Carla Torri

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.

Trafficking of adenosine A2A and dopamine D2 receptors

An interaction between adenosine A2A and dopamine D2 receptors has been demonstrated previously. It is generally found that agonist treatment internalizes receptors, including A2A and D2, whereas less is known of the long-term effects involved in the agonist-mediated trafficking of A2A and D2 receptors. Furthermore, the possible influence of the antagonists on receptor trafficking is still undefined. The present studies focus on the long-term effects of A2A and D2 agonist and D2 antagonist treatments on both A2A and D2 receptor trafficking studied at three different time intervals—3, 15, and 24 h. In addition, with the fluorescence resonance energy transfer technique, formation of heteromeric A2A and D2 receptor complexes was shown in the cotransfected CHO cell line. Confocal microscopy analysis showed that a 3-h treatment with the D2 agonist induced coaggregation of A2A/D2 receptors. These A2A/D2 receptor coaggregates internalized after 15 h with a recruitment of the receptors back to the cell membrane after 24 h. In contrast to the effects of the agonist treatment, a 3-h treatment with the D2-like antagonist raclopride increased both A2A and D2 immunoreactivity, indicating that the D2 antagonist stabilizes the D2 receptor and thereby reduces the internalization of both of the A2A and D2 receptors. Taken together, an activation of either A2A and D2 receptor or blockade of D2 receptors will cause long-lasting changes in A2A and D2 receptor trafficking.

Diet-induced changes in hypothalamic pro-opio-melanocortin mRNA in the rat hypothalamus

Hypothalamic mRNA and peptide levels of pro-opio-melanocortin (POMC) and other neuropeptides were studied in rats that either develop obesity (diet-induced obese, DIO), when fed a palatable and hypercaloric diet (cafeteria diet, caf) or do not develop obesity (diet resistant, DR), when fed the same diet. cafDIO rats showed a significant increase in POMC, but not in melanin concentrating hormone, mRNA levels as determined by semiquantitative in situ hybridization. cafDR and cafDIO rats showed no change in POMC-derived peptide levels, whereas neuropeptide Y immunoreactivity was significantly increased in cafDR rats. POMC mRNA levels were also studied in high-fat diet-fed rats but no significant change was observed. Altered hypothalamic transmission by POMC-derived peptides may contribute to the susceptibility of cafDIO rats to the weight promoting action of caf diet.

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.

Brain Oxidative Damage Following Acute Immobilization and Mild Emotional Stress

We studied the role of free radicals on brain oxidative damage in rats after acute immobilization stress (restraint) and mild emotional stress (handling). To investigate brain oxidative damage, CuZn and Mn dependent superoxide dismutase (CuZn SOD, Mn SOD) activities, lipid peroxidation (TBARs), Na+K+ATPase activity, protein carbonyl (PrC), and reduced and oxidized glutathione (GSH, GSSG) levels were measured in the cerebral cortex (CTX), hippocampus (HIP), and striatum (ST) of the animals after the two different stress stimuli. Because stress produces abnormalities in the hypothalamic-pituitary-adrenal axis, the intensity of the two stress conditions were measured by plasmatic corticosteroid (COR) levels: particularly, COR levels doubled in handled rats and increased 15-fold in restrained animals. The SOD activities increased in CTX and decreased in HIP of the handled rats, while in ST a significant decrease in handled animals but an increase in restrained animals occurred. TBARs, GSH, and GSSG levels remained unchanged, while an index of glutathione redox decreased significantly in ST of handled animals and in CTX of restrained ones. Na+K+ATPase activity increased significantly in the HIP and ST of both groups of stressed rats. The stress induced a remarkable increase in PrC levels in all studied cerebral areas. These findings provide evidence to support the idea that stress produces oxidants but that the oxidative damage in stress differs in cerebral areas and could contribute to the degenerative mechanism of aging.

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.

Protective effects of delapril combined with indapamide or hydrochlorothiazide in spontaneously hypertensive stroke-prone rats: a comparative dose-response analysis.

In previous articles, we have shown that the combination of the angiotensin-converting enzyme (ACE) inhibitor delapril (12 mg/kg/day) and the diuretic indapamide (1 mg/kg/ day) was able to prolong the life span significantly in salt-loaded stroke-prone spontaneously hypertensive rats (SHRsp). Because this finding was partly dependent on the antagonism of salt-loading effects by pharmacologic induction of diuresis, which prevented any increase in blood pressure values, we decided to evaluate whether lower doses of the combination could be equally protective without changing the progression of hypertension. Thus, we studied several treatments with progressively lower doses of delapril (6, 3, or 1.5 mg/kg/day) combined with indapamide (0.5, 0.25, or 0.125 mg/kg/day) or hydrochlorothiazide (2.5, 1.25, or 0.625 mg/kg/day) in salt-loaded SHRsp. Salt-loaded untreated animals were considered to be the control group. In agreement with previous experiments, control rats reached 50% mortality approximately 7 weeks after the beginning of salt loading. The combination of delapril and hydrochlorothiazide at the two lowest doses was not able to delay animal death significantly, whereas treatment with delapril and indapamide at the lowest dose was effective (50% survival rate, 15 weeks). The groups treated with the highest dose of delapril and hydrochlorothiazide or with the intermediate or highest dose of delapril and indapamide did not reach 50% mortality by the end of the experiment, at 44 weeks of treatment (i.e., when animals reached age 1 year). Only the highest delapril and indapamide doses were able to increase diuresis, but for a relatively short period. None of the treatments was able to lower or control blood pressure levels adequately. Therefore, blood pressure levels by themselves were not predictive of rat mortality. In contrast, the maximal value of proteinuria in the weeks preceding death was inversely correlated with the survival time. In conclusion, this study shows that low doses of an ACE inhibitor in combination with a diuretic can be effectively protective in a model of severe hypertension, independent of any change in blood pressure levels.

Oxidative Stress in the Early Phase of Traumatic Central Nervous System Injury: Clinical Data

Cerebrovascular and metabolic changes associated with traumatic injury to the CNS may be related, in part, with pathological alterations in the intracellular neurochemical system. These events may include alterations in neurotransmitter synthesis and release or changes in pre/or postsynaptic receptor activity. Other changes may include alterations in the synthesis and activity of endogenous neuroprotective compounds (e. g. enzymatic and non-enzymatic antioxidant) or pathological expression and release of endogenous “autodestructive” compounds associated with inflammation and/or with alterated oxidative metabohsm (e. g. free radicals). Although the timing of the precise cascade of neurochemical events following traumatic brain injury is poorly understood, there is now extensive experimental support for early occurrence and pathological importance of oxygen radical species (ROS) formation and cell membrane lipid peroxidation (LP) (1-4). The potential sources of oxygen radicals within the brain after trauma insult include the arachidonic acid cascade, catecholamine oxidation, electron “leakage” from the mitochondrial electron transfer chain, oxidation of extravasated hemoglobin and later infiltrating neutrophils (Figs. 1, 2). The radical dependent lipid peroxidation of nervous and vascular cell membranes and myelin is catalyzed by free iron released from hemoglobin, transferrin and ferritin by either lowered tissue pH or oxygen radicals. The LP is a geometrically progressing process that spreads over the surface of the cell membrane causing impairment to phospholipid-dependent enzymes, disruption of ionic gradients, membrane lysis and cell death. Criteria for the establishment of the pathophysiological significance of oxygen radical reactions include: a) the demonstration of increased post-traumatic levels of oxygen radicals and lipid peroxides after CNS trauma; b) the spatial and temporal correlation between oxygen radical formation and physiological and pathological alterations (e. g. decrease of oxygen consumption, energy failure, loss of microvasculature regulation, edema and progressive post-traumatic ischemia development); and c) a striking similarity between post-traumatic CNS pathology and experimental chemical peroxidative insult (e. g. ironmicroinjection).