Carla Café

Oxidative stress after acute and chronic application of β-amyloid fragment 25-35 in cortical cultures

The aim of this work was to investigate whether free radical reactions play a role in beta-amyloid neurotoxicity. Rat cortical neurons were exposed acutely (24 h) or chronically (3, 7 days) to beta-amyloid biologically active fragment beta 25-35 (50 microM). In these conditions, where only the longest exposure induced neuronal death, superoxide dismutase activity was increased after acute exposure but no change was detected after chronic treatments, whereas a different pattern was observed for glutathione peroxidase. In the basal condition, there was an eight-fold increase in dichlorofluoroscein, used as peroxide production marker, in neuronal cells after 7 days treatment with beta 25-35. Moreover, the intracellular peroxide production induced by Fe2+/ascorbate stimulation was amplified by beta 25-35, increasingly up to 7 days of exposure, by which time the dichlorofluoroscein-stimulated levels were 33 times higher than in controls. In conclusion, our results show that oxidative stress and free radical production are linked to beta 25-35 exposure and may contribute to neurodegenerative events associated with beta-amyloid deposits in Alzheimers disease.

Antioxidant enzymatic activities after experimental subarachnoid hemorrhage in rats.

Lipid peroxidation has been hypotesized as one of possible factors involved in the pathogenesis of neuronal damage and delayed vasospasm after subarachnoid hemorrhage. In the brain there are anti‐oxidant enzymatic systems which act as scavengers of superoxides and free radicals. In the present study the pattern of enzymatic anti‐oxidant activities (Cu‐Zn and Mn superoxide dismutase, and glutathione peroxidase) was investigated in an experimental model of subarachnoid hemorrhage in the rat in order to verify whether the hemorrhagic insult may be responsible for an impairment of such anti‐oxidant systems. Enzymatic activities were assayed in three different rat brain areas (cerebral cortex, hippocampus and brain stem) of sham‐operated and at 30 min, 1, 6 and 48 h after subarachnoid hemorrhage induction. After the hemorrhage induction the Cu‐Zn superoxide dismutase activity in cerebral cortex was significantly reduced at all the set times (p <.05), while Mn‐superoxide dismutase activity was significantly decreased since 1 h (p <.05) until 48 h (p <.05). Glutathione peroxidase activity was significantly reduced only in the late phase (48 h) of subarachnoid hemorrhage (p <.01). In the hippocampus, all enzymatic activities were significantly reduced in the late phase. In the brain stem Cu‐Zn superoxide dismutase was significantly impaired at 1 and 6 h (p <.05) after subarachnoid hemorrhage induction, while in the late phase (48 h) reached the control value. The mitochondrial Mn‐superoxide dismutase was significantly reduced since 1 h (p <.05) until 48 h (p <.02) after subarachnoid hemorrhage. Glutathione peroxidase activity in this area was impaired at 1, 6 (p <.01) and 48 h (p <.02). These results suggest that subarachnoid hemorrhage causes a significant reduction of anti‐oxidant enzymatic activities in brain compartment: Cu‐Zn and Mn superoxide dismutase, which are specific scavengers of superoxide radicals, show an early decrease, while glutathione peroxidase activity is significantly reduced in a delayed phase.

Experimental Parkinson's disease in monkeys. Effect of ergot alkaloid derivative on lipid peroxidation in different brain areas

The effects of the Parkinsonism induced by the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) were evaluated in four different monkey brain areas (frontal and occipital cortex, caudate putamen, substantia nigra). The basal and stimulated lipid peroxidation and the reduced glutathione (GSH) concentration were evaluated in three groups of maleMacaca fascicularis monkeys (6 animals/group): (a) controls; (b) MPTP-treated animals; (c) animals treated with MPTP and α-dihydroergocryptine (DEK; ergot alkaloid characterized by a dopaminergic agonist action). In MPTP-treated animals the GSH concentration was unchanged or decreased in a non-significant way in the frontal and occipital cortex, and in substantia nigra. The basal thiobabituric acid reactive substance (TBARS) concentrations were significantly higher in the caudate putamen and substantia nigra of MPTP-treated animals. In the MPTP-treated monkeys the DEK administration induced a restoration of basal TBARS values to nearly normal ones. By incubating tissue from different brain areas with FeSO4 plus ascorbic acid, the stimulation of lipid peroxidation decreased the TBARS production in the substantia nigra of the MPTP-treated animals. These results, taken together, may indicate that an increased lipid peroxidation could possibly play a role in producing the Parkinson-line syndrome by MPTP and that a free radical excess could be responsible for the degeneration of the substantia nigra. The treatment with an ergot alkaloid (i.e., α-dihydroergocryptine) partially antagonizes the MPTP-induced increase in basal TBARS concentration in caudate putamen.

Experimental subarachnoid hemorrhage : events related to anti-oxidant enzymatic systems and eicosanoid peroxide enhancement

Experimental and clinical studies have emphasized the role of free radicals in the pathogenesis of vasospasm and neurological dysfunction after subarachnoid hemorrhage (SAH). Increases in both enzymatic (arachidonic acid cascade and eicosanoid peroxide production) and non-enzymatic (tiobarbituric acid reactive substances production) lipid peroxidation were found, pointing out the key role of arachidonic acid cascade in impairing membrane functionality in the post-hemorrhage brain. The aim of this work is to investigate whether a correlation exists between time-dependent modifications of eicosanoid peroxide production (“ex vivo” release of leukotriene C4=LTC4) and antioxidant enzymatic systems in the brain after experimental subarachnoid hemorrhage in the rat. The release of the LTC4 is significantly enhanced at 1, 6 and 48 hours after SAH induction. Cu−Zn superoxide dismutase (SOD) activity is significantly reduced at 6 and 48 hours after SAH induction; Mn-SOD activity is significantly affected at 1, 6 and 48 hours after the hemorrhage. GSH-Px activity is significantly reduced only in the late phase (48 hours) after SAH. The linear regression of statistical analysis, performed to investigate any possible relationship among time-dependent modifications shows that the “ex vivo” release of LTC4 is significantly related to the decreasing trend of MnSOD activity (p<0.001). The present results suggest that after SAH, a deficit in endogenous anti-oxidant defenses may play a role in making the brain more susceptible to lipid peroxidative events.

Changes in non-synaptosomal and synaptosomal mitochondrial membrane-linked enzymatic activities after transient cerebral ischemia

Non-synaptosomal and synaptosomal mitochondrial membrane-linked enzymatic activities, NADH-cytochrome c reductase rotenone insensitive (marker of the outer membrane) and cytochrome oxidase (marker of the inner membrane), were measured in rat brain hippocampus and striatum immediately after and 1, 4, and 7 days following the induction of complete transient ischemia (15 min) by the four vessel occlusion method. Furthermore citrate synthetase activity was measured with and without Triton X-100 in order to qualitatively evaluate the membrane permeability. Nonsynaptosomal mitochondrial membranes showed reduction of both activities only in the late reperfusion phase: NADH-CCRRi decreased in striatal mitochondria after 4–7 days and only after 7 days in the hippocampus. COX activity decreased only in striatal mitochondria 7 days after ischemia. Non-synaptosomal mitochondrial membrane permeability did not show changes. Synaptosomal mitochondria showed a decrease of NADH-CCRRi only at 7 days of reperfusion both in hippocampus and striatum, while COX activity decreased only during ischemia and returned to normal levels in the following days in the two areas considered. In summary, free mitochondria showed insensitiveness to ischemia but they risulted damaged in the late reperfusion phase, while mitochondria from the synaptic terminal showed ischemic damage, partially restored during reperfusion. The striatal mitochondria showed a major susceptibility to ischemia/repefusion damage, showing changes earlier than the hippocampal ones.

Effect of high-dose methylprednisolone on anti-oxidant enzymes after experimental SAH

Lipid peroxidation has been considered one of the most important factors involved in the pathogenesis of neuronal damage following subarachnoid hemorrhage. In the brain, the protective systems most involved against peroxidative and free radicals generated reactions are superoxide-dismutase (SOD) and glutathione-peroxidase (GSH-Px). Since these activities are subjected to a significant reduction following experimental SAH induction in rats, we investigated in the present study if the beneficial effect of high-dose methylprednisolone (MP) in inhibiting lipid peroxidative processes in SAH is possibly linked to an influence on anti-oxidant enzymatic activities. In brain cortex, after MP treatment, Cu-Zn SOD activity in the early phase and more dramatically in the late phase after SAH was restored (4.06 +/- 0.06 and 4.07 +/- 0.14 enzymatic units/mg of protein, respectively) if compared to hemorrhagic non-treated controls (3.69 +/- 0.16 and 2.96 +/- 0.06 enzymatic U/mg of protein) while Mn-SOD and GSH-Px activities were improved in treated animals only in the early and late phases after SAH, respectively. In the hippocampus, in treated rats Cu-Zn activity was partially restored only at 6 h, while Mn-SOD activity recovered at 48 h after SAH; no significant changes in GSH-Px activity were found in treated animals at any time. In the brain stem, in treated animals, Cu-Zn SOD activity was restored in the early phase (3.86 +/- 0.12 enzymatic U/mg of protein) up to control values of non-hemorrhagic rats (3.44 +/- 0.30 enzymatic U/mg of protein), while GSH-Px activity recovered in the late phase.(ABSTRACT TRUNCATED AT 250 WORDS)

Superoxide dismutase activity in cisternal cerebrospinal fluid after aneurysmal subarachnoid haemorrhage.

SummaryIt has been recognised that the level of superoxide dismutase (SOD) significantly increases in CSF as the result of cerebral ischaemic damage. The aim of this study was to correlate the CSF levels of SOD enzymatic activity to the patterns of subarachnoid haemorrhage with regards to ischaemic complications due to vasospasm.A series of 78 patients operated on for intracranial aneurysms was studied; all patients were monitored with serial TCD measurements every second day after SAH. CSF samples were obtained at surgery by cisternal puncture of the subarachnoid cistern nearest to the aneurysm. SOD activity was assayed spectrophotometrically.Mean cisternal CSF level of SOD in 12 control cases (12.99±2.33 U/ml) is significantly higher (p < 0.01) than in 26 patients operated on between day 1 and 3 from last SAH episode (4.44±0.7 U/ml) and in 40 patients treated by delayed surgery (7.64±0.92 U/ml). In 13 patients presenting neurological deterioration related to arterial vasospasm mean cisternal SOD level was 12.23±1.86 U/ml; in 27 cases without vasospasm mean level was 5.43±0.7 U/ml (p < 001).The present results suggest that (a) cisternal CSF levels of SOD significantly decreases after SAH, probably in relation to an impaired synthesis in the brain compartment and that (b) a substantial elevation of SOD levels is evident in patients suffering ischaemic complications vasospasm-related. Biochemical events in the brain compartment could influence the expression and release of anti-oxidant enzymes in CSF after SAH.

SYNAPTOSOMAL IRON-DEPENDENT LIPID PEROXIDATION INHIBITION AFTER SUBARACHNOID HEMORRHAGE BY LAZAROID IN VIVO TREATMENT

The production of oxygen-free radicals and their subsequent peroxidative action on membrane unsaturated fatty acids could be enhanced after subarachnoid hemorrhage (SAH). We have studied the effects of the in vivo pharmacological treatment with a lazaroid (U78517F) after experimental SAH, on lipid peroxidative patterns in cortical synaptosomal preparations. U78517F is a lipid-soluble antioxidant with a potent action to inhibit iron-dependent lipid peroxidation. Experimental SAH was induced in anesthetized rats by slow injection of 0.3 mL of autologous arterial blood into cisterna magna. The hemorrhagic animals were treated with 5 mg/kg iv of U78517F immediately after surgical operation. The animals were sacrificed 1 d after the hemorrhage and the thiobarbituric acid reactive material (TBAR) was assayed in basal conditions and after 1, 3, 5, 10, and 20 min of incubation at 37 degrees C with a pro-oxidant mixture on three different rat groups: sham-operated (0.3 mL of mock cerebrospinal fluid (CSF) into cisterna magna), hemorrhagic (0.3 mL of autologous arterial blood into cisterna magna), and hemorrhagic-treated. The hemorrhagic event did not influence the membrane lipoperoxidation levels in basal conditions, whereas peroxidative stimulation in vitro caused significant increases in hemorrhagic animals compared to the sham-operated, and in hemorrhagic-treated animals, the synaptosomal TBARs were similar to controls. The pharmacological treatment showed its effectiveness only following incubations with pro-oxidants; therefore, U78517F seems to be protective for membranes in case of severe lipid peroxidative stress.