Absence of opioid stress-induced analgesia in mice lacking beta-endorphin by site-directed mutagenesis.

A physiological role for beta-endorphin in endogenous pain inhibition was investigated by targeted mutagenesis of the proopiomelanocortin gene in mouse embryonic stem cells. The tyrosine codon at position 179 of the proopiomelanocortin gene was converted to a premature translational stop codon. The resulting transgenic mice display no overt developmental or behavioral alterations and have a normally functioning hypothalamic-pituitary-adrenal axis. Homozygous transgenic mice with a selective deficiency of beta-endorphin exhibit normal analgesia in response to morphine, indicating the presence of functional mu-opiate receptors. However, these mice lack the opioid (naloxone reversible) analgesia induced by mild swim stress. Mutant mice also display significantly greater nonopioid analgesia in response to cold water swim stress compared with controls and display paradoxical naloxone-induced analgesia. These changes may reflect compensatory upregulation of alternative pain inhibitory mechanisms.

Acute stress-induced antinociception is cGMP-dependent but heme oxygenase-independent

Endogenous carbon monoxide (CO), which is produced by the enzyme heme oxygenase (HO), participates as a neuromodulator in physiological processes such as thermoregulation and nociception by stimulating the formation of 3′,5′-cyclic guanosine monophosphate (cGMP). In particular, the acute physical restraint-induced fever of rats can be blocked by inhibiting the enzyme HO. A previous study reported that the HO-CO-cGMP pathway plays a key phasic antinociceptive role in modulating noninflammatory acute pain. Thus, this study evaluated the involvement of the HO-CO-cGMP pathway in antinociception induced by acute stress in male Wistar rats (250-300 g; n=8/group) using the analgesia index (AI) in the tail flick test. The results showed that antinociception induced by acute stress was not dependent on the HO-CO-cGMP pathway, as neither treatment with the HO inhibitor ZnDBPG nor heme-lysinate altered the AI. However, antinociception was dependent on cGMP activity because pretreatment with the guanylate cyclase inhibitor 1H-[1,2,4] oxadiazolo [4,3-a] quinoxaline-1-one (ODQ) blocked the increase in the AI induced by acute stress.

Stress-induced neuroinflammation: mechanisms and new pharmacological targets

Stress is triggered by numerous unexpected environmental, social or pathological stimuli occurring during the life of animals, including humans, which determine changes in all of their systems. Although acute stress is essential for survival, chronic, long-lasting stress can be detrimental. In this review, we present data supporting the hypothesis that stress-related events are characterized by modifications of oxidative/nitrosative pathways in the brain in response to the activation of inflammatory mediators. Recent findings indicate a key role for nitric oxide (NO) and an excess of pro-oxidants in various brain areas as responsible for both neuronal functional impairment and structural damage. Similarly, cyclooxygenase-2 (COX-2), another known source of oxidants, may account for stress-induced brain damage. Interestingly, some of the COX-2-derived mediators, such as the prostaglandin 15d-PGJ2 and its peroxisome proliferator-activated nuclear receptor PPARγ, are activated in the brain in response to stress, constituting a possible endogenous anti-inflammatory mechanism of defense against excessive inflammation. The stress-induced activation of both biochemical pathways depends on the activation of the N-methyl-D-aspartate (NMDA) glutamate receptor and on the activation of the transcription factor nuclear factor kappa B (NFκB). In the case of inducible NO synthase (iNOS), release of the cytokine TNF-α also accounts for its expression. Different pharmacological strategies directed towards different sites in iNOS or COX-2 pathways have been shown to be neuroprotective in stress-induced brain damage: NMDA receptor blockers, inhibitors of TNF-α activation and release, inhibitors of NFκB, specific inhibitors of iNOS and COX-2 activities and PPARγ agonists. This article reviews recent contributions to this area addressing possible new pharmacological targets for the treatment of stress-induced neuropsychiatric disorders.

Proteasome Inhibition Represses Unfolded Protein Response and Nox4, Sensitizing Vascular Cells to Endoplasmic Reticulum Stress-Induced Death

Background: Endoplasmic reticulum (ER) stress has pathophysiological relevance in vascular diseases and merges with proteasome function. Proteasome inhibition induces cell stress and may have therapeutic implications. However, whether proteasome inhibition potentiates ER stress-induced apoptosis and the possible mechanisms involved in this process are unclear. Methodology/Principal Findings: Here we show that proteasome inhibition with MG132, per se at non-lethal levels, sensitized vascular smooth muscle cells to caspase-3 activation and cell death during ER stress induced by tunicamycin (Tn). This effect was accompanied by suppression of both proadaptive (KDEL chaperones) and proapoptotic (CHOP/GADD153) unfolded protein response markers, although, intriguingly, the splicing of XBP1 was markedly enhanced and sustained. In parallel, proteasome inhibition completely prevented ER stress-induced increase in NADPH oxidase activity, as well as increases in Nox4 isoform and protein disulfide isomerase mRNA expression. Increased Akt phosphorylation due to proteasome inhibition partially offset the proapoptotic effect of Tn or MG132. Although proteasome inhibition enhanced oxidative stress, reactive oxygen species scavenging had no net effect on sensitization to Tn or MG132-induced cell death. Conclusion/Relevance: These data indicate unfolded protein response-independent pathways whereby proteasome inhibition sensitizes vascular smooth muscle to ER stress-mediated cell death. This may be relevant to understand the therapeutic potential of such compounds in vascular disease associated with increased neointimal hyperplasia.

Giant Vesicles under Oxidative Stress Induced by a Membrane-Anchored Photosensitizer

We have synthesized the amphiphile photosensitizer PE-porph consisting of a porphyrin bound to a lipid head-group. We studied by optical microscopy the response to light irradiation of giant unilamellar vesicles of mixtures of unsaturated phosphatidylcholine lipids and PE-porph. In this configuration, singlet oxygen is produced at the bilayer surface by the anchored porphyrin. Under irradiation, the PE-porph decorated giant unilamellar vesicles exhibit a rapid increase in surface area with concomitant morphological changes. We quantify the surface area increase of the bilayers as a function of time and photosensitizer molar fraction. We attribute this expansion to hydroperoxide formation by the reaction of the singlet oxygen with the unsaturated bonds. Considering data from numeric simulations of relative area increase per phospholipid oxidized (15%), we measure the efficiency of the oxidative reactions. We conclude that for every 270 singlet oxygen molecules produced by the layer of anchored porphyrins, one eventually reacts to generate a hydroperoxide species. Remarkably, the integrity of the membrane is preserved in the full experimental range explored here, up to a hydroperoxide content of 60%, inducing an 8% relative area expansion.

Protective properties of quercetin against DNA damage and oxidative stress induced by methylmercury in rats

Aim of the study was to find out whether consumption of quercetin (QC), an abundant flavonoid in the human diet, protects against DNA damage caused by exposure to organic mercury. Therefore, rats were treated orally with methylmercury (MeHg) and the flavonoid with doses that reflect the human exposure. The animals received MeHg (30 mu g/kg/bw/day), QC (0.5-50 mg/kg/bw/day), or combinations of both over 45 days. Subsequently, the glutathione levels (GSH) and the activities of glutathione peroxidase (GPx) and catalase (CAT) were determined, and DNA damage was measured in hepatocytes and peripheral leukocytes in single cell gel electrophoresis assays. MeHg decreased the concentration of GSH and the activity of GPx by 17 and 12%, respectively and caused DNA damage to liver and blood cells, while with QC no such effects were seen. When the flavonoid was given in combination with MeHg, the intermediate and the highest concentrations (5.0 and 50.0 mg/kg/bw/day) were found to cause DNA protection; DNA migration was reduced by 54 and 65% in the hepatocytes and by 27 and 36% in the leukocytes; furthermore, the reduction in GSH and GPx levels caused by MeHg treatment was restored. In summary, our results indicate that consumption of QC-rich foods may protect Hg-exposed humans against the adverse health effects of the metal.

A quantitative chemiluminescent method for studying replicative and stress-induced premature senescence in cell cultures

beta-Galactosidase (beta-Gal) activity is a widely accepted biomarker to detect senescence both in situ and in vitro. A cytochemical assay based on production of a blue-dyed precipitate that results from the cleavage of the chromogenic substrate X-Gal is commonly used. Blue and nonblue cells are counted under the microscope and a semiquantitative percentage of senescent cells can be obtained. Here, we present a quantitative, fast, and easy to use chemiluminescent assay to detect senescence. The Galacton chemiluminescent method used to detect the prokaryotic beta-Gal reporter enzyme in transfection studies was adapted to assay mammalian beta-Gal. The assay showed linear production of luminescence in a time- and cell-number-dependent manner. The chemiluminescent assay showed significant correlation with the cytochemical assay in detecting replicative senescence (Pearson r = 0.8486, p < 0.005). Moreover, the chemiluminescent method (Galacton) also detected stress-induced senescence in cells treated with H2O2 similar to the cytochemical assay (X-Gal) (Galacton: control 25.207.3 +/- 6548.6. H2O, 52,487.4 +/- 16,284.9, p < 0.05; X-Gal: control 41.31 +/- 7.0%, H2O2 92.97 +/- 2.8%, p < 0.01). Thus, our method is well suited to the detection of replicative and stress-induced senescence in cell culture. (C) 2007 Elsevier Inc. All rights reserved.

Neuropharmacological basis of rTMS-induced analgesia: The role of endogenous opioids

We investigated the role of endogenous opioid systems in the analgesic effects induced by repetitive transcranial magnetic stimulation (rTMS). We compared the analgesic effects of motor cortex (M1) or dorsolateral prefrontal cortex (DLPFC) stimulation before and after naloxone or placebo treatment, in a randomized, double-blind crossover design, in healthy volunteers. Three groups of 12 volunteers were selected at random and given active stimulation (frequency 10 Hz, at 80% motor threshold intensity, 1500 pulses per session) of the right M1, active stimulation of the right DLPFC, or sham stimulation, during two experimental sessions 2 weeks apart. Cold pain thresholds and the intensity of pain induced by a series of fixed-temperature cold stimuli (5, 10, and 15 degrees C) were used to evaluate the analgesic effects of rTMS. Measurements were made at the left thenar eminence, before and 1 hour after the intravenous injection of naloxone (bolus of 0.1 mg/kg followed by a continuous infusion of 0.1 mg/kg/h until the end of rTMS) or placebo (saline). Naloxone injection significantly decreased the analgesic effects of M1 stimulation, but did not change the effects of rTMS of the DLPFC or sham rTMS. This study demonstrates, for the first time, the involvement of endogenous opioid systems in rTMS-induced analgesia. The differential effects of naloxone on M1 and DLPFC stimulation suggest that the analgesic effects induced by the stimulation of these 2 cortical sites are mediated by different mechanisms. (C) 2010 Published by Elsevier B.V. on behalf of International Association for the Study of Pain.

Effects of Lecithin and Vitamin E Supplementation on Liver Steatosis and Oxidative Stress Induced by Chronic Ethanol Consumption in Rats

Oxidative stress and lipid peroxidation, associated with ethanol, are considered important pathogenic mechanisms in the formation of hepatic steatosis. The objective of the present study was to assess the effects of supplementation with lecithin and vitamin E on the oxidatives stress and hepatic steatosis induced in rats by chronic ethanol consumption. Fifty-two Wistar rats were divided into 4 experimental groups: control (AIN-93 diet), ethanol group (control diet plus a 20% hydroalcoholic solution), ethanol + vitamin E group (addition of 0.6% vitamin E to the diet plus a 20% hydroalcoholic solution); ethanol + soy lecithin group (addition of 5 % soy lecithin to the diet plus a 20% hydroalcoholic solution). At the end of 4 weeks the animals were sacrificed. The results showed a significantly smaller number of animals (p < 0.05) classified as having a low degree of steatosis in the ethanol + vitamin E group and ethanol + soy lecithin group compared to the ethanol group. In addition, the ethanol + soy lecithin group had a significantly lower concentration of hepatic fat (p < 0.05) than the ethanol group. A significant reduction of hepatic TBARS concentration (p < 0.05) was detected in the ethanol + vitamin E group compared to the ethanol group. Hepatic carbonyl concentration was significantly lower in the ethanol + soy lecithin group. However, hepatic GSH was significantly lower in the ethanol + vitamin E and ethanol + soy lecithin groups compared to the control group. In conclusion, supplementation with lecithin and vitamin E attenuated the hepatotoxic effects of chronic ethanol intake and contributed to a reduction of the progression of steatosis status.

GABAergic mediation of stress-induced secretion of corticosterone and oxytocin, but not prolactin, by the hypothalamic paraventricular nucleus

The hypothalamus-pituitary-adrenal axis (HPA) participates in mediating the response to stressful stimuli. Within the HPA, neurons in the medial parvocellular region of paraventricular nucleus (PVN) of the hypothalamus integrate excitatory and inhibitory signals triggering secretion of corticotropin-releasing hormone (CRH), the main secretagogue of adrenocorticotropic hormone (ACTH). Stressful situations alter CRH secretion as well as other hormones, including prolactin and oxytocin. Most inputs to the PVN are of local origin, half of which are GABAergic neurons, and both GABA-A and GABA-B receptors are present in the PVN. The objective of the present study was to investigate the role of GABA-A and GABA-B receptors in the PVN`s control of stress-induced corticosterone, oxytocin and prolactin secretion. Rats Were microinjected with saline or different doses (0.5, 5 and 50 pmol) of GABA-A (bicuculine) or GABA-B (phaclofen) antagonists in the PVN. Ten minutes later, they were subjected to a stressor (ether inhalation) and blood samples were collected 30 min before and 10, 30, 60, 90 and 120 min after the stressful stimulus to measure hormone levels by radioimmunoassay. Our results indicate that GABA acts in the PVN to inhibit stress-induced corticosterone secretion via both its receptor subtypes, especially GABA-B. In contrast, GABA in the PVN stimulates oxytocin secretion through GABA-B receptors and does not alter prolactin secretion. (C) 2008 Elsevier Inc. All rights reserved.

Locus coeruleus mediates cold stress-induced polycystic ovary in rats

Previous reports about the rat ovary have shown that cold stress promotes ovarian morphological alterations related to a polycystic ovary (PCO) condition through activation of the ovarian sympathetic nerves. Because the noradrenergic nucleus locus coeruleus (LC) is activated by cold stress and synaptically connected to the preganglionic cell bodies of the ovarian sympathetic pathway, this study aimed to evaluate the LC`s role in cold stress-induced PCO in rats. Ovarian morphology and endocrine and sympathetic functions were evaluated after 8 wk of chronic intermittent cold stress (4 C, 3 h/d) in rats with or without LC lesion. The effect of acute and chronic cold stress upon the LC neuron activity was confirmed by Fos protein expression in tyrosine hydroxylase-immunoreactive neurons. Cold stress induced the formation of follicular cysts, type III follicles, and follicles with hyperthecosis alongside increased plasma estradiol and testosterone levels, irregular estrous cyclicity, and reduced ovulation. Considering estradiol release in vitro, cold stress potentiated the ovarian response to human chorionic gonadotropin. Ovarian norepinephrine (NE) was not altered after 8 wk of stress. However, LC lesion reduced NE activity in the ovary of cold-stressed rats, but not in controls, and prevented all the cold stress effects evaluated. Cold stress increased the number of Fos/tyrosine hydroxylase-immunoreactive neurons in the LC, but this effect was more pronounced for acute stress as compared with chronic stress. These results show that cold stress promotes PCO in rats, which apparently depends on ovarian NE activity that, under this condition, is regulated by the noradrenergic nucleus LC.

Relationship between stress-induced martensitic transformation and impact toughness in low carbon austenitic steels

The effect of test temperature, which controls the stability of austenite, on the impact toughness of a low carbon Fe-Ni-Mn-C austenitic steel and 304 stainless steel, has been investigated. Under impact conditions, stress-induced martensitic transformation occurred, in a region near the fracture surface, at test temperatures below 80degreesC for the Fe-Ni-Mn-C steel and below -25degreesC for 304 stainless steel. The former shows significant transformation toughening and the highest impact toughness was obtained at 10degreesC, which corresponds to the maximum amount of martensite formed by stress-induced transformation above the Ms temperature. The stress-induced martensitic transformation contributes negatively to the impact toughness in the 304 stainless steel. Increasing the amount of stress-induced transformation to martensite, lowered the impact toughness. The experimental results can be well explained by the Antolovich theory through the analysis of metallography and fractography. The different effect of stress-induced transformation on the impact toughness in Fe-Ni-Mn-C steel and 304 stainless steel has been further understood by applying the crystallographic model for stress-induced martensitic transformation to these two steels. (C) 2002 Kluwer Academic Publishers.

Role of oxidative stress-induced systemic and cavernosal molecular alterations in the progression of diabetic erectile dysfunction

Background Erectile dysfunction (ED) is a prevalent complication of diabetes, and oxidative stress is an important feature of diabetic ED. Oxidative stress-induced damage plays a pivotal role in the development of tissue alterations. However, the deleterious effects of oxidative stress in the corpus cavernosum with the progression of diabetes remain unclear. The aim of this study was to evaluate systemic and penile oxidative stress status in the early and late stages of diabetes. Methods Male Wistar streptozotocin-diabetic rats (and age-matched controls) were examined 2 (early) and 8 weeks (late) after the induction of diabetes. Systemic oxidative stress was evaluated by urinary H2O2 and the ratio of circulating reduced/oxidized glutathione (GSH/GSSG). Penile oxidative status was assessed by H2O2 production and 3-nitrotyrosine (3-NT) formation. Cavernosal endothelial nitric oxide synthase (eNOS) was analyzed by quantitative immunohistochemistry. Dual immunofluorescence was also performed for 3-NT and α-smooth muscle actin (α-SMA) and eNOS–α-SMA. Results There was a significant increase in urinary H2O2 levels in both diabetic groups. The plasma GSH/GSSG ratio was significantly augmented in late diabetes. In cavernosal tissue, H2O2 production was significantly increased in late diabetes. Reactivity for 3-NT was located predominantly in cavernosal smooth muscle (SM) and was significantly reduced in late diabetes. Quantitative immunohistochemistry revealed a significant decrease in eNOS levels in cavernosal SM and endothelium in late diabetes. Conclusions The findings indicate that the noxious effects of oxidative stress are more prominent in late diabetes. Increased penile protein oxidative modifications and decreased eNOS expression may be responsible for structural and/or functional deregulation, contributing to the progression of diabetes-associated ED.

Stress induced risk-aversion is reverted by D2/D3 agonist in the rat

Stress exposure triggers cognitive and behavioral impairments that influence decision-making processes. Decisions under a context of uncertainty require complex reward-prediction processes that are known to be mediated by the mesocorticolimbic dopamine (DA) system in brain areas sensitive to the deleterious effects of chronic stress, in particular the orbitofrontal cortex (OFC). Using a decision-making task, we show that chronic stress biases risk-based decision-making to safer behaviors. This decision-making pattern is associated with an increased activation of the lateral part of the OFC and with morphological changes in pyramidal neurons specifically recruited by this task. Additionally, stress exposure induces a hypodopaminergic status accompanied by increased mRNA levels of the dopamine receptor type 2 (Drd2) in the OFC; importantly, treatment with a D2/D3 agonist quinpirole reverts the shift to safer behaviors induced by stress on risky decision-making. These results suggest that the brain mechanisms related to risk-based decision-making are altered after chronic stress, but can be modulated by manipulation of dopaminergic transmission.

Early life stress-induced histone acetylations correlate with activation of the synaptic plasticity genes Arc and Egr1 in the mouse brain

Magdeburg, Univ., Fak. für Naturwiss., Diss., 2013