Interactions Between Chronic Stress and Chronic Consumption of Caffeine on the Enzymatic Antioxidant System

We studied the effect of chronic caffeine on parameters related to oxidative stress in different brain regions of stressed and non-stressed rats. Wistar rats were divided into three groups: control (receiving water), caffeine 0.3 g/L and caffeine 1.0 g/L (in the drinking water). These groups were subdivided into non-stressed and stressed (repeated restraint stress during 40 days). Lipid peroxide levels and the total radical-trapping potential were assessed, as well as antioxidant enzyme activities superoxide dismutase, gluthatione peroxidase, and catalase in hippocampus, striatum and cerebral cortex. Results showed interactions between stress and caffeine, especially in the cerebral cortex, since caffeine increased the activity of some antioxidant enzymes, but not in stressed animals. We concluded that chronic administration of caffeine led, in some cases, to increased activity of antioxidant enzymes. However, these effects were not observed in the stressed animals.     

Effects of Chronic Restraint Stress and Estradiol Replacement on Glutamate Release and Uptake in the Spinal Cord from Ovariectomized Female Rats

Glutamate is an excitatory neurotransmitter involved in neuronal plasticity and neurotoxicity. Chronic stress produces several physiological changes on the spinal cord, many of them presenting sex-specific differences, which probably involve glutamatergic system alterations. The aim of the present study was to verify possible effects of exposure to chronic restraint stress and 17β-estradiol replacement on [³H]-glutamate release and uptake in spinal cord synaptosomes of ovariectomized (OVX) rats. Female rats were subjected to OVX, and half of the animals received estradiol replacement. Animals were subdivided in controls and chronically stressed. Restraint stress or estradiol had no effect on [³H]-glutamate release. The chronic restraint stress promoted a decrease and 17β-estradiol induced an increase on [³H]-glutamate uptake, but the uptake observed in the restraint stress +17β-estradiol group was similar to control. Furthermore, 17β-estradiol treatment caused a significant increase in the immunocontent of the three glutamate transporters present in spinal cord. Restraint stress had no effect on the expression of these transporters, but prevented the 17β-estradiol effect. We suggest that changes in the glutamatergic system are likely to take part in the mechanisms involved in spinal cord plasticity following repeated stress exposure, and that 17β-estradiol levels may affect chronic stress effects in this structure.     

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.     

Opiate antagonist binding sites in discrete brain regions of spontaneously hypertensive and normotensive Wistar-Kyoto rats

The binding of 3H-naltrexone, an opiate receptor antagonist, to membranes of discrete brain regions and spinal cord of 10 week old spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats was determined. The brain regions examined were hypothalamus, amygdala, hippocampus, corpus striatum, pons and medulla, midbrain and cortex. 3H-Naltrexone bound to membranes of brain regions and spinal cord at a single high affinity site with an apparent dissociation constant value of 3 nM. The highest density of 3H-naltrexone binding sites were in hippocampus and lowest in the cerebral cortex. The receptor density (Bmax value) and apparent dissociation constant (Kd value) values of 3H-naltrexone to bind to opiate receptors on the membranes of amygdala, hippocampus, corpus striatum, pons and medulla, midbrain, cortex and spinal cord of WKY and SHR rats did not differ. The Bmax value of 3H-naltrexone binding to membranes of hypothalamus of SHR rats was 518% higher than WKY rats but the Kd values in the two strains did not differ. It is concluded that SHR rats have higher density of opiate receptors labeled with 3H-naltrexone in the hypothalamus only, in comparison with WKY rats, and that such a difference in the density of opiate receptors may be related to the elevated blood pressure in SHR rats.     

Clomipramine Treatment and Repeated Restraint Stress Alter Parameters of Oxidative Stress in Brain Regions of Male Rats

This study aimed to compare the effects of repeated restraint stress alone and the combination with clomipramine treatment on parameters of oxidative stress in cerebral cortex, striatum and hippocampus of male rats. Animals were divided into control and repeated restraint stress, and subdivided into treated or not with clomipramine. After 40 days of stress and 27 days of clomipramine treatment with 30 mg/kg, the repeated restraint stress alone reduced levels of Na⁺, K⁺-ATPase in all tissues studied. The combination of repeated restraint stress and clomipramine increased the lipid peroxidation, free radicals and CAT activity as well as decreased levels of NP-SH in the tissues studied. However, Na⁺, K⁺-ATPase level decreased in striatum and cerebral cortex and the SOD activity increased in hippocampus and striatum. Results indicated that clomipramine may have deleterious effects on the central nervous system especially when associated with repeated restraint stress and chronically administered in non therapeutic levels.     

Neuroprotective effect of mexiletine in the central nervous system of diabetic rats

Both experimental and clinical studies suggests that oxidative stress plays an important role in the pathogenesis of diabetes mellitus type 1 and type 2. Hyperglycaemia leads to free radical generation and causes neural degeneration. In the present study we investigated the possible neuroprotective effect of mexiletine against streptozotocin-induced hyperglycaemia in the rat brain and spinal cord.30 adult male Wistar rats were divided into three groups: control, diabetic, and diabetic-mexiletine treated group. Diabetes mellitus was induced by a single injection of streptozotocin (60 mg/kg body weight). Mexiletine (50 mg/kg) was injected intraperitoneally every day for six weeks. After 6 weeks the brain, brain stem and cervical spinal cord of the rats were removed and the hippocampus, cortex, cerebellum, brain stem and spinal cord were dissected for biochemical analysis (the level of Malondialdehide [MDA], Nitric Oxide [NO], Reduced Glutathione [GSH], and Xanthine Oxidase [XO] activity). MDA, XO and NO levels in the hippocampus, cortex, cerebellum, brain stem and spinal cord of the diabetic group increased significantly, when compared with control and mexiletine groups (P < 0.05). GSH levels in the hippocampus, cortex, cerebellum, brain stem and spinal cord of the diabetic group decreased significantly when compared with control and mexiletine groups (P < 0.05).This study demonstrates that mexiletine protects the neuronal tissue against the diabetic oxidative damage.     

Characteristics of central binding sites for [3H] DAMGO in spontaneously hypertensive rats

The binding of [3H] DAMGO, a highly selective ligand for mu-opiate receptors, to membranes of discrete brain regions and spinal cord of 10 week old spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats was determined. The brain regions examined were hypothalamus, amygdala, hippocampus, corpus striatum, pons and medulla, midbrain and cortex. [3H] DAMGO bound to membranes of brain regions and spinal cord at a single high affinity site. The receptor density (Bmax value) and apparent dissociation constant (Kd value) of [3H] DAMGO to bind to membranes of hippocampus, corpus striatum, pons and medulla, cortex and spinal cord of WKY and SHR rats did not differ. The Bmax value of [3H] DAMGO in membranes of hypothalamus and midbrain of SHR rats was significantly higher than in WKY rats but the Kd values in the two strains did not differ. On the other hand, the Bmax value of [3H] DAMGO in membranes of amygdala of SHR rats was lower than that of WKY rats but the Kd values in the two strains were similar. It is concluded that SHR rats have higher density of mu-opiate receptors in hypothalamus and midbrain but lower density in amygdala in comparison with WKY rats, and that such differences in the distribution of mu-opiate receptors may be related to the elevated blood pressure in SHR rats.     

Long-term effects of prenatal stress on dopamine and glutamate receptors in adult rat brain

Prenatal stress greatly influences the ability of an individual to manage stressful events in adulthood. Such vulnerability may result from abnormalities in the development and integration of forebrain dopaminergic and glutamatergic projections during the prenatal period. In this study, we assessed the effects of prenatal stress on the expression of selective dopamine and glutamate receptor subtypes in the adult offsprings of rats subjected to repeated restraint stress during the last week of pregnancy. Dopamine D₂-like receptors increased in dorsal frontal cortex (DFC), medial prefrontal cortex (MPC), hippocampal CA₁ region and core region of nucleus accumbens (NAc) of prenatally stressed rats compared to control subjects. Glutamate NMDA receptors increased in MPC, DFC, hippocampal CA₁, medial caudate-putamen, as well as in shell and core regions of NAc. Group III metabotropic glutamate receptors increased in MPC and DFC of prenatally stressed rats, but remained unchanged in all other regions examined. These results indicate that stress suffered during the gestational period has long lasting effects that extend into the adulthood of prenatally stressed offsprings. Changes in dopamine and glutamate receptor subtype levels in different forebrain regions of adult rats suggest that the development and formation of the corticostriatal and corticolimbic pathways may be permanently altered as a result of stress suffered prenatally. Maldevelopment of these pathways may provide a neurobiological substrate for the development of schizophrenia and other idiopathic psychotic disorders.     

Acute and chronic restraint stress alter the incidence of social conflict in male rats

Stress and elevated stress hormone levels are known to alter cognition, learning, memory, and emotional responses. Three weeks of chronic stress or glucocorticoid exposure is reported to alter neuronal morphology in the hippocampus, the amygdala, and the prefrontal cortex, and to decrease neurogenesis in the dentate gyrus. Here we examine the effects of acute and chronic restraint stress exposure on the incidence of emotional responses throughout a 3-week period among adult rat conspecifics. Our data indicate that acute restraint stress (i.e., a single 6-h exposure) results in a significant reduction in aggressive conflicts among stressed males compared to experimental controls. In contrast, on Days 14 and 21, repeatedly restrained rats exhibited significantly more aggressive behaviors than controls. Blood samples taken 18 h after the last restraint session indicate that plasma concentrations of the stress hormone corticosterone (CORT) in stressed rats were equivalent to those of unstressed rats; however, the number of individually initiated aggressive acts observed positively correlated with plasma CORT measures taken at the end of the study. In contrast to studies of psychosocial stress or intruder paradigms, here we observe spontaneous emotional responses to an uncontrollable stressor in the homecage. This study provides a novel examination of the effects of chronic restraint stress on emotional responses in the home environment among cagemates. These results indicate that acute and chronic restraint stress alter the incidence of aggression, and emphasize the relevance of this model of chronic stress to studies of stress-responsive disorders characterized by aggressive behavior.     

Influence of prenatal stress on the rat hypothalamic-pituitary-adrenal axis activity: the role of the brain glycocorticoid receptors

The effects of prenatal stress on the hypothalamic-pituitary-adrenal (HPA) axis activity and brain glycocorticoid receptors were studied in neonatal male and female offspring, as well as the influence of neonatal glycocorticoid receptors blockade on hormonal stress reactivity of adult rats. The results showed that there were sexual differences in plasma corticosterone level and corticosteroid binding in the cortex and hypothalamus of 5-day old control rats. Prenatal stress increased basal level of corticosterone in female rats, decreased corticosterone binding in hypothalamus and hippocampus of male and female rats, and increased corticosteroid receptor level in the male cortex. Neonatal administration of glycocorticoid receptor antagonist did not change plasma corticosterone level in 5-day old rats, but prolonged hormonal stress response of the HPA axis in adult male rats and increased hormonal stress response in female ones. The character of the IIPA axis activity of male and female rats with neonatal blockade of glycocorticoid receptors correspond to hormonal stress response of prenatal stressed rats. These data suggest that change of brain glycocorticoid receptors function in neonatal period of development might be one of the mechanisms of prenatal stress influence on the HPA axis activity in the adulthood.     

正常猕猴与树鼩神经系统和免疫系统组织中大麻素与阿片受体的表达

为应用猕猴和树鼩动物模型研究毒品成瘾对神经/免疫系统的影响提供基础数据,对大麻素及阿片受体在正常猕猴和树鼩神经系统和免疫系统的表达进行初步确定.采集正常猕猴和树鼩新鲜组织(皮质、小脑、脑干、海马、脊髓、脾脏),应用半定量逆转录PCR和实时定量PCR的方法检测大麻素与阿片受体mRNA在猕猴和树鼩各组织中的表达情况.猕猴脑部各区包括脾脏均表达大麻索受体1(CNR1),而大麻素受体2(CNR2)只表达于脾脏内.三类阿片受体中,mu(μ)受体表达最为广泛,在以上各组织中均有表达;delta(δ)受体表达的组织最少,只在海马表达;kappa(κ)受体表达介于两者之间,分别在皮质、小脑、脑干、脊髓中表达.在树鼩组织中,CNR1和CNR2表达于整个大脑重要脑区中,且CNR1表达量高于同一区域内CNR2表达的鼍:脾脏中CNR2的表达较高,而CNR1不表达.三类阿片受体只有检测到μ受体在脑部与脾脏表达,且在各个脑区的表达量明显高于脾脏的表达量;δ体和κ受体在被检各个组织中均无表达.总体而言,两种大麻素受体在猕猴和树鼩体内表达情况与人类和鼠的情况类似,而三类阿片受体在猕猴体内表达情况与人类吏为接近.猕猴和树鼩可能可用于人类毒品成瘾的研究;猕猴在某些神经受体的表达更接近人类,其在研究毒品成瘾的机理和对免疫系统的影响方面仍有不可替代的地位.     

产前束缚应激子代大鼠海马神经颗粒素表达降低

神经颗粒素（neurogranin,NG）是脑特异性突触后蛋白,参与在学习记忆功能中起核心作用的信号转导通路及突触可塑性。本研究旨在探讨产前束缚应激对子代大鼠海马NG表达的影响。连续7d对孕晚期大鼠进行束缚应激,建立产前束缚应激模型,分为对照雌、雄组,应激雌、雄组。采用免疫组化方法观察NG在产前束缚应激子代大鼠海马不同亚区的分布特点;采用蛋白免疫印迹方法检测产前束缚应激子代大鼠海马NG蛋白的表达。结果显示：各组子代大鼠海马各区均有NG蛋白表达,CA1和CA3区表达高于齿状回（dentate gyrus,DG）;应激组雌、雄子代大鼠海马NG的表达明显低于对照组（P〈0．01）,应激组雌性子代比雄性子代减少更显著,对照组雌、雄子代之间无差异。免疫组化与蛋白免疫印迹方法所得结果一致。上述结果表明,NG在产前束缚应激子代大鼠海马表达降低,并且雌性比雄性降低明显,NG对产前束缚应激子代大鼠有差异性调制,NG表达减少可能与产前束缚应激子代大鼠学习记忆能力下降有关。     

Gender differences in oxidative stress in spinal cord of rats submitted to repeated restraint stress

Behavioral and neurochemical gender-specific effects have been observed following repeated stress. The aim of this study is to verify the effects of repeated restraint stress on free radical production (evaluated by DCF test), lipoperoxidation (evaluated by TBARS levels), and total antioxidant reactivity (TAR) in the spinal cord of male and female rats. Results demonstrate no effect on lipoperoxidation; chronic stress decreased TAR both in male and female spinal cord. In addition, gender differences were observed both in TAR and in the production of free radicals, both being increased in females. These results may be relevant to the gender-specific differences observed after exposure to repeated stress.     

Pharmacology of descending control systems

In the cat there is no convincing evidence that a particular compound mediates a supraspinal control of spinal transmission of nociceptive information. There is good evidence that opioid peptides are released segmentally in response to nociceptive input to the spinal cord and that this acts to inhibit motoneurons and to reduce transmission of nociceptive information to supraspinal areas. In the cat there is no evidence that stimulation at supraspinal sites producing analgesia results in a spinal release of opioid peptides. In the rat evidence for the latter has been obtained but there are no data from other species. Tonically present supraspinal inhibition of spinal transmission of nociceptive information in the cat does not involve opioid peptides. Indirect evidence favours a role for 5-hydroxytryptamine and noradrenaline in supraspinal control of spinal processing of nociceptive transmission. Peripheral antagonists of 5-HT have reduced spinal inhibition from stimulation at supraspinal sites but the site of action is unknown. Progress with noradrenaline involvement has been hindered by lack of a suitable antagonist. Although the amino acids, glycine and GABA are involved in segmental inhibition of transmission of nociceptive information, no convincing evidence has indicated their involvement in supraspinal controls.     

Proteasomal Activity in Synaptosomes Obtained from the Cerebral Structures of Rats Subjected to Long-Lasting Immobilization Stress

We studied the proteasomal activity in synaptosomes obtained from tissues of the cerebral cortex, cerebellum, and hippocampus, as well as in the cytoplasm of cells of these brain structures, of rats subjected to long-lasting immobilization stress. It was demonstrated that the chymotrypsin-like activity of proteasomes in synaptosomes of the cerebral cortex and hippocampus of stressed animals was significantly higher (380 and 560%, respectively) as compared with that observed in control rats. The chymotrypsin-like and peptidylglutamyl peptide hydrolase activities of proteasomes in the cytoplasm of cortical cells under stress conditions also increased (210 and 180%, respectively). These data show that the activity of a multicatalytic proteolytic complex is sharply increased in synaptic terminals of cells of the cerebral cortex and hippocampus of stressed animals. The above complex plays a crucial role in the utilization of short-lived proteins whose molecules form receptors and ion channels; the amount of such proteins is especially great in synaptic terminals.     

Neuroprotective Effect of Etomidate in the Central Nervous System of Streptozotocin-Induced Diabetic Rats

It is well known that hyperglycaemia due to diabetes mellitus leads to oxidative stress in the central nervous system. Oxidative stress plays important role in the pathogenesis of neurodegenerative changes. In the present study we investigated the possible neuroprotective effect of etomidate against streptozotocin-induced (STZ-induced) hyperglycaemia in the rat brain and spinal cord. A total of 40 rats were used in this study. Rats were divided into four groups: sham-control, diabetic, diabetic-etomidate treated and vehicle for etomidate treatment group. Diabetes mellitus was induced by a single injection of streptozotocin (60 mg/kg body weight). Three days after streptoztocin injection, etomidate (2 mg/kg) was injected intraperitoneally for etomidate group and lipid emulsion (10%) for vehicle group was injected with corresponding amount intraperitoneally every day for 6 weeks. Six weeks after streptozotocin injection, seven rats from each group were killed and brain, brain stem and cervical spinal cord were removed. The hippocampus, cortex, cerebellum, brain stem and spinal cord were dissected for the biochemical analysis (the level of malondialdehyde [MDA], total nitrite, reduced glutathione [GSH], and xanthine oxidase [XO] activity). STZ-induced diabetes resulted in significantly elevation of MDA, XO and nitrite levels in the hippocampus, cortex, cerebellum, brain stem and spinal cord of the rats (P < 0.05) while etomidate treatment provided significantly lower values (P < 0.05). This study demonstrated that etomidate have neuroprotective effect on the neuronal tissue against the diabetic oxidative damage.     

Effect of Development of Habituation to Restraint Stress on Hypothalamic Noradrenaline Release and Adrenocorticotropin Secretion

Abstract: The effect of repeated stress has been studied on noradrenaline release in the hypothalamic paraventricular nucleus and on adrenocorticotropin levels. Rats were stressed by 20-min immobilization once a day for 5 days. On day 6 they were exposed to the same stress or to a different one (ether vapors for 2 min). Immobilization and ether stress increased noradrenaline release in naive rats (271 ± 43 and 197 ± 9%, respectively) and raised adrenocorticotropin levels, showing activation of the hypothalamus-pituitary axis. Repeated daily restraint did not modify basal noradrenaline or adrenocorticotropin levels. The further immobilization session on day 6 did not change noradrenaline levels at any observation time (20–120 min). The adrenocorticotropin response was still present, although significantly reduced. In repeatedly restrained rats, exposure to ether vapors induced a maximal increase in noradrenaline level similar to that observed in naive rats, although prolonged. In these rats the adrenocorticotropin response did not differ from that in acutely stressed rats. These results suggest that habituation may develop to a stressful stimulus leading to suppression of the hypothalamic noradrenergic response and that this phenomenon is stress specific. Moreover, modifications of noradrenaline release in the paraventricular nucleus are not solely responsible for the adrenocorticotropin response during stress, suggesting that other pathways and/or neurotransmitters are involved too.     

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.     

Role of hypothalamic inputs in maintaining pituitary-adrenal responsiveness in repeated restraint

The role of hypothalamic structures in the regulation of chronic stress responses was studied by lesioning the mediobasal hypothalamus or the paraventricular nucleus of hypothalamus (PVH). Rats were acutely (60 min) and/or repeatedly (for 7 days) restrained. In controls, a single restraint elevated the plasma adrenocorticotropin (ACTH), corticosterone, and prolactin levels. Repeated restraint produced all signs of chronic stress, including decreased body and thymus weights, increased adrenal weight, basal corticosterone levels, and proopiomelanocortin (POMC) mRNA expression in the anterior pituitary. Some adaptation to repeated restraint of the ACTH response, but not of other hormonal responses, was seen. Lesioning of the mediobasal hypothalamus abolished the hormonal response and POMC mRNA activation to acute and/or repeated restraint, suggesting that the hypothalamo-pituitary-adrenal axis activation during repeated restraint is centrally driven. PVH lesion inhibited the ACTH and corticosterone rise to the first restraint by approximately 50%. In repeatedly restrained rats with PVH lesion, the ACTH response to the last restraint was reduced almost to basal control levels, and the elevation of POMC mRNA level was prevented. PVH seems to be important for the repeated restraint-induced ACTH and POMC mRNA stimulation, but it appears to partially mediate other restraint-induced hormonal changes.