G. De Natale

Correlation between 1-methyl-4-phenylpyridinium ion (MPP+) levels, ascorbic acid oxidation and glutathione levels in the striatal synaptosomes of the 1-methyl-4-phenyl-1,2,3-6-tetrahydropyridine (MPTP)-treated rat

In 6-month-old male Wistar rats, levels of dopamine (DA), dihydroxyphenylacetic acid (DOPAC), ascorbic acid (AA), dehydroascorbic acid (DHAA), uric acid, glutathione (GSH) and 1-methyl-4-phenylpyridinium ion (MPP+) were determined by HPLC in the crude striatal synaptosomal fraction after single injections of MPTP 35 mg/kg i.p. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induced a 32.5% death rate within 15 min to 10 h. Groups of surviving rats were sacrificed 1, 3, 8 and 24 h after MPTP. MPTP significantly increased levels of DHAA and uric acid and decreased levels of DOPAC and GSH. Individual synaptosomal levels of MPP+ were correlated inversely with DOPAC (r = -0.601, P < 0.002) and GSH levels (r = -0.496, P < 0.02) and directly with levels of uric acid (r = +0.627, P < 0.001); these latter, in turn, were correlated with DHAA (r = +0.418, P < 0.05) and GSH levels (r = -0.357, P = 0.07). In conclusion, the response of the endogenous antioxidant system (increase in AA oxidation, decrease in GSH levels) correlates well with the MPTP-induced increase in uric acid levels and provides further evidence for a mechanism of MPTP neurotoxicity involving oxidative stress produced by xanthine oxidase.

Dopaminergic system activity and cellular defense mechanisms in the striatum and striatal synaptosomes of the rat subchronically exposed to manganese

In 6-month-old male Wistar rats, levels of dopamine (DA), dihydroxyphenylacetic acid (DOPAC), ascorbic acid (AA), dehydroascorbic acid (DHAA), uric acid and glutathione (GSH) were determined by HPLC in the striatum and striatal synaptosomes after subchronic oral exposure to MnCl2 50–100–150 mg/kg. Mn significantly decreased levels of DA and GSH and increased levels of DHAA and uric acid both in the striatum and synaptosomes. In synaptosomes, individual total Mn doses/rat were directly correlated with individual DOPAC/DA ratio values (r=+0.647), uric acid (r=+0.532) and DHAA levels (r=+0.889) and inversely correlated with DA (r=−0.757) and GSH levels (r=−0.608). In turn, GSH levels were inversely correlated with uric acid (r=−0.451) and DHAA levels (r=−0.460). In conclusion, the response of striatal cellular defense mechanisms (increase in AA oxidation, decrease in GSH levels) correlated well with changes in markers of dopaminergic system activity and increase in uric acid levels. The latter provides evidence of an Mn-induced oxidative stress mediated by xanthine oxidase.

Protective effect of deferoxamine on sodium nitroprusside-induced apoptosis in PC12 cells.

Reportedly, the generation of nitric oxide (NO) may lead to iron mobilization from ferritin disrupting intracellular iron homeostasis and increasing levels of reactive oxygen species. In the present study, we evaluated the role of endogenous iron in NO-induced apoptosis in PC12 cells. Apoptosis was tested by flow cytometry, fluorescence microscopy and terminal deoxynucleotidyl transferase-mediated 2-deoxy-uridine 5-triphosphate nick end labeling (TUNEL) technique. Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. When incubated with 0.5-0.75 mM sodium nitroprusside (SNP, a chemical NO donor), PC12 cells were shown to undergo apoptosis. In addition, SNP induced a time-dependent decrease in cell viability. Since deferoxamine (0.05-0.1 mM), a powerful iron chelator, inhibited both SNP-induced apoptosis and the decrease in cell viability, we suggest that these NO effects may be dependent upon iron mobilization within the cell.

Effects of ageing on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) neurotoxic effects on striatum and brainstem in the rat

In 3- and 18-month-old male Wistar rats, levels of dopamine (DA), dihydroxyphenylacetic acid (DOPAC), ascorbic acid (AA), dehydroascorbic acid (DHAA), noradrenaline (NA), uric acid, glutathione (GSH) and 1-methyl-4-phenylpyridinium ion (MPP+) were determined by HPLC in the striatum and/or in the brainstem 24 h after single injections of MPTP (12-35 mg/kg i.p.). Aged rats had lower baseline levels of AA and GSH, compared to young rats. In aged rats, MPTP 35 mg/kg induced a 70% death rate and a decrease in striatal DOPAC/DA ratio which was significantly correlated to MPP+ concentrations (r = -0.840, P < 0.005); in addition, MPTP did not increase AA oxidation. In the brainstem, the MPTP-induced decrease in NA levels and increase in uric acid levels were significantly correlated to the MPP+ concentrations (r = -0.709, P < 0.05, and r = +0.888, P < 0.001, respectively). In conclusion, evidence is given of a mechanism of toxicity of MPTP involving oxidative stress produced by xanthine oxidase; in addition, in aged rats the neuronal antioxidant system (levels of AA and GSH) is considerably lower than in young rats and may play an enabling role in the MPTP age-related neurotoxic effects on striatum and brainstem.

The effects of cortical ablation on d-amphetamine-induced changes in striatal dopamine turnover and ascorbic acid catabolism in the rat

Dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), ascorbic acid (AA) and dehydroascorbic acid (DHAA) levels were determined by HPLC in the striatal synaptosomal fraction and in the whole striatum of rats, whose fronto-parietal cortex had been bilaterally ablated, after a single injection of d-amphetamine (2.0 mg/kg i.p.). d-Amphetamine significantly increased the DHAA/AA ratio in unoperated and sham-operated rats, but failed to increase it in ablated rats, as compared to pertinent saline-treated groups. In the synaptosomal fraction, d-amphetamine significantly decreased the DHAA/AA ratio in unoperated, sham-operated and ablated rats. d-Amphetamine significantly decreased the DOPAC/DA ratio in the whole striatum and significantly increased it in the striatal synaptosomal fraction in all experimental groups. Cortical ablation greatly increased d-amphetamine-induced motor hyperactivity. We conclude that the d-amphetamine-induced increase in AA striatal oxidation requires integrity of the cortico-striatal glutamatergic pathways. Further, AA oxidation occurs in the extracellular space. The cortico-striatal glutamatergic pathways exert an inhibitory modulation on d-amphetamine behavioral effects.

Investigations into the relationship between the dopaminergic system and ascorbic acid in rat striatum.

Levels of dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), ascorbic acid (AA) and dehydroascorbic acid (DHAA) were determined by HPLC in the striatum of male Wistar rats after single or repeated injections of apomorphine (1 mg/kg/day s.c.) and/or haloperidol (1 mg/kg/day i.p.), and 24 h after the last drug administration. Apomorphine significantly reduced the DOPAC/DA ratio and increased the DHAA/AA ratio; these ratio changes were significantly correlated (r = -0.9969, P less than 0.0005). Haloperidol greatly increased the DOPAC/DA ratio; the DHAA/AA ratio was also slightly increased, but there was no significant correlation. When apomorphine was associated with haloperidol, the resulting DOPAC/DA ratio was significantly lower than after haloperidol alone; the DHAA/AA ratio was also significantly reduced in contrast to the effect of apomorphine alone. It is concluded that a non-selective DA receptor activation mediates, in a correlated way, both the inhibition of DA turnover and the increase of AA oxidation in the rat striatum.

Monoaminergic systems activity and cellular defense mechanisms in the brainstem of young and aged rats subchronically exposed to manganese

In 3- and 20-month-old male Wistar rats, levels of noradrenaline (NA), dopamine (DA), 5-hydroxytryptamine (5-HT), 5-hydroxyindoleacetic acid (5-HIAA), ascorbic acid (AA), dehydroascorbic acid (DHAA), uric acid and glutathione (GSH) were determined by HPLC in the brainstem after subchronic oral exposure to MnCl2 200 mg/kg (3-month-old) and 30-100-200 mg/kg (20-month-old). In aged rats, manganese (Mn) significantly decreased levels of NA, DA and GSH and increased 5-HIAA/5-HT ratio values and DHAA and uric acid levels. All these parameters were scarcely affected in young rats. In aged rats, individual total Mn doses/rat were inversely correlated with individual DA levels (r = -0.405) and GSH levels (r = -0.450). In conclusion, Mn induces changes in markers of monoaminergic systems activity in the brainstem of aged rats considerably greater than in young rats. The increase in AA oxidation and decrease in GSH levels are consistent with a Mn-induced increase in formation of reactive oxygen species. The increase in uric acid levels provides evidence that one of these species might arise from the activity of xanthine-oxidase on uric acid precursors.

Central nervous system

Cerebrospinal fl uid (CSF) evaluation is a mainstay in the diagnosis of central nervous system (CNS) disease because it is relatively simple to collect and has the potential to provide valuable information. Lesions of the CNS do not consistently cause CSF abnormalities related to the location and extent of the lesion. Although CSF evaluation infrequently provides a defi nitive diagnosis, it may be of benefi t in documenting normal or abnormal features and, in combination with other tests, determining a diagnosis or differential diagnoses (Bohn et al., 2006; Bush et al., 2002; Chrisman, 1992; Cook and DeNicola, 1988; Fenner, 1995; Rand, 1995). CSF collection is recommended as a part of virtually any diagnostic investigation of CNS disease of unknown cause when contraindications to its collection are not present. The submission of a properly collected specimen is necessary to obtain reliable and accurate information. Proper interpretation of the sample requires knowledge of the clinical presentation, collection site, and specimenhandling considerations. The presence of artifacts or contaminants may interfere with an appropriate interpretation unless the conditions surrounding the collection are known. Experience in interpretation of cytologic specimens from the species of interest and knowledge of the limitations of cytology or types of pathologic processes likely to be refl ected in CSF are also important and can only be gained by diligent study of the literature and specimens over time. Cerebrospinal fl uid is formed primarily by the ultrafi ltration and secretion through the choroid plexuses of the lateral, third, and fourth ventricles. Other sites that secrete CSF include the ependymal linings of the ventricles and blood vessels of the subarachnoid spaces and pia mater. Fluid then escapes from the fourth ventricle into the subarachnoid spaces and central canal of the spinal cord. It is then absorbed predominantly from the subarachnoid spaces via veins in the arachnoid and subarachnoid villi that project into subdural venous sinuses. Collection of Cerebrospinal Fluid