A microdialysis study investigating the mechanisms of hydroxyl radical formation in rat striatum exposed to glutamate.

Considerable evidence has linked hydroxyl radicals (.OH) to excitotoxicity. Glutamate infused through a microdialysis probe into rat striatum induced a massive .OH production, which was completely blocked by PBN and attenuated by dizocilpine, 2-amino-5-phosphonopentanoic acid (AP-5), NG-nitro-L-arginine methyl ester (L-NAME) and mepacrine. Thus, we suggest that the neurotoxic effects of glutamate in vivo may derive from an increased formation of .OH resulting from excessive activation of NMDA receptors and downstream enzymes such as NOS and PLA2.

Glutamate induces hydroxyl radical formation in vivo via activation of nitric oxide synthase in Sprague-Dawley rats.

It was recently reported that neuronal nitric oxide synthase (NOS) generates oxygen-derived free radicals in vitro at low concentrations of L-arginine. Using the microdialysis technique, we monitored both hydroxyl radical (.OH) and nitric oxide (.NO) formation in rat striatum perfused with glutamate (500 mM). .OH and .NO were quantitated in microdialysates by measuring the amounts of the non-enzymatic hydroxylation product of salicylate (2,3-dihydroxybenzoic acid) and the metabolites of .NO (nitrite + nitrate), respectively. .OH levels were dramatically increased during glutamate perfusion, while .NO generation was virtually abolished. .OH production was inhibited by the specific NOS blocker, NG-nitro-L-arginine methyl ester. This effect was not reversed but potentiated by L-arginine. Thus, it is likely that NOS generates oxygen-derived free radicals instead of .NO in brain subjected to highly excitotoxic conditions.

alpha-Phenyl-N-tert-butylnitrone attenuates excitotoxicity in rat striatum by preventing hydroxyl radical accumulation.

Various in vitro experiments have indicated that oxygen-derived free radicals may contribute to excitotoxic neuronal death. In the present study we induced excitotoxicity in rat striatum by perfusing glutamate at a high concentration through a microdialysis probe. We observed an increased formation of hydroxyl radicals (.OH) during the perfusion of the excitotoxin and an extensive striatal lesion 24 h after the insult. The spin trap, alpha-phenyl-N-tert-butylnitrone (PBN), attenuated both hydroxyl radical levels and the volume of the lesion. This result suggests that the neuroprotection may be due to a free radical scavenging mechanism. It also implies that PBN may be used in pathological situations involving excitotoxicity such as stroke, brain trauma, and chronic neurologic diseases.

Calcium-independent NO-synthase activity and nitrites/nitrates production in transient focal cerebral ischaemia in mice.

1. The temporal changes in constitutive NO-synthase (cNOS) and in calcium-independent NO-synthase activities were studied in mice subjected to 2 h of transient focal cerebral ischaemia. The changes in brain nitrites/nitrates (NOx) content were also studied. 2. NOS activities were measured by the conversion of L-[14C]-arginine to L-[14C]-citrulline. Brain NOx contents were investigated by the Griess colourimetric method. 3. cNOS activity in the infarcted cortical area was significantly reduced after 6 h of reperfusion and this activity remained attenuated for up to 10 days after ischaemia. A calcium-independent NOS activity began to increase 48 h after reperfusion, reached a maximum at 7 days and returned to baseline at 10 days. 4. There was a significant increase of brain NOx content beginning after 3 days of reperfusion. This increase was maximal at 7 days and returned to baseline at 10 days. 5. Thus, ischaemia followed by recirculation leads to a rapid, prolonged drop in cNOS activity in the infarcted cortex. There is also a substantial appearance of calcium-independent NOS activity in the later phase of transient ischaemia, leading to an important increase of NOx production.

Role of the L-arginine-nitric oxide pathway in the basal hydroxyl radical production in the striatum of awake rats as measured by brain microdialysis.

In the present study, using the microdialysis technique, we provided evidence of the existence of hydroxyl radicals (.OH) in the striatum of awake rats under physiological conditions. This .OH generation was virtually abolished by the N-methyl-D-aspartate (NMDA) receptor antagonist dizocilpine. On the contrary, it was significantly enhanced by the .NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME). The effect of L-NAME was completely reversed by L-arginine. These results suggest that the basal .OH production is largely the consequence of an NMDA receptor-mediated glutamatergic tone. Moreover, it is likely that endogenous .NO exerts an antioxidant activity in brain by preventing the rise in .OH levels.

Detection of hydroxyl radicals in rat striatum during transient focal cerebral ischemia: possible implication in tissue damage.

As increasing arguments suggest that the reperfusion phase following an ischemic insult may aggravate tissue injury by yielding hydroxyl radicals ('OH), we examined whether these oxyradicals are generated in rat striatum during transient focal cerebral ischemia. .OH were detected in dialysate samples by intrastriatal microdialysis coupled with the technique of salicylate hydroxylation. Ischemia was achieved by tandem occlusion of the left middle cerebral artery and common carotid arteries (45 min) followed by reperfusion. An .OH formation occurred both during ischemia and early reperfusion. Additionally, the volume of the striatal infarct induced by ischemia correlated positively with the amount of .OH produced during ischemia and reperfusion. Taken together, these results provide evidence of the formation of cytotoxic .OH in rat striatum which might participate in the ischemic injury of this structure.

Quantitative measurement of a new synthetic hetrazepine derivative, BN50730, in human plasma and urine by combined liquid chromatography-negative chemical ionization mass spectrometry using a particle beam interface.

A new simple and sensitive assay has been developed for the quantitative measurement of BN50730 at the picomole level in human plasma and urine. The drug and the internal standard (BN50765) were measured by combined liquid chromatography-negative chemical ionization mass spectrometry with methane as the reagent gas. A simple solid-liquid extraction procedure was used to isolate BN50730 from complex biological matrices. Mild operating conditions were required to assay the parent drug with a particle beam interface from Hewlett-Packard. The mass spectrometer was tuned to monitor the intense ion m/z 333, which was generated in the ion source by a dissociative capture process. This assay was performed with 1 ml of plasma or 0.1 ml of urine, and the quantification limit of the method was statistically calculated as 1 ng ml-1. The very low relative standard deviation and mean percentage of error calculated during the different within-day or between-day repeatability assays clearly demonstrate the ruggedness of the technique for the routine determination of BN50730 in the biological fluids. Some preliminary results on the pharmacokinetics of the drug are presented to illustrate the applicability of this new powerful LC-MS method.