Fernanda Urruth Fontella

Repeated Restraint Stress Induces Oxidative Damage in Rat Hippocampus

It has been shown that emotional stress may induce oxidative damage, and considerably change the balance between pro-oxidant and antioxidant factors in the brain. The aim of this study was to verify the effect of repeated restraint stress (RRS; 1 h/day during 40 days) on several parameters of oxidative stress in the hippocampus of adult Wistar rats. We evaluated the lipid peroxide levels (assessed by TBARS levels), the production of free radicals (evaluated by the DCF test), the total radical-trapping potential (TRAP) and the total antioxidant reactivity (TAR) levels, and antioxidant enzyme activities (SOD, GPx and CAT) in hippocampus of rats. The results showed that RRS induced an increase in TBARS levels and in GPx activity, while TAR was reduced. We concluded that RRS induces oxidative stress in the rat hippocampus, and that these alterations may contribute to the deleterious effects observed after prolonged stress.

Long-lasting delayed hyperalgesia after chronic restraint stress in rats—effect of morphine administration

Different effects upon the nociceptive response have been observed with exposure to acute and chronic stress in rats. In the present study we repeatedly submitted rats to restraint for 40 days, inducing hyperalgesia using the tail-flick test. A new session of acute stress was applied at the end of 40 days period, and the chronically-stressed animals demonstrated analgesia after forced swimming, but not after restraint. The effect of stress interruption for 14 or 28 days on the nociceptive threshold was then investigated. The basal tail-flick latency remained decreased for at least 28 days (hyperalgesic effect). Following the periods of suspension, the animals were submitted to new session of acute restraint, and stress-induced analgesia was observed only after 28 days of stress interruption. Thus, the mechanisms involved in the long-lasting hyperalgesia presented in this study are not exactly the same as those responsible for the analgesia induced by acute stressors. After 40 days of chronic stress treatment, morphine was injected i.p. (1.0, 5.0 mg/kg or saline). The repeatedly stressed rats displayed decreased morphine effects on nociception compared to unstressed controls. The tolerance of the response to morphine agrees with previous studies suggesting that chronic restraint stress could modify the activity of opioid systems.

Stimulation of lipid peroxidation in vitro in rat brain by the metabolites accumulating in maple syrup urine disease.

In this study we investigated the in vitro effects of the metabolites accumulating in maple syrup urine disease on lipid peroxidation in brain of young rats. Chemiluminescence and thiobarbituric acid-reactive substances were measured in brain homogenates from 7- and 30-day-old rats in the presence of 10 mM of the branched-chain amino acids L-leucine, L-isoleucine, or L-valine; their keto acids L-2-ketoisocaproic acid, L-2-keto-3-methylvaleric acid, or L-2-ketoisovaleric acid; or the hydroxy derivatives L-2-hydroxyisocaproic acid, L-2-hydroxy-3-methylvaleric acid, or L-2-hydroxyisovaleric acid separately added to the incubation medium. We observed that all amino acids, keto acids, and hydroxy acids accumulating in this disease stimulate to a variable degree the in vitro parameters of lipid peroxidation tested in homogenates of rat brain. The results indicate a possible participation of oxidative stress in the neuropathology of maple syrup urine disease patients, especially during a crisis, when the metabolites are highly increased, and point to the use of antioxidant drugs as a possible adjuvant therapy in such situations to improve the neurological status of the patients and to prevent sequelae.

Repeated restraint stress alters hippocampal glutamate uptake and release in the rat.

Glutamatergic mechanisms are thought to be involved in stress-induced changes of brain function, especially in the hippocampus. We hypothesized that alterations caused by the hormonal changes associated with chronic and acute stress may affect glutamate uptake and release from hippocampal synaptosomes in Wistar rats. It was found that [3H]glutamate uptake and release by hippocampal nerve endings, when measured 24 h after 1 h of acute restraint, presented no significant difference. The exposure to repeated restraint stress for 40 days increased neuronal presynaptic [3H]glutamate uptake as well as basal and K+-stimulated glutamate release when measured 24 h after the last stress session. Chronic treatment also caused a significant decrease in [3H]glutamate binding to hippocampal membranes. We suggest that changes in the glutamatergic system are likely to take part in the mechanisms involved in nervous system plasticity following repeated stress exposure.

Ascorbic acid prevents cognitive deficits caused by chronic administration of propionic acid to rats in the water maze

Propionic acidemia is an inherited neurometabolic disorder characterized by progressive neurological deterioration with psychomotor delay/mental retardation, convulsions and coma, and whose pathophysiology is poorly unknown. In the present study, we investigated the effect of chronic administration (from the 5th to the 28th days of life) of propionic acid (PA), the major metabolite accumulating in tissues of patients affected by propionic acidemia, on the cognitive performance of adult rats in the Morris water maze task. PA doses ranged from 1.44 to 1.92 micromol/g body weight as a function of animal age. Control rats were treated with saline in the same volumes. Chronic postnatal days (5-28) PA treatment had no effect on body weight. However, it impaired spatial performance in the water maze. We also determined the effect of ascorbic acid (AA) administered, alone or combined with PA, on the same behavioral parameters in order to test whether free radicals could be responsible for the behavioral alterations observed in PA-treated animals. AA was able to prevent the behavioral alterations provoked by PA, implying that oxidative stress may be involved in these effects. Furthermore, we also investigated the total radical-trapping antioxidant potential (TRAP) in the hippocampus of the animals. We observed that TRAP was significantly reduced in the brain of propionic acidemic rats and that co-administration of AA prevented this effect. The results provide evidence that early PA treatment induces long-lasting behavioral deficits, which are possibly caused by oxygen reactive species generation, and suggest that oxidative stress may be involved in the neuropathology of propionic acidemia.

Acute and repeated restraint stress influences cellular damage in rat hippocampal slices exposed to oxygen and glucose deprivation.

Several studies have shown that high corticosteroid hormone levels increase neuronal vulnerability. Here we evaluate the consequences of in vivo acute or repeated restraint stress on cellular viability in rat hippocampal slices suffering an in vitro model of ischemia. Cellular injury was quantified by measuring lactate dehydrogenase (LDH) and neuron-specific enolase released into the medium. Acute stress did not affect cellular death when oxygen and glucose deprivation (OGD) was applied both immediately or 24h after restraint. The exposure to OGD, followed by reoxygenation, resulted in increased LDH in the medium. Repeated stress potentiated the effect of OGD both, on LDH and neuron-specific enolase released to the medium. There was no effect of repeated stress on the release of S100B, an astrocytic protein. Additionally, no effect of repeated stress was observed on glutamate uptake by the tissue. These results suggest that repeated stress increases the vulnerability of hippocampal cells to an in vitro model of ischemia, potentiating cellular damage, and that the cells damaged by the exposure to repeated stress+OGD are mostly neurons. The uptake of glutamate was not observed to participate in the mechanisms responsible for rendering the neurons more susceptible to ischemic damage after repeated stress.

Estradiol protects against oxidative stress induced by chronic variate stress.

Neurochemical gender-specific effects have been observed following chronic stress. The aim of this study was to verify the effects of chronic variable stress on free radical production (evaluated by DCF test), lipoperoxidation (evaluated by TBARS levels), and total antioxidant reactivity (TAR) in three distinct structures of brain: hippocampus, cerebral cortex and hypothalamus of female rats, and to evaluate whether the replacement with estradiol in female rats exerts neuroprotection against oxidative stress. Results demonstrate that chronic stress had a structure-specific effect upon lipid peroxidation, since TBARS increased in hypothalamus homogenates of stressed animals, without alterations in the other structures analyzed. Estradiol replacement was able to counteract this effect. In hippocampus, estradiol induced a significant increase in TAR. No differences in DCF levels were observed. In conclusion, the hypothalamus is more susceptible to oxidative stress in female rats submitted to chronic variable stress, and this effect is prevented by estradiol treatment.

A chemically-induced acute model of maple syrup urine disease in rats for neurochemical studies.

We report a chemically-induced model of maple syrup urine disease (MSUD) in 10- and 30-day-old rats produced by subcutaneous administration of a branched-chain amino acids (BCAA) pool along with the analyses of plasma and brain amino acid levels by HPLC at 0-120 min after administration. We observed an increase of plasma leucine (Leu), isoleucine (Ile) and valine (Val) concentrations in both 10- and 30-day-old rats. These increases were accompanied by a concomitant reduction of plasma concentrations of methionine (Met), phenylalanine (Phe), tyrosine (Tyr), histidine (His), alanine (Ala), lysine (Lys), and ornithine (Orn) in 10-day-old rats and of Met, Phe, Tyr, tryptophan (Trp), and Orn in 30-day-old rats. These results are similar to those observed in MSUD patients during crises, when plasma levels of large neutral amino acids (LNAA) are also reduced when BCAA concentrations are increased. In the brain, increased concentrations of Leu, Ile and Val were achieved in 10-day-old rats at all times after injection. In contrast, no differences in cerebral concentrations of BCAA were observed in 30-day-old rats. In conclusion, the present MSUD model, using 10- rather than 30-day-old rats, has a similar amino acid profile to that of MSUD untreated patients and is suitable to investigate the mechanisms of brain damage characteristic of this disorder.

Nociceptive Response and Adenine Nucleotide Hydrolysis in Synaptosomes Isolated from Spinal Cord of Hypothyroid Rats

Purinergic system exerts a significant influence on the modulation of pain pathways at the spinal site. Adenosine has antinociceptive properties in experimental and clinical situations, while ATP exerts pronociceptive actions in different pain models. In this study we investigated the hydrolysis of ATP to adenosine in synaptosomes from spinal cord in parallel with the nociceptive response of rats at different ages after hypothyroidism induction. Hypothyroidism elicited a significant increase in AMP hydrolysis to adenosine in synaptosomes from spinal cord of rats subjected to neonatal hypothyroidism and in 420-day-old rats submitted to thyroidectomy. Accordingly, these rats presented an analgesic response as a consequence of hypothyroidism. In contrast, the ATP hydrolysis was decreased in the spinal cord of 60-day-old hypothyroid rats in parallel with a significant increase in nociceptive response. These results indicate the involvement of adenine nucleotides in the control of the hypothyroidism-induced nociceptive response during development.

Chronic administration of propionic acid reduces ganglioside N-acetylneuraminic acid concentration in cerebellum of young rats

Elevated levels of propionate comparable to those of human propionic acidaemia were achieved in the blood of young rats by injecting subcutaneously buffered propionic acid (PPA) twice a day at 8-h intervals from the 6th to the 28th day of life. A matched group of animals (controls) was treated with the same volumes of saline. The animals were weighed and sacrificed by decapitation at 28, 35 or 60 days of age. Cerebellum and cerebrum were weighed and their protein and ganglioside N-acetylneuraminic acid (G-NeuAc) contents determined. Body, cerebral and cerebellar weights were similar in both groups, suggesting that PPA per se neither alters the appetite of the rats nor causes malnutrition. Brain protein concentration was also not affected by chronic administration of PPA, in contrast to G-NeuAc concentration which was significantly reduced in the cerebellum. Since ganglioside concentration is closely related to the dendritic surface and indirectly reflects synaptogenesis, our results of an important ganglioside deficit in the brain of PPA-treated animals may be related to the neurologic dysfunction characteristic of propionic acidaemic patients.