N-tert-butylnitrone (PBN) is a spin trapping agent previously shown to exert a neuroprotective effect in infant rat brain during bacterial meningitis. In the present study, we investigated the effect of systemic PBN administration on nitric oxide (NO) production in a rat model of experimental meningitis induced by lipopolysaccharide (LPS). We assessed the NO concentration in rat brain tissues with an electron paramagnetic resonance (EPR) NO trapping technique. In this model, rats receiving intracisternal LPS administration showed symptoms of meningitis and cerebrospinal fluid (CSF) pleocytosis. The time course study indicated that the concentration of NO in the brain reached the maximum level 8.5h after injection of LPS, and returned to the control level 24 h after the injection. When various doses of PBN (125-400 mg/kg) were injected intraperitoneally 30 min prior to LPS, NO production in the brain was reduced with increasing PBN dose (250 mg/kg suppressed 80% at 8.5h after LPS injection), and white blood cells (WBC) in CSF were significantly decreased. We concluded that reduction of NO generation during bacterial meningitis contributes to the neuroprotective effect of PBN in addition to its possible direct scavenging of reactive oxygen intermediate (ROI).     

Spin trapping agent,phenyl N-tert-butylnitrone,reduces nitric oxide production in the rat brain during experimental meningitis

Phenyl N-tert-butylnitrone (PBN) is a spin trapping agent previously shown to exert a neuroprotective effect in infant rat brain during bacterial meningitis. In the present study, we investigated the effect of systemic PBN administration on nitric oxide (NO) production in a rat model of experimental meningitis induced by lipopolysaccharide (LPS). We assessed the NO concentration in rat brain tissues with an electron paramagnetic resonance (EPR) NO trapping technique. In this model, rats receiving intracisternal LPS administration showed symptoms of meningitis and cerebrospinal fluid (CSF) pleocytosis. The time course study indicated that the concentration of NO in the brain reached the maximum level 8.5h after injection of LPS, and returned to the control level 24 h after the injection. When various doses of PBN (125–400 mg/kg) were injected intraperitoneally 30 min prior to LPS, NO production in the brain was reduced with increasing PBN dose (250 mg/kg suppressed 80% at 8.5h after LPS injection), and white blood cells (WBC) in CSF were significantly decreased. We concluded that reduction of NO generation during bacterial meningitis contributes to the neuroprotective effect of PBN in addition to its possible direct scavenging of reactive oxygen intermediate (ROI).     

Matrix metalloproteinase-12 leads to elastin degradation in BALB/c mice with eosinophilic meningitis caused by Angiostrongylus cantonensis

The rat lugworm Angiostrongylus cantonensis can cause eosinophilic meningitis. The purpose of this study was to determine whether matrix metalloproteinase (MMP)-12 and its substrate elastin participate in this inflammatory response. We showed that the MMP-12/tissue inhibitor of metalloproteinase-1 ratio was significantly increased in the CSF of A. cantonensis-infected mice from day 10 p.i., and reached high levels on days 20 and 25 p.i. MMP-12 production was correlated with elastin degradation, eosinophil count, blood–CSF barrier permeability and pathological changes in the subarachnoid space. Also, MMP-12 might contribute to elastin degradation in the meningeal vessel of the subarachnoid space. Simultaneous administration of albendazole and doxycycline significantly reduced the levels of MMP-12, elastin and Evans blue in mice with meningitis. These results imply that MMP-12 contributes to the elastin degradation that occurs in angiostrongyliasis meningitis, and doxycycline can reverse related inflammatory events by inhibition of MMP-12.     

Dynamics of Nitric Oxide and Peroxynitrite During Global Brain Ischemia/Reperfusion in Rat Hippocampus: NO-sensor Measurement and Modeling Study

The dynamics of nitric oxide (NO) and peroxynitrite concentration changes during brain ischemia/reperfusion are poorly understood. In this paper, a NO-selective sensor was used to measure NO concentration changes in the rat brain hippocampus during global brain ischemia/reperfusion. Four-vessel occlusion model of transient global brain ischemia was used. Global cerebral ischemia was induced by occluding both common carotid arteries with artery nips (for 20 min) and reperfusion was induced by loosening the artery nips. Results showed that the changes of NO concentration during global brain ischemia/reperfusion could be divided into different stages. Together with the effects of O₂ tension changes and NO synthase (NOS) on nitric oxide levels, we determined five stages in the NO concentration profile: (1) acute O₂-limited decrease stage; (2) O₂-limited steady stage; (3) neuronal NOS activation stage; (4) acute O₂-recovery elevation stage; and (5) O₂-recovery steady stage. In addition, a chemical reaction network model was constructed to simulate the dynamics of peroxynitrite during the reperfusion stage, and the effects of a change in the NO formation rate on the dynamics of peroxynitrite were investigated specifically. Results show the rate of NO formation has a great influence on peroxynitrite dynamics.     

Gas Phase Oxidants of Cigarette Smoke Increase Nitric Oxide Synthase and Xanthine Oxidase Activities of Rabbit Brain Synaptosomes

In the present study we demonstrated that NO synthase and xanthine oxidase of synaptosomes isolated from rabbit brain cortex can be activated by the gas phase of cigarette smoke to produce nitric oxide and superoxide which react together to form peroxynitrite. Expose of synaptosomes, up to 3 hours, in the gas phase of cigarette smoke, a gradual increase in both nitric oxide and superoxide release that were inhibited by N-monomethyl-L-arginine (100 M) and oxypurinol (1 mM), respectively, was observed. NO synthase and xanthine oxidase activities were increased approximately three fold after treatment of synaptosomes with the gas phase of cigarette smoke as compared with the gas phase deprived of oxidants. Synaptosomes treated with the gas phase of cigarette smoke dramatically increased 3-nitrotyrosine production (used as an index of peroxynitrite formation). Synaptosomes treated with the gas phase of cigarette smoke, promptly increased malondialdehyde production with subsequent decrease of synaptosomal plasma membrane fluidity estimated by fluorescence anisotropy of 1,4-(trimethyl-amino-phenyl)-6-phenyl-hexa-1,3,5-triene. Gas phase deprived of oxidants showed a small but not statistically significant (p > 0.05) effect on both malondialdehyde and membrane fluidity. In summary, the present results indicate that activation of NO synthase and xanthine oxidase of brain cells by oxidants contained in the gas phase of cigarette smoke lead to the formation of peroxynitrite a causative factor in neurotoxicity.     

Redox thiol status plays a central role in the mobilization and metabolism of nitric oxide in human red blood cells

We assessed the redox thiol status influence on nitric oxide (NO) metabolism and efflux in erythrocytes stimulated with acetylcholinesterase substrate (acetylcholine, ACh) and inhibitor (velnacrine maleate, VM). Erythrocyte suspensions from healthy donors were incubated with increasing concentrations of dithiothreitol (1-50 μM), in the presence and absence of acetylcholine/velnacrine (10 μM). Levels of NO, nitrite/nitrate, S-nitrosohemoglobin, peroxynitrite and S-nitrosoglutathione were determined by spectrofluorimetric and spectrophotometric methods.Dithiothreitol significantly mobilized NO toward nitrite/nitrate and S-nitrosoglutathione, and decreased the amount of NO efflux. Both ACh/VM induce changes on the levels of erythrocyte nitrite/nitrate dependent on the DTT concentration. Higher levels of peroxynitrite and S-nitrosoglutathione were seen with velnacrine in presence of DTT 1 and 50 μM.We concluded that dithiothreitol-induced activation of erythrocyte thiol status decreases NO efflux and allows greater intracellular NO mobilization onto different derivative molecules, both in the absence and presence of acetylcholinesterase substrate and inhibitor.     

Oxidative stress in brain during experimental bacterial meningitis: differential effects of alpha-phenyl-tert-butyl nitrone and N-acetylcysteine treatment

Antioxidant treatment has previously been shown to be neuroprotective in experimental bacterial meningitis. To obtain quantitative evidence for oxidative stress in this disease, we measured the major brain antioxidants ascorbate and reduced glutathione, and the lipid peroxidation endproduct malondialdehyde in the cortex of infant rats infected with Streptococcus pneumoniae. Cortical levels of the two antioxidants were markedly decreased 22 h after infection, when animals were severely ill. Total pyridine nucleotide levels in the cortex were unaltered, suggesting that the loss of the two antioxidants was not due to cell necrosis. Bacterial meningitis was accompanied by a moderate, significant increase in cortical malondialdehyde. While treatment with either of the antioxidants alpha-phenyl-tert-butyl nitrone or N-acetylcysteine significantly inhibited this increase, only the former attenuated the loss of endogenous antioxidants. Cerebrospinal fluid bacterial titer, nitrite and nitrate levels, and myeloperoxidase activity at 18 h after infection were unaffected by antioxidant treatment, suggesting that they acted by mechanisms other than modulation of inflammation. The results demonstrate that bacterial meningitis is accompanied by oxidative stress in the brain parenchyma. Furthermore, increased cortical lipid peroxidation does not appear to be the result of parenchymal oxidative stress, because it was prevented by NAC, which had no effect on the loss of brain antioxidants.     

NO synthase and xanthine oxidase activities of rabbit brain synaptosomes: Peroxynitrite formation as a causative factor of neurotoxicity

In the present study we demonstrated that synaptosomes isolated from rabbit brain cortex contain NO synthase and xanthine oxidase that can be activated by ultraviolet B radiation and Ca²⁺ accumulation to produce nitric oxide and superoxide which react together to form peroxynitrite. Irradiation of synaptosomes with ultraviolet B (up to 100 mJ/cm²), or increase the intrasynaptosomal calcium concentration using various doses (up to 100 μM) of the calcium ionophore A 23187, a gradual increase in both nitric oxide and peroxynitrite release that was inhibited by N-monomethyl-L-arginine (100 μM) was observed. The rate of nitric oxide release and cyclic GMP production by NO synthase and soluble guanylate cyclase, both located in the soluble fraction of synaptosomes (synaptosol), were increased approximately eight fold after treatment of synaptosomes with Ultraviolet B radiation (100 mJ/cm²). In reconstitution experiments, when purified NO synthase isolated from synaptosol was added to xanthine oxidase, in the presence of the appropriate cofactors and substrates, a ten fold increase in peroxynitrite production at various doses (up to 20 mJ/cm²) of UVB radiation was observed. Ultraviolet B irradiated synaptosomes promptly increased malondialdehyde production with subsequent decrease of synaptosomal plasma membrane fluidity estimated by fluorescence anisotropy of 1-4-(trimethyl-amino-phenyl)-6-phenyl-hexa-1,3,5-triene. Desferrioxamine (100 μM) tested in Ultraviolet B-irradiated synaptosomes showed a decrease (approximately 80%) in malondialdehyde production with subsequent restoration of the membrane fluidity to that of non-irradiated (control) synaptosomes. Ca²⁺-stimulated ATPase activity was decreased after Ultraviolet B (100 mJ/cm²) radiation of synaptosomes indicating that the subsequent increase of intrasynaptosomal calcium promoted peroxynitrite production by a calmodulin-dependent increase of NO synthase and xanthine oxidase activities. Furthermore, it was shown that UVB-irradiated synaptosomes were subjected to higher oxidative stress by exogenous peroxynitrite (100 μM) compared to non-irradiated (control) synaptosomes. In summary, the present results indicate that activation of NO synthase and xanthine oxidase of brain cells lead to the formation of peroxynitrite providing important clues in the role of peroxynitrite as a causative factor in neurotoxicity.     

Urate Oxidation in CSF and Blood of Patients with Inflammatory Disorders of the Nervous System

Urate is largely excluded from the brain under non‐inflammatory conditions (concentration gradient serum:CSF about 10:1), but increases markedly in Guillain–Barré Syndrome and bacterial meningitis. The oxidation product allantoin is normally not passively distributed between blood and cerebrospinal fluid (gradient 3:1) and increases 5‐fold in CSF of patients with meningitis. Patients with multiple sclerosis had normal levels of urate and allantoin in blood and CSF.     

Nitrate tolerance does not increase production of peroxynitrite in the heart

Clinical studies have suggested that long-term nitrate treatment does not improve and may even worsen cardiovascular mortality, and the possible role of nitrate tolerance has been suspected. Nitrate tolerance has been recently shown to increase vascular superoxide and peroxynitrite production leading to vascular dysfunction. Nevertheless, nitrates exert direct cardiac effects independent from their vascular actions. Therefore, we investigated whether in vivo nitroglycerin treatment leading to vascular nitrate tolerance increases cardiac formation of nitric oxide (NO), reactive oxygen species, and peroxynitrite, thereby leading to cardiac dysfunction. Nitrate tolerance increased bioavailability of NO in the heart without increasing formation of reactive oxygen species. Despite elevated myocardial NO, neither cardiac markers of peroxynitrite formation nor cardiac mechanical function were affected by nitroglycerin treatment. However, serum free nitrotyrosine, a marker for systemic peroxynitrite formation, was significantly elevated in nitroglycerin-treated animals. This is the first demonstration that, although the systemic effects of nitroglycerin may be deleterious due to enhancement of extracardiac peroxynitrite formation, nitroglycerin does not result in oxidative damage in the heart.     

Hypoxia potentiates nitric oxide-mediated apoptosis in endothelial cells via peroxynitrite-induced activation of mitochondria-dependent and -independent pathways

Nitric oxide (NO*) at low concentrations is cytoprotective for endothelial cells; however, elevated concentrations of NO* (> or =1 micromol/liter), as may be achieved during inflammatory states, can induce apoptosis and cell death. Hypoxia is associated with tissue inflammation and ischemia and, therefore, may modulate the effects of NO* on endothelial function. To examine the influence of hypoxia on NO*-mediated apoptosis, we exposed bovine aortic endothelial cells (BAEC) to (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl) amino]diazen-1-ium-1,2-diolate (diethylenetriamine NONOate, DETA-NO) (1 mmol/liter) under normoxic or hypoxic conditions (pO2 = 35 mm of Hg) and measured the indices of apoptotic cell death. BAEC treated with DETA-NO under normoxic conditions demonstrated increased levels of histone-associated DNA fragments, which was confirmed by terminal dUTP nick-end labeling assay, and hypoxic conditions augmented this response. To determine whether mitochondrial dysfunction was one mechanism by which NO* initiated apoptosis under hypoxic conditions, we evaluated mitochondrial membrane potential in (Psim). Exposure to DETA-NO resulted in a decrease in Psim and concomitant release of cytochrome c and caspase-9 activation, which were enhanced by hypoxia. By utilizing Rho0 BAEC (Rho0-EC), which lack functional mitochondria, we demonstrated that dissipation of Psim was associated with increased reactive oxygen species generation and peroxynitrite formation. Moreover, in Rho0-EC we identified activation of caspase-8 as part of the mitochondrial-independent pathway of apoptosis. To establish that peroxynitrite mediated mitochondrial damage and apoptosis, we treated BAEC and Rho0-EC with the peroxynitrite scavenger uric acid and found that the indices of apoptosis were decreased significantly. These findings confirm that high flux of NO* under hypoxic conditions promotes cell death via mitochondrial damage and mitochondrial-independent mechanisms by peroxynitrite.     

Disruption of thiol homeostasis and nitrosative stress in the cerebrospinal fluid of patients with active multiple sclerosis: evidence for a protective role of acetylcarnitine

Recent studies suggest that NO and its reactive derivative peroxynitrite are implicated in the pathogenesis of multiple sclerosis (MS). Patients dying with MS demonstrate increased astrocytic inducible nitric oxide synthase activity, as well as increased levels of iNOS mRNA. Peroxynitrite is a strong oxidant capable of damaging target tissues, particularly the brain, which is known to be endowed with poor antioxidant buffering capacity. Inducible nitric oxide synthase is upregulated in the central nervous system (CNS) of animals with experimental allergic encephalomyelitis (EAE) and in patients with MS. We have recently demonstrated in patients with active MS a significant increase of NOS activity associated with increased nitration of proteins in the cerebrospinal fluid (CSF). Acetylcarnitine is proposed as a therapeutic agent for several neurodegenerative disorders. Accordingly, in the present study, MS patients were treated for 6 months with acetylcarnitine and compared with untreated MS subjects or with patients noninflammatory neurological conditions, taken as controls. Western blot analysis showed in MS patients increased nitrosative stress associated with a significant decrease of reduced glutathione (GSH). Increased levels of oxidized glutathione (GSSG) and nitrosothiols were also observed. Interestingly, treatment of MS patients with acetylcarnitine resulted in decreased CSF levels of NO reactive metabolites and protein nitration, as well as increased content of GSH and GSH/GSSG ratio. Our data sustain the hypothesis that nitrosative stress is a major consequence of NO produced in MS-affected CNS and implicate a possible important role for acetylcarnitine in protecting brain against nitrosative stress, which may underlie the pathogenesis of MS.     

Irradiation of the rabbit cornea with UVB rays stimulates the expression of nitric oxide synthases-generated nitric oxide and the formation of cytotoxic nitrogen-related oxidants

Until now, the role of nitric oxide (NO) in cornea irradiated with UVB rays remains unknown. Therefore, we investigated nitric oxide synthase isomers (NOS), enzymes that generate NO, nitrotyrosine (NT), a cytotoxic byproduct of NO, and malondialdehyde (MDA), a byproduct of lipid peroxidation, in rabbit corneas repeatedly irradiated with UVB rays (312 nm, 1x daily for 6 days, the dose per day 1.01 J/cm2) using immunohistochemical methods. The biochemical measurement of nitrite and nitrate has been used for the indirect investigation of NO concentration in the aqueous humor. Results show that in contrast to normal corneas, where of the NOS isomers only endothelial nitric oxide synthase (NOS3) was expressed in a significant amount (in the epithelium and endothelium), in irradiated corneas all NOS isomers (also brain nitric oxide synthase, NOS1, and inducible nitric oxide synthase, NOS2) as well as an indirect measure of ONOO-formation and MDA were gradually expressed, first in the epithelium, the endothelium and the keratocytes beneath the epithelium and finally in the cells of all corneal layers and the inflammatory cells that invaded the corneal stroma. This was accompanied by an elevated concentration of NO in the aqueous humor. In conclusion, repeated irradiation with UVB rays evoked the stimulation of NO production, peroxynitrite formation (demonstrated by NT residues) and lipid peroxidation (evaluated by MDA staining).     

Concentrations of hepatocyte growth factor in cerebrospinal fluid under normal and different pathological conditions

Hepatocyte growth factor (HGF) and its specific receptor, MET, are expressed in the developing and adult mammalian brain. Recent studies have shown a neurotrophic activity of HGF in the nervous system. The present study focused on HGF concentrations in the cerebrospinal fluid (CSF) and serum in normal persons and in different central nervous system (CNS) diseases considering blood-CSF barrier (BCB) function. Concentrations of HGF were analyzed using an enzyme-linked immunosorbent assay (ELISA). HGF was present in normal human CSF (346+/-126 pg/ml) representing approximately half of the HGF serum concentrations. The CSF HGF levels were not significantly changed in chronic CNS disease and in aseptic meningitis (419+/-71 pg/ml), but significantly increased in patients with bacterial meningitis (6101+/- 5200 pg/ml). The HGF levels in CSF were not influenced by increased serum concentrations in patients with normal or mildly affected BCB function. The results show that HGF is present in normal CSF and does not appear to cross the CSF barrier significantly unless it is severely disrupted. So far, strong increases of HGF concentration in CSF are only present in acute bacterial meningitis.