Early-Life Maternal Deprivation Predicts Stronger Sickness Behaviour and Reduced Immune Responses to Acute Endotoxaemia in a Pig Model.

Early-life adversity may have programming effects on neuroendocrine and immune adaptation mechanisms in humans and socially living animals. Using a pig model, we investigated the effect of daily 2-h maternal and littermate deprivation from postnatal days 2-15, either alone (DA) or in a group of littermates (DG) on the neuroendocrine, immunological and behavioural responses of piglets challenged with the bacterial endotoxin lipopolysaccharide (LPS) on day 42. LPS increased plasma concentrations of cortisol, tumour necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and interleukin-10 (IL-10) and induced typical signs of sickness in all piglets. DA+DG piglets showed stronger signs of sickness compared to control (C) piglets. Plasma TNF-α concentrations were significantly lower in DA+DG males. In addition, the TNF-α/IL-10 ratio was significantly lower in DA than in DG and C males. Gene expression analyses showed lower hypothalamic TNF-α mRNA expression and diminished mRNA expression of the mineralocorticoid receptor (MR) and IL-10 in the amygdala of DA+DG piglets in response to LPS. Interestingly, males showed a higher MR- and a lower IL-10 mRNA expression in the amygdala than females. The present data suggest that repeated maternal deprivation during early life may alter neuroendocrine and immune responses to acute endotoxaemia in a sex-specific manner.

Psychosocial stress sensitizes neuroendocrine and inflammatory responses to Escherichia coli challenge in domestic piglets.

Exposure to psychosocial stress can have a profound impact on immune reactivity and health mediated by hypothalamic-pituitaryadrenal (HPA) axis activation. However, current knowledge regarding the mechanisms involved in cross-sensitization between stress and the immune system is limited. Here, we investigated the effects of a single social isolation followed by repeated oral Escherichia coli (E. coli) applications on cortisol, tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), haptoglobin and C-reactive protein (CRP) concentrations in the blood; on clinical signs of disease; and on mRNA expression of the glucocorticoid receptor (GR), mineralocorticoid receptor (MR), 11ß-hydroxysteroid dehydrogenase 1 and 2 (11ß-HSD1 and 11ß-HSD2), TNF-α and IL-6 in the hypothalamus, prefrontal cortex (PFC) and spleen of 7-, 21- and 35-day-old piglets. Additionally, the protein levels of splenic TNF-α and IL-6 were analyzed. Non-isolated, E. coli-challenged piglets served as a control. Social isolation for 4 h induced a rise in the plasma cortisol concentrations immediately after social treatment and after repeated E. coli applications in isolated compared to non-isolated piglets. The circulating TNF-α concentration was not affected by social treatment. Furthermore, previously isolated piglets showed a higher frequency of signs of disease in response to E. coli challenge than non-isolated piglets, while the haptoglobin and CRP concentrations did not significantly differ between social treatments. In the brain, 11ß-HSD1, 11ß-HSD2 and IL-6 mRNA expression in the hypothalamus and GR, and 11ß-HSD1 and 11ß-HSD2 mRNA expression in the PFC were higher in isolated, E. coli-challenged piglets than in the corresponding controls. Moreover, isolated piglets also displayed higher MR, 11ß-HSD1 and IL-6 mRNA expression levels and TNF-α concentrations in the spleen. The stress-induced alterations in the hypothalamus and spleen were particularly pronounced in younger piglets. The present findings may contribute to a better understanding of the complex interplay between early psychological stress and an increased risk of disease and might also have implications on aspects of the health and welfare of farm animals and humans.

Quercetin Feeding in Newborn Dairy Calves Cannot Compensate Colostrum Deprivation: Study on Metabolic, Antioxidative and Inflammatory Traits.

Immaturity of the neonatal immune system is causative for high morbidity in calves and colostrum intake is crucial for acquiring passive immunity. Pathogenesis is promoted by reactive oxygen species accumulating at birth if counter-regulation is inadequate. The flavonol quercetin exerts antioxidative and anti-inflammatory effects that may enhance neonatal health. The aim of this work was to study effects of quercetin feeding on metabolic, antioxidative and inflammatory parameters in neonatal calves to investigate whether quercetin could compensate for insufficient colostrum supply. Twenty-eight newborn calves were assigned to two dietary groups fed colostrum or milk-based formula on day 1 and 2 and milk replacer thereafter. From day 2 onwards, 7 calves per diet group were additionally fed quercetin aglycone (50 mg/(kg body weight × day)). Blood samples were taken repeatedly to measure plasma concentrations of flavonols, glucose, lactate, total protein, albumin, urea, non-esterified fatty acids, triglycerides, cholesterol, insulin, glucagon, cortisol, immunoglobulins, fibrinogen, haptoglobin and serum amyloid A. Trolox equivalent antioxidative capacity, ferric reducing ability of plasma, thiobarbituric acid reactive species and F2-isoprostanes were analyzed to evaluate plasma antioxidative status. Expression of tumor necrosis factor, interleukin-1α, interleukin-1ß, serum amyloid A, haptoglobin, fibrinogen, C-reactive protein, catalase, glutathione peroxidase and superoxide dismutase mRNA were measured in liver tissue on day 8. Plasma flavonol concentrations were detectable only after quercetin-feeding without differences between colostrum and formula feeding. Plasma glucose, lactate, total protein, immunoglobulins, triglycerides, cholesterol, trolox equivalent antioxidative capacity and thiobarbituric acid reactive species were higher after colostrum feeding. Body temperature, fecal fluidity and plasma concentrations of cortisol and haptoglobin were higher in formula- than in colostrum-fed groups. Hepatic mRNA expression of tumor necrosis factor was higher after quercetin feeding and expression of C-reactive protein was higher after formula feeding. Data confirm that colostrum improves neonatal health and indicate that quercetin feeding cannot compensate for insufficient colostrum supply.

Social support modulates splenocyte glucocorticoid sensitivity in piglets exposed to social deprivation stress.

There is growing evidence that positive social interactions can attenuate the effects of stressful life experiences. However, little is known about the benefits of social partners on stress responses in farm animals. Therefore, in this study we examined the effects of social support on the endocrine and immune stress responses to a single 4h social deprivation in domestic piglets at 7, 21 or 35days of age. The piglets were socially deprived of their mother and littermates. They were left alone (DA) or in the presence of a familiar (DF) or unfamiliar (DU) age-matched piglet. Non-socially deprived piglets served as a control. DA piglets displayed elevated plasma cortisol levels, higher lipopolysaccharide (LPS)-stimulated proliferation of splenocytes and increased TNF-α and IL-6 production in splenocyte cultures than the control piglets. There were no significant buffering effects of social partners on stress-induced plasma cortisol levels and splenocyte proliferation in response to LPS. However, the presence of an age-matched conspecific diminished the IL-6 production by splenocytes in younger, socially deprived piglets, and it reduced the TNF-α release in the older piglets. Compared to the controls, LPS-stimulated splenocytes from DA piglets were more resistant to the inhibitory effects of cortisol, which was demonstrated by a higher proliferative response and increased production of pro-inflammatory cytokines. The dose-dependent cortisol resistance was attenuated by the presence of a familiar or an unfamiliar conspecific at each of the three age categories. Indeed, in the present study, the familiarity level of the social partners did not seem to play a role in the alleviation of social stress-induced inflammatory activity and splenocyte cortisol resistance. In addition, the buffering effect of social support provided by an age-matched conspecific was more pronounced in older piglets. Conclusively, these findings suggest that social support is an important factor for enhancing piglets' abilities to cope with stressful challenges, and it may be a key approach needed to improve the health and welfare of farm animals.

Activation of indoleamine 2,3-dioxygenase by LPS in a porcine model.

Indoleamine 2,3-dioxygenase (IDO) is a rate-limiting enzyme for the degradation of tryptophan (Trp) along the kynurenine (Kyn) pathway, and its increased activation is associated with immunologic disorders. Because the specific role of IDO activation is not yet completely clear, the aim of the present study was to establish a pig model of IDO activation for further research. The activation of IDO in pigs was induced experimentally by LPS stimulation in vivo and ex vivo. IDO activation was characterized by measuring Trp, Trp metabolites and IDO protein expression in blood, liver, lung, muscle and different brain areas. The results show that the in vivo LPS administration induced increased plasma concentrations of TNF-α and IL-10, a depletion of Trp and an increase of Kyn, indicating an elevated enzymatic activity of IDO. This was supported by an LPS-induced IDO protein expression in blood, liver and lung. The ex vivo LPS stimulation also resulted in increased TNF-α concentrations and an IDO activation, characterized by an increase of Trp metabolites and IDO protein expression. In conclusion, our data emphasize that the LPS stimulation is a suitable model for IDO activation in the domestic pig, which provides a basis for further research on immunoregulatory IDO functions.

Tumour necrosis factor receptor deficiency alters anxiety-like behavioural and neuroendocrine stress responses of mice.

Tumour necrosis factor (TNF)-alpha is known to be involved in anxiety and the regulation of the hypothalamic-pituitary-adrenal axis. To examine the role of its receptors in neuroendocrine immunomodulation, we studied behaviour, corticosterone production and T-cell activation in mice with a C57BL/6J background and deficient for one or both TNF receptors (TNFR1-/-, TNFR2-/-, and TNFR1+2-/-) compared to wildtype C57BL/6J mice with and without psychological stress. Stress was induced by social disruption (SDR), and anxiety-like behaviour was examined using the elevated plus maze (EPM). Anxiety of unstressed TNFR1+2-/- mice was increased compared to C57BL/6J mice as shown by reduced ratios of entries into open arms relatively to total entries. SDR-stressed TNFR1+2-/- mice showed reduced ratios of entries into open arms relatively to total entries, reduced ratios of distances walked in open relatively to distances walked in both arms and reduced time in open arms compared to C57BL/6J mice. Locomotor activity of unstressed and SDR-stressed TNFR1-/- and TNFR2-/- mice was reduced. Serum corticosterone concentrations of control mice do not differ between mouse strains. However, TNFR1+2-/- mice had significantly higher corticosterone concentrations than C57BL/6J mice after SDR. EPM testing significantly increased corticosterone concentrations in all strains. Mitogen-induced activation-marker expression was reduced in TNFR1-/- T-helper cells under control and stress conditions, while activation marker expression of TNFR2-/- and TNFR1+2-/- cells was only slightly affected by stress compared to C57BL/6J T cells. Our study suggests that both TNF receptors contribute to anxiety-like behaviour and corticosterone responses, whereas TNFR1 has a larger impact on T-cell activation.

High and low protein∶ carbohydrate dietary ratios during gestation alter maternal-fetal cortisol regulation in pigs.

Imbalanced maternal nutrition during gestation can cause alterations of the hypothalamic-pituitary-adrenal (HPA) system in offspring. The present study investigated the effects of maternal low- and high-protein diets during gestation in pigs on the maternal-fetal HPA regulation and expression of the glucocorticoid receptor (GR), mineralocorticoid receptor (MR), 11ß-hydroxysteroid dehydrogenase 1 and 2 (11ß-HSD1 and 11ß-HSD2) and c-fos mRNAs in the placenta and fetal brain. Twenty-seven German Landrace sows were fed diets with high (HP, 30%), low (LP, 6.5%) or adequate (AP, 12.1%) protein levels made isoenergetic by varying the carbohydrate levels. On gestational day 94, fetuses were recovered under general anesthesia for the collection of blood, brain and placenta samples. The LP diet in sows increased salivary cortisol levels during gestation compared to the HP and AP sows and caused an increase of placental GR and c-fos mRNA expression. However, the diurnal rhythm of plasma cortisol was disturbed in both LP and HP sows. Total plasma cortisol concentrations in the umbilical cord vessels were elevated in fetuses from HP sows, whereas corticosteroid-binding globulin levels were decreased in LP fetuses. In the hypothalamus, LP fetuses displayed an enhanced mRNA expression of 11ß-HSD1 and a reduced expression of c-fos. Additionally, the 11ß-HSD2 mRNA expression was decreased in both LP and HP fetuses. The present results suggest that both low and high protein∶carbohydrate dietary ratios during gestation may alter the expression of genes encoding key determinants of glucocorticoid hormone action in the fetus with potential long-lasting consequences for stress adaptation and health.

Alterations in anxiety-like behavior following knockout of the uncoupling protein 2 (ucp2) gene in mice.

AIMS: Uncoupling protein 2 (UCP2) is a mitochondrial protein that reduces oxidative stress and has a protective function in chronic inflammatory diseases such as multiple sclerosis, rheumatoid arthritis and systemic lupus erythematosus. UCP2 is strongly expressed in areas implicated in the central regulation of stress and anxiety. Therefore, we compared the neuroendocrine regulation of stress responses, immunity and behavior in UCP2-deficient and wildtype C57BL/6J mice under psychological stress. MAIN METHODS: Stress was induced by social disruption (SDR) and anxiety-like behavior was examined using the elevated plus-maze (EPM). Serum corticosterone was determined by radioimmunoassay and brain neurotransmitter concentrations were analyzed by HPLC of whole brain homogenates. T cell activation and tumor necrosis factor (TNF)-α production of mitogen-activated splenocytes were determined in vitro by flow cytometry staining of CD25, CD69 and CD71 on CD4 cells, and ELISA, respectively. The influence of corticosterone on UCP2 expression of splenocytes was analyzed by Western blot. KEY FINDINGS: At baseline, UCP2-deficient mice were significantly more anxious in the EPM than wildtype mice, and this phenotype was exacerbated after SDR stress. The corticosterone response after SDR+EPM was reduced in UCP2-deficient mice compared to wildtype mice. Corticosterone in turn downregulates UCP2 expression in splenocyte cultures of wildtype mice at physiological concentrations. Dopaminergic and serotonergic turnovers were increased in UCP2-deficient mice after SDR+EPM. While T-helper cell activation-marker expression was reduced, TNF-α production was increased in UCP2-deficient mice after SDR+EPM. SIGNIFICANCE: Our study shows that UCP2 is involved in anxiety-like behavior and modulates neuroendocrine and immune responses after stress.

Altered immunomodulation by glucocorticoids in neonatal pigs exposed to a psychosocial stressor.

Stressful early life experiences can have short- and long-term effects on neuroendocrine and immune mechanisms of adaptation, which are primarily modulated by glucocorticoids. This study aimed to examine how the stress and immune systems interact to cope with psychosocial stress induced by a single social isolation (4 h) in neonatal pigs at 7, 21, or 35 d of age. This social isolation provoked increased plasma ACTH and cortisol concentrations and reduced TNF-α levels but had no significant effect on IL-6 levels. Socially isolated piglets had a higher lipopolysaccharide (LPS)-stimulated proliferative response of peripheral blood mononuclear cells (PBMCs) than controls, whereas concanavalin A (ConA)-induced proliferation was not affected by isolation. A single social isolation also induced a dose-dependent cortisol resistance in ConA- and LPS-stimulated PBMCs compared with controls, which may be an adaptive response in the short term. Moreover, LPS-stimulated cultures from control piglets showed a reduction in cortisol sensitivity with increasing age. Conclusively, these findings provide stress-related measures for the psychophysiological assessment of livestock handling practices but might also have implications for stress and health studies in young animals and humans.

Study of enteral versus parenteral application of the gonadotropin releasing hormone agonist Gonadorelin[6-D-Phe] (D-Phe6-LHRH) on LH secretion in Goettinger miniature pigs.

With respect to the assessment of residue situation and as a part of preclinical trials to determine the biological activities of potential gonadotropin releasing hormone (GnRH) residues in porcine organisms the GnRH agonist Gonadorelin[6-D-Phe] (D-Phe(6)-LHRH) was administered either enterally or intramuscularly (i.m.) to female Goettinger miniature pigs in order to evaluate the GnRH-induced luteinizing hormone (LH) surge. Gilts received an (i) enteral application of 10 mg D-Phe(6)-LHRH via a probang (enteral group, n=7), (ii) i.m. injection of 0.1 mg D-Phe(6)-LHRH (parenteral group, n=5), or (iii) saline injection (control group, n=4). The GnRH and saline applications were repeated every second day with up to seven repetitions. Blood samples were collected via previously fitted jugular catheters immediately before injections, over an 8 h period in 1 h intervals beginning 2 h after injections, and at 24, 26, 28 and 30 h after applications. Enteral application of D-Phe(6)-LHRH induced an LH surge in 23 of 30 treatments. All gilts in the parenteral group exhibited LH release after each D-Phe(6)-LHRH application (P<0.05), whereas no LH surges were observed after saline injection in the control group. A significant (P<0.05) LH rise to mean maximum LH concentrations of 3.25 +/- 0.43 and 3.05 +/- 0.26 ng/ml occurred in both the enteral and parenteral groups, but there was no difference in the time interval after GnRH (2.6 +/- 0.3 vs. 2.3 +/- 0.3 h) and the mean duration of LH peak (6.5 +/- 0.4 and 6.8 +/- 0.3 h) between the treatment groups. In conclusion, (i) enteral application of 10 mg D-Phe(6)-LHRH induced LH release in a physiological range from the pituitary of female minipigs, and (ii) neither an accumulative effect nor a cumulative LH response were found after repeated GnRH application. Furthermore, (iii) in regard to consumer protection and gonadotropin secretion, D-Phe(6)-LHRH residues can be excluded from having long-term effects.

Early social isolation alters behavioral and physiological responses to an endotoxin challenge in piglets.

Psychosocial stress in the form of maternal deprivation and social isolation during early postnatal life induces persistent alterations in behavioral and physiological mechanisms of adaptation. One consequence may be an increased susceptibility to diseases in later life. Therefore, the aim of the present study was to investigate in domestic piglets the effects of a repeated social isolation (2 h daily from day 3 to day 11 of age) on behavioral, endocrine and immune responses to an endotoxin challenge with lipopolysaccharide (LPS) 1 day or 45 days after the isolation period. Peripheral LPS administration caused serious sickness behavior (somnolence, shivering, vomiting) and provoked profound increases in circulating tumor necrosis factor-alpha (TNF-alpha), ACTH and cortisol concentrations. The prior social isolation treatment enhanced signs of sickness and impaired suckling behavior. Early isolated piglets responded to LPS by an increase of shivering on day 12 and by increased vomiting on day 56 compared to controls. Further, there were considerable delays and reductions of time isolated piglets spent suckling on day 12. The repeated isolation stressor diminished TNF-alpha increases after LPS, whereas stress hormone levels were not significantly affected by isolation treatment. Finally, stronger relationships between signs of sickness and physiological measures were revealed in early isolated piglets. The duration of somnolence in isolated piglets was related to changes of cortisol and TNF-alpha concentrations, and the highest impact on duration of shivering was found for changes in cortisol and corticosteroid binding globulin levels. The present results suggest a sustained adaptive sensitization of coping with infection by social stress experience during early development in piglets.

Alteration of reproductive hormone levels in pregnant sows induced by repeated ACTH application and its possible influence on pre- and post-natal hormone secretion of piglets.

Prenatal stress has been seen as a reason for reproductive failures in pig offspring mostly originated or mediated by changed maternal functions. Experiments were conducted in pregnant gilts (n=32) to characterize effects of elevated maternal glucocorticoids on the secretion of reproductive hormones (LH, progesterone) during the 1st (EXP 1), 2nd (EXP 2) and 3rd (EXP 3) trimester of pregnancy (TP). Transiently elevated cortisol release was repeatedly achieved by application of 100 IU adenocorticotropic hormone (ACTH) (Synacthen Depot) six times every second day beginning either on day 28 (EXP 1), day 49 (EXP 2) or day 75 of pregnancy (EXP 3). Glucocorticoid concentrations were examined in umbilical blood vessels of fetuses which mothers were subjected to ACTH at 2nd and 3rd TP (EXP 4). Furthermore, the pituitary function of newborn piglets of EXP 2 was checked by a LH-RH challenge test. In sows, LH concentrations were at low basal level (0.1-0.2 ng/ml) but with pulsatory release pattern during each TP. The number of LH pulses/6 h (LSM +/- SE) of saline treated Controls increased with ongoing pregnancy and decreased to the 3rd TP (1.3 +/- 0.2 in EXP 1 vs. 2.0 +/- 0.1 in EXP 2 vs. 1.4 +/- 0.1 in EXP 3, p<0.05). After ACTH treatment the number of LH pulses left unchanged in Experiments 1 and 2 (1.3 +/- 0.2 and 1.5 +/- 0.1) and decreased in EXP 3 (0.8 +/- 0.2, p<0.05). Differences (p<0.05) were obtained comparing the LH pulse number of ACTH and saline treated sows at the 2nd and 3rd TP. Moreover, areas under the curve (AUC) of each LH pulse and of LH over baseline were significantly reduced by treatment. Levels of progesterone increased (p<0.05) for 150 to 170 min after each ACTH application both in EXP 1 and EXP 2, but not in EXP 3. The mean progesterone concentration was different between trimesters, and ACTH and Controls (1st TP: 30.0 +/- 0.9 and 24.4 +/- 0.7 ng/ml; 2nd TP: 35.5 +/- 0.9 and 29.1 +/- 1.0 ng/ml; 3rd TP: 13.6 +/- 0.2 and 13.1 +/- 0.1 ng/ml; p<0.05). In fetuses (n=87) recovered 3 h after ACTH or saline (EXP 4), the plasma cortisol concentrations were significantly increased in umbilical vein (93.7 +/- 5.5 vs. 47.0 +/- 5.3 nmol/l) and artery (95.7 +/- 5.4 vs. 66.4 +/- 5.4 nmol/l), and in periphery (46.8 +/- 5.3 vs. 27.1 +/- 5.3 nmol/l) compared to controls. Plasma ACTH concentrations, however, did not differ in fetuses of both treatment groups. Postnatal LH-RH challenge tests (1st and 28th day post partum) induced LH surges in female piglets (n=67) both of ACTH and saline treated sows, but did not differ between groups (1st day: 7.2 +/- 0.8 vs. 8.1 +/- 0.7 ng/ml; 28th day: 10.5 +/- 1.7 vs. 13.6 +/- 2.2 ng/ml). However, basal LH of piglets whose mothers were submitted to ACTH during 2nd TP was lower on 1st day (1.7 +/- 0.2 vs. 2.3 +/- 0.2 ng/ml, p<0.05) but not on 28th day (1.0 +/- 0.2 vs. 1.1 +/- 0.2 ng/ml). However in both groups, the basal LH was always higher on 1st as on 28th day (p<0.05). Thus, chronic intermittent ACTH administration is able to influence the release pattern of maternal reproductive hormones. However, these findings demonstrate that these effects are dependent on the stage of pregnancy. Furthermore, it was shown that maternal cortisol can cross the placenta during gestation and thus may affect maternal-fetal interactions and, as a result, reproductive function of offspring.

Stress-related gene expression in brain and adrenal gland of porcine fetuses and neonates.

This study was conducted to examine stress-induced effects on gene expression of specific markers for HPA axis and neuronal activity in fetuses and neonatal pigs. Brain, pituitary gland, and adrenal gland were obtained to determine the mRNA levels for corticotropin-releasing hormone (CRH), CRH receptor 1 (CRHR1), pro-opiomelanocortin (POMC), ACTH receptor (MC2R), c-jun and c-fos. The suitability of these molecular markers was determined in neonatal pigs which were maternally deprived for two hours. It was found that maternal deprivation caused significantly higher transcript levels of c-fos and CRH in brain accompanied by a down-regulation of CRHR1 mRNA and an up-regulation of c-jun in the pituitary gland. To determine the effect of elevated maternal cortisol levels on gene expression of these molecular markers in fetuses, pregnant sows were treated with 100 IU ACTH (Synacthen Depot) s.c. every two days between Day 49 and Day 75 of gestation (normal gestation length 114 days). Animals were killed 48 hours after the last ACTH administration and fetuses of each sow were isolated. The ACTH treatment of sows significantly increased mRNA expression of c-fos but not of CRH in the fetal brain, and significantly decreased MC2R mRNA expression in the adrenal gland. However, HPA axis seems not to be fully developed in Day 77-fetuses because fetal pituitary CRHR1 and POMC mRNA expression was low in most of the fetuses. Although the expression of endocrine regulatory factors was partially incomplete in fetuses at the beginning of the third-trimester, ACTH dependent activation of c-fos mRNA in brain indicates a stress-related increase of neuronal activity. Based on these results it is assumed that prenatal stress in pigs may also have effects on the activity of the HPA axis in the offspring.

Effects of postnatal social isolation on hormonal and immune responses of pigs to an acute endotoxin challenge.

Social stress during early postnatal life often results in long-term effects on neuroendocrine and immune adaptation mechanisms. Therefore, the aim of the present study was to determine the influence of a 2-h daily social isolation from Day 3 to Day 11 on the acute and long-term proinflammatory and neuroendocrine responses of piglets challenged with the bacterial endotoxin lipopolysaccharide (LPS; 100 microg/kg body weight). Peripheral LPS administration significantly increased plasma concentrations of tumor necrosis factor-alpha (TNF-alpha), ACTH and cortisol in isolated and control pigs. However, the activity of the hypothalamic-pituitary-adrenal (HPA) axis after LPS stimulation was not significantly affected by isolation treatment, whereas the prior social isolation diminished the plasma TNF-alpha response to LPS 1 day as well as 45 days after the isolation period. The hippocampal TNF-alpha concentration in response to LPS was also reduced in priorly isolated pigs compared to control animals. Furthermore, the significant increase of TNF-alpha in the spleen caused by LPS was associated with a dramatic decrease in glucocorticoid receptor (GR) binding. The GR binding in hippocampus was increased in isolated pigs and was significantly decreased after LPS injection. In addition, the repeated isolation stressor was shown to increase hippocampal levels of interleukin-1beta (IL-1beta). The present results indicate that repeated social isolation of neonatal pigs may cause long-term effects on proinflammatory regulation at the periphery and in the brain following immune challenge with particular importance of TNF-alpha in mediating these interactions.

Neuroendocrine and immune responses to acute endotoxemia in suckling and weaned piglets.

The objective of this study was to characterize effects of weaning stress on behavioural, endocrine and immune responses to acute peripheral lipopolysaccharide (LPS) challenge in neonatal pigs. Weaning in 28-day-old piglets was accompanied by a significant increase in ACTH concentrations (p = 0.0378) and an increase in basal cortisol level (p = 0.0135). There was also a significant suppressive effect on lymphocyte proliferation in response to concanavalin A (p = 0.0048) in newly weaned piglets. Peripheral administration of LPS induced vomiting, diarrhoea and somnolence in both suckling and weaned piglets. The frequency of these signs of sickness was significantly higher in weaned piglets compared with suckling piglets (p = 0.0049). Additionally, LPS significantly increased plasma concentrations of TNF-alpha, cortisol and ACTH. While weaned piglets reacted to LPS with a higher release of ACTH (p = 0.0239) and cortisol (p = 0.0015) than suckling piglets there was no significant effect of weaning on the magnitude of TNF-alpha. The present data indicate that weaning suppresses the lymphocyte function, causes changes in endocrine regulation and has a substantial effect on the behavioural and endocrine response to an acute peripheral LPS challenge; consequently it could increase disease susceptibility.