Role of astrocytes and altered regulation of spinal glutamatergic neurotransmission in stress-induced visceral hyperalgesia in rats

Glutamate (Glu) is the primary excitatory neurotransmitter in the central nervous system and plays a critical role in the neuroplasticity of nociceptive networks. We aimed to examine the role of spinal astroglia in the modulation of glutamatergic neurotransmission in a model of chronic psychological stress-induced visceral hyperalgesia in male Wistar rats. We assessed the effect of chronic stress on different glial Glu control mechanisms in the spinal cord including N-methyl-d-aspartate receptors (NMDARs), glial Glu transporters (GLT1 and GLAST), the Glu conversion enzyme glutamine synthetase (GS), and glial fibrillary acidic protein (GFAP). We also tested the effect of pharmacological inhibition of NMDAR activation, of extracellular Glu reuptake, and of astrocyte function on visceral nociceptive response in naive and stressed rats. We observed stress-induced decreased expression of spinal GLT1, GFAP, and GS, whereas GLAST expression was upregulated. Although visceral hyperalgesia was blocked by pharmacological inhibition of spinal NMDARs, we observed no stress effects on NMDAR subunit expression or phosphorylation. The glial modulating agent propentofylline blocked stress-induced visceral hyperalgesia, and blockade of GLT1 function in control rats resulted in enhanced visceral nociceptive response. These findings provide evidence for stress-induced modulation of glia-controlled spinal Glu-ergic neurotransmission and its involvement in chronic stress-induced visceral hyperalgesia. The findings reported in this study demonstrate a unique pattern of stress-induced changes in spinal Glu signaling and metabolism associated with enhanced responses to visceral distension.     

The activity of the monoaminergic systems of the rat hypothalamus in acute stress after chronic stressing]

The influence of chronic stress (footshock combined with randomized light flashes) on acute stress-induced (immobilization) release of noradrenaline, dopamine and serotonin in rat lateral hypothalamus was assessed by microdialysis. The chronic stress resulted in an increase and prolongation of the acute stress-induced release of noradrenaline but not of dopamine and serotonin. The increased rate of accumulation of dioxyphenylacetic acid and unchanged accumulation of homovanillic acid (dopamine metabolites) and dopamine during and after the acute stress in chronically stressed animals reflect a rise of synthetic activity of catecholaminergic systems in response to acute stress and reuptake increase. Marked stress-induced increase in hydroxyindoleacetic acid in chronically stressed rats without any changes in the ST dynamics may be regarded in a similar way. A significant increase in potassium-stimulated release of all the studied monoamines was found while their basal level remained unchanged. The conclusions was made that the hyperergic release of neurotransmitters may be the basis of an inadequate response of animals to acute stress, i.e., one of the neurotic symptoms.     

Melatonin and adrenal cortex steroid production: in vivo and in vitro studies.

The present study was designed to clarify the interaction between the pineal melatonin and adrenal cortex steroid production. Experiments with male rats under chronic stress conditions (sleep deprivation) revealed that melatonin circadian pattern was fully destroyed and daytime plasma concentration were significantly elevated. Constant illumination (2500 lux) during the nighttime was not able to suppress melatonin production in the stressed animals. Plasma concentration of corticosterone were increased in the stressed rats as well. The modulatory effect of melatonin on corticosterone and progesterone production by rat adrenals was studied in a superfusion system. During melatonin challenge progesterone secretion was two-three fold elevated with no effect on corticosterone content in the plasma samples. Pineal cytoplasmic glucocorticoid and progesterone receptors were investigated as well. A specific binding was not observed in that case. Presented data support the existence of direct communication between the pineal and adrenal glands.     

Prolactin inhibits the development of stress-induced ulcers in the rat

Hyperprolactinaemia, as induced by pituitary homografts under the kidney capsule, was accompanied by an inhibition of development of gastric ulcers following the application of cold-plus-restraint stress in male rats. This effect was mimicked by intracisternal administration of a low dose of the hormone. Peripheral injection of the dopamine receptor antagonist, domperidone, also inhibited the development of stress-induced ulcers. However, no effect was found after peripheral injection of another dopamine receptor antagonist, haloperidol. This latter drug appeared to antagonize the cytoprotective effect of prolactin (PRL) on stress-induced ulcers. Furthermore, peripheral injection of the prostaglandin synthesis inhibitor, indomethacin, increased the incidence of gastric ulcers in hyperprolactinaemic rats subjected to cold -plus-restraint stress. These data suggest that the cytoprotective effect of PRL on development of gastric ulcers in stressed animals may involve both central (i.e. dopamine transmission) and peripheral (i.e. prostaglandin synthesis) mechanisms.     

Inhibitory effects of stress-activated nitric oxide on antioxidant enzymes and testicular steroidogenesis

The messenger role of nitric oxide (NO) in immobilization stress-induced inhibition of testicular steroidogenesis has been previously suggested. In accord with this, here, we show that the intratesticular injection of isosorbide dinitrate (ISDN; 2x2.5 mg/testis), an NO donor, mimicked the action of stress on serum testosterone concentrations and hCG-stimulated testosterone production in rat testicular tissue. When added in vitro, ISDN inhibited testicular 3beta-hydroxysteroid dehydrogenase and 17alpha-hydroxylase/lyase. Immobilization stress and injections of ISDN also decreased the activity of catalase, glutathione peroxidase, glutathione transferase, and glutathione reductase in the interstitial compartment of testis. When stressed rats were treated concomitantly with bilateral intratesticular injections of N(omega)-nitro-L-arginine methyl ester, a non-selective NOS inhibitor (2x600 microg/testis), the activities of antioxidative enzymes, as well as serum testosterone concentration, were partially normalized. These results indicate that stress-induced stimulation of the testicular NO signalling pathway leads to inhibition of both steroidogenic and antioxidant enzymes.     

Water stress impact on young seedling growth of Acacia arabica

Drought is the major constraint in arid regions throughout the world and identification of drought-resistant plants is therefore of crucial importance. Since young seedling stage is especially sensitive to water stress, the present work analyzed the physiological behavior of seedling from Acacia arabica issued from a dry area, grown under controlled environmental conditions and subjected to progressive soil drying. Although soil gravimetric water content (g H₂O g^?1 soil dry weight) dropped from 80 % to less than 35 %, most plants remained alive until the end of the water stress. Seedlings were able to efficiently close their stomata to reduce water losses and accumulated high amounts of proline. Despite osmotic adjustment, turgor pressure decreased in stressed plants and could explain the stress-induced inhibition of plant growth. Decrease in net photosynthesis was related to stress-induced decrease in stomatal conductance and not to any impact on chlorophyll concentration or fluorescence-related parameter: both PSII efficiency and photochemical quenching remained unaffected by water stress while drought-induced increase in non-photochemical quenching should be regarded as a strategy to avoid over-energisation of the photosynthetic apparatus. Instantaneous water use efficiency increased in stressed plants comparative to controls. Oxidative stress estimated by malondialdehyde concentration was recorded only at the end of the treatment, suggesting that stressed plants remained able to cope with reactive oxygen species. Water stress induced an increase in anthocyanins, while aglycone flavonols decreased. Those compounds were not involved in the management of oxidative stress. It is concluded that A. arabica is a promising drought-resistant plant species for rehabilitation of dry areas.     

Clacium ion involvement in growth inhibition of mechanically stressed soybean (Glycine max) seedlings

A 40–50% reduction in soybean [ Glycine max (L.) Merr. cv. Century 84] hypocotyl elongation occurred 24 h after application of mechanical stress. Exogenous at 10 m M inhibited growth by 28% if applied with the Ca²⁺ ionophore A23187 to the zone of maximum hypocotyl elongation. La³⁺ was even more inhibitory than Ca²⁺, especially above 5 m M . Treatment with ethyleneglycol-bis-(β3-aminoethylether)-N, N, N', N'-tetraacetic acid (EGTA) alone had no effect on growth of non-stressed seedlings at the concentrations used but negated stress-induced growth reduction by 36% at 4 m M when compared to non-treated, stressed controls. Treatment with EDTA was ineffective in negating stress-induced growth inhibition. Calmodulin antagonists calmidazolium, chlorpromazine, and 48/80 also negated stress-induced growth reduction by 23, 50, and 35%, respectively.     

Stress by forced swimming in the rat: effects of mianserin and moclobemide on GABAergic-monoaminergic systems in the brain

The stress caused by forced swimming in male rats provoked a decrease in brain NA levels without changes in DA and 5-HT content, MAO and GABAergic activity. Acute or chronic treatment with mianserin did not modify the decrease in NA concentration in the brain of stressed rats. Acute treatment with moclobemide (IMAO) did not modify the decrease in NA content caused by stress; chronic treatment blocked the decrease in NA content in stressed rats.     

Neuroendocrine mechanism for tolerance to cerebral ischemia-reperfusion injury in male rats

Testosterone has been shown to exacerbate cerebral ischemia-reperfusion injury, which suggests that the well-known stress-induced testosterone reduction could be a protective response. We hypothesized that stress-induced testosterone reduction contributes to ischemia tolerance in cerebral ischemia-reperfusion injury in male rats. In intact male rats, stress was induced by brief anesthesia at 6 h before transient middle cerebral artery occlusion (MCAO). Testosterone levels were significantly decreased 6 h after stress. Testosterone reduction was associated with a 50% reduction in cerebral lesion volume in the stressed animals. Further, the stress-induced cerebral ischemia tolerance was eliminated by testosterone replacement in castrated males. Immunohistochemical staining showed that androgen receptors were up-regulated after cerebral ischemia-reperfusion injury and partially colocalized with terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) positive cells in the parietal cortex and extensively colocalized in the caudate putamen. Heat shock protein 70 (Hsp70) and 90 (Hsp90) are involved in ischemia tolerance, and were not colocalized with TUNEL in the immunohistochemical staining, suggesting an antiapoptotic role of Hsp's. To determine the effect of testosterone on MCAO-induced Hsp70 and -90 expression, a testosterone replacement or withdrawal paradigm was used. Testosterone-replaced animals exhibited a decrease in Hsp expression, whereas testosterone withdrawal (mimicking the stress-induced testosterone suppression) normalized this deficit. In summary, stress-induced testosterone reduction contributes to ischemia tolerance in cerebral ischemia-reperfusion injury in males, which could be related to the loss of inhibition by testosterone of Hsp70 and -90 expression.     

“Green odor” inhalation by stressed rat dams reduces behavioral and neuroendocrine signs of prenatal stress in the offspring

Chronic maternal stress during pregnancy results in the “prenatally stressed” offspring displaying behavioral and neuroendocrine alterations that persist into adulthood. We investigated how inhalation of green odor (a mixture of equal amounts of trans-2-hexenal and cis-3-hexenol) by stressed dams might alter certain indices of prenatal stress in their offspring. These indices were depression-like behavior (increased immobility time in the forced-swim test) and acute restraint stress-induced changes in hypothalamo-pituitary-adrenocortical (HPA) axis activity [plasma corticosterone (CORT) and ACTH levels and the number of Fos-immunoreactive cells in the hypothalamic paraventricular nucleus (an index of neuronal activity)]. Pregnant rats were exposed to restraint stress for 60 min/day for 10 days (gestational days 10-19). The prenatally stressed offspring exhibited significant increases in depression-like behavior and in restraint stress-induced ACTH, CORT, and Fos responses, unless their dam had been exposed to green odor. The behavioral effect of the odor was also seen in offspring that were fostered by unstressed dams. The results obtained in the dams themselves were as follows. In vehicle-exposed stressed dams, but not in green odor-exposed ones, total body and adrenal weights were significantly decreased or increased, respectively. Depression-like behavior was not observed in the vehicle-exposed stressed dams themselves. Green odor inhalation prevented the impairment of maternal behavior induced by restraint stress. Thus, exposure of dams to stress may affect both the fetal brain and fetal HPA axis, and also maternal behavior, leading to altered behavioral and neuroendocrine responses in the offspring. Such effects may be prevented by the stressed dams inhaling green odor.     

Atorvastatin suppresses oxidized LDL-induced dendritic cell-like differentiation of RAW264.7 cells regulated by the p38 MAPK pathway

The molecular signaling events leading to protection from oxidative stress-induced apoptosis upon contact inhibition have not been fully investigated. Previous research has indicated a role for mitogen-activated protein kinases (MAPKs) in the regulation of contact inhibition, and these proteins have also been associated with cell cycle regulation and stress-induced apoptosis. The potential role of the MAPK JNK-1 in the stress-response of actively proliferating and contact-inhibited cells was investigated. Actively proliferating normal fibroblasts (BJ) and fibrosarcoma cells (HT-1080) were stressed with H2O2, and levels of activated JNK-1 and cleaved PARP were ascertained. Similarly, these results were compared with levels of activated JNK-1 and cleaved PARP detected in H2O2-stressed confluent fibrosarcoma or contact-inhibited fibroblast cells. Contact-inhibited fibroblasts were protected from apoptosis in comparison to subconfluent fibroblasts, concurrent with decreased JNK-1 activation. Increased culture density of fibrosarcoma cells was not protective against apoptosis, and these cells did not demonstrate density-dependent alterations in the JNK-1 stress response. This decreased activation of JNK-1 in stressed, contact-inhibited cells did not appear to be dependent upon increased expression of MKP-1; however, over-expression of MKP-1 was sufficient to result in a slight decrease in H2O2-stimulated PARP cleavage. Increasing the antioxidant capacity of fibroblasts through NAC-treatment not only lessened H2O2-stimulated JNK-1 activation, but also did not influence the expression of MKP-1. Taken together, these results suggest that regulation of negative regulation of JNK-1 upon contact inhibition is protective against apoptosis, and that this regulation is independent of MKP-1.     

Translational repression of MCL-1 couples stress-induced eIF2 alpha phosphorylation to mitochondrial apoptosis initiation

The integrated stress response (ISR) integrates a broad range of environmental and endogenous stress signals to the phosphorylation of the alpha-subunit of eukaryotic translation initiation factor 2 (eIF2 alpha). Although intense or prolonged activation of this pathway is known to induce apoptosis, the molecular mechanisms coupling stress-induced eIF2 alpha phosphorylation to the cell death machinery have remained incompletely understood. In this study, we characterized apoptosis initiation in response to classical activators of the ISR (tunicamycin, UVC, elevated osmotic pressure, arsenite). We found that all applied stress stimuli activated a mitochondrial pathway of apoptosis initiation. Rapid and selective down-regulation of the anti-apoptotic BCL-2 family protein MCL-1 preceded the activation of BAX, BAK, and caspases. Stabilization of MCL-1 blocked apoptosis initiation, while cells with reduced MCL-1 protein content were strongly sensitized to stress-induced apoptosis. Stress-induced elimination of MCL-1 occurred with unchanged protein turnover and independently of MCL-1 mRNA levels. In contrast, stress-induced phosphorylation of eIF2 alpha at Ser(51) was both essential and sufficient for the down-regulation of MCL-1 protein in stressed cells. These findings indicate that stress-induced phosphorylation of eIF2 alpha is directly coupled to mitochondrial apoptosis regulation via translational repression of MCL-1. Down-regulation of MCL-1 enables but not enforces apoptosis initiation in stressed cells.     

Osmotic adjustment in indoor grown cassava in response to water stress

The water content-water potential relation in stressed and unstressed cassava ( Man-ihot species) was examined to ascertain (i) the magnitude of osmotic adjustment in response to water stress and (ii) the mechanisms of such adjustments.
Water stress resulted in a displacement of the water content-potential relation such that at any leaf water potential the water content was higher in the stressed plants. The osmotic potentials of turgid leaves (100% relative water content) were -0.97 and -1.00 MPa in the unstressed cultivars CMC 9 and MCOL 113 respectively. In the stressed plants, the values were-1.13 MPa (CMC 9) and-1.14 MPa (MCOL 113). The 0.14 to 0.16 MPa osmotic potential difference between the stressed and unstressed plants suggests that a stress-induced osmotic adjustment occurred in both cultivars. The biiSk volumetric elastic moduli at turgor pressures above 0.10 MPa were 9.84 MPa (CMC 9) and 13.58 MPa (MCOL 113) in the unstressed plants. Tbe higher values found in the stressed plants, 14.56 MPa in CMC 9 and 16.91 MPa in MCOL 113, suggest a stress-induced decrease in cell wall elasticity. Hence, the observed shift in the wafer content-potential relations in the cassava involved both an osmotic adjustment and a decrease in cell wall elasticity. Increasing the number of stress cycles per plant did not cause a further displacement of the water content-potential curves.     

Adrenergic response patterns in vas deferens isolated from rats under diurnal rhythms. Influence of stress

The aim of this study was to verify, by means of functional methods, whether the circadian rhythm changes adrenergic response patterns in the epididymal half of the vas deferens isolated from control rats as well as from rats submitted to acute stress. The experiments were performed at 9:00 a.m., 3:00 p.m., 9:00 p.m., and 3:00 a.m. The results showed a light-dark dependent variation of the adrenergic response pattern on organs isolated from control as well as from stressed rats. In the control group, only the phenylephrine sensitivity was changed throughout the circadian rhythm. Under the stress condition, both norepinephrine and phenylephrine response patterns were changed, mainly during darkness. The maximal contractile response to both alpha- and beta-agonist and alpha1-agonist was increased in the dark phase, corresponding to high plasmatic concentrations of endogenous melatonin. The vas deferens isolated from stressed rats during the light phase simultaneously incubated with exogenous melatonin showed the same pattern of response obtained in the dark phase, thus indicating a peripheric action of melatonin on this organ. Therefore, the circadian rhythms are important to the adrenergic response pattern in rat vas deferens from both control and stressed rats. In conclusion, we suggest a melatonin modulation on alpha1-postsynaptic adrenergic response in the rat vas deferens.     

5'AMP-activated protein kinase alpha deficiency enhances stress-induced apoptosis in BHK and PC12 cells

5'AMP-activated protein kinase (AMPK) activation occurs under a variety of stress conditions but the role of this enzyme in the promotion or inhibition of stress-induced cell death is unclear. To address this issue, we transformed two different cell lines with shRNA-expressing plasmids, targeting the alpha subunit of AMPK, and verified AMPKalpha downregulation. The cell lines were then stressed by exposure to medium without glucose (PC12 cells) or with the viral thymidine kinase-specific DNA replication inhibitors: acyclovir, penciclovir and ganciclovir (herpes simplex virus thymidine kinase-expressing Baby Hamster Kidney cells). In non-AMPK-downregulated cells, these stress treatments induced AMPK upregulation and phosphorylation, leaving open the question whether the association of AMPK activation with stress-induced cell death reflects a successful death-promoting or an ineffective death-inhibiting activity. In AMPKalpha-deficient cells (expressing AMPKalpha-specific shRNAs or treated with Compound C) exposure to low glucose medium or DNA replication inhibitors led to an enhancement of cell death, indicating that, under the conditions examined, the role of activated AMPK is not to promote, but to protect from or delay stress-induced cell death.     

Day-to-day variation in pineal serotonin N-acetyltransferase activity in stressed and non-stressed male Sprague-Dawley rats

Previous studies involving physical-immobilization stress in laboratory rats have yielded inconsistent results with respect to melatonin synthesis in the pineal gland. As melatonin formation undergoes circadian and infradian rhythms, the aim of the present study was to examine whether stress experiments exhibit day-to-day variation. Toward this end, groups of male Sprague-Dawley rats were stressed by physical immobilization on eight consecutive days, respectively, or left relatively undisturbed, and killed. The pineal gland was rapidly dissected out and serotonin N-acetyltransferase (NAT) activity and melatonin levels were measured. NAT activity was significantly depressed on experimental days 1, 3 and 5, and slightly depressed on day 7. In addition, both in control and experimental animals NAT activity exhibited statistically significant differences between experimental days. Pineal melatonin levels were less variable. On experimental days 3 and 6 immobilization led to a significant increase of pineal melatonin levels. These results show that day-to-day variation is an important factor that influences the outcome of stress experiments and represent another example that NAT activity and pineal melatonin levels do not always show corresponding changes.     

The counteracting effects of (-)-Epigallocatechin-3-Gallate on the immobilization stress-induced adverse reactions in rat pancreas

Many studies suggest that Epigallocatechin-3-Gallate (EGCG) has many protective effects. But little is known about its protective effects against chronic restraint stress-induced damage in rats. The aim was to demonstrate the potential protective effects of EGCG against harmful pancreatic damage to the immobilization stress in the rat model. Forty rats, 2 months old, were divided into four groups (n = 10): control group; EGCG group, rats received EGCG by gavage (100 mg/kg /day) for 30 days; stressed group, rats exposed to immobilization stress; and stressed with EGCG group, rats exposed to immobilization stress and received EGCG for 30 days. Glycemic status parameters, corticosterone, and inflammatory markers were investigated on the first day, 15th day, and the 30th day of the experiment. Pancreatic oxidative stress markers and cytokines were evaluated. Histological, immunohistological, and statistical studies were performed. On the 15th day, fasting blood glucose (FBG), fasting plasma insulin (FPI), homeostatic model assessment for insulin resistance (HOMA-IR), and fasting plasma corticosterone were significantly higher in the stressed group when compared with first and 30th day in the same group as well as when compared with control and stressed with EGCG groups. The stressed group revealed significantly higher pancreatic IL-1β, IL-6, TNF-α, MDA, and NO, serum amylase and serum lipase, and significantly lower GSH, SOD, and CAT when compared to control and stressed with EGCG groups. EGCG treatment attenuated the pancreatic stress-induced cellular degeneration, leucocytic infiltration, and cytoplasmic vacuolations; significantly decreased area percentage of collagen fibers; and significantly increased mean area percentage of insulin immunopositive cell as compared with stressed group. EGCG is a protective agent against immobilization stress because of its anti-diabetic, anti-inflammatory, and and anti-oxidative stress properties, as confirmed by biochemical and histological alterations.