Cortisol response and immune-related effects of Atlantic salmon (Salmo salar Linnaeus) subjected to short- and long-term stress

It is generally considered that stress causes decreased immune function in fish. In this study we examined in Atlantic salmon (Salmo salar Linnaeus) the effects of both short- (a single 15s out of water) and long-term (4 weeks of daily handling 15s out of water) stress on plasma cortisol (free and total) and glucose levels, expression of interleukin-1beta (IL-1beta) and survival of head kidney (HK) macrophages under culture with Aeromonas salmonicida. In the short-term study, samples were collected prior to the application of the stressor, and at 1, 3, 6, 12 and 24h post stress. Free and total plasma cortisol levels and the percentage of free cortisol increased significantly in the stressed group at 1 and 3h post stress. Plasma glucose levels were significantly higher than those of control fish at 1, 3 and 6h post stress. Constitutive expression of IL-1beta in macrophages isolated from head kidneys in stressed fish was significantly higher at 1 and 3h post stress. However, lipopolysaccharide (LPS) stimulated expression of IL-1beta in HK macrophages, exhibited significantly higher fold increases in unstressed fish compared to stressed fish. In the long-term study, with the exception of an increase in plasma glucose levels at 1 week, there were no significant differences in stress parameters between groups. There was a significantly higher constitutive IL-1beta expression in macrophages isolated from stressed fish over the first 2 weeks. At weeks 1, 2 and 3 the magnitude of IL-1beta response of isolated HK macrophages to LPS stimulation was reduced in >90% of the stressed fish. At 4 weeks there was no significant difference in inducible IL-1beta expression between the groups. Macrophages isolated from stressed fish also showed significantly decreased survival when exposed to A. salmonicida. This study shows a clear pattern from repeated handling stress, whereby effects on immune cells begin with increased constitutive expression of IL-1beta, followed by decreased stimulation of leucocytes by extracellular antigen, and finally decreased leukocyte survival when exposed to A. salmonicida. The implications of these changes in the immune system will be discussed with respect to the use of classical indicators of stress to predict possible effects on the immune system of fish.     

Hepatic iron content corresponds with the susceptibility of lymphocytes to oxidative stress in neonatal pigs

The pig is born with limited iron supplies. If not supplemented, piglets dramatically loose their body iron stores during the first few days of postnatal life. The aim of this study was to investigate the influence of hepatic iron content on susceptibility of blood cells to oxidative stress. Four 1-day-old and three 7-days-old animals were used in this study. The alkaline version of the comet assay was used to measure DNA damage. As expected, iron body stores of non-supplemented animals decrease significantly during the first 4 days of life. However, no difference in background DNA damage was found between untreated lymphocytes from these two groups of animals, despite the difference in their hepatic iron content. Interestingly, DNA damage induced by H2O2 and X-radiation in lymphocytes taken from 1-day-old piglets was significantly higher than in those taken from 7-days-old animals. In contrast, NaOCl or tert-butyl-hydroxide also induced significant amounts of DNA damage, but no differences between the two groups of piglets were found. Our data show that decreased hepatic iron content corresponds with decreased susceptibility of blood lymphocytes to oxidative stressors.     

Prolonged effect of stress (water and food deprivation) at weaning or in adult age on the triiodothyronine and histamine content of immune cells.

We used two days of total water and food deprivation as stress for female rats at weaning (three weeks old) and at adult age (two and a half months old). Triiodothyronine (T3) and histamine content of immune cells (lymphocytes, mast cells and monocyte-macrophage-granulocyte group in peritoneal fluid; lymphocytes, granulocytes and monocytes in blood; and lymphocytes in thymus) were studied three weeks after stress application using specific antibodies for flow cytometry and confocal microscopy. The stress at weaning increased T3 content of thymus lymphocytes. In case of adult T3, there was a cell type independent significant effect of stress, decreasing values in peritoneal fluid and slightly increasing effect in the blood. Histamine content of granulocytes was also significantly elevated. The experiments demonstrate that not only fetal or neonatal stress has long-lasting consequences, but also stress events in later periods of life in cells (organs) that are continuously differentiating. We will go on to discuss the importance of T3 and histamine in connection with stress and immunity.     

Restraint stress and anti-tumor immune response in mice

Psychological stress modulates the immune system through the hypothalamic-pituitary-adrenal axis, the sympatho-adrenomedullary axis and the opioid system. According to literature data, restraint stress increases the immune cell apoptosis, decreases the spleen and thymus cell content, the natural killer (NK) activity in the spleen, and it compromises the anti-tumor immune response in mice. We immobilized mice in two consecutive nights, and then determined the cell number, apoptosis, NK cell content, NK activity and the level of cytokine mRNAs (TNF-beta, TNF-alpha, IL-4, IL-5, IL-1alpha, IFN-gamma, IL-2, IL-6, IL-1beta and IL-3) in the thymus and spleen. No consistent changes were detected in any of the immune parameters either in C57Bl/6 or in DBA/2 mice. Stressed or control B6 mice were injected with B16 melanoma cells immediately after the immobilization or one week later. No significant differences were found in the growth of primary tumors and lung metastases in stressed and control animals. Taken together, our mice, kept in a general-purpose non-SPF animal house, seemed to be refractory to the stress-induced immunomodulation. Our interpretation is that stress-induced immunomodulation can occur only in mice isolated from any background stressors, or rather natural stimuli, throughout their life.     

Effect of repeated stress and administration of phenobarbital on the lymphoid tissue and on protein metabolism in the lymphocytes of infant and adult rats

Further study of the response to chronic stress stimulation in the early postnatal phase showed that the i.p. injection of physiological saline (stress stimulation) induced lymphopenia, a 50% decrease in the incorporation of 3H-leucine into isolated lymphocytes and a decrease in the weight of the thymus in 7-day-old male rats. No such changes were observed in adult animals. If repeated doses of phenobarbital were administered to stressed young rats, however, lymphopenia did not occur and the rate of the incorporation of 3H-leucine into isolated lymphocytes was not different from the control value; the protein content of the lymphocytes was significantly raised, however. In adult animals, phenobarbital increased the rate of incorporation of 3H-leucine into the lymphocytes. The repeated administration of phenobarbital reduced the weight of the thymus in both young and adult animals, but a decrease in spleen weight was recorded only in the young animals. A single i.p. injection of ACTH or dexamethasone caused lymphopenia and slowed down the incorporation of 3H-leucine into the lymphocytes of both young and adult animals. The results show that the striking decrease observed in the rate of the liver metabolism of corticosterone in suckling young rats not injured by repeated stress stimulation is accompanied by significant changes in the lymphoid tissue.     

Hepatic iron content corresponds with the susceptibility of lymphocytes to oxidative stress in neonatal pigs

The pig is born with limited iron supplies. If not supplemented, piglets dramatically loose their body iron stores during the first few days of postnatal life. The aim of this study was to investigate the influence of hepatic iron content on susceptibility of blood cells to oxidative stress. Four 1-day-old and three 7-days-old animals were used in this study. The alkaline version of the comet assay was used to measure DNA damage. As expected, iron body stores of non-supplemented animals decrease significantly during the first 4 days of life. However, no difference in background DNA damage was found between untreated lymphocytes from these two groups of animals, despite the difference in their hepatic iron content. Interestingly, DNA damage induced by H₂O₂ and X-radiation in lymphocytes taken from 1-day-old piglets was significantly higher than in those taken from 7-days-old animals. In contrast, NaOCl or tert-butyl-hydroxide also induced significant amounts of DNA damage, but no differences between the two groups of piglets were found. Our data show that decreased hepatic iron content corresponds with decreased susceptibility of blood lymphocytes to oxidative stressors.     

Changes in the ascorbic acid levels of peritoneal lymphocytes and macrophages of mice with endotoxin-induced oxidative stress.

Ascorbic acid (AA) is an important cytoplasmic antioxidant that mice synthesize in the liver, the intracellular levels of which decrease in an oxidative stress situation such as endotoxic shock. The present work deals with the changes in AA levels, that modulate the immune function, in the two main immune cells, namely macrophages and lymphocytes, from female BALB/c mice suffering endotoxic shock caused by intraperitoneal injection of Escherichia coli lipopolysaccharide (LPS) (100 mg/kg). The intake by cells of this antioxidant present in vitro at different concentrations was also studied. The animals show an oxidative stress, standardized in previous studies, that causes mortality at 30 h after LPS injection. The cells were obtained from the peritoneum at 2, 4, 12 and 24 h after LPS or PBS (control) injections and were incubated without or with AA at 0.01, 0.1 and 1 mM for 10, 30, 60, 120 or 180 min. The hepatic AA levels were also studied at 0, 2, 4, 12 and 24 h after LPS injection. The peritoneal cells obtained from animals injected with LPS showed increased AA levels in relation to the control cells at all times after LPS injection, with maximal effect at 12h. The AA levels decreased after this time, in agreement with changes in the AA hepatic levels. The increase was due to the AA of lymphocytes since macrophages showed a decrease in AA at different times after LPS injection. Both cells showed an increase in the intracellular levels of AA when this antioxidant was added in vitro. This takes place mainly at 30-60 min of incubation in cells from controls and at 10 min in cells from treated mice 12-24 h after LPS injection. The incorporation decreased at these times of endotoxic shock, a few hours before death. In all cases AA levels were higher in lymphocytes than in macrophages, and 1 mM was the most effective concentration. These results suggest that the immune cells need appropriate levels of antioxidants, such as AA, under oxidative stress conditions, and that while lymphocytes take and accumulate AA, macrophages use it.     

Prolonged effect of endorphin treatment during pregnancy in the rat on the histamine content of immune cells of F1 and F2 offspring generations

Female rats were treated with beta-endorphin on the 19th day of pregnancy and the histamine content of immune cells (blood lymphocytes; peritoneal lymphocytes, monocyte-macrophage-granulocyte group, mast cells; thymic lymphocytes) of the 7-week-old progenies (F1 generation) was studied using a flow-cytometric immunocytochemical technique. In an other group, female F1 progenies of endorphin-treated mothers were mated with control males and the F2 generation was monitored for histamine content similar to the F1. In the F1 generation each cell type, except peritoneal and blood lymphocytes, contained significantly more histamine than the control cells. In the F2 generation only mast cells contained significantly more histamine relative to the appropriate control. This means that the effect of endorphin (hormonal) imprinting is transmitted transgenerationally, but with decreasing intensity however. Mast cells retained the effect of imprinting for longer than the other cells. The results are compared with the levels of serotonin in similarly treated animals, studied in earlier experiments. As the endorphin level can be elevated during pregnancy (by pain, traumatization, or other stress conditions) this can the set biogenic amine content of adult immune cells.     

Effect of the N-terminal fragment of substance P1-4 on the somatic manifestations of the stress reaction and on the catecholamine content of the adrenals in rats

The antistress affect of the substance P1-4 N-terminal fragment (ARG-Pro-Lys-Pro, 100 mkg/kg, i.p.) has been studied on the model of immobilization stress in rats. It was ascertained that the preparation of protective effect is revealed to the greatest extent on the exhaustion stage (48 h of immobilization), which served to prevent the lymphoid organs mass reduction and ulcer development and also accounted for greater adrenaline and noradrenaline content preservation in tissues and chromaffin cells of adrenal glands in stressed animals.     

Relationship between occupied form of nuclear estrogen receptor and cytosolic progesterone receptor or DNA synthesis in uteri of estradiol implanted rats

The effect of 17 beta-estradiol (E2) implantation on the cytosolic progesterone receptor (RcP), DNA and occupied form of nuclear estrogen receptor (o- Rn) content in the uterus of ovariectomized adult rats, is described. Animals were implanted with oil or E2-oil solution in Silastic capsules. The latter group animals were divided into two subgroups: in subgroup (a), capsules remained in situ until decapitation time. In subgroup (b) they were removed 48 h after implantation. The E2 implantation caused a significant increase in uterine weight, RcP and o-Rn content 48 h later. However, the DNA content increased significantly only after 72 h, but there was no significant difference in the t-Rn concentration in relation to the non-estrogenized animals. In subgroup (a) animals, these values remained unchanged until 96 h. In subgroup (b), the removal of E2 implants 48 h later caused an almost complete return to the values before the E2 implantation in terms of uterine weight, RcP and o-Rn content. However the DNA concentration remained higher and the t-Rn level was lower than those values that were obtained for the non- estrogenized rats. These results suggest that the RcP and DNA synthesis induced by E2 would be connected to the level of o-Rn, although a closer dependency over time seems to exist between the o-Rn and RcP levels than between the o-Rn and DNA concentrations.     

Total leukocytes, T cell subpopulation and natural killer (NK) cell activity in rats exposed to restraint stress

Rats were stressed by immobilization for 3 hrs daily for 11 days and either sacrificed immediately after the last stress session (chronic stress group) or allowed to recover for 12 days and then sacrificed (recovery group). After 11 days of stress, leukocytes and lymphocytes were significantly decreased and neutrophils and large granular lymphocytes were markedly increased. The number of total, helper and suppressor T cells was significantly decreased but the percentage of T cells remained unchanged. Natural Killer (NK) cell activity was unaffected. After a 12 day recovery period from stress, the number of leukocytes returned to normal but the percentage of neutrophils was now below baseline whereas the percentages of lymphocytes and large granular lymphocytes had increased significantly. The number and percentage of total T cells and helper T cells was enhanced and NK cell activity tended to be increased. Thus, chronic stress as well as recovery from stress can affect individual components of the cellular immune system quite selectively and differently. In addition, a comparison of the effects of stress seen in this study with healthy rats with those seen under identical conditions in tumor bearing rats shows that stress or recovery from stress can affect the immune system differently in healthy or tumor bearing animals.     

Effect of prenatal stress on the hormonal response to acute and chronic stress and on immune parameters in the offspring

The effect of prenatal stress on the time course of the corticosterone response to acute and chronic stress and on hematological and immunological parameters in the offspring were analized in the present study. Pregnant Sprague-Dawley rats were stressed daily for 2 hours during the last week of gestation, and female and male off-spring were studied during adulthood. Corticosterone response to acute immobilization stress was not significantly different in either control or prenatally stressed rats. However, after 10 days of immobilization stress the corticosterone response completely disappeared in the control animals but not in the prenatally stressed group: high levels of corticosterone were found during the first hour of stress, although they were lower than those found in acutely stressed rats. Adrenal hypertrophy in response to prenatal stress was observed in females but not in male offspring, and chronic stress only increased adrenal weights in the male control group. Prenatal stress decreased the total peripheral leukocyte count, altered its diferential count decreasing lymphocytes and increasing neutrophil and eosinhophil counts, and significantly reduced the percentage of peripheral lymphocyte T CD8+ subset in male offspring. Chronic stress also reduced the percentage of the peripheral T CD8+ lymphocyte subset in the control group but not in the prenatally stressed group. These results suggest that the exposure to stress during pregnancy alters the adaptative response of the hypothalamus-pituitary-adrenocortical axis to chronic stress and presumably the immune competence in the offspring.     

Mechanism of tolerance to VI-antigen of Salmonella typhi induced by cyclophosphane

High dose Vi-antigen treatment and injection of cyclophosphamide 46 to 48 hours later induced in mice a state of immunological unresponsiveness remaining stable in adoptive transfer. Only low amounts of the antigen were revealed in the blood and spleen of tolerant animals 2 to 3 weeks after the tolerogenic treatment. No T-suppressors were found in the spleen of tolerant mice--the cells of tolerant mice failed to suppress the immune response of normal lymphocytes when transferred together to the irradiated recipients, or to induce tolerance in normal mice. Normal spleen cells restored partially the immune responsiveness in tolerant animals. The results obtained suggest that cyclophosphamide tolerance was due to deletion or the long-term inactivation of the immunocompetent cells.     

Prolonged effect of stress at weaning on the brain serotonin metabolism and sexuality of female rats.

Weanling female rats were stressed (by water and food deprivation for two days) and three months later the following indexes were studied: 5-HT and 5-HIAA levels in five brain regions, blood plasma and cerebrospinal fluid (CSF), sexual activity and nocistatin level of the plasma and CSF. The 5-HIAA content of hypothalamus and brainstem was significantly decreased (in the brainstem with one third) and in the striatum significantly increased. Plasma nocistatin level was significantly increased. Meyerson index and lordosis quotient were similar to control, but the estrus frequency almost doubled in the stressed animals. Much more defense reactions were observed in the stressed females during trials of mating. The results demonstrate that, 1) the perinatal period is not only sensitive to the remote-effects of stress but later could also be stress-sensitive critical periods, and 2) the continuously differentiating (e.g. bone marrow) cells are sensitive to late imprinting by stress, as well as to the brain and the sexual system.     

Changes in the ascorbic acid levels of peritoneal lymphocytes and macrophages of mice with endotoxin-induced oxidative stress

Ascorbic acid (AA) is an important cytoplasmic antioxidant that mice synthesize in the liver, the intracellular levels of which decrease in an oxidative stress situation such as endotoxic shock. The present work deals with the changes in AA levels, that modulate the immune function, in the two main immune cells, namely macrophages and lymphocytes, from female BALB/c mice suffering endotoxic shock caused by intraperitoneal injection of Escherichia coli lipopolysaccharide (LPS) (100 mg/kg). The intake by cells of this antioxidant present in vitro at different concentrations was also studied. The animals show an oxidative stress, standardized in previous studies, that causes mortality at 30h after LPS injection. The cells were obtained from the peritoneum at 2, 4, 12 and 24h after LPS or PBS (control) injections and were incubated without or with AA at 0.01, 0.1 and 1 mM for 10, 30, 60, 120 or 180 min. The hepatic AA levels were also studied at 0, 2, 4, 12 and 24h after LPS injection. The peritoneal cells obtained from animals injected with LPS showed increased AA levels in relation to the control cells at all times after LPS injection, with maximal effect at 12h. The AA levels decreased after this time, in agreement with changes in the AA hepatic levels. The increase was due to the AA of lymphocytes since macrophages showed a decrease in AA at different times after LPS injection. Both cells showed an increase in the intracellular levels of AA when this antioxidant was added in vitro. This takes place mainly at 30–60 min of incubation in cells from controls and at 10 min in cells from treated mice 12–24 h after LPS injection. The incorporation decreased at these times of endotoxic shock, a few hours before death. In all cases AA levels were higher in lymphocytes than in macrophages, and 1 mM was the most effective concentration. These results suggest that the immune cells need appropriate levels of antioxidants, such as AA, under oxidative stress conditions, and that while lymphocytes take and accumulate AA, macrophages use it.     

Effect of chronic psychological stress on insulin release from rat isolated pancreatic islets

Despite documented studies, the exact role of stress on diabetes is still unclear. The present study investigates the effect of chronic psychological stress on insulin release from isolated rat pancreatic islets. Male Wistar rats were divided into two groups of control and stressed (n=8/group). The animals of the stressed group were exposed to restraint stressors (1 h twice daily) for 15 or 30 consecutive days. At the beginning and end of the experimental periods, the animals were weighed and blood samples taken to determine the fasting plasma levels of glucose, insulin and corticosterone. On the following day the pancreatic islets of 5/group of the above animals were isolated and the static release of insulin in the presence of different glucose concentrations (2.8, 5.6, 8.3, 16.7 mM) was assessed. The results showed that in the stressed group, fasting plasma glucose levels were increased significantly on the 15th day as compared to the control group. However there was no significant increase on the 30th day. Fasting plasma insulin was significantly decreased on the 15th and 30th days of the experiment in the stressed group. Stressed rats showed significantly higher fasting plasma corticosterone levels, only on the 15th day, as compared to the control rats. In response to increasing concentrations of glucose, insulin release from islets of the stressed group was increased significantly on the 30th day of the experiment as compared to the control group. We conclude that chronic psychological stress could increase responsiveness of pancreatic beta cells to glucose, in vitro, and thus, low insulin levels of the stressed animals, in vivo, may be due to reason(s) other than the reduction of insulin releasing capacity of pancreatic beta cells.     

Lipids, carbohydrates and amino acids exuded from the axenic roots of rape seedlings exposed to water-deficit stress

Abstract. Rape ( Brassica napus [L.]) plants were cultivated for 25 d with axenic roots in a growth system with quartz sand and circulating nutrient solution. After a water stress period of 24h. fresh nutrient solution was added and root exudates were collected 3d later. The stress did not cause significant differences in the weights between the stressed and the control plants. The stressed plants tended to exude more soluble organic carbon than the control plants. This was not reflected in the amounts of low molecular weight carbohydrates exuded. A significantly lower proportion of the soluble organic carbon exuded by the stressed plants was made up of amino acids in comparison with the control plants, 7% and 28% respectively. Exuded hydrophobic substances could be recovered from the sand particles. The stressed plants exuded more sterols than the control plants, and an increased number of polar lipid types. The exudates of the stressed and control plants also differed regarding their fatty acid composition.     

Effects of diazepam and stress on lung inflammatory response in OVA-sensitized rats

The influence of stress and diazepam treatment on airway inflammation was investigated in ovalbumin (OVA)-sensitized rats. Animals were injected with OVA plus aluminum hydroxide intraperitoneally (day 0) and boosted with OVA subcutaneously (day 7). From the first to 13th day after sensitization, rats were treated with diazepam, and 1 h later they were placed in a shuttle box where they received 50 mild escapable foot shocks/day preceded by a sound signal (S). Response during the warning (S) canceled shock delivery and terminated the S. On day 14, rats were submitted to a single session of 50 inescapable foot shocks preceded by S and then were challenged with OVA. High levels of stress were detected in shocked animals, manifested as ultrasonic vocalizations. Morphometric analysis of stressed animals revealed a significant increase in both edema and lymphomononucleated cells in airways compared with controls. Diazepam treatment reduced edema in stressed and nonstressed rats. No differences were found in polymorphonucleated cell infiltration. Diazepam treatment reduced lymphomononucleated cell infiltration in stressed animals. These data suggest that stress and diazepam treatment play relevant roles in edema and lymphomononucleated airway inflammation in OVA-sensitized rats.     

A chronic stress that impairs reactivity in rats also decreases dopaminergic transmission in the nucleus accumbens: a microdialysis study

Chronic stress induces in rats a decreased reactivity toward noxious stimuli (escape deficit), which can be reverted by antidepressant treatments. The present study reports that this condition of behavioral deficit is accompanied by a decreased level of extracellular dopamine in the nucleus accumbens shell. To assess whether this finding was the result of a decreased release or of an enhanced removal of dopamine, we acutely administered cocaine, and 2 h later d-amphetamine, to stressed and control rats. The increases in dopamine output observed in stressed animals after cocaine administration were significantly lower than those observed in control rats; whereas the total amount of dopamine released after d-amphetamine administration was similar in both groups of rats. These data suggest a reduced activity of dopaminergic neurons as the possible mechanism underlying dopamine basal level reduction in stressed animals. It is interesting that the stress group showed a locomotor response to cocaine not different from control rats, thus suggesting a condition of sensitization to dopamine receptor stimulation. Imipramine administered daily concomitantly with stress exposure completely reverted the escape deficit condition of chronically stressed rats. Moreover, stressed rats treated with imipramine showed basal and cocaine stimulated levels of extraneuronal dopamine similar to those observed in control animals.     

束缚应激大鼠血清淋巴细胞转化抑制因子的研究

为研究应激对淋巴细胞转化的影响,将 SD 大鼠四肢束缚于支架上,仰卧位,室温(20℃)下维持20h,对照组留置原饲养笼中,不予惊动。然后在乙醚轻麻下穿刺心脏取血,肝素化后密度梯度离心分离淋巴细胞,或待凝后分离血清。结果表明,应激大鼠外周血淋巴细胞对刀豆素(Con A)诱导的转化反应明显下降(p<0.01,n=8,ANOVA),而且应激大鼠血清可明显抑制正常小鼠淋巴细胞转化,这提示应激大鼠血清中可能存在某种具有抑制淋巴细胞转化的活性物质。进一步的分析实验表明,这种血清经加热56℃(30min),30%甲醇或透析(透析袋孔径阻滞分子量为6000)处理,抑制活性均不受影响;但经加热100℃(3min),80%甲醇或胰蛋白酶(64/μg/ml)处理,抑制活性丧失。提示这种抑制活性物质很可能是一类蛋白质。